Molecular Brain

The glutamate delta 1 receptor (GluD1) represents a unique subtype of ionotropic glutamate receptors that is strongly expressed in the mammalian striatum. Disruptions of the GRID1 gene, which encodes GluD1, have been associated with neuropsychiatric disorders, including schizophrenia and autism spectrum disorder; however, the role of GluD1 in the brain remains poorly understood. Previous studies in mice have demonstrated that the knockout of striatal GluD1 led to fear-conditioning deficits and depressive-like behaviors. Furthermore, these mice exhibited reduced excitatory input to the striatum due to a loss of thalamostriatal innervation, whereas corticostriatal innervation was unaffected. In this study, we examined whether changes in synapse morphology contribute to the observed functional deficits. We found that the ablation of GluD1 does not affect synaptic targeting patterns of corticostriatal and thalamostriatal terminals, using transmission electron microscopy. We further utilized three-dimensional reconstruction to obtain quantitative data on synapse ultrastructure and found no significant changes in corticostriatal and thalamostriatal synaptic components, including the presynaptic terminal volume, postsynaptic density area and morphology, and postsynaptic dendritic spine volume. These findings support a model in which GluD1 regulates input-specific circuit organization and synaptic connectivity rather than the structural morphology of individual synapses.

Parkinsons disease (PD) is associated with motor impairment and cortical synaptic dysfunction, which involve altered glutamate receptor trafficking, yet the underlying mechanisms remain incompletely understood. VPS26B, a component of the retromer complex, regulates GluA1 recycling in the trans-entorhinal cortex region. However, its role in the primary motor cortex (M1) under Parkinsonian conditions has not been explored. Here, we show that VPS26B levels are reduced in the M1 of an MPTP-induced PD mouse model, accompanied by decreased surface GluA1 and synaptic protein levels. VPS26B overexpression partially attenuated these alterations. In the accelerating rotarod test, VPS26B-deficient mice exhibited unstable motor performance following MPTP administration, whereas VPS26B overexpression was associated with improved performance in both wild-type and knockout mice. These findings suggest that cortical VPS26B may contribute to maintaining glutamate receptor surface expression and synaptic protein levels, especially under Parkinsonian conditions, with potential implications for motor learning.

The cerebellar nuclei form the main output structures of the cerebellum and are composed of a deeply conserved set of cell types. Two excitatory cell classes, Class-A and -B, are present in each cerebellar nucleus and mediate all excitatory output of the cerebellum. To provide genetic access to these cell types, here we identified Acan as a marker gene for Class-B cells and generated a knock-in Acan-P2A-Cre mouse line. We demonstrate that this Acan-Cre line selectively labels Class-B neurons in the cerebellar nuclei and validate its use in viral projection tracing. This new mouse line provides a valuable genetic tool to study cerebellar nuclei organization and function.

Lysosomal trafficking and homeostasis are biological functions that are pivotal for DRG neurons, given their metabolic demands and extremely long axons. Previous studies indicate that lysosomal signaling is altered in a mouse model of chemotherapy-induced peripheral neuropathy (CIPN) and that blocking mitogen activated protein kinase-associated kinase (MNK1/2) signaling can alleviate pain behaviors in CIPN. Here, we investigated lysosome dynamics and lysosome-associated signaling in a mouse model of CIPN induced by paclitaxel (PTX), a chemotherapeutic agent used for various types of cancer. Using spinning disk super-resolution microscope (SPINSR), we demonstrate that PTX treatment in vivo causes reduced lysosome motility observed in vitro. PTX likewise drives the accumulation of Sequestosome 1 (SQSTM1), also known as P62, in cultured mouse DRG neurons, indicating lysosomal dysfunction in DRG neurons. The transcription factor EB (TFEB), a master regulator of lysosomal biogenesis, was also upregulated in the nucleus of cultured mouse DRG neurons treated with PTX. In line with this, increased lysosomal-associated membrane protein 1 (LAMP1) expression was observed in PTX-treated mice. Given that our previous work demonstrated PTX treatment increases MNK1/2-eIF4E signaling in DRG neurons, we examined whether MNK1/2 inhibition could rescue lysosomal dysfunction. Treatment with Tomivosertib (eFT508), a potent MNK1/2 inhibitor, restored P62 levels in DRG neurons of PTX-treated mice and reduced TFEB in DRG treated in vitro. To establish translation relevance, we further show that PTX elevates phosphorylated eiF4E (p-eIF4E) in human DRG neurons, and concurrent eFT508 administration attenuates this effect. Collectively, these findings indicated that PTX disrupts lysosome trafficking and biogenesis, and that MNK inhibition with eFT508 restores lysosomal signaling and can serve as a neuroprotective strategy for CIPN.

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.

Memory impairment is a common and sometimes overlooked feature of major depressive disorder, and cognitive deficits may precede the onset of depressive symptoms in some cases. However, the cognitive benefits of first-line treatments such as SSRIs are mixed. Tianeptine is an atypical antidepressant and cognitive enhancer that neither interacts with monoamine receptors nor inhibits the reuptake of their neurotransmitters. Its antidepressant efficacy in animal models requires activation of the mu-opioid receptor (mu-OR) and phosphorylation of the AMPA receptor. However, the receptors that mediate its memory enhancing actions have never been investigated. We therefore tested the ability of tianeptine to improve spatial memory in a cross-maze task in wild-type (WT) mice compared to its effects in mice with global knockout of either the mu-OR or delta-OR. In parallel, we assessed the effects of tianeptine on hippocampal oscillatory activity and spontaneous locomotion in the same genotypes. Adult male and female WT, mu -/-, and delta -/- mice on a C57BL/6J background were implanted with hippocampal electrodes for the recording of local field potential (LFP) oscillations. Consistent with our previous observations in anaesthetised rats, injection of tianeptine (10 mg/kg and 30 mg/kg SC) caused a dose-dependent increase in beta-frequency power in WT mice that was maximal at circa 25 Hz. The same effect was observed in delta -/- mice, but the increase in beta was completely absent in mu -/- animals. As others have reported previously, tianeptine also caused a mu-OR-dependent increase in spontaneous locomotor activity, but with a time-course that was distinct from the increase in beta power. Separate groups of WT, mu -/-, and delta -/- mice were tested for their ability to learn a food-rewarded spatial memory task in a cross-maze. Over a 20-day training period, sub-groups of each genotype received either tianeptine (10 mg/kg SC) or vehicle injection 30 min before testing. Tianeptine increased the percentage of correct trials and the number of allocentric (place) responses in WT mice, but did not enhance memory in either mu -/- or delta -/- mice, even though both genotypes were able to learn the task. These results indicate that the ability of tianeptine to drive hippocampal beta oscillations is dependent on the mu-OR, whereas its memory-enhancing actions require the presence of both mu- and delta-ORs. The latter result is consistent with the actions of tianeptine on postsynaptic AMPA receptors, and we are currently exploring the signalling pathways involved in this process.

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

Melanin is a pigment found in the skin and cuticle of animals. Oculocutaneous albinism (OCA) is a group of autosomal recessive disorders defined by reduced melanin in skin and eyes, and is associated with visual defects such as foveal hypoplasia and infantile nystagmus. Sleep disturbance has been documented in children with OCA, and oca2 loss-of-function in cavefish causes constitutive sleep loss, indicating a sleep regulatory function of OCA-associated genes in the visual system. To test potential roles of OCA-associated genes in regulating sleep and vision through evolutionarily conserved mechanisms, we used Drosophila melanogaster, a high-throughput phenotyping system to screen sleep and visual phenotypes for genetic mutants of OCA-associated genes. Among the OCA-associated genes, bidirectional DRSC Integrative Ortholog Prediction Tool identified Drosophila orthologues for OCA2, SLC45A2, SLC24A5 and LRMDA. RNAi-mediated knockdown in developing Drosophila eye tissue identified the OCA2 orthologues hoe1, hoe2, and the SLC45A2 orthologue lovit as candidate gene required for normal sleep. Moreover, hoe1, hoe2 and lovit1 null alleles reduced sleep and circadian rhythmicity, and showed altered photoreceptor neurotransmission. Collectively the data indicate evolutionarily conserved neuronal function of OCA2 and SLC45A2 orthologues that regulates sleep and photoreceptor neurotransmission. Author summaryOculocutaneous albinism (OCA) is a pigmentation disorder of the skin and eyes accompanied by reduced visual acuity and nystagmus. Children with albinism also report sleep disturbance, and the mechanism is unclear. We tested whether genes mutated in albinism regulate sleep in the fruit fly Drosophila melanogaster, an organism that has a divergent melanin synthesis pathway and lacks tyrosinase, the principal pigmentation enzyme in mammals. We screened fly orthologues of seven human OCA-associated genes and identified two that regulate sleep: the OCA2 orthologues hoe1 and hoe2, and the SLC45A2 orthologue lovit. Mutants of these genes show reduced sleep and circadian rhythmicity; electrical recordings show that photoreceptors fail to signal normally to downstream neurons, placing the sleep defect within a defined visual circuit. Because none of the above mutation cause pigmentation defect, our finding shows that OCA2 and SLC45A2 have a neuronal function that is separate from pigment synthesis. Pigment synthesis cells and neurons share a common embryonic origin, which may explain how the same transporter genes came to function in both cell types, and the data are consistent with the proposal that this pleiotropic neuronal function underlies the sleep and vision symptoms in albinism.

PurposeEndophthalmitis is a serious complication of intraocular surgery due to the risk of irreversible retinal damage. Because retinal cell populations are highly heterogenous, single-cell resolution is required to uncover the detailed mechanisms of infection response. Using a mouse model of bacterial endophthalmitis, we investigated transcriptional changes across resident and infiltrating cell types in the retina. MethodsA methicillin-sensitive strain of Staphylococcus aureus was isolated from a patient with endophthalmitis. Adult C57Bl/6J mice received an intravitreal injection of phosphate-buffer solution (PBS) with or without 5000 CFU S. aureus (n=3 per group). 24 hours later, retinas were isolated and single-cell suspensions were sent to the Penn Genomic Core for sequencing with an Illumina NovaSeq 6000. After standard pre-processing of the data, differential genes and pathways were identified for each cell type (adjusted p < 0.01, log2FC > 1 or < -1). ResultsOur analysis identified all expected retinal cell types, including a population of infiltrating neutrophils in the infected retinas. We surveyed genes known to be upregulated at the bulk-retina level in this model (e.g. Tlr2, Nlrp3, Il1b), and found that infiltrating cells mainly drove this expression. Several genes were altered across nearly all retinal cell types, including upregulation of Hsph1 and Stat3. Muller glia downregulated Gpx4 while upregulating Acsl4 and iron importers Tfrc, Zip14, and Dmt1. Top pathways for macrophages/microglia included chemotaxis, cell-cell adhesion, and wound healing. Vascular cells upregulated angiogenesis-related genes. Cellular respiration was a commonly affected pathway across several neuronal populations, with most genes decreasing. ConclusionsThis study advances our understanding of the pathobiology of bacterial endophthalmitis. Muller glia appear to be undergoing ferroptosis, potentially while activating a program to sequester iron away from bacteria. Decreased cellular respiration may indicate hypoxia among neurons. Our results reveal several trends in the retinal response to infection, including iron dysregulation and hypoxia. Understanding these cell-type-specific responses to endophthalmitis may help design therapies to combine with antibiotics.

Adeno-associated viral (AAV) vectors are foundational tools for dissecting brain structure-function relationships, but AAV serotype tropism varies across brain regions and species, requiring empirical validation to inform experimental design. This need is especially important in non-model organisms, where molecular neuroscience tools remain underdeveloped and access to research subjects is often limited. The Arctic ground squirrel (AGS, Urocitellus parryii) is a valuable model for studying extreme physiology, including metabolic suppression during hibernation and resistance to cerebral ischemia/reperfusion, yet no studies have evaluated AAV performance in the AGS brain. Here, we investigated the ability of AAV serotypes 1, 8, 9, and DJ to transduce the AGS hypothalamus using the human synapsin (hSyn) promoter and directly compared cellular transduction rates in a region implicated in thermoregulation and hibernation. To maximize data collection from a limited experimental population, we used a within-animal, contralateral stereotaxic injection design. Recombinant AAV vectors expressing enhanced green fluorescent protein or mCherry were delivered bilaterally, and reporter expression was analyzed four weeks later. All tested serotypes produced clear and reproducible reporter expression, establishing AAV as a viable molecular tool in the AGS hypothalamus. AAV1 produced significantly greater cellular transduction rates than AAV-DJ (17.2% {+/-} 3.5% vs 8.4% {+/-} 2.9%, paired t-test, p = 0.032). AAV8 and AAV9 showed transduction rates of 22.8% {+/-} 0.6% and 20.1% {+/-} 1.5%, respectively; however, with only two biological replicates per serotype, formal statistical comparison was not performed. These findings provide the first direct characterization of AAV-mediated gene delivery in the AGS brain and establish a foundation for future molecular interrogation of hypothalamic circuits in this extreme mammalian hibernator.

Disease variants in GABR genes encoding {gamma}-Aminobutyric acid type A receptor (GABAAR) subunits are major causes of developmental and epileptic encephalopathies (DEEs). There is no effective treatment for these DEEs although the GABAAR is a major target for antiseizure drugs. We previously identified the therapeutic effect of 4-phenyl-butyrate (PBA) in Gabrg2+/Q390X knockin DEE mice and now test the effect of the drug in GABRA1 variants that encode the 1 subunit. We used a multidisciplinary approach including in silico structural modeling, flow cytometry, patch clamp recordings and bio-chemistry in conjunction with differential tagging of the wild-type and the mutant alleles to evaluate the effect of PBA on rescue of GABAAR subunit expression, surface trafficking, and function in vitro in heterologous HEK293T cell model and in vivo in Gabra1+/A322D mice. We found that both total and cell surface 1 expression was reduced when the variant 1 protein was present; suggesting reduced functional receptor on the cell membrane and synapse. Patch clamp recordings identified 1 variants reduced GABA-evoked current amplitude. In silico prediction indicated reduced protein stability for GABRA1 variants indicated by negative {Delta}{Delta}G values. PBA increased both total and surface expression of wildtype 1 and 1 variants; and improved expression of both wildtype and variant 1 alleles when these were co-expressed. Importantly, PBA also increased the GABAAR expression in the thalamus of the Gabra1+/A322D mice. This study indicates that PBA is a promising treatment option for DEEs associated with GABRA1 mutations. Our previous work has demonstrated that PBA improves proteostasis by enhancing expression of the wildtype allele, repairing the mutant allele, and reducing endoplasmic reticulum stress. Therefore, it can mitigate seizures and improve neurobehavioral phenotypes at behavioral levels. Based on this and our previous work on GABRG2 and SLC6A1 mutations, we propose that PBA holds promise as a common medicine for multiple genetic neurologic disorders that share the proteostasis pathology with a broad clinical application in DEEs.

Cognitive impairments significantly impact the daily life of people with Down syndrome (DS). Overinhibition mediated by interneurons in the central nervous system was proposed as a key pathophysiological mechanism. Previous studies demonstrated cognitive rescue in the Ts65Dn mouse model using 5IA, a negative allosteric modulator of the 5 subunit-containing GABAA receptors. Here, we evaluated the effect of this drug in a mouse model carrying a more accurate duplication of the orthologous region to the human chromosome 21, namely the Dp(16)1Yey mouse model. First, we expanded the phenotypic characterization of Dp(16)1Yey mice using translationally more relevant behavioral tests. We confirmed spatial memory deficits in Dp(16)1Yey mice in the Barnes maze, and highlighted robust learning deficits in the pattern dissociation task and impairments in motor coordination. Next, we evaluated the effect of 5IA treatment on cognitive and motor performance. While 5IA treatment improved motor coordination in the Dp(16)1Yey mice, it failed to restore cognitive performance in the Barnes maze or in the pattern dissociation task. These findings could suggest divergent pathophysiological mechanisms between the Dp(16)1Yey and the Ts65Dn models. Potentially, it could explain the limited efficacy of similar pharmacological intervention in clinical trials for DS. Further preclinical studies should prioritize refined behavioral paradigms and probably the use of more complex DS models to enhance the translational potential of candidate therapies.

The endoplasmic reticulum (ER) and mitochondria maintain a dynamic structural partnership essential for pancreatic {beta}-cell homeostasis, yet the high-resolution 3D remodeling of these networks under stress conditions remains poorly defined. We employed Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) to perform 3D reconstructions of INS1E cells subjected to mitochondrial respiratory chain inhibition, uncoupling, and exogenous oxidative stress. Quantitative analysis revealed that mitochondrial dysfunction induces profound ultrastructural transitions, characterized by significant luminal swelling of the ER, expansion of the perinuclear space, and mitochondrial diameter enlargement. 3D volume imaging identified a coordinated fragmentation of both ER and mitochondrial networks into discrete, spatially separated structures--a phenomenon distinct from the reticular morphology observed in control cells. The similarity between respiratory inhibition- and H2O2-induced phenotypes, together with preservation of ER structure following mitochondrial uncoupling, suggests a potential contribution of reactive oxygen species to the observed remodeling process. Despite this extensive organelle breakdown, interorganelle membrane contact sites were not only preserved but expanded under stress conditions. We further provide a quantitative description of nuclear envelope-mitochondria contact sites (NAMs), demonstrating their selective remodeling during mitochondrial dysfunction. Our findings provide a high-resolution structural framework for organelle remodeling in {beta}-cells, demonstrating that membrane contact sites are actively preserved and reorganized despite profound organelle fragmentation. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/727587v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@4f63b5org.highwire.dtl.DTLVardef@1b3dc8org.highwire.dtl.DTLVardef@7527fcorg.highwire.dtl.DTLVardef@1944f2c_HPS_FORMAT_FIGEXP M_FIG C_FIG

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

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

Leucine-Rich Repeat Kinase 2 (LRRK2) is a signaling molecule involved in Parkinsons disease pathomechanisms. In disease, the LRRK2 protein displays both a toxic gain of kinase function and a loss of phosphorylation at heterophosphosites found in an extended loop of the LRR domain. RAB GTPases, such as RAB29, have been identified as upstream activators of LRRK2. Indeed, co-expression of LRRK2 with RAB29 induces a hyperactivation of LRRK2 kinase activity, however the role of the LRRK2 heterologous phosphorylation status in its activation remains unknown. Here, our aim was to determine the role of LRRK2 heterologous phosphorylation on its activation by RAB29. Using single and compound phosphodead or phosphomimetic mutants of LRRK2 we show differential sensitivity of LRRK2 phosphomutants to activation by RAB29, with phosphodead mutants being more susceptible to be activated than phosphomimetic mutants. Interestingly, we find that the single phosphodead S910A LRRK2 mutant displays an activation of LRRK2 kinase activity similar to that observed for the compound phosphodead 6xS>A LRRK2 mutant (S860A/S910A/S935A/S955A/S973A/S976A). Time-course analysis revealed that phosphodead mutants displayed higher but also faster activation by RAB29. In addition, both physical interaction between LRRK2 and RAB29 as well as RAB29-induced recruitment of LRRK2 to the trans-Golgi network (TGN) was enhanced by phosphodead compared to phosphomimetic mutants. To confirm effects on native LRRK2, we tested a panel of ten nanobodies targeting LRRK2 that stabilized LRRK2 phosphorylation at varying levels. Nanobodies stabilizing LRRK2 at low S935 phosphorylation levels showed enhanced RAB29-induced activation compared to nanobodies not affecting pS935 LRRK2. Finally, we tested whether LRRK2 heterologous phosphorylation could affect centrosome cohesion deficits, a phenotype that has been linked to LRRK2 hyperactivation, and found that both the phosphodead LRRK2 as well as a nanobody stabilizing dephosphorylated LRRK2 enhanced the centrosome cohesion deficit. Our findings indicate that hyperactivability of LRRK2 is directly related to its heterologous phosphorylation status, with dephosphorylation leading to strong hyperactivation of LRRK2 by upstream activating RABs, and phosphorylated LRRK2 showing the opposite. This implies that strategies favoring LRRK2 phosphorylation will have therapeutic benefit.

Cystatin-related epididymal spermatogenic (CRES) is a member of a reproductive subgroup of family 2 cystatins and part of a functional amyloid-containing extracellular matrix (ECM) with host defense functions in the epididymal lumen. CRES and the endogenous epididymal amyloid matrix exhibit high plasticity, forming distinct amyloid structures, with antimicrobial functions tailored to specific bacterial strains. We recently demonstrated that CRES/CRES amyloid is also present in the mammalian brain ECM. In this study, we utilized a CRES knockout (KO) mouse model to determine if the loss of CRES altered the structure of the hippocampal and cortical ECM and impacted brain function. We report that CRES KO male and female mice exhibited sex-specific structural changes in both the loose/interstitial and perineuronal net (PNN) ECM. Additionally, young CRES KO males--but not females--displayed deficits in learning, memory and executive functioning when compared to WT controls. In contrast, older CRES KO males did not exhibit behavioral deficits, a finding that correlated with the observation of a similar PNN ECM structure when compared to WT mice. Our data suggest that CRES functions as a regulatory or structural component of the brain ECM, contributing to its sex-specific structural organization and plasticity, affecting sex-specific behaviors of learning and memory.

Disruption of glutamate homeostasis is believed to contribute to the early progression of Alzheimers disease (AD) and associated neurodegeneration. Soluble amyloid-{beta} oligomers impair excitatory amino acid transporters (EAATs), reducing glutamate clearance, while also enhancing glutamate release from neurons and astrocytes. Together, these effects produce persistent glutamatergic dysregulation that disrupts synaptic and network function. Here, we asked whether the effects of EAAT attenuation can be mitigated through ion-channel modulation. TBOA, a selective EAAT inhibitor, was used to model early-stage glutamatergic dysregulation. TBOA reduced the local field potential amplitude of hippocampal sharp-wave ripples (SWRs) in mouse hippocampal slices, suggesting that glutamate accumulation disrupts network synchrony. Calcium imaging further showed that TBOA diminished SWR-associated population calcium transients while promoting spontaneous calcium transients in individual neurons, indicating a shift from coordinated population activity toward disorganized cellular activity. KCNQ-channel openers ML213 and ICA-27243 partially restored the TBOA-induced decline in SWR amplitude. In contrast, similar restorative effects were not observed following modulation of other ion channels, including blockade of AMPA and NMDA receptors or HCN/Ih channels, or activation of large-conductance Ca2+-activated K+ (BK) channels and G-protein-activated inwardly rectifying potassium (GIRK) channels. Together, these findings suggest that KCNQ-channel openers may occupy a unique position in mitigating glutamate-related hyperexcitability during early AD-associated network dysfunction.

BackgroundDirect conversion of human somatic cells into functional neurons could offer a faster way to generate patient-specific neurons for use in regenerative medicine, disease modelling, and drug development. Although it has been reported that neuronal direct reprogramming bypasses the intermediate pluripotent state, no reports have included time-lapse experiments, potentially overlooking transient intermediate states. Recent studies have shown that the conversion of human mesenchymal stromal cells (hMSCs) into neuron-like cells involves a transition through a transient intermediate state. Therefore, further research is needed to fully understand the process by which human somatic cells can become neurons without cell division. In this study we investigates whether direct neuronal reprogramming of human bone marrow-derived MSC (hBM-MSCs), dental pulp-derived MSC (hDP-MSCs), and adult human dermal fibroblasts (HDFa), involves a transient intermediate state, and sought to further validate the neuronal identity of hMSC-derived induced neurons. MethodsIn this study, we conducted time-lapse experiments to observe the transformation of hBM-MSCs, hDP-MSCs and HDFa, into neurons using a small-molecule-based direct reprogramming protocol. Cellular and ultrastructural changes were further characterized by confocal and electron microscopy. ResultsDirect conversion of hBM-MSCs, hDP-MSCs and HDFa into neuron-like cells occurred rapidly and in absence of cell division. Time-lapse analyses revealed that reprogramming proceeds through a transient intermediate state characterized by distinct morphological changes and dynamic nuclear remodelling. Furthermore, we found that neuron-like cells derived from hBM-MSCs and hDP-MSCs exhibit neuronal polarization, expressed specific neuronal and synaptic markers, formed interconnected cellular networks, and exhibited functional plasticity, providing further evidence that hMSCs can become functional neurons. ConclusionsThis study provides clear evidence that the direct neuronal reprogramming process involves a transition through an intermediate, transient state. Our findings also provide further evidence that hMSCs can become functional neurons. In summary, our work provides new insights into the direct neuronal reprogramming process, which is essential for advancing both developmental biology and regenerative medicine.

AO_SCPLOWBSTRACTC_SCPLOWO_ST_ABSBackgroundC_ST_ABSExcessive or unregulated iron in the brain can lead to toxicity via ferroptosis and related mechanisms. Iron accumulation in the substantia nigra (SN) occurs with Parkinsons disease (PD) progression and has been hypothesized to be an etiological mechanism. ObjectiveBased on emerging clinical observations, we tested the hypothesis that iron accumulation in the SN is a consequence of levodopa administration and is treatment-related rather than an intrinsic etiological mechanism. MethodsWe used both unilaterally lesioned 6-OHDA and unlesioned rats. We administered levodopa to rats at doses that were allometrically calculated to be similar to those used in mid-stages of PD. Iron-sensitive MRI (R2*) was used to quantify iron in the brain. Both group and intra-subject analyses were done using paired t-tests and linear mixed models. ResultsExperiment 1 used the unilateral 6-OHDA model to take advantage of the almost complete lack of dopamine neurons on the lesioned side. This permitted testing if levodopa-induced iron accumulation occurred in and/or depended on dopamine neurons. Fifteen days of levodopa treatment caused a marked increase in Fe in both the lesioned (p = 0.042) and unlesioned sides (p = 0.005), showing that iron accumulation does not depend on the presence of dopamine neurons. Based on these data, in experiment 2 unlesioned rats were administered levodopa daily for four months, and iron (R2*) values were assessed at baseline, 1, 2, and 4 months. In these normal rats, the levodopa-treated group had significantly increased Fe (R2*) in the substantia nigra compared to the vehicle group (p = 0.013). Interestingly, these effects were limited to the striatum, with no increases seen in the striatum, ventral tegmental area, or frontal cortex ConclusionLevodopa triggers processes that increase iron deposition in the substantia nigra, but this process may not depend on dopamine neurons. The underlying mechanisms and the effect on PD progression are important to elucidate and may transform how we understand PD and related neurodegenerative disorders