ASN Neuro

Oligodendrocytes (OL) are myelin forming glial cells in the central nervous system. In vitro primary OL culture models provide the benefit of a more readily controlled environment facilitating the evaluation of diverse OL stages and convoluted dynamics. Conventional methods of primary OL culture do exist, but their performance in terms of efficiency and simplicity has room for improvement. We present a novel method of primary OL culture, namely the E3 (Easy, efficient, and effective) method, which greatly improves cell yield and reduces time required to oligodendrocyte progenitor cell (OPC) acquisition and maturation into OLs. We also provide optimal media compositions for augmentation of OPC proliferation and a more robust maturation into myelin forming OLs. In vitro characteristics of the OL lineage discovered during the development of the E3 method present implications for further research on OL physiology and pathophysiology.

The g-ratio, calculated as the axon diameter divided by the total myelinated fiber diameter, is widely used to assess the degree of myelination in the central and peripheral nervous systems. Changes in g-ratios accompany demyelinating, hypomyelinating, and remyelinating conditions, and can also result from activity-dependent, adaptive myelination. Although manual tracing from electron micrographs has been the gold-standard to measure g-ratios, this process is laborious and limits throughput. To address this challenge, we developed SnapG, an automated and user-friendly software tool to streamline g-ratio analysis from electron micrographs. SnapG implements an end-to-end pipeline for axon and myelin segmentation on EM images. The tool integrates robust segmentation and post-processing steps to extract axon and fiber diameters and generates both per-image and cohort-level summary statistics. SnapG produces g-ratio distributions that agree well with expert manual annotations and established tools across imaging modalities. It reduces user intervention, supports large image and batch processing, and achieves faster processing times while delivering accurate, reproducible measurements. SnapGs automated reporting thereby facilitates high-throughput analyses and improves consistency across experiments. SnapG can accelerate studies of myelin biology and provides a practical alternative to existing tools.

Recent advances in experimental strategies that promote axon regeneration in adult mammals lay the foundation for future therapies. Reliable and unbiased quantification of regenerated axons is challenging, yet essential for comparing the efficacy of individual treatments and identification of most efficacious combinatorial therapies. Here, we introduce AxoMetric, a user-friendly and freely available software for the rapid quantification of regenerated axons in longitudinal nerve tissue sections. AxoMetric automatically identifies and traces regenerated axons, generating quantitative measurements that closely match conventional manual quantification but with significantly greater speed. Key features include length-dependent axon quantification at defined intervals from the injury site and normalization of axon density to nerve diameter to account for anatomical variability. To facilitate high-throughput analysis, the software includes an image queuing function. Additional features of AxoMetric allow quantification of a range of labeled cellular structures. As a proof of concept, we demonstrate accurate quantification of regenerated axons in the optic nerve, retinal ganglion cells density in retinal flat-mounts, and regenerated axon bundles in injured sciatic nerves. Collectively, we introduce a new platform that is expected to streamline and standardize regenerative outcome assessments across diverse experimental conditions and laboratories. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=102 SRC="FIGDIR/small/662816v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@162d692org.highwire.dtl.DTLVardef@1f52ed4org.highwire.dtl.DTLVardef@1d58f76org.highwire.dtl.DTLVardef@1bdabb1_HPS_FORMAT_FIGEXP M_FIG C_FIG

In the present study, we report that the concentration of myelin debris-like myelin basic protein-immunoreactive particles was observed in the distal (anterior)-most part of the myelinated region in the normal rat optic nerve. These particles were visualized using fluorescent immunohistochemistry with a mouse monoclonal anti-human myelin basic protein (MBPh) antibody (clone SMI-99). Fluorescent double immunohistochemistry, employing both the rat monoclonal anti-cow myelin basic protein (MBPc) antibody (clone 12) and the anti-MBPh antibody, revealed that the myelin basic protein immunoreactive-particles detected by the anti-MBPc antibody nearly completely overlapped with those immunostained by the anti-MBPh antibody. Since these antibodies target different sites, it can be concluded that these particles contain authentic myelin basic protein. We hypothesized that the MBPh-immunoreactive particles represent myelin debris-like structures in the normal rat optic nerve. Quantitative morphological analyses indicated that only 2 out of 6 differences in size and shape descriptors between the particles and the myelin debris observed in the damaged optic nerve of the glaucoma rat were statistically significant. Glial fibrillary acidic protein-immunoreactivity and glutamine synthetase-immunoreactivity were observed in the particles. Most of these particles were isolated from ionized calcium-binding adapter molecule 1-labeled microglia. These findings demonstrate that the myelin debris-like MBPh-immunoreactive particles are concentrated in the distal-most part of the myelinated region. This evidence suggests that the distal-most part is under physiologically stressed conditions. Furthermore, these findings may provide valuable insights into the pathophysiological mechanisms that induce localized vulnerability of the myelin sheaths. Key pointsO_LIThis article demonstrates that myelin basic protein-immunoreactive particles are densely distributed in the distal (anterior)-most part of the myelinated region in the normal rat optic nerve. C_LIO_LIThese particles exhibit morphological characteristics akin to myelin debris observed in the damaged optic nerve of the glaucoma rat. (45 words) C_LI Graphical Abstract Image O_FIG O_LINKSMALLFIG WIDTH=148 HEIGHT=200 SRC="FIGDIR/small/643597v2_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@13d6c62org.highwire.dtl.DTLVardef@1969690org.highwire.dtl.DTLVardef@176fd11org.highwire.dtl.DTLVardef@e08fd9_HPS_FORMAT_FIGEXP M_FIG C_FIG

Parkinsonism-Dementia Complex (PDC) is one phenotype of a disappearing neurodegenerative disease (Guam ALS-PDC) that shows clinical and neuropathological relationships with amyotrophic lateral sclerosis (ALS), atypical parkinsonism and Alzheimers disease. ALS-PDC has been linked with exposure to environmental factors (notably cycad plant neurotoxins), but evidence from human and animal studies is inconclusive. Patient-derived induced pluripotent stem cells (iPSCs) provide a powerful in vitro system to explore the underlying cause of PDC. iPSC lines were derived from lymphocytes of a PDC-affected Guamanian Chamorro female patient and an age- and gender-matched healthy Chamorro resident of PDC-unaffected Saipan using non-integrating episomal plasmids. iPSCs derived from both patients expressed pluripotency markers (Oct4, SSEA-4, TRA-1-60, Sox2) prior to the generation of neuroprogenitor cells (hNPCs), neurons and astrocytes. An embryoid body protocol was used to derive hNPCs from both iPSC lines while a differentiation media was used to generate neurons from hNPCs. hNPCs derived from both iPSC patients lines displayed established neuroprogenitor markers (nestin, Sox2), while the differentiated hNPCs exhibited both neuronal (beta-tubulin III, Map2, doublecortin) and synaptic (synaptophysin, PSD-95) markers. Expression of these protein markers in hNPCs and neurons by dot blotting was also observed for both lines. Astrocyte progenitor cells and mature astrocytes with appropriate markers were also developed from the hNPCs of both lines using commercial kits. Development of these patient-derived iPSCs provides a human model for evaluating the role of environmental (e.g., cycad toxins) and genetic factors in ALS-PDC and possibly other related neurodegenerative diseases.

Neuronal ceroid lipofuscinosis (NCL), type 6 (CLN6) is a neurodegenerative disorder associated with progressive neurodegeneration leading to dementia, seizures, and retinopathy. CLN6 encodes a resident-ER protein involved in trafficking lysosomal proteins to the Golgi. CLN6p deficiency results in lysosomal dysfunction and deposition of storage material comprised of Nile Red+ lipids/proteolipids that include subunit C of the mitochondrial ATP synthase (SUBC). White matter involvement has been recently noted in several CLN6 animal models and several CLN6 subjects had neuroimaging was consistent with leukodystrophy. CLN6 patient-derived induced pluripotent stem cells (IPSCs) were generated from several of these subjects. IPSCs were differentiated into oligodendroglia or neurons using well-established small-molecule protocols. A doxycycline-inducible transgenic system expressing neurogenin-2 (the I3N-system) was also used to generate clonal IPSC-lines (I3N-IPSCs) that could be rapidly differentiated into neurons (I3N-neurons). All CLN6 IPSC-derived neural cell lines developed significant storage material, CLN6-I3N-neuron lines revealed significant Nile Red+ and SUBC+ storage within three and seven days of neuronal induction, respectively. CLN6-I3N-neurons had decreased tripeptidyl peptidase-1 activity, increased Golgi area, along with increased LAMP1+ in cell bodies and neurites. SUBC+ signal co-localized with LAMP1+ signal. Bulk-transcriptomic evaluation of control- and CLN6-I3N-neurons identified >1300 differentially-expressed genes (DEGs) with Gene Ontogeny (GO) Enrichment and Canonical Pathway Analyses having significant changes in lysosomal, axonal, synaptic, and neuronal-apoptotic gene pathways. These findings indicate that CLN6-IPSCs and CLN6-I3N-IPSCs are appropriate cellular models for this disorder. These I3N-neuron models may be particularly valuable for developing therapeutic interventions with high-throughput drug screening assays and/or gene therapy.

Glial cells play a critical role in the maintenance of neuronal activity in the optic nerve. The present study reports the distribution of glial structural proteins (GFAP: glial fibrillary acidic protein; MBP: myelin basic protein), a glial functional protein (GS: glutamine synthetase), and of a cell nuclear marker (bisBenzimide) in the various myelinated regions of the normal rat optic nerve. Fourteen optic nerves from 12 male Sprague-Dawley rats were used. Immunohistochemistry and confocal microscopy were employed to investigate the distribution of GFAP, MBP, GS, and of bisBenzimide along the longitudinal plane of the myelinated region. GFAP-immunoreactivity and GS-immunoreactivity were strong in the distal (anterior)-most part but weak in the proximal (posterior) part, demonstrating a significant decrease in strength along the longitudinal plane of the myelinated region. bisBenzimide labeling was also strong in the distal-most part but weak in the proximal part, indicating a significant difference in strength across the myelinated region. MBP-immunoreactive particles and cell nuclei were densely distributed in the distal-most part; however, they were sparsely dispersed in the proximal part, showing a significant difference. The heterogeneous distribution of GFAP, GS, bisBenzimide, cell nuclei, and of MBP-immunoreactive particles along the longitudinal plane may represent an important functional adaptation reflecting the non-uniform nature of the physiological and structural environment of the myelinated region. Notably, the concentrations of GFAP, GS, and of MBP-immunoreactive particles in the distal-most part of the myelinated region suggest that this area is under physiologically stressed conditions in the normal rat optic nerve. Key pointsO_LIThe present study reports the distribution of glial structural proteins (GFAP: glial fibrillary acidic protein; MBP: myelin basic protein), and of a glial functional protein (GS: glutamine synthetase) in the various myelinated regions of the normal rat optic nerve. C_LIO_LIGFAP-immunoreactivity and GS-immunoreactivity were strong in the distal (anterior)-most part but weak in the proximal (posterior) part. C_LIO_LIMBP-immunoreactive particles were densely distributed in the distal-most part; however, they were sparsely dispersed in the proximal part. C_LIO_LIThese results suggest that the distal-most part is not under physiological but rather under physiologically stressed conditions in the normal rat optic nerve. C_LI Graphical AbstractThe present study reports the distribution of glial structural pro-teins (GFAP: glial fibrillary acidic protein; MBP: myelin basic protein), and of a glial functional protein (GS: glutamine syn-thetase) in the various myelinated regions of the normal rat optic nerve. GF AP-immunoreactivity and GS-immunoreactivity were strong in the distal (anterior)-most part but weak in the proximal (poste-rior) part. MBP-immunoreactive particles were densely distributed in the distal-most part; however, they were sparsely dispersed in the proximal part. These results suggest that the distal-most part is not under physi-ological but rather under physiologically stressed conditions in the normal rat optic nerve. O_FIG O_LINKSMALLFIG WIDTH=197 HEIGHT=200 SRC="FIGDIR/small/643607v2_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@54c666org.highwire.dtl.DTLVardef@1ad8f3borg.highwire.dtl.DTLVardef@196870borg.highwire.dtl.DTLVardef@17c1641_HPS_FORMAT_FIGEXP M_FIG C_FIG

Gliomas are primary brain tumors that develop from glial cells within the central nervous system and are among the deadliest human cancers. Glioblastoma (GBM) is the most malignant form of glioma. NLRX1 is an innate immune pattern recognition receptor that exhibits tumor-suppressive and tumor-promoting effects that may be cancer or cell-type, context-dependent, aided by differences in the microenvironment. Here, we report that NLRX1 is differentially expressed in microglia, astrocytes, GBM cell lines, and glioma patient tissues. siRNA-mediated silencing of Nlrx1 decreases the ability of the GBM cell line, LN-229, to proliferate and migrate. Nlrx1-/- GBM cells exhibit attenuated ability to generate 3D spheroids and enhanced capability to form tunneling nanotubes. Moreover, Nlrx1-/- GBM cells show decreased expression of autophagy markers, suggesting that NLRX1 plays a role in maintaining autophagy in GBM. In summary, our findings indicate that NLRX1 may modulate GBM pathophysiology by regulating GBM cell proliferation, migration, and metabolism. We believe our understanding of NLRX1 in GBM pathophysiology paves the potential development of GBM-targeting therapeutics that may delay disease progression and/or improve survival.

The tubulin protein fulfills a variety of cellular functions that range from chromosomal separation to locomotion. Functional diversity is achieved through the expression of specific tubulin isotypes in different cell types or developmental time periods. Post-translational modifications (PTMs) of tubulin also are vital for specific intracellular tasks, such as binding and recruiting motor proteins. In neurons, the isotypic expression profile for tubulin is well characterized, and the importance of PTMs for proper neuronal function has gained recent attention due to their implication in neurodegenerative disorders. In contrast, the role of tubulin specializations in the specification of neural cell fate has received minimal attention and studies of tubulin PTMs and isotypes in neuroglia such as astrocytes are relatively few. To bridge this knowledge gap, we undertook an analysis of PTMs in neurons and astrocytes derived from the federally approved H9 hESC-derived human neural stem cell (hNSC) line. In hNSCs, basal cells can be directed to assume neural fate as neurons or astrocytes by specifying different media growth conditions. Immunocytochemical methods, fluorescent antibody probes, and confocal microscopy facilitated image acquisition of fluorescent signals from class III {beta}-tubulin ({beta}III-tubulin), acetylated tubulin, and polyglutamylated tubulin. Fluorescent probe intensities were assessed with the EBImage package for the statistical programming language R, and compared using Students t-tests. Qualitative analysis indicated that {beta}III-tubulin, acetylated tubulin, and polyglutamylated tubulin were expressed to some degree in basal hNSCs and their media-differentiated hNSC neuronal and astroglial progeny. In media-differentiated hNSC astrocyte progeny, quantification and statistical analysis of fluorescence probe intensity showed that acetylated tubulin/ {beta}III-tubulin ratios were greater than the ratio for polyglutamylated tubulin/ {beta}III-tubulin. These findings represent a snapshot of the dynamic and varied changes in the tubulin expression profile during the specification of neural cell fate. Results imply that investigations of tubulin PTMs have the potential to advance our understanding of the generation and regeneration of nervous tissue.

Myelinating Schwann cell (SC)- dorsal root ganglion (DRG) neuron cocultures have been an important technique over the last four decades in understanding cell-cell signalling and interactions during peripheral nervous system (PNS) myelination, injury, and regeneration. While methods using rat SCs and rat DRG neurons are commonplace, there are no established protocols in the field describing the use of mouse SCs with mouse DRG neurons in dissociated myelinating cocultures. There is a great need for such a protocol as this would allow the use of cells from many different transgenic mouse lines. Here we describe a protocol to coculture dissociated mouse SCs and DRG neurons and induce robust myelination. Use of microfluidic chambers permits fluidic isolation for drug treatments, allows cultures to be axotomised to study injury responses, and cells can readily be transfected with lentiviruses to permit live imaging. We used this model to quantify the rate of degeneration after traumatic axotomy in the presence and absence of myelinating SCs and axon aligned SCs that were not induced to myelinate. We find that SCs, irrespective of myelination status, are axo-protective and delay axon degeneration early on. At later time points after injury, we use live imaging of cocultures to show that once axonal degeneration has commenced SCs break up, ingest, and clear axonal debris. Summary statementA novel compartmentalised dissociated mouse myelinating SC-DRG coculture system reveals distinct axo-protective and axo-destructive phases of Schwann cells on axon integrity after trauma.

Primary mixed glial cultures are key tools to isolate and study astrocytes, microglia and oligodendrocytes. Cell-substrate adhesion is critical for neural cell survival and differentiation. Cationic polymers like poly-D-lysine (PDL) are widely used to promote cell adhesion to cell culture substrates, however, PDL is not stable long-term, with cultured cells often detaching (peeling) after 2-3 weeks. Dendritic polyglycerol amine (dPGA) is a synthetic polycationic non-protein polymer biomimetic of poly-lysine that is highly resistant to degradation by cellular proteases. Substrates coated with dPGA promote cell adhesion and improve survival in long-term neuronal cultures. Here we assessed dPGA as a substrate coating to provide long-term support for mixed glial cultures. Oligodendrocyte precursor cells (OPCs) were isolated weekly by differential adhesion from cultures grown in T75 flasks with PDL or dPGA-coated substrates. Following two "shake-off" isolations, the cell layer in most PDL-coated flasks fully detached, rendering these flasks unusable for further culture. In contrast, dPGA-coated flasks consistently yielded cells for six or more sequential isolations over seven weeks in culture. dPGA-coated flasks produced more cells, a greater percentage of O4+ cells, and maintained similar proportions of OPCs and MBP-positive cells as when isolated from a PDL-coated substrate. dPGA is cyto-compatible, functionally superior, easy to use, low cost and a stable alternative to conventional cell substrate coatings. The enhanced long-term stability of mixed glial cultures grown on a dPGA substrate has the capacity to increase cellular yield, reduce animal use, and facilitate studies of oligodendrocyte cell biology.

In adult mammalian central nervous system (CNS) neurons, axon regeneration after injury remains limited due to unfavorable gene regulatory programs. Factors enabling comprehensive epigenetic and transcriptional transitions, for instance, pivotal transcription factors that mediate neurogenesis and morphogenesis may be sufficient to promote CNS axon regeneration. Based on the analyses of multiple public whole-genome RNA and chromatin accessibility sequencing dataset of mouse retina development, as well as previous functional studies on the regeneration-capable dorsal root ganglion neurons, we hypothesize that the overexpression of pioneer transcription factor Achaete-Scute homolog 1 (Ascl1) would promote axon regeneration in the adult mammalian CNS neurons. We employed the optic nerve crush in mice, a common model for studying CNS axon regeneration, neuron survival and glaucoma, to investigate the effect of Ascl1 overexpression on the post-injury optic nerve regeneration. We found that Ascl1 could sufficiently promote regenerated axons past the crush site and significantly preserve the survival of retinal ganglion cells. Mechanistically, we revealed that effects of Ascl1 was mediated by known pro-regeneration factor Sox11 but not others. Together, our study established an effective workflow combined with the integrated computational inference and experimental validation for discovering functionally important target for promoting CNS neuron axon regeneration and survival.

Multiple sclerosis (MS) is an autoimmune inflammatory disease of the central nervous system. The cause of the disease is unknown but both genetic and environmental factors are strongly implicated in its pathogenesis. We derived cerebral and spinal cord organoids from induced pluripotent stem cells (iPSC) from healthy controls as well as from primary progressive MS (PPMS), secondary progressive MS (SPMS) and relapsing-remitting MS (RRMS) patients to investigate and compare oligodendrocyte differentiation and myelination capacity in healthy subjects and MS subtypes. In MS organoids, particularly in PPMS, we observed a decrease in p21 expression associated with a dysregulation of PAK1 and E2F1 expression. In parallel, a decrease in oligodendrocyte maturation was detected in long-term cultured cerebral and spinal cord organoids, especially in PPMS, leading to a reduced myelination capacity. Disruption of astrocyte and neuronal populations was also observed. Our findings demonstrate that in MS, inherent deficits in the p21 pathway may alter glial and neuronal cell populations and may contribute to the disease pathogenesis by reducing the capacity for myelin repair. Summary StatementUsing cerebral and spinal cord organoids derived from multiple sclerosis patients, we found an innate disruption of oligodendrocyte differentiation and myelination capacity as well as excitotoxicity, associated with PAK1 and E2F1-induced p21 dysregulation.

Myelination is the terminal step in a complex and precisely timed program that orchestrates the proliferation, migration and differentiation of oligodendroglial cells. It is thought that Sonic Hedgehog (Shh) acting on Smoothened (Smo) participates in regulating this process, but that these effects are highly context dependent. Here, we investigate oligodendroglial development and remyelination from three specific transgenic lines: NG2-CreERT2 (control), Smofl/fl/NG2-CreERT2 (loss of function) and SmoM2/NG2-CreERT2 (gain of function), as well as pharmacological manipulation that enhance or inhibit the Smo pathway (SAG or cyclopamine treatment respectively). To explore the effects of Shh/Smo on differentiation and myelination in vivo, we developed a highly quantifiable model by transplanting OPCs in the retina. We find that myelination is greatly enhanced upon cyclopamine treatment and hypothesize that Shh/Smo could promote OPC proliferation to subsequently inhibit differentiation. Consistent with this hypothesis, we find that the genetic activation of Smo significantly increased numbers of OPCs and decreased oligodendrocyte differentiation when we examined the corpus callosum during development and after cuprizone demyelination and remyelination. However, upon loss of function with the conditional ablation of Smo, myelination in the same scenarios are unchanged. Taken together, our present findings suggest that the Shh pathway is sufficient to maintain OPCs in an undifferentiated state, but is not necessary for myelination and remyelination.

Parkinsons disease (PD) is characterized by the loss of A9 midbrain dopaminergic neurons and the accumulation of alpha-synuclein aggregates in remaining neurons. Many studies of the molecular and cellular basis of neurodegeneration in PD have made use of iPSC-derived neurons from patients with familial PD mutations. However, approximately half of the cells in the brain are glia, and their role facilitating neurodegeneration is unclear. We developed a novel serum-free protocol to generate midbrain astrocytes from patient-derived iPSCs harbouring the pathogenic p.A30P, p.A53T mutations in SNCA, as well as duplication and triplication of the SNCA locus. In our cellular model, aggregates of alpha-synuclein occurred only within the GFAP+ astrocytes carrying the pathogenic SNCA mutations. Assessment of spontaneous cytosolic calcium (Ca2+) release using Fluo4 revealed that SNCA mutant astrocytes released excess Ca2+ compared to controls. Unbiased evaluation of 3D mitochondrial morphometric parameters showed that these SNCA mutant astrocytes had increased mitochondrial fragmentation and decreased mitochondrial connectivity compared to controls, and reduced mitochondrial bioenergetic function. This comprehensive assessment of different pathogenic SNCA mutations derived from PD patients using the same cellular model enabled assessment of the mutation effect, showing that p.A53T and triplication astrocytes were the most severely affected. Together, our results indicate that astrocytes harbouring the familial PD mutations in SNCA are dysfunctional, suggesting a contributory role for dysfunctional astrocytes in the disease mechanism and pathogenesis of PD. Table of Contents Image O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=89 SRC="FIGDIR/small/053470v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@974d72org.highwire.dtl.DTLVardef@15d9da0org.highwire.dtl.DTLVardef@1177d4eorg.highwire.dtl.DTLVardef@123c0c2_HPS_FORMAT_FIGEXP M_FIG C_FIG Main PointsO_LIWe used a novel serum-free protocol to generate midbrain-specific functional astrocytes from Parkinsons disease patients carrying pathological mutations in SNCA C_LIO_LIPatient-derived astrocytes show morphological and functional impairments C_LI

Retinal ganglion cell axons provide the only link between the light sensitive and photon transducing neural retina and visual centers of the brain. Retinal ganglion cell axon degeneration occurs in a number of blinding diseases and the ability to stimulate axon regeneration from surviving ganglion cells could provide the anatomic substrate for restoration of vision. OTX2 is a homeoprotein transcription factor expressed in the retina and previous studies showed that, in response to stress, exogenous OTX2 increases the in vitro and in vivo survival of retinal ganglion cells. The present results show that, in addition to promoting adult retinal ganglion cell survival, OTX2 also stimulates the regeneration of their axons in vitro and in vivo. This dual activity of OTX2 on retinal ganglion cell survival and regeneration is of potential interest for degenerative diseases affecting this cell type.

We previously described a process referred to as transmitophagy where mitochondria shed by retinal ganglion cell (RGC) axons are transferred to and degraded by surrounding astrocytes in the optic nerve head of mice. Since the mitophagy receptor Optineurin (OPTN) is one of few large- effect glaucoma genes and axonal damage occurs at the optic nerve head in glaucoma, here we explored whether OPTN mutations perturb transmitophagy. Live-imaging of Xenopus laevis optic nerves revealed that diverse human mutant but not wildtype OPTN increase stationary mitochondria and mitophagy machinery and their colocalization within, and in the case of the glaucoma-associated OPTN mutations also outside of, RGC axons. These extra-axonal mitochondria are degraded by astrocytes. Our studies support the view that in RGC axons under baseline conditions there are low levels of mitophagy, but that glaucoma-associated perturbations in OPTN result in increased axonal mitophagy involving the shedding and astrocytic degradation of the mitochondria. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=157 HEIGHT=200 SRC="FIGDIR/small/542507v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@c1d3d4org.highwire.dtl.DTLVardef@13a33e6org.highwire.dtl.DTLVardef@2544beorg.highwire.dtl.DTLVardef@c456a5_HPS_FORMAT_FIGEXP M_FIG C_FIG

Misfolded protein accumulation in demyelinating disorders or injury can trigger internal endoplasmic reticulum (ER) stress alleviation mechanisms, namely the unfolded protein response (UPR) and cholesterol biosynthesis pathways. Here we demonstrate that the ER stress-associated transcription factor Luman/CREB3, herein called Luman, shown to drive axon regeneration by UPR-dependent regulation of adaptive low-level stress, also positively regulates rat Schwann cell (SC) survival, myelination, the UPR, and cholesterol production in vitro. siRNA knockdown of SC Luman expression decreased SC viability and increased apoptosis 48 hours post-transfection. Dorsal root ganglion (DRG) neuron/SC co-cultures where SCs overexpressed Luman exhibited increased myelination. Simulation of unstressed, mild and moderate ER stress by tunicamycin-mediated UPR induction in SCs, allowed examination of Luman on expression of known UPR regulators, including Xbp1, Xbp1s, CHOP, and pIRE1. They collectively demonstrate a cytoprotective role for Luman under manageable ER stress. Total cholesterol levels and sterol precursor Srebf1 expression, key to myelination, also decreased following Luman knockdown. Finally, levels of mature brain-derived neurotrophic factor (mBDNF), a positive regulator of myelination and also regulated by the UPR, decreased with Luman knockdown. In contrast, pro-apoptotic BDNF precursor (proBDNF) levels increased in Luman-deficient SCs at higher ER stress levels, indicating that any protection Luman confers at moderate ER stress levels is lost upon its reduced expression. In conclusion, a connection between Luman and adaptive beneficial ER stress pathways linked to survival and myelination capacity in SCs exists. These Luman-driven cytoprotective mechanisms including survival and myelination open avenues for targeting this pathway in nerve trauma and myelinating disorders.

Myelin formation by oligodendrocytes is essential for the regulation of the conduction velocity and proper brain function. To ensure accurate information processing in response to experiences such as sensory stimuli and learning, oligodendrocytes adjust their number and morphology. In addition, oligodendrocyte morphology changes with senescence and in the presence of neurodegenerative diseases. Thus, visualizing oligodendrocytes and analyzing their morphology is crucial for understanding how our brains change under such conditions. Herein, we describe the methods for labeling and analyzing the morphologies of individual oligodendrocytes in mouse white matter at the light microscopic level. PDGFRa-CreERT2:Tau-mGFP and PLP-CreERT2:Tau-mGFP mice enable us to visualize and analyze later-born or early-born oligodendrocyte morphology. In addition, sparse oligodendrocyte labeling with attenuated rabies virus expressing GFP enables the visualization and morphological analysis of individual oligodendrocytes in various brain white matter regions without the need for transgenic animals. Furthermore, the combination with immunostaining in thick tissues enables the identification of labeled oligodendrocytes and myelin sheaths, as well as their interactions with neuronal axons. These methods are suitable for revealing how oligodendrocytes adapt their morphologies depending on environmental stimuli or pathological conditions.

The actin/spectrin membrane-associated periodic skeleton (MPS), is a cytoskeletal structure that supports axonal integrity and function. Lower spinal motor neurons (MNs) are characterized by exceptionally long axons and are particularly susceptible to degeneration in a wide range of hereditary neuromuscular disorders, including amyotrophic lateral sclerosis (ALS). Using confocal and super-resolution imaging, we characterized the spatial distribution {beta}II-spectrin and the assembly pattern of the MPS in human MN axons derived from induced pluripotent stem cells (iPSCs). We discovered a striking gap-and-patch pattern in the medial axon, where sharply demarcated {beta}II-spectrin gaps alternate with patches containing a well-organized MPS. The pattern is acutely induced by the kinase inhibitor staurosporine and pharmacological inhibition of actin polymerization prevents patch formation, indicating a requirement for actin nucleation in MPS assembly. Our data supports a model in which spectrin incorporation into nascent MPS patches depletes neighboring regions, producing long-range gaps-and-patches patterns. Key words: actin, spectrin, MPS, cytoskeleton, motor neurons, iPSCs, staurosporine.