Molecular Neurobiology

Tau is a microtubule-associated protein important for neuronal structure and function, with abnormal phosphorylation associated with neuronal dysfunction and degeneration. While tau pathology has been extensively studied in neurodegenerative diseases and traumatic brain injury, its role in spinal cord injury (SCI) remains unclear. This study investigates changes in the levels of tau isoforms and phosphorylation following a high lateral C2 hemisection (C2Hx) in adult female rats. Western blot analysis was performed on spinal cord tissue collected caudal to the lesion at 4 weeks post-injury. Low-molecular-weight (LMW) tau and high-molecular-weight, Big tau did not differ significantly between injured and control groups. In contrast, phosphorylated tau (p-tau), detected using the AT8 antibody, was significantly elevated after injury, suggesting a shift in tau functional state. Assessment of microtubule stability markers revealed increased acetylated tubulin and decreased tyrosinated tubulin after SCI, indicating a shift toward a more stable cytoskeletal state. Immunohistochemistry was performed on spinal cord sections at 6 weeks post-injury and dorsal root ganglia (DRG) at 8 weeks post-injury. These analyses demonstrated increased p-tau immunoreactivity within the ipsilateral dorsal horn of the injured spinal cord and a higher proportion of p-tau-positive neurons in DRGs from injured animals compared with controls. Together, these findings demonstrate that cervical SCI induces persistent tau phosphorylation without altering total tau levels, and that this modification extends beyond the lesion site into distal spinal regions and beyond the acute phase of injury. These alterations may influence neural plasticity and remodeling in the injured spinal cord.

The FoxP family of transcription factors has been linked to neurodevelopmental disorders. FoxP1 and FoxP2 were identified as genes associated with autism spectrum disorders (ASD). FoxP1, FoxP2, and FoxP4 are expressed in many brain regions, including the cerebellum that has been of particular interest in the context of ASD. However, their role in the development of the peripheral nervous system (PNS) has been neglected. In view of the fact that a majority of ASD patients show some signs of sensory perception deficits that could go beyond the central nervous system, we focused on neural circuit formation in the PNS. Our in vivo and in vitro studies revealed a role of all three FoxP transcription factors in neural circuit formation in the hindlimb of the chicken embryo. Silencing of FoxP genes altered motor axon guidance and also impacted neural crest cell migration. This in turn resulted in aberrant formation of dorsal root ganglia and deficits in neural circuits in the PNS. Taken together, our results are in line with the hypothesis that some of the sensory perception deficits in ASD patients, either hyper- or hyposensitivity, are due to aberrant neural circuits in the PNS.

Purpose. Neuroblastoma is the most common malignancy of infancy, and cancer cells strongly depend on iron metabolism for proliferation. Whether sub-cytotoxic iron load reprograms mitochondrial and redox metabolism, and how this relates to the malignant behavior of neuroblastoma cells, is poorly defined. We hypothesized that exposure of human neuroblastoma cells to a non-toxic iron overload perturbs mitochondrial and redox metabolism, thereby affecting cancer cell properties. Methods. SH-SY5Y neuroblastoma cells were exposed to 100 M iron sulfate for 24 h. The resulting changes were monitored using complementary methods, including cell viability, cytotoxicity, proliferation and migration assays, intracellular iron quantification by ICP-MS, confocal imaging of mitochondrial morphology and membrane potential ({Delta}{Psi}m), high-resolution respirometry with flux control ratios, probe-based oxidant and H2O2 measurements, and label-free quantitative proteomics with Ingenuity Pathway Analysis. Results. Non-toxic iron loading raised intracellular iron about four-fold, promoted cell proliferation and reduced migration. Iron induced mitochondrial fragmentation and {Delta}{Psi}m depolarization while preserving mitochondrial content and OXPHOS coupling efficiency. Among respiratory states, only proton-leak respiration increased, paralleled by a higher leak/ETS coupling ratio. Total cellular oxidant levels and H2O2 output were strongly reduced. The proteome showed downregulation of ribosomal proteins and translation/RNA-processing components and upregulation of histones and ferritin. Conclusion. Mild iron exposure reprograms mitochondrial and redox metabolism in neuroblastoma cells. The observed selective increase in proton leak lowers {Delta}{Psi}m to a range that biophysically limits oxidant production while OXPHOS coupling is preserved. Interfering with this adaptive, proton leak-dependent and preventive antioxidant program may offer a way to alter neuroblastoma progression.

Metabotropic glutamate receptors (mGluR) are G-protein coupled receptors that regulate neuroplasticity and have been shown to play a role in auditory processing. However, not much is known about the expression of mGluRs during cochlear nucleus development in the context of Fragile X syndrome (FXS). FXS is the most common heritable form of intellectual disability and autism and is associated with auditory processing deficits including hypersensitivity. Audiogenic seizure is a notable feature of the Fmr1 knock-out (KO) mouse model for FXS, in which mGluR5 has been shown to be necessary. In this study, we investigated the expression of eight metabotropic glutamate receptors (mGluRs) in the cochlear nucleus at both young and adult ages and in wildtype and Fmr1 KO mice. While it is established that mGluR1 is the predominantly expressed mGluR in adult cochlear nucleus, we report the following new finding that mGluR5 is the more abundant receptor during early development. The expression of Group II and III mGluRs (2,3,4,6,7,8) was relatively stable during development. We show that mGluR1 and mGluR5 are co-expressed across multiple neuron types. Fmr1 genotype did not have statistically significant impact on the expression pattern of mGluRs in cochlear nucleus. Although hearing onset occurs during this mGluR5 to 1 transition period, mGluR expression patterns were not impacted by cochlear activity. We conclude that there is an mGluR5 is to mGluR1 transition that occurs within cochlear nucleus neurons during the critical period closure that is intrinsic to neuronal development.

Microglial activation is a central component of neuroinflammatory responses in many brain pathologies. Increasing evidence indicates that microglial phenotype is tightly linked to cellular metabolism, with pro-inflammatory activation associated with enhanced glycolytic flux. Lactate, traditionally considered a metabolic substrate, has recently emerged as a signaling molecule capable of modulating immune responses. However, its direct impact on microglial inflammatory activation remains incompletely understood. In the present study, we investigated the effects of lactate on microglial phenotype under inflammatory conditions using primary rat microglial cultures stimulated with lipopolysaccharide (LPS). Microglial activation was assessed through the expression of phenotypic markers, cytokine production, and secreted chemokine profiles. LPS stimulation induced a strong pro-inflammatory response characterized by increased CD86 expression, elevated TNF-alpha secretion, and enhanced release of several pro-inflammatory chemokines. Post-treatment with sodium L-lactate significantly attenuated these inflammatory responses, reducing pro-inflammatory marker expression and cytokine secretion, while restoring the anti-inflammatory marker CD206. To explore the relevance of these findings in a pathological context, the effects of lactate were further examined in a neonatal rat model of hypoxia-ischemia. Sodium L-lactate administration after injury reduced microglial activation and promoted a shift toward an anti-inflammatory phenotype in cortical regions, whereas hippocampal microglia showed a more limited response. Together, these results demonstrate that lactate directly modulates microglial inflammatory activation and cytokine production in vitro and suggest that lactate-mediated metabolic signaling may contribute in vivo to the regulation of neuroinflammatory responses.

Background and Purpose: Neonatal hypoxia-ischemia (NHI) is a leading cause of mortality and long-term neurodevelopmental disability. Therapeutic hypothermia (TH) is currently the only established treatment for NHI; however, its efficacy remains limited, underscoring the need for adjunctive neuroprotective strategies. Lactate is now regarded as a major metabolic substrate of the brain and has previously been shown to exert neuroprotective effects following experimental NHI. We investigated whether lactate enhances hypothermia-induced neuroprotection and evaluated the optimal timing of administration and potential underlying mechanisms. Methods: Postnatal day 7 Wistar rats were subjected to NHI using the Rice-Vannucci model and treated with TH alone (2h at 32{degrees}C), sodium L-lactate alone, or combined L-lactate-TH. Animals underwent longitudinal magnetic resonance imaging (MRI) and behavioral assessments, complemented by histological, immunohistochemical, and Western blot analyses, including assessment of protein lactylation. The effect of sodium L-lactate administration timing relative to TH was also evaluated. Results: Lactate significantly enhanced the neuroprotective effects of TH, particularly when administered before hypothermia, resulting in greater lesion recovery, reduced neuroinflammation and apoptotic cell death, preserved neuronal integrity, and improved long-term motor and cognitive outcomes. Combined treatment preserved neuronal lactate transporter, prevented NHI-induced astrocytic glutamate transporter upregulation, modulated lactylation markers, supporting coordinated metabolic and epigenetic adaptations following NHI. Conclusions: Early lactate administration enhances the neuroprotective efficacy of TH following NHI. These findings identify lactate as a promising adjunct to TH and support its translational potential as a metabolic strategy to improve outcomes following NHI brain injury.

Myelin is an essential and highly specialized membrane in the central and peripheral nervous systems that enwraps axons to accelerate electrical transmission and support neuronal health. The generation of this multilamellar structure requires a tightly coordinated synthesis of specific proteins and lipids. Among these are sphingolipids (SLs), which are major components of myelin. SL production is initiated by the serine palmitoyltransferase (SPT) enzyme complex, the rate-limiting enzyme in the de novo SL biosynthesis pathway. ORMDL proteins (ORMDL1-3) are the regulatory subunits of SPT, which, by sensing ceramide levels, tune SL flux. Although ORMDL3 has been linked to asthma and peripheral myelination, its role in CNS myelination and the CNS myelin-making oligodendrocytes (OLGs) remains unclear. We therefore assessed the function of ORMDL3 in OLs by generating a Cnp-Cre-driven, oligodendrocyte-specific Ormdl3 conditional knockout (cKO) mouse model. Loss of Ormdl3 selectively increased myelin SLs, particularly long-chain sulfatides, without altering ceramide or galactosylceramide abundance. These changes are most prominent around postnatal day 35, a developmental period of active myelin turnover. Ultrastructural analysis of optic nerves shows a thicker myelin sheath and increased axon caliber in cKO mice. Unexpectedly, deletion of Ormdl3 also increases levels of major myelin proteins (MBP, MOG, and PLP) and is accompanied by dynamic, region- and sex-dependent regulation of enzymes involved in sulfatide biosynthesis, without altering OLGs number or maturation. Together, for the first time, these findings identify Ormdl3 as a key regulator of SL homeostasis during developmental myelination and suggest that it helps synchronize lipid synthesis with myelin protein expression.

Heavy metal contamination of aquatic environments poses persistent risks to ecosystems and human health. Aquatic macrophytes such as Pistia stratiotes are increasingly used for phytoremediation due to their capacity to accumulate contaminants without displaying symptoms of abiotic stress. However, the molecular mechanisms underlying their metal tolerance remain poorly understood. Metallothioneins (MTs) are cysteine-rich proteins involved in metal binding and detoxification in plants. In this study, genome-wide analysis was performed to identify and characterise candidate MT genes in P. stratiotes and to assess their transcriptional response to copper exposure. Six novel MT genes (PsMT2f, PsMT3a-d, and PsMT4) were identified and classified into MT2, MT3, and MT4 subfamilies based on conserved cysteine motifs and domain architecture. Quantitative real-time PCR showed differential and tissue-specific expression of MT genes in roots and leaves following exposure to copper for up to 4 h. These findings provide the first comprehensive characterisation of the MT gene family in P. stratiotes and demonstrate their potential role in copper tolerance, supporting the application of this species in the phytoremediation of metal-contaminated aquatic systems.

Peripheral neurons exhibit robust regeneration after nerve injury in adulthood; a process linked to the activation of regeneration-associated genes (RAGs). However, the mechanisms underlying nerve regeneration during early postnatal development remain poorly understood. In a previous work, we observed that motoneurons axotomized at early postnatal stages regenerate without overexpressing RAGs, contrasting with the expected RAG activation observed in postnatal day 30 (P30) mice. To determine whether this phenomenon extends to sensory neurones, we investigated two paradigmatic dorsal root ganglia populations: proprioceptive (PV) and nociceptive (TrpV1) neurones, which exhibit distinct regenerative capacities in adult mice. Following sciatic nerve crush injury at P4, P10, and P30, both neuronal subtypes demonstrated robust axonal regeneration across all postnatal stages, with nociceptors exhibiting significantly superior regeneration compared to proprioceptors. Remarkably, no substantial neuronal death was observed at any developmental timepoint. However, RNA sequencing revealed that P4 and P10 sensory neurones failed to upregulate RAGs following injury, whereas P30 neurones exhibited the expected pro-regenerative transcriptional programme. Pathway enrichment analysis confirmed that classical regeneration-associated signalling pathways were activated exclusively in young adults, whilst developmental and inflammatory pathways predominated in early postnatal stages. Using the conditioning lesion paradigm in primary dorsal root ganglia cultures, we demonstrated that only P30 neurones exhibited enhanced neurite outgrowth following conditioning lesion, whilst P4 and P10 neurones showed no conditioning effect or reduced outgrowth, respectively. These findings indicate that early postnatal sensory neurones possess intrinsic growth capacity independent of RAG activation, suggesting fundamentally distinct regenerative mechanisms across developmental stages.

Hearing loss and deafness as a result of hair cell loss cannot be restored due to the incapacity of spontaneous regeneration of these cells. Compounds that manipulate key signaling pathways can potentially regenerate cochlear hair cells. In order to test different compounds that promote hair cell regeneration in vitro models can be used. Three-dimensional cultures have allowed the expansion and experimentation of human and mouse inner ear organoids. This is mainly performed in the embryonic or the early postnatal developmental stage. However, since the majority of patients with hearing loss are adult, it is crucial to understand the adult inner ear regenerative capacity. Here, we evaluated progenitor cell markers in the adult human and mouse inner ear and the generation, expansion and differentiation of cochlear organoids derived from the adult human and mouse inner ear. Cochlear and vestibular sensory epithelium from adult humans; and from adult and aged mice express the progenitor markers SOX2 and LGR5; and can generate organoids in vitro. By optimizing the culture conditions, organoids derived from the cochlea and vestibular organ from adult human and adult and aged mouse differentiated to Myosin 7A positive cells. This indicates that the adult inner ear has regenerative capacity. These findings are encouraging for future regenerative therapies to cure hair cell-related hearing loss and deafness.

Cannabis is one of the most widely consumed substances in the world. Consumers seek out cannabis cultivars with varying levels of phytocannabinoids, primarily delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD). The effects of THC, CBD or the combination of THC:CBD have distinct outcomes on cognitive processes, cellular functions, and phytocannabinoid pharmacokinetics. The majority of research on cannabis effects on the brain has focussed on neurons, and few studies have investigated the impact of different cannabis cultivars on glia. In particular, the impact of varying levels of THC:CBD on oligodendrocyte lineage cells, which play numerous support roles in the brain essential to proper neuronal communication, is relatively unknown. This study set out to examine the acute impact of different cultivars of vaporized cannabis on oligodendrocyte lineage cells in the forceps minor of adult male and female mice. Mice were exposed to vapor from cannabis flower high in THC, high in CBD or balanced in THC:CBD over 15 minutes, and brains were fixed 30 minutes post-cannabis onset. Using immunofluorescence microscopy, we observed significant changes to oligodendrocyte progenitor cell (OPC) morphology in mice exposed to balanced cannabis, and, using correlative light and electron microscopy, we observed alterations to OPC mitochondria. The alterations observed (i.e., enlarged soma and nucleus volume, reduced density and increased area of mitochondria in the soma) in OPCs due to balanced cannabis are reminiscent of the very early changes seen during OPC differentiation. This study highlights the differing effects of cannabis cultivars on OPCs and the rapidity of the OPC response to inhaled phytocannabinoids.

Background: Parkinson's disease (PD) is characterized by the progressive loss of midbrain dopaminergic (DA) neurons and aberrant alpha-synuclein (alpha-syn) aggregation, yet disease-modifying therapies that simultaneously retard neurodegeneration and foster regeneration remain lacking. Methods: In this study, we isolated extracellular vesicles (TH and FOXA2 double-positive cell-derived EVs) from TH and FOXA2 double-positive differentiated neural cells, a distinct midbrain floor-plate-derived neural cell population that co-expresses the floor-plate transcription factor FOXA2 together with TH and should therefore not be equated with conventional mature midbrain dopaminergic neurons, and systematically evaluated their therapeutic potential across three complementary models: A53T transgenic mice, 6-OHDA-lesioned rats, and 6-OHDA-treated human midbrain organoids. Integrative transcriptomic and single-cell sequencing analyses, together with toxicological evaluations in rodents and non-human primates, were performed to investigate the underlying mechanisms and safety profile. These TH and FOXA2 double-positive cells were generated using a fully suspension-based, matrix-free, and chemically defined differentiation system devoid of N2/B27 supplements and fetal bovine serum (FBS). This approach offers a simple, controllable, low-cost, and scalable platform with high batch-to-batch consistency for EV manufacturing. Throughout this study, the term "TH and FOXA2 double-positive cells" refers to this distinct TH and FOXA2 double-positive cell population and should not be interpreted as conventional terminally differentiated dopaminergic neurons. Results: Intranasal administration of TH and FOXA2 double-positive cell-derived EVs in A53T mice and 6-OHDA-lesioned rats significantly ameliorated motor deficits, increased nigral tyrosine hydroxylase (TH)-positive neuron counts, reduced alpha-syn aggregation, and suppressed gliosis. In human midbrain organoids, TH and FOXA2 double-positive cell-derived EVs preserved DA neuronal morphology and reduced GFAP and alpha-syn expression. Notably, when EVs were administered simultaneously with 6-OHDA modeling rather than after modeling was completed, the protective effect was stronger. Multi-omics analysis revealed that TH and FOXA2 double-positive cell-derived EVs reversed pathological signatures of interferon-responsive microglia and oxidative stress-adapted astrocytes, while restoring WNT- and non-canonical WNT-mediated intercellular communications. Safety evaluations showed no discernible organ toxicity. Conclusions: TH and FOXA2 double-positive cell-derived EVs exert neuroprotective effects across multiple PD models by modulating specific glial subpopulations and reinstating development-associated signaling pathways. Together with a fully suspension-based, matrix-free, and N2/B27-free differentiation process that enables low-cost, scalable, and highly reproducible manufacturing, TH and FOXA2 double-positive cell-derived EVs represent a promising therapeutic candidate for Parkinson's disease.

Background: Low-density lipoprotein cholesterol (LDL-C) is an established risk factor for atherosclerotic cerebrovascular disease, but its effects on the cerebral microcirculation are poorly defined. We investigated whether LDL-C is associated with pericyte dysfunction in acute ischemic stroke (AIS) and explored underlying mechanisms. Methods: Serum soluble platelet-derived growth factor receptor-{beta} (sPDGFR{beta}) was measured in two independent AIS cohorts (n=97 and n=86). Cerebral pericyte transcriptional responses to LDL-C were analyzed by single-cell RNA sequencing in Apoe-knockout mice fed control or high-fat diet (n=4 [male/female: 2/2]/group), and cultured human brain pericytes were exposed to purified human LDL cholesterol with or without N-acetyl-L-cysteine. Results: Higher serum sPDGFR{beta} was associated with greater neurological deficit and circulating neurofilament light chain. LDL-C positively correlated with sPDGFR{beta} in both clinical cohorts. High-fat diet induced coordinated suppression of type-I interferon pathways, and a positive shift in reactive oxygen species-related transcriptional programs in mouse pericytes; exploratory sex-by-diet analyses suggested larger transcriptional responses in females. In cultured human pericytes, LDL-C induced oxidative-stress, lipid-handling, and ferroptosis-associated transcriptional programs while suppressing type-I interferon signaling. Cross-species enrichment analysis showed that LDL-C-induced and -repressed signatures in cultured pericytes aligned with the sex-by-diet interaction in Apoe-knockout mice. LDL-C also reduced cell-surface PDGFR{beta} and increased sPDGFR{beta} release in cultured pericytes, which were significantly attenuated by N-acetyl-L-cysteine, whereas LDH activity increased numerically but not significantly. Conclusions: Clinical and experimental findings support an LDL-pericyte axis linking atherogenic lipid stress to cerebral microvascular dysfunction, with possible sex-dependent modulation of oxidative stress and type-I interferon signaling as potential mechanisms.

Axonal degeneration is a principal driver of irreversible neurological disability in multiple sclerosis (MS), yet current treatments fail to target this neurodegenerative phase directly. Sterile alpha and TIR domain-containing protein 1 (SARM1) has emerged as an executioner of programmed axon destruction and is overexpressed in grey and white matter MS tissue, making its inhibition a promising therapeutic strategy. However, the effects of SARM1 inhibition on localized neurodegeneration versus systemic and central inflammation remain poorly understood in complex autoimmune environments like MS. Here, we investigated SARM1 pathology using global SARM1-/- knockout (SARM1-/-) mice, AAV-mediated CRISPR knockdown and overexpression of SARM1 in retinal ganglion cells (RGCs), and the small-molecule SARM1 inhibitor 5-iodoisoquinoline (5IIQ), across optic nerve crush (ONC) and experimental autoimmune encephalomyelitis (EAE) models. While global SARM1-/- did not alter the overall clinical course of EAE, it revealed a complex phenotype characterized by an altered peripheral inflammatory cytokine profile and persistent CNS immune infiltration, alongside preserved axonal and myelin integrity. RGC-restricted SARM1 knockdown partially preserved RGCs and axons during EAE, whereas SARM1 overexpression worsened both retinal function and axonal injury. Pharmacological inhibition with 5IIQ preserved axonal integrity and restored visual function in both the ONC and EAE models, as confirmed by electrophysiology, and reduced serum neurofilament light chain (NfL) in EAE, without affecting demyelination. Together, these findings demonstrate that SARM1 inhibition uncouples axonal self-destruction from demyelination and gross neuroinflammation while providing robust structural and functional neuroprotection. This study establishes SARM1 as a viable target for neuroprotective co-therapies designed to complement existing immunomodulatory and remyelinating regimens in MS and related neurodegenerative disorders.

Axonal projections and precise synapse formation are essential for the establishment of functional neural circuits, and their dysregulation contributes to various psychiatric and neurodegenerative disorders. Although numerous extracellular cues secreted from surrounding tissues regulate axon guidance and synapse formation, the mechanisms by which neurons locally regulate terminal patterning within their projection fields remain poorly understood. Bone morphogenetic protein 4 (BMP4) is a classical morphogen that is involved in early neural tube patterning during development. However, its postnatal function remains unclear. Previous in vitro studies using hippocampal glutamatergic neurons demonstrated that BMP4 can act locally on axons to suppress synapse formation, raising the possibility that BMP4 regulates terminal differentiation after target innervation. Here, we investigated the role of BMP4 in postnatal cholinergic circuit development using cholinergic neuron-specific Bmp4 conditional knockout (Bmp4 cKO) mice, with adult analyses performed in males. Basal forebrain cholinergic neurons project extensively throughout the neocortex and are essential for higher brain functions, including arousal and attention. Bmp4 cKO mice exhibited increased cholinergic axon terminals in the neocortex, hippocampus, and amygdala, as well as increased axonal ramification in the neocortex without affecting cytoarchitectonic organization or cholinergic neuron numbers in the basal forebrain. These findings demonstrate that BMP4 functions as a negative regulator of cholinergic terminal formation and axonal arborization, suggesting that BMP4 may act locally to restrict cholinergic axonal expansion during neural circuit maturation.

Background: Understanding fetal brain responses to hypoxia-ischemia is of growing interest, as it remains unclear to what extent these responses differ from those of newborns and whether developmental adaptations during gestation contribute to such differences. Materials and methods: Using a combination of broadband near-infrared spectroscopy (bNIRS) and diffuse correlation spectroscopy (DCS), we measured brain microvascular oxygen saturation (StO2), total hemoglobin concentration (THC), relative cerebral blood flow (rCBF), and the oxidation state of cytochrome c oxidase (oxCCO) during an acute ischemic challenge in the brains of five rabbit fetuses in utero. Simultaneous measurements were acquired from four control fetuses located in the contralateral uterine horn. Results: During the hypoxia-ischemia phase, we observed a rapid reduction of CBF (47% +/- 29%) and StO2, (from 64.7% +/- 1.5% at baseline to 40.6% +/- 2.0%) together with an increase of oxCCO (3.7 uM +/- 0.5 uM). Conclusion: The simultaneous increase in oxCCO and decrease in rCBF and StO2 during hypoxia-ischemia reveals a cerebral response pattern that differs from that commonly reported in the newborn brain. These findings support the hypothesis that unique developmental adaptations influence fetal cerebral metabolism during hypoxic-ischemic stress.

Dietary magnesium (Mg2+) supplement enhances memory in mammals and Drosophila, but whether and how Mg2+ changes molecular states of neurons, remains unknown. Here we used single-cell RNA sequencing to map cell-type specific transcriptional responses to memory-enhancing dietary Mg2+. These analyses revealed changes in Hippo-pathway gene expression in memory-relevant {beta} Kenyon Cells (KCs) of the mushroom bodies. Targeted knock-down of Merlin, hippo (hpo) and warts demonstrated requirement in {beta} KCs for Mg2+-enhanced, but not baseline memory. Further study of Hippo signaling identified Yorkie (yki) transcriptional coactivator and DNA-binding partner Scalloped (sd) to be required for baseline and Mg2+-enhanced memory. Moreover, releasing Yki::Sd of inhibition from Tondu-domain containing growth inhibitor (Tgi) specifically in {beta} KCs enhanced memory, independent of diet. Analyses of Yki::Sd target genes identified several required in {beta} KCs for baseline memory, including crb and Glut1 which are also Mg2+-induced in a Hippo-dependent manner in {beta} KCs. These genes suggest that aiding the metabolic demands of long-term memory and facilitating structural neuronal plasticity are key mediators of Mg2+-induced memory enhancement. We propose pharmacological enhancement of Sd-dependent transcription, and activity of particular Sd-target genes may have therapeutic potential for memory disorders.

Extracellular cold-inducible RNA-binding protein (eCIRP) is a crucial neuroinflammatory mediator in ischemic stroke and alcohol-induced memory impairment. We have recently discovered that amyloid{beta} causes microglia to release eCIRP, prompting us to investigate its role in Alzheimer's disease (AD). We found that eCIRP levels were significantly elevated in the cerebrospinal fluid (CSF) and plasma of AD patients compared with age-matched non-AD subjects, as well as in hTau.P301S mice (a model of AD tauopathy) compared with wildtype control mice. Plasma eCIRP strongly correlated with astrocyte activation marker glial fibrillary acidic protein (GFAP) in AD patients. eCIRP induced neurotoxic astrocyte-specific genes and astrocyte release of proinflammatory and neurotoxic factors in C8-D1a cells, primary murine astrocytes and intracerebroventricular eCIRP-injected C57BL/6 mice brains. In particular, eCIRP increased Complement 3, an astrocytic marker involved in neuroinflammation-associated neurodegeneration. Primary astrocytes from TREM-1 knockout mice were resistant to eCIRP induction of neurotoxic astrocytes. eCIRP increased TREM-1 expression and activation in astrocytes. Blocking CIRP/TREM-1 interaction using peptide M3, effectively attenuated eCIRP's induction of neurotoxic astrocytes. Thus, eCIRP strongly correlates with astrocyte reactivity in AD patients, and eCIRP induces neurotoxic astrocytes via TREM-1, which is attenuated by M3, suggesting a novel therapeutic opportunity to target neurotoxic astrocytes in AD.

Introduction: In both humans and experimental animals, obesity is associated with hypothalamic inflammation and neuronal metabolic dysfunction, which contribute to disrupting body energy homeostasis. Although ciliary neurotrophic factor (CNTF) is a neuroprotective cytokine with recognized anti-obesity effects in peripheral metabolic tissues, its role in obesity-associated hypothalamic dysfunction is still poorly characterized. We investigated whether CNTF exerts any direct effects on mHypoE-N41 (N41) hypothalamic neurons and whether it modulates the effects induced by palmitate (PA), an established model of obesity-associated neuronal metabolic dysfunction. Methods: N41 cells were administered CNTF alone or combined with PA to assess whether CNTF affects PA-induced lipotoxic stress. Gene and protein expression was assessed by qRT-PCR, western blotting, and immunofluorescence analysis. Results: In N41 cells, CNTF activated the STAT3, ERK and AMPK signaling pathways. It also upregulated genes involved in mitochondrial metabolism and fatty acid oxidation, including Ucp2, Cpt1 and Pgc1, as well as the proliferation-associated markers Ki-67 and PCNA. PA treatment induced inflammatory and endoplasmic reticulum stress responses and impaired insulin signaling. In PA-treated N41 cells, CNTF significantly attenuated the expression of pro-inflammatory markers Tnf, Mcp1 and Nfkb, restored insulin-induced AKT phosphorylation, and partially reversed PA-induced changes in the expression of metabolic genes Ucp2 and Cpt1. Conclusions: Collectively, these findings indicate that CNTF exerts direct protective metabolic effects on hypothalamic N41 neurons subjected to lipotoxic stress and supports a role for it in the preservation of neuronal metabolic function under obesogenic conditions.

Ogerin, a positive regulator of GPR68, reduces infarct volume and attenuates blood-brain barrier (BBB) leakage post transient middle cerebral artery occlusion (tMCAO). These data support the potential use of Ogerin for ischemic protection. For therapeutic interventions, tissue distribution, kinetics, and brain penetration are important characteristics for a compound. For these reasons, we performed liquid chromatography-mass spectrometry (LC-MS) analysis of Ogerin in mice. At 3 hours following intraperitoneal (IP) injection, liver exhibited highest level of Ogerin among all major organs assessed. We further analyzed time-dependent changes in Ogerin concentration in serum following intravenous delivery. Brain Ogerin level was ~140 ng/g at 2 hour and ~0.06ng/g at 24 hours. The brain-to-serum ratio of Ogerin was ~0.6 at 5 hr. To determine the effect of Ogerin on signaling, we collected samples 24 hours after tMCAO with or without Ogerin delivered and performed Western blotting. Consistent with the diminished level of Ogerin in brain at 24 hour, tMCAO increased similar changes in phosphorylation of PKA substrates and PKC substrates in vehicle- and Ogerin-treated groups. The pharmacokinetic data suggests that Ogerin is effective mainly during acute hours after injection. This finding generates important insights on the design of effective protocol for Ogerin delivery to potentiate GPR68 function in vivo.