Neurochemistry International

BackgroundNeurulation is a fundamental process in the formation of the central nervous system (CNS). The process begins with the folding and fusion of the neural plate to form the neural tube which subsequently gives rise to the development of the brain and spinal cord. Environmental and genetic factors that disrupt neurulation can induce neural tube defects (NTDs) and consequently cause additional developmental complications, including motor impairments. Purinergic signaling is a conserved form of extracellular communication (i.e. paracrine, synaptic signaling) that plays a role in early development. This signaling is mediated by purine nucleotides and nucleosides, which activate metabotropic P2Y and ionotropic P2X purinoceptors, respectively. Distinct patterns of intracellular calcium dynamics are observed throughout vertebrate development, from fertilization through organogenesis, including neurulation. Among P2X receptors, P2X4 is an ATP-modulated, Ca2+-permeable, ligand-gated ion channel characterized by having the highest Ca2+ permeability and is known to be modulated by ivermectin (IVM). ObjectiveOur investigation focuses on assessing the effects of IVM treatment during neurulation and evaluating the impact of this drug on phenotype, motor behavior and neuromuscular junction (NMJ) structure at tadpole stage. These results were compared with those obtained following separate treatments with compounds that specifically block glycine, GABA(A) and nACh receptors, all which have been described as IVM targets. ResultsIn this study we demonstrate the transcriptional expression for both P2X and P2Y purinergic receptors during neurulation, as well as the expression of P2X4. Following IVM neurula-treatments, we observed neural tube defects (NTDs), pigmentation changes, motor paralysis and alterations in neuromuscular junction (NMJ) structure, particularly affecting axonal branching. In contrast, treatment with the blockers strychnine, bicuculline and -bungarotoxin, used to assess the involvement of GlyR, GABA(A)R and 7nAChR, respectively, failed to show similar outcomes. ConclusionsIn summary, our results highlight the critical role of purinergic signaling during early development, particularly P2X4 receptor mediated signaling during neurulation which may account for the pharmacological effects induced by the positive allosteric modulator ivermectin.

While cognitive and behavioral manifestations of obsessive-compulsive disorder (OCD) are well known, the neuronal dynamics underlying these symptoms remain poorly understood. Theoretical work suggests that changes in the attractor dynamics of neuronal networks towards increased stability and decreased flexibility might cause behavioral and cognitive symptoms of OCD. We used chronic treatment with the D2 and D3 dopamine receptor agonist quinpirole as a rat model of the disease. In this model, we examined changes in behavioral dynamics and, in a parallel experiment, changes in organization of neuronal activity in the hippocampus and anterior cingulate cortex (ACC). At the behavioral level, we observed increased locomotion and repetitive stereotypical trajectories in quinpirole-treated rats, with frequency of repetitions increasing over the course of the session. At the level of neuronal activity, a gradual increase in the firing rate of ACC neurons within a session paralleled the dynamics of behavioral stereotypy after quinpirole treatment. In quinpirole-treated rats, we observed increased stability in the temporal organization of hippocampal neuronal firing, but no increase in the stability of the spatial organization of discharge. The increased stability of hippocampal firing was observed at both the level of single neurons and coordinated activity of neuronal pairs, and was connected to modulation of activity by theta rhythm. Studying neuronal activity changes underlying behavioral and cognitive manifestations of brain disorders is crucial for understanding and treating brain pathologies. HIGHLIGHTSO_LIDynamics of behavior and neuronal activity was characterized in rats after chronic quinpirole treatment, which is considered a model of obsessive-compulsive disorder. C_LIO_LIThe quinpirole treatment led to repetitive stereotypical trajectories, with increasing frequency of repetitions over the course of a session. C_LIO_LIThe quinpirole treatment was associated with more stable theta modulation of single-cell hippocampal firing within experimental sessions. C_LIO_LIQuinpirole increased stability in cell-pair correlations of hippocampal units within and between sessions. C_LIO_LIQuinpirole led to more stable coordination of local field potential activity between the hippocampus and anterior cingulate cortex at theta frequencies. C_LI

Temozolomide is the gold standard chemotherapeutic agent used in the treatment of glioblastoma multiforme. Yet its pharmacological use has been linked to the emergence of depressive- and/or anxiety-like behaviors, probably through the inhibition of hippocampal neurogenesis. Since prior studies reporting these negative effects were based on prolonged treatment paradigms (i.e., from 2 weeks to up to 6 months), and given the few reports that have included female rodents in their studies, our approach aimed at further characterizing the behavioral effects induced by temozolomide (25 mg/kg, 1 or 2 cycles, 5 days/cycle) in a mixed-sex cohort of adult rats. To do so, rats were scored across time through specific behavioral tests that capture diverse manifestations of affective-like responses (forced-swim, open field, novelty-suppressed feeding and sucrose preference) or cognitive performance (Barnes maze). At the neurochemical level, we ascertained the effects of 2 cycles of temozolomide on hippocampal neurogenesis (neural progenitors with NeuroD) and other potential neuroplasticity targets (i.e., FADD, BDNF). The main results showed that temozolomide induced unexpected antidepressant-like responses in a treatment-duration manner while decreased hippocampal FADD, a neuroplastic marker previously associated with the acute and repeated actions of most antidepressants. These results break the prior dogma linking increased hippocampal neurogenesis with antidepressant-like efficacy, and suggest that other mechanisms of action, such as the one described through the neuroplastic molecule FADD, might be responsible for the antidepressant-like actions of temozolomide, even in the presence of impaired neurogenesis. Our results, in conjunction with the prior data, suggested cycle- and/or length-dependent treatment effects in terms of temozolomides antidepressant- vs. depressant-like profile, while proposing a novel biomarker of its treatment response.

Lack of social interaction results in various behavioral abnormalities in rodents, including increased anxiety levels, altered sociability, and impaired cognitive ability. Epigenetic factors regulate gene expression, however, how they contribute to juvenile social isolation (jSI)-induced behavioral alterations remains largely unknown. Here, we focused on the nucleus accumbens (NAc), a critical brain region of the reward system that regulates motivation-related behaviors. We first performed RNA-seq on neuronal nuclei and found alterations in genes related to neuronal function, as well as in transcriptional and epigenetic regulation. Protein-protein interaction (PPI) analysis of differentially expressed genes (DEGs) showed that top key nodes among down-regulated genes include membrane receptors (Ntrk2, Grin3a, and Grik1) and an apoptosis regulator (Bcl2). To further investigate whether jSI-induced gene expression alterations are mediated by histone modifications, we next performed CUT&Tag for four histone modifications (H3K4me1, H3K4me3, H3K27ac, and H3K27me3), and the results implied that epigenetic alterations may also play a role in neuronal function as well as transcriptional regulation. Reanalysis of previously published RNA-seq data on the manipulation of histone modification-associated factors (including Kdm6b, Brd4, and Setd1a) suggested that these enzymes were probably involved in jSI-induced gene expression alterations. Taken together, our comprehensive analysis implies the involvement of histone modification regulation in jSI-related alterations of gene expression in NAc.

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.

Abstract/SummaryRetroelements, including retrotransposons, endogenous retroviruses, and their fragments, as well as rare co-opted or domesticated retroelements, can contribute to neurodegenerative disorders and aging through modulation of gene expression and induction of neuroinflammation. Paternally Expressed Gene 10 (PEG10) is a retroelement-derived human gene that has recently been identified as a putative driver of Amyotrophic Lateral Sclerosis (ALS) and Angelmans Syndrome. PEG10 has been reported to bind nucleic acid and undergoes a complex self-processing pathway that results in gene expression changes when the protein accumulates in cells. Here, we report that PEG10 has selectivity for binding U/G-rich RNAs and influences widespread gene expression changes. PEG10 overexpression mimics the loss of TDP-43 in broad changes to gene expression, including dysregulation of mRNA splicing pathways. Specific changes to mRNA splicing were largely unique between TDP-43 knockdown and PEG10 overexpression, as classic TDP-43 targets including STMN2 were not altered by PEG10. Instead, we identified a unique role for PEG10 in regulating splicing of neuregulin 3 (NRG3), a ligand for the neuronal receptor ERBB4. In SH-SY5Y cells and in human neurons overexpressing PEG10, NRG3 protein levels were decreased along cellular processes, suggesting that these cells are less competent at signaling through the NRG3/ERBB4 axis. Using human patient data, we observed similar changes to NRG3 splicing in UBQLN2-mediated ALS, where PEG10 is accumulated, as well as in some cases of sporadic ALS. In conclusion, the retroelement-derived gene PEG10 plays an unexpected role in regulating splicing of neuronal transcripts, which mimics some of the transcript changes observed in human ALS patient samples. Ultimately, this work has implications for the study of PEG10, and mRNA splicing in neurological diseases associated with elevated PEG10 abundance. HighlightsO_LIPEG10 NC expression influences abundance of transcripts implicated in ALS C_LIO_LIPEG10 NC expression leads to an exon skipping event in neuregulin 3 (NRG3) C_LIO_LINRG3 expression is decreased along dendrites of PEG10 NC expressing human neurons C_LIO_LIExpression of PEG10 NC mimics changes observed in human ALS C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/727000v1_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@1a957d2org.highwire.dtl.DTLVardef@c4b15corg.highwire.dtl.DTLVardef@15825faorg.highwire.dtl.DTLVardef@25533d_HPS_FORMAT_FIGEXP M_FIG C_FIG

Social behavior is a fundamental phenotype across vertebrates. Zebrafish (Danio rerio) have emerged as a valuable translational model for investigating the neurobiological mechanisms underlying sociability, particularly due to their robust shoaling behavior and experimental tractability. However, the literature presents issues of reproducibility and inconsistent findings regarding the modulation of social preference and shoal cohesion in adult zebrafish. We conducted a systematic review and meta-analysis to synthesize studies evaluating the effects of pharmacological interventions that modulate the central nervous system and stress-related interventions on social behavior in adult zebrafish and, when available, anxiety-like behavior. The literature search was performed in three databases (Embase, PubMed, and Web of Science), followed by a two-step screening process based on inclusion/exclusion criteria. The included studies underwent extraction of qualitative and quantitative data, as well as risk of bias assessment. Interventions from the included studies (n = 108) were categorized according to their nature, mechanism of action, and/or therapeutic purpose, resulting in seven, four, and five meta-analyses for social preference, shoal cohesion, and anxiety-related tests, respectively. Ethanol, NMDA antagonists, pro-dopaminergic agents, and stress-related interventions decreased social preference, while stress-related interventions increased shoal cohesion. The fact that stress produced opposite effects suggests that these paradigms measure distinct sociability constructs, or perhaps are differentially modulated by confounding factors, like anxiety for example. The studies presented high heterogeneity, with prediction intervals compatible with effects in both directions, as well as methodological limitations and deficiencies in data reporting, as evidenced by the risk of bias assessment. These findings emphasize the need for well-designed new studies to validate the findings and expand the evidence on interventions that currently lack sufficient studies for quantitative synthesis.

BackgroundHuntington disease (HD) is a neurological disorder caused by an aberrant CAG expansion in the HTT gene, producing a mutant protein (mHTT). Although HD is classically characterized by adult-onset cortical and striatal degeneration, accumulating evidence suggests that altered cortical development may also contribute to disease pathogenesis. ObjectiveWe sought to investigate the impact of mHTT on neocortical patterning, which is a largely unexplored aspect of HD. MethodsUsing the HdhQ140 HD knock-in mouse model, we performed immunofluorescence and in situ hybridization to analyze the patterning of the cortex from embryonic day 10 to postnatal day 7. ResultsDuring embryogenesis, HTT expression exhibited a high medial-to-low lateral gradient in the neocortex, like that observed for key transcription factors involved in cortical patterning. Notably, HTT expression was absent from the cortical hem, a critical patterning center. In HD, the protein gradient remained unchanged whereas the expression in medial pallium seemed increased. During the early development of the cerebral hemispheres, the expression of morphogens and signaling pathways, including Shh, Fgf8, and Wnt/BMP genes, were disrupted in organizing centers, leading to altered expression of major neocortical transcription factors. At postnatal stages, the motor and somatosensory cortical areas were misplaced. These developmental alterations were associated with postnatal sensorimotor deficits relevant to HD. ConclusionsOur findings demonstrate that HD-related neurodevelopmental alterations arise as early as embryonic day 10 in mice. This supports previous work suggesting that defects in brain development contribute to HD pathogenesis prior to clinical onset.

Vitamin D3 (VD) deficiency is a global health concern, and its supplementation has been shown to alleviate inflammation and oxidative stress across numerous neurological disorders. However, the beneficial effect of this common nutraceutical in schizophrenia (SCZ) remains inadequately explored. The present study investigated the presupplementation effects of VD on positive and cognitive symptoms in a MK-801induced mouse model of SCZ. MK-801, a non-competitive NMDA receptor antagonist, is a widely used drug that mimics some of the psychotic symptoms associated with SCZ. The repeated administration of a single dose of MK-801 (0.5mg/kg; intraperitoneally) for two weeks produced hyperlocomotion, anxiety- like behavior, and working memory deficits in MK-801-induced SCZ-like mice. These behavioral abnormalities were significantly attenuated in VS5 mice (SCZ mice presupplemented with 500 IU/kg/day of VD). At the molecular level, VD rescued gene expression of major NMDA receptor subunits (NR1, NR2A, NR2B), 7 nicotinic acetylcholine receptors (7nAChRs), and neurotrophin factors (NGF and BDNF). A restoration of PSD-95 protein expression, accompanied by downregulation of calcineurin, was also observed in the prefrontal cortex (PFC) of VS5 mice, suggesting protective effects of VD on synaptic communication and function in SCZ. In vitro studies showed that calcitriol (1 M) treatment of HEK-293 T cells transfected with 7nAChRs potentiated the single-channel current amplitude and demonstrated a direct modulatory effect of this nutraceutical on 7nAChRs expression and function. In silico JASPAR analysis further identified putative Vitamin D response elements (VDREs) within the promoter regions of various target genes, supporting the genomic action of VD. Additionally, VD deficiency was observed in Indian SCZ patients, highlighting its potential clinical relevance. Together with our previous findings (Manjari et al., 2022, 2023), the present study also demonstrates anti-inflammatory, anti-cholinesterase, neurotrophic, and synaptic-enhancing effects of VD, deepening our understanding of the multifaceted neuroprotective effects of the "D3" neurosteroid in neuropsychiatric disorders such as SCZ. HighlightsO_LIVD presupplementation improves the behavioral deficits in MK-801 induced SCZ mice. C_LIO_LINutraceutical intervention normalizes the gene expression of major NMDARs subunits namely, NR1, NR2A, NR2B, in the PFC of SCZ mice. C_LIO_LIVD mediates a restoration in the expression and function of 7nAChRs in SCZ mice. C_LIO_LIVD exhibits neuroprotective, neurotrophic, synaptoprotective, anti-inflammatory and anti-acetylcholinesterase effects, highlighting its therapeutic potential in SCZ. C_LI

RationaleAdolescence is a period of heightened vulnerability to nicotine reinforcement. While zebrafish are a valuable model for investigating drug reward, standard conditioned place preference (CPP) assays typically test subjects in isolation. In this highly social species, solitary testing may act as an environmental stressor that confounds behavioral readouts. ObjectivesThis study examined how social context during testing (isolated vs. grouped) affects experimental attrition, behavioral stability, and nicotine CPP expression in late juvenile zebrafish. MethodsZebrafish housed in groups of four were tested either individually (isolated) or in their housing groups (grouped) during daily 20-minute sessions. Following baseline preference assessments, subjects underwent six days of conditioning pairing their initially non-preferred compartment with fish water or nicotine (0.5, 1.6, or 5.0 {micro}mol/L). Place preference, locomotion, and thigmotaxis were assessed on a drug-free test day. ResultsIsolated testing reduced distance traveled, decreased swimming speed, and increased time spent near tank walls, indicating heightened anxiety-like behavior. Experimental attrition was significantly higher in isolated (38.9%) than grouped (2.5%) subjects. Grouped subjects developed significant place preference at 1.6 and 5.0 {micro} mol/L nicotine, whereas preference was not detectable in isolated subjects. ConclusionsSolitary testing acts as a stressor that increases experimental attrition and masks place preference. Conversely, testing in the presence of conspecifics stabilizes behavior and facilitates the detection of nicotine reward in late juvenile zebrafish.

Abstract Objectives: Amyotrophic lateral sclerosis (ALS) is a clinically heterogeneous neurodegenerative disease requiring reliable biomarkers to improve patient stratification and trial design. While serum neurofilament light chain (sNfL) reflects neuroaxonal stress and disease aggressiveness, troponin T (TnT) may capture complementary aspects of neuromuscular involvement. We assessed the associations of TnT and sNfL with D50-derived measures of disease aggressiveness (D50) and disease accumulation (rD50) in ALS. Material and Methods: In this retrospective observation, TnT and sNfL levels from ALS patients in two independent German cohorts were analyzed using the D50 disease progression model; discovery cohort (Essen, n =433) and validation cohort (Bonn, n =185). Results: In both cohorts TnT demonstrated a robust correlation with rD50-defined phases across all aggressiveness subgroups (p<0.001). There was no consistent pattern regarding sNfL and the rD50 phases. sNfL concentrations demonstrated a significant and inverse correlation with D50 applied for all disease aggressiveness subgroups (p<0.001). Correlations of TnT levels with D50 disease aggressiveness groups were generally less strong and inconsistent between the two cohorts. In the discovery cohort only low aggressiveness subgroups correlated significantly (p<0.001), intermediate aggressiveness subgroups showed only a weak correlation (p<0.05) with TnT levels. High disease aggressiveness subgroups showed no significant correlation with TnT. Conclusion: In application of the D50 disease progression model, TnT was strongly associated with disease accumulation (rD50) across all disease phases, independent of disease aggressiveness (D50), whereas sNfL robustly reflected disease aggressiveness but not overall disease burden. These complementary biomarker profiles highlight the value of an integrated approach for refined disease stratification in ALS. Combining TnT and sNfL may enhance clinical decision-making, improve monitoring of disease progression and treatment response, and support optimized clinical trial design.

BackgroundAmyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by progressive motor neuron (MN) loss, muscle atrophy and paralysis. Although traditionally considered a MN-specific disease, accumulating evidence supports a crucial contribution of skeletal muscle pathology to disease onset and progression. Except for specific mutations, to date there is no effective treatment for ALS. FOXO transcription factors regulate programs of atrophy, metabolism and stress response in skeletal muscle, and their inhibition has shown beneficial effects in cellular and Drosophila models of ALS. MethodsIn this study, we investigated whether pharmacological FOXO inhibition (iFOXO) could modify disease progression and muscle pathology in female hSOD1G93A mice. Mice received daily oral administration of iFOXO starting at presymptomatic (P50; n=5 per group) or symptomatic (P90; n=9 mice per group) stages until end-stage. Body weight was monitored longitudinally, and motor performance was evaluated using grip strength and hanging-wire tests. Tibialis anterior and soleus muscles, representing fast- and slow-twitch muscles respectively, were analyzed by histology and immunofluorescence to assess fiber atrophy, fibrosis, lipid accumulation, satellite cell pool and fiber type composition. Quadriceps muscles (n=3 per group) were used for RNA-seq analysis. ResultsWhile histological analyses revealed severe fiber atrophy and increased fibrosis in hSOD1G93A mice, satellite cell numbers were preserved or mildly increased in a muscle and treatment onset dependent manner. iFOXO treatment did not improve motor performance, survival or attenuate muscle atrophy. Transcriptomic profiling indicated that genotype was the predominant driver of gene expression changes, while iFOXO produced only subtle, treatment onset dependent effects on pathways related to oxidative stress responses, mitochondrial function and adaptive metabolism. ConclusionOverall, FOXO inhibition alone showed limited therapeutic benefit in the hSOD1G93A ALS mouse model. These findings highlight the dominant influence of ALS driven molecular alterations over pharmacological modulation and emphasize the need for combinatorial therapeutic strategies targeting multiple disease mechanisms, including those preserving nerve health.

Background and PurposeEpilepsy is a neurological condition characterized by recurring seizures and neuronal hyperexcitability. Cell-based high-throughput screening applications have been essential for drug development and discovering novel biological processes. However, cell-based screens do not provide information on how drug-targeted pathways are integrated into a whole animal. Our objective was to develop and evaluate a screening application using zebrafish larvae to identify signalling mechanisms that modulate neural activity. Experimental ApproachWe developed an in vivo automated high-content screening assay using zebrafish larvae expressing the calcium sensor CaMPARI (calcium-modulated photoactivatable ratiometric integrator) in neurons. This assay can quantify neural activity of multiple individual larvae per well in a 96-well format. We quantified neural activity in 8725 individual larvae, in response to 1292 different drugs to identify molecules that protect against convulsant-induced neuronal hyperexcitability. Key ResultsThe assay was effective at identifying drugs that target diverse neurotransmitter signalling systems. While some commonly used anti-convulsants (e.g. phenytoin, carbamazepine, valproic acid) had poor activity in the assay, Kv7 potassium channel activators were consistently effective (ICA-069673, ICA-27243, ICA-110381, retigabine, and ML213). Many compounds approved for treatment of other conditions, including amitriptyline (depression), cyclobenzaprine (muscle spasm), clomipramine (obsessive-compulsive disorder) and ganaxolone (seizures), also strongly suppressed excitability in the assay. Conclusion and ImplicationsNeuronal CaMPARI expression in zebrafish larvae is a powerful tool for plate-based compound library screening to identify drugs that suppress hyperexcitability in vivo. Bullet Point SummaryO_ST_ABSWhat is already knownC_ST_ABSO_LICaMPARI is an integrative Ca2+ sensor that can be used to identify active neurons. C_LIO_LIKv7 activators (retigabine, ML213, and ICA-069673) are effective at reducing convulsant-induced (4-AP) neuronal hyperexcitability. C_LI What this study addsO_LIAn automated in vivo high-content drug screening assay to quantify neural activity. C_LIO_LIA series of drug targets that influence convulsant-induced hyperexcitability. C_LI Clinical significanceO_LIOur new tool will help identify novel compounds and signalling mechanisms that could be pursued as therapeutic targets for diseases involving electrical hyperexcitability. C_LI

Bisphenol S (BPS), a widely used substitute for bisphenol A, is increasingly detected in aquatic environments; however, its neurodevelopmental effects remain insufficiently understood. This study investigated whether developmental exposure to an environmentally relevant concentration of BPS disrupts social behaviour and underlying neurobiological pathways in zebrafish (Danio rerio). At 21 days post-fertilization, BPS-exposed larvae exhibited a significant reduction in social preference, indicating impaired conspecific interactions. Neurochemical analysis revealed a marked increase in serotonin (5-HT) levels, whereas lipid peroxidation (MDA) remained unchanged, suggesting the absence of overt oxidative damage. Gene expression profiling demonstrated a dysregulated antioxidant response, suppression of apoptotic signaling, and pronounced upregulation of serotonergic receptors and transporters. To resolve system-level mechanisms, protein-protein interaction (PPI) network analysis identified BDNF and CREB1 as dominant regulatory hubs, with the serotonergic synapse pathway as the most significantly enriched term. Molecular docking further demonstrated direct binding of BPS to multiple serotonergic targets, including HTR1A and TPH2, supporting receptor-level interference. Expanded network and pathway analyses revealed coordinated enrichment of monoamine GPCR, oxidative stress, and inflammatory pathways. These findings demonstrate that BPS induces serotonergic dysregulation and network-level reprogramming rather than significant oxidative damage, leading to behavioural impairment. This study provides a multi-scale mechanistic framework linking molecular perturbations to neurobehavioural outcomes, identifying serotonergic signaling and BDNF-CREB1 pathways as central targets of BPS neurotoxicity.

Mitochondrial morphology and function are critical determinants of neuronal function and survival, with disruptions in mitochondrial dynamics often preceding the overt neuronal dysfunction seen in neurodegenerative diseases such as Alzheimers disease, Huntingtons disease and Parkinsons disease. The kynurenine pathway accounts for 95% of dietary tryptophan catabolism and many of the metabolites are neuroactive, including redox-active 3-hydroxykynurenine (3-HK). 3-HK is present under normal physiological conditions in the central nervous system (CNS) and is elevated during inflammation. While supraphysiological levels of 3-HK have been associated with neurotoxicity, the effects of physiological concentrations on neuronal cells, and specifically their mitochondria, remain poorly understood. Here we assessed viability, ATP levels and redox status to determine cellular health and function in neuronal cells exposed to physiological levels of 3-HK, alongside confocal imaging and transcriptomic profiling, finding significant alterations in mitochondrial function and morphology. Interestingly, a biphasic influence of 3-HK on mitochondrial morphology was observed, with an elongated network as well as decreased surface area and volume being observed only at the lowest concentration of 3-HK, reflecting normal physiological levels. At the highest 3-HK concentration tested, reflecting an inflammatory situation, an increased number of mitochondria were present, accompanied by increased activation of caspase-3/7 and enhanced production of mitochondrial superoxide. These results highlight a previously unknown role for 3-HK in regulating mitochondrial function and structure, possibly through altered fission and fusion events, suggesting that subtle changes in kynurenine pathway metabolism may contribute to early mitochondrial dysfunction in neurological disease.

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

Statins, widely used cholesterol-lowering drugs, inhibit the mevalonate pathway and reduce coenzyme Q (CoQ) biosynthesis, potentially impairing mitochondrial function. Because astrocytes are essential for maintaining brain redox homeostasis, statin-induced mitochondrial dysfunction in these cells may contribute to CNS pathology. We examined the effects of a six-day statin exposure on mitochondrial bioenergetics in rat astrocytes, focusing on mitochondrial CoQ (mtCoQ) deficiency. Treatment with 200 nM atorvastatin or simvastatin decreased the total mtCoQ pool (mtCoQ9 + mtCoQ10) by 30-35% and decreased the antioxidant pool mtCoQH2 by 40%, whereas the levels of mitochondrial antioxidant proteins, including superoxide dismutase 2 and uncoupling proteins, remained unchanged. Mitochondria of statin-treated astrocytes showed decreased respiratory activity, membrane potential, and ATP synthesis, and increased mtCoQ reduction leading to increased H2O2 production during the oxidation of complex I (CI) and CII substrates. Statin treatment also altered the organization of the respiratory chain, leading to a downregulation of the CI+CIII2+CIV and CIII2+CIV supercomplexes and decreased protein levels and activity of all respiratory chain complexes. Furthermore, a decrease in cytochrome a + a3 content was accompanied by a reduction in the maximum activity of CIV. CoQ10 supplementation elevated mtCoQ levels, restored respiratory function, and decreased H2O2 production in the mitochondria of statin-treated astrocytes. Prolonged statin exposure alters mtCoQ redox homeostasis and impairs mitochondrial bioenergetic function in astrocytes. CoQ10 supplementation attenuates these changes, supporting its potential role in protecting astrocyte mitochondria from statin-induced dysfunction.

BackgroundAlcohol use disorder (AUD) is linked to increased neuroinflammation. Alcohol (ethanol) may activate toll-like receptors, which leads to the release of inflammatory molecules that could influence AUD-related behaviors, such as increased alcohol intake. Activation of toll-like receptor 3 (TLR3) by Polyinosinic:polycytidylic acid (Poly(I:C) or PIC) is associated with escalation of alcohol consumption in male, but not female F1 hybrid mice from reciprocal crosses between FVB/NJ (FVB) and C57BL/6J (B6) strains. Little is known about the underlying mechanisms of these sex-specific behavioral effects. In this study, we investigated the effects of TLR3 activation by PIC on temporal profiles of several pro- and anti-inflammatory molecules in the blood and brain of FVB/B6 F1 hybrid male and female mice at multiple time points. We hypothesized that TLR3 - dependent immune profiles would differ between males and females, which may, at least in part, explain the observed differences in drinking behavior. MethodsMale and female FVB/B6 F1 hybrid alcohol-naive mice were injected intraperitoneally with PIC (10 mg/kg) or saline. Blood and perfused brain tissues from the prefrontal cortex (PFC) and striatum were collected at 6-, 24-, and 48-hours post-injection. The expressions of Ccl2, Ccl5, Tnf, Il-6, Il-1{beta}, Ifng, Ifnb1, and Mmp9 genes were analyzed using qPCR. Protein levels of a subset of these molecules and IL-17r/a, IL-4, and IL-10 were measured in striatal samples from the same animals using ELISA. ResultsActivation of TLR3 by PIC triggered time-dependent, sex- and tissue-specific responses in immune genes and their proteins. PIC induced a time-dependent increase in expression of majority of the genes peaking at the 6 hr time point. Temporal immune profiles for pro-inflammatory chemokines, Ccl2 and Ccl5 differed between males and females in the PFC and striatum, suggesting possible sex-specific effects of these molecules on behavior. Protein levels of CCL2, CCL5, and IL-6 increased in the striatum of both sexes and correlated strongly with gene expression, with females showing somewhat higher protein fold changes. MMP-9, a key regulator of blood-brain barrier (BBB) permeability and synaptic plasticity, showed an increase in protein levels, but not mRNA levels in striatum. This pattern suggests altered blood-brain barrier (BBB) permeability, although this would require further investigation. ConclusionOur results revealed distinct TLR3-dependent immune gene and protein expression profiles in blood and brain between males and females and suggested different roles for these molecules in regulating alcohol consumption. We identified CCL2, CCL5 and MMP-9 as target molecules for investigating sex-specific behavior in the immune modulation of alcohol consumption.

It is becoming increasingly clear that chronic exposure to lower levels of ethanol impact learning and behavior. To determine the impact of chronic low-dose ethanol exposure on sensitivity to changes in stimulus value, a conditioned taste aversion procedure was used. Adult male and female mice underwent a sucrose two bottle-choice drinking paradigm. Each day, mice received an injection of either low-dose ethanol (0.5g/kg) or saline two hours after sucrose access for 20 days. This was followed by a lithium chloride (LiCl)-induced conditioned taste aversion (CTA) paradigm in which 0.15M LiCl or vehicle injection was administered immediately after sucrose consumption for three days. On the fourth day, changes in sucrose consumption were analyzed. Chronic exposure to low-dose ethanol did not affect sucrose consumption in either female of male mice during two-bottle choice. In female mice, a history of chronic low-dose ethanol exposure blocked the development of LiCl-induced CTA. A history of chronic low-dose ethanol did not impact LiCl-induced CTA in male mice as both ethanol-naive and -exposed male mice who underwent LiCl pairing reduced sucrose consumption. This suggests that low-dose ethanol alters aversion-related learning in female mice which may have implication for development of aberrant behavior and risk for alcohol use disorder (AUD).

ImportanceWhile prior work in other neurodegenerative disorders link white matter hyperintensities (WMHs) to disease severity and progression, they remain unexplored in ALS. ObjectiveTo investigate the relationship between presence and progression of WMHs, disease severity, survival, and medication efficacy in ALS. DesignThis retrospective study uses data from the Canadian ALS Neuroimaging Consortium (CALSNIC), containing prospectively acquired multicentre longitudinal (three time points over one year) MRI and clinical assessments between 2014 and 2022. SettingMulticentre study across 9 North American sites. ParticipantsParticipants with a diagnosis of possible, probable, laboratory-supported probable or definite ALS and healthy controls were included. Participants with prior brain trauma were excluded; controls with cognitive impairment or stroke were also excluded. Main Outcome(s) and Measure(s)The main outcomes were differences in baseline and progression of WMHs in ALS patients compared to controls. Secondary outcomes included associations between WMH progression and ALS progression, and subgroup differences (short versus long survival, treatment vs non-treatment groups). ResultsFollowing exclusion criteria, 204 ALS (mean [SD] age, 59.7 [10.4] years; 71 females [34.8%]) and 165 control (mean [SD] age, 55.8 [9.64] years; 70 females [42.8%]) participants were included. ALS patients showed 35.7% greater WMH burden at baseline (p<0.005) and experienced 0.9 cubic centimeters (CCs) more WMH progression over one year (p<0.0001) compared to age and sex matched controls. ALS patients experienced 2 and 4 point drops in ALSFRS-R (p<0.0005) and ECAS-ALS (p<0.005) scores respectively for every 1 CC of WMH progression they experienced. The short survival group (N = 51) experienced faster WMH progression (0.690 CC per year, p<0.05) than the long survival group (N = 75). Patients taking edaravone (N = 181) and riluzole (N = 112) experienced slower WMH progression (0.764 and 0.924 CC per year, respectively, p<0.0005) than those who did not take these medications (N = 23 and N = 90, respectively). Conclusions and RelevanceWMH burden and progression were associated with ALS disease severity, progression, and survival. Edaravone and riluzole treatments were associated with slower WMH progression. Key PointsO_ST_ABSQuestionC_ST_ABSIs the burden of white matter hyperintensities (WMH), and their progression, linked to ALS diagnosis, clinical progression, survival, and medication treatment? FindingThis retrospective study revealed significantly greater WMH burden and progression in ALS compared to healthy controls, as well as links between WMH progression and clinical progression and differences across survival and treatment groups. MeaningWMHs may be utilized as a biomarker for ALS, and should be integrated into prognostic modeling and clinical trial design.