Molecular and Cellular Neuroscience

Traumatic brain injury (TBI) impacts millions of individuals annually causing death, disability, and a heightened risk for long-term neurological and neuropsychiatric disorders. In recent years the fruit fly, Drosophila melanogaster has become a valuable model organism to study the cellular and molecular responses following TBI. AP-1 mediated transcriptional responses to TBI have previously been identified in Drosophila using pan-glial approaches. Fruit flies possess multiple glial subtypes which vary greatly in both cellular morphology and function, including glia of the blood hemolymph barrier, cortex, astrocyte-like, and ensheathing glia. By generating and utilizing a nuclear localized AP-1 transcriptional reporter, we identified glial subtype-specific differences in the extent of AP-1 activation following injury. Our findings identify a strong AP-1 response in the blood hemolymph barrier and ensheathing glia, a moderate response in cortex glia and little to no AP-1 activation in astrocyte-like glia. In addition, we inhibited AP-1 signaling in each glial subtype and tested the effect on acute survival. We found that inhibition of the AP-1 response in neuropil ensheathing glia leads to increased mortality following mild and moderate TBI. These results show that AP-1 activation levels vary across glial subtypes after TBI, with activation in neuropil ensheathing glia having a particularly important role in promoting post-injury survival. ARTICLE SUMMARYUsing Drosophila as a model organism, we investigated the early molecular and cellular response to traumatic brain injury. Our findings substantiate the requirement of a functional glial associated AP-1 transcriptional activation response for survival. Using colocalization studies, we characterized the AP-1 glial response in six morphologically and functionally distinct glia subtypes. After TBI, we find high levels of AP-1 activation in glia of the hemolymph brain barrier, cortex glia, and ensheathing glia. We further show the importance of AP-1 transcription within the neuropil ensheathing glia subtype for optimal survival following TBI.

Exposure to chemical cues released by predator or pathogen can evoke anxiety or fear responses in prey/host animals such as fight, flight or freeze both at behavioral and molecular levels. Freezing is a fundamental anxiety response when fighting or fleeing arent feasible. Despite the potential relevance of freezing as a stress-coping mechanism, its behavioral and molecular underpinnings are not understood yet. At molecular level danger cues are perceived by chemosensory receptors expressed in sensory neurons which may further regulate the animals behavioral responses(Ye et al., 2024){Citation}. 2-nonanone (2-NA) is one of the principal volatile organic compounds secreted by many pathogenic bacteria infecting Caenorhabditis elegans as well as humans and may signal danger to worms. Here, we show that olfactory exposure to threat-associated cue 2-NA induces a reversible fear-like freezing response characterized by immobility and halted feeding in C. elegans. With the application of in silico and behavioral approaches we showed that 2-NA is one of the ligands for an olfactory G-protein Coupled Receptor (GPCR) STR-211 and RNAi knockdown of the receptor leads to a defect in 2-NA induced avoidance behavior in worms. We next discovered that STR-211 is required for immediate behavioral changes in C. elegans during freezing response against 2-NA. The study proposes an environment relevant animal model to mimic human anxiety and fear-like behavior, along with the identification of one of the olfactory GPCRs mediating this behavior. The model may help in understanding the neuromolecular basis of freezing response in human anxiety, contributing towards treatment of mental health disorders.

Epilepsy affects approximately 30% of individuals with autism spectrum disorder (ASD). Consistent with these observations, while PRICKLE mutations are primarily linked with epilepsy, there is an enrichment of pathogenic DNA sequence variants in PRICKLE genes carried by individuals with ASD. Nonetheless, a connection between PRICKLE function and ASD warrants further investigation. Here, we show that a seizure-prone Drosophila prickle mutant (prickle-spiny-legs, or pksple) exhibits learning and memory deficits, increased pain sensitivity, both communication and social interaction difficulties, and restrictive repetitive grooming behaviors, all of which are strongly correlated with ASD, while a non-seizure prone prickle mutant (prickle-prickle, or pkpk) does not, thereby providing a direct genetic connection between epilepsy and ASD through prickle. Comparing headed versus headless pksple mutants, we also show that the excessive grooming requires higher level cognitive processing from the brain. Finally, both pksple and pkpk mutants exhibit circadian rhythm defects, another feature correlated with ASD, as well as distinct yet overlapping neurological anomalies in processes that include innate immune response, oxidative stress response, neuronal cell death, neurodegeneration, motor dysfunction and reduced lifespan, likely reflecting the unique isoform expression patterns observed in the developing CNS. Collectively, this study highlights the broadscale effects of PRICKLE mutations that extend beyond the primary clinical features of epilepsy to include several of the core features of ASD.

Major depressive disorder (MDD) is a leading cause of global morbidity and mortality. Unfortunately, a substantial proportion of patients do not respond adequately to currently available therapies, highlighting the need for new therapeutic targets. Here, we investigated the role of volume-regulated anion channel (VRAC) in depression-related phenotypes using zebrafish. Disruption of VRAC function, either by pharmacological inhibition or morpholino-mediated knockdown of lrrc8aa, the zebrafish ortholog of mammalian LRRC8A, the obligatory subunit required for VRAC function, induced anxiety- and depression-like behaviours in zebrafish larvae and altered the expression of genes associated with affective disorders. Transcriptomic analysis of lrrc8aa-deficient larvae revealed dysregulation of pathways involved in neuronal signalling and cellular stress responses. Conversely, pharmacological activation of VRAC with zinc pyrithione (ZPT) improved behavioural abnormalities and partially restored altered gene expression. In adult zebrafish subjected to chronic unpredictable stress, ZPT produced antidepressant- and anxiolytic-like effects comparable to those of imipramine and normalized elevated monoamine oxidase (mao) expression. Together, these findings indicate that reduced VRAC function contributes to depression- and anxiety-related behavioural and molecular phenotypes and identify VRAC as a potential target for the development of novel antidepressant therapies. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=149 SRC="FIGDIR/small/740018v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@d81884org.highwire.dtl.DTLVardef@131b8e9org.highwire.dtl.DTLVardef@162d1d0org.highwire.dtl.DTLVardef@bcdb82_HPS_FORMAT_FIGEXP M_FIG C_FIG

Defects in Drosophila Cu2+/Zn2+ superoxide dismutase (encoded by the gene Sod1) lead to elevated oxidative stress and a drastically shortened lifespan. To contrast the effects of aging and oxidative stress on nerve conduction, synaptic transmission, and muscle excitability, we developed an easily accessible adult abdominal neuromuscular preparation, utilizing the male-specific Muscle of Lawrence (MOL) in Drosophila. The large size of MOL facilitated analyses of presynaptic nerve signals and postsynaptic responses that could result in sizable excitatory junctional potentials (EJPs) evoking full-blown muscle action potentials (APs) which were terminated rapidly by a characteristic afterhyperpolarization (AHP). Aged wild-type (WT) individuals (80 days or older) exhibited diminished neuromuscular transmission, mainly reflecting declines in motor axon conduction, with synaptic transmission remaining largely intact (since robust EJPs could still be evoked when nerve terminals were directly stimulated electrotonically). Additionally, muscle APs showed enhanced depolarizing peaks and weakened AHPs during current injection, suggesting weakening in repolarizing K+ currents. Chronologically younger Sod1 mutants (up to 30 days) displayed similar trends of neuromuscular changes, confirming a major role of oxidative stress in aging. However, certain distinctions exist in muscle membrane properties and transmitter release machinery. A clear increase in muscle membrane resistance was seen in Sod1 but not in aged WT. Additionally, unlike normal spontaneous release of synaptic vesicles leading to miniature EJPs (mEJPs), extremely enlarged spontaneous transmitter discharges occurred in aged WT but was never seen in Sod1, indicating a distinct, aging-specific alteration in transmitter release regulation. Notably, our work revealed considerable variation among individuals, ranging from transmission failure to largely intact neuromuscular functions, demonstrating the stochastic nature of functional declines due to aging and oxidative stress. Moreover, this study uncovered a well-defined common vulnerability, i.e. weakening of the Ca2+-activated BK current that caused drastic reduction in AHP in both aged WT and Sod1 mutants, as confirmed by their diminishing sensitivity to the BK channel blocker paxilline, which caused striking alterations in the AHP in WT control.

Metabolic alterations commonly accompany neurodevelopmental disorders and may contribute to their pathophysiology. Neurofibromatosis type 1 (OMIM 162200) is a genetic disorder that results from mutations in the NF1 gene and its encoded neurofibromin protein (Nf1). The disorder is multisystemic, affecting multiple aspects of development, physiology, and brain function. In addition, recent evidence suggests that Nf1 deficiency alters metabolic function in both humans and animal models. Whether the metabolic alterations result from changes in neurodevelopment is not known. Here we approach this question in Drosophila melanogaster, which expresses a conserved NF1 gene, exhibits phenotypes reminiscent of the human disease, and shares key developmental mechanisms with humans. Flies with nf1 mutations or RNAi-mediated knockdown exhibit altered metabolism in adulthood. Conditional Nf1 inactivation revealed that the adult metabolic phenotype resulted from loss of Nf1 in neurons during a developmental critical period (third instar larva/pupa), which corresponded to the period of nervous system maturation. Prior to the developmental critical period, nf1 mutants did not exhibit metabolic difference - rather, the metabolic alterations appeared only after the critical period. This suggests that the adult phenotype results from the onset of the developmental alteration during the critical period. High-resolution respirometry on adult mitochondria revealed no differences in complex I/II function or fatty acid oxidation between nf1 mutants and controls, suggesting that the metabolic alterations localize upstream of the electron transport chain at the cellular level. Overall, these data suggest that loss of Nf1 alters adult metabolism via effects during a critical period of nervous system development.

While a growing number of studies have linked environmental exposures and Parkinsons disease (PD)1-3, the impact of many pesticides remains understudied4,5; for example, neonicotinoids are the most used insecticides in the world, but research into their contribution to PD is limited to a handful of studies6-8. Newer pesticides, such as the butenolide flupyradifurone (FPF), specifically developed to overcome increased pest resistance9 and spurred on by tighter restrictions on neonicotinoids such as imidacloprid (IMI)10, are even less studied. New approach methodologies (NAMs) that allow for rapid evaluation of pesticide exposures are needed to evaluate potential links between the growing number of pesticides and PD11. To this end, we exposed the model nematode Caenorhabditis elegans12 to IMI and FPF. Due to its high degree of tractability, and conservation of many genetic, neuronal, and toxic mode of action processes, C. elegans has been invaluable in both elucidating mechanisms and novel therapeutic targets for PD that can be validated in other models13, and as a complementary tool for early toxicity screening14. Along this line, we found that exposure to IMI, and to a greater extent FPF, in young adult animals causes significant dendritic blebbing, an early sign of neurodegeneration, exclusively in dopaminergic neurons. Blebbing was accompanied by impairment of dopamine-mediated behaviors, changes in neuronal mitochondrial morphology, and elevation of pathways related to reactive oxygen species (ROS). We were able to reduce the blebbing caused by IMI and FPF two ways: 1) pharmacologically via administration of the antioxidant N-acetyl cysteine (NAC); 2) genetically via knockout of a MAP kinase (MAPK) stress response pathway. This suggests that oxidative stress is a key mediator of this insecticide-induced dopaminergic neurodegeneration.

Parkinsons disease is a progressive neurodegenerative disorder associated with mitochondrial dysfunction, oxidative stress, impaired autophagy, and dysregulated cellular signaling pathways. Although Glycyrrhiza glabra has been reported to exhibit neuroprotective properties, the early phosphorylation-mediated signaling mechanisms underlying its protective effects remain poorly understood. In this study, we employed a Tandem Mass Tag (TMT)-based temporal quantitative phosphoproteomic approach to investigate early signaling events associated with Glycyrrhiza glabra-mediated neuroprotection in a rotenone-induced in vitro PD model. Differentiated IMR-32 neuronal cells were treated with rotenone alone or in combination with Glycyrrhiza glabra extract, and phosphoproteomic alterations were analyzed at 2, 5, 15, and 30 minutes using liquid chromatography coupled with tandem mass spectrometer. Temporal phosphoproteomic analysis identified 6,424 phosphopeptides corresponding to 2,368 phosphoproteins and 5,468 phosphorylation sites. Comparative analysis revealed extensive phosphorylation rewiring induced by rotenone and restoration of several dysregulated phosphorylation events following Glycyrrhiza glabra co-treatment. More than 130 phosphoproteins and multiple kinase-associated signaling pathways were dynamically regulated across the temporal conditions. Kinase enrichment analysis identified restoration of several critical kinases, including AKT1, MTOR, MAPK1/3, PRKACA, PRKCD, and GSK3A/B, which are associated with neuronal survival, stress adaptation, and autophagy. Integrated pathway and kinase-substrate interaction analyses further revealed enrichment of AMPK signaling, FOXO signaling, receptor tyrosine kinase signaling, RNA processing, and cell-cycle regulatory pathways. Notably, several spliceosome-associated phosphoproteins demonstrated dynamic phosphorylation changes during the early neuroprotective response. Collectively, this study provides a detailed temporal phosphoproteomic landscape of early signaling events associated with Glycyrrhiza glabra-mediated neuroprotection and highlights kinase-driven signaling pathways that may represent potential therapeutic targets in Parkinsons disease.

Pigment epithelial-derived factor (PEDF) is a multifunctional protein produced predominantly by the retinal pigment epithelium and expressed in many tissues, including the brain, highlighting its participation in crucial processes, such as neuroprotection and angiogenesis. Some neurodevelopmental disorders, such as ASD, are characterized by neurodevelopmental abnormalities, including altered neurite formation, spine formation, and neuronal activities. Many efforts have been made to resolve NDDs, but until now, some symptoms remain untargeted. PEDF is involved in many steps of neurodevelopment. The treatment of PEDF peptide might improve the outcome of NDD symptoms by altering neuronal morphologies. We used PEDF peptides that contain different functional domains to study the effect of administering PEDF peptides on neuronal morphology in a prenatal valproic acid (VPA)-exposed mouse model. We identified that the treatment with PEDF peptides rectified the abnormalities in neurite formation and spine formation in VPA-exposed cortical neurons. In vitro calcium imaging showed abnormalities in the spontaneous activity in VPA-exposed cortical neurons. Treatment of a short PEDF peptide normalized intracellular calcium response to the control level. Accordingly, PEDF peptides have the prospect of serving as potential treatments for patients with neurodevelopmental disorders, such as ASD.

The neuropathology of Parkinson's disease is characterized by -synuclein (-syn) aggregation and dopaminergic (DAergic) neurodegeneration. While neuronal loss in C. elegans -syn-induced neurodegeneration models is temporally age-dependent, prior research indicates it is uncoupled from the organismal aging process. Here we examined transgenic C. elegans expressing human A53T -syn in DAergic neurons to determine the impact of localized DA metabolism on both neurodegeneration and organismal lifespan. Increasing endogenous DA levels through overexpression of tyrosine hydroxylase (CAT-2) exacerbated A53T-induced DAergic degeneration, whereas DA depletion via{Delta} cat-2 mutation rescued neuronal survival. By mutating a DA interaction motif within -syn, neurodegeneration was rendered insensitive to DA manipulation, thus confirming a structural basis for in vivo toxicity. We identified a DA--syn interaction that acts as a common upstream bridge whereby localized stress induces physiological responses in C. elegans. Genetically, this biochemical interaction acts as a pleiotropic trigger driving two compartmentalized responses: localized DAergic neurodegeneration via oxidative stress, and organism-wide, TFEB/hlh-30-dependent proteostatic remodeling that extends lifespan. Modulating autophagy, without exacerbating DA-mediated oxidative stress, represents a promising strategy to preserve adaptive systemic remodeling while limiting targeted neuronal damage.

Noise-induced hearing loss (NIHL) is a leading cause of permanent hearing impairment worldwide, yet no pharmacological therapies are currently available to prevent or treat this disorder. Although inflammation is increasingly recognized as a key contributor to cochlear degeneration, the therapeutic potential of targeting early inflammatory signaling remains poorly understood. Here, we combined phenotypic screening in zebrafish with mechanistic and functional validation in complementary mouse models to identify quinoxaline derivatives with otoprotective activity following acoustic trauma. Lead compounds preserved cochlear synapses and auditory function after moderate noise exposure, while one derivative also protected sensory hair cells in a model of permanent hearing loss. Mechanistic analyses demonstrated that this protection was associated with attenuation of early NF-{kappa}B signaling and modulation of the cochlear inflammatory response toward a reparative state, consistent with suppression of pathogenic innate immune activation before irreversible tissue damage occurred. Together, these findings identify early NF-{kappa}B-dependent inflammatory signaling as a therapeutically actionable mechanism in NIHL and establish quinoxaline derivatives as promising candidates for pharmacological intervention. More broadly, this work demonstrates the utility of a cross-species discovery platform for identifying therapies that preserve sensory function by targeting early inflammatory pathways.

Parkinsons disease is predominantly characterized by dopaminergic neurodegeneration linked to toxic aggregation of -synuclein. Genipin, a bioactive iridoid, was previously shown to improve the motility and survival deficits caused by pan-neuronal expression of native -synuclein in a transgenic Drosophila melanogaster model system. We show that expression of -synuclein causes sleep deficits and that genipin treatment rescued these sleep deficits, increasing total sleep and consolidating nighttime sleep relative to untreated -synuclein-expressing fruit flies. Our findings extend genipins protective profile in Drosophila melanogaster and highlight sleep regulation as an additional phenotype responsive to -synuclein-targeted interventions.

Although prenatal alcohol exposure (PAE) has been proposed as an early-life risk factor for Alzheimers disease and related dementias (AD/ADRD), the mechanistic underpinnings are underexplored. Mutations in the Presenilin genes contribute to AD/ADRD, with Presenilin 1 acting as the catalytic subunit of the {gamma}-secretase complex responsible for cleaving Notch and amyloid precursor protein (APP). We hypothesized that PAE disrupts {gamma}-secretase activity during brain development, which persists and is associated with behavioral deficits later in life. Pregnant wild-type B6129 and 3xTg-AD mice were fed an ethanol-containing liquid diet during gestational days 13-15. From birth to adulthood, PAE increased APP C-terminal fragments and Notch intracellular domain (NICD) levels in cortical lysates. These changes were associated with impaired hippocampal-dependent learning and memory in wild-type mice at 3 and 6 months of age and exacerbated behavioral deficits in 4-month-old 3xTg-AD mice. Our findings provide the first mechanistic insight linking PAE to AD/ADRD vulnerability.

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

Transactive response DNA-binding protein 43 (TDP-43) aggregation and loss of function are hallmark features of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) among other neurodegenerative diseases. Despite epidemiological evidence linking environmental exposures to neurodegeneration, few toxicants have been directly associated with neurodegeneration. Here, we performed a high-content imaging screen, using a library of over a thousand chemical compounds that are considered high risk for human exposure and identified 21 toxicants that drive TDP-43 aggregation. Among the top chemical hits, five belonged to the dithiocarbamate (DTC) class of thiol-reactive compounds including the agricultural pesticides thiram and ziram. Thiram directly promoted TDP-43 cysteine oxidation and intermolecular crosslinking, whereas ziram induced TDP-43 aggregation via zinc imbalance and enhanced oxidative stress, suggesting DTCs disrupt redox homeostasis. In primary neurons and human iPSC-derived neurons, DTCs led to TDP-43 aggregation and prominent splicing defects consistent with loss of TDP-43 function. In exposed zebrafish, DTCs impaired TDP-43 function and triggered widespread transcriptional changes reflected by perturbed stress response and metabolic signatures. By combining TDP-43 loss of function mutations with chemical exposures, we observed accelerated TDP-43 loss of function and chemical-induced aggregation, supporting a multiple hit mechanism driving TDP-43 dysfunction. Together, these findings identify DTCs, particularly those used as agricultural pesticides, as dominant modifiers of TDP-43 proteostasis and identify redox imbalance and zinc homeostasis as a central molecular mechanism linking toxicant exposure to TDP-43 proteinopathy.

Traumatic brain injury (TBI) induces rapid neuroinflammatory responses not only in the brain but also in anatomically and immunologically connected central nervous system (CNS) compartments, including the retina. In our study, we investigated retinal microglial activation, retinal ganglion cell (RGC) calcium dynamics, and caspase-3 activation in adult mice subjected to severe traumatic brain injury using the Marmarou impact-acceleration model at 24 and 48 h post-injury. Carrying out Ca{superscript 2}-imaging, immunohistochemistry, and ex vivo time-lapse microscopy, we found robust microglial activation in both the superficial and deep retinal layers following TBI, accompanied by increased microglial motility. RGCs exhibited a transient surge in degeneration-induced spontaneous activity at 24 h, followed by a marked reduction below control levels at 48 h, consistent with early degenerative changes. Activated caspase-3 levels were significantly elevated in both microglia and other retinal cell types at both time points, indicating ongoing apoptotic effects. Together, these findings demonstrate that TBI rapidly triggers inflammatory and apoptotic mechanisms in the retina, which are detectable within the first 48 hours. Our results highlight the retina as a sensitive indicator of early CNS pathology after traumatic injury and underscore the potential of retinal analysis for monitoring TBI-induced neurodegeneration for future clinical implementation. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/734783v1_ufig1.gif" ALT="Figure 1"> View larger version (14K): org.highwire.dtl.DTLVardef@5bc694org.highwire.dtl.DTLVardef@14a4ce4org.highwire.dtl.DTLVardef@fe2d32org.highwire.dtl.DTLVardef@149419d_HPS_FORMAT_FIGEXP M_FIG C_FIG

Loss of estrogens at menopause is linked to impaired brain metabolism and increased risk of Alzheimers disease (AD). However, estrogen replacement therapies are limited due to the deleterious effects of estrogen on peripheral organs and increased risk of vascular dementia. We have developed a non-steroidal estrogenic compound, STX, which does not bind to the classical estrogen receptors and {beta}, but mimics estrogenic signaling in the central nervous system (CNS) without the peripheral reproductive actions. STX is protective against neurodegeneration in stroke and AD models, but its molecular targets are unknown. Here, we identified and validated STX neural targets using chemoproteomic, molecular biological, electrophysiological and metabolic assays of hypothalamic proopiomelanocortin (POMC) neurons. Chemoproteomic profiling identified voltage dependent anion channels (VDAC1-3) as major intracellular binding partners in mHypo43 (POMC) cells. Based on quantitative single-cell PCR, Vdac2 was identified as the dominant isoform in female hypothalamic POMC neurons. Seahorse metabolic flux analyses showed that STX potently increased glycolysis, oxidative respiration and mitochondrial ATP production in mHypo43 cells. Nanomolar concentrations of STX enhanced VDAC2 voltage-dependent gating in reconstituted lipid membranes and shifted the low-conductance states toward anion selectivity, consistent with increased ATP flux. Together, these findings reveal a mechanism for the neuroprotective effects of STX through enhancing mitochondrial bioenergetics and modulating VDAC channel properties, potentially increasing cellular energy stores. Therefore, this work identifies previously unrecognized estrogenic mitochondrial targets and provides a mechanistic basis for the neuroprotective actions of STX relevant to menopause-associated brain vulnerability.

Tubulin polymerization promoting proteins (TPPPs) are known for their cytoskeletal regulation across species; however, emerging evidence suggests broader cellular functions, including potential roles in mitochondrial biology. Here, we identify the Drosophila homolog of human TPPP, Ringer, as a previously unrecognized regulator of mitochondrial bioenergetics and electron transport chain complex I (CI) function. Ringer is enriched in the mitochondrial matrix, and its loss results in reduced levels of multiple CI subunits and assembly factors and a significant decrease in CI enzymatic activity. Notably, similar deficits are observed in postmortem human Parkinsons disease (PD) brain tissues, underscoring the translational relevance of our Drosophila model and highlighting conserved, disease-associated mechanisms. Pharmacological administration of the CI-specific reactive oxygen species (ROS) scavenger, resveratrol, ameliorates superoxide levels and improves CI enzymatic activity and ATP production in ringer mutants, demonstrating that targeted antioxidant therapeutics can improve bioenergetic function with Ringer loss. Together, these findings establish Ringer as a key regulator of mitochondrial bioenergetics and reveal CI instability as a potential mechanism underlying PD-associated mitochondrial dysfunction, providing a robust and translationally meaningful framework for future therapeutic exploration.

Tau pathology is central to Alzheimers disease and related tauopathies, yet mechanisms driving neuronal dysfunction and degeneration downstream of pathological changes in tau remain poorly understood. Drosophila melanogaster models provide a genetically tractable system with an intact nervous system and short lifespan that allows investigation of mechanisms of many diseases. However, in Drosophila, developmental expression of human tau frequently causes lethality and developmental phenotypes, limiting the study of neurodegenerative disease processes. Further, sex is rarely considered in Drosophila studies of tau pathology despite clear sex differences being observed in many aspects of human tauopathies. Here, we used an inducible, pan-neuronal GeneSwitch system to express human tau isoforms exclusively in adulthood, enabling the dissection of tau toxicity independent of development. We combined longitudinal behavioural monitoring with lifespan and neurodegeneration analyses, and performed a targeted genetic screen to identify modifiers of tau-induced dysfunction. Adult-onset tau expression produced striking, sexually dimorphic effects on survival and behaviour. Neuronal expression of the human tau isoform with 4 microtubule binding repeats and neither alternatively spliced N-terminal exon (0N4R tau) caused pronounced neurodegeneration and reduced lifespan, which was exacerbated in flies expressing the phospho-mimetic 0N4R-TauE14 variant. Tau expression produced sexually dimorphic effects on survival and behaviour, with females exhibiting a greater reduction in lifespan, while the induction-dependent increase in vacuolar neurodegeneration was broadly comparable between sexes. Behaviourally, tau expression induced elevated daytime inactivity in females, whereas males exhibited hyperactivity, revealing opposing functional outcomes between sexes. A targeted genetic screen further identified modifiers of tau-dependent behavioural impairment. APOE2 expression in glia, syndecan overexpression in neurons, and increased global expression of the chaperone heat shock protein 90 all reduced 0N4R-TauE14-induced behavioural changes. Seventeen candidate perturbations enhanced the TauE14-induced behavioural phenotype, including manipulations of APOE3, CLU, INPP5D/INPP5K, BIN1/Amph, synaptogyrin, LRP1, NPC1, and Hsp90 pathways. Together, these findings establish an adult-onset Drosophila model of tauopathy that uncouples neurotoxicity from development, reveals sex as a major determinant of tau-induced behavioural outcomes in flies, and uncovers genetic modulators of tau-induced dysfunction. This work highlights the importance of incorporating sex as a biological variable and provides a platform for mechanistic and translational studies of tauopathy.

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