ASN Neuro

Spinal muscular atrophy (SMA) is increasingly recognized as a multisystem disorder involving non-neuronal cells, yet the role of Schwann cells (SCs) in late-onset SMA (loSMA) remains unclear. We investigated age-dependent peripheral nerve pathology in a four-copy SMN2 mouse model of loSMA. Sciatic nerves from wild-type and loSMA mice were analyzed at postnatal (P) days 20, 35, 70, and >100 using semi-thin morphometry, immunofluorescence for MBP, Sox10, Sox2, and F4/80, and nerve conduction studies. loSMA nerves showed reduced myelin thickness at all time points and smaller axon diameters at P20 and P35. G- ratios were reduced at P20 but increased from P35 onward, indicating progressively altered axon-myelin relationships. MBP immunofluorescence intensity, compound muscle action potential amplitude, and nerve conduction velocity were reduced in loSMA mice at P>100. The proportion of Sox2+ SCs increased from P35 onward, while Sox10+ cell abundance increased at later stages. F4/80+ macrophages were transiently elevated at P35 and correlated with Sox2+ cell numbers at this stage. These findings demonstrate age-dependent myelin abnormalities, altered SC states, and transient accumulation of macrophages in loSMA peripheral nerves. Whether these changes are SC-autonomous or secondary to chronic axonal dysfunction remains to be determined.

Neuronal dendrites can be injured by a number of insults, but the cellular mechanism by which dendrites respond to tissue injury and undergo repair is poorly understood. Much of the fields progress has evaluated dendrite regeneration following laser injury. While precise, laser injury does not accurately model the real-world damage to surrounding tissue that would accompany neuronal injury. Here, we modify a pinch injury technique to injure both the dendrites and their surrounding tissues in Drosophila melanogaster larvae, more similar to what is observed in real-world injury. We refined this technique such that only half of a sensory neurons dendrites are injured, leaving the other half uninjured. Our data indicate that both dynamic and stable dendritic arbors regrow dendrites following pinch injury. Neurons primarily engage in compensatory regeneration whereby new branches are added on the uninjured half of the arbor. Comparing the regenerative response following pinch versus laser injury revealed that dendrites preferentially regrew into areas where the surrounding tissue was left intact, and not into areas where the surrounding tissue was damaged by pinch. These results prompted us to evaluate the damage sustained to surrounding tissue. In examining non-neuronal tissues after pinch injury, we found damage to epidermal cells and the ECM, but not glia. We also observed a robust immune response on the pinched half of the arbor. We conclude that the sustained damage to surrounding tissue and the initiation of an immune response create a non-permissive environment for dendrite regeneration following pinch injury. Significance StatementNeuronal dendrites are injured in clinical conditions, such as stroke, traumatic brain injury, and neonatal hypoxia. Dendrites also degenerate in the early stages of a number of neurodegenerative diseases. The role of surrounding tissues in dendrite regeneration is poorly characterized, especially considering that neuronal injury is typically accompanied by broad tissue damage. Our data evaluates dendrite regeneration following an injury that better mirrors real-world conditions and demonstrates that broad tissue damage diminishes a neurons capacity to regenerate its dendrites. Our findings show that neurons preferentially regrow into intact, undamaged tissue environments, addressing a large gap in the fields knowledge: how damage to the surrounding tissue limits neuron regeneration after injury. Visual Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=160 SRC="FIGDIR/small/738747v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@1b3f41forg.highwire.dtl.DTLVardef@160284dorg.highwire.dtl.DTLVardef@1f5f6b5org.highwire.dtl.DTLVardef@118208d_HPS_FORMAT_FIGEXP M_FIG C_FIG

Microglia are the brain-resident macrophages and key regulators of the brain tumor microenvironment. Although induced pluripotent stem cell-derived microglia (iMG) provide a valuable model for studying human microglial, systematic comparisons of differentiation protocols are limited, and their utility for modeling microglia-tumor cell interactions remains underexplored. Here, we analyzed 54 public RNA-seq datasets representing 22 iMG differentiation protocols, including embryoid body (EB)-based, two-dimensional (2D), transcription factor-induced, and coculture-based approaches. Most iMG closely resembled primary human microglia, although substantial protocol-dependent differences were observed. iMG generated using EB-based protocols showed higher TMEM119 expression, whereas those generated using 2D-based protocols showed higher P2RY12 expression. A widely adopted EB-based protocol showed the highest phagocytosis gene signature. Using this protocol, we generated iMG that efficiently phagocytosed patient-derived glioma stem-like cells and upregulated inflammatory and immunoregulatory genes following phagocytosis. These findings provide a transcriptomic benchmark for current iMG models and support their use in investigating microglia-glioma interactions. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/739939v1_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@10d7abeorg.highwire.dtl.DTLVardef@1f55289org.highwire.dtl.DTLVardef@fdbd12org.highwire.dtl.DTLVardef@880f34_HPS_FORMAT_FIGEXP M_FIG C_FIG

Uveal Melanoma (UM) is the most common eye cancer, with a metastatic mortality rate of 80%. Only 1-3% of patients have detectable UM at metastasis, and UM exhibits punctuated early growth. Doxycycline has recently been shown to inhibit metabolic processes exploited by cancer cells and reduce cancer cell growth in models of liver cancer. We hypothesized doxycycline may also be effective in UM and therefore tested doxycycline treatment in an eye organoid model of uveal melanoma. Using a stem cell line whereby BAP1 can be knocked down with a tetracycline-inducible system, we differentiated this line into a whole eye organoid model termed self-formed ectodermal autonomous multi-zone of ocular cells (SEAM). We found an enhanced proliferation in neural crest cells within the SEAM colonies. To identify the neural crest cells, we conducted single-cell RNA sequencing (scRNA-seq) analysis utilizing the Seurat R toolkit to pinpoint genes within neural crest clusters. To confirm the results of the in silico scRNA-seq analysis, genes with notable functions and differential expression in the neural crest cluster in relation to UM proliferation, angiogenesis, and oxidative phosphorylation were analyzed through immunofluorescence and RT-qPCR. Based on the scRNA-seq analysis, immunofluorescence, and RT-qPCR, the novel BAP1 KD (UM phenotype) model was found to replicate UM-relevant gene and protein expressions effectively, so the BAP1 KD (UM phenotype) was then treated with doxycycline to evaluate its effect on UM metastasis. Subsequent analysis found that doxycycline significantly inhibited UM growth, angiogenesis, and oxidative phosphorylation in the BAP1 KD (UM phenotype) model more than that of the control model, perhaps due to doxycycline targeting higher regions with more mitochondrial activity, indicating doxycyclines therapeutic potential in treating UM.

Myotonic Dystrophy type 1 (DM1) is an autosomal multisystem disorder manifested due to unstable CTG nucleotide repeat expansion within the 3'-untranslated region of the dystrophia myotonica protein kinase (DMPK) gene. Although progress towards understanding of molecular pathogenesis in muscle and heart has been made, the pathways that affect the brain in DM1 is fundamentally unknown. In addition, the congenital DM1 manifest even more complicated brain abnormalities. Despite the wealth of existing cellular and animal models, iPSCs based studies are being fostered as they replicate the human model more closely to the disease. In view of this context, we set out to characterize the differentiation potential of congenital DM1 patient derived iPSC lines towards neuronal cells. Using neurogenin2 (NGN2) induced direct reprogramming of iPSCs into neurons and chemically defined media-induced neural induction protocol, we find that congenital DM1 mutant iPSC derived neurons exhibited precocious differentiation, as evidenced by their expression of pan-neuronal markers TUJ1 and Map2, along with increased processes extension and neurite length. Moreover, unbiased RNA sequencing analyses and qPCR validation revealed precocious and enhanced expression of several neurogenic transcription factors including, Ascl1, NeuroG2, and NeuroD1. Furthermore, immunofluorescence imaging of MBNL1 and MBNL2, RNA-splicing factors, displayed enhanced nuclear aggregations, a hallmark of the DM1 disease, in the mutant lines. Moreover, investigation of RNA splicing events identified mis-splicing in many important genes/transcripts including RMST, ANK3 and MBD1 during the neural conversion of congenital DM1 lines. These studies reveal novel paradigms that may contribute to neurological pathogenesis in CDM1 patients. These studies also provide a strong foundation for future mechanistic investigation aimed at understanding CDM1 pathology and may open new avenues for the development of gene therapy approaches for individuals with DM1.

The inability of oligodendrocyte progenitor cells (OPCs) to mature into myelin-making oligodendrocytes (OLs) is a major contributor to disease and disability in multiple sclerosis (MS). Oligodendrocyte maturation is a tightly controlled process with a strong reliance on epigenetic regulation involving DNA methylation and hydroxymethylation. We have previously shown that one carbon metabolism is dysregulated in MS, specifically the methyl donor betaine is depleted in the MS brain. Betaine donates methyl groups to betaine homocysteine methyltransferase (BHMT) in the methionine cycle to increase S-adenosylmethionine (SAM) for epigenetic methylation processes. In the present study we tested the effects of activating the BHMT methylation pathway on preventing MS pathology. We describe a novel mechanism mediated by BHMT and the Ten-eleven translocator enzyme (TET1) that converts 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC). We show that this pathway supports oligodendrocyte metabolism to enhance myelin and reduce clinical disability in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS. ChIP-seq studies show that BHMT is enriched at genes involved in OPC metabolism and proximal ligation assays (PLAs) demonstrate that BHMT interacts with TET1 on chromatin. This interaction regulates gene expression programs that support a shift in OPC metabolism to glycolysis during neuroinflammatory processes. These data highlight the critical role of methionine metabolism in supporting myelination and have important implications for the development of new therapeutic strategies for MS and other neurodegenerative diseases.

ObjectiveTo systematically review automated nerve morphometry tools and independently benchmark their performance on independent optic nerve datasets. DesignSystematic review and comparative benchmarking study. ControlsBenchmarking was performed using paraphenylenediamine-stained mouse (n = 85) and rat (n = 44) optic nerve images with manually annotated axon counts as ground truth. MethodsPublished studies describing automated or semi-automated neural tissue morphometry tools were identified through systematic searches of PubMed, Embase, and Scopus through January 2026 following PRISMA guidelines. Data extraction covered 70 fields across tool capabilities, imaging modality, species, automation level, and validation approach. Eighteen eligible tools (8 deep learning [DL], 10 classical computer vision [CV]) were benchmarked on both mouse and rat independent datasets. Main Outcome MeasuresPerformance was assessed by mean absolute percentage error (MAPE), Pearson correlation, and median predicted-to-ground-truth ratio. Tools were ranked per image and compared using Friedman tests with Nemenyi post-hoc analysis. ResultsSeventy-one studies met inclusion criteria, spanning from 1999 to 2026. Deep learning methods represented 38% (27/71) of studies, increasing from 0% before 2017 to over 55% of publications after 2020. Axon counting was the most common output (73%, 52/71), while only 35% (25/71) reported g-ratio. Among benchmarked tools, Marina (CV, 2010) achieved the lowest average MAPE (32.9%). The top five tools (MAPE ranging from 32.9 to 44.8%) included both CV and DL methods and were statistically indistinguishable by Friedman-Nemenyi analysis (p > 0.05). Performance varied substantially across datasets: AxonJ (CV) achieved the second best MAPE on rat images (27.7%) but the worst on mouse images (438.6%). ConclusionsNo single tool demonstrated consistently superior performance across both datasets. Classical and deep learning approaches achieved comparable accuracy for axon counting. Tool selection should be guided by target species, tissue preparation protocol, and desired morphometric outputs. This systematic review and independent benchmarking study provide an evidence base for tool selection in optic nerve research.

Background/ObjectivesGlioblastoma is an aggressive primary brain tumor that develops within a chronically low-oxygen microenvironment, yet most preclinical studies are performed under atmospheric oxygen conditions that poorly reflect in vivo physiology. This study investigated how sustained culture under physiological oxygen tension (physioxia; 5% O{square}) influences glioblastoma cell behavior, signaling, and therapeutic response. MethodsMultiple patient-derived glioblastoma models were cultured under normoxia (21% O{square}) or sustained physioxia (5% O{square}) for at least seven days before experimentation. Cell migration, proliferation, cell cycle distribution, expression of the epithelial-to-mesenchymal transition-associated transcription factor Slug (SNAI2), PDGFR{beta}-associated signaling, and sensitivity to 5-fluorouracil were evaluated using transwell migration assays, cell counting, flow cytometry, RT-qPCR, immunoblotting, and BrdU incorporation assays. Additional patient-derived cultures established and maintained continuously under physioxia were used to examine the effects of oxygen history. ResultsSustained physioxia consistently increased migration across all glioblastoma models while reducing proliferation in normoxia-adapted cell lines through increased G0/G1 cell cycle arrest. Physioxia significantly increased Slug expression in all models and enhanced PDGFR{beta}, AKT, and ERK phosphorylation in a cell line-dependent manner. Therapeutic sensitivity to 5-fluorouracil was also altered, with physioxia conferring increased resistance in selected glioblastoma models but not universally. Patient-derived cultures maintained continuously under physioxia retained enhanced migratory capacity and exhibited increased proliferation compared with normoxia, indicating that prior oxygen exposure influences proliferative responses while the pro-migratory phenotype remains conserved. ConclusionsPhysiological oxygen tension is a major regulator of glioblastoma cell behavior, influencing migration, proliferation, signaling, and therapeutic response. These findings demonstrate that conventional normoxic culture conditions can obscure biologically relevant phenotypes and support incorporating physioxia into experimental design to improve the physiological and translational relevance of preclinical glioblastoma research.

Malignant brain tumors are among the most aggressive and difficult to treat human cancers. Glioblastomas (World Health Organization grade IV gliomas) are particularly lethal and refractory to treatment. Few drugs exist that are even somewhat effective. Our investigation of the physiologic role of fatty acid (FA) activating enzymes (acyl-CoA synthetase; ACS) identified an ACS that was widely expressed in gliomas but not in normal glial cells. Depletion of this enzyme, ACSVL3 (very long-chain ACS3), by knockdown or knockout decreased the malignant behavior of several glioma cell models including U87MG and Mayo-22 cells both in culture and when grown as xenografts. Hypothesizing that ACSVL3 is a potential therapeutic target in glioma, we conducted a search for inhibitors of this enzyme and found that CB5 (grassofermata) was a promising candidate. Treating U87MG glioma cells with CB5 slowed growth in monolayer culture; the growth rate was similar to that seen in cells in which ACSVL3 was either knocked down or knocked out. CB5 inhibited growth in a dose-dependent manner over a narrow range, and concentrations above 10 M were toxic. Treatment at the lower dose of 3 M inhibited growth of U87MG cells but was reversible, suggesting that this dose was not toxic. CB5- treated U87MG cells exhibited an altered morphology with a larger size and longer projections. In contrast, normal human fibroblasts treated with 10 M CB5, a concentration that was toxic to U87MG cells, showed no effect on either growth rate or morphology. Treating U87MG cells with 3 M CB5 induced differentiation as shown by increased expression of the astrocyte-specific marker glial fibrillary acidic protein (GFAP). In contrast, GFAP levels remained low in ACSVL3 knockdown cells. CB5- treated U87MG cells were less invasive, and thus less malignant, than either untreated cells or ACSVL3 knockout cells when assessed by a scratch wound healing assay. Acute treatment of U87MG cells with 3 M CB5 decreased the ability of these cells to degrade FA of differing chain lengths from 16-24 carbons by {beta}-oxidation, suggesting that decreased ACS enzyme activity contributes at least in part to the drugs mechanism of action. NOD/SCID mice receiving up to 32 mg/kg/day CB5 by intraperitoneal injection showed no obvious side effects, suggesting that the drug was well-tolerated. Xenografts induced by subcutaneous injection of U87MG cells in the flanks of NOD/SCID mice were allowed to grow for 8 days after which half of the mice were treated with 2 mg/kg/day CB5. After 7 days of treatment, xenograft growth slowed in the treated mice and by 12 days tumor size had begun to decrease, suggesting therapeutic efficacy. When a similar study was done using xenografts induced by subcutaneous injection of Mayo-22 cells, which are maintained as subcutaneous tumors in mice rather than in cell culture, the effect of CB5 on tumor growth or weight at sacrifice was not statistically significant. The results of these studies suggest that CB5 may have therapeutic value in malignant glioma. Additional studies using other glioma models and other drugs chemically related to CB5 seem warranted.

Three-dimensional (3D) tumor models exhibit drug responses that differ from conventional 2D cultures. However, how cellular metabolism dynamically evolves across culture models and during drug treatment remains poorly understood. We compared the responses of MCF-7 cells in 2D and 3D environments to 5-fluorouracil (5-FU), combining {superscript 1}H NMR cellular metabolomics with viable cell counts, the GLUT1-positive population, gene expression, ATP activity, and morphology. Principal component analysis revealed that culture dimensionality, rather than 5-FU treatment, was the primary driver of metabolic flux variation. 3D spheroids exhibited higher glycolytic flux at 72h. Importantly, this elevated glycolysis reflected a higher per-cell flux in larger spheroids rather than an increased cell number. We further observed a higher proportion of GLUT1-positive cells and increased HK2 expression in 3D culture, together with an epithelial phenotype characterized by increased CDH1 and decreased VIM expression. Functionally, 3D displayed maintaining higher cell viability, ATP activity following treatment. Together, these findings suggest that 3D architecture promotes a metabolically defensive phenotype and cellular metabolic behaviors is associated with morphology, which may inform future drug screening model selection. BlurbTime-resolved NMR metabolomics reveals that 3D culture architecture, rather than 5-FU treatment, defines the metabolic phenotype of MCF-7 cells, linking spheroid morphology with per-cell glycolytic activity, ATP preservation, and reduced chemotherapy sensitivity. Synopsis O_FIG O_LINKSMALLFIG WIDTH=197 HEIGHT=200 SRC="FIGDIR/small/740505v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@cf6b74org.highwire.dtl.DTLVardef@197a4aeorg.highwire.dtl.DTLVardef@bcb5f8org.highwire.dtl.DTLVardef@14ec1ac_HPS_FORMAT_FIGEXP M_FIG C_FIG Graphical abstract -- Time-resolved metabolic and phenotypic responses of 2D and 3D MCF-7 cultures to 5-fluorouracil. Bullet pointsO_LITime-resolved {superscript 1}H NMR cellular metabolomics combined with multilevel phenotypic readouts reveals that culture dimensionality, rather than 5-FU chemotherapy, is the dominant determinant of metabolic phenotype in MCF-7 breast cancer cells. C_LIO_LI3D spheroids develop a glycolysis-dominant metabolic state with markedly elevated GLUT1 populations and HK2 transcript, accompanied by substantially reduced sensitivity to 5-FU compared with 2D monolayers. C_LIO_LISpheroid morphology correlates with cellular metabolic flux: larger and more elongated spheroids exhibit higher glycolytic activity on a per cell, independent of spheroid cell number. C_LIO_LIThe 3D-defined metabolic state buffers ATP under 5-FU stress and reinforces an epithelial transcriptional program (CDH1{uparrow}, VIM{downarrow}), arguing that culture architecture must be considered when interpreting preclinical drug responses. C_LI

The success of stem cell-derived brain repair for Parkinsons is limited by the variable survival and poor maturation of dopaminergic progenitors after transplantation into the Parkinsonian brain. One approach that has been developed to improve this is engraftment of the cells within a neurotrophin-enriched collagen hydrogel. Although this has been shown to improve progenitor survival and maturation in athymic nude rats, the same beneficial effects of the hydrogel were not seen in cyclosporine immunosuppressed rats. To determine the reasons for these differences, the aim of this study was to assess the local and systemic immune responses to progenitor transplantation in these two recipient groups. To do so, human induced pluripotent stem cell-derived dopaminergic progenitors were transplanted into 6-hydroxydopamine-lesioned striatum of athymic or cyclosporine immunosuppressed rats. The cells were transplanted either alone, with the neurotrophins GDNF and BDNF, in an unloaded collagen hydrogel, or in a neurotrophin-loaded collagen hydrogel. Post-mortem assessment included both graft site and blood analysis of immune cell populations. As expected, nude rats showed a pronounced innate immune cell response at the graft site but no T-cell recruitment or activation locally or systemically. In contrast, while the immunosuppressed rats also showed the expected innate immune cells response to the transplant, there was also infiltration of CD4+ and CD8+ T cells at the site of transplantation as well as circulating activated T-cells. Thus, this study suggests that the benefits of the hydrogel that were seen in the athymic nude rats did not manifest in the cyclosporine immunosuppressed rats due to incomplete immunosupression. This study shows the importance of careful optimisation of the immunosuppressive regime chosen before xenotransplantation experiments.

Valproic acid (VPA) is a widely used antiepileptic drug that also increases the risk of neurodevelopmental disorders in the offspring of exposed mothers. Prenatal exposure to VPA is a widely used rodent model of autism spectrum disorder (ASD). Anatomical, functional and molecular alterations in the retinas of various ASD model animals have been described in the literature, but the impact on the neural composition of the retina remains unclear. We examined whether and how the density and spatial regularity of selected retinal neurons are altered in the VPA induced model of ASD. Whole-mount retinas of 2-month-old VPA-treated and control animals were immunolabeled for S-cones, horizontal cells, AII amacrine cells, and parvalbumin-positive wide-field amacrines (PV-wfACs), and the positions of labelled cells mapped in various regions of interest (n = 39 for treated, n = 32 for control animals) across the retinas. Multivariate analysis of variance revealed a significant overall effect of VPA on cell densities (p = 6.1x10-7, 2 = 0.43), driven mainly by reduced AII amacrine density, while horizontal cells showed a modest reduction and S-cones were unaffected. After adjusting for retinal location, analysis of covariance indicated a 7% decrease in AII cells and a 15% increase in PV-wfACs. Regularity indices calculated from nearest neighbor distances or Voronoi-domain areas of cell mosaics were largely unchanged. These findings suggest that prenatal VPA exposure selectively alters inhibitory inner retinal circuitry in the rat ASD model at the time of cell differentiation, but self-organizing mechanisms responsible for spatial order are not affected. Lay SummaryValproic acid (VPA) is a medicine for epilepsy, but it can also raise the risk of autism in children when taken during pregnancy. In rats exposed to VPA before birth, we found changes in certain nerve cells of the retina: one type of cell important for night vision was reduced, while another type increased slightly, while most other cells stayed the same. This suggests that the changes in development that lead to autism may also be reflected in the structure and function of the eye.

Amyotrophic lateral sclerosis (ALS) is a terminal disease caused by motor neuron loss. Schwann cells, the myelinating cells of the peripheral nervous system, metabolically and structurally support neurons. ALS patients exhibit Schwann cell pathology, such as TDP-43 proteinopathy, therefore Schwann cell dysfunction may contribute to disease progression. Here, we have characterised myelinating Schwann cell pathology in a TDP-43Q331K (TDP-43) transgenic mouse model of ALS. We also crossed the floxxed TDP-43 mouse with a myelin protein zero (P0)-cre mouse to excise the transgene from Schwann cells alone (P0-cre/TDP-43) to assess rescue. Compared to wild-type (WT) littermates, 10 mo TDP-43 mice exhibited changes to myelin architecture, including loss of myelin binding proteins at the paranodes, decreased node of Ranvier length, and non-compact, degenerating myelin. In P0-cre/TDP-43 mice these myelin disruptions were rescued. However, this improved histology did not lead to a functional rescue, with both P0-cre/TDP-43 and TDP-43 mice exhibiting slowed sciatic nerve conduction and worsened motor behaviour. Further histological analyses revealed that Bungner Schwann cells, a subtype of Schwann cells triggered by neuronal injury, were activated in both TDP-43 and P0-cre/TDP-43 mice. Activation of Bungner Schwann cells can trigger damaging inflammation through the recruitment of macrophages, which can hinder motor and electrophysiological performance, potentially underpinning the lack of functional rescue in the P0-cre/TDP-43. We established that the rescue of Schwann cells indeed protects myelin in this ALS model, however understanding how Bungner Schwann cells exacerbate neuronal pathology is essential for developing effective therapeutics that can improve functional output. Significance StatementAmyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with no cure and limited treatments. Given that the average patient life expectancy is 3-5 years following diagnosis, finding novel treatment targets is of the utmost importance. Recent research has revealed that non-neuronal cells, such as Schwann cells, contribute to the disease, however the extent of their pathology remains elusive. Investigating Schwann cell and peripheral myelin pathology in ALS may lead to the identification of previously unrecognized disease mechanisms, opening novel avenues for therapeutic development. Identifying approaches through which to target glial and neuronal pathology concurrently would enable more holistic treatment of the various aspects of ALS pathobiology to improve patient outcomes.

The ultimate cause of blindness in glaucoma is the death of retinal ganglion cells, and understanding the mechanism behind retinal ganglion cell loss during glaucoma could lead to the development of novel treatments for glaucoma. Endothelin-1 has been shown to mediate retinal ganglion cell death during glaucoma through impairment of mitochondrial function. Retinal ganglion cells are highly metabolically active, and susceptible to oxidative damage and decreased respiratory capacity. Mitophagy is the process whereby damaged mitochondria are degraded to prevent further propagation of oxidative damage. The current study evaluates the effect of endothelin-1 on mitophagy in retinal ganglion cells. Electron microscopy revealed endothelin-1 administration lead to a decrease in healthy mitochondria in the optic nerve. The MitoQC mouse was used to evalute mitophagy in response to endothelin-1, along with immunohistochemical analysis of mitophagy proteins. Mitophagy follows different trends in the optic nerve and retinal ganglion cell bodies following endothelin-1 administration, mitophagy was increased in the optic nerve but decreased in the retina following endothelin administration. With elevation of intraocular pressure, mitophagy was increased in the retina but decreased in the optic nerve. In retinal ganglion cells, parkin expression and activation was unchanged 24 hours after endothelin-1 administration, but was decreased 72 hours following endothelin-1 administration. Taken together, these results suggest that endothelin-1 impacts mitophagy through parkin-independent mechanisms in retinal ganglion cell bodies, and the ganglion cell bodies and optic nerve appear to have different responses to endothelin-1.

Synaptojanin 1 is a brain enriched phosphoinositide phosphatase implicated in endocytosis at the synapse. A mutation (R258Q) that selectively impairs its Sac1 phosphatase domain causes early onset familial Parkinsonism. Neurons of mice with this mutation display synaptic vesicle traffic defects across the brain, but selective dystrophic changes in a subset of dopaminergic axons in the dorsolateral striatum. Using correlative light microscopy-FIB-SEM of mutant mouse striata to visualize in 3D these abnormal structures we show that they represent clusters of focal axonal dilations harboring massive, onion-like DAT enriched plasma membrane infoldings, generally localized next to cell bodies of neighboring cells, often engulfing evaginations of such cells. This dysmorphia was associated with a deficit in dopamine release in the same striatal region. Given the involvement of Synj1 in endocytic mechanisms, these structures may reflect an imbalance between exocytosis and endocytosis. Their occurrence only in a subset of axons suggest a vulnerability threshold of these axons beyond which the expansion of the plasma membrane is not counteracted by compensatory mechanisms.

This study examined the role of ST3GAL3 as a regulator of excitatory/inhibitory (E/I) synaptic homeostasis using a human iPSC-based model. Neurodevelopmental disorders (NDDs) are increasingly linked to disruptions in the E/I balance, yet the molecular determinants remain poorly defined. ST3GAL3, a sialyltransferase associated with both rare monogenic disorders, including intellectual disability and infantile epilepsy, and complex polygenic conditions, such as ADHD, represents a strong candidate gene for involvement in synaptic regulation. To investigate this, isogenic ST3GAL3 knockout (ST3GAL3 KO) and wildtype (WT) iPSC lines were generated through CRISPR/Cas9 editing and differentiated into cortical neurons using both directed and induced protocols. This dual strategy enabled robust comparisons across cellular contexts and minimised methodological bias. To this end, we conducted functional characterisation using microelectrode array (MEA) technology alongside transcriptomic profiling through RNA sequencing (RNAseq), directly comparing ST3GAL3 KO-derived neurons with their isogenic controls. Functional assays using MEA revealed aberrant bursting patterns, particularly prolonged burst durations and heightened variability in S3GAL3KO neurons. Complementary transcriptomic profiling performed via RNAseq demonstrated downregulation in ST3GAL3 KO lines of genes involved in cognition, memory, as well as glutamatergic and GABAergic synaptic plasticity and functionality, providing molecular evidence for widespread synaptic dysregulation. Together, these findings establish ST3GAL3 as a key regulator of E/I balance in the cortices, advancing current knowledge on the pathophysiological involvement of ST3GAL3 deficiencies in the development of NDDs.

Friedreich Ataxia (FRDA) is an autosomal recessive neurodegenerative disorder characterized by progressive loss of cerebellar and proprioceptive neurons that control movement and coordination. In most patients, FRDA is caused by homozygous GAA trinucleotide repeat expansions in the first intron of the frataxin (FXN) gene, resulting in reduced expression of frataxin, a mitochondrial protein essential for biogenesis of iron-sulfur clusters and mitochondrial function. Although recent therapeutic advances have provided modest clinical benefit, effective disease-modifying treatments remain lacking. Increasing evidence indicates that microglial cell dysfunction contributes to FRDA pathogenesis, highlighting these cells as potential therapeutic targets. However, the molecular mechanisms underlying FXN-deficient microglial dysfunction remain poorly understood. Here, we show that microglia generated from FRDA patient-derived iPSCs exhibit a cell-autonomous pro-inflammatory phenotype in the absence of exogenous inflammatory stimuli. This phenotype is characterized by coordinated activation of immune transcriptional programs, dysregulated secretion of neuroinflammatory proteins, impaired autophagy-lysosomal function, and activation of inflammasomes pathways involving NLRP2 and NLRP3. These findings demonstrate that FXN deficiency is sufficient to induce intrinsic microglial activation and identify molecular pathways that may represent attractive targets for future FRDA therapies.

Myelin is classically viewed as a uniform axon-insulating membrane, yet its molecular composition may differ between species and even within one species. Fatty acid binding protein-8 (FABP8/PMP2) was previously identified in CNS myelin of humans but not mice. Here we show that FABP8/PMP2 is a defining feature of CNS myelin in humans and old-world-monkeys, but absent from CNS myelin in other mammals, indicating evolutionary neofunctionalization of this lipid-binding protein in the primate lineage. In the human CNS, FABP8/PMP2 marks a subset of myelin sheaths that preferentially ensheath large-diameter axons, revealing sheath-to-sheath molecular heterogeneity correlated with axonal geometry. Chromatin is accessible at the PMP2/Pmp2 gene locus in oligodendrocytes of humans but not mice. Human oligodendrocytes intrinsically express FABP8/PMP2 when transplanted into mouse brains, demonstrating species-specific competence independent of environmental cues. Humanized transgenic mice expressing FABP8/PMP2 in oligodendrocytes form morphologically normal but developmentally transiently thicker myelin sheaths, and show elevated cholesterol content in purified myelin. Because FABP8/PMP2 binds cholesterol, we propose that its emergence in primate CNS myelin contributes to the cholesterol enrichment of human myelin. Thus, CNS myelin protein composition is evolvable and modular, with relevance for myelin lipids and morphology, and previously unrecognized complexity in neuron-glia co-adaptation. Main points- Fatty acid binding protein 8 (FABP8/PMP2) is present in CNS myelin of humans and old-world monkeys - PMP2 defines sheath-to-sheath heterogeneity in the human CNS - PMP2-immunopositive myelin ensheaths large-diameter axons - Human oligodendrocytes intrinsically express PMP2 upon transplantation into mice - Humanized PMP2-transgenic mice show thicker myelin and altered myelin lipid composition

Traumatic optic neuropathy (TON) occurs due to direct or indirect injury to the optic nerve and is a significant cause of visual disability. So far, there is no effective treatment. The lack of understanding of the cellular mechanisms by which trauma induces inflammation and damage in retinal neurons is a critical knowledge gap in developing effective therapies. We have studied the role of the arginase 1 (A1) enzyme in this pathology. We have found previously that treatment with a long-acting form of human recombinant A1, pegylated A1 (PEG-A1) after optic nerve crush limits activation of retinal microglia and macrophages (M{Phi}) and reduces inflammation, thereby decreasing injury and protecting visual function. Here we report on studies designed to demonstrate the therapeutic efficacy of PEG-A1 in mouse models of direct and indirect TON and to elucidate the underlying mechanisms. We used ONC to model direct TON and sonication-induced trauma to the supraorbital rim to model indirect TON (SI-TON). At different times after injury, mice were treated with PEG-A1 which was delivered systemically by i.p. injection or locally by intravitreal injection. In order to assess the role of A1-induced activation of the ornithine/polyamine pathway in the protective effects of PEG-A1, some mice were treated with the ornithine decarboxylase (ODC) inhibitor, difluoromethylornithine (DFMO) immediately after the PEG-A1 treatment. Retinal function was determined by OptoMotry and electroretinography. Retinal injury and microglia/M{Phi} activation were assessed by immunofluorescence imaging. Expression of inflammatory cytokines was determined by Western blotting and quantitative RT PCR. Liquid chromatography mass spectrometry was used to analyze changes in arginase/ODC pathway metabolites. Results showed that PEG-A1 treatment improved neuronal survival and visual function whether delivered systemically or intravitreally. This neuroprotection was associated with decreased microglia/M{Phi} activation, decreased inflammatory cytokine expression, and increased formation of L-ornithine and putrescine. Furthermore, DFMO treatment blocked these effects, indicating that PEG-A1 limits retinal injury and preserves vision after ocular injury by activating ODC. ODC processes the arginase product L-ornithine to form polyamines which are known to promote reparative functions. Thus, PEG-A1 therapy offers a new strategy to limit trauma-induced vision loss and promote repair after TON.

PurposeTo investigate whether rapamycin can regulate microglial activation and polarization via mTOR and its downstream signals via autophagy both in vivo and in vitro. MethodsThe in vivo study used wild type C57BL/6 mice that were intraperitoneally injected with rapamycin (2 mg/kg) plus ONC. The BV2 cell line was used in the in vitro study and the cells were incubated with rapamycin (50 nM) or transfected with a specific mTOR-targeting small interfering RNA (si-mTOR). Immunohistochemical staining was used to observe the changes in the morphology and cell surface area of microglia and Weste blotting analysis was used for detection of the changes in the proteins related autophagy, microglia polarization and mTOR pathway after the retinal tissue or the cell samples were collected. ResultsThese results indicate that rapamycin increases autophagy and M2 polarization by inhibiting p-mTOR in wild-type C57BL/6 mice in vivo. In the BV2 cell line, rapamycin and si-mTOR can enhance autophagy and promote M2 polarization by inhibiting the p-mTOR/p-Unc-51-like kinase 1 (p-ULK1) pathway. ConclusionsIn conclusion, this work contributes to the understanding of the complex interplay among rapamycin, autophagy and microglial activation/polarization, highlights the downstream signaling pathway of mTOR, and highlights the potential therapeutic effects of autophagy-modulating drugs in retinal neuroinflammation and neurodegeneration after TON.