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Neuronal Class III beta-Tubulin (TUJ1) Monoclonal Antibody, Purified

RRID:AB_2313773

Antibody ID

AB_2313773

Target Antigen

Neuronal Class III beta-Tubulin (TUJ1) Purified mammalian, other mammalian, hamster, sheep, bovine, horse, rabbit, guinea pig, human, non-human primate, donkey, feline, goat, porcine, canine, mouse, rat

Proper Citation

(Covance Cat# MMS-435P, RRID:AB_2313773)

Clonality

monoclonal antibody

Comments

Discontinued; This product is discontinued at Covance (documented 4/21/2016) and the same as AB_2313773, but was at one point sold in the 250uL quantity; and it is currently offered by BioLegend under the catalog numbers: 801201 and 801202Consolidation on 9/2016: AB_10063408

Clone ID

Clone TUJ1

Host Organism

mouse

Vendor

Covance

Cat Num

MMS-435P also MMS-435P-250

Publications that use this research resource

Changes in Corticotrope Gene Expression Upon Increased Expression of Peptidylglycine α-Amidating Monooxygenase.

  • Mains RE
  • Endocrinology
  • 2018 Jul 1

Literature context:


Abstract:

Throughout evolution, secretion has played an essential role in the ability of organisms and single cells to survive in the face of a changing environment. Peptidylglycine α-amidating monooxygenase (PAM) is an integral membrane monooxygenase, first identified for its role in the biosynthesis of neuroendocrine peptides released by the regulated secretory pathway. PAM was subsequently identified in Chlamydomonas reinhardtii, a unicellular green alga, where it plays an essential role in constitutive secretion and in ciliogenesis. Reduced expression of C. reinhardtii PAM resulted in significant changes in secretion and ciliogenesis. Hence, a screen was performed for transcripts and proteins whose expression responded to changes in PAM levels in a mammalian corticotrope tumor cell line. The goal was to identify genes not previously known to play a role in secretion. The screen identified transcription factors, peptidyl prolyl isomerases, endosomal/lysosomal proteins, and proteins involved in tissue-specific responses to glucose and amino acid availability that had not previously been recognized as relevant to the secretory pathway. Perhaps reflecting the dependence of PAM on molecular oxygen, many PAM-responsive genes are known to be hypoxia responsive. The data highlight the extent to which the performance of the secretory pathway may be integrated into a wide diversity of signaling pathways.

Funding information:
  • Medical Research Council - G0501957(United Kingdom)

MicroRNAs Overcome Cell Fate Barrier by Reducing EZH2-Controlled REST Stability during Neuronal Conversion of Human Adult Fibroblasts.

  • Lee SW
  • Dev. Cell
  • 2018 Jul 2

Literature context:


Abstract:

The ability to convert human somatic cells efficiently to neurons facilitates the utility of patient-derived neurons for studying neurological disorders. As such, ectopic expression of neuronal microRNAs (miRNAs), miR-9/9∗ and miR-124 (miR-9/9∗-124) in adult human fibroblasts has been found to evoke extensive reconfigurations of the chromatin and direct the fate conversion to neurons. However, how miR-9/9∗-124 break the cell fate barrier to activate the neuronal program remains to be defined. Here, we identified an anti-neurogenic function of EZH2 in fibroblasts that acts outside its role as a subunit of Polycomb Repressive Complex 2 to directly methylate and stabilize REST, a transcriptional repressor of neuronal genes. During neuronal conversion, miR-9/9∗-124 induced the repression of the EZH2-REST axis by downregulating USP14, accounting for the opening of chromatin regions harboring REST binding sites. Our findings underscore the interplay between miRNAs and protein stability cascade underlying the activation of neuronal program.

Funding information:
  • NHLBI NIH HHS - P50 HL077107(United States)

Generation of six multiple sclerosis patient-derived induced pluripotent stem cell lines.

  • Miquel-Serra L
  • Stem Cell Res
  • 2018 Jul 3

Literature context:


Abstract:

Multiple sclerosis (MS) is considered a chronic autoimmune disease of the central nervous system that leads to gliosis, demyelination, axonal damage and neuronal death. The MS disease aetiology is unknown, though a polymorphism of the TNFRSF1A gene, rs1800693, is known to confer an increased risk for MS. Using retroviral delivery of reprogramming transgenes, we generated six MS patient-specific iPSC lines with two distinct genotypes, CC or TT, of the polymorphism rs1800693. iPSC lines had normal karyotype, expressed pluripotency genes and differentiated into the three germ layers. These lines offer a good tool to study MS pathomechanisms and for drug testing.

Funding information:
  • NIDDK NIH HHS - DK075386(United States)

The neuroregenerative capacity of olfactory stem cells is not limitless: implications for aging.

  • Child KM
  • J. Neurosci.
  • 2018 Jun 22

Literature context:


Abstract:

The olfactory epithelium (OE) of vertebrates is a highly regenerative neuroepithelium, maintained under normal condition by a population of stem and progenitor cells - globose basal cells (GBCs) that also contribute to epithelial reconstitution after injury. However, aging of the OE often leads to neurogenic exhaustion - the disappearance of both GBCs and olfactory sensory neurons (OSNs). Aneuronal tissue may remain as olfactory, with an uninterrupted sheet of apically arrayed microvillar-capped sustentacular cell, or may undergo respiratory metaplasia. We have generated a transgenic mouse model for neurogenic exhaustion using OMP-driven Tet-off regulation of the A subunit of Diphtheria toxin such that the death of mature OSNs is accelerated. As early as 2 months of age the epithelium of transgenic mice, regardless of sex, recapitulates what is seen in the aged OE of humans and rodents. Areas of the epithelium completely lack neurons and GBCs, while the horizontal basal cells, a reserve stem cell population, show no evidence of activation. Surprisingly, other areas that were olfactory undergo respiratory metaplasia. The impact of accelerated neuronal death and reduced innervation on the olfactory bulb (OB) is also examined. Constant neuronal turnover leaves glomeruli shrunken and impacts the dopaminergic interneurons in the periglomerular layer. Moreover, the acceleration of OSN death can be reversed in those areas where some GBCs persist. However, the projection onto the OB recovers incompletely and the reinnervated glomeruli are markedly altered. Thus, the capacity for OE regeneration is tempered when GBCs disappear.SIGNIFICANCE STATEMENTA large percentage of humans lose or suffer a significant decline in olfactory function as they age. Consequently, quality of life suffers, and safety and nutritional status are put at risk. With age, the OE apparently becomes incapable of fully maintaining the neuronal population of the epithelium despite its well-known capacity for recovering from most forms of injury when younger which may contribute to age-related olfactory loss. Efforts to identify the mechanism by which olfactory neurogenesis becomes exhausted with age require a powerful model for accelerating age-related tissue pathology. The current OMP-tTA;TetO-DTA transgenic mouse model, in which olfactory neurons die when they reach maturity and accelerated death can be aborted to assess the capacity for structural recovery, satisfies that need.

Funding information:
  • NICHD NIH HHS - R01 HD008188-36(United States)
  • NIDCD NIH HHS - R01 DC014217(United States)

Evolution of Cortical Neurogenesis in Amniotes Controlled by Robo Signaling Levels.

  • Cárdenas A
  • Cell
  • 2018 Jun 20

Literature context:


Abstract:

Cerebral cortex size differs dramatically between reptiles, birds, and mammals, owing to developmental differences in neuron production. In mammals, signaling pathways regulating neurogenesis have been identified, but genetic differences behind their evolution across amniotes remain unknown. We show that direct neurogenesis from radial glia cells, with limited neuron production, dominates the avian, reptilian, and mammalian paleocortex, whereas in the evolutionarily recent mammalian neocortex, most neurogenesis is indirect via basal progenitors. Gain- and loss-of-function experiments in mouse, chick, and snake embryos and in human cerebral organoids demonstrate that high Slit/Robo and low Dll1 signaling, via Jag1 and Jag2, are necessary and sufficient to drive direct neurogenesis. Attenuating Robo signaling and enhancing Dll1 in snakes and birds recapitulates the formation of basal progenitors and promotes indirect neurogenesis. Our study identifies modulation in activity levels of conserved signaling pathways as a primary mechanism driving the expansion and increased complexity of the mammalian neocortex during amniote evolution.

Funding information:
  • Wellcome Trust - (United Kingdom)

Repurposing HAMI3379 to Block GPR17 and Promote Rodent and Human Oligodendrocyte Differentiation.

  • Merten N
  • Cell Chem Biol
  • 2018 Jun 21

Literature context:


Abstract:

Identification of additional uses for existing drugs is a hot topic in drug discovery and a viable alternative to de novo drug development. HAMI3379 is known as an antagonist of the cysteinyl-leukotriene CysLT2 receptor, and was initially developed to treat cardiovascular and inflammatory disorders. In our study we identified HAMI3379 as an antagonist of the orphan G protein-coupled receptor GPR17. HAMI3379 inhibits signaling of recombinant human, rat, and mouse GPR17 across various cellular backgrounds, and of endogenous GPR17 in primary rodent oligodendrocytes. GPR17 blockade by HAMI3379 enhanced maturation of primary rat and mouse oligodendrocytes, but was without effect in oligodendrocytes from GPR17 knockout mice. In human oligodendrocytes prepared from inducible pluripotent stem cells, GPR17 is expressed and its activation impaired oligodendrocyte differentiation. HAMI3379, conversely, efficiently favored human oligodendrocyte differentiation. We propose that HAMI3379 holds promise for pharmacological exploitation of orphan GPR17 to enhance regenerative strategies for the promotion of remyelination in patients.

Funding information:
  • NIGMS NIH HHS - GM30186(United States)

Increased Microglial Activity, Impaired Adult Hippocampal Neurogenesis, and Depressive-like Behavior in Microglial VPS35-Depleted Mice.

  • Appel JR
  • J. Neurosci.
  • 2018 Jun 27

Literature context:


Abstract:

Vacuolar sorting protein 35 (VPS35) is a critical component of retromer, which is essential for selective endosome-to-Golgi retrieval of membrane proteins. VPS35 deficiency is implicated in neurodegenerative disease pathology, including Alzheimer's disease (AD). However, exactly how VPS35 loss promotes AD pathogenesis remains largely unclear. VPS35 is expressed in various types of cells in the brain, including neurons and microglia. Whereas neuronal VPS35 plays a critical role in preventing neurodegeneration, the role of microglial VPS35 is largely unknown. Here we provide evidence for microglial VPS35's function in preventing microglial activation and promoting adult hippocampal neurogenesis. VPS35 is expressed in microglia in various regions of the mouse brain, with a unique distribution pattern in a brain region-dependent manner. Conditional knocking out of VPS35 in microglia of male mice results in regionally increased microglial density and activity in the subgranular zone of the hippocampal dentate gyrus (DG), accompanied by elevated neural progenitor proliferation, but decreased neuronal differentiation. Additionally, newborn neurons in the mutant DG show impaired dendritic morphology and reduced dendritic spine density. When examining the behavioral phenotypes of these animals, microglial VPS3S-depleted mice display depression-like behavior and impairment in long-term recognition memory. At the cellular level, VPS35-depleted microglia have grossly enlarged vacuolar structures with increased phagocytic activity toward postsynaptic marker PSD95, which may underlie the loss of dendritic spines observed in the mutant DG. Together, these findings identify an important role of microglial VPS35 in suppressing microglial activation and promoting hippocampal neurogenesis, which are both processes involved in AD pathogenesis.SIGNIFICANCE STATEMENT The findings presented here provide the first in vivo evidence that Vacuolar sorting protein 35 (VPS35)/retromer is essential for regulating microglial function and that when microglial retromer mechanics are disrupted, the surrounding brain tissue can be affected in a neurodegenerative manner. These findings present a novel, microglial-specific role of VPS35 and raise multiple questions regarding the mechanisms underlying our observations. These findings also have myriad implications for the field of retromer research and the role of retromer dysfunction in neurodegenerative pathophysiology. Furthermore, they implicate a pivotal role of microglia in the regulation of adult hippocampal neurogenesis and the survival/integration of newborn neurons in the adult hippocampus.

Funding information:
  • Canadian Institutes of Health Research - (Canada)

Generation of gene-corrected iPSC line from Parkinson's disease patient iPSC line with alpha-SNCA A53T mutation.

  • Lee SY
  • Stem Cell Res
  • 2018 Jun 9

Literature context:


Abstract:

Parkinson's disease (PD) is the second most common age-related neurodegenerative disorder. PD can result from a mutation of alpha-synuclein (α-SNCA), such as α-SNCA A53T. Using episomal vectors, induced pluripotent stem cells (iPSCs) were generated from skin fibroblasts with the α-SNCA A53T mutation. A huge bacterial artificial chromosome (BAC) harboring the normal α-SNCA gene successfully corrected the α-SNCA A53T-mutant iPSCs. Melting curve analysis for allelic composition indicated that the BAC DNA was precisely targeted to the α-SNCA A53T mutation allele, without random integration. The corrected PD-iPSCs displayed the normal karyotype and pluripotency, with the capability to differentiate to any cell type.

Funding information:
  • Medical Research Council - (United Kingdom)

TRPV1 Agonist, Capsaicin, Induces Axon Outgrowth after Injury via Ca2+/PKA Signaling.

  • Frey E
  • eNeuro
  • 2018 Jun 2

Literature context:


Abstract:

Preconditioning nerve injuries activate a pro-regenerative program that enhances axon regeneration for most classes of sensory neurons. However, nociceptive sensory neurons and central nervous system neurons regenerate poorly. In hopes of identifying novel mechanisms that promote regeneration, we screened for drugs that mimicked the preconditioning response and identified a nociceptive ligand that activates a preconditioning-like response to promote axon outgrowth. We show that activating the ion channel TRPV1 with capsaicin induces axon outgrowth of cultured dorsal root ganglion (DRG) sensory neurons, and that this effect is blocked in TRPV1 knockout neurons. Regeneration occurs only in NF200-negative nociceptive neurons, consistent with a cell-autonomous mechanism. Moreover, we identify a signaling pathway in which TRPV1 activation leads to calcium influx and protein kinase A (PKA) activation to induce a preconditioning-like response. Finally, capsaicin administration to the mouse sciatic nerve activates a similar preconditioning-like response and induces enhanced axonal outgrowth, indicating that this pathway can be induced in vivo. These findings highlight the use of local ligands to induce regeneration and suggest that it may be possible to target selective neuronal populations for repair, including cell types that often fail to regenerate.

Funding information:
  • NIDDK NIH HHS - 1R01DK097087(United States)

ER Lipid Defects in Neuropeptidergic Neurons Impair Sleep Patterns in Parkinson's Disease.

  • Valadas JS
  • Neuron
  • 2018 Jun 27

Literature context:


Abstract:

Parkinson's disease patients report disturbed sleep patterns long before motor dysfunction. Here, in parkin and pink1 models, we identify circadian rhythm and sleep pattern defects and map these to specific neuropeptidergic neurons in fly models and in hypothalamic neurons differentiated from patient induced pluripotent stem cells (iPSCs). Parkin and Pink1 control the clearance of mitochondria by protein ubiquitination. Although we do not observe major defects in mitochondria of mutant neuropeptidergic neurons, we do find an excess of endoplasmic reticulum-mitochondrial contacts. These excessive contact sites cause abnormal lipid trafficking that depletes phosphatidylserine from the endoplasmic reticulum (ER) and disrupts the production of neuropeptide-containing vesicles. Feeding mutant animals phosphatidylserine rescues neuropeptidergic vesicle production and acutely restores normal sleep patterns in mutant animals. Hence, sleep patterns and circadian disturbances in Parkinson's disease models are explained by excessive ER-mitochondrial contacts, and blocking their formation or increasing phosphatidylserine levels rescues the defects in vivo.

Funding information:
  • NIGMS NIH HHS - GM073874(United States)

Alpha protocadherins and Pyk2 kinase regulate cortical neuron migration and cytoskeletal dynamics via Rac1 GTPase and WAVE complex in mice.

  • Fan L
  • Elife
  • 2018 Jun 18

Literature context:


Abstract:

Diverse clustered protocadherins are thought to function in neurite morphogenesis and neuronal connectivity in the brain. Here we report that the protocadherin alpha (Pcdha) gene cluster regulates neuronal migration during cortical development and cytoskeletal dynamics in primary cortical culture through the WAVE (Wiskott-Aldrich syndrome family verprolin homologous protein, also known as WASP or Wasf) complex. In addition, overexpression of proline-rich tyrosine kinase 2 (Pyk2, also known as Ptk2b, Cakb, Raftk, Fak2, and Cadtk), a non-receptor cell-adhesion kinase and scaffold protein downstream of Pcdha, impairs cortical neuron migration via inactivation of the small GTPase Rac1. Thus, we define a molecular Pcdha/WAVE/Pyk2/Rac1 axis from protocadherin cell-surface receptors to actin cytoskeletal dynamics in cortical neuron migration in mouse brain.

Funding information:
  • Ministry of Science and Technology of the People's Republic of China - 2017YFA0504203()
  • National Natural Science Foundation of China - 31470820()
  • National Natural Science Foundation of China - 31630039()
  • National Natural Science Foundation of China - 91640118()
  • NIGMS NIH HHS - GM052872(United States)

Palladin Is a Neuron-Specific Translational Target of mTOR Signaling That Regulates Axon Morphogenesis.

  • Umegaki Y
  • J. Neurosci.
  • 2018 May 23

Literature context:


Abstract:

The mTOR signaling pathway regulates protein synthesis and diverse aspects of neuronal morphology that are important for brain development and function. To identify proteins controlled translationally by mTOR signaling, we performed ribosome profiling analyses in mouse cortical neurons and embryonic stem cells upon acute mTOR inhibition. Among proteins whose translation was significantly affected by mTOR inhibition selectively in neurons, we identified the cytoskeletal regulator protein palladin, which is localized within the cell body and axons in hippocampal neurons. Knockdown of palladin eliminated supernumerary axons induced by suppression of the tuberous sclerosis complex protein TSC1 in neurons, demonstrating that palladin regulates neuronal morphogenesis downstream of mTOR signaling. Our findings provide novel insights into an mTOR-dependent mechanism that controls neuronal morphogenesis through translational regulation.SIGNIFICANCE STATEMENT This study reports the discovery of neuron-specific protein translational responses to alterations of mTOR activity. By using ribosome profiling analysis, which can reveal the location and quantity of translating ribosomes on mRNAs, multiple aspects of protein translation were quantitatively analyzed in mouse embryonic stem cells and cortical neurons upon acute mTOR inhibition. Neurons displayed distinct patterns of ribosome occupancy for each codon and ribosome stalling during translation at specific positions of mRNAs. Importantly, the cytoskeletal regulator palladin was identified as a translational target protein of mTOR signaling in neurons. Palladin operates downstream of mTOR to modulate axon morphogenesis. This study identifies a novel mechanism of neuronal morphogenesis regulated by mTOR signaling through control of translation of the key protein palladin.

Funding information:
  • Medical Research Council - (United Kingdom)
  • NINDS NIH HHS - R01 NS051255()

The homeodomain transcription factor Prox1 is a direct target of SoxC proteins during developmental vertebrate neurogenesis.

  • Jacob A
  • J. Neurochem.
  • 2018 May 11

Literature context:


Abstract:

The high-mobility-group-domain containing SoxC transcription factors Sox4 and Sox11 are expressed and required in the vertebrate central nervous system in neuronal precursors and neuroblasts. To identify genes that are widely regulated by SoxC proteins during vertebrate neurogenesis we generated expression profiles from developing mouse brain and chicken neural tube with reduced SoxC expression and found the transcription factor Prox1 strongly downregulated under both conditions. This led us to hypothesize that Prox1 expression depends on SoxC proteins in the developing central nervous system of mouse and chicken. By combining luciferase reporter assays and overexpression in the chicken neural tube with in vivo and in vitro binding studies, we identify the Prox1 gene promoter and two upstream enhancers at -44 kb and -40 kb relative to the transcription start as regulatory regions that are bound and activated by SoxC proteins. This argues that Prox1 is a direct target gene of SoxC proteins during neurogenesis. Electroporations in the chicken neural tube furthermore show that Prox1 activates a subset of SoxC target genes, whereas it has no effects on others. We propose that the transcriptional control of Prox1 by SoxC proteins may ensure coupling of two types of transcription factors that are both required during early neurogenesis, but have at least in part distinct functions. This article is protected by copyright. All rights reserved.

Funding information:
  • NCRR NIH HHS - S10 RR024615(United States)

Generation of a human induced pluripotent stem cell line from a patient with a rare A673T variant in amyloid precursor protein gene that reduces the risk for Alzheimer's disease.

  • Lehtonen Š
  • Stem Cell Res
  • 2018 May 19

Literature context:


Abstract:

An amyloid precursor protein (APP) A673T mutation was found to be protective against Alzheimer's disease (AD) and cognitive decline in the Icelandic population and to associate with decreased levels of plasma β-amyloid in a Finnish population-based cohort. Human fibroblasts from a Finnish male individual carrying the protective mutation were used to generate integration-free induced pluripotent stem cell (iPSCs) line by Sendai virus technology. The iPSC line retained the mutation and expressed pluripotency markers, had a normal karyotype and differentiated into all three germ layers.

Funding information:
  • NCI NIH HHS - CA131967(United States)

A homozygous loss-of-function CAMK2A mutation causes growth delay, frequent seizures and severe intellectual disability.

  • Chia PH
  • Elife
  • 2018 May 22

Literature context:


Abstract:

Calcium/calmodulin-dependent protein kinase II (CAMK2) plays fundamental roles in synaptic plasticity that underlies learning and memory. Here, we describe a new recessive neurodevelopmental syndrome with global developmental delay, seizures and intellectual disability. Using linkage analysis and exome sequencing, we found that this disease maps to chromosome 5q31.1-q34 and is caused by a biallelic germline mutation in CAMK2A. The missense mutation, p.His477Tyr is located in the CAMK2A association domain that is critical for its function and localization. Biochemically, the p.His477Tyr mutant is defective in self-oligomerization and unable to assemble into the multimeric holoenzyme.In vivo, CAMK2AH477Y failed to rescue neuronal defects in C. elegans lacking unc-43, the ortholog of human CAMK2A. In vitro, neurons derived from patient iPSCs displayed profound synaptic defects. Together, our data demonstrate that a recessive germline mutation in CAMK2A leads to neurodevelopmental defects in humans and suggest that dysfunctional CAMK2 paralogs may contribute to other neurological disorders.

Funding information:
  • Agency for Science, Technology and Research - GODAFIT Strategic Positioning Fund()
  • NIGMS NIH HHS - GM080399(United States)

Derivation and characterization of the NIH registry human stem cell line NYSCF100 line under defined feeder-free conditions.

  • Sevilla A
  • Stem Cell Res
  • 2018 Apr 10

Literature context:


Abstract:

The human embryonic stem cell line NYSCFe001-A was derived from a day 6 blastocyst in feeder-free and antibiotic free conditions. The blastocyst was voluntarily donated for research as surplus after in vitro fertilization treatment following informed consent. The NYSCFe001-A line, registered as NYSCF100 on the NIH registry, presents normal karyotype, is mycoplasma free, expresses all the pluripotency markers and has the potential to differentiate into all three germ layers in vitro.

Funding information:
  • NCI NIH HHS - R01CA138998(United States)

Generation and characterization of two human iPSC lines from patients with methylmalonic acidemia cblB type.

  • Richard E
  • Stem Cell Res
  • 2018 Apr 17

Literature context:


Abstract:

Two human induced pluripotent stem cell (iPSC) lines were generated from fibroblasts of two siblings with methylmalonic acidemia cblB type carrying mutations in the MMAB gene: c.287T➔C (p.Ile96Thr) and a splicing loss-of-function variant c.584G➔A affecting the last nucleotide of exon 7 in MMAB (p.Ser174Cysfs*23). Reprogramming factors OCT3/4, SOX2, KLF4 and c-MYC were delivered using a non-integrative method based on the Sendai virus. Once established, iPSCs have shown full pluripotency, differentiation capacity and genetic stability.

Funding information:
  • Telethon - GGP08051(Italy)

Genetic disruption of fractalkine signaling leads to enhanced loss of cochlear afferents following ototoxic or acoustic injury.

  • Kaur T
  • J. Comp. Neurol.
  • 2018 Apr 1

Literature context:


Abstract:

Cochlear hair cells are vulnerable to a variety of insults like acoustic trauma and ototoxic drugs. Such injury can also lead to degeneration of spiral ganglion neurons (SGNs), but this occurs over a period of months to years. Neuronal survival is necessary for the proper function of cochlear prosthetics, therefore, it is of great interest to understand the mechanisms that regulate neuronal survival in deaf ears. We have recently demonstrated that selective hair cell ablation is sufficient to attract leukocytes into the spiral ganglion, and that fractalkine signaling plays a role in macrophage recruitment and in the survival of auditory neurons. Fractalkine (CX3 CL1), a chemokine that regulates adhesion and migration of leukocytes is expressed by SGNs and signals to leukocytes via its receptor CX3 CR1. The present study has extended the previous findings to more clinically relevant conditions of sensorineural hearing loss by examining the role of fractalkine signaling after aminoglycoside ototoxicity or acoustic trauma. Both aminoglycoside treatment and acoustic overstimulation led to the loss of hair cells as well as prolonged increase in the numbers of cochlear leukocytes. Lack of CX3 CR1 did not affect macrophage recruitment after injury, but resulted in increased loss of SGNs and enhanced expression of the inflammatory cytokine interleukin-1β, when compared to mice with intact CX3 CR1. These data indicate that the dysregulation of macrophage response caused by the absence of CX3 CR1 may contribute to inflammation-mediated neuronal loss in the deafened ear, suggesting a key role for inflammation in the long-term survival of target-deprived afferent neurons.

Funding information:
  • FDA HHS - BB/I004823/1(United States)
  • NIDCD NIH HHS - R01 DC006283()
  • NIDCD NIH HHS - R03 DC015320()

Generation of the human induced pluripotent stem cell (hiPSC) line PSMi003-A from a patient affected by an autosomal recessive form of Long QT Syndrome type 1.

  • Mura M
  • Stem Cell Res
  • 2018 Apr 24

Literature context:


Abstract:

We generated human induced pluripotent stem cells (hiPSCs) from dermal fibroblasts of a 51years old female patient homozygous for the mutation c.535 G>A p.G179S on the KCNQ1 gene, causing a severe form of autosomal recessive Long QT Syndrome type 1 (AR-LQT1), not associated with deafness. The hiPSCs, generated using four retroviruses each encoding for a reprogramming factor OCT4, SOX2, KLF4, cMYC, are pluripotent and can differentiate into spontaneously beating cardiomyocytes (hiPSC-CMs).

Funding information:
  • NCI NIH HHS - K22 CA130984(United States)

Generation of 3 spinocerebellar ataxia type 1 (SCA1) patient-derived induced pluripotent stem cell lines LUMCi002-A, B, and C and 2 unaffected sibling control induced pluripotent stem cell lines LUMCi003-A and B.

  • Buijsen RAM
  • Stem Cell Res
  • 2018 Apr 16

Literature context:


Abstract:

Spinocerebellar ataxia type 1 (SCA1) is a hereditary neurodegenerative disease caused by a CAG repeat expansion in exon 8 of the ATXN1 gene. We generated induced pluripotent stem cells (hiPSCs) from a SCA1 patient and his non-affected sister by using non-integrating Sendai Viruses (SeV). The resulting hiPSCs are SeVfree, express pluripotency markers, display a normal karyotype, retain the mutation (length of the CAG repeat expansion in the ATXN1 gene) and are able to differentiate into the three germ layers in vitro.

Funding information:
  • NIAID NIH HHS - T32 AI007638-09(United States)

Generation of the human induced pluripotent stem cell (hiPSC) line PSMi002-A from a patient affected by the Jervell and Lange-Nielsen syndrome and carrier of two compound heterozygous mutations on the KCNQ1 gene.

  • Mura M
  • Stem Cell Res
  • 2018 Apr 21

Literature context:


Abstract:

We report the generation of human induced pluripotent stem cells (hiPSCs) from dermal fibroblasts of a female patient carrier of the two compound heterozygous mutations c.568 C>T p.R190W (maternal allele), and c.1781 G>A p.R594Q (paternal allele) on the KCNQ1 gene, causing Jervell and Lange-Nielsen Syndrome (JLNS). To obtain hiPSCs, we used the classical approach of the four retroviruses each encoding for a reprogramming factor OCT4, SOX2, KLF4, cMYC. The obtained hiPSC clones display pluripotent stem cell characteristics, and differentiate into spontaneously beating cardiomyocytes (hiPSC-CMs).

Funding information:
  • NIDDK NIH HHS - P30 DK019525(United States)

Dissecting the Functional Consequences of De Novo DNA Methylation Dynamics in Human Motor Neuron Differentiation and Physiology.

  • Ziller MJ
  • Cell Stem Cell
  • 2018 Apr 5

Literature context:


Abstract:

The somatic DNA methylation (DNAme) landscape is established early in development but remains highly dynamic within focal regions that overlap with gene regulatory elements. The significance of these dynamic changes, particularly in the central nervous system, remains unresolved. Here, we utilize a powerful human embryonic stem cell differentiation model for the generation of motor neurons (MNs) in combination with genetic mutations in the de novo DNAme machinery. We quantitatively dissect the role of DNAme in directing somatic cell fate with high-resolution genome-wide bisulfite-, bulk-, and single-cell-RNA sequencing. We find defects in neuralization and MN differentiation in DNMT3A knockouts (KO) that can be rescued by the targeting of DNAme to key developmental loci using catalytically inactive dCas9. We also find decreased dendritic arborization and altered electrophysiological properties in DNMT3A KO MNs. Our work provides a list of DNMT3A-regulated targets and a mechanistic link between de novo DNAme, cellular differentiation, and human MN function.

Funding information:
  • NCATS NIH HHS - UL1 TR000457(United States)

Insm1 Induces Neural Progenitor Delamination in Developing Neocortex via Downregulation of the Adherens Junction Belt-Specific Protein Plekha7.

  • Tavano S
  • Neuron
  • 2018 Mar 21

Literature context:


Abstract:

Delamination of neural progenitor cells (NPCs) from the ventricular surface is a crucial prerequisite to form the subventricular zone, the germinal layer linked to the expansion of the mammalian neocortex in development and evolution. Here, we dissect the molecular mechanism by which the transcription factor Insm1 promotes the generation of basal progenitors (BPs). Insm1 protein is most highly expressed in newborn BPs in mouse and human developing neocortex. Forced Insm1 expression in embryonic mouse neocortex causes NPC delamination, converting apical to basal radial glia. Insm1 represses the expression of the apical adherens junction belt-specific protein Plekha7. CRISPR/Cas9-mediated disruption of Plekha7 expression suffices to cause NPC delamination. Plekha7 overexpression impedes the intrinsic and counteracts the Insm1-induced, NPC delamination. Our findings uncover a novel molecular mechanism underlying NPC delamination in which a BP-genic transcription factor specifically targets the integrity of the apical adherens junction belt, rather than adherens junction components as such.

Funding information:
  • Intramural NIH HHS - ZIA BC010763-05(United States)

Purification and Characterization of Schwann Cells from Adult Human Skin and Nerve.

  • Stratton JA
  • eNeuro
  • 2018 Mar 20

Literature context:


Abstract:

Despite its modest capacity for regeneration, peripheral nervous system injury often results in significant long-term disability. Supplementing peripheral nervous system injury with autologous Schwann cells (SCs) may serve to rejuvenate the postinjury environment to enhance regeneration and ultimately improve functional outcomes. However, human nerve-derived SC (hN-SC) collection procedures require invasive surgical resection. Here, we describe the characterization of SCs from adult human skin (hSk-SCs) of four male donors ranging between 27 and 46 years old. Within five weeks of isolating and culturing adherent mixed skin cells, we were able to obtain 3-5 million purified SCs. We found that hSk-SCs appeared transcriptionally indistinguishable from hN-SCs with both populations exhibiting expression of SC genes including: SOX10, SOX9, AP2A1, CDH19, EGR1, ETV5, PAX3, SOX2, CX32, DHH, NECL4, NFATC4, POU3F1, S100B, and YY1. Phenotypic analysis of hSk-SCs and hN-SCs cultures revealed highly enriched populations of SCs indicated by the high percentage of NES+ve, SOX10+ve, s100+ve and p75+ve cells, as well as the expression of a battery of other SC-associated proteins (PAX3, CDH19, ETV5, SOX2, POU3F1, S100B, EGR2, and YY1). We further show that both hSk-SCs and hN-SCs are capable of promoting axonal growth to similar degrees and that a subset of both associate with regenerating axons and form myelin following transplantation into the injured mouse sciatic nerve. Interestingly, although the majority of both hSk-SCs and hN-SCs maintained SOX10 immunoreactivity following transplant, only a subset of each activated the promyelinating factor, POU3F1, and were able to myelinate. Taken together, we demonstrate that adult hSk-SCs are genetically and phenotypically indistinguishable to hN-SCs.

Differential Expression of NF2 in Neuroepithelial Compartments Is Necessary for Mammalian Eye Development.

  • Moon KH
  • Dev. Cell
  • 2018 Jan 8

Literature context:


Abstract:

The optic neuroepithelial continuum of vertebrate eye develops into three differentially growing compartments: the retina, the ciliary margin (CM), and the retinal pigment epithelium (RPE). Neurofibromin 2 (Nf2) is strongly expressed in slowly expanding RPE and CM compartments, and the loss of mouse Nf2 causes hyperplasia in these compartments, replicating the ocular abnormalities seen in human NF2 patients. The hyperplastic ocular phenotypes were largely suppressed by heterozygous deletion of Yap and Taz, key targets of the Nf2-Hippo signaling pathway. We also found that, in addition to feedback transcriptional regulation of Nf2 by Yap/Taz in the CM, activation of Nf2 expression by Mitf in the RPE and suppression by Sox2 in retinal progenitor cells are necessary for the differential growth of the corresponding cell populations. Together, our findings reveal that Nf2 is a key player that orchestrates the differential growth of optic neuroepithelial compartments during vertebrate eye development.

Funding information:
  • NEI NIH HHS - R01 EY013760()
  • NIAMS NIH HHS - R01 AR050772-09(United States)

Intrinsic Immunity Shapes Viral Resistance of Stem Cells.

  • Wu X
  • Cell
  • 2018 Jan 25

Literature context:


Abstract:

Stem cells are highly resistant to viral infection compared to their differentiated progeny; however, the mechanism is mysterious. Here, we analyzed gene expression in mammalian stem cells and cells at various stages of differentiation. We find that, conserved across species, stem cells express a subset of genes previously classified as interferon (IFN) stimulated genes (ISGs) but that expression is intrinsic, as stem cells are refractory to interferon. This intrinsic ISG expression varies in a cell-type-specific manner, and many ISGs decrease upon differentiation, at which time cells become IFN responsive, allowing induction of a broad spectrum of ISGs by IFN signaling. Importantly, we show that intrinsically expressed ISGs protect stem cells against viral infection. We demonstrate the in vivo importance of intrinsic ISG expression for protecting stem cells and their differentiation potential during viral infection. These findings have intriguing implications for understanding stem cell biology and the evolution of pathogen resistance.

Funding information:
  • NIAID NIH HHS - R01 AI091707()
  • NIAID NIH HHS - U19 AI111825()
  • NIDDK NIH HHS - R01 DK100810()
  • NINDS NIH HHS - R01 NS046789-09S1(United States)

Reversible Conformational Conversion of α-Synuclein into Toxic Assemblies by Glucosylceramide.

  • Zunke F
  • Neuron
  • 2017 Dec 20

Literature context:


Abstract:

α-Synuclein (α-syn) aggregation is a key event in Parkinson's disease (PD). Mutations in glycosphingolipid (GSL)-degrading glucocerebrosidase are risk factors for PD, indicating that disrupted GSL clearance plays a key role in α-syn aggregation. However, the mechanisms of GSL-induced aggregation are not completely understood. We document the presence of physiological α-syn conformers in human midbrain dopamine neurons and tested their contribution to the aggregation process. Pathological α-syn assembly mainly occurred through the conversion of high molecular weight (HMW) physiological α-syn conformers into compact, assembly-state intermediates by glucosylceramide (GluCer), without apparent disassembly into free monomers. This process was reversible in vitro through GluCer depletion. Reducing GSLs in PD patient neurons with and without GBA1 mutations diminished pathology and restored physiological α-syn conformers that associated with synapses. Our work indicates that GSLs control the toxic conversion of physiological α-syn conformers in a reversible manner that is amenable to therapeutic intervention by GSL reducing agents.

Funding information:
  • NCI NIH HHS - CA90668(United States)
  • NCRR NIH HHS - P20 RR017677()
  • NINDS NIH HHS - P30 NS081774()
  • NINDS NIH HHS - R01 NS092823()

The dynamics of native Atoh7 protein expression during mouse retinal histogenesis, revealed with a new antibody.

  • Miesfeld JB
  • Gene Expr. Patterns
  • 2017 Dec 12

Literature context:


Abstract:

The Atoh7 transcription factor catalyzes the rate-limiting step in the specification of retinal ganglion cells (RGCs). As a tool to study vertebrate retinal development, we validate an antibody that recognizes human and mouse Atoh7 polypeptide, using informative knockout and transgenic mouse tissues and overexpression experiments. The transient features of Atoh7 protein expression during retinal neurogenesis match the expected pattern at the tissue and cellular level. Further, we compare endogenous Atoh7 to established RGC markers, reporter mouse lines and cell cycle markers, demonstrating the utility of the antibody to investigate molecular mechanisms of retinal histogenesis.

Funding information:
  • NEI NIH HHS - R01 EY013612()
  • NEI NIH HHS - R01 EY014259()
  • NEI NIH HHS - R01 EY019497()
  • NEI NIH HHS - T32 EY015387()
  • NINDS NIH HHS - NS08952(United States)

A Chemical-Genetic Approach Reveals the Distinct Roles of GSK3α and GSK3β in Regulating Embryonic Stem Cell Fate.

  • Chen X
  • Dev. Cell
  • 2017 Dec 4

Literature context:


Abstract:

Glycogen synthase kinase 3 (GSK3) plays a central role in diverse cellular processes. GSK3 has two mammalian isozymes, GSK3α and GSK3β, whose functions remain ill-defined because of a lack of inhibitors that can distinguish between the two highly homologous isozymes. Here, we show that GSK3α and GSK3β can be selectively inhibited in mouse embryonic stem cells (ESCs) using a chemical-genetic approach. Selective inhibition of GSK3β is sufficient to maintain mouse ESC self-renewal, whereas GSK3α inhibition promotes mouse ESC differentiation toward neural lineages. Genome-wide transcriptional analysis reveals that GSK3α and GSK3β have distinct sets of downstream targets. Furthermore, selective inhibition of individual GSK3 isozymes yields distinct phenotypes from gene deletion, highlighting the power of the chemical-genetic approach in dissecting kinase catalytic functions from the protein's scaffolding functions. Our study opens new avenues for defining GSK3 isozyme-specific functions in various cellular processes.

Funding information:
  • NICHD NIH HHS - T32 HD060549()
  • NINDS NIH HHS - R01 NS048276(United States)

The Glia-Neuron Lactate Shuttle and Elevated ROS Promote Lipid Synthesis in Neurons and Lipid Droplet Accumulation in Glia via APOE/D.

  • Liu L
  • Cell Metab.
  • 2017 Nov 7

Literature context:


Abstract:

Elevated reactive oxygen species (ROS) induce the formation of lipids in neurons that are transferred to glia, where they form lipid droplets (LDs). We show that glial and neuronal monocarboxylate transporters (MCTs), fatty acid transport proteins (FATPs), and apolipoproteins are critical for glial LD formation. MCTs enable glia to secrete and neurons to absorb lactate, which is converted to pyruvate and acetyl-CoA in neurons. Lactate metabolites provide a substrate for synthesis of fatty acids, which are processed and transferred to glia by FATP and apolipoproteins. In the presence of high ROS, inhibiting lactate transfer or lowering FATP or apolipoprotein levels decreases glial LD accumulation in flies and in primary mouse glial-neuronal cultures. We show that human APOE can substitute for a fly glial apolipoprotein and that APOE4, an Alzheimer's disease susceptibility allele, is impaired in lipid transport and promotes neurodegeneration, providing insights into disease mechanisms.

Funding information:
  • NIAID NIH HHS - P30 AI036211()
  • NIGMS NIH HHS - R01 GM067858()
  • NIGMS NIH HHS - R01 GM084947()
  • NIGMS NIH HHS - R01 GM120033()

Suppression of MAPK11 or HIPK3 reduces mutant Huntingtin levels in Huntington's disease models.

  • Yu M
  • Cell Res.
  • 2017 Nov 21

Literature context:


Abstract:

Most neurodegenerative disorders are associated with accumulation of disease-relevant proteins. Among them, Huntington disease (HD) is of particular interest because of its monogenetic nature. HD is mainly caused by cytotoxicity of the defective protein encoded by the mutant Huntingtin gene (HTT). Thus, lowering mutant HTT protein (mHTT) levels would be a promising treatment strategy for HD. Here we report two kinases HIPK3 and MAPK11 as positive modulators of mHTT levels both in cells and in vivo. Both kinases regulate mHTT via their kinase activities, suggesting that inhibiting these kinases may have therapeutic values. Interestingly, their effects on HTT levels are mHTT-dependent, providing a feedback mechanism in which mHTT enhances its own level thus contributing to mHTT accumulation and disease progression. Importantly, knockout of MAPK11 significantly rescues disease-relevant behavioral phenotypes in a knockin HD mouse model. Collectively, our data reveal new therapeutic entry points for HD and target-discovery approaches for similar diseases.

Funding information:
  • NIGMS NIH HHS - R35 GM118110(United States)

Autophagy inhibition in endogenous and nutrient-deprived conditions reduces dorsal root ganglia neuron survival and neurite growth in vitro.

  • Clarke JP
  • J. Neurosci. Res.
  • 2017 Nov 29

Literature context:


Abstract:

Peripheral neuropathies can result in cytoskeletal changes in axons, ultimately leading to Wallerian degeneration and cell death. Recently, autophagy has been studied as a potential target for improving axonal survival and growth during peripheral nerve damage. This study investigates the influence of autophagy on adult dorsal root ganglia (DRG) neuron survival and axonal growth under control and nutrient deprivation conditions. Constitutive autophagy was modulated with pharmacological activators (rapamycin; Rapa) and inhibitors (3-methyladenine, bafilomycin A1) in conjunction with either a nutrient-stable environment (standard culture medium) or a nutrient-deprived environment (Hank's balanced salt solution + Ca(2+) /Mg(2+) ). The results demonstrated that autophagy inhibition decreased cell viability and reduced neurite growth and branching complexity. Although autophagy was upregulated with nutrient deprivation compared with the control, it was not further activated by rapamycin, suggesting a threshold level of autophagy. Overall, both cellular and biochemical approaches combined to show the influence of autophagy on adult DRG neuron survival and growth. © 2016 Wiley Periodicals, Inc.

Transplantation of feeder-free human induced pluripotent stem cell-derived cortical neuron progenitors in adult male Wistar rats with focal brain ischemia.

  • Hermanto Y
  • J. Neurosci. Res.
  • 2017 Nov 8

Literature context:


Abstract:

The use of human induced pluripotent stem cells (hiPSCs) eliminates the ethical issues associated with fetal or embryonic materials, thus allowing progress in cell therapy research for ischemic stroke. Strict regulation of cell therapy development requires the xeno-free condition to eliminate clinical complications. Maintenance of hiPSCs with feeder-free condition presents a higher degree of spontaneous differentiation in comparison with conventional cultures. Therefore, feeder-free derivation might be not ideal for developing transplantable hiPSC derivatives. We developed the feeder-free condition for differentiation of cortical neurons from hiPSCs. Then, we evaluated the cells' characteristics upon transplantation into the sham and focal brain ischemia on adult male Wistar rats. Grafts in lesioned brains demonstrated polarized reactivity toward the ischemic border, indicated by directional preferences in axonal outgrowth and cellular migration, with no influence on graft survival. Following the transplantation, forelimb asymmetry was better restored compared with controls. Herein, we provide evidence to support the use of the xeno-free condition for the development of cell therapy for ischemic stroke.

Fibroblast growth factor 2 regulates activity and gene expression of human post-mitotic excitatory neurons.

  • Gupta S
  • J. Neurochem.
  • 2017 Nov 24

Literature context:


Abstract:

Many neuropsychiatric disorders are thought to result from subtle changes in neural circuit formation. We used human embryonic stem cells and induced pluripotent stem cells (hiPSCs) to model mature, post-mitotic excitatory neurons and examine effects of fibroblast growth factor 2 (FGF2). FGF2 gene expression is known to be altered in brain regions of major depressive disorder (MDD) patients and FGF2 has anti-depressive effects in animal models of depression. We generated stable inducible neurons (siNeurons) conditionally expressing human neurogenin-2 (NEUROG2) to generate a homogenous population of post-mitotic excitatory neurons and study the functional as well as the transcriptional effects of FGF2. Upon induction of NEUROG2 with doxycycline, the vast majority of cells are post-mitotic, and the gene expression profile recapitulates that of excitatory neurons within 6 days. Using hES cell lines that inducibly express NEUROG2 as well as GCaMP6f, we were able to characterize spontaneous calcium activity in these neurons and show that calcium transients increase in the presence of FGF2. The FGF2-responsive genes were determined by RNA-Seq. FGF2-regulated genes previously identified in non-neuronal cell types were up-regulated (EGR1, ETV4, SPRY4, and DUSP6) as a result of chronic FGF2 treatment of siNeurons. Novel neuron-specific genes were also identified that may mediate FGF2-dependent increases in synaptic efficacy including NRXN3, SYT2, and GALR1. Since several of these genes have been implicated in MDD previously, these results will provide the basis for more mechanistic studies of the role of FGF2 in MDD.

Generation and characterization of a human iPSC line SANi005-A containing the gray platelet associated heterozygous mutation p.Q287* in GFI1B.

  • Hansen M
  • Stem Cell Res
  • 2017 Oct 22

Literature context:


Abstract:

Peripheral blood mononuclear cells were isolated from an individual harboring a heterozygous c.859C→T p.Q287* mutation in GFI1B, causing an autosomal dominant bleeding disorder, platelet type, 17 (BDPLT17). PBMCs were differentiated to erythroblasts and reprogrammed by lentiviral delivery of a self-silencing hOKSM polycistronic vector. Pluripotency of iPSC line was confirmed by expression of associated markers and by in vitro spontaneous differentiation towards the 3 germ layers. Normal karyotype confirmed the genomic integrity of iPSCs and the presence of disease causing mutation was shown by Sanger sequencing. The generated iPSCs can be used to study BDPLT17 pathophysiology and basic functions of GFI1B.

Generation of human erythroblast-derived iPSC line using episomal reprogramming system.

  • Varga E
  • Stem Cell Res
  • 2017 Oct 19

Literature context:


Abstract:

Peripheral blood mononuclear cells were isolated and cultured to a pure pro-EBL population and reprogrammed using episomal plasmids. The pluripotency of transgene-free induced pluripotent stem cell (iPSC) line was verified by the expression of pluripotency-associated markers and by in vitro spontaneous differentiation towards the 3 germ layers. The iPSC line showed normal karyotype. Peripheral blood is a non-invasive easy accessible cell source and combined with EBL outgrowth in vitro, a routine process obtaining sufficient amount of homogenous cells can be obtained within a week. Using episomal delivery, pro-EBLs can be reprogrammed in a transgene-free, cost effective system.

Paclitaxel Reduces Axonal Bclw to Initiate IP3R1-Dependent Axon Degeneration.

  • Pease-Raissi SE
  • Neuron
  • 2017 Oct 11

Literature context:


Abstract:

Chemotherapy-induced peripheral neuropathy (CIPN) is a debilitating side effect of many cancer treatments. The hallmark of CIPN is degeneration of long axons required for transmission of sensory information; axonal degeneration causes impaired tactile sensation and persistent pain. Currently the molecular mechanisms of CIPN are not understood, and there are no available treatments. Here we show that the chemotherapeutic agent paclitaxel triggers CIPN by altering IP3 receptor phosphorylation and intracellular calcium flux, and activating calcium-dependent calpain proteases. Concomitantly paclitaxel impairs axonal trafficking of RNA-granules and reduces synthesis of Bclw (bcl2l2), a Bcl2 family member that binds IP3R1 and restrains axon degeneration. Surprisingly, Bclw or a stapled peptide corresponding to the Bclw BH4 domain interact with axonal IP3R1 and prevent paclitaxel-induced degeneration, while Bcl2 and BclxL cannot do so. Together these data identify a Bclw-IP3R1-dependent cascade that causes axon degeneration and suggest that Bclw-mimetics could provide effective therapy to prevent CIPN.

Funding information:
  • NCI NIH HHS - R01 CA205255()
  • NCI NIH HHS - R35 CA197583()
  • NCI NIH HHS - R50 CA211399()
  • NINDS NIH HHS - R01 NS050674()

Generation and characterization of human iPSC lines SANi001-A and SANi002-A from mobilized peripheral blood derived megakaryoblasts.

  • Hansen M
  • Stem Cell Res
  • 2017 Oct 22

Literature context:


Abstract:

Mobilized peripheral blood (MPB) CD34+ cells were differentiated to CD34+/CD41+ megakaryoblasts. Cells were sorted to obtain a pure megakaryoblast population which was reprogrammed with a hOKSM self-silencing polycistronic lentiviral vector. Resulting iPSC showed normal karyotype and expression of pluripotency associated markers and in vitro spontaneous differentiation towards the 3 germ layers confirmed pluripotency of iPSC lines. Besides normal iPSC applications, these lines can be used as a control line for other megakaryoid origin iPSC and could be applied for epigenetic based research.

YAP/TAZ and Hedgehog Coordinate Growth and Patterning in Gastrointestinal Mesenchyme.

  • Cotton JL
  • Dev. Cell
  • 2017 Oct 9

Literature context:


Abstract:

YAP/TAZ are the major mediators of mammalian Hippo signaling; however, their precise function in the gastrointestinal tract remains poorly understood. Here we dissect the distinct roles of YAP/TAZ in endodermal epithelium and mesenchyme and find that, although dispensable for gastrointestinal epithelial development and homeostasis, YAP/TAZ function as the critical molecular switch to coordinate growth and patterning in gut mesenchyme. Our genetic analyses reveal that Lats1/2 kinases suppress expansion of the primitive mesenchymal progenitors, where YAP activation also prevents induction of the smooth muscle lineage through transcriptional repression of Myocardin. During later development, zone-restricted downregulation of YAP/TAZ provides the positional cue and allows smooth muscle cell differentiation induced by Hedgehog signaling. Taken together, our studies identify the mesenchymal requirement of YAP/TAZ in the gastrointestinal tract and highlight the functional interplays between Hippo and Hedgehog signaling underlying temporal and spatial control of tissue growth and specification in developing gut.

Nogo-B is the major form of Nogo at the floor plate and likely mediates crossing of commissural axons in the mouse spinal cord.

  • Wang L
  • J. Comp. Neurol.
  • 2017 Sep 1

Literature context:


Abstract:

Using Nogo antibodies with defined binding specificity, Nogo-B, but not Nogo-A, was localized on radial glia in the floor plate of mouse embryos. The presence of Nogo-B was confirmed in Nogo-A knockout mice. In explant cultures of embryonic day (E) 11 and E12 spinal cord, blocking of NgR function with antagonist peptide NEP1-40 reduced the crossing of newly arrived commissural axons, resulting in an accumulation of growth cones in the floor plate. Analysis of growth cone morphology demonstrated an increase in size of growth cones in the floor plate after peptide treatment, which was not detected in axons growing toward the midline. In knockout embryos, midline crossing was not affected by absence of Nogo-A. In co-culture experiments using collagen gel, floor plate showed a strong inhibitory effect on the extension of post-commissural neurites from the spinal cord. This effect was abolished by NEP1-40, and was observed neither in pre-commissural neurites, nor in post-commissural neurites grown with floor plate derived from Nogo-A knockout embryo. Furthermore, western blot analysis of conditioned medium from floor plates showed a truncated form of Nogo with molecular weight of 37 kDa, which could mediate the diffusible effect to axon growth. We conclude that Nogo-B is expressed in the floor plate of mouse embryo, which probably mediates axon crossing in the spinal cord by repelling axons out of the midline when they start upregulate NgR. Nogo acts on axon growth not only through a contact-mediated mechanism, but also through a diffusible mechanism.

Human Astrocyte Maturation Captured in 3D Cerebral Cortical Spheroids Derived from Pluripotent Stem Cells.

  • Sloan SA
  • Neuron
  • 2017 Aug 16

Literature context:


Abstract:

There is significant need to develop physiologically relevant models for investigating human astrocytes in health and disease. Here, we present an approach for generating astrocyte lineage cells in a three-dimensional (3D) cytoarchitecture using human cerebral cortical spheroids (hCSs) derived from pluripotent stem cells. We acutely purified astrocyte-lineage cells from hCSs at varying stages up to 20 months in vitro using immunopanning and cell sorting and performed high-depth bulk and single-cell RNA sequencing to directly compare them to purified primary human brain cells. We found that hCS-derived glia closely resemble primary human fetal astrocytes and that, over time in vitro, they transition from a predominantly fetal to an increasingly mature astrocyte state. Transcriptional changes in astrocytes are accompanied by alterations in phagocytic capacity and effects on neuronal calcium signaling. These findings suggest that hCS-derived astrocytes closely resemble primary human astrocytes and can be used for studying development and modeling disease.

Structure and Cooperativity of the Cytosolic Domain of the CorA Mg2+ Channel from Escherichia coli.

  • Lerche M
  • Structure
  • 2017 Aug 1

Literature context:


Abstract:

Structures of the Mg2+ bound (closed) and apo (open) states of CorA suggests that channel gating is accomplished by rigid-body motions between symmetric and asymmetric assemblies of the cytosolic portions of the five subunits in response to ligand (Mg2+) binding/unbinding at interfacial sites. Here, we structurally and biochemically characterize the isolated cytosolic domain from Escherichia coli CorA. The data reveal an Mg2+-ligand binding site located in a novel position between each of the five subunits and two Mg2+ ions trapped inside the pore. Soaking experiments show that cobalt hexammine outcompetes Mg2+ at the pore site closest to the membrane. This represents the first structural information of how an analog of hexa-hydrated Mg2+ (and competitive inhibitor of CorA) associates to the CorA pore. Biochemical data on the isolated cytoplasmic domain and full-length protein suggests that gating of the CorA channel is governed cooperatively.

The Subventricular Zone Response to Stroke Is Not a Therapeutic Target of Anti-Nogo-A Immunotherapy.

  • Shepherd DJ
  • J. Neuropathol. Exp. Neurol.
  • 2017 Aug 1

Literature context:


Abstract:

Ischemic stroke is a leading cause of adult disability with no pharmacological treatments to promote the recovery of lost function. Neutralizing antibodies against the neurite outgrowth inhibitor Nogo-A have emerged as a promising treatment for subacute and chronic stroke in animal models; however, whether anti-Nogo-A treatment affects poststroke neurogenesis remains poorly understood. In this study, we confirmed expression of Nogo-A by neuroblasts in the adult rat subventricular zone (SVZ), a major neurogenic niche; however, we found no evidence that Nogo-A was expressed at the surface of these cells. In vitro migration assays demonstrated that Nogo-A signaling induced a modest reduction in neuroblast migration speed, while anti-Nogo-A antibodies had no effect on motility properties. Using a permanent distal middle cerebral artery occlusion model of cortical stroke, we found that the number of proliferating cells in the SVZ was unaffected in response to stroke, while neuroblast mobilization from the SVZ toward the stroke lesion correlated positively with lesion size. However, we found no evidence that proliferation or neuroblast mobilization were affected by anti-Nogo-A antibody treatment. Our results suggest that the SVZ is not a therapeutic target of anti-Nogo-A immunotherapy, and contribute to our understanding of the SVZ response to cortical stroke.

Funding information:
  • NINDS NIH HHS - K08NS048858(United States)

Boosting CNS axon regeneration by harnessing antagonistic effects of GSK3 activity.

  • Leibinger M
  • Proc. Natl. Acad. Sci. U.S.A.
  • 2017 Jul 3

Literature context:


Abstract:

Implications of GSK3 activity for axon regeneration are often inconsistent, if not controversial. Sustained GSK3 activity in GSK3S/A knock-in mice reportedly accelerates peripheral nerve regeneration via increased MAP1B phosphorylation and concomitantly reduces microtubule detyrosination. In contrast, the current study shows that lens injury-stimulated optic nerve regeneration was significantly compromised in these knock-in mice. Phosphorylation of MAP1B and CRMP2 was expectedly increased in retinal ganglion cell (RGC) axons upon enhanced GSK3 activity, but, surprisingly, no GSK3-mediated CRMP2 inhibition was detected in sciatic nerves, thus revealing a fundamental difference between central and peripheral axons. Conversely, genetic or shRNA-mediated conditional KO/knockdown of GSK3β reduced inhibitory phosphorylation of CRMP2 in RGCs and improved optic nerve regeneration. Accordingly, GSK3β KO-mediated neurite growth promotion and myelin disinhibition were abrogated by CRMP2 inhibition and largely mimicked in WT neurons upon expression of constitutively active CRMP2 (CRMP2T/A). These results underscore the prevalent requirement of active CRMP2 for optic nerve regeneration. Strikingly, expression of CRMP2T/A in GSK3S/A RGCs further boosted optic nerve regeneration, with axons reaching the optic chiasm within 3 wk. Thus, active GSK3 can also markedly promote axonal growth in central nerves if CRMP2 concurrently remains active. Similar to peripheral nerves, GSK3-mediated MAP1B phosphorylation/activation and the reduction of microtubule detyrosination contributed to this effect. Overall, these findings reconcile conflicting data on GSK3-mediated axon regeneration. In addition, the concept of complementary modulation of normally antagonistically targeted GSK3 substrates offers a therapeutically applicable approach to potentiate the regenerative outcome in the injured CNS.

Enhanced Functional Genomic Screening Identifies Novel Mediators of Dual Leucine Zipper Kinase-Dependent Injury Signaling in Neurons.

  • Welsbie DS
  • Neuron
  • 2017 Jun 21

Literature context:


Abstract:

Dual leucine zipper kinase (DLK) has been implicated in cell death signaling secondary to axonal damage in retinal ganglion cells (RGCs) and other neurons. To better understand the pathway through which DLK acts, we developed enhanced functional genomic screens in primary RGCs, including use of arrayed, whole-genome, small interfering RNA libraries. Explaining why DLK inhibition is only partially protective, we identify leucine zipper kinase (LZK) as cooperating with DLK to activate downstream signaling and cell death in RGCs, including in a mouse model of optic nerve injury, and show that the same pathway is active in human stem cell-derived RGCs. Moreover, we identify four transcription factors, JUN, activating transcription factor 2 (ATF2), myocyte-specific enhancer factor 2A (MEF2A), and SRY-Box 11 (SOX11), as being the major downstream mediators through which DLK/LZK activation leads to RGC cell death. Increased understanding of the DLK pathway has implications for understanding and treating neurodegenerative diseases.

Microglia Are Irrelevant for Neuronal Degeneration and Axon Regeneration after Acute Injury.

  • Hilla AM
  • J. Neurosci.
  • 2017 Jun 21

Literature context:


Abstract:

The role of microglia in degenerative and regenerative processes after damage of the nervous system remains ambiguous, partially due to the paucity of appropriate investigative methods. Here, we show that treatment with the pharmacological colony stimulating factor 1 receptor inhibitor PLX5622 specifically eliminated microglia in murine retinae and optic nerves with high efficiency. Interestingly, time course and extent of retinal ganglion cell (RGC) degeneration after optic nerve crush remained unaffected upon microglia depletion, although remnants of prelabeled apoptotic RGCs were not cleared from the retina in these animals. In addition, microglia depletion neither affected the induction of regeneration associated genes upon optic nerve injury nor the increased regenerative potential of RGCs upon lens injury (LI). However, although the repopulation of the optic nerve lesion site by astrocytes was significantly delayed upon microglia depletion, spontaneous and LI-induced axon regeneration were unaffected by PLX5622 treatment or peripheral macrophage depletion by clodronate liposome treatment. Only concurrent double depletion of microglia and infiltrated macrophages slightly, but significantly, compromised optic nerve regeneration. Therefore, microglia are not essentially involved in RGC degeneration or axonal regeneration after acute CNS injury.SIGNIFICANCE STATEMENT The roles of microglia, the phagocytosing cells of the CNS, and invading macrophages in degenerative and regenerative processes after injury are still controversial and insufficiently characterized. Here, we show that application of a CSF1R inhibitor eliminated virtually all microglia from the visual system, whereas macrophages were spared. Specific microglia depletion impaired the removal of dead labeled retinal ganglion cells after optic nerve crush, but remarkable had no influence on their degeneration. Similarly, optic nerve regeneration was completely unaffected, although repopulation of the lesion site by astrocytes was delayed significantly. Therefore, contrary to previous reports, this experimental approach revealed that microglia seemingly neither promote nor inhibit neuronal degeneration or axonal regrowth within the injured visual system.

Uncoupling of UNC5C with Polymerized TUBB3 in Microtubules Mediates Netrin-1 Repulsion.

  • Shao Q
  • J. Neurosci.
  • 2017 Jun 7

Literature context:


Abstract:

Modulation of microtubule (MT) dynamics is a key event of cytoskeleton remodeling in the growth cone (GC) during axon outgrowth and pathfinding. Our previous studies have shown that the direct interaction of netrin receptor DCC and DSCAM with polymerized TUBB3, a neuron-specific MT subunit in the brain, is required for netrin-1-mediated axon outgrowth, branching, and attraction. Here, we show that uncoupling of polymerized TUBB3 with netrin-1-repulsive receptor UNC5C is involved in netrin-1-mediated axonal repulsion. TUBB3 directly interacted with UNC5C and partially colocalized with UNC5C in the peripheral area of the GC of primary neurons from the cerebellar external granule layer of P2 mouse pups of both sexes. Netrin-1 reduced this interaction as well as the colocalization of UNC5C and TUBB3 in the GC. Results from the in vitro cosedimentation assay indicated that UNC5C interacted with polymerized TUBB3 in MTs and netrin-1 decreased this interaction. Knockdown of either TUBB3 or UNC5C blocked netrin-1-promoted axon repulsion in vitro and caused defects in axon projection of DRG toward the spinal cord in vivo Furthermore, live-cell imaging of end-binding protein 3 tagged with EGFP (EB3-GFP) in primary external granule layer cells showed that netrin-1 differentially increased MT dynamics in the GC with more MT growth in the distal than the proximal region of the GC during repulsion, and knockdown of either UNC5C or TUBB3 abolished the netrin-1 effect. Together, these data indicate that the disengagement of UNC5C with polymerized TUBB3 plays an essential role in netrin-1/UNC5C-mediated axon repulsion.SIGNIFICANCE STATEMENT Proper regulation of microtubule (MT) dynamics in the growth cone plays an important role in axon guidance. However, whether guidance cues modulate MT dynamics directly or indirectly is unclear. Here, we report that dissociation of UNC5C and polymerized TUBB3, the highly dynamic β-tubulin isoform in neurons, is essential for netrin-1/UNC5C-promoted axon repulsion. These results not only provide a working model of direct modulation of MTs by guidance cues in growth cone navigation but also help us to understand molecular mechanisms underlying developmental brain disorders associated with TUBB3 mutations.

Distinct Requirements for FGFR1 and FGFR2 in Primitive Endoderm Development and Exit from Pluripotency.

  • Molotkov A
  • Dev. Cell
  • 2017 Jun 5

Literature context:


Abstract:

Activation of the FGF signaling pathway during preimplantation development of the mouse embryo is known to be essential for differentiation of the inner cell mass and the formation of the primitive endoderm (PrE). We now show using fluorescent reporter knockin lines that Fgfr1 is expressed in all cell populations of the blastocyst, while Fgfr2 expression becomes restricted to extraembryonic lineages, including the PrE. We further show that loss of both receptors prevents the development of the PrE and demonstrate that FGFR1 plays a more prominent role in this process than FGFR2. Finally, we document an essential role for FGFRs in embryonic stem cell (ESC) differentiation, with FGFR1 again having a greater influence than FGFR2 in ESC exit from the pluripotent state. Collectively, these results identify mechanisms through which FGF signaling regulates inner cell mass lineage restriction and cell commitment during preimplantation development.

Funding information:
  • NCI NIH HHS - P30 CA196521()
  • NIDCR NIH HHS - R01 DE022778()

A Brain-Region-Specific Neural Pathway Regulating Germinal Matrix Angiogenesis.

  • Ma S
  • Dev. Cell
  • 2017 May 22

Literature context:


Abstract:

Intimate communication between neural and vascular cells is critical for normal brain development and function. Germinal matrix (GM), a key primordium for the brain reward circuitry, is unique among brain regions for its distinct pace of angiogenesis and selective vulnerability to hemorrhage during development. A major neonatal condition, GM hemorrhage can lead to cerebral palsy, hydrocephalus, and mental retardation. Here we identify a brain-region-specific neural progenitor-based signaling pathway dedicated to regulating GM vessel development. This pathway consists of cell-surface sphingosine-1-phosphate receptors, an intracellular cascade including Gα co-factor Ric8a and p38 MAPK, and target gene integrin β8, which in turn regulates vascular TGF-β signaling. These findings provide insights into region-specific specialization of neurovascular communication, with special implications for deciphering potent early-life endocrine, as well as potential gut microbiota impacts on brain reward circuitry. They also identify tissue-specific molecular targets for GM hemorrhage intervention.

Funding information:
  • NINDS NIH HHS - R01 NS076729()

A Gene Regulatory Network Balances Neural and Mesoderm Specification during Vertebrate Trunk Development.

  • Gouti M
  • Dev. Cell
  • 2017 May 8

Literature context:


Abstract:

Transcriptional networks, regulated by extracellular signals, control cell fate decisions and determine the size and composition of developing tissues. One example is the network controlling bipotent neuromesodermal progenitors (NMPs) that fuel embryo elongation by generating spinal cord and trunk mesoderm tissue. Here, we use single-cell transcriptomics to identify the molecular signature of NMPs and reverse engineer the mechanism that regulates their differentiation. Together with genetic perturbations, this reveals a transcriptional network that integrates opposing retinoic acid (RA) and Wnt signals to determine the rate at which cells enter and exit the NMP state. RA, produced by newly generated mesodermal cells, provides feedback that initiates NMP generation and induces neural differentiation, thereby coordinating the production of neural and mesodermal tissue. Together, the data define a regulatory network architecture that balances the generation of different cell types from bipotential progenitors in order to facilitate orderly axis elongation.

Using DNA Methylation Profiling to Evaluate Biological Age and Longevity Interventions.

  • Petkovich DA
  • Cell Metab.
  • 2017 Apr 4

Literature context:


Abstract:

The DNA methylation levels of certain CpG sites are thought to reflect the pace of human aging. Here, we developed a robust predictor of mouse biological age based on 90 CpG sites derived from partial blood DNA methylation profiles. The resulting clock correctly determines the age of mouse cohorts, detects the longevity effects of calorie restriction and gene knockouts, and reports rejuvenation of fibroblast-derived iPSCs. The data show that mammalian DNA methylomes are characterized by CpG sites that may represent the organism's biological age. They are scattered across the genome, they are distinct in human and mouse, and their methylation gradually changes with age. The clock derived from these sites represents a biomarker of aging and can be used to determine the biological age of organisms and evaluate interventions that alter the rate of aging.

Funding information:
  • NIA NIH HHS - DP1 AG047745()
  • NIA NIH HHS - P01 AG047200()
  • NIA NIH HHS - P30 AG024824()
  • NIA NIH HHS - R01 AG019899()

iPSC-Derived Human Microglia-like Cells to Study Neurological Diseases.

  • Abud EM
  • Neuron
  • 2017 Apr 19

Literature context:


Abstract:

Microglia play critical roles in brain development, homeostasis, and neurological disorders. Here, we report that human microglial-like cells (iMGLs) can be differentiated from iPSCs to study their function in neurological diseases, like Alzheimer's disease (AD). We find that iMGLs develop in vitro similarly to microglia in vivo, and whole-transcriptome analysis demonstrates that they are highly similar to cultured adult and fetal human microglia. Functional assessment of iMGLs reveals that they secrete cytokines in response to inflammatory stimuli, migrate and undergo calcium transients, and robustly phagocytose CNS substrates. iMGLs were used to examine the effects of Aβ fibrils and brain-derived tau oligomers on AD-related gene expression and to interrogate mechanisms involved in synaptic pruning. Furthermore, iMGLs transplanted into transgenic mice and human brain organoids resemble microglia in vivo. Together, these findings demonstrate that iMGLs can be used to study microglial function, providing important new insight into human neurological disease.

Funding information:
  • NCATS NIH HHS - UL1 TR001414()
  • NIA NIH HHS - P01 AG000538()
  • NIA NIH HHS - P50 AG016573()
  • NIA NIH HHS - R01 AG054025()
  • NIA NIH HHS - RF1 AG048099()

Nociceptive DRG neurons express muscle lim protein upon axonal injury.

  • Levin E
  • Sci Rep
  • 2017 Apr 4

Literature context:


Abstract:

Muscle lim protein (MLP) has long been regarded as a cytosolic and nuclear muscular protein. Here, we show that MLP is also expressed in a subpopulation of adult rat dorsal root ganglia (DRG) neurons in response to axonal injury, while the protein was not detectable in naïve cells. Detailed immunohistochemical analysis of L4/L5 DRG revealed ~3% of MLP-positive neurons 2 days after complete sciatic nerve crush and maximum ~10% after 4-14 days. Similarly, in mixed cultures from cervical, thoracic, lumbar and sacral DRG ~6% of neurons were MLP-positive after 2 days and maximal 17% after 3 days. In both, histological sections and cell cultures, the protein was detected in the cytosol and axons of small diameter cells, while the nucleus remained devoid. Moreover, the vast majority could not be assigned to any of the well characterized canonical DRG subpopulations at 7 days after nerve injury. However, further analysis in cell culture revealed that the largest population of MLP expressing cells originated from non-peptidergic IB4-positive nociceptive neurons, which lose their ability to bind the lectin upon axotomy. Thus, MLP is mostly expressed in a subset of axotomized nociceptive neurons and can be used as a novel marker for this population of cells.

Dual leucine zipper kinase-dependent PERK activation contributes to neuronal degeneration following insult.

  • Larhammar M
  • Elife
  • 2017 Apr 25

Literature context:


Abstract:

The PKR-like endoplasmic reticulum kinase (PERK) arm of the Integrated Stress Response (ISR) is implicated in neurodegenerative disease, although the regulators and consequences of PERK activation following neuronal injury are poorly understood. Here we show that PERK signaling is a component of the mouse MAP kinase neuronal stress response controlled by the Dual Leucine Zipper Kinase (DLK) and contributes to DLK-mediated neurodegeneration. We find that DLK-activating insults ranging from nerve injury to neurotrophin deprivation result in both c-Jun N-terminal Kinase (JNK) signaling and the PERK- and ISR-dependent upregulation of the Activating Transcription Factor 4 (ATF4). Disruption of PERK signaling delays neurodegeneration without reducing JNK signaling. Furthermore, DLK is both sufficient for PERK activation and necessary for engaging the ISR subsequent to JNK-mediated retrograde injury signaling. These findings identify DLK as a central regulator of not only JNK but also PERK stress signaling in neurons, with both pathways contributing to neurodegeneration.

Tridimensional Visualization and Analysis of Early Human Development.

  • Belle M
  • Cell
  • 2017 Mar 23

Literature context:


Abstract:

Generating a precise cellular and molecular cartography of the human embryo is essential to our understanding of the mechanisms of organogenesis in normal and pathological conditions. Here, we have combined whole-mount immunostaining, 3DISCO clearing, and light-sheet imaging to start building a 3D cellular map of the human development during the first trimester of gestation. We provide high-resolution 3D images of the developing peripheral nervous, muscular, vascular, cardiopulmonary, and urogenital systems. We found that the adult-like pattern of skin innervation is established before the end of the first trimester, showing important intra- and inter-individual variations in nerve branches. We also present evidence for a differential vascularization of the male and female genital tracts concomitant with sex determination. This work paves the way for a cellular and molecular reference atlas of human cells, which will be of paramount importance to understanding human development in health and disease. PAPERCLIP.

Hallmarks of Alzheimer's Disease in Stem-Cell-Derived Human Neurons Transplanted into Mouse Brain.

  • Espuny-Camacho I
  • Neuron
  • 2017 Mar 8

Literature context:


Abstract:

Human pluripotent stem cells (PSCs) provide a unique entry to study species-specific aspects of human disorders such as Alzheimer's disease (AD). However, in vitro culture of neurons deprives them of their natural environment. Here we transplanted human PSC-derived cortical neuronal precursors into the brain of a murine AD model. Human neurons differentiate and integrate into the brain, express 3R/4R Tau splice forms, show abnormal phosphorylation and conformational Tau changes, and undergo neurodegeneration. Remarkably, cell death was dissociated from tangle formation in this natural 3D model of AD. Using genome-wide expression analysis, we observed upregulation of genes involved in myelination and downregulation of genes related to memory and cognition, synaptic transmission, and neuron projection. This novel chimeric model for AD displays human-specific pathological features and allows the analysis of different genetic backgrounds and mutations during the course of the disease.

Small-Molecule Stabilization of 14-3-3 Protein-Protein Interactions Stimulates Axon Regeneration.

  • Kaplan A
  • Neuron
  • 2017 Mar 8

Literature context:


Abstract:

Damaged central nervous system (CNS) neurons have a poor ability to spontaneously regenerate, causing persistent functional deficits after injury. Therapies that stimulate axon growth are needed to repair CNS damage. 14-3-3 adaptors are hub proteins that are attractive targets to manipulate cell signaling. We identify a positive role for 14-3-3s in axon growth and uncover a developmental regulation of the phosphorylation and function of 14-3-3s. We show that fusicoccin-A (FC-A), a small-molecule stabilizer of 14-3-3 protein-protein interactions, stimulates axon growth in vitro and regeneration in vivo. We show that FC-A stabilizes a complex between 14-3-3 and the stress response regulator GCN1, inducing GCN1 turnover and neurite outgrowth. These findings show that 14-3-3 adaptor protein complexes are druggable targets and identify a new class of small molecules that may be further optimized for the repair of CNS damage.

Differential Routing of Mindbomb1 via Centriolar Satellites Regulates Asymmetric Divisions of Neural Progenitors.

  • Tozer S
  • Neuron
  • 2017 Feb 8

Literature context:


Abstract:

Unequal centrosome maturation correlates with asymmetric division in multiple cell types. Nevertheless, centrosomal fate determinants have yet to be identified. Here, we show that the Notch pathway regulator Mindbomb1 co-localizes asymmetrically with centriolar satellite proteins PCM1 and AZI1 at the daughter centriole in interphase. Remarkably, while PCM1 and AZI1 remain asymmetric during mitosis, Mindbomb1 is associated with either one or both spindle poles. Asymmetric Mindbomb1 correlates with neurogenic divisions and Mindbomb1 is inherited by the prospective neuron. By contrast, in proliferative divisions, a supplementary pool of Mindbomb1 associated with the Golgi apparatus in interphase is released during mitosis and compensates for Mindbomb1 centrosomal asymmetry. Finally, we show that preventing Mindbomb1 centrosomal association induces reciprocal Notch activation between sister cells and promotes symmetric divisions. Thus, we uncover a link between differential centrosome maturation and Notch signaling and reveal an unexpected compensatory mechanism involving the Golgi apparatus in restoring symmetry in proliferative divisions.

Isoform-specific subcellular localization and function of protein kinase A identified by mosaic imaging of mouse brain.

  • Ilouz R
  • Elife
  • 2017 Jan 12

Literature context:


Abstract:

Protein kinase A (PKA) plays critical roles in neuronal function that are mediated by different regulatory (R) subunits. Deficiency in either the RIβ or the RIIβ subunit results in distinct neuronal phenotypes. Although RIβ contributes to synaptic plasticity, it is the least studied isoform. Using isoform-specific antibodies, we generated high-resolution large-scale immunohistochemical mosaic images of mouse brain that provided global views of several brain regions, including the hippocampus and cerebellum. The isoforms concentrate in discrete brain regions, and we were able to zoom-in to show distinct patterns of subcellular localization. RIβ is enriched in dendrites and co-localizes with MAP2, whereas RIIβ is concentrated in axons. Using correlated light and electron microscopy, we confirmed the mitochondrial and nuclear localization of RIβ in cultured neurons. To show the functional significance of nuclear localization, we demonstrated that downregulation of RIβ, but not of RIIβ, decreased CREB phosphorylation. Our study reveals how PKA isoform specificity is defined by precise localization.

DNA-PKcs, ATM, and ATR Interplay Maintains Genome Integrity during Neurogenesis.

  • Enriquez-Rios V
  • J. Neurosci.
  • 2017 Jan 25

Literature context:


Abstract:

The DNA damage response (DDR) orchestrates a network of cellular processes that integrates cell-cycle control and DNA repair or apoptosis, which serves to maintain genome stability. DNA-PKcs (the catalytic subunit of the DNA-dependent kinase, encoded by PRKDC), ATM (ataxia telangiectasia, mutated), and ATR (ATM and Rad3-related) are related PI3K-like protein kinases and central regulators of the DDR. Defects in these kinases have been linked to neurodegenerative or neurodevelopmental syndromes. In all cases, the key neuroprotective function of these kinases is uncertain. It also remains unclear how interactions between the three DNA damage-responsive kinases coordinate genome stability, particularly in a physiological context. Here, we used a genetic approach to identify the neural function of DNA-PKcs and the interplay between ATM and ATR during neurogenesis. We found that DNA-PKcs loss in the mouse sensitized neuronal progenitors to apoptosis after ionizing radiation because of excessive DNA damage. DNA-PKcs was also required to prevent endogenous DNA damage accumulation throughout the adult brain. In contrast, ATR coordinated the DDR during neurogenesis to direct apoptosis in cycling neural progenitors, whereas ATM regulated apoptosis in both proliferative and noncycling cells. We also found that ATR controls a DNA damage-induced G2/M checkpoint in cortical progenitors, independent of ATM and DNA-PKcs. These nonoverlapping roles were further confirmed via sustained murine embryonic or cortical development after all three kinases were simultaneously inactivated. Thus, our results illustrate how DNA-PKcs, ATM, and ATR have unique and essential roles during the DDR, collectively ensuring comprehensive genome maintenance in the nervous system. SIGNIFICANCE STATEMENT: The DNA damage response (DDR) is essential for prevention of a broad spectrum of different human neurologic diseases. However, a detailed understanding of the DDR at a physiological level is lacking. In contrast to many in vitro cellular studies, here we demonstrate independent biological roles for the DDR kinases DNA-PKcs, ATM, and ATR during neurogenesis. We show that DNA-PKcs is central to DNA repair in nonproliferating cells, and restricts DNA damage accumulation, whereas ATR controls damage-induced G2 checkpoint control and apoptosis in proliferating cells. Conversely, ATM is critical for controlling apoptosis in immature noncycling neural cells after DNA damage. These data demonstrate functionally distinct, but cooperative, roles for each kinase in preserving genome stability in the nervous system.

Funding information:
  • NCI NIH HHS - P01 CA096832()
  • NCI NIH HHS - P30 CA021765()
  • NIA NIH HHS - R01 AG027376()
  • NINDS NIH HHS - R01 NS037956()
  • NINDS NIH HHS - R56 NS037956()

ApoE2, ApoE3, and ApoE4 Differentially Stimulate APP Transcription and Aβ Secretion.

  • Huang YA
  • Cell
  • 2017 Jan 26

Literature context:


Abstract:

Human apolipoprotein E (ApoE) apolipoprotein is primarily expressed in three isoforms (ApoE2, ApoE3, and ApoE4) that differ only by two residues. ApoE4 constitutes the most important genetic risk factor for Alzheimer's disease (AD), ApoE3 is neutral, and ApoE2 is protective. How ApoE isoforms influence AD pathogenesis, however, remains unclear. Using ES-cell-derived human neurons, we show that ApoE secreted by glia stimulates neuronal Aβ production with an ApoE4 > ApoE3 > ApoE2 potency rank order. We demonstrate that ApoE binding to ApoE receptors activates dual leucine-zipper kinase (DLK), a MAP-kinase kinase kinase that then activates MKK7 and ERK1/2 MAP kinases. Activated ERK1/2 induces cFos phosphorylation, stimulating the transcription factor AP-1, which in turn enhances transcription of amyloid-β precursor protein (APP) and thereby increases amyloid-β levels. This molecular mechanism also regulates APP transcription in mice in vivo. Our data describe a novel signal transduction pathway in neurons whereby ApoE activates a non-canonical MAP kinase cascade that enhances APP transcription and amyloid-β synthesis.

Funding information:
  • NIA NIH HHS - RF1 AG048131()

Surface topography during neural stem cell differentiation regulates cell migration and cell morphology.

  • Czeisler C
  • J. Comp. Neurol.
  • 2016 Dec 1

Literature context:


Abstract:

We sought to determine the contribution of scaffold topography to the migration and morphology of neural stem cells by mimicking anatomical features of scaffolds found in vivo. We mimicked two types of central nervous system scaffolds encountered by neural stem cells during development in vitro by constructing different diameter electrospun polycaprolactone (PCL) fiber mats, a substrate that we have shown to be topographically similar to brain scaffolds. We compared the effects of large fibers (made to mimic blood vessel topography) with those of small-diameter fibers (made to mimic radial glial process topography) on the migration and differentiation of neural stem cells. Neural stem cells showed differential migratory and morphological reactions with laminin in different topographical contexts. We demonstrate, for the first time, that neural stem cell biological responses to laminin are dependent on topographical context. Large-fiber topography without laminin prevented cell migration, which was partially reversed by treatment with rock inhibitor. Cell morphology complexity assayed by fractal dimension was inhibited in nocodazole- and cytochalasin-D-treated neural precursor cells in large-fiber topography, but was not changed in small-fiber topography with these inhibitors. These data indicate that cell morphology has different requirements on cytoskeletal proteins dependent on the topographical environment encountered by the cell. We propose that the physical structure of distinct scaffolds induces unique signaling cascades that regulate migration and morphology in embryonic neural precursor cells. J. Comp. Neurol. 524:3485-3502, 2016. © 2016 Wiley Periodicals, Inc.

Funding information:
  • NCI NIH HHS - CA92880(United States)

The Mammalian-Specific Protein Armcx1 Regulates Mitochondrial Transport during Axon Regeneration.

  • Cartoni R
  • Neuron
  • 2016 Dec 21

Literature context:


Abstract:

Mitochondrial transport is crucial for neuronal and axonal physiology. However, whether and how it impacts neuronal injury responses, such as neuronal survival and axon regeneration, remain largely unknown. In an established mouse model with robust axon regeneration, we show that Armcx1, a mammalian-specific gene encoding a mitochondria-localized protein, is upregulated after axotomy in this high regeneration condition. Armcx1 overexpression enhances mitochondrial transport in adult retinal ganglion cells (RGCs). Importantly, Armcx1 also promotes both neuronal survival and axon regeneration after injury, and these effects depend on its mitochondrial localization. Furthermore, Armcx1 knockdown undermines both neuronal survival and axon regeneration in the high regenerative capacity model, further supporting a key role of Armcx1 in regulating neuronal injury responses in the adult central nervous system (CNS). Our findings suggest that Armcx1 controls mitochondrial transport during neuronal repair.

Funding information:
  • NEI NIH HHS - P30 EY012196()
  • NEI NIH HHS - R01 EY021242()
  • NEI NIH HHS - R01 EY021526()
  • NICHD NIH HHS - P30 HD018655()
  • NICHD NIH HHS - U54 HD090255()
  • NIGMS NIH HHS - R01 GM069808()
  • NINDS NIH HHS - R01 NS077929()

MEF2C regulates cortical inhibitory and excitatory synapses and behaviors relevant to neurodevelopmental disorders.

  • Harrington AJ
  • Elife
  • 2016 Oct 25

Literature context:


Abstract:

Numerous genetic variants associated with MEF2C are linked to autism, intellectual disability (ID) and schizophrenia (SCZ) - a heterogeneous collection of neurodevelopmental disorders with unclear pathophysiology. MEF2C is highly expressed in developing cortical excitatory neurons, but its role in their development remains unclear. We show here that conditional embryonic deletion of Mef2c in cortical and hippocampal excitatory neurons (Emx1-lineage) produces a dramatic reduction in cortical network activity in vivo, due in part to a dramatic increase in inhibitory and a decrease in excitatory synaptic transmission. In addition, we find that MEF2C regulates E/I synapse density predominantly as a cell-autonomous, transcriptional repressor. Analysis of differential gene expression in Mef2c mutant cortex identified a significant overlap with numerous synapse- and autism-linked genes, and the Mef2c mutant mice displayed numerous behaviors reminiscent of autism, ID and SCZ, suggesting that perturbing MEF2C function in neocortex can produce autistic- and ID-like behaviors in mice.

Funding information:
  • NIGMS NIH HHS - R01GM080477(United States)

Maintenance of age in human neurons generated by microRNA-based neuronal conversion of fibroblasts.

  • Huh CJ
  • Elife
  • 2016 Sep 20

Literature context:


Abstract:

Aging is a major risk factor in many forms of late-onset neurodegenerative disorders. The ability to recapitulate age-related characteristics of human neurons in culture will offer unprecedented opportunities to study the biological processes underlying neuronal aging. Here, we show that using a recently demonstrated microRNA-based cellular reprogramming approach, human fibroblasts from postnatal to near centenarian donors can be efficiently converted into neurons that maintain multiple age-associated signatures. Application of an epigenetic biomarker of aging (referred to as epigenetic clock) to DNA methylation data revealed that the epigenetic ages of fibroblasts were highly correlated with corresponding age estimates of reprogrammed neurons. Transcriptome and microRNA profiles reveal genes differentially expressed between young and old neurons. Further analyses of oxidative stress, DNA damage and telomere length exhibit the retention of age-associated cellular properties in converted neurons from corresponding fibroblasts. Our results collectively demonstrate the maintenance of age after neuronal conversion.

Organ of Corti explants direct tonotopically graded morphology of spiral ganglion neurons in vitro.

  • Smith FL
  • J. Comp. Neurol.
  • 2016 Aug 1

Literature context:


Abstract:

The spiral ganglion is a compelling model system to examine how morphological form contributes to sensory function. While the ganglion is composed mainly of a single class of type I neurons that make simple one-to-one connections with inner hair cell sensory receptors, it has an elaborate overall morphological design. Specific features, such as soma size and axon outgrowth, are graded along the spiral contour of the cochlea. To begin to understand the interplay between different regulators of neuronal morphology, we cocultured neuron explants with peripheral target tissues removed from distinct cochlear locations. Interestingly, these "hair cell microisolates" were capable of both increasing and decreasing neuronal somata size, without adversely affecting survival. Moreover, axon characteristics elaborated de novo by the primary afferents in culture were systematically regulated by the sensory endorgan. Apparent peripheral nervous system (PNS)-like and central nervous system (CNS)-like axonal profiles were established in our cocultures allowing an analysis of putative PNS/CNS axon length ratios. As predicted from the in vivo organization, PNS-like axon bundles elaborated by apical cocultures were longer than their basal counterparts and this phenotype was methodically altered when neuron explants were cocultured with microisolates from disparate cochlear regions. Thus, location-dependent signals within the organ of Corti may set the "address" of neurons within the spiral ganglion, allowing them to elaborate the appropriate tonotopically associated morphological features in order to carry out their signaling function. J. Comp. Neurol. 524:2182-2207, 2016. © 2015 Wiley Periodicals, Inc.

Developmental Inhibition of Gsk3 Rescues Behavioral and Neurophysiological Deficits in a Mouse Model of Schizophrenia Predisposition.

  • Tamura M
  • Neuron
  • 2016 Mar 2

Literature context:


Abstract:

While the genetic basis of schizophrenia is increasingly well characterized, novel treatments will require establishing mechanistic relationships between specific risk genes and core phenotypes. Rare, highly penetrant risk genes such as the 22q11.2 microdeletion are promising in this regard. Df(16)A(+/-) mice, which carry a homologous microdeletion, have deficits in hippocampal-prefrontal connectivity that correlate with deficits in spatial working memory. These mice also have deficits in axonal development that are accompanied by dysregulated Gsk3β signaling and can be rescued by Gsk3 antagonists. Here we show that developmental inhibition of Gsk3 rescues deficits in hippocampal-prefrontal connectivity, task-related neural activity, and spatial working memory behavior in Df(16)A(+/-) mice. Taken together, these results provide mechanistic insight into how the microdeletion results in cognitive deficits, and they suggest possible targets for novel therapies.

Control of Polyamine Biosynthesis by Antizyme Inhibitor 1 Is Important for Transcriptional Regulation of Arginine Vasopressin in the Male Rat Hypothalamus.

  • Greenwood MP
  • Endocrinology
  • 2015 Oct 6

Literature context:


Abstract:

The polyamines spermidine and spermine are small cations present in all living cells. In the brain, these cations are particularly abundant in the neurons of the paraventricular (PVN) and supraoptic nuclei (SON) of the hypothalamus, which synthesize the neuropeptide hormones arginine vasopressin (AVP) and oxytocin. We recently reported increased mRNA expression of antizyme inhibitor 1 (Azin1), an important regulator of polyamine synthesis, in rat SON and PVN as a consequence of 3 days of dehydration. Here we show that AZIN1 protein is highly expressed in both AVP- and oxytocin-positive magnocellular neurons of the SON and PVN together with antizyme 1 (AZ1), ornithine decarboxylase, and polyamines. Azin1 mRNA expression increased in the SON and PVN as a consequence of dehydration, salt loading, and acute hypertonic stress. In organotypic hypothalamic cultures, addition of the irreversible ornithine decarboxylase inhibitor DL-2-(difluoromethyl)-ornithine hydrochloride significantly increased the abundance of heteronuclear AVP but not heteronuclear oxytocin. To identify the function of Azin1 in vivo, lentiviral vectors that either overexpress or knock down Azin1 were stereotaxically delivered into the SON and/or PVN. Azin1 short hairpin RNA delivery resulted in decreased plasma osmolality and had a significant effect on food intake. The expression of AVP mRNA was also significantly increased in the SON by Azin1 short hairpin RNA. In contrast, Azin1 overexpression in the SON decreased AVP mRNA expression. We have therefore identified AZIN1, and hence by inference, polyamines as novel regulators of the expression of the AVP gene.

Funding information:
  • NIMH NIH HHS - 5P50MH086383-04(United States)

Prostacyclin prevents pericyte loss and demyelination induced by lysophosphatidylcholine in the central nervous system.

  • Muramatsu R
  • J. Biol. Chem.
  • 2015 May 1

Literature context:


Abstract:

Pericytes play pivotal roles in physiological and pathophysiological conditions in the central nervous system. As pericytes prevent vascular leakage, they can halt neuronal damage stemming from a compromised blood-brain barrier. Therefore, pericytes may be a good target for the treatment of neurodegenerative disorders, although evidence is lacking. In this study, we show that prostacyclin attenuates lysophosphatidylcholine (LPC)-mediated vascular dysfunction through pericyte protection in the adult mouse spinal cord. LPC decreased the number of pericytes in an in vitro blood-brain barrier model, and this decrease was prevented by iloprost treatment, a prostacyclin analog. Intrathecal administration of iloprost attenuated vascular barrier disruption after LPC injection in the mouse spinal cord. Furthermore, iloprost treatment diminished demyelination and motor function deficits in mice injected with LPC. These results support the notion that prostacyclin acts on pericytes to maintain vascular barrier integrity.

Funding information:
  • Canadian Institutes of Health Research - 89904(Canada)
  • NEI NIH HHS - R01-EY18005(United States)

Characterization of the trunk neural crest in the bamboo shark, Chiloscyllium punctatum.

  • Juarez M
  • J. Comp. Neurol.
  • 2013 Oct 1

Literature context:


Abstract:

The neural crest is a population of mesenchymal cells that after migrating from the neural tube gives rise to structure and cell types: the jaw, part of the peripheral ganglia, and melanocytes. Although much is known about neural crest development in jawed vertebrates, a clear picture of trunk neural crest development for elasmobranchs is yet to be developed. Here we present a detailed study of trunk neural crest development in the bamboo shark, Chiloscyllium punctatum. Vital labeling with dioctadecyl tetramethylindocarbocyanine perchlorate (DiI) and in situ hybridization using cloned Sox8 and Sox9 probes demonstrated that trunk neural crest cells follow a pattern similar to the migratory paths already described in zebrafish and amphibians. We found shark trunk neural crest along the rostral side of the somites, the ventromedial pathway, the branchial arches, the gut, the sensory ganglia, and the nerves. Interestingly, C. punctatum Sox8 and Sox9 sequences aligned with vertebrate SoxE genes, but appeared to be more ancient than the corresponding vertebrate paralogs. The expression of these two SoxE genes in trunk neural crest cells, especially Sox9, matched the Sox10 migratory patterns observed in teleosts. Also of interest, we observed DiI cells and Sox9 labeling along the lateral line, suggesting that in C. punctatum, glial cells in the lateral line are likely of neural crest origin. Although this has been observed in other vertebrates, we are the first to show that the pattern is present in cartilaginous fishes. These findings demonstrate that trunk neural crest cell development in C. punctatum follows the same highly conserved migratory pattern observed in jawed vertebrates.

Global expression profiling of globose basal cells and neurogenic progression within the olfactory epithelium.

  • Krolewski RC
  • J. Comp. Neurol.
  • 2013 Mar 1

Literature context:


Abstract:

Ongoing, lifelong neurogenesis maintains the neuronal population of the olfactory epithelium in the face of piecemeal neuronal turnover and restores it following wholesale loss. The molecular phenotypes corresponding to different stages along the progression from multipotent globose basal cell (GBC) progenitor to differentiated olfactory sensory neuron are poorly characterized. We used the transgenic expression of enhanced green fluorescent protein (eGFP) and cell surface markers to FACS-isolate ΔSox2-eGFP(+) GBCs, Neurog1-eGFP(+) GBCs and immature neurons, and ΔOMP-eGFP(+) mature neurons from normal adult mice. In addition, the latter two populations were also collected 3 weeks after olfactory bulb ablation, a lesion that results in persistently elevated neurogenesis. Global profiling of mRNA from the populations indicates that all stages of neurogenesis share a cohort of >2,100 genes that are upregulated compared to sustentacular cells. A further cohort of >1,200 genes are specifically upregulated in GBCs as compared to sustentacular cells and differentiated neurons. The increased rate of neurogenesis caused by olfactory bulbectomy had little effect on the transcriptional profile of the Neurog1-eGFP(+) population. In contrast, the abbreviated lifespan of ΔOMP-eGFP(+) neurons born in the absence of the bulb correlated with substantial differences in gene expression as compared to the mature neurons of the normal epithelium. Detailed examination of the specific genes upregulated in the different progenitor populations revealed that the chromatin modifying complex proteins LSD1 and coREST were expressed sequentially in upstream ΔSox2-eGFP(+) GBCs and Neurog1-eGFP(+) GBCs/immature neurons. The expression patterns of these proteins are dynamically regulated after activation of the epithelium by methyl bromide lesion.

Funding information:
  • NHLBI NIH HHS - U54-HL108460(United States)

Transgenic quail as a model for research in the avian nervous system: a comparative study of the auditory brainstem.

  • Seidl AH
  • J. Comp. Neurol.
  • 2013 Jan 1

Literature context:


Abstract:

Research performed on transgenic animals has led to numerous advances in biological research. However, using traditional retroviral methods to generate transgenic avian research models has proved problematic. As a result, experiments aimed at genetic manipulations on birds have remained difficult for this popular research tool. Recently, lentiviral methods have allowed the production of transgenic birds, including a transgenic Japanese quail (Coturnix coturnix japonica) line showing neuronal specificity and stable expression of enhanced green fluorescent protein (eGFP) across generations (termed here GFP quail). To test whether the GFP quail may serve as a viable alternative to the popular chicken model system, with the additional benefit of genetic manipulation, we compared the development, organization, structure, and function of a specific neuronal circuit in chicken (Gallus gallus domesticus) with that of the GFP quail. This study focuses on a well-defined avian brain region, the principal nuclei of the sound localization circuit in the auditory brainstem, nucleus magnocellularis (NM), and nucleus laminaris (NL). Our results demonstrate that structural and functional properties of NM and NL neurons in the GFP quail, as well as their dynamic properties in response to changes in the environment, are nearly identical to those in chickens. These similarities demonstrate that the GFP quail, as well as other transgenic quail lines, can serve as an attractive avian model system, with the advantage of being able to build on the wealth of information already available from the chicken.

Funding information:
  • NCI NIH HHS - U24 CA143848(United States)

Developmental dynamics of PAFAH1B subunits during mouse brain development.

  • Escamez T
  • J. Comp. Neurol.
  • 2012 Dec 1

Literature context:


Abstract:

Platelet-activating factor (PAF) mediates an array of biological processes in the mammalian central nervous system as a bioactive lipid messenger in synaptic function and dysfunction (plasticity, memory, and neurodegeneration). The intracellular enzyme that deacetylates the PAF (PAFAH1B) is composed of a tetramer of two catalytic subunits, ALPHA1 (PAFAH1B3) and ALPHA2 (PAFAH1B2), and a regulatory dimer of LIS1 (PAFAH1B1). We have investigated the mouse PAFAH1B subunit genes during brain development in normal mice and in mice with a hypomorphic allele for Lis1 (Lis1/sLis1; Cahana et al. [2001] Proc Natl Acad Sci U S A 98:6429-6434). We have analyzed quantitatively (by means of real-time polymerase chain reaction) and qualitatively (by in situ hybridization techniques) the amounts and expression patterns of their transcription in developing and postnatal brain, focusing mainly on differences in two laminated encephalic regions, the forebrain (telencephalon) and hindbrain (cerebellum) separately. The results revealed significant differences in cDNA content between these two brain subdivisions but, more importantly, between the LIS1 complex subunits. In addition, we found significant spatial differences in gene expression patterns. Comparison of results obtained with Lis1/sLis1 analysis also revealed significant temporal and spatial differences in Alpha1 and Lis1 expression levels. Thus, small changes in the amount of the Lis1 gene may differentially regulate expression of Alpha1 and Alpha2, depending on the brain region, which suggests different roles for each LIS1 complex subunit during neural differentiation and neural migration.

Funding information:
  • European Research Council - 250128(International)
  • NINDS NIH HHS - R01 NS032385(United States)

Synaptic proteins are tonotopically graded in postnatal and adult type I and type II spiral ganglion neurons.

  • Flores-Otero J
  • J. Comp. Neurol.
  • 2011 Jun 1

Literature context:


Abstract:

Inherent in the design of the mammalian auditory system is the precision necessary to transduce complex sounds and transmit the resulting electrical signals to higher neural centers. Unique specializations in the organ of Corti are required to make this conversion, such that mechanical and electrical properties of hair cell receptors are tailored to their specific role in signal coding. Electrophysiological and immunocytochemical characterizations have shown that this principle also applies to neurons of the spiral ganglion, as evidenced by distinctly different firing features and synaptic protein distributions of neurons that innervate high- and low-frequency regions of the cochlea. However, understanding the fine structure of how these properties are distributed along the cochlear partition and within the type I and type II classes of spiral ganglion neurons is necessary to appreciate their functional significance fully. To address this issue, we assessed the localization of the postsynaptic AMPA receptor subunits GluR2 and GluR3 and the presynaptic protein synaptophysin by using immunocytochemical labeling in both postnatal and adult tissue. We report that these presynaptic and postsynaptic proteins are distributed oppositely in relation to the tonotopic map and that they are equally distributed in each neuronal class, thus having an overall gradation from one end of the cochlea to the other. For synaptophysin, an additional layer of heterogeneity was superimposed orthogonal to the tonotopic axis. The highest anti-synaptophysin antibody levels were observed within neurons located close to the scala tympani compared with those located close to the scala vestibuli. Furthermore, we noted that the protein distribution patterns observed in postnatal preparations were largely retained in adult tissue sections, indicating that these features characterize spiral ganglion neurons in the fully developed ear.

Funding information:
  • NIDA NIH HHS - R01 DA014546(United States)

Expression and vesicular localization of mouse Trpml3 in stria vascularis, hair cells, and vomeronasal and olfactory receptor neurons.

  • Castiglioni AJ
  • J. Comp. Neurol.
  • 2011 Apr 15

Literature context:


Abstract:

TRPML3 is a member of the mucolipin branch of the transient receptor potential cation channel family. A dominant missense mutation in Trpml3 (also known as Mcoln3) causes deafness and vestibular impairment characterized by stereocilia disorganization, hair cell loss, and endocochlear potential reduction. Both marginal cells of the stria vascularis and hair cells express Trpml3 mRNA. Here we used in situ hybridization, quantitative RT-qPCR, and immunohistochemistry with several antisera raised against TRPML3 to determine the expression and subcellular distribution of TRPML3 in the inner ear as well as in other sensory organs. We also use Trpml3 knockout tissues to distinguish TRPML3-specific from nonspecific immunoreactivities. We find that TRPML3 localizes to vesicles of hair cells and strial marginal cells but not to stereociliary ankle links or pillar cells, which nonspecifically react with two antisera raised against TRPML3. Upon cochlear maturation, TRPML3 protein is redistributed to perinuclear vesicles of strial marginal cells and is augmented in inner hair cells vs. outer hair cells. Mouse somatosensory neurons, retinal neurons, and taste receptor cells do not appear to express physiologically relevant levels of TRPML3. Finally, we found that vomeronasal and olfactory sensory receptor cells do express TRPML3 mRNA and protein, which localizes to vesicles in their somas and dendrites as well as at apical dendritic knobs.

Funding information:
  • NIEHS NIH HHS - U01 ES017154(United States)

Identification of cerebellin2 in chick and its preferential expression by subsets of developing sensory neurons and their targets in the dorsal horn.

  • Yang M
  • J. Comp. Neurol.
  • 2010 Jul 15

Literature context:


Abstract:

The cerebellins are a family of four secreted proteins, two of which, Cbln1 and Cbln3, play an important role in the formation and maintenance of parallel fiber-Purkinje cell synapses. We have identified the chicken homologue of Cbln2 and, through the use of in situ hybridization, shown that it is expressed by specific subsets of neurons in the dorsal root ganglia (DRGs) and spinal cord starting shortly after those neurons are generated. In the developing spinal cord, Cbln2 is highly expressed by dI1, dI3, dI5, and dILB dorsal interneurons and to a lesser extent by dI2, dI4, dI6, and dILA dorsal interneurons, but not by ventral (v0-v3) interneurons. After the spinal cord has matured and neurons have migrated to their final destinations, Cbln2 is abundant in the dorsal horn. In the DRGs, Cbln2 is expressed by TrkB+ and TrkC+ sensory neurons, but not by TrkA+ sensory neurons. Interestingly, regions of the spinal cord where TrkB+ and TrkC+ afferents terminate (i.e., laminae II, III, IV, and VI) exhibit the highest levels of Cbln2 expression. Cbln2 is also expressed by preganglionic sympathetic neurons and their targets in the sympathetic chain ganglia. Thus, the results show that Cbln2 is frequently expressed by synaptically connected neuronal populations. This, in turn, raises the possibility that if Cbln2, like Cbln1, plays a role in the formation and maintenance of synapses, it may somehow mediate bi-directional communication between discrete populations of neurons and their appropriate neuronal targets.

Funding information:
  • Medical Research Council - R01AG036039(United Kingdom)

Patterned assembly and neurogenesis in the chick dorsal root ganglion.

  • George L
  • J. Comp. Neurol.
  • 2010 Feb 15

Literature context:


Abstract:

The birth of small-diameter TrkA+ neurons that mediate pain and thermoreception begins approximately 24 hours after the cessation of neural crest cell migration from progenitors residing in the nascent dorsal root ganglion. Although multiple geographically distinct progenitor pools have been proposed, this study is the first to comprehensively characterize the derivation of small-diameter neurons. In the developing chick embryo we identify novel patterns in neural crest cell migration and colonization that sculpt the incipient ganglion into a postmitotic neuronal core encapsulated by a layer of proliferative progenitor cells. Furthermore, we show that this outer progenitor layer is composed of three spatially, temporally, and molecularly distinct progenitor zones, two of which give rise to distinct populations of TrkA+ neurons.

Fezf1 is required for penetration of the basal lamina by olfactory axons to promote olfactory development.

  • Watanabe Y
  • J. Comp. Neurol.
  • 2009 Aug 10

Literature context:


Abstract:

In the development of the olfactory system, olfactory receptor neurons (ORNs) project their axons from the olfactory epithelium (OE) to the olfactory bulb (OB). The surface of the OB is covered by the central nervous system (CNS) basal lamina. To establish this connection, pioneer axons of the ORNs penetrate the CNS basal lamina at embryonic day 12.5 in mice. The importance of this penetration is highlighted by the Kallmann syndrome. However, little has been known about the molecular mechanism underlying this penetration process. Fezf1 (also called as Fez, Zfp312-like, and 3110069A13Rik) is a C2H2-type zinc-finger gene expressed in the OE and hypothalamic region in mice. In Fezf1-deficient mice, ORN axons (olfactory axons) do not reach the OB. Here we demonstrate that Fezf1-deficient olfactory axons do not penetrate the CNS basal lamina in vivo, and the penetration activity of the axons in Matrigel is impaired in vitro. Coculture experiments using the OE and OB reveal that axonal projection of ORNs is rescued in Fezf1-deficient mice in which the meninges including the CNS basal lamina are removed from the mutant OB. These data indicate that Fezf1 is required for the penetration of olfactory axons through the CNS basal lamina before they innervate the OB.

The dynamics of long-term transgene expression in engrafted neural stem cells.

  • Lee JP
  • J. Comp. Neurol.
  • 2009 Jul 1

Literature context:


Abstract:

To assess the dynamics and confounding variables that influence transgene expression in neural stem cells (NSCs), we generated distinct NSC clones from the same pool of cells, carrying the same reporter gene transcribed from the same promoter, transduced by the same retroviral vector, and transplanted similarly at the same differentiation state, at the same time and location, into the brains of newborn mouse littermates, and monitored in parallel for over a year in vivo (without immunosuppression). Therefore, the sole variables were transgene chromosomal insertion site and copy number. We then adapted and optimized a technique that tests, at the single cell level, persistence of stem cell-mediated transgene expression in vivo based on correlating the presence of the transgene in a given NSC's nucleus (by fluorescence in situ hybridization [FISH]) with the frequency of that transgene's product within the same cell (by combined immunohistochemistry [IHC]). Under the above-stated conditions, insertion site is likely the most contributory variable dictating transgene downregulation in an NSC after 3 months in vivo. We also observed that this obstacle could be effectively and safely counteracted by simple serial infections (as few as three) inserting redundant copies of the transgene into the prospective donor NSC. (The preservation of normal growth control mechanisms and an absence of tumorigenic potential can be readily screened and ensured ex vivo prior to transplantation.) The combined FISH/IHC strategy employed here for monitoring the dynamics of transgene expression at the single cell level in vivo may be used for other types of therapeutic and housekeeping genes in endogenous and exogenous stem cells of many organs and lineages.

Ultrastructure of the subventricular zone in Macaca fascicularis and evidence of a mouse-like migratory stream.

  • Gil-Perotin S
  • J. Comp. Neurol.
  • 2009 Jun 10

Literature context:


Abstract:

Recent publications have shown that the lateral wall of the lateral ventricles in the Macaca fascicularis brain, in particular the subventricular zone (SVZ), contains neural stem cells throughout adulthood that migrate through a migratory pathway (RMS) to the olfactory bulb (OB). To date, a detailed and systematic cytoarchitectural and ultrastructural study of the monkey SVZ and RMS has not been done. We found that the organization of the SVZ was similar to that of humans, with the ependymal layer surrounding the lateral ventricles, a hypocellular GAP layer formed by astrocytic and ependymal expansions, and the astrocyte ribbon, composed of astrocytic bodies. We found no cells corresponding to the type C proliferating precursor of the rodent brain. Instead, proliferating cells, expressed as Ki-67 immunoreactivity, were predominantly young neurons concentrated in the anterior regions, and occasional astrocytes of the ribbon. We observed displaced ependymal cells of still unknown significance. New neurons tended to organize in chain-like structures, which were surrounded by astrocytes. This pattern was highly reminiscent of that observed in rodent RMS, but not in humans. These chains spread from the frontal SVZ along a GAP-like layer, uniquely composed of astrocytic expansions, to the olfactory bulb (OB). The number of neuronal chains and the number of chain-forming cells decreased gradually upon reaching the OB. The purpose of this work is to provide a reference for future studies in the field of adult neurogenesis that may lead to an understanding of the fate and functionality of newborn neurons in primates, and ultimately in humans.

Funding information:
  • NINDS NIH HHS - R01 NS053976-01(United States)

Developmental and spatial expression pattern of alpha-taxilin in the rat central nervous system.

  • Sakakibara S
  • J. Comp. Neurol.
  • 2008 Nov 1

Literature context:


Abstract:

Alpha-taxilin has been identified as a binding partner of syntaxin family members and thus has been proposed to function in syntaxin-mediated intracellular vesicle trafficking. However, the lack of detailed information concerning the cellular and subcellular localization of alpha-taxilin impedes an understanding of the role of this protein. In the present study, we characterized alpha-taxilin-expressing cells in the rat CNS with a specific antibody. During embryonic development, alpha-taxilin was prominently expressed in nestin-positive neural stem cells in vivo and in vitro. As CNS development proceeded, the alpha-taxilin expression level was rapidly down-regulated. In the postnatal CNS, alpha-taxilin expression was almost confined to the neuronal lineage, with the highest levels of expression in motor neurons within the brainstem nuclei and spinal cord and in primary sensory neurons in mesencephalic trigeminal nucleus. At the cellular level, alpha-taxilin was preferentially located in Nissl substance-like structures with a tigroid or globular morphology within the soma and proximal to dendrites, but it was excluded from terminals. Combined staining with propidium iodide demonstrated that alpha-taxilin distribution overlapped with the cytoplasmic compartment enriched in RNA species, suggesting a close association of alpha-taxilin with actively translating ribosomes or polysomes in neurons. In agreement with this, a recent study indicated the preferential binding of alpha-taxilin to the nascent polypeptide-associated complex (alphaNAC), a dynamic component of the ribosomal exit tunnel in eukaryotic cells. Taken together, these findings suggest that alpha-taxilin plays multiple roles in the generation and maintenance of neurons through modulation of the NAC-mediated translational machinary and/or the syntaxin-mediated vesicle traffic in the soma.

Altered olfactory epithelial structure and function in feline models of mucopolysaccharidoses I and VI.

  • Lischka FW
  • J. Comp. Neurol.
  • 2008 Nov 20

Literature context:


Abstract:

The mucopolysaccharidoses (MPS) are a family of lysosomal storage diseases resulting in developmental defects and, in some types, mental retardation and other neurological symptoms. To gain insight into the neurological dysfunction in MPS, we examined the morphology of olfactory epithelia (OE) and physiology of olfactory receptor neurons (ORNs) in cat models of MPS I, a type in which neuronal lesions are prominent, and MPS VI, in which they are essentially absent. Histopathology showed that both groups of MPS-affected cats had significantly thinner OE than controls. Although immature and mature ORNs were present in both MPS I and VI affected OE, the OE of MPS I-affected cats was structurally disorganized. ORN function was assessed with calcium imaging and patch-clamp recording. Few viable ORNs were recovered from MPS VI cats, but these exhibited normal responses to odors and pharmacological stimuli. In contrast, viable ORNs were as prevalent in MPS I as in controls but were significantly less likely to respond to odor stimuli, although other responses were normal. Disrupted OE organization and impaired ORN function in MPS I, but not MPS VI, corresponds to the central nervous system lesions found in MPS I but not MPS VI. These data represent the first neurophysiological correlate of this correspondence and have implications both for understanding the role of glycosaminoglycans in maintenance of the OE and for targeting further research into the basis for and treatment of the neurological consequences of MPS disorders.

ERM proteins regulate growth cone responses to Sema3A.

  • Mintz CD
  • J. Comp. Neurol.
  • 2008 Oct 1

Literature context:


Abstract:

Axonal growth cones initiate and sustain directed growth in response to cues in their environment. A variety of events such as receptor internalization, kinase activation, and actin rearrangement can be stimulated by guidance cues and are essential for mediating targeted growth cone behavior. Surprisingly little is known about how such disparate actions are coordinated. Our data suggest that ezrin, radixin, and moesin (ERMs), a family of highly homologous, multifunctional proteins may be able to coordinate growth cone responses to the guidance cue Semaphorin 3A (Sema3A). We show that active ERMs concentrate asymmetrically in neocortical growth cones, are rapidly and transiently inactivated by Sema3A, and are required for Sema3A-mediated growth cone collapse and guidance. The FERM domain of active ERMs regulates internalization of the Sema3A receptor, Npn1, and its coreceptor, L1CAM, while the ERM C-terminal domain binds and caps F-actin. Our data support a model in which ERMs can coordinate membrane and actin dynamics in response to Sema3A.

Espin actin-cytoskeletal proteins are in rat type I spiral ganglion neurons and include splice-isoforms with a functional nuclear localization signal.

  • Sekerková G
  • J. Comp. Neurol.
  • 2008 Aug 20

Literature context:


Abstract:

The espins are Ca(2+)-resistant actin-bundling proteins that are enriched in hair cell stereocilia and sensory cell microvilli. Here, we report a novel localization of espins to a large proportion of rat type I spiral ganglion neurons (SGNs) and their projections to the cochlear nucleus (CN). Moreover, we show that a fraction of these espins is in the nucleus of SGNs owing to the presence of splice-isoforms that contain a functional nuclear localization signal (NLS). Espin antibody labeled approximately 83% of type I SGNs, and the labeling intensity increased dramatically during early postnatal development. Type II SGNs and vestibular ganglion neurons were unlabeled. In the CN, espin-positive auditory nerve fibers showed a projection pattern typical of type I SGNs, with intense labeling in the nerve root region and posteroventral CN (PVCN). The anteroventral CN (AVCN) showed moderate labeling, whereas the dorsal CN showed weak labeling that was restricted to the deep layer. Espin-positive synaptic terminals were enriched around nerve root neurons and octopus cells in the PVCN and were also found on globular bushy cells and multipolar neurons in the PVCN and AVCN. SGNs expressed multiple espin transcripts and proteins, including splice-isoforms that contain a nonapeptide, which is rich in positively charged amino acids and creates a bipartite NLS. The nonapeptide was necessary to target espin isoforms to the nucleus and was sufficient to target an unrelated protein to the nucleus when joined with the upstream di-arginine-containing octapeptide. The presence of cytoplasmic and nuclear espins in SGNs suggests additional roles for espins in auditory neuroscience.

Early involvement of synapsin III in neural progenitor cell development in the adult hippocampus.

  • Kao HT
  • J. Comp. Neurol.
  • 2008 Apr 20

Literature context:


Abstract:

Synapsin III is a synaptic vesicle-associated protein that is expressed in cells of the subgranular layer of the hippocampal dentate gyrus, a brain region known to sustain substantial levels of neurogenesis into adulthood. Here we tested the hypothesis that synapsin III plays a role in adult neurogenesis with synapsin III knockout and wild-type mice. Immunocytochemistry of the adult hippocampal dentate gyrus revealed that synapsin III colocalizes with markers of neural progenitor cell development (nestin, PSA-NCAM, NeuN, and Tuj1) but did not colocalize with markers of mitosis (Ki67 and PCNA). Because neurogenesis consists of a number of stages, the proliferation, survival, and differentiation of neural progenitor cells were systematically quantitated in the hippocampal dentate gyrus of adult synapsin III knockout and wild-type mice. We found a 30% decrease in proliferation and a 55% increase in survival of neural progenitor cells in synapsin III knockout mice. We also observed a 6% increase in the number of neural progenitor cells that differentiated into neurons. No difference in the volume of the dentate gyrus was observed between synapsin III knockout and wild-type mice. Collectively, our results demonstrate a novel role for synapsin III in regulating the proliferation of neural progenitor cells in the adult hippocampal dentate gyrus. These findings suggest a distinct function for this synaptic vesicle protein, in addition to its role in neurotransmission.

Funding information:
  • Howard Hughes Medical Institute - (United States)

Temporal and spatial regulation of alpha6 integrin expression during the development of the cochlear-vestibular ganglion.

  • Davies D
  • J. Comp. Neurol.
  • 2007 Jun 10

Literature context:


Abstract:

The neurons of the cochlear-vestibular ganglion (CVG) that innervate the sensory hair cells of the inner ear are derived from the otic epithelium early in development. Neuroblasts detach from neighboring cells, migrate into the mesenchyme where they coalesce to form the ganglion complex, then send processes back into the epithelium. Cell migration and neuronal process formation involve changes in cellular interactions with other cells and proteins in the extracellular matrix that are orchestrated by cell surface-expressed adhesion molecules, including the integrins. I studied the expression pattern of the alpha6 integrin subunit during the early development of the CVG using immunohistochemistry and reverse-transcriptase polymerase chain reaction (RT-PCR) in murine tissue sections, otocyst, and ganglion explants. At embryonic day (E)10.5 alpha6 integrin was expressed in the otic epithelium but not in migrating neuroblasts. Importantly, the loss of alpha6 was associated with exit from the epithelium, not neuronal determination, revealing differentiation cues acutely associated with the cellular environment. Markers of glial and neuronal phenotype showed that alpha6-expressing cells present in the CVG at this stage were glia of neural crest origin. By E12.5 alpha6 expression in the ganglion increased alongside the elaboration of neuronal processes. Immunohistochemistry applied to otocyst cultures in the absence of glia revealed that neuronal processes remained alpha6-negative at this developmental stage and confirmed that alpha6 was expressed by closely apposed glia. The spatiotemporal modulation of alpha6 expression suggests changing roles for this integrin during the early development of inner ear innervation.

Funding information:
  • NICHD NIH HHS - Z01-HD008776(United States)

Localization of the transcriptional coactivator PGC-1alpha to GABAergic neurons during maturation of the rat brain.

  • Cowell RM
  • J. Comp. Neurol.
  • 2007 May 1

Literature context:


Abstract:

The transcriptional coactivator peroxisome proliferator activated receptor gamma coactivator 1alpha (PGC-1alpha) can activate a number of transcription factors to regulate mitochondrial biogenesis and cell-specific responses to cold, fasting, and exercise. Recent studies indicate that PGC-1alpha knockout mice exhibit behavioral abnormalities and progressive vacuolization in various brain regions. To investigate the roles for PGC-1alpha in the nervous system, we evaluated the temporal and cell-specific expression of PGC-1alpha in the normal developing rat brain. Western blot of whole brain homogenates with a PGC-1alpha-specific antibody revealed that PGC-1alpha protein was most abundant in the embryonic and early postnatal forebrain and cerebellum. Using quantitative reverse-transcriptase polymerase chain reaction (RT-PCR), we determined that PGC-1alpha mRNA expression increased most markedly between postnatal days 3 (P3) and 14 in the cortex, striatum, and hippocampus. Immunohistochemical and immunofluorescence analyses of brain tissue indicated that while PGC-1alpha was found in most neuronal populations from embryonic day 15 to P3, it was specifically concentrated in GABAergic populations from P3 to adulthood. Interestingly, PGC-1alpha colocalized with the developmentally regulated chemoattractant reelin in the cortex and hippocampus, and the survival-promoting transcription factor myocyte enhancing factor 2 was highly concentrated in GABAergic populations in the striatum and cerebellum at times of PGC-1alpha expression. These results implicate PGC-1alpha as a regulator of metabolism and/or survival in GABAergic neurons during a phase of mitochondrial and synaptic changes in the developing brain and suggest that PGC-1alpha may be a good target for increasing metabolism in GABAergic populations in neurodevelopmental and neurodegenerative disorders.

Funding information:
  • NINDS NIH HHS - R15 NS087606(United States)

Expression of GATA3 and tenascin in the avian vestibular maculae: normative patterns and changes during sensory regeneration.

  • Warchol ME
  • J. Comp. Neurol.
  • 2007 Feb 1

Literature context:


Abstract:

Sensory receptors in the vestibular organs of birds can regenerate after ototoxic injury. Notably, this regenerative process leads to the restoration of the correct patterning of hair cell phenotype and afferent innervation within the repaired sensory epithelium. The molecular signals that specify cell phenotype and regulate neuronal guidance during sensory regeneration are not known, but they are likely to be similar to the signals that direct these processes during embryonic development. The present study examined the recovery of hair cell phenotype during regeneration in the avian utricle, a vestibular organ that detects linear acceleration and head orientation. First, we show that Type I hair cells in the avian vestibular maculae are immunoreactive for the extracellular matrix molecule tenascin and that treatment with the ototoxic antibiotic streptomycin results in a nearly complete elimination of tenascin immunoreactivity. Cells that express tenascin begin to recover after about 2 weeks and are then contacted by calyx terminals of vestibular neurons. In addition, our previous work had shown that the zinc finger transcription factor GATA3 is uniquely expressed within the striolar reversal zone of the utricle (Hawkins et al. [2003] Hum Mol Genet 12:1261-1272), and we show here that this regionalized expression of GATA3 is maintained after severe hair cell lesions and after transplantation of the sensory epithelium onto a chemically defined substrate. In contrast, the expression of three other supporting cell markers--alpha- and beta-tectorin and SCA--is reduced following ototoxic injury. These observations suggest that GATA3 expression may maintain positional information in the maculae during sensory regeneration.

Funding information:
  • Biotechnology and Biological Sciences Research Council - BBF0083091(United Kingdom)

Aligned neurite bundles of granule cells regulate orientation of Purkinje cell dendrites by perpendicular contact guidance in two-dimensional and three-dimensional mouse cerebellar cultures.

  • Nagata I
  • J. Comp. Neurol.
  • 2006 Nov 10

Literature context:


Abstract:

To identify structures that determine the 90 degree orientation of thin espalier dendritic trees of Purkinje cells with respect to parallel fibers (axonal neurite bundles of granule cells) in the cerebellar cortex, we designed five types of two-dimensional and three-dimensional cell and tissue cultures of cerebella from postnatal mice and analyzed the orientation of Purkinje cell dendrites with respect to neurite bundles and astrocyte fibers by immunofluorescence double or triple staining. We cultured dissociated cerebellar cells on micropatterned substrates and preformed neurite bundles of a microexplant culture two-dimensionally and in matrix gels three-dimensionally. Dendrites, but not axons, of Purkinje cells extended toward the neurites of granule cells and oriented at right angles two-dimensionally to aligned neurite bundles in the three cultures. In a more organized explant proper of the microexplant culture, Purkinje cell dendrites extended toward thin aligned neurite bundles not only consistently at right angles but also two-dimensionally. However, in the "organotypic microexplant culture," in which three-dimensionally aligned thick neurite bundles mimicking parallel fibers were produced, Purkinje cell dendrites often oriented perpendicular to the thick bundles three-dimensionally. Astrocytes were abundant in all cultures, and there was no definite correlation between the presence of and orientation to Purkinje cell dendrites, although their fibers were frequently associated in parallel with dendrites in the organotypic microexplant culture. Therefore, Purkinje cells may grow their dendrites to the newly produced neurite bundles of parallel fibers in the cerebellar cortex and be oriented at right angles three-dimensionally mainly via "perpendicular contact guidance."

Funding information:
  • NIMH NIH HHS - MH-61869(United States)

Postnatal cellular contributions of the hippocampus subventricular zone to the dentate gyrus, corpus callosum, fimbria, and cerebral cortex.

  • Navarro-Quiroga I
  • J. Comp. Neurol.
  • 2006 Aug 10

Literature context:


Abstract:

The rodent dentate gyrus (DG) is formed in the embryo when progenitor cells migrate from the dentate neuroepithelium to establish a germinal zone in the hilus and a secondary germinal matrix, near the fimbria, called the hippocampal subventricular zone (HSVZ). The developmental plasticity of progenitors within the HSVZ is not well understood. To delineate the migratory routes and fates of progenitors within this zone, we injected a replication-incompetent retrovirus, encoding the enhanced green fluorescent protein (EGFP), into the HSVZ of postnatal day 5 (P5) mice. Between P6 and P45, retrovirally-infected EGFP(+) of progenitors migrated into the DG, established a reservoir of progenitor cells, and differentiated into neurons and glia. By P6-7, EGFP(+) cells were observed migrating into the DG. Subsets of these EGFP(+) cells expressed Sox2 and Musashi-1, characteristic of neural stem cells. By P10, EGFP(+) cells assumed positions within the DG and expressed immature neuronal markers. By P20, many EGFP(+) cells expressed the homeobox prospero-like protein Prox1, an early and specific granule cell marker in the CNS, and extended mossy fiber projections into the CA3. A subset of non-neuronal EGFP(+) cells in the dentate gyrus acquired the morphology of astrocytes. Another subset included EGFP(+)/RIP(+) oligodendrocytes that migrated into the fimbria, corpus callosum, and cerebral cortex. Retroviral injections on P15 labeled very few cells, suggesting depletion of HSVZ progenitors by this age. These findings suggest that the early postnatal HSVZ progenitors are multipotent and migratory, and contribute to both dentate gyrus neurogenesis as well as forebrain gliogenesis.

Funding information:
  • NIDDK NIH HHS - R01 DK065806(United States)

Sox3 expression identifies neural progenitors in persistent neonatal and adult mouse forebrain germinative zones.

  • Wang TW
  • J. Comp. Neurol.
  • 2006 Jul 1

Literature context:


Abstract:

Neural precursors persist throughout life in the rodent forebrain subventricular zone (SVZ) and hippocampal dentate gyrus. The regulation of persistent neural stem cells is poorly understood, in part because of the lack of neural progenitor markers. The Sox B1 subfamily of HMG-box transcription factors (Sox1-3) is expressed by precursors in the embryonic nervous system, where these factors maintain neural progenitors in an undifferentiated state while suppressing neuronal differentiation. Sox2 expression persists in germinative zones of the adult rodent brain, but Sox3 expression in the postnatal brain remains largely unexplored. Here we examine Sox3 expression in the neonatal and adult mouse brain to gain insight into its potential involvement in regulating persistent neural stem cells and neurogenesis. We also investigate Sox3 expression during expansion and neural differentiation of postnatal mouse SVZ neural stem cell and human embryonic stem cell (hESC) cultures. We find that Sox3 is expressed transiently by proliferating and differentiating neural progenitors in the SVZ-olfactory bulb pathway and dentate gyrus. Sox3 immunoreactivity also persists in specific postmitotic neuronal populations. In vitro, high Sox3 protein expression levels in undifferentiated, SVZ-derived neurospheres decline markedly with differentiation. Sox3 immunoreactivity in hESCs appears upon differentiation to neural progenitors and then decreases as cells differentiate further into neurons. These findings suggest that Sox3 labels specific stages of hESC-derived and murine neonatal and adult neural progenitors and are consistent with a role for Sox3 in neural stem cell maintenance. Persistent Sox3 expression in some mature neuronal populations suggests additional undefined roles for Sox3 in neuronal function.

Funding information:
  • NIH HHS - U42 OD011175(United States)