ASN Neuro

Secondary degeneration following optic nerve crush (ONC) is driven in part by mitochondrial dysfunction and microglial activation. Inspired by hibernation, where reduced succinate oxidation limits reactive oxygen species (ROS) production, we tested whether pharmacological inhibition of this pathway confers neuroprotection. Using in vivo ONC models and in vitro microglial assays, we evaluated the effects of dimethyl malonate (DMM), an inhibitor of succinate dehydrogenase, and a cell-permeable succinate analog (succinate-NV). Succinate-NV increased pro-inflammatory cytokine expression (IL-1{beta}) and reduced anti-inflammatory IL-10, whereas non-permeable succinate had no effect, indicating that intracellular succinate can drive microglial activation. In hibernating animals, succinate-NV disrupted neuroprotection and reduced retinal ganglion cell (RGC) survival following optic nerve injury. Although DMM partially reduced select inflammatory cytokines, it failed to normalize IL-1{beta} or IL-10 and suppressed microglial phagocytosis while exhibiting cytotoxic effects. In vivo, DMM-treated animals showed reduced IBA1{square} microglia but increased CD68{square} activation and accumulation of DAPI{square} cells at 7 days post-injury at the crush site. RGC somas persisted but were Caspase3+ consistent with impaired clearance. Astrocyte reactivity increased at lesion borders, while reduced and fragmented GFAP at the lesion site indicated localized astrocyte loss. Collectively, these findings demonstrate that inhibition of succinate oxidation alone is insufficient for neuroprotection and underscore the need for coordinated metabolic and immune regulation that cannot be achieved through single-pathway pharmacological intervention.

BackgroundThe optic nerve serves as a vital conduit for visual signaling, and its degeneration in optic neuropathy results in irreversible vision loss. It is also a widely used model for studying central nervous system (CNS) injury and repair. Although adeno-associated virus (AAV) and lentivirus are extensively applied in CNS research, their transduction efficiency and cell-type specificity within the optic nerve remain poorly characterized. This study aimed to identify the most effective viral vector, serotype, and promoter for direct gene delivery to the adult rat optic nerve. MethodsSprague-Dawley rats (7-10 weeks) received intra-optic nerve injections of lentiviral or AAV vectors encoding GFP under different promoters (CAG, CMV, or GFAP). Two to three weeks post-injection, optic nerves were collected for immunohistochemistry with markers of oligodendrocytes (Olig2), astrocytes (GFAP, Sox9), and microglia (IBA1). Transduction efficiency and cell-type specificity were assessed using confocal microscopy. ResultsAAV2, AAV5, and lentivirus showed minimal transduction, with only sparse GFP-positive cells observed near injection sites. In contrast, AAV-PHP.eB carrying the CAG promoter yielded robust and widespread GFP expression near the injection site. Quantitative analysis revealed that approximately 90% of transduced cells were Olig2-positive oligodendrocytes, indicating strong tropism for this glial population. ConclusionAAV-PHP.eB driven by the CAG promoter enables efficient gene delivery to the optic nerve, with a predominant tropism for oligodendrocytes. This targeted intra-optic nerve injection approach offers a reliable platform for manipulating oligodendrocytes and investigating mechanisms of CNS development, injury, and repair relevant to both optic neuropathies and other CNS diseases.

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.

BackgroundDysregulated peptidyl deiminase 2 (PAD2) and aberrant protein citrullination (PC), a posttranslational modification (PTM), are involved in various inflammatory and neurodegenerative diseases. We previously showed in transgenic mice and postmortem human tissues that PC and PAD2 are altered in amyotrophic lateral sclerosis (ALS), a neurodegenerative disease characterized by motor neurons loss, paralysis, and death. Herein, we investigated the role of PAD2 in ALS by PAD2 knockout in a SOD1-ALS mouse model. MethodsTo investigate the role of PAD2-induced citrullination in ALS pathogenesis, we generated PAD2 knockout (PAD2KO) in SOD1G93A ALS mouse model and investigated the consequent modulation on the neuropathology and clinical symptoms, using molecular biology techniques such as qPCR, Western blotting, confocal microscopy, and electron microscopy. Additionally, we identified C3 as being citrullinated in human ALS using ionFinder. ResultsOur results show that PAD2KO blocked the increased PC and reduced myelin basic protein (MBP) aggregates in the ALS model. PAD2KO also improved motor neuron survival and the integrity of myelin, axons, and neuromuscular junctions, and reduced microgliosis in the white matter and C3 protein levels in astrocytes. Clinically, data from monitoring the body weight changes suggests that PAD2KO modulates the course of the disease in the ALS mouse model, accelerating the onset while slowing the progression after the onset, and modestly extending the survival of male mice. ConclusionThese results show that PAD2 is responsible for the increased PC in ALS and PC contributes to neuroinflammation and degeneration of motor neurons and myelinated axons. The modest modulation of the disease phenotype suggests that the role of PC in ALS is complex, involving altered PC in numerous proteins and in multiple cell types. Future studies are needed to investigate how PC modulates individual protein functions in various cell types to understand the contribution of PC to ALS pathogenesis.

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

Homeostatic plasticity of intrinsic excitability (IE) in the visual system has been essentially shown at the cortical level but whether thalamic nuclei also express homeostatic plasticity of IE is unknown. We show here that 4 days of monocular deprivation (MD) at eye opening induces a homeostatic change in IE in dorsal lateral geniculate nucleus (dLGN) neurons. Neurons recorded in the dLGN region activated by the deprived eye are more excitable than neurons recorded in the dLGN region activated by the open eye. No significant changes were observed following 7 days of MD, however. Enhanced excitability in neurons from the deprived side after 4 days of MD was associated with a reduced Kv1-dependent LTP-IE, a smaller voltage ramp, and a reduced inter-spike interval, suggesting that Kv1 channels are down-regulated in deprived dLGN neurons. Furthermore, the ankyrin G signal of the axon initial segment was larger in deprived dLGN neurons compared with open ones, indicating that Nav1 channel number also undergoes homeostatic regulation, and Kv1.1 channel signals were lower in deprived neurons compared to open ones. In addition, electrical coupling was found to be strengthened in neurons displaying enhanced IE following either brief (4 days) or long (10 days) MD. These results suggest that homeostatic and Hebbian plasticity in the dLGN share common expression mechanisms involving the regulation of Kv1 channels, Nav1 channels and electrical coupling between relay neurons.

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.

1.Cholesterol in the central nervous system (CNS) is largely unesterified (>99%) and is predominantly present in the myelin sheath ([~]70% of total CNS cholesterol). Damage to the myelin sheath can result in the conversion of cholesterol to cholesterol esters, which occurs in many neurological diseases, including multiple sclerosis. In this study, we measured longitudinal CNS free cholesterol and cholesterol ester levels in a genetic mouse model during postnatal myelination, demyelination, and remyelination using gas chromatography-mass spectrometry with single ion monitoring technique (GC-MS-SIM) and liquid chromatography mass spectrometry (LC-MS). Cholesterol levels in healthy mouse brains increased up to 38 weeks. In contrast, cholesterol in the healthy spinal cord increased during postnatal timepoints, but then remained steady out to 38 weeks. Interestingly, cholesterol esters in the spinal cord were highest at P1 and drastically reduced by P42, while the brain had similar levels during all postnatal time points. During demyelination, both brain and spinal cord cholesterol levels were significantly reduced as compared to healthy mice and failed to return to normal cholesterol levels even during remyelination. Absolute quantification of cholesterol esters during peak demyelination revealed that cholesterol esters comprise 19% of the total cholesterol pool in the brain and 65% in the spinal cord. The lack of recovery in CNS cholesterol levels after demyelination suggests that healthy de novo cholesterol synthesis pathways are disrupted in this model. Absolute quantification of CNS cholesterol is critical for revealing mechanisms of cholesterol regulation during disease and identifying targets for restoring cholesterol to promote myelin repair.

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.

Mutations in SCN1A, which encodes the voltage-gated sodium channel Nav1.1 (I subunit), are the major cause of Dravet syndrome, a severe developmental and epileptic encephalopathy. Although Nav1.1 haploinsufficiency preferentially impairs inhibitory interneuron function, the region-specific contributions of distributed brain circuits to seizure susceptibility, particularly in subcortical structures that have received less attention than the neocortex and hippocampus, remain unclear. Here, we examined the effects of region-specific Nav1.1 deficiency on hyperthermia-induced seizures by selectively deleting Scn1a in the neocortex, nucleus accumbens (NAc), and dorsal striatum (caudate-putamen, CPu) of adult mice using an adeno-associated virus-mediated Cre-loxP approach. Contrary to expectations based on prior cortical studies, homozygous Scn1a deletion in the neocortex produced only modest effects on seizure generalization. In contrast, homozygous deletion in the NAc and CPu induced generalized seizures to varying degrees. Notably, heterozygous Scn1a deletion in the CPu alone was sufficient to trigger generalized seizures, whereas similar manipulations in the neocortex or NAc were not. Seizure threshold temperatures were largely comparable across regions. These findings identify the dorsal striatum as particularly vulnerable to partial Nav1.1 loss and reveal functional heterogeneity within striatal circuits. Our results underscore a previously underappreciated role of striatal inhibitory networks in hyperthermia-induced seizure susceptibility and provide new insights into the circuit mechanisms underlying Dravet syndrome.

In the zebrafish visual system, accurate retinotopic mapping occurs throughout life as new neurons are generated and integrated into existing circuitry in both the retina and optic tectum (OT). To explore how OT development changes relative to innervation from the retina, we examined cell death and proliferation in genetic and surgical models that disrupt retinal innervation of the OT. Specifically, we compared lakritz (lak) mutants, which have no optic nerves due to a lesion in the atoh7 gene, with either wild-type or one-eyed fish generated through surgical eye removal. We observed elevated cell death, fewer proliferating progenitors, and fewer sox2+ OT neuroepithelial stem cells in lak mutant and denervated OT lobes. To examine whether light-mediated vision contributes to proliferation and survival in the optic tectum, we reared fish in constant darkness and then compared survival and proliferation of OT cells in innervated and non-innervated tecta. We found that OT cells were still more likely to survive and proliferate in the presence of optic nerve innervation even when fish were reared in the dark. To identify molecular pathways that could regulate OT growth, we examined the expression of known mitogens in the zebrafish optic tectum and found evidence that Wnt/{beta}-catenin pathway activity could promote innervation-dependent proliferation in lak mutant tecta. Expression of both wnt3a and the Wnt/{beta}-catenin target gene axin2, as detected by in situ hybridization and RT-qPCR, is decreased in non-innervated tectal lobes. Further supporting an innervation-dependent role for Wnt/{beta}-catenin pathway activation in the zebrafish OT, we found that lak mutants treated with a Wnt-pathway agonist, BIO, exhibited levels of OT cell proliferation that were indistinguishable from wild-type. Together these findings suggest that progenitor cells in the optic tectum produce Wnt3a in response to innervation by the optic nerve, providing new insight into how a vertebrate visual system coordinates growth across its sensory and recipient tissues.

The effect of sortilin inhibition on acute inner retinal neurodegeneration induced by optic nerve crush was investigated. Pharmacological sortilin inhibition using intravitreal delivery of a polyclonal antibody or a small-molecule inhibitor was evaluated in C57BL/6JRj male mice subjected to unilateral crush. Inner retinal thickness was evaluated by optical coherence tomography, and retinal ganglion cell density was determined in retinal flat mounts. Furthermore, the effect of constitutive sortilin deficiency was examined using Sort1-/- mice. Changes in protein and mRNA levels of sortilin, p75NTR, and associated injury markers were analyzed. Neither pharmacological inhibition or constitutive loss of sortilin protected against inner retinal thinning or retinal ganglion cell loss following optic nerve crush. A transient 1.4-fold increase in p75NTR mRNA was observed early after injury, accompanied by a two-fold increase in protein levels. While sortilin expression remained largely unchanged, sortilin deficiency was associated with an altered baseline retinal state, including increased GFAP, p75NTR, and proBDNF levels. Following optic nerve crush, the induction of p75NTR was significantly attenuated in sortilin-deficient retinas compared with wild type, without affecting the extent of RGC degeneration. In summary, sortilin inhibition does not preserve inner retinal structure following optic nerve crush, but modulates glial activation, inflammatory signaling, and proneurotrophin dynamics. These findings indicate that sortilin-dependent pathways are not key drivers of optic nerve crush-induced neurodegeneration but may be more relevant in disease contexts characterized by chronic stress and neuroinflammation.

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.

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

Assessing reproducibility across different molecular profiling studies is a persistent methodological challenge (Zhang et al., 2009; Sweeney et al., 2017; Ioannidis, 2005). Differences in platform technology, cohort composition, analytical pipelines, and feature definitions often make it difficult to interpret cross-study comparisons based solely on gene-identity overlap. In this study, we conducted a retrospective computational analysis of seven publicly available analytical datasets (including alternative analytical pipelines applied to the same cohort) derived from five biologically independent peripheral blood transcriptomic and DNA methylation cohorts, comprising 3,487 samples (1,824 Parkinsons disease cases and 1,663 controls). Reproducibility was evaluated using gene-identity overlap, enrichment-based comparisons, and a permutation-based framework to assess directional consistency of effect estimates across datasets. We also tested the robustness of results by varying false discovery rate thresholds and applying alternative probe-to-gene collapsing strategies. All analyses were performed using reproducible workflows implemented in R and Python with fixed random seeds. Across independent cohorts, gene-identity overlap was generally limited, with enrichment ratios close to one, especially when datasets were generated using different platforms. In several datasets, limited numbers of statistically significant features further constrained overlap-based comparisons. In contrast, directional consistency showed greater stability. High levels of directional consistency were observed across independent cohort comparisons when restricted to overlapping statistically significant features and remained stable across statistical thresholds (90.0% at FDR < 0.05 and 82.8% at FDR < 0.10). When evaluated across the full shared gene universe without conditioning on statistical significance, directional consistency was substantially lower ([~]30 to 32%) but remained significantly above permutation-based null expectations. Permutation testing confirmed that the observed directional consistency exceeded what would be expected by chance. A combined analysis including methodological replicates (n [&ge;] 3 datasets) showed 98.3% directional consistency; however, this estimate includes non-independent analytical pipelines applied to the same cohort and reflects analytical stability rather than independent biological replication. Rather than introducing a new statistical method, this study examines how commonly used reproducibility metrics behave under crossstudy heterogeneity and identifies their practical limitations and appropriate use boundaries.

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

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.

BackgroundMicroglia have become a cell type of interest in the neurodegenerative field given both genetic and pathological evidence for their role in disease development and progression. There has been a rapid growth of studies using iPSC-derived microglial models to understand the molecular mechanisms driving these neurological diseases. However, it remains difficult to transduce myeloid cells effectively which is critical when aiming to study the role of disease associated genes and pathways. Current methods require exposure to multiple viruses which is not suitable for all experimental paradigms. We have therefore sought and characterised a high efficiency promoter and plasmid design to allow high transduction efficacy with a single lentivirus. ResultsUsing the spleen focus-forming virus (SFFV) promoter in combination with central polypurine tract (cPPT) and Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) plasmid elements gave significantly higher transduction efficiency and transgene expression than was achieved with commonly used promoters CMV and EF1. This could then be further improved if required to over 90% transduction efficiency with the removal of lentivirus restriction factor SAM and HD domain-containing protein 1 (SAMHD1) by adding VPX. ConclusionsOur findings allow for a simpler, more efficient and streamlined approach to transgene expression in iPSC-derived microglia and macrophages using only a single lentivirus. This minimises potential unintended side effects such as additional cellular activation and increased cell death.