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Biochimica Et Biophysica Acta.... Jun 2024The regulation of protein degradation through the ubiquitin-proteasome system is essential for normal brain development, axon growth, synaptic growth and plasticity. The...
The regulation of protein degradation through the ubiquitin-proteasome system is essential for normal brain development, axon growth, synaptic growth and plasticity. The E3 ubiquitin ligase RFWD2 plays a key role in the onset and development of neurological diseases, including the pathogenesis of Alzheimer's disease (AD), but the mechanisms controlling the homeostasis of neuronal synaptic proteins are still poorly understood. Here, we showed that the expression level of RFWD2 gradually decreased with the age of the rats and was negatively correlated with the development of cerebral cortical neurons and dendrites in vivo. RFWD2 was shown to localize to presynaptic terminals and some postsynaptic sides of both excitatory synapses and inhibitory synapses via colocalization with neuronal synaptic proteins (SYN, PSD95, Vglut1 and GAD67). Overexpression of RFWD2 promoted dendrite development and dendritic spine formation and markedly decreased the expression of synaptophysin and PSD95 by reducing the expression of ETV1, ETV4, ETV5 and c-JUN in vitro. Furthermore, the whole-cell membrane slice clamp results showed that RFWD2 overexpression resulted in greater membrane capacitance in neuronal cells, inadequate cell repolarization, and a longer time course for neurons to emit action potentials with decreased excitability. RFWD2 regulates dendritic development and plasticity, dendritic spine formation and synaptic function in rat cerebral cortex neurons by activating the ERK/PEA3/c-Jun pathway via a posttranslational regulatory mechanism and can be used as an efficient treatment target for neurological diseases.
PubMed: 38909848
DOI: 10.1016/j.bbadis.2024.167319 -
Scientific Reports Jun 2024Nicotinic acetylcholine receptors (nAChRs) in the medial habenula (MHb)-interpeduncular nucleus (IPN) pathway play critical roles in nicotine-related behaviors. This...
Nicotinic acetylcholine receptors (nAChRs) in the medial habenula (MHb)-interpeduncular nucleus (IPN) pathway play critical roles in nicotine-related behaviors. This pathway is particularly enriched in nAChR α3 and β4 subunits, both of which are genetically linked to nicotine dependence. However, the cellular and subcellular expression of endogenous α3β4-containing nAChRs remains largely unknown because specific antibodies and appropriate detection methods were unavailable. Here, we successfully uncovered the expression of endogenous nAChRs containing α3 and β4 subunits in the MHb-IPN pathway using novel specific antibodies and a fixative glyoxal that enables simultaneous detection of synaptic and extrasynaptic molecules. Immunofluorescence and immunoelectron microscopy revealed that both subunits were predominantly localized to the extrasynaptic cell surface of somatodendritic and axonal compartments of MHb neurons but not at their synaptic junctions. Immunolabeling for α3 and β4 subunits disappeared in α5β4-knockout brains, which we used as negative controls. The enriched and diffuse extrasynaptic expression along the MHb-IPN pathway suggests that α3β4-containing nAChRs may enhance the excitability of MHb neurons and neurotransmitter release from their presynaptic terminals in the IPN. The revealed distribution pattern provides a molecular and anatomical basis for understanding the functional role of α3β4-containing nAChRs in the crucial pathway of nicotine dependence.
Topics: Animals; Receptors, Nicotinic; Habenula; Interpeduncular Nucleus; Mice; Mice, Knockout; Neurons; Synapses; Mice, Inbred C57BL; Male
PubMed: 38902419
DOI: 10.1038/s41598-024-65076-3 -
Cells Jun 2024Presynaptic Ca influx through voltage-gated Ca channels (VGCCs) is a key signal for synaptic vesicle release. Synaptic neurexins can partially determine the strength of...
Presynaptic Ca influx through voltage-gated Ca channels (VGCCs) is a key signal for synaptic vesicle release. Synaptic neurexins can partially determine the strength of transmission by regulating VGCCs. However, it is unknown whether neurexins modulate Ca influx via all VGCC subtypes similarly. Here, we performed live cell imaging of synaptic boutons from primary hippocampal neurons with a Ca indicator. We used the expression of inactive and active Cre recombinase to compare control to conditional knockout neurons lacking either all or selected neurexin variants. We found that reduced total presynaptic Ca transients caused by the deletion of all neurexins were primarily due to the reduced contribution of P/Q-type VGCCs. The deletion of neurexin1α alone also reduced the total presynaptic Ca influx but increased Ca influx via N-type VGCCs. Moreover, we tested whether the decrease in Ca influx induced by activation of cannabinoid receptor 1 (CB1-receptor) is modulated by neurexins. Unlike earlier observations emphasizing a role for β-neurexins, we found that the decrease in presynaptic Ca transients induced by CB1-receptor activation depended more strongly on the presence of α-neurexins in hippocampal neurons. Together, our results suggest that neurexins have unique roles in the modulation of presynaptic Ca influx through VGCC subtypes and that different neurexin variants may affect specific VGCCs.
Topics: Animals; Calcium; Presynaptic Terminals; Hippocampus; Mice; Mice, Knockout; Calcium Channels; Neurons; Receptor, Cannabinoid, CB1; Calcium Signaling; Gene Knockout Techniques; Neurexins
PubMed: 38891114
DOI: 10.3390/cells13110981 -
Proceedings of the National Academy of... Jun 2024Synapses containing γ-aminobutyric acid (GABA) constitute the primary centers for inhibitory neurotransmission in our nervous system. It is unclear how these synaptic...
Synapses containing γ-aminobutyric acid (GABA) constitute the primary centers for inhibitory neurotransmission in our nervous system. It is unclear how these synaptic structures form and align their postsynaptic machineries with presynaptic terminals. Here, we monitored the cellular distribution of several GABAergic postsynaptic proteins in a purely glutamatergic neuronal culture derived from human stem cells, which virtually lacks any vesicular GABA release. We found that several GABA receptor (GABAR) subunits, postsynaptic scaffolds, and major cell-adhesion molecules can reliably coaggregate and colocalize at even GABA-deficient subsynaptic domains, but remain physically segregated from glutamatergic counterparts. Genetic deletions of both Gephyrin and a Gephyrin-associated guanosine di- or triphosphate (GDP/GTP) exchange factor Collybistin severely disrupted the coassembly of these postsynaptic compositions and their proper apposition with presynaptic inputs. Gephyrin-GABAR clusters, developed in the absence of GABA transmission, could be subsequently activated and even potentiated by delayed supply of vesicular GABA. Thus, molecular organization of GABAergic postsynapses can initiate via a GABA-independent but Gephyrin-dependent intrinsic mechanism.
Topics: Humans; Membrane Proteins; gamma-Aminobutyric Acid; Receptors, GABA-A; Carrier Proteins; Presynaptic Terminals; Synapses; Synaptic Transmission; Rho Guanine Nucleotide Exchange Factors
PubMed: 38889143
DOI: 10.1073/pnas.2315100121 -
Neuroscience Jun 2024Entorhinal cortex (EC) LIII and LII glutamatergic neurons make monosynaptic connections onto distal apical dendrites of CA1 and CA2 pyramidal neurons (PNs) in the...
Entorhinal cortex (EC) LIII and LII glutamatergic neurons make monosynaptic connections onto distal apical dendrites of CA1 and CA2 pyramidal neurons (PNs) in the hippocampus, respectively, through perforant path (PP) projections. We previously reported that a brief train of PP stimuli evokes strong supralinear temporal summation of excitatory postsynaptic response (EPSP) in CA1 PNs that requires NMDAR activation, with relatively little summation in CA2 PNs in mice of either sex. Here we provide evidence from combined immunogold electron microscopy, cell-type specific genetic deletion and pharmacology that the NMDARs required for supralinear temporal summation of the CA1 PP EPSP are presynaptic, located in the PP terminals. Moreover, we found that the number of NMDARs in PP terminals innervating CA1 PNs is significantly greater than that found in PP terminals innervating CA2 PNs, providing a potential explanation for the difference in temporal summation in these two classes of hippocampal PNs.
PubMed: 38878815
DOI: 10.1016/j.neuroscience.2024.06.005 -
Cold Spring Harbor Protocols Jun 2024The neuromuscular junction (NMJ) is an excellent model for studying vertebrate glutamatergic synapses. Researchers have uncovered fundamental mechanisms at the fly NMJ...
The neuromuscular junction (NMJ) is an excellent model for studying vertebrate glutamatergic synapses. Researchers have uncovered fundamental mechanisms at the fly NMJ that are conserved in higher-order organisms. To gain molecular and structural insight into these and other structures, immunolabeling is invaluable. In this protocol, we describe how to use immunolabeling to visualize embryonic/larval presynaptic and postsynaptic structures at the NMJ. We also include details about amplification of weak immunohistochemistry signals and how to use these signals to quantify synaptic growth via bouton counting. Boutons are bead-like structures at motor axon terminals that house synapses, and the number of boutons reflects the size of the NMJ. We also describe how to identify the different bouton types.
PubMed: 38866543
DOI: 10.1101/pdb.prot108500 -
ENeuro Jun 2024Synapsins are highly abundant presynaptic proteins that play a crucial role in neurotransmission and plasticity via the clustering of synaptic vesicles. The synapsin III...
Synapsins are highly abundant presynaptic proteins that play a crucial role in neurotransmission and plasticity via the clustering of synaptic vesicles. The synapsin III isoform is usually downregulated after development, but in hippocampal mossy fiber boutons it persists in adulthood. Mossy fiber boutons express presynaptic forms of short- and long-term plasticity, which are thought to underlie different forms of learning. Previous research on synapsins at this synapse focused on synapsin isoforms I and II. Thus, a complete picture regarding the role of synapsins in mossy fiber plasticity is still missing. Here, we investigated presynaptic plasticity at hippocampal mossy fiber boutons by combining electrophysiological field recordings and transmission electron microscopy in a mouse model lacking all synapsin isoforms. We found decreased short-term plasticity - i.e. decreased facilitation and post-tetanic potentiation - but increased long-term potentiation in male synapsin triple knockout mice. At the ultrastructural level, we observed more dispersed vesicles and a higher density of active zones in mossy fiber boutons from knockout animals. Our results indicate that all synapsin isoforms are required for fine regulation of short- and long-term presynaptic plasticity at the mossy fiber synapse. Synapsins cluster vesicles at presynaptic terminals and shape presynaptic plasticity at giant hippocampal mossy fiber boutons. Deletion of all synapsin isoforms results in decreased short- but increased long-term plasticity.
PubMed: 38866497
DOI: 10.1523/ENEURO.0330-23.2024 -
FASEB Journal : Official Publication of... Jun 2024Subclinical hypothyroidism (SCH) in pregnancy is the most common form of thyroid dysfunction in pregnancy, which can affect fetal nervous system development and increase...
Subclinical hypothyroidism (SCH) in pregnancy is the most common form of thyroid dysfunction in pregnancy, which can affect fetal nervous system development and increase the risk of neurodevelopmental disorders after birth. However, the mechanism of the effect of maternal subclinical hypothyroidism on fetal brain development and behavioral phenotypes is still unclear and requires further study. In this study, we constructed a mouse model of maternal subclinical hypothyroidism by exposing dams to drinking water containing 50 ppm propylthiouracil (PTU) during pregnancy and found that its offspring were accompanied by severe cognitive deficits by behavioral testing. Mechanistically, gestational SCH resulted in the upregulation of protein expression and activity of HDAC1/2/3 in the hippocampus of the offspring. ChIP analysis revealed that H3K9ac on the neurogranin (Ng) promoter was reduced in the hippocampus of the offspring of SCH, with a significant reduction in Ng protein, leading to reduced expression levels of synaptic plasticity markers PSD95 (a membrane-associated protein in the postsynaptic density) and SYN (synaptophysin, a specific marker for presynaptic terminals), and impaired synaptic plasticity. In addition, administration of MS-275 (an HDAC1/2/3-specific inhibitor) to SCH offspring alleviated impaired synaptic plasticity and cognitive dysfunction in offspring. Thus, our study suggests that maternal subclinical hypothyroidism may mediate offspring cognitive dysfunction through the HDAC1/2/3-H3K9ac-Ng pathway. Our study contributes to the understanding of the signaling mechanisms underlying maternal subclinical hypothyroidism-mediated cognitive impairment in the offspring.
Topics: Animals; Neurogranin; Hypothyroidism; Female; Pregnancy; Mice; Cognitive Dysfunction; Histone Deacetylase 2; Prenatal Exposure Delayed Effects; Histone Deacetylase 1; Down-Regulation; Hippocampus; Male; Histone Deacetylases; Mice, Inbred C57BL; Neuronal Plasticity
PubMed: 38865202
DOI: 10.1096/fj.202400389R -
Learning & Memory (Cold Spring Harbor,... May 2024The intricate molecular and structural sequences guiding the formation and consolidation of memories within neuronal circuits remain largely elusive. In this study, we...
The intricate molecular and structural sequences guiding the formation and consolidation of memories within neuronal circuits remain largely elusive. In this study, we investigate the roles of two pivotal presynaptic regulators, the small GTPase Rab3, enriched at synaptic vesicles, and the cell adhesion protein Neurexin-1, in the formation of distinct memory phases within the mushroom body Kenyon cells. Our findings suggest that both proteins play crucial roles in memory-supporting processes within the presynaptic terminal, operating within distinct plasticity modules. These modules likely encompass remodeling and maturation of existing active zones (AZs), as well as the formation of new AZs.
Topics: Animals; Mushroom Bodies; Presynaptic Terminals; Drosophila Proteins; Memory; rab3 GTP-Binding Proteins; Nerve Tissue Proteins; Drosophila; Synaptic Vesicles
PubMed: 38862173
DOI: 10.1101/lm.054013.124 -
Acta Neuropathologica Jun 2024Widespread cortical accumulation of misfolded pathological tau proteins (ptau) in the form of paired helical filaments is a major hallmark of Alzheimer's disease....
Widespread cortical accumulation of misfolded pathological tau proteins (ptau) in the form of paired helical filaments is a major hallmark of Alzheimer's disease. Subcellular localization of ptau at various stages of disease progression is likely to be informative of the cellular mechanisms involving its spread. Here, we found that the density of ptau within several distinct rostral thalamic nuclei in post-mortem human tissue (n = 25 cases) increased with the disease stage, with the anterodorsal nucleus (ADn) consistently being the most affected. In the ADn, ptau-positive elements were present already in the pre-cortical (Braak 0) stage. Tau pathology preferentially affected the calretinin-expressing subpopulation of glutamatergic neurons in the ADn. At the subcellular level, we detected ptau immunoreactivity in ADn cell bodies, dendrites, and in a specialized type of presynaptic terminal that expresses vesicular glutamate transporter 2 (vGLUT2) and likely originates from the mammillary body. The ptau-containing terminals displayed signs of degeneration, including endosomal/lysosomal organelles. In contrast, corticothalamic axon terminals lacked ptau. The data demonstrate the involvement of a specific cell population in ADn at the onset of the disease. The presence of ptau in subcortical glutamatergic presynaptic terminals supports hypotheses about the transsynaptic spread of tau selectively affecting specialized axonal pathways.
Topics: Humans; tau Proteins; Female; Male; Aged; Aged, 80 and over; Alzheimer Disease; Middle Aged; Neurons; Vesicular Glutamate Transport Protein 2; Glutamic Acid; Anterior Thalamic Nuclei; Calbindin 2; Neurofibrillary Tangles; Presynaptic Terminals
PubMed: 38861157
DOI: 10.1007/s00401-024-02749-3