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.

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.

Oxidative phosphorylation (OxPhos) and substrate-level phosphorylation (SLP) are the primary biochemical mechanisms for ATP generation in mammalian cells. Increased dependency on SLP can arise from either transient (e.g., Crabtree effect) or chronic (e.g., Warburg effect) suppression of OxPhos. We evaluated the steady-state bioenergetics of quiescent and proliferating mouse cells matched in genetic background and cell biology, including primary thioglycollate-elicited peritoneal macrophages, malignant VM-M3 glioma with macrophage characteristics, and malignant CT-2A astrocytoma. Data are presented showing: 1) OxPhos insufficiency was the primary bioenergetic hallmark of proliferating malignant cells. 2) Dysregulated proliferation was associated with lower metabolic flexibility under nutrient stress than was growth arrest or regulated proliferation. 3) The Crabtree effect was present in macrophages and involved a glucose- and time-dependent suppression of OxPhos coupled to ATP production through SLP and resistance to mitochondrial inhibitors; lowering glucose concentration or substituting glucose with respiratory fuels increased OxPhos and restored sensitivity to mitochondrial inhibitors. 4) The Warburg effect in tumor cells involved a persistent SLP dependency and resistance to mitochondrial inhibitors under equivalent glucose-replete conditions. 5) OxPhos could sustain the regulated proliferation of non-tumor cells in the absence of a Warburg effect. Collectively, our findings emphasize that comparisons between normal and cancer cell metabolism require long-term experimental endpoints that ensure steady-state ATP sufficiency.

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.

Vesicular glutamate transporters 1 and 2 (VGluT1 and VGluT2) exhibit largely complementary distributions and have been proposed as molecular markers of descending modulatory and ascending driver-like pathways, respectively. However, this correspondence has rarely been tested directly in anatomically identified projections. We combined anterograde Phaseolus vulgaris leucoagglutinin tracing with simultaneous VGluT1 and VGluT2 immunofluorescence to characterize glutamatergic boutons arising from the primary and secondary visual cortices, lateral geniculate nucleus, lateral posterior thalamic nucleus, and superior colliculus in adult mice. Among 2,178 singly labelled boutons, VGluT1 predominated in corticothalamic, corticopontine, corticotectal, corticostriatal, and corticocortical feedback projections. Conversely, thalamocortical, tectothalamic, thalamostriatal, and tectopontine projections were almost exclusively VGluT2-positive. Projection direction did not fully predict transporter phenotype: ascending corticocortical projections remained predominantly VGluT1-positive, whereas the descending tectopontine projection was exclusively VGluT2-positive. Only 12 of 2,190 VGluT-immunoreactive boutons exhibited detectable VGluT1/VGluT2 colocalization. Morphometric analyses further revealed an interaction between VGluT isoform and projection direction. Within ascending projections, VGluT2-positive boutons and puncta were larger than their VGluT1-positive counterparts, whereas no isoform-related difference in bouton area was detected within descending projections. These findings demonstrate an association between VGluT phenotype and projection class in the mouse visual system. VGluT isoforms therefore provide informative markers of pathway organization, but neither transporter identity nor terminal size alone constitutes an invariant molecular indicator of projection direction or driver-modulator function.

Background: Succinate dehydrogenase (SDH) is a heterotetrameric enzyme in both the tricarboxylic acid (TCA) cycle and the electron transport chain. SDH-deficiency has been shown to drive the oncogenesis of cancer types including gastrointestinal stromal tumors (GIST), paraganglioma/pheochromocytoma, and clear cell renal cell carcinoma. Previous work on SDH loss has shown increases in succinate, decreases in fumarate, as well as increase in the succinate to -ketoglutarate ratio, and correlated these changes to metabolite signaling and epigenetic dysregulation. Alterations to other TCA intermediates, including succinyl-CoA, the acyl-donor for succinylation, and the resulting cellular effects has not been fully explored. Methods: We used liquid chromatography high resolution mass spectrometry to quantify TCA cycle metabolites across the NCI-60 panel of cell lines, three SDH-competent and SDH-deficient model systems, and human tumor samples. We measured histone post-translational modifications in SHD-deficient and SDH-competent models and examined the correlation and potential pharmacological targeting of described desuccinylase enzymes by protein and cell assays. Results: We found an increase in succinyl-CoA in multiple SDH-deficient models, but no correlation between succinate to fumarate ratio and succinyl-CoA concentrations in the NCI-60. Interestingly, we found that while there was an increase in succinyl-CoA in the SDH-deficient cells, there was no corresponding increase in histone succinylation. We also observed a decrease in lysine malonylation and glutarylation, two other anionic lysine modifications. Cell viability and clonogenic assays revealed very weak or no selective effect of desuccinylase targeting in SDH-deficient models. Similarly, using SDH-deficient versus SDH-competent GIST patient samples, we examined both protein expression and metabolite abundance at the bulk tumor level and found no significant increase in succinyl-CoA or desuccinylase expression, despite the increase in succinate to fumarate ratio. Conclusions: We found that SDH-deficiency led to altered central carbon metabolism including increases to succinate and succinyl-CoA. Despite data that suggested the upregulation of desuccinylases may mediate a disconnect between succinyl-CoA concentration and histone succinylation, we found weak or no reductions in cell viability in response to pharmacological inhibition of desuccinylases. Additionally, no correlation of SDH loss with desuccinylase expression and succinyl-CoA in bulk tumor samples was observed.

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

Midbrain organoids (MOs) can be powerful tools to study brain diseases, yet one shortcoming is the lack of microglia due to their mesodermal developmental origin. Previous studies have incorporated iPSC-derived microglia (iMG), but their immature phenotype restricts the applicability to model age-related neuroinflammatory processes relevant to diseases such as Parkinson's Disease (PD). To address this, we have incorporated MOs with human postmortem PD microglia, iPSC-derived microglial progenitors (iMP), and iMG. Postmortem microglia had a higher infiltration capacity resulting in more microglia per organoid, while iMP had a higher migration capacity reaching the core of the organoid. Compared to iPSC-derived cells, more infiltrated postmortem PD microglia were HLA Class II positive and CD68 positive. This novel approach of integrating postmortem microglia into organoids opens new avenues for modelling age-related neuroinflammatory diseases.Ongoing methodological advancements like our study will offer the field a valuable tool for studying human microglia biology in both health and disease.

Patients with NF2-related schwannomatosis (NF2-SWN) present with hallmark bilateral vestibular schwannomas, but also schwannomas on other cranial, spinal, and peripheral nerves, as well as meningiomas and ependymomas, caused by germline mutations in the tumor suppressor gene NF2. Current therapies involving surgery and radiosurgery are effective for individual tumors but are not always a viable option for patients with multiple tumors, and harbor significant risk of neurological deficits and morbidity. Gene replacement therapy is becoming a promising new treatment strategy for several neurologic diseases. This study aims to understand if restoration of a functional merlin protein, gene product of the Nf2 gene, in Nf2-deficient tumor cells, can provide preclinical therapeutic efficacy in a NF2-SWN genetically engineered mouse model. We have developed a new Nf2 allele (Nf2FRT) that allows to conditionally restore Nf2 expression by activation with the Flp recombinase. We generated a new mouse model using the Nf2FRT allele in combination with Nf2flox and Postn-Cre alleles. Using the new conditional schwannoma mouse model Postn-Cre;Nf2FRT/flox;R26FlpoER, we validated the hypothesis that restoration of Nf2 reduces the growth of schwannoma. Demonstrating that merlin restoration effectively controls schwannoma growth is a crucial first step toward developing this concept as a new therapeutic strategy for potentially treating schwannomas. The new mouse model will be used to better understand the cellular and molecular mechanisms involved in stopping the growth of Nf2-deficient tumors.

Oligodendrocytes play an essential role in axonal metabolic support and myelination, and their dysfunction is associated with amyotrophic lateral sclerosis (ALS). LINGO1 is a major inhibitor of oligodendrocyte differentiation and axonal regeneration, identified as a promising therapeutic targeted for inflammatory demyelinating diseases. Here, we investigated the expression pattern of LINGO1 in the well-established SOD1(G93A) transgenic mouse model of ALS. We show that LINGO1 protein expression is significantly elevated in both grey and white matter of the mutant spinal cord during both the symptomatic and end stages of the disease. LINGO1 is also notably co-expressed with astroglial and neuronal markers. However, neither the levels of LINGO1 transcripts, nor levels of microRNAs predicted to regulate its expression were altered in the mutant spinal cord, suggesting the involvement of alternative regulatory mechanisms. Our analysis of microRNAs revealed in the mutant a significant downregulation of miR-138, a microRNA known to promote myelination. The differential expression of LINGO1 and miR-138 in ALS mice thus may drive deleterious processes during disease progression in adulthood. Moreover, our data show that white-matter pathology arises earlier than initially thought, as evidenced by significant alterations of oligodendrogenesis that manifest as early as postnatal day 5. Study of the molecular determinants of this developmental defect shows increased miR-138 expression, which may promote oligodendrocyte progenitor cell maturation and involve compensatory mechanisms. Overall, our work identifies disrupted expression of LINGO1 and/or miR-138 in brain during early oligodendrogenesis and at symptomatic disease stages. These findings open the way to further investigations that may consider miR-138 as a potential presymptomatic biomarker and LINGO1 as a therapeutic target to promote remyelination in the context of ALS.

Suspension mass cytometry can measure 50 or more markers per cell and is not impacted by cellular autofluorescence, making it an appealing platform for comprehensive phenotyping of single cells in tissue with high innate fluorescence, such as brain. However, this organ is challenging to reduce to a single cell suspension: it is a deeply interconnected tissue rich in extracellular material and contains a wide variety of cell types including neurons, their supporting glia, and hematopoietic cells from peripheral circulation, any or all of which may have competing requirements for survival through the dissociation process. This study compares approaches for producing single-cell preparations from white and gray matter enriched regions of adult mouse brain, testing simple mechanical dissociation, Accutase, and three gentle enzymes plus DNase: papain, collagenase II, and collagenase plus dispase, all with or without Percoll gradient centrifugation to separate intact cells from debris. Live cells were counted by hemocytometer and Trypan blue exclusion to assess yield and compare across enzyme conditions. Mass cytometry was used to assess cellular composition after representative dissociation, debris removal, and viable cryopreservation. Key proteins measured included beta III-tubulin for neurons, glial fibrillary acidic protein (GFAP) for astrocytes, oligodendrocyte transcription factor (OLIG2) for oligodendrocytes, CD31 for endothelial cells, CD24 for ependymal cells, purinergic receptor P2Y12 (P2RY12) for microglia, and CD45 for leukocytes. Mechanical disaggregation paired with enzymatic dissociation by papain, Collagenase/Disapse, or Collagenase II plus DNase produced approximately 400,000 live cells per 100 grams of tissue. Cell yield was not significantly different between conditions and yields were similar between white and grey matter. Addition of Percoll gradient centrifugation substantially reduced the number of viable cells recovered (average decrease of 88%) and resulted in viable cell numbers that were insufficient for robust analysis of less common cell subsets. In addition to neurons, mass cytometry identified six major populations of non-neuronal cells: oligodendrocytes, astrocytes, endothelial cells, ependymal cells, microglia, and leukocytes. The relative abundance of these six populations was generally unchanged by cryopreservation in both grey matter (r = 0.95), and white matter (r = 0.90), though loss of oligodendrocytes did occur in both tissues. Proliferation, as measured by Ki67 positivity, was non-existent; consistent with this, levels of phosphorylated STAT1 (p-STAT1) and p-STAT3 proteins were low in all cell populations and unchanged by cryopreservation. A modest increase in p-S6(S235/236) after cryopreservation was consistently observed only in microglia. Together, these results establish standardized procedures for generating viable single cell suspensions for cytometry that preserve the cellular diversity of the adult murine brain with minimal perturbation of intracellular signaling cascades.

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.

Mutations in mitochondrial DNA (mtDNA) compromise ETC activity and impair oxidative phosphorylation. Since eukaryotic cells contain multiple copies of mtDNA, the resulting phenotype depends on the proportion of mutant mitochondrial genomes (the heteroplasmy level). Using isogenic cell lines carrying similar mtDNA deletions, a linear decline in cellular respiration was observed as mitochondrial DNA heteroplasmy increased. Despite this, cellular redox imbalance did not change until heteroplasmy exceeded 50%. As heteroplasmy increased past 70%, cells also exhibited an integrated stress response (ISR) and impaired translation was observed. These defects were reversed by either addition of asparagine or overexpression of pyruvate carboxylase (PC). The dependence on exogenous asparagine in other respiration-deficient cells was found to correlate inversely with the PC expression level. For example, patient-derived thyroid tumor cells, harboring high heteroplasmy for a Complex I mtDNA mutation and low levels of PC, exhibited asparagine auxotrophy, and L-asparaginase treatment suppressed tumor growth. Together, these findings demonstrate a role for mitochondrial pyruvate carboxylase in cellular asparagine synthesis under conditions of compromised respiratory activity.

Cell confluency is a major determinant of cell-cell communication, protein interactions, access to nutrients, and signalling dynamics, thereby significantly impacting biological outcomes. Lung cancer cells like A549 are widely used as screening models for scientific studies wherein their growth in vitro progress from non-confluent to confluent growth. In this study, we investigated the transcriptomic adaptations associated with the transition of A549 cells from baseline non-confluent to confluent growth. Comparative transcriptomic analysis between confluent and cells at baseline identified 815 upregulated and 671 downregulated transcripts. Pathway enrichment analysis of deregulated transcripts in confluent cells revealed enhanced cholesterol and sterol biosynthetic pathways, along with suppression of chromosomal segregation and mitotic pathways. At confluency, an increased expression of glucose transporters (SLC2, SLC60, and SL37 families) and glycolytic pathways, and a decrease in amino acid transporters (SLC1, SLC7, SLC38, and SLC36) and amino acid metabolic pathways is observed. A reduced one-carbon metabolic signature (SHMT2, DHFR, and MTHFD2) and enhanced fatty acid precursor synthesis (HMGCLL1, ALDH6A1, and AASS) were also observed at confluency. 1H NMR profiling of culture media revealed higher glucose and glutamine utilisation with lactate accumulation during culture maturation. Collectively, the data suggest transcriptome-level rewiring in A549 cells with preferential biosynthesis of lipids and sterols at confluency and underscore the importance of considering culture maturity in cancer biology, metabolism, and therapeutic studies.

Purpose: Retinal ganglion cell (RGC) death following optic nerve injury is driven by transcriptional programs involved in apoptosis, neuroinflammation, and other regulated cell death pathways; however, the early transcriptomic changes initiating these processes remain poorly defined. We used rRNA-depleted total RNA sequencing to characterize early alterations in protein-coding genes, long noncoding RNAs (lncRNAs), and alternative splicing events in mouse retinas following optic nerve crush (ONC) and silicone oil-induced ocular hypertension under-detected (SOHU), prior to substantial RGC loss. Methods: Whole retinas were collected 6 hours after ONC and 5 days after SOHU induction. Differentially expressed protein-coding genes and lncRNA-classified transcripts were identified using total RNA sequencing, followed by functional enrichment, lncRNA-associated pathway analysis, alternative splicing analysis, and fluorescent in situ hybridization (FISH) validation. Results: In SOHU retinas, hundreds of protein-coding genes and lncRNA-classified transcripts were differentially expressed, with enrichment of immune activation, extracellular matrix remodeling, complement, phagocytic signaling, and suppression of mitochondrial energy metabolism pathways. Similarly, in ONC retinas, many protein-coding genes and lncRNA-classified transcripts were differentially expressed, with a prominent chemokine, cytokine-dominant inflammatory response. FISH validation supported injury-associated upregulation of Prss56 and Lif. Both injury models exhibited substantial transcript-level alterations, with widespread alternative splicing changes detected. Conclusions: Early optic nerve injury induces rapid transcriptional changes involving protein-coding genes, lncRNAs, and transcript isoform regulation before substantial RGC loss. These findings highlight early alterations in both coding and noncoding RNA landscapes following retinal injury and provide a valuable resource of candidate genes and pathways for future validation in RGC degeneration and neuroprotection.

Retinal neurons rely on continuous oxygen supply to maintain function, yet some species, like goldfish, withstand prolonged hypoxia due to adaptations that remain unclear. Horizontal cells (HCs) are inhibitory interneurons of the retina that express ionotropic glutamate receptors (iGluRs) and secrete gama-aminobutyric acid (GABA), allowing them to shape visual signalling by feeding back to photoreceptors. We investigated whether iGluRs in dissociated goldfish HCs are suppressed during hypoxia, and whether this process is mediated by oxygen-sensitive activation of mitochondrial ATP-sensitive K+ (mKATP) channels. Through perforated patch-clamp electrophysiology, we determined that 20 min of hypoxia reduced peak inward current density associated with glutamate application by ~25% (P = 0.0059). When mKATP channels were selectively blocked with 100 microM glibenclamide, hypoxic suppression of iGluRs was abolished. Inhibiting mitochondrial Ca2+ uptake via the mitochondrial Ca2+ uniporter with ruthenium red, or blocking Ca2+ release through ryanodine receptors in the endoplasmic reticulum, also abolished the hypoxia-induced suppression. Similarly, inhibition of calmodulin with calmidazolium, or of protein phosphatase 1 and 2A (PP1/PP2A) activity with calyculin A, prevented the hypoxia-induced suppression of iGluRs. Taken together, these results indicate that mKATP activation is required to mediate an intracellular Ca2+ signal that downregulates iGluR activity during hypoxia. Through the reduction of iGluR activity, HCs may lower their metabolic demand, allowing them to maintain cell function during hypoxia. Additionally, GABA release from HCs, as measured by mass spectrometry, was not affected by hypoxia, and we propose that downregulation of iGluR may account for hypoxic suppression of the retina in hypoxia-tolerant organisms.

Microglia are essential immune cells of the brain parenchyma which are responsible for maintaining homeostasis and responding to brain injury. When activated, they induce immune signaling and become phagocytic. In the context of neurodegenerative diseases, they exhibit both pro- and anti-inflammatory properties. Human endogenous retroviruses (HERVs) are the remnants of ancient retroviral germline infections that integrated into the human genome, and their expression has been directly linked to microglial-mediated neurodegenerative diseases. Despite this, HERV expression is not consistently included in analyses characterizing microglia. Investigating the function of microglia across different diseases is critical for elucidating mechanisms of disease progression and for the development of effective therapeutic interventions. Approaches to studying microglia include isolation from post-mortem brain tissues, differentiation from peripheral blood monocytes or differentiation of induced pluripotent stem cells (iPSC). Ex vivo brain derived microglia (BdMG) are considered most physiologically relevant, but constraints including the unpredictable timing of availability, labor-intensive protocols, and low yields reduce their practicality. Existing alternatives are iPSC-derived microglia (iMG) and monocyte-derived microglia (MDMi) models that circumvent sample accessibility and scalability issues. Here, we explored the transcriptome and proteome of these three microglia sources and identified a common microglia surface marker expression profile, establishing that MDMi and iMG exhibit similar phenotypes relative to BdMG. We also identified a common HERV expression profile across BdMG, iMG and MDMi, indicating deeper similarities across different sources. Altogether, our findings indicate that given the phenotypic similarities of the 2 models, MDMi represent a practical and reliable model for investigating microglial function with iMG models serving for more molecular based approaches, such as CRISPRa/i.

The glymphatic system is the waste clearance pathway in the central nervous system. Although its dysfunction has been associated with several neurological disorders, its role in multiple sclerosis (MS) remains poorly understood. This system is thought to be particularly active during sleep, yet the relationship between sleep metrics and MRI-derived markers of glymphatic activity has been scarcely explored. In this study, we aimed to perform a multimodal magnetic resonance imaging (MRI) assessment of glymphatic function in MS and investigate its association with sleep parameters, clinical disability, disease duration and MRI-derived structural markers. The study included 22 patients (34.3{+/-}10.1 years; 16 female) with relapsing-remitting MS and 26 healthy controls (32.2{+/-}5.7 years; 18 female), all of whom underwent 3.0 Tesla MRI. Glymphatic function was assessed using diffusion tensor image analysis along the perivascular space (DTI-ALPS) index and global blood-oxygen-level-dependent (BOLD) signal-cerebrospinal fluid (CSF) coupling. In addition, sleep micro- and macrostructural features, clinical disability, disease duration, along with mean diffusivity (MD), lateral ventricular, choroid plexus volumes and lesion load were evaluated. Patients showed reduced DTI-ALPS index, though this difference became only a trend after controlling for MD. No significant group differences were observed in global BOLD signal-CSF coupling. However, in patients, the weaker coupling was associated with longer total sleep time, lower arousal index, greater disability and lateral ventricle enlargement, whereas the DTI-ALPS index showed no significant correlations with any of the measures. Together, these findings challenge the assumption that DTI-ALPS can serve as a reliable marker of glymphatic function in demyelinating diseases such as MS, where disease-related alterations in white matter microstructure can confound diffusion-based measures. In contrast, gBOLD-CSF coupling showed associations with sleep, clinical disability and structural changes in patients, despite the absence of a significant group-level difference. This finding suggests that gBOLD-CSF coupling may capture individual variability in glymphatic-related dynamics relevant to sleep and the clinical expression of MS.

Axons of substantia nigra dopaminergic neurons "die back" in Parkinson's disease (PD), exhibiting early degeneration relative to cell bodies, a key feature also observed in PD models. A critical step towards understanding the process of axonal neurodegeneration is to determine the physiological properties of dopaminergic axons that are altered during the early stages of cellular dysfunction. Here, we compared the electrophysiology and calcium dynamics of dopaminergic axons in the MitoPark model of PD with age-matched controls. Our experiments show a pronounced reduction in action potential (AP)-evoked calcium signals in MitoPark axons that emerges well before the onset of dopaminergic fiber loss and motor deficits. In addition, calcium imaging and direct axon recording revealed deficits in AP propagation accompanied by alterations in the axonal AP waveform. Therefore, our experimental findings identify the progression of axon-specific physiological deficits that occur long before neurodegeneration, more precisely defining the period that precedes axonal loss.

Background Gliomas are primary brain tumors that integrate into the surrounding brain via neuron-glioma synapses and disrupt normal neuronal functioning. Recent work links tumor-brain connectivity to clinical measures such as patients' functional status. However, few experimental systems allow the visualization of tumor and resident brain cell architecture together in human tissue at the scale needed to connect cellular organization to clinical outcomes. To address this gap, we developed an experimental framework for investigating the neuron-glioma network in organotypic human cortex from glioma patients, designed to support future correlation of cellular architecture to clinical outcomes such as functional status and survival. Methods Brain tissue samples containing cortex from seven glioma patients were collected during tumor resection and cultured for seven days as organotypic slices. We established several multiplexed immunohistochemistry panels to stain for glioma cells, various types of resident brain cells including neurons and cells from the oligodendrocyte lineage, as well as axonal networks and myelination patterns. Results We demonstrated the presence of tumor cells in all samples using SOX2, a tumor cell marker. NeuN+ neuronal cells were also successfully identified, as were cells from the oligodendrocyte lineage using SOX10 (pan-lineage marker), BCAS1 (pre-myelinating oligodendrocyte marker), and ASPA (mature oligodendrocyte marker). The neuronal network and myelin were successfully visualized using MAP2 (neurons and dendrites), SMI312 (axons), and PLP1 (myelin). Conclusions High-quality visualization of tumor cells, resident brain cells and the (myelinated) neuronal network in organotypic human cortex from glioma patients is possible after seven days of culturing. Future studies could further optimize culture conditions and should study stability of all cellular components in longer term slice cultures. Preliminary analyses indicate that cellular measures derived from human organotypic slices can be linked to patients' functional status, demonstrating the capacity to generate data suitable for large-scale investigation of the effects of glioma-brain network architecture on clinical outcomes.