Current differentiation protocols for generating mesencephalic dopaminergic (mesDA) neurons from human pluripotent stem cells result in grafts containing only a small proportion of mesDA neurons when transplanted in vivo. In this study, we develop lineage-restricted undifferentiated stem cells (LR-USCs) from pluripotent stem cells, which enhances their potential for differentiating into caudal midbrain floor plate progenitors and mesDA neurons. Using a ventral midbrain protocol, 69% of LR-USCs become bona fide caudal midbrain floor plate progenitors, compared to only 25% of human embryonic stem cells (hESCs). Importantly, LR-USCs generate significantly more mesDA neurons under midbrain and hindbrain conditions in vitro and in vivo. We demonstrate that midbrain-patterned LR-USC progenitors transplanted into 6-hydroxydopamine-lesioned rats restore function in a clinically relevant non-pharmacological behavioral test, whereas midbrain-patterned hESC-derived progenitors do not. This strategy demonstrates how lineage restriction can prevent the development of undesirable lineages and enhance the conditions necessary for mesDA neuron generation.

Herpes simplex encephalitis (HSE) is the most common form of acute viral encephalitis in industrialized countries. Type I interferon (IFN) is important for control of herpes simplex virus (HSV-1) in the central nervous system (CNS). Here we show that microglia are the main source of HSV-induced type I IFN expression in CNS cells and these cytokines are induced in a cGAS-STING-dependent manner. Consistently, mice defective in cGAS or STING are highly susceptible to acute HSE. Although STING is redundant for cell-autonomous antiviral resistance in astrocytes and neurons, viral replication is strongly increased in neurons in STING-deficient mice. Interestingly, HSV-infected microglia confer STING-dependent antiviral activities in neurons and prime type I IFN production in astrocytes through the TLR3 pathway. Thus, sensing of HSV-1 infection in the CNS by microglia through the cGAS-STING pathway orchestrates an antiviral program that includes type I IFNs and immune-priming of other cell types.

The aim of this study was to test the hypothesis that long-term fetal valproic acid (VPA) exposure at doses relevant to the human clinic interferes with normal brain development. Pregnant rats were given intraperitoneal injections of VPA (20mg/kg or 100mg/kg) continuously during the last 9-12 days of pregnancy and during the lactation period until sacrifice on the 23rd postnatal day. Total number of neocortical neurons was estimated using the optical fractionator and frontal cortical thicknesses were sampled in VPA exposed pups compared with an unexposed control group. We found that pups exposed to 20mg/kg and 100mg/kg doses of VPA had statistically significant higher total number of neurons in neocortex by 15.8% and 12.3%, respectively (p<0.05) compared to controls amounting to 15.5×10(6) neocortical neurons (p<0.01). There was no statistical difference between the two VPA groups. Pups exposed to100mg/kg, but not to 20mg/kg VPA displayed a significant (p<0.05) broader (7.5%) of frontal cortical thickness compared to controls. Our results support the hypothesis that fetal exposure of VPA may interfere with normal brain development by disturbing neocortical organization, resulting in overgrowth of frontal lobes and increased neuronal cell numbers. The results indirectly suggest that prenatal VPA may contribute as a causative factor in the brain developmental disturbances equivalent to those seen in human autism spectrum disorders. We therefore suggest that this version of the VPA model may provide a translational model of autism.

Carbamazepine (CBZ) is an anticonvulsant drug used to treat epilepsy and mood disorders. However, it can cause birth defects like reduced head circumference. It was recently shown to protect against brain overgrowth and seizure-induced abnormal plasticity in the megalencephalic mice Kv1.1(mceph/mceph), (mceph/mceph) despite remaining seizures. The mceph/mceph mouse displays two-fold enlarged hippocampus due to more neurons and astrocytes. Using stereology, we found that CBZ normalized the number of neurons and astrocytes in mceph/mceph hippocampus. To characterize CBZ's protective ability on brain growth we studied the gene expression profile of mceph/mceph and wild type hippocampus, with and without CBZ treatment. Microarray analysis revealed transcripts involved in proliferation, differentiation and apoptosis including; NPY, Penk, Vgf, Mlc1, Sstr4, ApoD, Ndn, Aatk, Rgs2 and Gabra5, where Vgf may be of particular interest. The results also support CBZ's effect on synaptic transmission through GABA A receptors, which could promote apoptotic neurodegeneration, affecting cell number.

SorCS1, SorCS2 and SorCS3 belong to the Vps10p-domain family of multiligand receptors. Genetic and functional studies have linked SorCS receptors to psychiatric disorders, Alzheimer's disease and type 2 diabetes, demonstrating critical roles in neuronal functionality and metabolic control. Surprisingly, their structural composition has so far not been studied. Here we have characterized SorCS1, SorCS2 and SorCS3 using biochemical methods and electron microscopy. We found that their purified extracellular domains co-exist in stable dimeric and monomeric populations. This was supported by co-immunoprecipitation experiments, where membrane-bound dimers were successfully pulled down from cell lysate. While dimers were virtually unbreakable, dimerization of the monomeric population was promoted through enzymatic deglycosylation. We conclude that post-translational modifications, specifically the degree and pattern of glycosylation, regulate the oligomeric state of the protein. Hence, cells may dictate ligand specificity by controlling the ratio between monomers and dimers and, therefore, regulate the multiple functions of SorCS receptors.

Schwann cell reprogramming and differentiation are crucial prerequisites for neuronal regeneration and re-myelination to occur following injury to peripheral nerves. The neurotrophin receptor p75NTR has been identified as a positive modulator for Schwann cell myelination during development and implicated in promoting nerve regeneration after injury. However, most studies base this conclusion on results obtained from complete p75NTR knockout mouse models and cannot dissect the specific role of p75NTR expressed by Schwann cells. In this present study, a conditional knockout model selectively deleting p75NTR expression in Schwann cells was generated, where p75NTR expression is replaced with that of an mCherry reporter. Silencing of Schwann cell p75NTR expression was confirmed in the sciatic nerve in vivo and in vitro, without altering axonal expression of p75NTR. No difference in sciatic nerve myelination during development or following sciatic nerve crush injury was observed, as determined by quantification of both myelinated and unmyelinated nerve fiber densities, myelinated axonal diameter and myelin thickness. However, the absence of Schwann cell p75NTR reduced motor nerve conduction velocity after crush injury. Our data indicate that the absence of Schwann cell p75NTR expression in vivo is not critical for axonal regrowth or remyelination following sciatic nerve crush injury, but does play a key role in functional recovery. Overall, this represents the first step in redefining the role of p75NTR in the peripheral nervous system, suggesting that the Schwann cell-axon unit functions as a syncytium, with the previous published involvement of p75NTR in remyelination most likely depending on axonal/neuronal p75NTR and/or mutual glial-axonal interactions.

Schwann cells (SCs) are the main glial cells of the peripheral nervous system (PNS) and are known to be involved in various pathophysiological processes, such as diabetic neuropathy and nerve regeneration, through neurotrophin signaling. Such glial trophic support to axons, as well as neuronal survival/death signaling, has previously been linked to the p75 neurotrophin receptor (p75NTR) and its co-receptor Sortilin. Recently, SC-derived extracellular vesicles (EVs) were shown to be important for axon growth and nerve regeneration, but cargo of these glial cell-derived EVs has not yet been well-characterized. In this study, we aimed to characterize signatures of small RNAs in EVs derived from wild-type (WT) SCs and define differentially expressed small RNAs in EVs derived from SCs with genetic deletions of p75NTR (Ngfr-/-) or Sortilin (Sort1-/-). Using RNA sequencing, we identified a total of 366 miRNAs in EVs derived from WT SCs of which the most highly expressed are linked to the regulation of axonogenesis, axon guidance and axon extension, suggesting an involvement of SC EVs in axonal homeostasis. Signaling of SC EVs to non-neuronal cells was also suggested by the presence of several miRNAs important for regulation of the endothelial cell apoptotic process. Ablated p75NTR or sortilin expression in SCs translated into a set of differentially regulated tRNAs and miRNAs, with impact in autophagy and several cellular signaling pathways such as the phosphatidylinositol signaling system. With this work, we identified the global expression profile of small RNAs present in SC-derived EVs and provided evidence for a regulatory function of these vesicles on the homeostasis of other cell types of the PNS. Differentially identified miRNAs can pave the way to a better understanding of p75NTR and sortilin roles regarding PNS homeostasis and disease.

Depression is a debilitating mental disease, characterized by persistent low mood and anhedonia. Stress represents a major environmental risk factor for depression; the complex interaction of stress with genetic factors results in different individual vulnerability or resilience to the disorder. Dysfunctions of the glutamate system have a primary role in depression. Clinical neuroimaging studies have consistently reported alterations in volume and connectivity of cortico-limbic areas, where glutamate neurons and synapses predominate. This is confirmed by preclinical studies in rodents, showing that repeated stress induces morphological and functional maladaptive changes in the same brain regions altered in humans. Confirming the key role of glutamatergic transmission in depression, compelling evidence has shown that the non-competitive NMDA receptor antagonist, ketamine, induces, at sub-anesthetic dose, rapid and sustained antidepressant response in both humans and rodents. We show here that the Chronic Mild Stress model of depression induces, only in stress-vulnerable rats, depressed-like anhedonic behavior, together with impairment of glutamate/GABA presynaptic release, BDNF mRNA trafficking in dendrites and dendritic morphology in hippocampus. Moreover, we show that a single administration of ketamine restores, in 24 h, normal behavior and most of the cellular/molecular maladaptive changes in vulnerable rats. Interestingly, ketamine treatment did not restore BDNF mRNA levels reduced by chronic stress but rescued dendritic trafficking of BDNF mRNA. The present results are consistent with a mechanism of ketamine involving rapid restoration of synaptic homeostasis, through re-equilibration of glutamate/GABA release and dendritic BDNF for synaptic translation and reversal of synaptic and circuitry impairment.

SORL1, the gene encoding the large multidomain SORLA protein, has emerged as only the fourth gene that when mutated can by itself cause Alzheimer's disease (AD), and as a gene reliably linked to both the early- and late-onset forms of the disease. SORLA is known to interact with the endosomal trafficking regulatory complex called retromer in regulating the recycling of endosomal cargo, including the amyloid precursor protein (APP) and the glutamate receptor GluA1. Nevertheless, SORLA's precise structural-functional relationship in endosomal recycling tubules remains unknown. Here, we address these outstanding questions by relying on crystallographic and artificial-intelligence evidence to generate a structural model for how SORLA folds and fits into retromer-positive endosomal tubules, where it is found to dimerize via both SORLA's fibronectin-type-III (3Fn)- and VPS10p-domains. Moreover, we identify a SORLA fragment comprising the 3Fn-, transmembrane, and cytoplasmic domains that has the capacity to form a dimer, and to enhance retromer-dependent recycling of APP by decreasing its amyloidogenic processing. Collectively, these observations generate a model for how SORLA dimer (and possibly polymer) formation can function in stabilizing and enhancing retromer function at endosome tubules. These findings can inform investigation of the many AD-associated SORL1 variants for evidence of pathogenicity and can guide discovery of novel drugs for the disease.

Peripheral nerve regeneration relies on the ability of Schwann cells to support the regrowth of damaged axons. Schwann cells re-differentiate when reestablishing contact with the sprouting axons, with large fibers becoming remyelinated and small nociceptive fibers ensheathed and collected into Remak bundles. We have previously described how the receptor sortilin facilitates neurotrophin signaling in peripheral neurons via regulated trafficking of Trk receptors. This study aims to characterize the effects of sortilin deletion on nerve regeneration following sciatic crush injury. We found that Sort1 - / - mice displayed functional motor recovery like that of WT mice, with no detectable differences in relation to nerve conduction velocities and morphological aspects of myelinated fibers. In contrast, we found abnormal ensheathment of regenerated C-fibers in injured Sort1 - / - mice, demonstrating a role of sortilin for Remak bundle formation following injury. Further studies on Schwann cell signaling pathways showed a significant reduction of MAPK/ERK, RSK, and CREB phosphorylation in Sort1 - / - Schwann cells after stimulation with neurotrophin-3 (NT-3), while Schwann cell migration and myelination remained unaffected. In conclusion, our results demonstrate that loss of sortilin blunts NT-3 signaling in Schwann cells which might contribute to the impaired Remak bundle regeneration after sciatic nerve injury.

Neuropathological observations in neurodegenerative synucleinopathies, including Parkinson disease, implicate a pathological role of α-synuclein accumulation in extranigral sites during the prodromal phase of the disease. In a transgenic mouse model of peripheral-to-central neuroinvasion and propagation of α-synuclein pathology (via hindlimb intramuscular inoculation with exogenous fibrillar α-synuclein: the M83 line, expressing the mutant human Ala53Thr α-synuclein), we studied the development and early-stage progression of α-synuclein pathology in the CNS of non-symptomatic (i.e. freely mobile) mice. By immunohistochemical analyses of phosphroylated α-synuclein on serine residue 129 (p-S129), our data indicate that the incipient stage of pathological α-synuclein propagation could be categorized in distinct phases: (i) initiation phase, whereby α-synuclein fibrillar inoculum induced pathological lesions in pools of premotor and motor neurons of the lumbar spinal cord, as early as 14 days post-inoculation; (ii) early central phase, whereby incipient α-synuclein pathology was predominantly detected in the reticular nuclei of the brainstem; and (iii) late central phase, characterized by additional sites of lesions in the brain including vestibular nuclei, deep cerebellar nuclei and primary motor cortex, with coincidental emergence of a sensorimotor deficit (mild degree of hindlimb clasping). Intriguingly, we also detected progressive α-synuclein pathology in premotor and motor neurons in the thoracic spinal cord, which does not directly innervate the hindlimb, as well as in the oligodendroglia within the white matter tracts of the CNS during this prodromal phase. Collectively, our data provide crucial insights into the spatiotemporal propagation of α-synuclein pathology in the nervous system of this rodent model of α-synucleinopathy following origin in periphery, and present a neuropathological context for the progression from pre-symptomatic stage to an early deficit in sensorimotor coordination. These findings also hint towards a therapeutic window for targeting the early stages of α-synuclein pathology progression in this model, and potentially facilitate the discovery of mechanisms relevant to α-synuclein proteinopathies. In a rodent model of synucleinopathy, Ferreira et al., delineate the spatiotemporal progression of incipient α-synuclein pathology (of peripheral origin) in the CNS. The authors show early affection of brainstem reticular nuclei in non-paralyzed mice, and pathological white matter lesions in relation to the neuronal pathology.

Stress plays a crucial role in the pathogenesis of psychiatric disorders and affects neuronal plasticity in different brain regions. We have previously found that acute foot-shock (FS) stress elicits fast and long-lasting functional and morphological remodeling of excitatory neurons in the prefrontal cortex (PFC), which were partly prevented by the pretreatment with antidepressants. Here we investigated, whether acute stress and pretreatment with desipramine (DMI) interfere in hippocampal dendritic remodeling. Male Sprague-Dawley rats were subjected to acute FS-stress, followed by measurement of time-dependent (1, 7 and 14 days) structural plasticity (dendritic arborization, spine number and morphology) in hippocampal CA1 pyramidal neurons and expression patterns of molecular markers implicated in neuronal plasticity. We found that acute stress significantly decreased spine number, dendritic length, and altered spine morphometric parameters at all time points evaluated after stress. This was paralleled by changes in the gene expression of Spinophilin and Cdc42, and protein expression of homer1. Pretreatment with DMI prevented the stress-induced dendritic atrophy and spine loss 14 days after acute FS. However, DMI treatment without stress differentially affected the expression patterns of spine-related genes and proteins. In conclusion, acute FS-stress and pretreatment with DMI significantly changed dendritic morphology, including number and morphology of spines, and the length of dendrites in hippocampal CA1 pyramidal cells as early as 1 day, and sustained up to 14 days after acute FS. The findings were paralleled by changes in gene and protein expression of actin binding and cytoskeletal proteins, Rho GTPases, and postsynaptic scaffolding proteins.

Stroke is a major cause of death and severe disability, but effective treatments are limited. Neuroglobin, a neuronal heme-globin, has been advocated as a novel pharmacological target in combating stroke and neurodegenerative disorders based on cytoprotective properties. Using thoroughly validated antibodies and oligos, we give a detailed brain anatomical characterization of transgenic mice over expressing Neuroglobin. Moreover, using permanent middle artery occlusion the effect of elevated levels of Neuroglobin on ischemic damage was studied. Lastly, the impact of mouse strain genetic background on ischemic damage was investigated.

Techniques involving three-dimensional (3D) tissue structure reconstruction and analysis provide a better understanding of changes in molecules and function. We have developed AutoCUTS-LM, an automated system that allows the latest advances in 3D tissue reconstruction and cellular analysis developments using light microscopy on various tissues, including archived tissue. The workflow in this paper involved advanced tissue sampling methods of the human cerebral cortex, an automated serial section collection system, digital tissue library, cell detection using convolution neural network, 3D cell reconstruction, and advanced analysis. Our results demonstrated the detailed structure of pyramidal cells (number, volume, diameter, sphericity and orientation) and their 3D spatial organization are arranged in a columnar structure. The pipeline of these combined techniques provides a detailed analysis of tissues and cells in biology and pathology.

Autonomic dysfunction is a common complication of small-fiber neuropathy (SFN). In this study we aimed to assess the applicability of autonomic microvascular indices as a potential marker for SFN assessment.

The schizophrenia-associated gene, BRD1, encodes an epigenetic regulator in which chromatin interactome is enriched with genes implicated in mental health. Alterations in histone modifications and epigenetic regulation contribute to brain transcriptomic changes in affective disorders and preclinical data supports a role for BRD1 in psychopathology. However, the implication of BRD1 on affective pathology remains poorly understood. In this study, we assess affective behaviors and associated neurobiology in Brd1+/- mice along with their responses to Fluoxetine and Imipramine. This involves behavioral, neurostructural, and neurochemical characterizations along with regional cerebral gene expression profiling combined with integrative functional genomic analyses. We report behavioral changes in female Brd1+/- mice with translational value to depressive symptomatology that can be alleviated by the administration of antidepressant medications. Behavioral changes are accompanied by altered brain morphometry and imbalances in monoaminergic systems. In accordance, gene expression changes across brain tissues reveal altered neurotransmitter signaling and cluster in functional pathways associated with depression including 'Adrenergic-, GPCR-, cAMP-, and CREB/CREM-signaling'. Integrative gene expression analysis specifically links changes in amygdaloid intracellular signaling activity to the behavioral treatment response in Brd1+/- mice. Collectively, our study highlights the importance of BRD1 as a modulator of affective pathology and adds to our understanding of the molecular mechanisms underlying affective disorders and their treatment response.

Neuropathic pain is a major incurable clinical problem resulting from peripheral nerve trauma or disease. A central mechanism is the reduced expression of the potassium chloride cotransporter 2 (KCC2) in dorsal horn neurons induced by brain-derived neurotrophic factor (BDNF), causing neuronal disinhibition within spinal nociceptive pathways. Here, we demonstrate how neurotensin receptor 2 (NTSR2) signaling impairs BDNF-induced spinal KCC2 down-regulation, showing how these two pathways converge to control the abnormal sensory response following peripheral nerve injury. We establish how sortilin regulates this convergence by scavenging neurotensin from binding to NTSR2, thus modulating its inhibitory effect on BDNF-mediated mechanical allodynia. Using sortilin-deficient mice or receptor inhibition by antibodies or a small-molecule antagonist, we lastly demonstrate that we are able to fully block BDNF-induced pain and alleviate injury-induced neuropathic pain, validating sortilin as a clinically relevant target.