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Journal of Translational Medicine Aug 2023The nucleotide-binding oligomeric domain (NOD)-like receptor protein 3 (NLRP3) inflammasome is believed to be a key mediator of neuroinflammation and subsequent...
Inhibiting NLRP3 inflammasome signaling pathway promotes neurological recovery following hypoxic-ischemic brain damage by increasing p97-mediated surface GluA1-containing AMPA receptors.
BACKGROUND
The nucleotide-binding oligomeric domain (NOD)-like receptor protein 3 (NLRP3) inflammasome is believed to be a key mediator of neuroinflammation and subsequent secondary brain injury induced by ischemic stroke. However, the role and underlying mechanism of the NLRP3 inflammasome in neonates with hypoxic-ischemic encephalopathy (HIE) are still unclear.
METHODS
The protein expressions of the NLRP3 inflammasome including NLRP3, cysteinyl aspartate specific proteinase-1 (caspase-1) and interleukin-1β (IL-1β), the α-amino-3-hydroxy-5-methyl-4-isoxazole-propionicacid receptor (AMPAR) subunit, and the ATPase valosin-containing protein (VCP/p97), were determined by Western blotting. The interaction between p97 and AMPA glutamate receptor 1 (GluA1) was determined by co-immunoprecipitation. The histopathological level of hypoxic-ischemic brain damage (HIBD) was determined by triphenyltetrazolium chloride (TTC) staining. Polymerase chain reaction (PCR) and Western blotting were used to confirm the genotype of the knockout mice. Motor functions, including myodynamia and coordination, were evaluated by using grasping and rotarod tests. Hippocampus-dependent spatial cognitive function was measured by using the Morris-water maze (MWM).
RESULTS
We reported that the NLRP3 inflammasome signaling pathway, such as NLRP3, caspase-1 and IL-1β, was activated in rats with HIBD and oxygen-glucose deprivation (OGD)-treated cultured primary neurons. Further studies showed that the protein level of the AMPAR GluA1 subunit on the hippocampal postsynaptic membrane was significantly decreased in rats with HIBD, and it could be restored to control levels after treatment with the specific caspase-1 inhibitor AC-YVAD-CMK. Similarly, in vitro studies showed that OGD reduced GluA1 protein levels on the plasma membrane in cultured primary neurons, whereas AC-YVAD-CMK treatment restored this reduction. Importantly, we showed that OGD treatment obviously enhanced the interaction between p97 and GluA1, while AC-YVAD-CMK treatment promoted the dissociation of p97 from the GluA1 complex and consequently facilitated the localization of GluA1 on the plasma membrane of cultured primary neurons. Finally, we reported that the deficits in motor function, learning and memory in animals with HIBD, were ameliorated by pharmacological intervention or genetic ablation of caspase-1.
CONCLUSION
Inhibiting the NLRP3 inflammasome signaling pathway promotes neurological recovery in animals with HIBD by increasing p97-mediated surface GluA1 expression, thereby providing new insight into HIE therapy.
Topics: Mice; Animals; Rats; Inflammasomes; NLR Family, Pyrin Domain-Containing 3 Protein; Receptors, AMPA; Hypoxia-Ischemia, Brain; Signal Transduction; Caspase 1; Brain
PubMed: 37620837
DOI: 10.1186/s12967-023-04452-5 -
Frontiers in Physiology 2023The plus-end directed actin-dependent motor protein, myosin Va, is of particular relevance for outward vesicular protein trafficking and for restraining specific cargo... (Review)
Review
The plus-end directed actin-dependent motor protein, myosin Va, is of particular relevance for outward vesicular protein trafficking and for restraining specific cargo vesicles within the actin cortex. The latter is a preferred site of cAMP production, and the specificity of cAMP signaling is largely mediated through the formation of microdomains that spatially couple localized metabotropic receptor activity and cAMP production to selected effectors and downstream targets. This review summarizes the core literature on the role of myosin Va for the creation of such a cAMP microdomain at the mammalian nerve-muscle synapse that serves the activity-dependent recycling of nicotinic acetylcholine receptors (nAChRs)-a principal ligand-gated ion channel which is imperative for voluntary muscle contraction. It is discussed that i) the nerve-muscle synapse is a site with a unique actin-dependent microstructure, ii) myosin Va and protein kinase A regulatory subunit Iα as well as nAChR and its constitutive binding partner, rapsyn, colocalize in endocytic/recycling vesicles near the postsynaptic membrane, and iii) impairment of myosin Va or displacement of protein kinase A regulatory subunit Iα leads to the loss of nAChR stability. Regulation of this signaling process and underlying basic pieces of machinery were covered in previous articles, to which the present review refers.
PubMed: 38239886
DOI: 10.3389/fphys.2023.1342994 -
Biophysical Journal Aug 2023Recently, cellular biomolecular condensates formed via phase separation have received considerable attention. While they can be formed either in cytosol (denoted as 3D)...
Recently, cellular biomolecular condensates formed via phase separation have received considerable attention. While they can be formed either in cytosol (denoted as 3D) or beneath the membrane (2D), the underlying difference between the two has not been well clarified. To compare the phase behaviors in 3D and 2D, postsynaptic density (PSD) serves as a model system. PSD is a protein condensate located under the postsynaptic membrane that influences the localization of glutamate receptors and thus contributes to synaptic plasticity. Recent in vitro studies have revealed the formation of droplets of various soluble PSD proteins via liquid-liquid phase separation. However, it is unclear how these protein condensates are formed beneath the membrane and how they specifically affect the localization of glutamate receptors in the membrane. In this study, focusing on the mixture of a glutamate receptor complex, AMPAR-TARP, and a ubiquitous scaffolding protein, PSD-95, we constructed a mesoscopic model of protein-domain interactions in PSD and performed comparative molecular simulations. The results showed a sharp contrast in the phase behaviors of protein assemblies in 3D and those under the membrane (2D). A mixture of a soluble variant of the AMPAR-TARP complex and PSD-95 in the 3D system resulted in a phase-separated condensate, which was consistent with the experimental results. However, with identical domain interactions, AMPAR-TARP embedded in the membrane formed clusters with PSD-95, but did not form a stable separated phase. Thus, the cluster formation behaviors of PSD proteins in the 3D and 2D systems were distinct. The current study suggests that, more generally, stable phase separation can be more difficult to achieve in and beneath the membrane than in 3D systems.
Topics: Neuronal Plasticity; Biomolecular Condensates; Nerve Tissue Proteins; Receptors, Glutamate; Cell Membrane; Disks Large Homolog 4 Protein; Computer Simulation; Cytosol; Phase Separation; Protein Interaction Maps; Models, Chemical
PubMed: 37496268
DOI: 10.1016/j.bpj.2023.07.015 -
Targeting the vascular endothelial growth factor A/neuropilin 1 axis for relief of neuropathic pain.Pain Jul 2023Vascular endothelial growth factor A (VEGF-A) is a pronociceptive factor that causes neuronal sensitization and pain. We reported that blocking the interaction between...
Vascular endothelial growth factor A (VEGF-A) is a pronociceptive factor that causes neuronal sensitization and pain. We reported that blocking the interaction between the membrane receptor neuropilin 1 (NRP1) and VEGF-A-blocked VEGF-A-mediated sensory neuron hyperexcitability and reduced mechanical hypersensitivity in a rodent chronic neuropathic pain model. These findings identified the NRP1-VEGF-A signaling axis for therapeutic targeting of chronic pain. In an in-silico screening of approximately 480 K small molecules binding to the extracellular b1b2 pocket of NRP1, we identified 9 chemical series, with 6 compounds disrupting VEGF-A binding to NRP1. The small molecule with greatest efficacy, 4'-methyl-2'-morpholino-2-(phenylamino)-[4,5'-bipyrimidin]-6(1H)-one, designated NRP1-4, was selected for further evaluation. In cultured primary sensory neurons, VEGF-A enhanced excitability and decreased firing threshold, which was blocked by NRP1-4. In addition, NaV1.7 and CaV2.2 currents and membrane expression were potentiated by treatment with VEGF-A, and this potentiation was blocked by NRP1-4 cotreatment. Neuropilin 1-4 reduced VEGF-A-mediated increases in the frequency and amplitude of spontaneous excitatory postsynaptic currents in dorsal horn of the spinal cord. Neuropilin 1-4 did not bind to more than 300 G-protein-coupled receptors and receptors including human opioids receptors, indicating a favorable safety profile. In rats with spared nerve injury-induced neuropathic pain, intrathecal administration of NRP1-4 significantly attenuated mechanical allodynia. Intravenous treatment with NRP1-4 reversed both mechanical allodynia and thermal hyperalgesia in rats with L5/L6 spinal nerve ligation-induced neuropathic pain. Collectively, our findings show that NRP1-4 is a first-in-class compound targeting the NRP1-VEGF-A signaling axis to control voltage-gated ion channel function, neuronal excitability, and synaptic activity that curb chronic pain.
Topics: Rats; Humans; Animals; Vascular Endothelial Growth Factor A; Hyperalgesia; Neuropilin-1; Chronic Pain; Neuralgia; Spinal Cord Dorsal Horn; Sensory Receptor Cells
PubMed: 36729125
DOI: 10.1097/j.pain.0000000000002850 -
Neuropharmacology Aug 2023Early life stress (ELS) alters the excitation-inhibition-balance (EI-balance) in various rodent brain areas and may be responsible for behavioral impairment later in...
Early life stress (ELS) alters the excitation-inhibition-balance (EI-balance) in various rodent brain areas and may be responsible for behavioral impairment later in life. The EI-balance is (amongst others) influenced by the switch of GABAergic transmission from excitatory to inhibitory, the so-called "GABA-switch". Here, we investigated how ELS affects the GABA-switch in mouse infralimbic Prefrontal Cortex layer 2/3 neurons, using the limited-nesting-and-bedding model. In ELS mice, the GABA-switch occurred already between postnatal day (P) 6 and P9, as opposed to P15-P21 in controls. This was associated with increased expression of the inward chloride transporter NKCC1, compared to the outward chloride transporter KCC2, both of which are important for the intracellular chloride concentration and, hence, the GABA reversal potential (Erev). Chloride transporters are not only important for regulating chloride concentration postsynaptically, but also presynaptically. Depending on the Erev of GABA, presynaptic GABA receptor stimulation causes a depolarization or hyperpolarization, and thereby enhanced or reduced fusion of glutamate vesicles respectively, in turn changing the frequency of miniature postsynaptic currents (mEPSCs). In accordance, bumetanide, a blocker of NKCC1, shifted the Erev GABA towards more hyperpolarized levels in P9 control mice and reduced the mEPSC frequency. Other modulators of chloride transporters, e.g. VU0463271 (a KCC2 antagonist) and aldosterone -which increases NKCC1 expression-did not affect postsynaptic Erev in ELS P9 mice, but did increase the mEPSC frequency. We conclude that the mouse GABA-switch is accelerated after ELS, affecting both the pre- and postsynaptic chloride homeostasis, the former altering glutamatergic transmission. This may considerably affect brain development.
Topics: Animals; Mice; Acceleration; Chlorides; gamma-Aminobutyric Acid; Membrane Transport Proteins; Receptors, GABA-A; Symporters; Stress, Physiological
PubMed: 37061088
DOI: 10.1016/j.neuropharm.2023.109543 -
Journal of Neuroscience Research Nov 2023One group of the K ion channels, the small-conductance Ca -activated potassium channels (K 2.x, also known as SK channels family), is widely expressed in neurons as well... (Review)
Review
One group of the K ion channels, the small-conductance Ca -activated potassium channels (K 2.x, also known as SK channels family), is widely expressed in neurons as well as the heart, endothelial cells, etc. They are named small-conductance Ca -activated potassium channels (SK channels) due to their comparatively low single-channel conductance of about ~10 pS. These channels are insensitive to changes in membrane potential and are activated solely by rises in the intracellular Ca . According to the phylogenic research done on the K 2.x channels family, there are three channels' subtypes: K 2.1, K 2.2, and K 2.3, which are encoded by KCNN1, KCNN2, and KCNN3 genes, respectively. The K 2.x channels regulate neuronal excitability and responsiveness to synaptic input patterns. K 2.x channels inhibit excitatory postsynaptic potentials (EPSPs) in neuronal dendrites and contribute to the medium afterhyperpolarization (mAHP) that follows the action potential bursts. Multiple brain regions, including the hippocampus, express the K 2.2 channel encoded by the KCNN2 gene on chromosome 5. Of particular interest, rat cerebellar Purkinje cells express K 2.2 channels, which are crucial for various cellular processes during development and maturation. Patients with a loss-of-function of KCNN2 mutations typically exhibit extrapyramidal symptoms, cerebellar ataxia, motor and language developmental delays, and intellectual disabilities. Studies have revealed that autosomal dominant neurodevelopmental movement disorders resembling rodent symptoms are caused by heterozygous loss-of-function mutations, which are most likely to induce KCNN2 haploinsufficiency. The K 2.2 channel is a promising drug target for spinocerebellar ataxias (SCAs). SCAs exhibit the dysregulation of firing in cerebellar Purkinje cells which is one of the first signs of pathology. Thus, selective K 2.2 modulators are promising potential therapeutics for SCAs.
Topics: Rats; Animals; Potassium Channels; Endothelial Cells; Neurons; Membrane Potentials; Purkinje Cells
PubMed: 37466411
DOI: 10.1002/jnr.25233 -
The Neuroscientist : a Review Journal... Dec 2023Startle disease (SD) is characterized by enhanced startle responses, generalized muscle stiffness, unexpected falling, and fatal apnea episodes due to disturbed feedback... (Review)
Review
Startle disease (SD) is characterized by enhanced startle responses, generalized muscle stiffness, unexpected falling, and fatal apnea episodes due to disturbed feedback inhibition in the spinal cord and brainstem of affected individuals. Mutations within the glycine receptor (GlyR) subunit and glycine transporter 2 (GlyT2) genes have been identified in individuals with SD. Impaired inhibitory neurotransmission in SD is due to pre- and/or postsynaptic GlyR or presynaptic GlyT2 dysfunctions. Previous research has focused on mutated GlyRs and GlyT2 that impair ion channel/transporter function or trafficking. With insights provided by recently solved cryo-electron microscopy and X-ray structures of GlyRs, a detailed picture of structural transitions important for receptor gating has emerged, allowing a deeper understanding of SD at the molecular level. Moreover, studies on novel SD mutations have demonstrated a higher complexity of SD, with identification of additional clinical signs and symptoms and interaction partners representing key players for fine-tuning synaptic processes. Although our knowledge has steadily improved during the last years, changes in synaptic localization and GlyR or GlyT2 homeostasis under disease conditions are not yet completely understood. Combined proteomics, interactomics, and high-resolution microscopy techniques are required to reveal alterations in receptor dynamics at the synaptic level under disease conditions.
Topics: Humans; Cryoelectron Microscopy; Receptors, Glycine; Nervous System Diseases; Glycine Plasma Membrane Transport Proteins; Mutation
PubMed: 35754344
DOI: 10.1177/10738584221104724 -
Neuroscience Aug 2023Astrocytes have been increasingly acknowledged to play active roles in regulating synaptic transmission and plasticity. Through a variety of metabotropic and ionotropic...
Astrocytes have been increasingly acknowledged to play active roles in regulating synaptic transmission and plasticity. Through a variety of metabotropic and ionotropic receptors expressed on their surface, astrocytes detect extracellular neurotransmitters, and in turn, release gliotransmitters to modify synaptic strength, while they can also alter neuronal membrane excitability by modulating extracellular ionic milieu. Given the seemingly large repertoire of synaptic modulation, when, where and how astrocytes interact with synapses remain to be fully understood. Previously, we have identified a role for astrocyte NMDA receptor and L-VGCCs signaling in heterosynaptic presynaptic plasticity and promoting the heterogeneity of presynaptic strengths at hippocampal synapses. Here, we have sought to further clarify the mode by which astrocytes regulate presynaptic plasticity by exploiting a reduced culture system to globally evoke NMDA receptor-dependent presynaptic plasticity. Recording from a postsynaptic neuron intracellularly loaded with BAPTA, briefly bath applying NMDA and glycine induces a stable decrease in the rate of spontaneous glutamate release, which requires the presence of astrocytes and the activation of A1 adenosine receptors. Upon preventing astrocyte calcium signaling or blocking L-VGCCs, NMDA + glycine application triggers an increase, rather than a decrease, in the rate of spontaneous glutamate release, thereby shifting the presynaptic plasticity to promote an increase in strength. Our findings point to a crucial and surprising role of astrocytes in controlling the polarity of NMDA receptor and adenosine-dependent presynaptic plasticity. Such a pivotal mechanism unveils the power of astrocytes in regulating computations performed by neural circuits and is expected to profoundly impact cognitive processes.
Topics: Astrocytes; Calcium Signaling; Receptors, N-Methyl-D-Aspartate; N-Methylaspartate; Synaptic Transmission; Synapses; Glutamates; Glycine; Calcium; Neuronal Plasticity
PubMed: 37295597
DOI: 10.1016/j.neuroscience.2023.05.032 -
Toxicology Aug 2023It has been widely reported that long-term exposure to copper increases the prevalence and mortality of Parkinson's disease. Our previous study showed that CuSO exposure...
It has been widely reported that long-term exposure to copper increases the prevalence and mortality of Parkinson's disease. Our previous study showed that CuSO exposure induced a significant increase in the expression of cleaved Caspase1 proteins and the loss of dopaminergic neurons in the SNpc of mice. In this study, the effects of copper(Ⅱ) on cAMP/PKA/CREB pathway and pyroptosis-related proteins in MN9D cells were investigated by setting up copper(Ⅱ) exposure groups with different concentration gradients, to provide possible molecular evidence for studying the mechanism of copper(Ⅱ)-induced degeneration of dopaminergic neurons. We found that after 48 h of copper(Ⅱ) exposure, the cu content in MN9D cells increased in a dose-dependent manner, and the proliferation activity decreased significantly. In addition, copper(Ⅱ) exposure caused up-regulation of PDE4D and down-regulation of D1R, cAMP, PKA and p-CREB/CREB. Simultaneously, we proved that copper(Ⅱ) exposure induced oxidative stress in MN9D cells, including decreased GSH-Px content, Keap1 expression and mitochondrial membrane potential, increased malondialdehyde content, ROS intensity, and Nrf2, NQO1, HO-1, HSP-70 expression, further causing up-regulation of inflammasome and GSDMD protein. After pretreatment with Roflupram, the level of copper(Ⅱ)-induced oxidative damage decreased, the expression of inflammasome and GSDMD proteins were down-regulated. However, the protective effects of ROF were blocked by H-89. In summary, copper(Ⅱ) treatment induced oxidative stress and inflammasome-mediated pyroptosis in MN9D cells, which may be related to copper(Ⅱ)-induced postsynaptic cAMP, PKA, and CREB signal transduction disorders.
Topics: Mice; Animals; Up-Regulation; Pyroptosis; Copper; Kelch-Like ECH-Associated Protein 1; Inflammasomes; NF-E2-Related Factor 2; Oxidative Stress; Signal Transduction; Dopaminergic Neurons; Cyclic AMP Response Element-Binding Protein
PubMed: 37353053
DOI: 10.1016/j.tox.2023.153582