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Cell Reports May 2024Recent findings show that effective integration of novel information in the brain requires coordinated processes of homo- and heterosynaptic plasticity. In this work, we...
Recent findings show that effective integration of novel information in the brain requires coordinated processes of homo- and heterosynaptic plasticity. In this work, we hypothesize that activity-dependent remodeling of the peri-synaptic extracellular matrix (ECM) contributes to these processes. We show that clusters of the peri-synaptic ECM, recognized by CS56 antibody, emerge in response to sensory stimuli, showing temporal and spatial coincidence with dendritic spine plasticity. Using CS56 co-immunoprecipitation of synaptosomal proteins, we identify several molecules involved in Ca signaling, vesicle cycling, and AMPA-receptor exocytosis, thus suggesting a role in long-term potentiation (LTP). Finally, we show that, in the CA1 hippocampal region, the attenuation of CS56 glycoepitopes, through the depletion of versican as one of its main carriers, impairs LTP and object location memory in mice. These findings show that activity-dependent remodeling of the peri-synaptic ECM regulates the induction and consolidation of LTP, contributing to hippocampal-dependent memory.
Topics: Animals; Extracellular Matrix; Long-Term Potentiation; Mice; Neuronal Plasticity; Memory; Synapses; Mice, Inbred C57BL; Male; CA1 Region, Hippocampal; Hippocampus
PubMed: 38676925
DOI: 10.1016/j.celrep.2024.114112 -
Genes Apr 2024Down syndrome (DS) is the most common form of inherited intellectual disability caused by trisomy of chromosome 21, presenting with intellectual impairment, craniofacial...
Down syndrome (DS) is the most common form of inherited intellectual disability caused by trisomy of chromosome 21, presenting with intellectual impairment, craniofacial abnormalities, cardiac defects, and gastrointestinal disorders. The Ts65Dn mouse model replicates many abnormalities of DS. We hypothesized that investigation of the cerebral cortex of fluoxetine-treated trisomic mice may provide proteomic signatures that identify therapeutic targets for DS. Subcellular fractionation of synaptosomes from cerebral cortices of age- and brain-area-matched samples from fluoxetine-treated vs. water-treated trisomic and euploid male mice were subjected to HPLC-tandem mass spectrometry. Analysis of the data revealed enrichment of trisomic risk genes that participate in regulation of synaptic vesicular traffic, pre-synaptic and post-synaptic development, and mitochondrial energy pathways during early brain development. Proteomic analysis of trisomic synaptic fractions revealed significant downregulation of proteins involved in synaptic vesicular traffic, including vesicular endocytosis (CLTA, CLTB, CLTC), synaptic assembly and maturation (EXOC1, EXOC3, EXOC8), anterograde axonal transport (EXOC1), neurotransmitter transport to PSD (SACM1L), endosomal-lysosomal acidification (ROGDI, DMXL2), and synaptic signaling (NRXN1, HIP1, ITSN1, YWHAG). Additionally, trisomic proteomes revealed upregulation of several trafficking proteins, involved in vesicular exocytosis (Rab5B), synapse elimination (UBE3A), scission of endocytosis (DBN1), transport of ER in dendritic spines (MYO5A), presynaptic activity-dependent bulk endocytosis (FMR1), and NMDA receptor activity (GRIN2A). Chronic fluoxetine treatment of Ts65Dn mice rescued synaptic vesicular abnormalities and prevented abnormal proteomic changes in adult Ts65Dn mice, pointing to therapeutic targets for potential treatment of DS.
Topics: Animals; Fluoxetine; Mice; Down Syndrome; Male; Proteomics; Synaptic Vesicles; Disease Models, Animal; Proteome; Cerebral Cortex; Synaptosomes; Trisomy
PubMed: 38674386
DOI: 10.3390/genes15040452 -
Brain Communications 2024We have previously characterized the molecular mechanisms for variants in γ-aminobutyric acid transporter 1-encoding solute carrier family 6-member 1 () and concluded...
We have previously characterized the molecular mechanisms for variants in γ-aminobutyric acid transporter 1-encoding solute carrier family 6-member 1 () and concluded that a partial or complete loss of γ-aminobutyric acid uptake due to impaired protein trafficking is the primary aetiology. Impairment of γ-aminobutyric acid transporter 1 function could cause compensatory changes in the expression of γ-aminobutyric acid receptors, which, in turn, modify disease pathophysiology and phenotype. Here we used different approaches including radioactive H γ-aminobutyric acid uptake in cells and synaptosomes, immunohistochemistry and confocal microscopy as well as brain slice surface protein biotinylation to characterize and mice, representative of a partial or a complete loss of function of mutations, respectively. We employed the γ-aminobutyric acid transporter 1-specific inhibitor [H]tiagabine binding and GABA receptor subunit-specific radioligand binding to profile the γ-aminobutyric acid transporter 1 and GABA receptor expression in major brain regions such as cortex, cerebellum, hippocampus and thalamus. We also determined the total and surface expression of γ-aminobutyric acid transporter 1, γ-aminobutyric acid transporter 3 and expression of GABA receptor in the major brain regions in the knockin mice. We found that γ-aminobutyric acid transporter 1 protein was markedly reduced in cortex, hippocampus, thalamus and cerebellum in both mutant mouse lines. Consistent with the findings of reduced γ-aminobutyric acid uptake for both γ-aminobutyric acid transporter 1(A288V) and γ-aminobutyric acid transporter 1(S295L), both the total and the γ-aminobutyric acid transporter 1-mediated H γ-aminobutyric acid reuptake was reduced. We found that γ-aminobutyric acid transporter 3 is only abundantly expressed in the thalamus and there was no compensatory increase of γ-aminobutyric acid transporter 3 in either of the mutant mouse lines. γ-Aminobutyric acid transporter 1 was reduced in both somatic regions and nonsomatic regions in both mouse models, in which a ring-like structure was identified only in the mouse, suggesting more γ-aminobutyric acid transporter 1 retention inside endoplasmic reticulum in the mouse. The [H]tiagabine binding was similar in both mouse models despite the difference in γ-aminobutyric acid uptake function and γ-aminobutyric acid transporter 1 protein expression for both mutations. There were no differences in GABA receptor subtype expression, except for a small increase in the expression of α5 subunits of GABA receptor in the hippocampus of homozygous mice, suggesting a potential interaction between the expression of this GABA receptor subtype and the mutant γ-aminobutyric acid transporter 1. The study provides the first comprehensive characterization of the mutations in two representative mouse models. Because both γ-aminobutyric acid transporter 1 and GABA receptors are targets for anti-seizure medications, the findings from this study can help guide tailored treatment options based on the expression and function of γ-aminobutyric acid transporter 1 and GABA receptor in mutation-mediated neurodevelopmental and epileptic encephalopathies.
PubMed: 38650830
DOI: 10.1093/braincomms/fcae110 -
Toxicology in Vitro : An International... Jun 2024The action of calix[4]arenes C-424, C-425 and C-1193 has been investigated on suspended cholesterol/egg phosphatidylcholine lipid bilayer in a voltage-clamp mode....
Substitution of bridge carbons for sulphur in calix[4]arene-bis-α-hydroxymethylphosphonic acid transformed mobile carrier into ionic channel accompanied with evoked muscle contraction and impaired neurotransmission powered by membrane action of resulting...
The action of calix[4]arenes C-424, C-425 and C-1193 has been investigated on suspended cholesterol/egg phosphatidylcholine lipid bilayer in a voltage-clamp mode. Comparative analysis with the membrane action by calix[4]arene-bis-α-hydroxymethylphosphonic acid (C-99) has shown that the substitution of bridge carbons for sulphur and addition of another methyl group to two alkyl tales in the lower rim of former dipropoxycalix[4]arene C-99 transformed mobile carrier that C-99 created in lipid bilayer (Shatursky et al., 2014) into a transmembrane pore as exposure of the bilayer membrane to sulphur-containing derivative dibutoxythiocalix[4]arene C-1193 resulted in microscopic transmembrane current patterns indicative of a channel-like mode of facilitated diffusion. Within all calix[4]arenes tested a net steady-state voltage-dependent transmembrane current was readily achieved only after addition of calix[4]-arene C-1193. In comparison with the membrane action of C-99 the current induced by calix[4]-arene C-1193 exhibited a much weakened anion selectivity passing slightly more current at positive potentials applied from the side of bilayer membrane to which the calix[4]-arene was added. Testing C-1193 for the membrane action against smooth muscle cells of rat uterus or swine myometrium and synaptosomes of rat brain nerve terminals revealed an increase in intracellular concentration of Ca with reduction of the effective hydrodynamic diameter of the smooth muscle cells and enhanced basal extracellular level of neurotransmitters (glutamate and γ-aminobutyric acid) after C-1193-induced depolarization of the nerve terminals.
Topics: Animals; Calixarenes; Synaptic Transmission; Lipid Bilayers; Muscle Contraction; Ion Channels; Sulfur; Rats; Female; Organophosphonates; Male; Phenols; Rats, Wistar
PubMed: 38636607
DOI: 10.1016/j.tiv.2024.105815 -
The Journal of Physiology May 2024Hypertension is a major adverse effect of calcineurin inhibitors, such as tacrolimus (FK506) and cyclosporine, used clinically as immunosuppressants. Calcineurin...
Hypertension is a major adverse effect of calcineurin inhibitors, such as tacrolimus (FK506) and cyclosporine, used clinically as immunosuppressants. Calcineurin inhibitor-induced hypertension (CIH) is linked to augmented sympathetic output from the hypothalamic paraventricular nucleus (PVN). GluA2-lacking, Ca-permeable AMPA receptors (CP-AMPARs) are a key feature of glutamatergic synaptic plasticity, yet their role in CIH remains elusive. Here, we found that systemic administration of FK506 in rats significantly increased serine phosphorylation of GluA1 and GluA2 in PVN synaptosomes. Strikingly, FK506 treatment reduced GluA1/GluA2 heteromers in both synaptosomes and endoplasmic reticulum-enriched fractions from the PVN. Blocking CP-AMPARs with IEM-1460 induced a larger reduction of AMPAR-mediated excitatory postsynaptic current (AMPAR-EPSC) amplitudes in retrogradely labelled, spinally projecting PVN neurons in FK506-treated rats than in vehicle-treated rats. Furthermore, FK506 treatment shifted the current-voltage relationship of AMPAR-EPSCs from linear to inward rectification in labelled PVN neurons. FK506 treatment profoundly enhanced physical interactions of α2δ-1 with GluA1 and GluA2 in the PVN. Inhibiting α2δ-1 with gabapentin, α2δ-1 genetic knockout, or disrupting α2δ-1-AMPAR interactions with an α2δ-1 C terminus peptide restored GluA1/GluA2 heteromers in the PVN and diminished inward rectification of AMPAR-EPSCs in labelled PVN neurons induced by FK506 treatment. Additionally, microinjection of IEM-1460 or α2δ-1 C terminus peptide into the PVN reduced renal sympathetic nerve discharges and arterial blood pressure elevated in FK506-treated rats but not in vehicle-treated rats. Thus, calcineurin in the hypothalamus constitutively regulates AMPAR subunit composition and phenotypes by controlling GluA1/GluA2 interactions with α2δ-1. Synaptic CP-AMPARs in PVN presympathetic neurons contribute to augmented sympathetic outflow in CIH. KEY POINTS: Systemic treatment with the calcineurin inhibitor increases serine phosphorylation of synaptic GluA1 and GluA2 in the PVN. Calcineurin inhibition enhances the prevalence of postsynaptic Ca-permeable AMPARs in PVN presympathetic neurons. Calcineurin inhibition potentiates α2δ-1 interactions with GluA1 and GluA2, disrupting intracellular assembly of GluA1/GluA2 heterotetramers in the PVN. Blocking Ca-permeable AMPARs or α2δ-1-AMPAR interactions in the PVN attenuates sympathetic outflow augmented by the calcineurin inhibitor.
Topics: Animals; Receptors, AMPA; Calcineurin; Male; Tacrolimus; Rats; Neurons; Rats, Sprague-Dawley; Paraventricular Hypothalamic Nucleus; Calcium; Excitatory Postsynaptic Potentials; Calcineurin Inhibitors; Synapses
PubMed: 38630836
DOI: 10.1113/JP286081 -
Methods in Molecular Biology (Clifton,... 2024Postsynaptic density (PSD) is a morphologically and functionally specialized postsynaptic membrane structure of excitatory synapses. It contains hundreds of proteins...
Postsynaptic density (PSD) is a morphologically and functionally specialized postsynaptic membrane structure of excitatory synapses. It contains hundreds of proteins such as neurotransmitter receptors, adhesion molecules, cytoskeletal proteins, and signaling enzymes. The study of the molecular architecture of the PSD is one of the most intriguing issues in neuroscience research. The isolation of the PSD from the brain of an animal is necessary for subsequent biochemical and morphological analyses. Many laboratories have developed methods to isolate PSD from the animal brain. In this chapter, we present a simple method to isolate PSD from the mouse brain using sucrose density gradient-based purification of synaptosomes followed by detergent extraction.
Topics: Animals; Mice; Post-Synaptic Density; Synaptic Membranes; Brain; Cytoskeletal Proteins; Laboratories
PubMed: 38630221
DOI: 10.1007/978-1-0716-3810-1_7 -
British Journal of Pharmacology Apr 2024An estimated 40% of patients with erectile dysfunction have a poor prognosis for improvement with currently available treatments. The present study investigated whether...
BACKGROUND AND PURPOSE
An estimated 40% of patients with erectile dysfunction have a poor prognosis for improvement with currently available treatments. The present study investigated whether a newly developed monoamine transport inhibitor, IP2015, improves erectile function.
EXPERIMENTAL APPROACH
We investigated the effects of IP2015 on monoamine uptake and binding, erectile function in rats and diabetic mice and the effect on corpus cavernosum contractility.
KEY RESULTS
IP2015 inhibited the uptake of 5-HT, noradrenaline and dopamine by human monoamine transporters expressed in cells and in rat brain synaptosomes. Intracavernosal pressure measurement in anaesthetized rats revealed that IP2015 dose-dependently increased the number and the duration of spontaneous erections. Whereas pretreatment with the dopamine D-like receptor antagonists, clozapine and (-)-sulpiride, or cutting the cavernosal nerve inhibited IP2015-induced erectile responses, the phosphodiesterase type 5 inhibitor sildenafil further enhanced the IP2015-mediated increase in intracavernosal pressure. IP2015 also increased the number of erections in type 2 diabetic db/db mice. Direct intracavernosal injection of IP2015 increased penile pressure, and in corpus cavernosum strips, IP2015 induced concentration-dependent relaxations. These relaxations were enhanced by sildenafil and blunted by endothelial cell removal, a nitric oxide synthase inhibitor, N-nitro-l-arginine and a D-like receptor antagonist, SCH23390. Quantitative polymerase chain reaction (qPCR) showed the expression of the dopamine transporter in the rat corpus cavernosum.
CONCLUSION AND IMPLICATIONS
Our findings suggest that IP2015 stimulates erectile function by a central mechanism involving dopamine reuptake inhibition and direct NO-mediated relaxation of the erectile tissue. This novel multi-modal mechanism of action could offer a new treatment approach to erectile dysfunction.
PubMed: 38604613
DOI: 10.1111/bph.16362 -
Experimental Neurology Jun 2024Sleep-related hypermotor epilepsy (SHE) is a focal epilepsy syndrome characterized by seizures that predominantly occur during sleep. The pathogenesis of these seizures...
OBJECTIVE
Sleep-related hypermotor epilepsy (SHE) is a focal epilepsy syndrome characterized by seizures that predominantly occur during sleep. The pathogenesis of these seizures remains unclear. We previously detected rare variants in GABRG2, which encodes the γ subunit of γ-aminobutyric acid type A receptor (GABAR), in patients with SHE and demonstrated that these variants impaired GABAR function in vitro. However, the mechanisms by which GABRG2 variants contribute to seizure attacks during sleep remain unclear.
METHODS
In this study, we designed a knock-in (KI) mouse expressing the mouse Gabrg2 T316N variant, corresponding to human GABRG2 T317N variant, using CRISPR/Cas9. Continuous video-electroencephalogram monitoring and in vivo multichannel electrophysiological recordings were performed to explore seizure susceptibility to pentylenetetrazol (PTZ), alterations in the sleep-wake cycle, spontaneous seizure patterns, and synchronized activity in the motor thalamic nuclei (MoTN) and secondary motor cortex (M2). Circadian variations in the expression of total, membrane-bound, and synaptic GABAR subunits were also investigated.
RESULTS
No obvious changes in gross morphology were detected in Gabrg2 mice compared to their wild-type (Gabrg2) littermates. Gabrg2 mice share key phenotypes with patients, including sleep fragmentation and spontaneous seizures during sleep. Gabrg2 mice showed increased susceptibility to PTZ-induced seizures and higher mortality after seizures. Synchronization of the local field potentials between the MoTN and M2 was abnormally enhanced in Gabrg2 mice during light phase, when sleep dominates, accompanied by increased local activities in the MoTN and M2. Interestingly, in Gabrg2 mice, GABAR γ2 subunits showed a circadian increase on the neuronal membrane and synaptosomes in the transition from dark phase to light phase, which was absent in Gabrg2 mice.
CONCLUSION
We generated a new SHE mouse model and provided in vivo evidence that rare variants of GABRG2 contribute to seizure attacks during sleep in SHE.
Topics: Animals; Female; Male; Mice; Cerebral Cortex; Electroencephalography; Epilepsy; Gene Knock-In Techniques; Mice, Inbred C57BL; Mice, Transgenic; Phenotype; Receptors, GABA-A; Sleep; Thalamus
PubMed: 38604438
DOI: 10.1016/j.expneurol.2024.114775 -
Stem Cell Research & Therapy Apr 2024Microglia, the brain's resident immune cells, play vital roles in brain development, and disorders like Alzheimer's disease (AD). Human iPSC-derived microglia (iMG)...
BACKGROUND
Microglia, the brain's resident immune cells, play vital roles in brain development, and disorders like Alzheimer's disease (AD). Human iPSC-derived microglia (iMG) provide a promising model to study these processes. However, existing iMG generation protocols face challenges, such as prolonged differentiation time, lack of detailed characterization, and limited gene function investigation via CRISPR-Cas9.
METHODS
Our integrated toolkit for in-vitro microglia functional genomics optimizes iPSC differentiation into iMG through a streamlined two-step, 20-day process, producing iMG with a normal karyotype. We confirmed the iMG's authenticity and quality through single-cell RNA sequencing, chromatin accessibility profiles (ATAC-Seq), proteomics and functional tests. The toolkit also incorporates a drug-dependent CRISPR-ON/OFF system for temporally controlled gene expression. Further, we facilitate the use of multi-omic data by providing online searchable platform that compares new iMG profiles to human primary microglia: https://sherlab.shinyapps.io/IPSC-derived-Microglia/ .
RESULTS
Our method generates iMG that closely align with human primary microglia in terms of transcriptomic, proteomic, and chromatin accessibility profiles. Functionally, these iMG exhibit Ca2 + transients, cytokine driven migration, immune responses to inflammatory signals, and active phagocytosis of CNS related substrates including synaptosomes, amyloid beta and myelin. Significantly, the toolkit facilitates repeated iMG harvesting, essential for large-scale experiments like CRISPR-Cas9 screens. The standalone ATAC-Seq profiles of our iMG closely resemble primary microglia, positioning them as ideal tools to study AD-associated single nucleotide variants (SNV) especially in the genome regulatory regions.
CONCLUSIONS
Our advanced two-step protocol rapidly and efficiently produces authentic iMG. With features like the CRISPR-ON/OFF system and a comprehensive multi-omic data platform, our toolkit equips researchers for robust microglial functional genomic studies. By facilitating detailed SNV investigation and offering a sustainable cell harvest mechanism, the toolkit heralds significant progress in neurodegenerative disease drug research and therapeutic advancement.
Topics: Humans; Microglia; Proteomics; Amyloid beta-Peptides; Neurodegenerative Diseases; Genomics; Alzheimer Disease; Chromatin
PubMed: 38600587
DOI: 10.1186/s13287-024-03700-9 -
Aging Brain 2024Defective brain glucose utilization is a hallmark of Alzheimer's disease (AD) while Type II diabetes and elevated blood glucose escalate the risk for AD in later life....
Defective brain glucose utilization is a hallmark of Alzheimer's disease (AD) while Type II diabetes and elevated blood glucose escalate the risk for AD in later life. Isolating contributions of normal aging from coincident metabolic or brain diseases could lead to refined approaches to manage specific health risks and optimize treatments targeted to susceptible older individuals. We evaluated metabolic, neuroendocrine, and neurobiological differences between young adult (6 months) and aged (24 months) male rats. Compared to young adults, blood glucose was significantly greater in aged rats at the start of the dark phase of the day but not during the light phase. When challenged with physical restraint, a potent stressor, aged rats effected no change in blood glucose whereas blood glucose increased in young adults. Tissues were evaluated for markers of oxidative phosphorylation (OXPHOS), neuronal glucose transport, and synapses. Outright differences in protein levels between age groups were not evident, but circadian blood glucose was inversely related to OXPHOS proteins in hippocampal synaptosomes, independent of age. The neuronal glucose transporter, GLUT3, was positively associated with circadian blood glucose in young adults whereas aged rats tended to show the opposite trend. Our data demonstrate aging increases daily fluctuations in blood glucose and, at the level of individual differences, negatively associates with proteins related to synaptic OXPHOS. Our findings imply that glucose dyshomeostasis may exacerbate metabolic aspects of synaptic dysfunction that contribute to risk for age-related brain disorders.
PubMed: 38596458
DOI: 10.1016/j.nbas.2024.100116