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.

Mitochondrial dysfunction underlies numerous age-related pathologies. In an effort to uncover how the detrimental effects of mitochondrial dysfunction might be alleviated, we examined how the nematode C. elegans not only adapts to disruption of the mitochondrial electron transport chain, but in many instances responds with extended lifespan. Studies have shown various retrograde responses are activated in these animals, including the well-studied ATFS-1-dependent mitochondrial unfolded protein response (UPRmt). Such processes fall under the greater rubric of cellular surveillance mechanisms. Here we identify a novel p38 signaling cascade that is required to extend life when the mitochondrial electron transport chain is disrupted in worms, and which is blocked by disruption of the Mitochondrial-associated Degradation (MAD) pathway. This novel cascade is defined by DLK-1 (MAP3K), SEK-3 (MAP2K), PMK-3 (MAPK) and the reporter gene Ptbb-6::GFP. Inhibition of known mitochondrial retrograde responses does not alter induction of Ptbb-6::GFP, instead induction of this reporter often occurs in counterpoint to activation of SKN-1, which we show is under the control of ATFS-1. In those mitochondrial bioenergetic mutants which activate Ptbb-6::GFP, we find that dlk-1, sek-3 and pmk-3 are all required for their life extension.

TDP-1 is the Caenorhabditis elegans ortholog of mammalian TDP-43, which is strongly implicated in the etiology of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). We discovered that deletion of the tdp-1 gene results in enhanced nuclear RNA interference (RNAi). As nuclear RNAi in C. elegans involves chromatin changes moderated by HPL-2, a homolog of heterochromatin protein 1 (HP1), we investigated the interaction of TDP-1 and HPL-2. We found that TDP-1 and HPL-2 interact directly and that loss of TDP-1 dramatically alters the chromatin association of HPL-2. We showed previously that deletion of the tdp-1 gene results in transcriptional alterations and the accumulation of double-stranded RNA (dsRNA). These molecular changes are replicated in an hpl-2 deletion strain, consistent with HPL-2 acting in consort with TDP-1 to modulate these aspects of RNA metabolism. Our observations identify novel mechanisms by which HP1 homologs can be recruited to chromatin and by which nuclear depletion of human TDP-43 may lead to changes in RNA metabolism that are relevant to disease.

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.

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.

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.

Aging is the primary risk factor for most neurodegenerative diseases, including Alzheimer's disease. Major hallmarks of brain aging include neuroinflammation/immune activation and reduced neuronal health/function. These processes contribute to cognitive dysfunction (a key risk factor for Alzheimer's disease), but their upstream causes are incompletely understood. Age-related increases in transposable element (TE) transcripts might contribute to reduced cognitive function with brain aging, as the reverse transcriptase inhibitor 3TC reduces inflammation in peripheral tissues and TE transcripts have been linked with tau pathology in Alzheimer's disease. However, the effects of 3TC on cognitive function with aging have not been investigated. Here, in support of a role for TE transcripts in brain aging/cognitive decline, we show that 3TC: (a) improves cognitive function and reduces neuroinflammation in old wild-type mice; (b) preserves neuronal health with aging in mice and Caenorhabditis elegans; and (c) enhances cognitive function in a mouse model of tauopathy. We also provide insight on potential underlying mechanisms, as well as evidence of translational relevance for these observations by showing that TE transcripts accumulate with brain aging in humans, and that these age-related increases intersect with those observed in Alzheimer's disease. Collectively, our results suggest that TE transcript accumulation during aging may contribute to cognitive decline and neurodegeneration, and that targeting these events with reverse transcriptase inhibitors like 3TC could be a viable therapeutic strategy.

Multiple gene expression alterations have been linked to Alzheimer's disease (AD), implicating multiple metabolic pathways in its pathogenesis. However, a clear distinction between AD-specific gene expression changes and those resulting from nonspecific responses to toxic aggregating proteins has not been made. We investigated alterations in gene expression induced by human beta-amyloid peptide (Aβ) in a Caenorhabditis elegans AD model. Aβ-induced gene expression alterations were compared with those caused by a synthetic aggregating protein to identify Aβ-specific effects. Both Aβ-specific and nonspecific alterations were observed. Among Aβ-specific genes were those involved in aging, proteasome function, and mitochondrial function. An intriguing observation was the significant overlap between gene expression changes induced by Aβ and those induced by Cry5B, a bacterial pore-forming toxin. This led us to hypothesize that Aβ exerts its toxic effect, at least in part, by causing damage to biological membranes. We provide in vivo evidence consistent with this hypothesis. This study distinguishes between Aβ-specific and nonspecific mechanisms and provides potential targets for therapeutics discovery.

Epidemiological studies have associated estrogen replacement therapy with a lower risk of developing Alzheimer's disease, but a higher risk of developing breast cancer and certain cardiovascular disorders. The neuroprotective effect of estrogen prompted us to determine potential therapeutic impact of soy-derived estrogenic compounds. Transgenic C. elegans, that express human beta amyloid (Abeta), were fed with soy derived isoflavones genistein, daidzein and glycitein (100 microg/ml) and then examined for Abeta-induced paralysis and the levels of reactive oxygen species.

The amyloid beta (Aβ) peptide is believed to play a central role in Alzheimer's disease (AD), the most common age-related neurodegenerative disorder. However, the natural, evolutionarily selected functions of Aβ are incompletely understood. Here, we report that nanomolar concentrations of Aβ act synergistically with known cytokines to promote pro-inflammatory activation in primary human astrocytes (a cell type increasingly implicated in brain aging and AD). Using transcriptomics (RNA-seq), we show that Aβ can directly substitute for the complement component C1q in a cytokine cocktail previously shown to induce astrocyte immune activation. Furthermore, we show that astrocytes synergistically activated by Aβ have a transcriptional signature similar to neurotoxic "A1" astrocytes known to accumulate with age and in AD. Interestingly, we find that this biological action of Aβ at low concentrations is distinct from the transcriptome changes induced by the high/supraphysiological doses of Aβ often used in in vitro studies. Collectively, our results suggest an important, cytokine-like function for Aβ and a novel mechanism by which it may directly contribute to the neuroinflammation associated with brain aging and AD.

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.