Molecular and Cellular Neuroscience

Exposure to cyanotoxins has been linked to neurodegenerative diseases, including amyotrophic lateral sclerosis, Alzheimers, and Parkinsons disease. While the cyanotoxin {beta}-methylamino-L-alanine (BMAA) has received much attention, cyanobacteria produce many cyanotoxic compounds, several of which have been detected in nature alongside BMAA including 2,4-diaminobutyric acid (2,4-DAB), and N-(2-aminoethyl)glycine (AEG). Thus, the question of whether DAB and AEG also cause neurotoxic effects in vivo is of great interest, as is the question of whether they interact to enhance toxicity. Here, we evaluate the toxic and neurotoxic effects of these cyanotoxins alone or in combination by measuring zebrafish larval viability and behavior after exposure. 2,4-DAB was the most potent cyanotoxin as it decreased larval viability by approximately 50% at 6 days post fertilization, while BMAA and AEG decreased viability by just 16% and 8%, respectively. Although we only observed minor neurotoxic effects on spontaneous locomotion, BMAA and AEG enhanced acoustic startle sensitivity, and they interacted in an additive manner to exert their effects. 2,4-DAB, however, only modulated the startle kinematics, an indication of motor dysfunction. To investigate the mechanisms of 2,4-DABs effects, we analyzed the protein profile of larval zebrafish exposed to 500M 2,4-DAB at two time points and identified molecular signatures consistent with neurodegeneration, including disruption of metabolic pathways and downregulation of the ALS-associated genes SOD1 and UBQLN4. Together, our data demonstrate that BMAA and its isomers AEG and 2,4-DAB cause neurotoxic effects in vivo, with 2,4-DAB as the most potent of the three in the zebrafish model.

Mutations in Swiss-cheese (SWS) or its vertebrate ortholog PNLPA6, also called Neuropathy Target Esterase (NTE), cause progressive neuronal degeneration in Drosophila and mice and several complex syndromes in humans. These include mental retardation, spastic paraplegia, ataxia and blindness and several other symptoms. SWS and PNPLA6 are widely expressed in neurons and in several glial cell types in Drosophila and mice and both cell types require SWS/PNPLA6 function autonomously. SWS and PNPLA6 are structurally and functionally conserved because expression of human or mouse PNPLA6 can replace SWS in flies. These orthologues share several domains, including the highly conserved phospholipase domain that mediates its function in deacetylating phosphatidylcholine (PC) to lysophosphatidylcholine and glycerophosphocholine. In addition, they share three cyclic nucleotide binding sites and although about 10% of the known disease-causing mutations occur in these sites, their function is still unknown. We therefore generated mutations in these sites in SWS to address what consequences this has for the function of the protein. Mutating only one site (SWSG558E) results in a partially functional protein that rescues the sws knockdown and that decreases PC when overexpressed. However, mutating all three sites (SWS{Delta}CNB) renders SWS non-functional and results in an increase of PC when overexpressed, suggesting that cyclic nucleotide binding can regulate the phospholipase function.

Neuroinflammation is a pathological hallmark of Alzheimers disease and related neurodegenerative diseases. However, signaling molecules that regulate glial activation status are not fully understood. Microtubule affinity-regulating kinase 2 (MARK2) has been implicated in both immune responses and AD pathology. Here, we report that MARK2 negatively regulates glial immune responses, which protects against neurodegeneration. We found that MARK2 knockdown in the BV2 murine microglial cell line enhanced IL-6 expression in response to LPS. MARK2 knockdown enhanced IL-6 expression induced by TLR7 agonist but not stimulation of RLR pathways and cGAS-STING. In the brains of PS19 tauopathy mice, MARK2 was elevated in homeostatic microglia but reduced in activated microglia. In Drosophila expressing human tau in the retina, expression of AMP downstream of the Toll pathway in the pigment glia enhances degeneration of photoreceptor neurons. Glial knockdown of Par-1, the Drosophila ortholog of MARK2, enhanced Toll-mediated AMP expression and neurodegeneration, whereas overexpression of Par-1 in the pigment glia suppressed them. These results suggest that MARK2/Par-1 in glia negatively regulates Toll pathway-driven inflammation and protects against tau-induced neurodegeneration. These findings provide insight into the molecular underpinnings of glial inflammation in neurodegenerative conditions and highlight MARK2 as a potential therapeutic target for modulating neuroinflammatory responses.

Nature-derived bioactive compounds have emerged as promising candidates for the prevention and treatment of diverse chronic illnesses, including neurodegenerative diseases. However, the exact molecular mechanisms underlying their neuroprotective effects remain unclear. Most studies focus solely on the antioxidant activities of natural products which translate to poor outcome in clinical trials. Current therapies against neurodegeneration only provide symptomatic relief thereby underscoring the need for novel strategies to combat disease onset and progression. We have employed an environmental toxin-induced Drosophila Parkinsons disease (PD) model as an inexpensive in vivo screening platform to explore neuroprotective potential of selected dietary flavonoids. We have identified a specific group of flavonoids known as flavones displaying protection against paraquat (PQ)-induced neurodegenerative phenotypes, involving reduced survival, mobility defects and enhanced oxidative stress. Interestingly, the other groups of investigated flavonoids, namely, the flavonones and flavonols failed to provide protection indicating a requirement of specific structural features that confer protection against PQ-mediated neurotoxicity in Drosophila. Based on our screen, the neuroprotective flavones lack a functional group substitution at the C3 and contain ,{beta}-unsaturated carbonyl group. Furthermore, flavones-mediated neuroprotection is not solely dependent on antioxidant properties but also involves regulation of neuroinflammatory responses. Our data identify specific structural features of selected flavonoids that provide neuroprotection against environmental toxin-induced PD pathogenesis that can be explored for novel therapeutic interventions. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=158 SRC="FIGDIR/small/494711v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@130933aorg.highwire.dtl.DTLVardef@11e959org.highwire.dtl.DTLVardef@1eef5fforg.highwire.dtl.DTLVardef@1585a2a_HPS_FORMAT_FIGEXP M_FIG C_FIG

Organisms have evolved protective strategies that are geared toward limiting cellular damage and enhancing organismal survival in the face of environmental stresses, but how these protective mechanisms are coordinated remains unclear. Here, we define a requirement for neural activity in mobilizing the antioxidant defenses of the nematode Caenorhabditis elegans both during chronic oxidative stress and prior to its onset. We show that acetylcholine-deficient mutants are particularly vulnerable to chronic oxidative stress. We find that extended oxidative stress mobilizes a broad transcriptional response which is strongly dependent on both cholinergic signaling and activation of the muscarinic G-protein acetylcholine coupled receptor (mAChR) GAR-3. Gene enrichment analysis revealed a lack of upregulation of proteasomal proteolysis machinery in both cholinergic-deficient and gar-3 mAChR mutants, suggesting that muscarinic activation is critical for stress-responsive upregulation of protein degradation pathways. Further, we find that GAR-3 overexpression in cholinergic motor neurons prolongs survival during chronic oxidative stress. Our studies demonstrate neuronal modulation of antioxidant defenses through cholinergic activation of G protein-coupled receptor signaling pathways, defining new potential links between cholinergic signaling, oxidative damage, and neurodegenerative disease.

Lipid transfer proteins mediate the transfer of lipids between organelle membranes in eukaryotes and loss of function in these has been linked to neurodegenerative disorders. However, the mechanism by which loss of lipid transfer protein function leads to neurodegeneration is not understood. In Drosophila photoreceptors, depletion of Retinal Degeneration B (RDGB), a phosphatidylinositol transfer protein localized to endoplasmic reticulum-plasma membrane contact sites leads to defective phototransduction and retinal degeneration but the mechanism by which RDGB function is regulated and the process by which loss of this activity leads to retinal degeneration is not understood. RDGB is localized to membrane contact sites (MCS) and this depends in the interaction of its FFAT motif with the ER integral protein VAP. To identify regulators of RDGB function in vivo, we depleted more than 300 VAP interacting proteins and identified a set of 52 suppressors of rdgB. The molecular identity of these suppressors indicates a role for novel lipids in regulating RDGB function and for transcriptional and ubiquitination processes in mediating retinal degeneration in rdgB. The human homologs of several of these molecules have been implicated in neurodevelopmental diseases underscoring the importance of VAP mediated processes in these disorders.

Alzheimers disease (AD) is a progressive neurodegenerative disorder, accounting for most dementia cases worldwide. Current therapies for AD have limited effectiveness in slowing disease progression or delivering a cure. As such, there is an immediate need for ongoing research and innovative strategies to tackle this multifaceted disease. Recently, several studies have implicated the renin-angiotensin system (RAS), known to regulate blood pressure, as a possible therapeutic target for AD. RAS-inhibiting drugs, including angiotensin-converting enzyme inhibitors (ACE-Is), have been shown to reduce the incidence and progression of AD. However, the literature describing their beneficial effects is inconsistent, with contradictory findings reporting no effects. How these drugs may function in AD remains poorly understood. Our previous work in Drosophila models expressing AD-related transgenes investigated the benefits of captopril, an ACE-I, and found it effectively rescued AD-related phenotypes including cognitive performance independent of A{beta}42 changes. Importantly, our study implicated Acer, a homolog of mammalian ACE, as a key player. In our current study, we demonstrate that the beneficial outcomes of Acer inhibition depend on preventing its catalytic activity and downstream target processing. We identify CG2233 as a prospective target and reveal its functional interaction with Acer. Furthermore, we show CG2233 is implicated in AD-related pathways in A{beta}42 expressing flies. Together, these findings provide a new avenue to study the role of ACE in AD. Significance StatementAD is a devastating neurodegenerative disorder with limited therapeutic success. Emerging research highlights the potential of inhibiting the renin-angiotensin system (RAS) in AD. Epidemiological findings and experimental studies have shown promising outcomes with RAS-targeting drugs including angiotensin-converting enzyme inhibitors (ACE-Is). Our previous work in Drosophila AD models revealed the efficacy of captopril, an ACE-I, in improving AD-related phenotypes. Moreover, we identified Acer as a key player in these mechanisms. Our current study further elucidates the role of Acer, identifies CG2233 as a potential target, and uncovers their functional interaction, shedding light on pathways relevant to AD phenotypes. This research underscores the significance of investigating ACE and ACE-I mechanisms in AD, offering potential innovative means for AD therapy.

Autism spectrum disorder (ASD) is a common neurodevelopmental condition for which there are no pharmacological therapies that effectively target its core symptomatology. Animal models of syndromic forms of ASD, such as neurofibromatosis type 1, may be of use in screening for such treatments. Drosophila larvae lacking Nf1 expression exhibit tactile hypersensitivity following mechanical stimulation, proposed to mirror the sensory sensitivity issues comprising part of the ASD diagnostic criteria. Such behaviour is associated with synaptic dysfunction at the neuromuscular junction (NMJ). Both phenotypes may thus provide tractable outputs with which to screen for potential ASD therapies. In this study, we demonstrate that, while loss of Nf1 expression within the embryo is sufficient to impair NMJ synaptic transmission in the larva, constitutive Nf1 knockdown is required to induce tactile hypersensitivity, suggesting that a compound must be administered throughout development to rescue this behaviour. With such a feeding regime, we identify two compounds from a targeted, low-throughput screen that significantly and consistently reduce, but do not fully rescue, tactile hypersensitivity in Nf1P1 larvae. These are the HMG-CoA reductase inhibitor simvastatin, and the BKCa channel activator BMS-204352. At the NMJ, both compounds induce a significant reduction in the enhanced spontaneous transmission frequency of Nf1P1 larvae, though again not to the level of vehicle-treated controls. However, both compounds fully rescue the increased quantal size of Nf1P1 mutants, with simvastatin also fully rescuing their reduced quantal content. Thus, the further study of both compounds as potential ASD interventions is warranted. Significance StatementNo therapies currently exist that consistently and effectively target the core symptoms of autism spectrum disorder (ASD), which include altered responses to sensory stimuli. Previously it was shown that Drosophila larvae lacking expression of ASD-associated Nf1 display a heightened response to a mechanical stimulus and increased neuronal excitability, likely due to excessive Ras activity. Here, out of a screen for compounds targeting such mechanisms, we identified simvastatin and BMS-204352 to reduce the likelihood of a response in Nf1-/- larvae following mechanical stimulation. These compounds also improved synaptic transmission defects at the neuromuscular junction. Such findings support the further study of these drugs as potential ASD therapies in the clinic.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcriptional activator of antioxidant response elements (ARE), which function to increase expression of antioxidant enzymes. Recent works suggests Nrf2 activation is a promising protective mechanism against the progressive neurodegeneration in Parkinsons disease (PD). While Nrf2 inducers show some promising results in animal models of PD, the response is limit in dopaminergic neurons with the protection mostly conferred by surrounding glia. The present study characterizes ARE transcriptional repressors Bach1 and Bach 2 (Broad complex- Tramtrack-Bric-a-brac and Cap n Collar homology, basic leucine zipper transcription factors 1 and 2) as a potential explanation for limited Nrf2 activity in these neurons. The current work identified Bach1 and Bach2 in dopaminergic neurons of the human substantia nigra by immunocytochemical analyses. We further identified Bach2 as a more robust inhibitor of Nrf2 responses. The effects of both Bach1 and Bach2 were dependent on their DNA-binding domains, but the DNA-binding domains did not entirely explain the differences in their relative repressor activities. Using IMR-32 neuroblastoma cells, we found differentiation into a dopaminergic neuronal phenotype resulted in increased Bach2 expression, decreased full-length Bach1 expression, increased truncated Bach1, and blunted Nrf2-mediated responses. Bach inhibitors cobalt protoporphyrin or cadmium chloride, which were more effective against Bach1 than Bach2, did not rescue Nrf2 responses. These results provide a novel mechanism for the lack of antioxidant responses and sensitivity of dopaminergic neurons to oxidative damage, in addition to proposing Bach2 as a novel drug target for the treatment of PD.\n\nSignificanceOxidative stress and damage are unifying contributors to explain the progressive loss of dopaminergic neurons in Parkinsons disease (PD). Although increased transcription of antioxidant enzymes by nuclear factor erythroid 2-related factor 2 (Nrf2) shows potential in combating oxidative damage, Nrf2 protection is mostly conferred by surrounding glia with a relative inability of neurons to mount a protective response. This work characterizes transcriptional repressors Bach1 and Bach2 that inhibit Nrf2 responses and prevent antioxidant enzyme production. This study supports Bach2 as a major factor in preventing antioxidant production in neurons and as a novel drug target to protect against PD.

The proper functioning of the nervous system is dependent on the establishment and maintenance of intricate networks of neurons that form functional neural circuits. Once neural circuits are assembled during development, a distinct set of molecular programs is likely required to maintain their connectivity throughout the lifetime of the organism. Here, we demonstrate that Fasciclin 3 (Fas3), an axon guidance cell adhesion protein, is necessary for the maintenance of the olfactory circuit in adult Drosophila. We utilized the TARGET system to spatiotemporally knockdown Fas3 in selected populations of adult neurons. Our findings show that Fas3 knockdown results in the death of olfactory circuit neurons and reduced survival of adults. We also demonstrated that Fas3 knockdown activates caspase-3 mediated cell death in olfactory local interneurons, which can be rescued by overexpressing p35, an anti-apoptotic protein. This work adds to the growing set of evidence indicating a critical role for axon guidance proteins in the maintenance of neuronal circuits in adults. SUMMARY STATEMENTLittle is known about the maintenance of adult neural circuits. We show that the continuous expression of Fasciclin 3, a cell adhesion protein involved in axon guidance, is required for neuronal survival in the adult olfactory circuit.

The reactive oxygen species (ROS)-scavenging enzyme Cu/Zn superoxide dismutase (SOD1) is an evolutionarily conserved mechanism for the maintenance of oxidative homeostasis, and missense mutations in the human SOD1 gene are associated with the motor neuron degenerative disease amyotrophic lateral sclerosis (ALS). Mutations in the Drosophila melanogaster SOD1 gene (Sod) shorten fly lifespan, attenuate motor function, and induce developmental mortality. We have previously found morphological and physiological defects at the neuromuscular junctions (NMJs) of hypomorphic Sodn108 and ALS model SodG85R mutant larvae. Here, we report genetic interactions causing striking modifications of Sod mutant phenotypes by mutations in the gene Prickle (Pk), which are linked to planar cell polarity, epilepsy, and axonal transport disruptions. Pk is expressed as two isoforms prickle-spiny-legs (sple) and prickle-prickle (pk), which are each suppressed by specific hypomorphic mutations (sple1 and pk1). Interestingly, Sod phenotypes are distinctly modified depending on both the Pk isoform suppressed, and whether said suppression is heterozygous or homozygous. Heterozygous sple1 and pk1 improved the developmental survival of Sod mutants, whereas homozygous sple1 and pk1 drastically increased mortality. Further, only heterozygous sple1 and pk1 clearly ameliorated morphological defects at the neuromuscular junctions of Sod mutant larvae. Pharmacological treatment of Sod mutants reveals allele-specific motor neuron terminal hyperexcitability, characterized by synaptic transmissions of extended duration and abnormal presynaptic Ca2+ transients. Heterozygous sple1 mutation suppresses this Sod mutant terminal hyperexcitability, but pk1 does not. We reversed this suppressive effect of sple1 by pharmacological blockade of Ca2+-activated K+ channel slowpoke. Altogether, this study builds on our prior knowledge of Sod mutant development and physiology to show that axonal transport-linked gene mutations strikingly modify Sod mutant phenotypes, providing strong evidence for a role of intracellular transport in alterations of neuromuscular morphology and physiology by SOD1 mutations.

Neurodegeneration arising from aging, injury or disease has devastating health consequences. Whereas neuronal survival and axon degeneration have been studied extensively, much less is known about how neurodegeneration impacts dendrites. To develop an assay for dendrite degeneration and repair in the Drosophila peripheral nervous system, we used photo-switchable caspase-3 (caspase-LOV) to induce neuronal damage with tunable severity by adjusting illumination duration, thereby revealing cell type-specific responses to caspase-3 induced dendrite degeneration in dendrite arborization (da) neurons. To ask whether mechanisms underlying axon degeneration also govern dendrite degeneration, we tested the involvement of the Wallerian degeneration pathway by examining the effects of expressing the mouse Wallerian degeneration slow (WldS) protein and knockdown of the Drosophila sterile alpha/Armadillo/Toll-Interleukin receptor homology domain protein (dSarm1) and Axundead (Axed) in class 4 da neurons. Here we report WldS expression or knockdown of dSarm1 improved dendrite repair following caspase- 3 induced dendrite degeneration. Whereas both dSarm1 and Axed were required for thermal nocifensive behavior in uninjured animals, WldS expression improved the recovery of thermal nocifensive behavior that was impaired by chronic low-level of caspase-LOV activity. By establishing ways to induce graded dendrite degeneration, we uncover a protective role of WldS in caspase-3 induced dendrite degeneration and repair.

Brain inflammation contributes to the pathogenesis of neurodegenerative diseases such as Alzheimers disease (AD). Glucose hypometabolism and glial activation are pathological features seen in AD brains; however, the connection between the two is not fully understood. Using a Drosophila model of AD, we identified that glucose metabolism in glia plays a critical role in neuroinflammation under disease conditions. Expression of human Tau in the retinal cells, including photoreceptor neurons and pigment glia, causes photoreceptor degeneration accompanied by inclusion formation and swelling of the lamina cortex. We found that inclusions are formed by glial phagocytosis, and swelling of the laminal cortex correlates with the expression of antimicrobial peptides. Co-expression of human glucose transporter 3 (GLUT3) with Tau in the retina does not affect tau levels but suppresses these inflammatory responses and photoreceptor degeneration. We also found that expression of GLUT3, specifically in the pigment glia, is sufficient to suppress inflammatory phenotypes and mitigate photoreceptor degeneration in the tau-expressing retina. Our results suggest that glial glucose metabolism contributes to inflammatory responses and neurodegeneration in tauopathy. Summary StatementGlucose uptake into pigment glia suppresses inflammatory responses and photoreceptor degeneration in the fly model of tauopathy.

Hypoxic-ischemic injury is a major cause of olfactory dysfunction, yet the cellular and morphological mechanisms underlying this sensory loss remain poorly understood. Here, we investigated the structural, cellular, and functional effects of acute hypoxic exposure on the olfactory system of adult zebrafish (Danio rerio) of both sexes, a model organism with remarkable neuroregenerative capacity. Fish were subjected to 15 minutes of acute severe hypoxia (0.8 mg/L dissolved oxygen) and assessed at 1 and 5 days post-hypoxia (dph). We evaluated olfactory function by means of cadaverine-evoked aversive behavioral assays. Structural and morphological integrity and inflammation of the olfactory epithelium (OE) and olfactory bulb (OB) were characterized using immunohistochemistry, histological stainings, and a 2,3,5-triphenyltetrazolium chloride (TTC) colorimetric assay. Acute hypoxic exposure impaired olfactory-mediated behaviors without affecting locomotion or exploratory behavior. In the peripheral OE, hypoxia caused neurodegeneration, disruption of the nasal mucus layer, and robust leukocytic infiltration. We observed reduced mitochondrial dehydrogenase activity in the olfactory bulb (OB) along with reactive astrogliosis. Olfactory function recovered by 5 days, coinciding with full restoration of OE morphology, and supported by a strong proliferative response. These findings reveal a coordinated degenerative and regenerative response to hypoxia across the olfactory axis, with implications for understanding hypoxia-induced sensory loss and neural repair. SIGNIFICANCEThis work addresses an important gap in knowledge regarding the mechanisms linking hypoxic insult and olfactory dysfunction. By using adult zebrafish, an extraordinarily regenerative vertebrate, it also provides insight into neuronal repair and regenerative processes supporting olfactory recovery. The novelty of our study resides in that, to our knowledge, there are no studies that provide a comprehensive characterization of the effects of hypoxia in the olfactory system across molecular, histological, and functional levels. These findings advance our understanding of hypoxia-induced sensory neurodegeneration and regeneration, and highlight the zebrafish olfactory system as a powerful model for investigating neural repair mechanisms relevant to hypoxic-ischemic brain injury.

Alzheimers disease (AD) is a memory related neurodegenerative disorder mainly associated with older adults. In this study, transgenic Drosophila AD model has been employed to investigate the tau associated proteome. Tau expression was specifically induced in the eye tissues and diseased fly heads were considered for proteomic studies with appropriate controls. We have identified 6 novel proteins from tau induced AD group by 2D and PD Quest analyses and further characterized them by in-vivo and in-silico approaches. The novel Tau interactors, [Obp44a Isoform A, Pglym Isoform A, IP15846p (Adh variant), RE45450p (mRpL2), Retinin, and Glob1 Isoform B], identified by MALDI-TOF/MS were validated through q-RT-PCR. The altered metabolic, behavioral and mitochondrial dynamics associated with Tau over expressing AD flies could be due to the altered expression of Odorant binding protein 44a (Obp44a) and mitochondrial ribosomal protein L2 (mRpL2), respectively. Further, we have showed for the first time the newly identified protein interaction with tau and other regulatory proteins through protein-protein docking, biocomputational classification and evolutionary relationship using in silico studies. Moreover, the study highlights the plausible role of these novel proteins in pathophysiology of phospho-tau induced AD flies and further help unraveling molecular pathways implicated in tauopathies.

Restless legs syndrome (RLS) is a common neurological disorder associated with iron deficiency and dopaminergic (DAergic) neuronal dysfunction. BTBD9 is a genetic risk factor for RLS. However, its molecular function remains largely unknown. Here, we report the interaction between BTBD9, manganese (Mn) and insulin/insulin-like growth factor (IGF) signaling in Caenorhabditis elegans, mouse Neuro2a cells and humans. We found that elevated Mn downregulated BTBD9 mRNA levels; in turn, BTBD9 expression attenuated Mn-induced cellular stress and dopaminergic neurodegeneration. As Mn is a known co-factor for insulin receptor and IGF-1 receptor, which activates IGF signaling, we posited that BTBD9 negatively regulates IGF signaling. Our results showed that the protective effects of BTBD9 against Mn toxicity were dependent on the forkhead box O (FOXO) protein. Furthermore, BTBD9 overexpression significantly elevated FOXO level and decreased PKB level, while phosphoinositide-dependent kinase-1 (PDK1) level remained unchanged. We conclude that BTBD9 acts as a key component in the IGF signaling pathway. Meanwhile, the roles of Mn in DAergic neurotoxicity and regulating BTBD9 shed new light on the etiology of RLS.

Neuronal polarization is a fundamental process in the formation of functional neural circuits, relying on the precise coordination between cytoskeletal regulatory signals and mechanisms that sustain cellular integrity. Disruption of these processes compromises neuronal differentiation and survival, and various neurotoxic compounds, including certain pesticides, have been associated with such dysfunctions. In this context, identifying molecules that counteract these detrimental effects is of significant therapeutic interest. Neuronal polarization is essential for the establishment of functional neural circuits and relies on coordinated regulation of actin cytoskeleton dynamics, RhoA/ROCK signaling, and mitochondrial function. Here, we investigated the neuroprotective and neurorestorative potential of the ganglioside GM1 and its oligosaccharide derivative, osGM1, in primary hippocampal pyramidal neurons exposed to the mitochondrial neurotoxin rotenone. Rotenone induced a marked arrest of neuronal development, impaired axonal elongation, and disrupted mitochondrial organization and membrane potential. Both GM1 and osGM1 promoted recovery of neuronal polarity and axonal growth, exerting protective and restorative effects even under continuous toxin exposure, with osGM1 showing superior efficacy. Notably, osGM1 also reversed axonal growth deficits caused by pathological actin stabilization. Mechanistically, osGM1 normalized rotenone-induced hyperactivation of the RhoA/ROCK pathway without altering basal signaling and partially restored mitochondrial network integrity and function. Collectively, these findings identify osGM1 as a multi-target modulator of cytoskeletal and mitochondrial dysfunction and support its translational potential as a therapeutic strategy to counteract neurotoxin-induced neuronal damage.

With the global rise in aging populations, the increasing incidence of neurodegenerative diseases underscores concerns about brain health, with pesticides like rotenone, emerging as key environmental hazardous. The precise mechanism by which chronic environmental concentration of rotenone exposure causes Parkinson-like phenotype is not understood. Previous studies showed that rotenone induces depletion of dopaminergic neurons by influencing mitochondrial functions. Mitochondrial dysfunction alters lipid homeostasis; therefore, brain lipids can be potential targets for the early risk assessment and prognosis of Parkinsons disease (PD). However, the specific lipidome changes and associated biomarkers of chronic rotenone in vivo exposure causing PD are largely unknown. This study investigates the lipid profile disruptions and biomarkers induced by environmentally relevant concentrations of chronic rotenone exposure using the neuro-model Drosophila melanogaster. An untargeted LC-HRAMS-based lipidomics identified that lipid classes, GP, SP, FA and GL were significantly altered. Furthermore, system-wide loss of cross talk of mitochondrial and peroxisome lipids by altering their redox homeostasis causing PD was observed. Additionally, lipid oxidative stress markers, and behavior abnormalities correlated with altered lipids linked to PD. The findings highlight the rotenone induced complex metabolic trade-offs, prioritizing brains neural integrity at the expense of peripheral lipid levels, leading to PD. Environmental implicationThis study highlights the environmental risks of chronic rotenone exposure, commonly used in agriculture. The findings show that even low concentrations of rotenone disrupt lipid metabolism, particularly in the brain, affecting mitochondrial and peroxisomal functions, contributing to depleting dopaminergic neurons, which are linked to neurodegenerative diseases like Parkinsons. The study also identify lipid markers linked to rotenone induced PD and reveals metabolic trade-offs, where the brain prioritizes neural integrity over peripheral lipid balance. These lipidome changes and redox-driven shifts threaten both individual health and ecosystem stability, emphasizing the need for policies to regulate pesticide use and minimize exposure risks. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=145 SRC="FIGDIR/small/637453v1_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@145133aorg.highwire.dtl.DTLVardef@1f1612forg.highwire.dtl.DTLVardef@2360f0org.highwire.dtl.DTLVardef@b161f9_HPS_FORMAT_FIGEXP M_FIG Graphical abstract C_FIG

Amyotrophic Lateral Sclerosis (ALS) is a debilitating and incurable neurodegenerative disease with unsolved etiology. Due to the large proportion of patients lacking direct disease inheritance, understanding the environmental factors that contribute to ALS development is of high priority. Epidemiological studies have implicated pesticides and other environmental exposures as possible contributors to ALS pathogenesis. Recently, our group determined that the organochlorine pesticide cis-chlordane is toxic to human motor neurons in a dose-dependent manner, causing an ALS-like phenotype in culture and animals with a mode of action independent of its known GABAA antagonism. Here, we aimed to characterize downstream motor neuron phenotypes associated with cis-chlordane treatment. We performed bulk RNA sequencing, live imaging, immunofluorescent labeling, and real-time metabolic assays on stem cell-derived motor neurons to assess chlordane-associated phenotypes in vitro. We demonstrate that cis-chlordane treatment causes a highly altered mitochondrial phenotype in motor neurons, including increased production of reactive oxygen species, decreased OCR and ATP production, and loss of mitochondrial membrane potential. We further implicate cis-chlordane as a possible mediator of potent motor neuron damage, with exposure to the pesticide inducing mitochondrial phenotypes akin to those seen in ALS. We suggest that future studies investigating the role of pesticides in ALS development center upon the organochlorine molecules.

MAPK8IP3-related neurodevelopmental disorders are a spectrum of rare conditions caused by de novo mutations in the MAPK8IP3 gene that encodes the JIP3 protein. These disorders are associated with a spectrum of neurodevelopmental symptoms that manifest in children and cause brain abnormalities, profound intellectual disabilities, movement disorders, and developmental delays. JIP3 is required for axonal transport of proteins and organelles between the soma and the synaptic terminal of neurons, a process critical for normal brain development and function. Homozygous loss-of-function mutations in JIP3 lead to impaired axonal transport and aggregation of cargo, which result in axonal swelling and stunted elongation. Despite these severe outcomes, disease mechanisms are poorly understood, and no current treatments are available. Here we conduct thorough morphological, behavioral, and motility phenotyping in the JIP3 knockout zebrafish and identify locomotor deficits and morphological abnormalities. To identify treatment options, we used insights from expert clinicians and the artificial intelligence tool, mediKanren, to identify drug candidates hypothesized to improve patient symptoms or compensate for the loss of JIP3 at the molecular level. We then prioritized drugs that are FDA-approved, safe for children, and readily available. These collective efforts identified amantadine and levodopa as candidate therapies and rescued motor phenotypes associated with JIP3 loss-of-function in zebrafish.