ASN Neuro

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.

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.

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.

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.

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.

Achromatopsia (ACHM) is a rare autosomal recessive retinal disorder characterized by absent cone photoreceptor function from early life, leading to severe visual impairment. Mutations in genes involved in the cone phototransduction cascade frequently result in elevated cyclic guanosine monophosphate (cGMP) levels and activation of stress pathways, including endoplasmic reticulum (ER) stress and the unfolded protein response. Targeting common downstream mechanisms rather than individual mutations may provide a broadly applicable therapeutic strategy. Here, we investigated whether pharmacological inhibition of valosin-containing protein (VCP), a key regulator of ER and protein homeostasis, can prevent cone degeneration in the spontaneous cone photoreceptor function loss 1 (cpfl1) mouse model of ACHM. Organotypic culture of retinal explants from cpfl1 mice were treated with the selective VCP inhibitor ML240. Cone survival, cell death, opsin expression and localization were assessed by TUNEL assay, immunohistochemistry, and quantitative image analysis. ML240 treatment significantly increased cone density and improved cone opsin expression and trafficking to the outer segments (OSs) in cpfl1 explants compared to controls. Importantly, rhodopsin trafficking in rod photoreceptors was unaffected, indicating that VCP inhibition did not impair normal rod phototransduction. These findings demonstrate that VCP inhibition by ML240 effectively preserves cone photoreceptors and improves cone-specific functional markers in the cpfl1 model. Targeting VCP may represent a mutation-independent therapeutic strategy for preventing cone death in ACHM.

Axonal action potential propagation critically depends on tightly regulated intra and extracellular electrolyte concentrations and acid base homeostasis. Experimental studies have demonstrated that changes in extracellular sodium, potassium, and calcium concentrations alter axonal excitability and conduction properties. However, it remains unknown whether small variations in electrolyte concentrations within the physiological range are associated with measurable changes in nerve conduction velocity in humans. In this study, data from neurologically healthy children who underwent both nerve conduction studies and blood gas analysis were analyzed to investigate the relationship between electrolyte concentrations and median motor nerve conduction velocity. Nerve conduction velocity was significantly negatively correlated with ionized calcium concentration and positively correlated with sodium concentration, whereas no significant correlation was observed for potassium. In an a priori defined multiple linear regression model, ionized calcium, sodium, and bicarbonate remained significantly associated with nerve conduction velocity after correction for multiple testing. These findings indicate that even small variations in electrolyte concentrations within the physiological range are associated with measurable differences in peripheral nerve conduction velocity and suggest an additional contribution of acid base status to nerve conduction.

The field of microglial research has evolved throughout the years. Microglia, the immune cells of the central nervous system, are recognized as highly heterogeneous and dynamic cells, known for modifying their structure and function based on the local context. Investigating microglial density, spatial distribution, and morphological states is important for uncovering their distinct functional states in health and pathology. However, quantifying these features accurately is often a methodological challenge. Fully automated computational approaches often fail to capture subtle biological nuances and complex structural variations. Also, the high diversity of image sets makes it difficult to maintain consistent reliability. In contrast, entirely manual quantification is labor-intensive and prone to observer bias. To bridge this gap, we propose a semi-automated framework using the open-source software CellProfiler. Our workflow is divided into two distinct pipelines designed to combine automation of batch analysis with targeted user oversight, allowing for manual intervention when necessary to ensure maximum accuracy. Both pipelines are capable of recognizing microglial soma and tracing their processes. In the density workflow, it automatically calculates cell density and provides spatial distribution measurements, such as closest-neighbor distance and spacing index. For morphological profiling, it yields extensive structural data, including area, perimeter, convex area, form factor, and various shape descriptors. Furthermore, we demonstrate how researchers can optimize the pipeline settings to accommodate varied image datasets and experimental conditions. We hope this open-source framework can standardize microglial density, distribution, and morphological analysis and reduce systematic bias, providing researchers with a robust tool to better characterize their heterogeneity.

Background: Nervous system regeneration requires creation of new neurons and their integration into new and existing networks. How new neurons find their targets and reconnect in the environment of regenerating tissue remains a major gap in knowledge. The planarian Schmidtea mediterranea is well suited to the study of neural regeneration due to the remarkable ability of planarians to regenerate their entire body, including the nervous system. However, we have not yet been able to explore synaptogenesis in regeneration using planarians due to a lack of tools to study synapses. Here we investigate potential players in planarian synapse, focusing on the conserved syntaxin protein family, which functions in neurotransmitter vesicle fusion at the synapse. Results: We found that reduction of either syntaxin-1 (stx1) or syntaxin-binding protein 1-1 (stxbp1-1) decreased locomotion and impeded brain regeneration, specifically in synapse-rich regions. Conclusions: Our observations support STX-1 and STXBP1-1 as bona fide synapse components, providing a starting point for studies neuronal connectivity during homeostatic remodeling and regeneration.

Myelin formation requires extensive membrane lipid synthesis, yet how myelinating glia acquire the choline needed for this process remains incompletely understood. Choline transporter-like protein 1 (CTL1; SLC44A1) is highly expressed in Schwann cells, and CTL1 deficiency in oligodendrocytes impairs CNS myelination. Here, we examined the role of CTL1 in peripheral nerve myelination using Schwann cell-specific Ctl1 knockout mice. Unexpectedly, loss of Ctl1 did not alter Schwann cell differentiation, the number of myelinated axons, or myelin thickness, but increased myelin abnormalities, including in-foldings and out-foldings. Lipidomic analysis revealed selective alterations in myelin lipid composition, particularly among long-chain lipid species, together with triglyceride accumulation in whole nerves. CTL1 deficiency also increased mTORC1-associated S6 and mTORC2-associated AKT S473 phosphorylation. Transcriptomic analysis revealed downregulation of gene programs associated with fatty acid {beta}-oxidation, triglyceride catabolism, and oxidative phosphorylation. Following peripheral nerve injury, Ctl1-deficient Schwann cells generated a normal repair response and efficiently remyelinated regenerated axons despite altered mTOR signaling. Together, these findings demonstrate that CTL1 is not required for overall peripheral myelin production but contributes to maintaining normal myelin lipid composition and architecture, revealing an unexpected ability of Schwann cells to sustain myelination despite disruption of CTL1-dependent choline metabolism

Peripheral neurons exhibit robust regeneration after nerve injury in adulthood; a process linked to the activation of regeneration-associated genes (RAGs). However, the mechanisms underlying nerve regeneration during early postnatal development remain poorly understood. In a previous work, we observed that motoneurons axotomized at early postnatal stages regenerate without overexpressing RAGs, contrasting with the expected RAG activation observed in postnatal day 30 (P30) mice. To determine whether this phenomenon extends to sensory neurones, we investigated two paradigmatic dorsal root ganglia populations: proprioceptive (PV) and nociceptive (TrpV1) neurones, which exhibit distinct regenerative capacities in adult mice. Following sciatic nerve crush injury at P4, P10, and P30, both neuronal subtypes demonstrated robust axonal regeneration across all postnatal stages, with nociceptors exhibiting significantly superior regeneration compared to proprioceptors. Remarkably, no substantial neuronal death was observed at any developmental timepoint. However, RNA sequencing revealed that P4 and P10 sensory neurones failed to upregulate RAGs following injury, whereas P30 neurones exhibited the expected pro-regenerative transcriptional programme. Pathway enrichment analysis confirmed that classical regeneration-associated signalling pathways were activated exclusively in young adults, whilst developmental and inflammatory pathways predominated in early postnatal stages. Using the conditioning lesion paradigm in primary dorsal root ganglia cultures, we demonstrated that only P30 neurones exhibited enhanced neurite outgrowth following conditioning lesion, whilst P4 and P10 neurones showed no conditioning effect or reduced outgrowth, respectively. These findings indicate that early postnatal sensory neurones possess intrinsic growth capacity independent of RAG activation, suggesting fundamentally distinct regenerative mechanisms across developmental stages.

Oligodendrocyte (OLG) injury and loss are increasingly recognized as major determinants of demyelination and remyelination failure in multiple sclerosis (MS), alongside conventional immune-mediated processes. However, the mechanism underlying OLG injury and remyelination failure remains incompletely understood. Astrocyte-oligodendrocyte interactions mediated by Connexin (Cx)-43-Cx47 gap junction (GJ) communication and leukemia inhibitory factor (LIF)-LIFR/gp130 signaling are critical for oligodendroglial survival, homeostasis, and myelination. Despite their established role, how these pathways are altered during neuroinflammatory diseases remains unclear. To address this, we integrated a murine {beta}-coronavirus (RSA59)-induced in vivo model with enriched primary oligodendrocyte precursor cell (OPC) and mature OLG cultures to investigate the effect of RSA59 infection on OLGs, Cx47-mediated GJ communication, and LIF-LIFR/gp130 Signaling. We observed that RSA59 directly targets oligodendroglial lineage cells, including OPCs and mature OLGs, and induces apoptosis in mature OLGs. Despite increased OPC abundance, RSA59 infection reduces the expression of mature and myelinating markers CNPase and MBP, raising the possibility of impaired oligodendrocyte maturation and myelination. RSA59 infection also differentially regulates Cx47-mediated GJ communication and LIF-LIFR/gp130 signaling, crucial for OLG survival and homeostasis. Furthermore, our studies demonstrate that extracellular signal-regulated kinase (ERK), a downstream effector of LIF signaling, phosphorylates the Cx47 C-terminal domain (Cx47CT) at Serine-372 residue, identifying a previously unreported mechanism. Collectively, these findings reveal that RSA59 infection disrupts interconnected oligodendroglial communication and survival pathways, providing mechanistic insights into virus-induced chronic demyelination pathology. The study further identifies Cx47-mediated GJ communication and LIF-LIFR/gp130 signaling as crucial therapeutic targets, and highlights ERK-dependent Cx47CT phosphorylation as a potential regulatory mechanism.

Charcot Marie Tooth type 1A (CMT1A) is the most common inherited peripheral neuropathy, caused by duplication of the peripheral myelin protein 22 (PMP22) gene and characterized by dysmyelination and defects in myelin maturation. Hypercellularity is a prominent feature of the disease, although its origin, persistence, and underlying mechanisms remain controversial. Here, we performed a longitudinal analysis of sciatic nerves from wild-type (WT) and CMT1A rats between 2 and 12 months of age. We demonstrated that CMT1A nerves exhibit persistent cellular proliferation extending into adulthood. This phenotype is confirmed by bulk RNA-seq data showing sustained enrichment of proliferation-associated gene signatures. Cell type signature analysis identifies overrepresentation of immature, neural crest related, and epithelial mesenchymal transitional (EMT) transcriptional programs in CMT1A nerves. Among the most upregulated genes, RUNX2 showed strong and prolonged expression at both transcript and protein levels. Consistently with the key role of RUNX2 in the activation of the injury response, CMT1A nerves also showed a marked enrichment in SOX10+JUN+ cells, that only partially overlap with SOX10+RUNX2+ cells, indicating a maladaptive activation of the injury-like Schwann cell (SC) program. Interestingly, we detected robust upregulation of SMOC2, an extracellular matrix protein selectively expressed by endoneurial fibroblasts, pointing to activation of non-canonical cell types and altered extracellular matrix remodeling. Together, these data demonstrate that CMT1A peripheral nerves are characterized by chronic hypercellularity driven by persistent proliferation, EMT, and dysregulated injury transcriptional programs, collectively pointing at progressive and multicellular nerve remodeling as a relevant pathomechanism in the disease.

Tau is a microtubule-associated protein important for neuronal structure and function, with abnormal phosphorylation associated with neuronal dysfunction and degeneration. While tau pathology has been extensively studied in neurodegenerative diseases and traumatic brain injury, its role in spinal cord injury (SCI) remains unclear. This study investigates changes in the levels of tau isoforms and phosphorylation following a high lateral C2 hemisection (C2Hx) in adult female rats. Western blot analysis was performed on spinal cord tissue collected caudal to the lesion at 4 weeks post-injury. Low-molecular-weight (LMW) tau and high-molecular-weight, Big tau did not differ significantly between injured and control groups. In contrast, phosphorylated tau (p-tau), detected using the AT8 antibody, was significantly elevated after injury, suggesting a shift in tau functional state. Assessment of microtubule stability markers revealed increased acetylated tubulin and decreased tyrosinated tubulin after SCI, indicating a shift toward a more stable cytoskeletal state. Immunohistochemistry was performed on spinal cord sections at 6 weeks post-injury and dorsal root ganglia (DRG) at 8 weeks post-injury. These analyses demonstrated increased p-tau immunoreactivity within the ipsilateral dorsal horn of the injured spinal cord and a higher proportion of p-tau-positive neurons in DRGs from injured animals compared with controls. Together, these findings demonstrate that cervical SCI induces persistent tau phosphorylation without altering total tau levels, and that this modification extends beyond the lesion site into distal spinal regions and beyond the acute phase of injury. These alterations may influence neural plasticity and remodeling in the injured spinal cord.

Background: Parkinson's disease (PD) is characterized by the progressive loss of midbrain dopaminergic (DA) neurons and aberrant alpha-synuclein (alpha-syn) aggregation, yet disease-modifying therapies that simultaneously retard neurodegeneration and foster regeneration remain lacking. Methods: In this study, we isolated extracellular vesicles (TH and FOXA2 double-positive cell-derived EVs) from TH and FOXA2 double-positive differentiated neural cells, a distinct midbrain floor-plate-derived neural cell population that co-expresses the floor-plate transcription factor FOXA2 together with TH and should therefore not be equated with conventional mature midbrain dopaminergic neurons, and systematically evaluated their therapeutic potential across three complementary models: A53T transgenic mice, 6-OHDA-lesioned rats, and 6-OHDA-treated human midbrain organoids. Integrative transcriptomic and single-cell sequencing analyses, together with toxicological evaluations in rodents and non-human primates, were performed to investigate the underlying mechanisms and safety profile. These TH and FOXA2 double-positive cells were generated using a fully suspension-based, matrix-free, and chemically defined differentiation system devoid of N2/B27 supplements and fetal bovine serum (FBS). This approach offers a simple, controllable, low-cost, and scalable platform with high batch-to-batch consistency for EV manufacturing. Throughout this study, the term "TH and FOXA2 double-positive cells" refers to this distinct TH and FOXA2 double-positive cell population and should not be interpreted as conventional terminally differentiated dopaminergic neurons. Results: Intranasal administration of TH and FOXA2 double-positive cell-derived EVs in A53T mice and 6-OHDA-lesioned rats significantly ameliorated motor deficits, increased nigral tyrosine hydroxylase (TH)-positive neuron counts, reduced alpha-syn aggregation, and suppressed gliosis. In human midbrain organoids, TH and FOXA2 double-positive cell-derived EVs preserved DA neuronal morphology and reduced GFAP and alpha-syn expression. Notably, when EVs were administered simultaneously with 6-OHDA modeling rather than after modeling was completed, the protective effect was stronger. Multi-omics analysis revealed that TH and FOXA2 double-positive cell-derived EVs reversed pathological signatures of interferon-responsive microglia and oxidative stress-adapted astrocytes, while restoring WNT- and non-canonical WNT-mediated intercellular communications. Safety evaluations showed no discernible organ toxicity. Conclusions: TH and FOXA2 double-positive cell-derived EVs exert neuroprotective effects across multiple PD models by modulating specific glial subpopulations and reinstating development-associated signaling pathways. Together with a fully suspension-based, matrix-free, and N2/B27-free differentiation process that enables low-cost, scalable, and highly reproducible manufacturing, TH and FOXA2 double-positive cell-derived EVs represent a promising therapeutic candidate for Parkinson's disease.

In our past study on human cilia from primary ciliary dyskinesia (PCD) patients using cryo-electron tomography and mass spectrometry, defects in the DNAH5 gene were proven to cause the loss of the entire outer dynein arm along with other proteins. This phenomenon was not observed in the unicellular green alga Chlamydomonas. In this study, we examined the loss of ciliary components caused by defects in various genes, based on proteomic and structural analyses of motile cilia from multiple PCD patients. The absence of single outer arm dynein genes causes a loss or significant decrease in other inner and outer arm dyneins. Interestingly, some patients with outer dynein defects show decreased intraflagellar transport (IFT) proteins, suggesting an influence of cargo components or assembly on transport. These phenomena were not observed in Chlamydomonas, suggesting a more complex mechanism of ciliogenesis in humans despite the high similarity in the final 3D architecture of the 9+2 axoneme. Defects in certain central pair proteins unexpectedly reduced components on the doublets as well. Our results demonstrate protein-protein interactions during stages of human motile ciliogenesis that are more complex than those in unicellular organisms.