ASN Neuro

Photoreceptor outer segments are sensory cilia whose maintenance depends on a balance between basal disc renewal and tip shedding, controlled by intraflagellar transport and axonemal microtubule organization. Microtubule plus-end proteins regulate microtubule dynamics and are strong candidates for roles in this process. In this study, mCherry-tagged EB1, EB3, and DCX were overexpressed in zebrafish (Danio rerio) cone photoreceptors under a cone-specific promoter. Eyes were examined at 5 and 10 dpf, and eyecup depth, diameter, and cone photoreceptor area were quantified relative to uninjected controls. At 5 dpf, all three constructs produced eyes indistinguishable from those of controls. By 10 dpf, all three constructs significantly increased eye cup depth and cone photoreceptor area. EB1 and DCX also significantly increased eye cup diameter. EB1 and, more severely, EB3 also caused retinal holes, mainly in the retinal pigment epithelium and at the outer nuclear/outer plexiform layer, along with misshapen cells near the inner plexiform layer. DXC did not cause retinal holes, but, like EB1 and EB3, produced enlarged, bulbous cone outer segments. The results show that overexpression of any of the three +TIPs results in a similar eye and photoreceptor overgrowth phenotype, while also producing construct-specific defects: EB1 and EB3 disrupt the broader retinal architecture, whereas DCX produces enlarged eyes. The shared outer segment hypertrophy suggests an imbalance between cargo delivery at the basal end and shedding of the distal tips. The organomegaly may reflect altered progenitor signaling in the ciliary marginal zone.

Neural stem cell (NSC) transplantation is a promising strategy for repairing the injured spinal cord, but transplanted cells typically require immunosuppressive therapy to prevent rejection, even for induced pluripotent stem cell (iPSC)-derived autologous grafts. However, the effects of immunosuppressive drugs on neurite outgrowth and axonal regeneration, processes critical for neural circuit reconstruction, have not been fully characterized. In this study, we tested nine clinically relevant immunosuppressants on human iPSC-derived neurons and primary human spinal cord NSCs in vitro at concentrations approximating clinical exposure levels. The drug panel included FK-506 (tacrolimus), cyclosporine A (CsA), rapamycin, belatacept (Nulojix), etanercept (Enbrel), mycophenolate mofetil (CellCept), cyclophosphamide (Cytoxan), prednisone, and azathioprine (Imuran). Neurite outgrowth was quantified via automated high-content imaging. Multiple agents, including CsA, Imuran, Nulojix, and CellCept, induced significant reductions in neurite outgrowth in a cell type- and dose-dependent manner, with CsA producing the most robust and consistent inhibition across both cell lines. In contrast, FK-506 showed no significant effect on neurite extension at clinically relevant concentrations. Consistent with the in vitro results, human neural progenitor cell grafts in a rodent spinal cord injury model exhibited significantly reduced graft-derived axon extension in the host spinal cord when hosts were treated with CsA rather than FK-506. These findings demonstrate that immunosuppressant choice can profoundly influence neural graft integration and axonal regeneration. Our study underscores the importance of preclinical evaluation of immunosuppressive regimens and suggests that selecting agents such as FK-506 over CsA may improve outcomes in future stem cell-based therapeutic trials for spinal cord injury and related disorders of the central nervous system.

Glaucoma is a group of eye diseases that affects 4 million people in the US and is one of the leading causes of vision loss due to damage to the eyes optic nerve (ON) which is composed of axons from retinal ganglion cells (RGCs) that transmit visual information to the brain. Injury to the ON often triggers RGC death and subsequent loss of visual function. Despite its increasing prevalence worldwide, effective therapies for glaucoma remain elusive. Notably, the thirteen-lined ground squirrel (TLGS) exhibits intrinsic neuroprotection during hibernation; however, reproducing this protective state pharmacologically has proven challenging. To elucidate the metabolic mechanisms underlying this resilience, we conducted untargeted metabolomic analyses on TLGS retinas at 6 hours, 3 days, and 7 days following ON crush. Retinas from awake and hibernating animals were compared to identify temporal and state-dependent metabolic signatures. Distinct metabolomic profiles were observed in hibernating animals relative to their awake counterparts. Pathway analyses revealed coordinated regulation of amino acid, lipid, and purine metabolism that likely contributes to hibernation-induced resilience. Furthermore, our findings indicate that hibernating TLGS retinas increase exosome biogenesis, prompting in vitro validation using TLGS-derived exosomes, which demonstrated robust neuroprotective and anti-inflammatory effects. Proteomic and transcriptomic characterization of exosomal cargo identified conserved miRNAs, mRNAs, and proteins implicated in redox balance, cytoskeletal stabilization, and stress-response regulation. Collectively, these data support the hypothesis that metabolic reprogramming and exosome-mediated intercellular signaling underlie hibernation-associated neuroprotection. Modulating these pathways may provide a blueprint for novel therapeutic strategies to mitigate neurodegeneration and promote recovery following optic nerve injury. Graphical AbstractIllustration depicting state-dependent metabolic responses to optic nerve crush (ONC) injury in Thirteen-lined Ground Squirrels (TLGS). In Awake animals, injury triggers enhanced ATP production through the TCA cycle, leading to excessive reactive oxygen species (ROS) generation and subsequent retinal ganglion cell (RGC) death. In contrast, Hibernating animals shift toward lipid metabolism and utilize ATP for the biosynthesis of ceramides and sphingolipids, promoting membrane integrity and exosomal signaling. Additionally, a range of metabolites associated with hibernation-linked neuroprotection are elevated, contributing to enhanced RGC survival. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/733742v1_ufig1.gif" ALT="Figure 1"> View larger version (81K): org.highwire.dtl.DTLVardef@1ef3a7eorg.highwire.dtl.DTLVardef@e9293dorg.highwire.dtl.DTLVardef@192729forg.highwire.dtl.DTLVardef@1a32cb5_HPS_FORMAT_FIGEXP M_FIG C_FIG

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.

A critical step in visual cortical maturation is refinement of receptive field (RF) size, producing higher acuity vision. This was previously studied using species with well-developed vision (e.g., carnivores, primates), in which visual experience was necessary for refinement but not maintenance of RFs in visual cortex. In contrast, in Syrian hamsters, a crepuscular species with low visual acuity, dark rearing had no effect on RF refinement in juveniles, but RFs re-enlarged in adulthood, resulting in reduced acuity. These inter-species differences raise the question of whether the need for visual experience is primarily related to the phylogenetic position of the species or to its ecological niche. Here we report that dark rearing had no effect on development or maintenance of RF properties of visual cortical neurons in nocturnal mice. Mice with lifelong visual deprivation refined and maintained their RF size over time. Furthermore, the development of stimulus direction tuning was unaffected by dark rearing. In contrast, surround suppression, orientation tuning and the sharpness of direction tuning were abnormal in dark reared mice. These and our previous results from hamsters show that species living in an ecological niche with minimal daylight exposure require little to no visual experience to develop and maintain refined RFs. This study is an important step in developing a better understanding of the role of visual experience in the development of visual processing circuitry and suggests that diurnal mammals may be a better model for human visual cortical development than mice.

Cell replacement represents a potential treatment modality for retinal disorders characterized by photoreceptor loss. However, photoreceptor replacement approaches have not been clinically established. To take this forward, the main goal of this study was to systematically compare human photoreceptors of different ages and identify those that enable functional integration into the degenerative retina. Donor cells were isolated from iPSC-derived retinal organoids generated by a GMP-compliant protocol at differentiation days 120, 150, or 200 and transplanted subretinally into cone photoreceptor function loss 1 (Cpfl1) recipients, an inherited mouse model of cone degeneration. While younger photoreceptors showed slightly improved transplantation outcomes, donor photoreceptors of all culture stages displayed long-term survival, cone identity, structural integration into the host retina, and tight interactions with host Mueller glia, including formation of a continuous outer limiting membrane. Transplanted photoreceptors showed signs of advanced maturation, including correct polarization with generation of apical inner- and outer segments, while basal synapses were formed with host bipolar cells. Electrophysiological assessment of host retinal ganglion cells revealed light-evoked responses in transplant-containing regions, providing evidence for functional incorporation of human photoreceptors into the mouse neuro-retinal circuitry. Thus, GMP-compliant human iPSC-derived photoreceptors are stable over a wide range of differentiation stages and constitute a robust cell source for retinal transplantation and functional repair. The findings provide important prerequisites for the development of standardized procedures towards clinical translation of photoreceptor replacement in the retina.

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.

Background. L-Dopa remains the primary treatment for Parkinson's disease (PD), but chronic administration frequently leads to L-Dopa-induced dyskinesia (LID). While D1 and D2 medium spiny neuron (MSN) specific structural changes on the spine level have been observed in the striatum of PD and LID, studying microglia mediated synapse loss has not been done to date. Methods. Here we generated novel reporter mice by crossing floxed PSD95c(mCherry/eGFP) mice with D1-Cre and D2-Cre lines, producing D1-PSD95-EGFP and D2-PSD95-EGFP strains for MSN-specific synapse visualization. Using the 6-OHDA mouse model of PD and LID we assessed microglia mediated MSN subtype specific synapse loss in these mice while PLX3397 was used to investigate effects of microglia depletion and repopulation on LID development and synapse loss. Results. Both D1- and D2-MSNs exhibited significant PSD95 synapse loss in PD, with D1-MSN loss further exacerbated in LID. Microglia displayed increased phagocytic activity and accumulated PSD95 material within lysosomes, particularly in LID. PLX3397-mediated microglial depletion reduced LID severity and preserved D1-MSN synapses. A depletion and repopulation paradigm attenuated LID severity, preserved D1-MSN synapses, and reduced synaptic material within microglia. Conclusions. Microglia-mediated synapse loss in MSN subtypes contributes to PD and LID pathogenesis. Pharmacological microglial depletion and repopulation mitigate synapse loss and dyskinesia, highlighting microglial turnover as a promising therapeutic strategy for LID.

STRUCTURED ABSTRACT Purpose of Research: The generation of iPSC lines expressing 21, 56 and 79 glutamine repeats within the HTT protein and homozygous KO of HTT in the KOLF2.1J background as an additional disease series within the iPSC Neurodegenerative Disease Initiative (iNDI) collection. Major Findings: All iPSCs, even those expressing long repeats of 79Q or HTT KO, were capable of differentiating to striatal and cortical neurons, astrocytes and microglia using established protocols. General quality control stains and morphological analyses are described for each differentiation. A selected set of assays were carried out on differentiated cells; expanded repeat expressing astrocytes showed altered expression of astrocyte protein markers and morphological characteristics, and striatal neurons showed altered DARPP-32/CTIP2 colocalization. mRNAseq carried out for striatal neurons showed high similarities in gene expression changes between 79Q and KO lines compared to the unexpanded repeat. Conclusions: The KOLF2.1J isogenic CAG repeat series serves as a community resource to study HD mechanisms with the potential for direct comparison across other neurodegenerative diseases through the iNDI collection.

Vision depends on the transfer of photoreceptor signals through the retina and then to many CNS visual nuclei. While the most common inherited retinal diseases (IRDs) involve defects in rod and/or cone function, another group (referred to as congenital stationary night blindness (CSNB)) results from defects in glutamate release from photoreceptors, or conversion of the glutamatergic signal in bipolar cells. One example results from mutations in the CACNA2D4 gene, which encodes a subunit of the voltage-gated calcium channel that is critical for glutamate release from both rod and cone photoreceptors. Mutations in CACNA2D4 result in a range of phenotypes in human patients, from incomplete CSNB to rod-cone dystrophy. In the CACNA2D4 knockout mouse (2{delta}4-/-), there is slow photoreceptor degeneration, the photoreceptor-to-bipolar cell synapse is disorganized, and the retina lacks scotopic and photopic full-field electroretinogram b-waves; this also results in low visual acuity. Using adult 2{delta}4-/- mice, we show that recombinant adeno-associated virus (rAAV)-mediated gene therapy directed to rod photoreceptors prevents rod degeneration, restores synaptic organization, retinal function, and improves visual acuity under both light- and dark-adapted conditions. This rescue was maintained for up to 14 months post-treatment. Together, our results demonstrate that synaptic structure and function can be restored in the mature mouse retina in a model of complete synaptic disorganization. The results highlight the neuroprotective potential of targeting synaptic organizing proteins in retinal gene therapy.

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.

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.

Parkinsons disease (PD) is characterised by progressive dopaminergic neurodegeneration in the substantia nigra, leading to debilitating motor dysfunction. Current treatments remain largely symptomatic, highlighting the need for disease-modifying therapies. {beta}1 integrin, implicated in neuroinflammation and trophic signalling, represents a candidate therapeutic target. We investigated whether allosteric {beta}1 integrin modulation could attenuate motor asymmetry in the unilateral 6-hydroxydopamine (6-OHDA) mouse model of PD. Adult male C57BL/6 mice received intracerebral 6-OHDA into the substantia nigra. The anti-{beta}1 integrin antibody JB1a (50 {micro}g) was administered prophylactically (3 days pre-lesion) or therapeutically (3 or 7 days post-lesion). Motor asymmetry was assessed through spontaneous circling (5 min) and apomorphine-induced (0.5 mg/kg s.c.) circling (30 min). 6-OHDA induced dose-dependent contralateral circling, confirming nigrostriatal lesion. Pre-treatment with JB1a (3 days before 6-OHDA) reduced apomorphine-induced circling, although this did not reach statistical significance (28.5 {+/-} 12.8, n = 4 versus 38.6 {+/-} 7.5, n = 8; p>0.05). Post-treatment at 3 days post-lesion produced no statistically significant change in either spontaneous or apomorphine-induced circling (p>0.05). Post-treatment at 7 days post-lesion reduced apomorphine-induced circling by approximately 50%, with values returning to those of sham-operated controls (n =8-9; p<0.01). These findings, obtained in a murine 6-OHDA model, indicate that allosteric {beta}1 integrin modulation attenuates lesion-induced motor asymmetry with apparent temporal specificity. As apomorphine-induced rotation reflects post-synaptic dopamine receptor supersensitivity rather than direct neuronal preservation, and as histological confirmation of dopaminergic integrity was not obtainable in this study, the present data should be interpreted as proof-of-concept behavioural evidence requiring further mechanistic and translational validation in models incorporating -synuclein pathology. The findings are not directly generalizable to human Parkinsons disease. The histological confirmation of lesion extent was not available and as such the behavioural findings are correspondingly interpreted as a proof-of-concept observation requiring histological replication.

Aggregation of -synuclein protein is a characteristic of Parkinsons disease pathology that relates to the degeneration of vulnerable dopaminergic neurons and motor symptoms of the disease. However, -synuclein pathology can contribute to neuronal dysfunction by disrupting several processes within the cell, including intracellular calcium balance, mitochondrial function, and synaptic function. Here, we use a preformed fibril (PFF) model of synucleinopathy to examine effects of striatal -synuclein seeding on dopamine neurons of the substantia nigra pars compacta (SNc). The SNc is heterogeneous and contains dopaminergic neurons with differential vulnerability to Parkinsons disease pathology. We found that intrastriatal injections of PFFs differentially affect these SNc neuron subtypes by increasing the excitability of resilient SNc neurons, while altering tonic firing patterns and T-type calcium currents in vulnerable SNc neurons. In addition, we performed comprehensive electrophysiological analyses and neural morphology reconstructions on SNc neurons from PFF and monomer injected mice. These findings provide insights to the selective vulnerability of SNc neuron subtypes and further our understanding of the role of -synuclein in Parkinsons disease progression and circuit dysfunction.

Hereditary spastic paraplegia type 7 (SPG7) is a neurodegenerative disorder characterized by progressive motor impairment and cerebellar dysfunction. Mutations in the SPG7 gene, encoding the mitochondrial metalloprotease paraplegin, disrupt mitochondrial homeostasis and lead to neuronal vulnerability and deficits in motor coordination. Recent studies have identified defective flickering of the mitochondrial permeability transition pore (mPTP) in SPG7 models, suggesting that altered pore dynamics may represent a functional biomarker of mitochondrial dysfunction. Here, we investigated whether pharmacological modulation of mPTP activity could improve mitochondrial function and motor performance in SPG7 models. Mitochondrial flickering was assessed in vitro, while motor behavior was evaluated in vivo following chronic treatment with berberine, a natural isoquinoline alkaloid known to modulate mitochondrial bioenergetics. Spg7-/- mice and age-matched Spg7+/ littermate controls received daily oral berberine administration for several weeks, and motor coordination was assessed using the accelerating rotarod test. Untreated Spg7-/- mice exhibited reduced rotarod performance compared with controls, indicating impaired motor coordination. Berberine treatment significantly improved motor performance in pre-symptomatic mutant mice. These findings indicate that pharmacological modulation of mitochondrial permeability transition pore dynamics can ameliorate motor dysfunction associated with SPG7 deficiency and highlight mPTP flickering as a functional readout of mitochondrial health.

Neurodegeneration is a major determinant of disability progression in multiple sclerosis (MS), yet the pathophysiological mechanisms associating inflammation to neuronal insult remain poorly understood. We recently identified Dendritic Syntaphilin Intrusion (DSI), a novel excitoxicity pathway in which the axonal mitochondrial anchor syntaphilin (SNPH) aberrantly translocates into dendrites, causing neurodegeneration in a non-inflammatory model of MS. However, whether this protein intrudes abruptly into dendrites in inflammatory MS pathology is still not clear. Here, we investigated the role of synaptic and extrasynaptic NMDA receptors (NMDAR) in regulating the intrusion of Syntaphilin into dendrites. Using primary hippocampal neuronal cultures, we examined how the balance between synaptic GluN2A-containing and extrasynaptic GluN2B-containing NMDARs influences DSI under inflammatory conditions. Pharmacological and viral-mediated approaches were employed to manipulate NMDAR subtype activity and evaluate their impact on DSI. Inflammatory cytokines discernibly sensitized neurons to DSI. Our results revealed that blockade of synaptic NMDARs significantly increased DSI, whereas inhibition of extrasynaptic NMDARs reduced DSI. These findings demonstrate opposing roles of NMDAR subtypes, with GluN2A-containing synaptic receptors inhibiting DSI and fostering neuronal survival, while GluN2B-containing extrasynaptic receptors enhancing DSI and neurodegenerative signaling. Manipulation of the GluN2A/GluN2B balance showed opposite effect on DSI, suggesting a relationship between NMDAR subtype signaling and SNPH mislocalization. Overall, our findings extend the relevance of DSI from non-inflammatory MS to inflammatory MS and identify DSI as a downstream convergence point linking inflammatory cytokines and excitotoxic NMDAR signaling to neuronal insult. These results reveal DSI as a potential mechanistic link between inflammatory signaling and excitotoxic neuronal injury and indicate that modulation of GluN2B-dependent pathways warrants further investigation in inflammatory neurodegenerative disorders.

In contrast to many other developing systems, in which axon pathfinding and synaptogenesis are separated in time, the pioneering axon of the individually identifiable caudal primary motor neuron in embryonic zebrafish forms en passant synapses during its stereotypical ventral growth. How simultaneous synaptic differentiation and axon pathfinding are coordinated is not fully understood. Here we ask what the role of the tac1 gene, coding for the synaptic tachykinin neuropeptides, is in this unique axon differentiation process. The gene is expressed during axon outgrowth and its disruption results in increased branch length of CaP axons and subtle morphological defects of the pre-synapse. These abnormalities are accompanied by a robust [~]1.5-fold increase in motor neuron activity and in spontaneous early contractions in tac1-deficient embryos. Furthermore, pharmacological inhibition of the tachykinin receptor (Tacr1) leads to altered CaP axonal morphology, mimicking the axonal phenotype observed in tac1-deficient zebrafish. These findings suggest that tachykinin neuropeptides modulate formation and activity of en passant synapses and prevent aberrant axon branching during growth of zebrafish motor axons. HIGHLIGHTS- tac1 refines CaP primary motor axon development in zebrafish - Loss of tac1 disrupts presynaptic maturation at the horizontal myoseptum - tac1 mutants show elevated motor neuron activity and spontaneous contractions

BackgroundBiallelic loss-of-function mutations in PRKN gene (encoding Parkin protein) cause early-onset Parkinsons disease (EOPD). Parkin is a crucial component of PINK1-Parkin pathway, which marks damaged mitochondria for degradation via mitophagy. Without functional Parkin, damaged mitochondria accumulate, causing oxidative stress and neurodegeneration. ObjectiveInvestigate Parkin gene replacement via AAV gene therapy as a potential treatment for Parkin-dependent EOPD. MethodsWe initially validated phosphorylated ubiquitin Ser65 (pUbSer65) as an indicator of Parkin-mediated mitophagy initiation. We evaluated AAV-mediated PRKN replacement (hereafter, AAV-Parkin) in a Parkin knockout neuroblastoma cell line (SH-SY5Y cells) and feasibility of delivery in mouse and rat models. ResultsOur research showed pUbSer65 signal was reduced in Parkin-KO SH-SY5Y cells when compared to wild-type cells after mitochondrial stress, indicating deficiency in initiation of mitophagy. AAV-mediated human PRKN gene replacement successfully restored these pUbSer65 levels in knockout cells. We saw restoration in patient-derived fibroblasts following AAV-Parkin overexpression. We developed a translatable gene therapy approach using rodents. We demonstrated the feasibility of delivering AAV-Parkin directly into the substantia nigra (SN) of wild-type rats. Using an AAV1 capsid with Ef1a promoter, we achieved dose-dependent Parkin expression and identified a well-tolerated dose. We also evaluated multiple promoters in a proprietary Spark100 capsid, finding Ef1a and Synapsin1 (Syn1) were most effective for transducing dopaminergic neurons in the SN of mice without causing adverse effects. These findings established a well-tolerated vector dose and an optimal capsid-promoter combination. ConclusionsOur results support the potential of AAV-Parkin gene therapy as a disease-modifying approach for Parkin-deficient EOPD. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=132 SRC="FIGDIR/small/737487v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@16dd13corg.highwire.dtl.DTLVardef@c3dfcdorg.highwire.dtl.DTLVardef@19a310dorg.highwire.dtl.DTLVardef@a66f2_HPS_FORMAT_FIGEXP M_FIG C_FIG

Huntingtons disease (HD), caused by a CAG repeat expansion in the huntingtin (HTT) gene, is characterized by progressive neurodegeneration and accumulation of DNA damage with multiple disease-modifier genes involved in DNA repair pathways. Previous studies have implicated ataxia telangiectasia mutated (ATM) signaling in the regulation of genomic stability and DNA damage repair (DDR) pathways in HD. ATM has also been linked to the WW domain-containing oxidoreductase (WWOX), a protein involved in DNA repair and maintenance of genomic stability, through the E3 ubiquitin ligase ITCH. However, whether this signaling pathway contributes to HD pathogenesis remains unknown. Here, we investigated the role of ATM-ITCH-WWOX signaling in HD. Our results revealed no significant alterations in total ATM, phosphorylated ATM (pATM-S1981), or ITCH in HD post-mortem prefrontal cortex (PFC) compared to controls. Although treatment of human neuroblastoma SH-SY5Y cells with HD PFC lysates did not alter pATM-S1981 levels, it increased histone H2AX phosphorylation at S139 ({gamma}-H2AX), a marker of DNA double-strand breaks. This finding suggested the presence of persistent DNA damage signaling independent of canonical ATM activation. Conversely, WWOX levels were increased in both HD PFC and HD embryonic stem cell-derived cortical neurons. Additionally, treatment of SH-SY5Y cells with recombinant human WWOX protein or WWOX overexpression increased {gamma}-H2AX levels, supporting a role for WWOX in promoting DNA damage. To determine whether WWOX contributed to DNA damage in HD, SH-SY5Y cells were treated with HD PFC lysates that were depleted of WWOX. Immuno-depletion of WWOX reduced the ability of HD PFC lysates to increase {gamma}-H2AX, suggesting that WWOX contributes to DNA damage in HD. Finally, overexpression of WWOX in RPE1-AAVS1-CAG115 cells did not affect somatic CAG repeat instability, despite persistent increases in {gamma}-H2AX levels. Collectively, our findings identify WWOX as a contributor to DNA damage in HD, acting independently of the ATM pathway.