Chronic α1-Na/K-ATPase inhibition reverses the elongation of the axon initial segment of the hippocampal CA1 pyramidal neurons in Angelman syndrome model mice.
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ABSTRACT:
SUBMITTER: Rayi PR
PROVIDER: S-EPMC8027375 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The axon initial segment (AIS) is a region of the neuron that is critical for action potential generation as well as for the regulation of neural activity. This specialized structure-characterized by the expression of different types of ion channels as well as adhesion, scaffolding and cytoskeleton proteins-is subjected to morpho-functional plastic changes in length and position upon variations in neural activity or in pathological conditions. In the present study, using immunocytochemistry with the AT8 antibody (phospho-tau S202/T205) and 3D confocal microscopy reconstruction techniques in brain tissue from Alzheimer's disease patients, we found that around half of the cortical pyramidal neurons with hyperphosphorylated tau showed changes in AIS length and position in comparison with AT8-negative neurons from the same cortical layers. We observed a wide variety of AIS alterations in neurons with hyperphosphorylated tau, although the most common changes were a proximal shift or a lengthening of the AISs. Similar results were found in neocortical tissue from non-demented cases with neurons containing hyperphosphorylated tau. These findings support the notion that the accumulation of phospho-tau is associated with structural alterations of the AIS that are likely to have an impact on normal neuronal activity, which might contribute to neuronal dysfunction in AD.
| S-EPMC9130508 | biostudies-literature
Project description:The N-terminal caveolin-binding motif (CBM) in Na/K-ATPase (NKA) α1 subunit is essential for cell signaling and somitogenesis in animals. To further investigate the molecular mechanism, we have generated CBM mutant human-induced pluripotent stem cells (iPSCs) through CRISPR/Cas9 genome editing and examined their ability to differentiate into skeletal muscle (Skm) cells. Compared with the parental wild-type human iPSCs, the CBM mutant cells lost their ability of Skm differentiation, which was evidenced by the absence of spontaneous cell contraction, marker gene expression, and subcellular myofiber banding structures in the final differentiated induced Skm cells. Another NKA functional mutant, A420P, which lacks NKA/Src signaling function, did not produce a similar defect. Indeed, A420P mutant iPSCs retained intact pluripotency and ability of Skm differentiation. Mechanistically, the myogenic transcription factor MYOD was greatly suppressed by the CBM mutation. Overexpression of a mouse Myod cDNA through lentiviral delivery restored the CBM mutant cells' ability to differentiate into Skm. Upstream of MYOD, Wnt signaling was demonstrated from the TOPFlash assay to have a similar inhibition. This effect on Wnt activity was further confirmed functionally by defective induction of the presomitic mesoderm marker genes BRACHYURY (T) and MESOGENIN1 (MSGN1) by Wnt3a ligand or the GSK3 inhibitor/Wnt pathway activator CHIR. Further investigation through immunofluorescence imaging and cell fractionation revealed a shifted membrane localization of β-catenin in CBM mutant iPSCs, revealing a novel molecular component of NKA-Wnt regulation. This study sheds light on a genetic regulation of myogenesis through the CBM of NKA and control of Wnt/β-catenin signaling.
| S-EPMC8943859 | biostudies-literature
Project description:Hyperphosphorylated tau has a critical role in tauopathies such as Alzheimer's disease and frontotemporal dementia, impairing neuronal function and eventually leading to neurodegeneration. A critical role for tau is supported by studies in transgenic mouse models that express the P301L tau mutation found in cases of familial frontotemporal dementia, with the accumulation of hyperphosphorylated tau in the hippocampus causing reductions in hippocampal long-term potentiation and impairments in spatial learning and memory. However, what has remained unexplored is the role of hyperphosphorylated tau in reducing neuronal excitability. Here, we show in two complementary P301L tau transgenic mouse models that hyperphosphorylated tau induces a more depolarized threshold for action potential initiation and reduces firing in hippocampal CA1 neurons, which was rescued by the suppression of transgenic tau. Furthermore, using mutagenesis and primary hippocampal neuronal cultures, we reveal that this reduction in neuronal excitability results from the relocation of the axon initial segment (AIS) down the axon in a tau phosphorylation-dependent manner. We also demonstrate that this effect is microtubule-dependent. In addition, pharmacological stabilization was found to prevent both the structural and functional deficits caused by tau hyperphosphorylation. Finally, we demonstrate that the AIS of neurons from tau transgenic mice is further down the axon, which correlates with a reduction in excitability. We therefore propose that a reduction in hippocampal excitability due to a tau-mediated distal relocalization of the AIS contributes to the hippocampal dysfunction observed in tauopathies.
| S-EPMC5389999 | biostudies-literature
Project description:AimHighly prevalent diseases such as insulin resistance and heart failure are characterized by reduced metabolic flexibility and reserve. We tested whether Na/K-ATPase (NKA)-mediated regulation of Src kinase, which requires two NKA sequences specific to the α1 isoform, is a regulator of metabolic capacity that can be targeted pharmacologically.MethodsMetabolic capacity was challenged functionally by Seahorse metabolic flux analyses and glucose deprivation in LLC-PK1-derived cells expressing Src binding rat NKA α1, non-Src-binding rat NKA α2 (the most abundant NKA isoform in the skeletal muscle), and Src binding gain-of-function mutant rat NKA α2. Mice with skeletal muscle-specific ablation of NKA α1 (skα1-/-) were generated using a MyoD:Cre-Lox approach and were subjected to treadmill testing and Western diet. C57/Bl6 mice were subjected to Western diet with or without pharmacological inhibition of NKA α1/Src modulation by treatment with pNaKtide, a cell-permeable peptide designed by mapping one of the sites of NKA α1/Src interaction.ResultsMetabolic studies in mutant cell lines revealed that the Src binding regions of NKA α1 are required to maintain metabolic reserve and flexibility. Skα1-/- mice had decreased exercise endurance and mitochondrial Complex I dysfunction. However, skα1-/- mice were resistant to Western diet-induced insulin resistance and glucose intolerance, a protection phenocopied by pharmacological inhibition of NKA α1-mediated Src regulation with pNaKtide.ConclusionsThese results suggest that NKA α1/Src regulatory function may be targeted in metabolic diseases. Because Src regulatory capability by NKA α1 is exclusive to endotherms, it may link the aerobic scope hypothesis of endothermy evolution to metabolic dysfunction.
| S-EPMC8570534 | biostudies-literature
Project description:The microdomain that orchestrates action potential initiation in neurons is the axon initial segment (AIS). It has long been considered to be a rather homogeneous domain at the very proximal axon hillock with relatively stable length, particularly in cortical pyramidal cells. However, studies in other brain regions paint a different picture. In hippocampal CA1, up to 50% of axons emerge from basal dendrites. Further, in about 30% of thick-tufted layer V pyramidal neurons in rat somatosensory cortex, axons have a dendritic origin. Consequently, the AIS is separated from the soma. Recent in vitro and in vivo studies have shown that cellular excitability is a function of AIS length/position and somatodendritic morphology, undermining a potentially significant impact of AIS heterogeneity for neuronal function. We therefore investigated neocortical axon morphology and AIS composition, hypothesizing that the initial observation of seemingly homogeneous AIS is inadequate and needs to take into account neuronal cell types. Here, we biolistically transfected cortical neurons in organotypic cultures to visualize the entire neuron and classify cell types in combination with immunolabeling against AIS markers. Using confocal microscopy and morphometric analysis, we investigated axon origin, AIS position, length, diameter as well as distance to the soma. We find a substantial AIS heterogeneity in visual cortical neurons, classified into three groups: (I) axons with somatic origin with proximal AIS at the axon hillock; (II) axons with somatic origin with distal AIS, with a discernible gap between the AIS and the soma; and (III) axons with dendritic origin (axon-carrying dendrite cell, AcD cell) and an AIS either starting directly at the axon origin or more distal to that point. Pyramidal cells have significantly longer AIS than interneurons. Interneurons with vertical columnar axonal projections have significantly more distal AIS locations than all other cells with their prevailing phenotype as an AcD cell. In contrast, neurons with perisomatic terminations display most often an axon originating from the soma. Our data contribute to the emerging understanding that AIS morphology is highly variable, and potentially a function of the cell type.
| S-EPMC5684645 | biostudies-literature
Project description:The Na(+)/K(+) ATPase (NKA) is an essential membrane protein underlying the membrane potential in excitable cells. Transmembrane ion transport is performed by the catalytic α subunits (α1-4). The predominant subunits in neurons are α1 and α3, which have different affinities for Na(+) and K(+), impacting on transport kinetics. The exchange rate of Na(+)/K(+) markedly influences the activity of the neurons expressing them. We have investigated the distribution and function of the main isoforms of the α subunit expressed in the mouse spinal cord. NKAα1 immunoreactivity (IR) displayed restricted labeling, mainly confined to large ventral horn neurons and ependymal cells. NKAα3 IR was more widespread in the spinal cord, again being observed in large ventral horn neurons, but also in smaller interneurons throughout the dorsal and ventral horns. Within the ventral horn, the α1 and α3 isoforms were mutually exclusive, with the α3 isoform in smaller neurons displaying markers of γ-motoneurons and α1 in α-motoneurons. The α3 isoform was also observed within muscle spindle afferent neurons in dorsal root ganglia with a higher proportion at cervical versus lumbar regions. We confirmed the differential expression of α subunits in motoneurons electrophysiologically in neonatal slices of mouse spinal cord. γ-Motoneurons were excited by bath application of low concentrations of ouabain that selectively inhibit NKAα3 while α-motoneurons were insensitive to these low concentrations. The selective expression of NKAα3 in γ-motoneurons and muscle spindle afferents, which may affect excitability of these neurons, has implications in motor control and disease states associated with NKAα3 dysfunction.
| S-EPMC3722489 | biostudies-literature
Project description:Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related mortality worldwide, with an estimate of 0.84 million cases every year. In Western countries, because of the obesity epidemic, non-alcoholic steatohepatitis (NASH) has become the major cause of HCC. Intriguingly, the molecular mechanisms underlying tumorigenesis of HCC from NASH are largely unknown. We hypothesized that the growing uncoupled metabolism during NASH progression to HCC, manifested by lower cell redox status and an apoptotic 'switch' activity, follows a dysregulation of α1-Na/K-ATPase (NKA)/Src signalosome. Our results suggested that in NASH-related malignancy, α1-NKA signaling causes upregulation of the anti-apoptotic protein survivin and downregulation of the pro-apoptotic protein Smac/DIABLO via the activation of the PI3K → Akt pro-survival pathway with concomitant inhibition of the FoxO3 circuit, favoring cell division and primary liver carcinogenesis. Signalosome normalization using an inhibitory peptide resets apoptotic activity in malignant cells, with a significant decrease in tumor burden in vivo. Therefore, α1-NKA signalosome exercises in HCC the characteristic of a tumor suppressor, suggesting α1-NKA as a putative target for clinical therapy.
| S-EPMC9266688 | biostudies-literature
Project description:Through their ion-pumping and non-ion-pumping functions, Na(+)-K(+)-ATPase protein complexes at the plasma membrane are critical to intracellular homeostasis and to the physiological and pharmacological actions of cardiotonic steroids. Alteration of the abundance of Na(+)-K(+)-ATPase units at the cell surface is one of the mechanisms for Na(+)-K(+)-ATPase regulation in health and diseases that has been closely examined over the past few decades. We here summarize these findings, with emphasis on studies that explicitly tested the involvement of defined regions or residues on the Na(+)-K(+)-ATPase α1 polypeptide. We also report new findings on the effect of manipulating Na(+)-K(+)-ATPase membrane abundance by targeting one of these defined regions: a dileucine motif of the form [D/E]XXXL[L/I]. In this study, opossum kidney cells stably expressing rat α1 Na(+)-K(+)-ATPase or a mutant where the motif was disrupted (α1-L499V) were exposed to 30 min of substrate/coverslip-induced-ischemia followed by reperfusion (I-R). Biotinylation studies suggested that I-R itself acted as an inducer of Na(+)-K(+)-ATPase internalization and that surface expression of the mutant was higher than the native Na(+)-K(+)-ATPase before and after ischemia. Annexin V/propidium iodide staining and lactate dehydrogenase release suggested that I-R injury was reduced in α1-L499V-expressing cells compared with α1-expressing cells. Hence, modulation of Na(+)-K(+)-ATPase cell surface abundance through structural determinants on the α-subunit is an important mechanism of regulation of cellular Na(+)-K(+)-ATPase in various physiological and pathophysiological conditions, with a significant impact on cell survival in face of an ischemic stress.
| S-EPMC3023198 | biostudies-literature
Project description:BackgroundWe have demonstrated that cardiotonic steroids, such as ouabain, signaling through the Na/K-ATPase, regulate sodium reabsorption in the renal proximal tubule. By direct carbonylation modification of the Pro222 residue in the actuator (A) domain of pig Na/K-ATPase α1 subunit, reactive oxygen species are required for ouabain-stimulated Na/K-ATPase/c-Src signaling and subsequent regulation of active transepithelial (22)Na(+) transport. In the present study we sought to determine the functional role of Pro222 carbonylation in Na/K-ATPase signaling and sodium handling.Methods and resultsStable pig α1 knockdown LLC-PK1-originated PY-17 cells were rescued by expressing wild-type rat α1 and rat α1 with a single mutation of Pro224 (corresponding to pig Pro222) to Ala. This mutation does not affect ouabain-induced inhibition of Na/K-ATPase activity, but abolishes the effects of ouabain on Na/K-ATPase/c-Src signaling, protein carbonylation, Na/K-ATPase endocytosis, and active transepithelial (22)Na(+) transport.ConclusionsDirect carbonylation modification of Pro224 in the rat α1 subunit determines ouabain-mediated Na/K-ATPase signal transduction and subsequent regulation of renal proximal tubule sodium transport.
| S-EPMC5079028 | biostudies-literature
Project description:Hippocampal pyramidal cells (PCs) express many GABAAR subunit types and receive GABAergic inputs from distinct interneurons. Previous experiments revealed input-specific differences in ?1 and ?2 subunit densities in perisomatic synapses, suggesting distinct IPSC decay kinetics. However, IPSC decays evoked by axo-axonic, parvalbumin- or cholecystokinin-expressing basket cells were found to be similar. Using replica immunogold labeling, here we show that all CA1 PC somatic and AIS synapses contain the ?1, ?2, ?1, ?2, ?3 and ?2 subunits. In CA3 PCs, 90% of the perisomatic synapses are immunopositive for the ?1 subunit and all synapses are positive for the remaining five subunits. Somatic synapses form unimodal distributions based on their immunoreactivity for these subunits. The ?2 subunit densities in somatic synapses facing Cav2.1 (i.e. parvalbumin) or Cav2.2 (cholecystokinin) positive presynaptic active zones are comparable. We conclude that perisomatic synapses made by three distinct interneuron types have similar GABAA receptor subunit content.
| S-EPMC4990423 | biostudies-literature