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Proceedings of the National Academy of... Jun 2024Ca/calmodulin (CaM)-dependent kinase II (CaMKII) plays a critical role in long-term potentiation (LTP), a well-established model for learning and memory through the...
Ca/calmodulin (CaM)-dependent kinase II (CaMKII) plays a critical role in long-term potentiation (LTP), a well-established model for learning and memory through the enhancement of synaptic transmission. Biochemical studies indicate that CaMKII catalyzes a phosphotransferase (kinase) reaction of both itself (autophosphorylation) and of multiple downstream target proteins. However, whether either type of phosphorylation plays any role in the synaptic enhancing action of CaMKII remains hotly contested. We have designed a series of experiments to define the minimal requirements for the synaptic enhancement by CaMKII. We find that autophosphorylation of T286 and further binding of CaMKII to the GluN2B subunit are required both for initiating LTP and for its maintenance (synaptic memory). Once bound to the NMDA receptor, the synaptic action of CaMKII occurs in the absence of target protein phosphorylation. Thus, autophosphorylation and binding to the GluN2B subunit are the only two requirements for CaMKII in synaptic memory.
Topics: Calcium-Calmodulin-Dependent Protein Kinase Type 2; Phosphorylation; Animals; Receptors, N-Methyl-D-Aspartate; Long-Term Potentiation; Memory; Synapses; Rats; Mice
PubMed: 38889145
DOI: 10.1073/pnas.2402783121 -
Acta Neuropathologica Jun 2024Myasthenia gravis is a chronic antibody-mediated autoimmune disease disrupting neuromuscular synaptic transmission. Informative biomarkers remain an unmet need to...
Myasthenia gravis is a chronic antibody-mediated autoimmune disease disrupting neuromuscular synaptic transmission. Informative biomarkers remain an unmet need to stratify patients with active disease requiring intensified monitoring and therapy; their identification is the primary objective of this study. We applied mass spectrometry-based proteomic serum profiling for biomarker discovery. We studied an exploration and a prospective validation cohort consisting of 114 and 140 anti-acetylcholine receptor antibody (AChR-Ab)-positive myasthenia gravis patients, respectively. For downstream analysis, we applied a machine learning approach. Protein expression levels were confirmed by ELISA and compared to other myasthenic cohorts, in addition to myositis and neuropathy patients. Anti-AChR-Ab levels were determined by a radio receptor assay. Immunohistochemistry and immunofluorescence of intercostal muscle biopsies were employed for validation in addition to interactome studies of inter-alpha-trypsin inhibitor heavy chain H3 (ITIH3). Machine learning identified ITIH3 as potential serum biomarker reflective of disease activity. Serum levels correlated with disease activity scores in the exploration and validation cohort and were confirmed by ELISA. Lack of correlation between anti-AChR-Ab levels and clinical scores underlined the need for biomarkers. In a subgroup analysis, ITIH3 was indicative of treatment responses. Immunostaining of muscle specimens from these patients demonstrated ITIH3 localization at the neuromuscular endplates in myasthenia gravis but not in controls, thus providing a structural equivalent for our serological findings. Immunoprecipitation of ITIH3 and subsequent proteomics lead to identification of its interaction partners playing crucial roles in neuromuscular transmission. This study provides data on ITIH3 as a potential pathophysiological-relevant biomarker of disease activity in myasthenia gravis. Future studies are required to facilitate translation into clinical practice.
Topics: Humans; Myasthenia Gravis; Biomarkers; Male; Female; Middle Aged; Adult; Aged; Autoantibodies; Receptors, Cholinergic; Proteomics; Cohort Studies; Young Adult; Proteinase Inhibitory Proteins, Secretory; Machine Learning
PubMed: 38888758
DOI: 10.1007/s00401-024-02754-6 -
Molecular Brain Jun 2024Areca nut, the seed of Areca catechu L., is one of the most widely consumed addictive substances in the world after nicotine, ethanol, and caffeine. The major effective...
Areca nut, the seed of Areca catechu L., is one of the most widely consumed addictive substances in the world after nicotine, ethanol, and caffeine. The major effective constituent of A. catechu, arecoline, has been reported to affect the central nervous system. Less is known if it may affect pain and its related emotional responses. In this study, we found that oral application of arecoline alleviated the inflammatory pain and its induced anxiolytic and anti-depressive-like behavior. Arecoline also increased the mechanical nociceptive threshold and alleviated depression-like behavior in naïve mice. In the anterior cingulate cortex (ACC), which acts as a hinge of nociception and its related anxiety and depression, by using the multi-electrode field potential recording and whole-cell patch-clamp recording, we found that the evoked postsynaptic transmission in the ACC of adult mice has been inhibited by the application of arecoline. The muscarinic receptor is the major receptor of the arecoline in the ACC. Our results suggest that arecoline alleviates pain, anxiety, and depression-like behavior in both physiological and pathological conditions, and this new mechanism may help to treat patients with chronic pain and its related anxiety and disorder in the future.
Topics: Animals; Synaptic Transmission; Anxiety; Arecoline; Male; Depression; Behavior, Animal; Nociception; Mice, Inbred C57BL; Gyrus Cinguli; Mice; Cerebral Cortex
PubMed: 38886822
DOI: 10.1186/s13041-024-01106-5 -
Journal of Pharmacy & Bioallied Sciences Apr 2024The synaptic contacts play an important role in central nervous system (CNS) functioning. Ultrastructural features of synapses in CNS are not studied in naphthalene...
INTRODUCTION
The synaptic contacts play an important role in central nervous system (CNS) functioning. Ultrastructural features of synapses in CNS are not studied in naphthalene neurotoxicity model.
MATERIALS AND METHODOLOGY
In the present work, transmission electron microscopy was used for studying the ultrastructural features of synapses in the hippocampus of Sprague Dawley rat brain, on subsequent exposure to naphthalene balls. The ultrastructural changes were observed for naphthalene low dose (200 mg), high dose (400 mg) after the treatment for 28 days, and post-delayed toxicity phase after 14 days in Sprague Dawley rats.
RESULTS
In comparison with different groups of naphthalene exposure including control and satellite, axon degeneration, axonal demyelination and abnormal synapses was observed in high dose naphthalene administration group. In the post-delayed naphthalene toxicity group, degeneration of synaptic contacts was observed.
CONCLUSIONS
This exploration of ultrastructural variations in the synapses of Hippocampus gives information that will be valued in naphthalene neurotoxicological research.
PubMed: 38882886
DOI: 10.4103/jpbs.jpbs_1184_23 -
Neuron Jun 2024NMDA receptors (NMDARs) are ionotropic receptors crucial for brain information processing. Yet, evidence also supports an ion-flux-independent signaling mode mediating...
NMDA receptors (NMDARs) are ionotropic receptors crucial for brain information processing. Yet, evidence also supports an ion-flux-independent signaling mode mediating synaptic long-term depression (LTD) and spine shrinkage. Here, we identify AETA (Aη), an amyloid-β precursor protein (APP) cleavage product, as an NMDAR modulator with the unique dual regulatory capacity to impact both signaling modes. AETA inhibits ionotropic NMDAR activity by competing with the co-agonist and induces an intracellular conformational modification of GluN1 subunits. This favors non-ionotropic NMDAR signaling leading to enhanced LTD and favors spine shrinkage. Endogenously, AETA production is increased by in vivo chemogenetically induced neuronal activity. Genetic deletion of AETA production alters NMDAR transmission and prevents LTD, phenotypes rescued by acute exogenous AETA application. This genetic deletion also impairs contextual fear memory. Our findings demonstrate AETA-dependent NMDAR activation (ADNA), characterizing AETA as a unique type of endogenous NMDAR modulator that exerts bidirectional control over NMDAR signaling and associated information processing.
PubMed: 38878768
DOI: 10.1016/j.neuron.2024.05.027 -
Molecular Autism Jun 2024Mutations in the X-linked gene cyclin-dependent kinase-like 5 (CDKL5) cause a severe neurological disorder characterised by early-onset epileptic seizures, autism and...
BACKGROUND
Mutations in the X-linked gene cyclin-dependent kinase-like 5 (CDKL5) cause a severe neurological disorder characterised by early-onset epileptic seizures, autism and intellectual disability (ID). Impaired hippocampal function has been implicated in other models of monogenic forms of autism spectrum disorders and ID and is often linked to epilepsy and behavioural abnormalities. Many individuals with CDKL5 deficiency disorder (CDD) have null mutations and complete loss of CDKL5 protein, therefore in the current study we used a Cdkl5 rat model to elucidate the impact of CDKL5 loss on cellular excitability and synaptic function of CA1 pyramidal cells (PCs). We hypothesised abnormal pre and/or post synaptic function and plasticity would be observed in the hippocampus of Cdkl5 rats.
METHODS
To allow cross-species comparisons of phenotypes associated with the loss of CDKL5, we generated a loss of function mutation in exon 8 of the rat Cdkl5 gene and assessed the impact of the loss of CDLK5 using a combination of extracellular and whole-cell electrophysiological recordings, biochemistry, and histology.
RESULTS
Our results indicate that CA1 hippocampal long-term potentiation (LTP) is enhanced in slices prepared from juvenile, but not adult, Cdkl5 rats. Enhanced LTP does not result from changes in NMDA receptor function or subunit expression as these remain unaltered throughout development. Furthermore, Ca permeable AMPA receptor mediated currents are unchanged in Cdkl5 rats. We observe reduced mEPSC frequency accompanied by increased spine density in basal dendrites of CA1 PCs, however we find no evidence supporting an increase in silent synapses when assessed using a minimal stimulation protocol in slices. Additionally, we found no change in paired-pulse ratio, consistent with normal release probability at Schaffer collateral to CA1 PC synapses.
CONCLUSIONS
Our data indicate a role for CDKL5 in hippocampal synaptic function and raise the possibility that altered intracellular signalling rather than synaptic deficits contribute to the altered plasticity.
LIMITATIONS
This study has focussed on the electrophysiological and anatomical properties of hippocampal CA1 PCs across early postnatal development. Studies involving other brain regions, older animals and behavioural phenotypes associated with the loss of CDKL5 are needed to understand the pathophysiology of CDD.
Topics: Animals; Long-Term Potentiation; Receptors, N-Methyl-D-Aspartate; Receptors, AMPA; Spasms, Infantile; Disease Models, Animal; Rats; Protein Serine-Threonine Kinases; Hippocampus; Pyramidal Cells; Male; CA1 Region, Hippocampal; Epileptic Syndromes; Genetic Diseases, X-Linked; Synapses; Excitatory Postsynaptic Potentials
PubMed: 38877552
DOI: 10.1186/s13229-024-00601-9 -
Frontiers in Neuroscience 2024Abnormal hippocampal neurodevelopment, particularly in the dentate gyrus region, may be a key mechanism of attention-deficit/hyperactivity disorder (ADHD). In this...
OBJECTIVES
Abnormal hippocampal neurodevelopment, particularly in the dentate gyrus region, may be a key mechanism of attention-deficit/hyperactivity disorder (ADHD). In this study, we investigate the effect of the most commonly used Chinese herb for the treatment of ADHD, Rehmanniae Radix Preparata (RRP), on behavior and hippocampal neurodevelopment in spontaneously hypertensive rats (SHR).
METHODS
Behavior tests, including Morris water maze (MWM) test, open field test (OFT) and elevated plus maze (EPM) test were performed to assess the effect of RRP on hyperactive and impulsive behavior. Hippocampal neurodevelopment was characterized by transmission electron microscopy, immunofluorescence, Golgi staining and Nissl staining approaches. Regulatory proteins such as Trkb, CDK5, FGF2/FGFR1 were examined by Western blot analysis.
RESULTS
The results showed that RRP could effectively control the impulsive and spontaneous behavior and improve the spatial learning and memory ability. RRP significantly reduced neuronal loss and increased the number of hippocampal stem cells, and promoted synaptic plasticity. In addition, FGF/FGFR signaling was upregulated after RRP treatment.
CONCLUSION
RRP can effectively reduce impulsive and spontaneous behavior and ameliorate hippocampal neurodevelopmental abnormalities in ADHD rat model.
PubMed: 38872946
DOI: 10.3389/fnins.2024.1402056 -
ENeuro Jun 2024Glutamatergic synapses exhibit significant molecular diversity but circuit-specific mechanisms that underlie synaptic regulation are not well characterized. Prior...
Glutamatergic synapses exhibit significant molecular diversity but circuit-specific mechanisms that underlie synaptic regulation are not well characterized. Prior reports show that RhoGEF Tiam1 regulates perforant path-dentate gyrus (DG) granule neuron synapses. In the present study, we report Tiam1's homolog Tiam2 is implicated in glutamatergic neurotransmission at CA1 pyramidal neurons. We find that Tiam2 regulates evoked excitatory glutamatergic currents via a post-synaptic mechanism mediated by the catalytic Dbl-homology domain. Overall, we present evidence for RhoGEF Tiam2's role in glutamatergic synapse function at Schaffer-collateral-CA1 pyramidal neuron synapses. Glutamatergic synapses are known to vary in composition and function but how this heterogeneity is established to create input-specific synaptic diversity is not well understood. In the present study we show Tiam2 regulates glutamatergic neurotransmission at Schaffer-collateral-CA1 pyramidal neuron synapses. We find that this function is dependent on its catalytic domain. By contrast we did not observe a role for Tiam2 in synaptic transmission at perforant path-DG granule neuron synapses. We also find that Tiam1 and Tiam2 are individually dispensable for functional synaptic plasticity in CA1 pyramidal neurons. To our knowledge, this is the first evidence of the RhoGEF Tiam2's role in regulating glutamatergic synapses.
PubMed: 38871458
DOI: 10.1523/ENEURO.0500-21.2024 -
ENeuro Jun 2024CRISPR/Cas9 gene editing represents an exciting avenue to study genes of unknown function, and can be combined with genetically-encoded tools such as fluorescent...
CRISPR/Cas9 gene editing represents an exciting avenue to study genes of unknown function, and can be combined with genetically-encoded tools such as fluorescent proteins, channelrhodopsins, DREADDs, and various biosensors to more deeply probe the function of these genes in different cell types. However, current strategies to also manipulate or visualize edited cells are challenging due to the large size of Cas9 proteins and the limited packaging capacity of adeno-associated viruses (AAVs). To overcome these constraints, we developed an alternative gene editing strategy using a single AAV vector and mouse lines that express Cre-dependent Cas9 to achieve efficient cell-type specific editing across the nervous system. Expressing Cre-dependent Cas9 from a genomic locus affords space to package guide RNAs for gene editing together with Cre-dependent, genetically encoded tools to manipulate, map, or monitor neurons using a single virus.We validated this strategy with three common tools in neuroscience: ChRonos, a channelrhodopsin, for studying synaptic transmission using optogenetics; GCaMP8f for recording Ca2+ transients using photometry, and mCherry for tracing axonal projections. We tested these tools in multiple brain regions and cell types, including GABAergic neurons in the nucleus accumbens, glutamatergic neurons projecting from the ventral pallidum to the lateral habenula, dopaminergic neurons in the ventral tegmental area, and proprioceptive neurons in the periphery. This flexible approach could help identify and test the function of novel genes affecting synaptic transmission, circuit activity, or morphology with a single viral injection. Our CRISPR/Cas9 approach is the first to use a single vector to both knock-down genes of interest and express tools to monitor, map, and manipulate neurons. We demonstrate its utility in the central nervous system and describe the first systemic CRISPR/Cas9 gene editing with co-expressed reporters in the peripheral nervous system. Our approach fills a significant gap in the neuronal gene editing toolkit, allowing high-throughput study of genes of unknown function in the nervous system, and has broad utility for loss-of-function studies in other biological fields. This tool has great translational potential: it can be used to screen risk factor genes identified through genome-wide association studies, or knock-down native gene expression and reintroduce mutant variants identified in clinical settings.
PubMed: 38871457
DOI: 10.1523/ENEURO.0438-23.2024 -
Biological Research Jun 2024Spreading depression (SD) is an intriguing phenomenon characterized by massive slow brain depolarizations that affect neurons and glial cells. This phenomenon is...
BACKGROUND
Spreading depression (SD) is an intriguing phenomenon characterized by massive slow brain depolarizations that affect neurons and glial cells. This phenomenon is repetitive and produces a metabolic overload that increases secondary damage. However, the mechanisms associated with the initiation and propagation of SD are unknown. Multiple lines of evidence indicate that persistent and uncontrolled opening of hemichannels could participate in the pathogenesis and progression of several neurological disorders including acute brain injuries. Here, we explored the contribution of astroglial hemichannels composed of connexin-43 (Cx43) or pannexin-1 (Panx1) to SD evoked by high-K stimulation in brain slices.
RESULTS
Focal high-K stimulation rapidly evoked a wave of SD linked to increased activity of the Cx43 and Panx1 hemichannels in the brain cortex, as measured by light transmittance and dye uptake analysis, respectively. The activation of these channels occurs mainly in astrocytes but also in neurons. More importantly, the inhibition of both the Cx43 and Panx1 hemichannels completely prevented high K-induced SD in the brain cortex. Electrophysiological recordings also revealed that Cx43 and Panx1 hemichannels critically contribute to the SD-induced decrease in synaptic transmission in the brain cortex and hippocampus.
CONCLUSIONS
Targeting Cx43 and Panx1 hemichannels could serve as a new therapeutic strategy to prevent the initiation and propagation of SD in several acute brain injuries.
Topics: Animals; Astrocytes; Connexins; Cortical Spreading Depression; Synaptic Transmission; Connexin 43; Male; Nerve Tissue Proteins; Cerebral Cortex; Neurons; Hippocampus; Rats, Sprague-Dawley; Rats; Potassium
PubMed: 38867288
DOI: 10.1186/s40659-024-00519-9