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Frontiers in Neurology 2022Neuroinvasive infection is the most common cause of meningoencephalitis in people living with human immunodeficiency virus (HIV), but autoimmune etiologies have been...
Neuroinvasive infection is the most common cause of meningoencephalitis in people living with human immunodeficiency virus (HIV), but autoimmune etiologies have been reported. We present the case of a 51-year-old man living with HIV infection with steroid-responsive meningoencephalitis whose comprehensive pathogen testing was non-diagnostic. Subsequent tissue-based immunofluorescence with acute-phase cerebrospinal fluid revealed anti-neural antibodies localizing to the axon initial segment (AIS), the node of Ranvier (NoR), and the subpial space. Phage display immunoprecipitation sequencing identified ankyrinG (AnkG) as the leading candidate autoantigen. A synthetic blocking peptide encoding the PhIP-Seq-identified AnkG epitope neutralized CSF IgG binding to the AIS and NoR, thereby confirming a monoepitopic AnkG antibody response. However, subpial immunostaining persisted, indicating the presence of additional autoantibodies. Review of archival tissue-based staining identified candidate AnkG autoantibodies in a 60-year-old woman with metastatic ovarian cancer and seizures that were subsequently validated by cell-based assay. AnkG antibodies were not detected by tissue-based assay and/or PhIP-Seq in control CSF ( = 39), HIV CSF ( = 79), or other suspected and confirmed neuroinflammatory CSF cases ( = 1,236). Therefore, AnkG autoantibodies in CSF are rare but extend the catalog of AIS and NoR autoantibodies associated with neurological autoimmunity.
PubMed: 36756346
DOI: 10.3389/fneur.2022.1102484 -
The Journal of Neuroscience : the... Mar 2023Brain pH is a critical factor for determining neuronal activity, with alkalosis increasing and acidosis reducing excitability. Acid shifts in brain pH through the...
Brain pH is a critical factor for determining neuronal activity, with alkalosis increasing and acidosis reducing excitability. Acid shifts in brain pH through the breathing of carbogen (5% CO/95% O) reduces seizure susceptibility in animal models and patients. The molecular mechanisms underlying this seizure protection remain to be fully elucidated. Here, we demonstrate that male and female mice exposed to carbogen are fully protected from thermogenic-triggered seizures. Whole-cell patch-clamp recordings revealed that acid shifts in extracellular pH (pHo) significantly reduce action potential firing in CA1 pyramidal neurons but did not alter firing in hippocampal inhibitory interneurons. In real-time dynamic clamp experiments, acidification reduced simulated action potential firing generated in hybrid model neurons expressing the excitatory neuron predominant Na1.2 channel. Conversely, acidification had no effect on action potential firing in hybrid model neurons expressing the interneuron predominant Na1.1 channel. Furthermore, knockdown of mRNA using antisense oligonucleotides reduced the protective effects of carbogen on seizure susceptibility. Both carbogen-mediated seizure protection and the reduction in CA1 pyramidal neuron action potential firing by low pHo were maintained in an knock-out mouse ruling out this acid-sensing channel as the underlying molecular target. These data indicate that the acid-mediated reduction in excitatory neuron firing is mediated, at least in part, through the inhibition of Na1.2 channels, whereas inhibitory neuron firing is unaffected. This reduction in pyramidal neuron excitability is the likely basis of seizure suppression caused by carbogen-mediated acidification. Brain pH has long been known to modulate neuronal excitability. Here, we confirm that brain acidification reduces seizure susceptibility in a mouse model of thermogenic seizures. Extracellular acidification reduced excitatory pyramidal neuron firing while having no effect on interneuron firing. Acidification also reduced dynamic clamp firing in cells expressing the Na1.2 channel but not in cells expressing Na1.1 channels. knockdown of Scn2a mRNA reduced seizure protection of acidification. In contrast, acid-mediated seizure protection was maintained in the Asic1a knock-out mouse. These data suggest Na1.2 channel as an important target for acid-mediated seizure protection. Our results have implications on how natural variations in pH can modulate neuronal excitability and highlight potential antiseizure drug development strategies based on the Na1.2 channel.
Topics: Mice; Male; Animals; Female; Carbon Dioxide; Acidosis, Respiratory; Axon Initial Segment; Seizures; Pyramidal Cells; Action Potentials; Mice, Knockout; RNA, Messenger
PubMed: 36732074
DOI: 10.1523/JNEUROSCI.1387-22.2022 -
The Journal of Neuroscience : the... Mar 2023The amyloid precursor protein (APP) is linked to the genetics and pathogenesis of Alzheimer's disease (AD). It is the parent protein of the β-amyloid (Aβ) peptide, the...
The amyloid precursor protein (APP) is linked to the genetics and pathogenesis of Alzheimer's disease (AD). It is the parent protein of the β-amyloid (Aβ) peptide, the main constituent of the amyloid plaques found in an AD brain. The pathways from APP to Aβ are intensively studied, yet the normal functions of APP itself have generated less interest. We report here that glutamate stimulation of neuronal activity leads to a rapid increase in gene expression. In mouse and human neurons, elevated APP protein changes the structure of the axon initial segment (AIS) where action potentials are initiated. The AIS is shortened in length and shifts away from the cell body. The GCaMP8f Ca reporter confirms the predicted decrease in neuronal activity. NMDA antagonists or knockdown of block the glutamate effects. The actions of APP on the AIS are cell-autonomous; exogenous Aβ, either fibrillar or oligomeric, has no effect. In culture, APP (a familial AD mutation) induces larger AIS changes than wild type APP. Ankyrin G and βIV-spectrin, scaffolding proteins of the AIS, both physically associate with APP, more so in AD brains. Finally, in humans with sporadic AD or in the R1.40 AD mouse model, both females and males, neurons have elevated levels of APP protein that invade the AIS. as , this increased APP is associated with a significant shortening of the AIS. The findings outline a new role for the APP and encourage a reconsideration of its relationship to AD. While the amyloid precursor protein (APP) has long been associated with Alzheimer's disease (AD), the normal functions of the full-length Type I membrane protein have been largely unexplored. We report here that the levels of APP protein increase with neuronal activity. and , modest amounts of excess APP alter the properties of the axon initial segment. The β-amyloid peptide derived from APP is without effect. Consistent with the observed changes in the axon initial segment which would be expected to decrease action potential firing, we show that APP expression depresses neuronal activity. In mouse AD models and human sporadic AD, APP physically associates with the scaffolding proteins of the axon initial segment, suggesting a relationship with AD dementia.
Topics: Male; Female; Mice; Humans; Animals; Amyloid beta-Protein Precursor; Alzheimer Disease; Axon Initial Segment; Amyloid beta-Peptides; Membrane Proteins; Mice, Transgenic; Disease Models, Animal
PubMed: 36717226
DOI: 10.1523/JNEUROSCI.0172-22.2023 -
BMC Neuroscience Jan 2023Autism spectrum condition or 'autism' is associated with numerous genetic risk factors including the polygenic 16p11.2 microdeletion. The balance between excitatory and...
BACKGROUND
Autism spectrum condition or 'autism' is associated with numerous genetic risk factors including the polygenic 16p11.2 microdeletion. The balance between excitatory and inhibitory neurons in the cerebral cortex is hypothesised to be critical for the aetiology of autism making improved understanding of how risk factors impact on the development of these cells an important area of research. In the current study we aim to combine bioinformatics analysis of human foetal cerebral cortex gene expression data with anatomical and electrophysiological analysis of a 16p11.2 rat model to investigate how genetic risk factors impact on inhibitory neuron development.
METHODS
We performed bioinformatics analysis of single cell transcriptomes from gestational week (GW) 8-26 human foetal prefrontal cortex and anatomical and electrophysiological analysis of 16p11.2 rat cerebral cortex and hippocampus at post-natal day (P) 21.
RESULTS
We identified a subset of human interneurons (INs) first appearing at GW23 with enriched expression of a large fraction of risk factor transcripts including those expressed from the 16p11.2 locus. This suggests the hypothesis that these foetal INs are vulnerable to mutations causing autism. We investigated this in a rat model of the 16p11.2 microdeletion. We found no change in the numbers or position of either excitatory or inhibitory neurons in the somatosensory cortex or CA1 of 16p11.2 rats but found that CA1 Sst INs were hyperexcitable with an enlarged axon initial segment, which was not the case for CA1 pyramidal cells.
LIMITATIONS
The human foetal gene expression data was acquired from cerebral cortex between gestational week (GW) 8 to 26. We cannot draw inferences about potential vulnerabilities to genetic autism risk factors for cells not present in the developing cerebral cortex at these stages. The analysis 16p11.2 rat phenotypes reported in the current study was restricted to 3-week old (P21) animals around the time of weaning and to a single interneuron cell-type while in human 16p11.2 microdeletion carriers symptoms likely involve multiple cell types and manifest in the first few years of life and on into adulthood.
CONCLUSIONS
We have identified developing interneurons in human foetal cerebral cortex as potentially vulnerable to monogenic autism risk factors and the 16p11.2 microdeletion and report interneuron phenotypes in post-natal 16p11.2 rats.
Topics: Humans; Rats; Animals; Interneurons; Autistic Disorder; Neurons; Cerebral Cortex; Risk Factors
PubMed: 36658491
DOI: 10.1186/s12868-022-00771-3 -
The Journal of Neuroscience : the... Jan 2023The structural plasticity of the axon initial segment (AIS) contributes to the homeostatic control of activity and optimizes the function of neural circuits; however,...
The structural plasticity of the axon initial segment (AIS) contributes to the homeostatic control of activity and optimizes the function of neural circuits; however, the underlying mechanisms are not fully understood. In this study, we prepared a slice culture containing nucleus magnocellularis from chickens of both sexes that reproduces most features of AIS plasticity , regarding its effects on characteristics of AIS and cell-type specificity, and revealed that microtubule reorganization via activation of CDK5 underlies plasticity. Treating the culture with a high-K medium shortened the AIS and reduced sodium current and membrane excitability, specifically in neurons tuned to high-frequency sound, creating a tonotopic difference in AIS length in the nucleus. Pharmacological analyses revealed that this AIS shortening was driven by multiple Ca pathways and subsequent signaling molecules that converge on CDK5 via the activation of ERK1/2. AIS shortening was suppressed by overexpression of dominant-negative CDK5, whereas it was facilitated by the overexpression of p35, an activator of CDK5. Notably, p35(T138A), a phosphorylation-inactive mutant of p35, did not shorten the AIS. Moreover, microtubule stabilizers occluded AIS shortening during the p35 overexpression, indicating that CDK5/p35 mediated AIS shortening by promoting disassembly of microtubules at distal AIS. This study highlights the importance of microtubule reorganization and regulation of CDK5 activity in structural AIS plasticity and the tuning of AIS characteristics in neurons. The structural plasticity of AIS has a strong impact on the output of neurons and plays a fundamental role in the physiology and pathology of the brain. However, the mechanisms linking neuronal activity to structural changes in AIS are not well understood. In this study, we prepared an organotypic culture of avian auditory brainstem, reproducing most AIS plasticity features , and we revealed that activity-dependent AIS shortening occurs through the disassembly of microtubules at distal AIS via activation of CDK5/p35 signals. This study emphasizes the importance of microtubule reorganization and regulation of CDK5 activity in structural AIS plasticity and tonotopic differentiation of AIS structures in the brainstem auditory circuit.
Topics: Animals; Female; Male; Axon Initial Segment; Chickens; Cyclin-Dependent Kinase 5; Microtubules; Neurons; Phosphorylation
PubMed: 36639893
DOI: 10.1523/JNEUROSCI.0917-22.2022 -
Molecular Psychiatry Apr 2023Copy number variations (CNVs) are associated with psychiatric and neurodevelopmental disorders (NDDs), and most, including the recurrent 15q13.3 microdeletion disorder,...
Copy number variations (CNVs) are associated with psychiatric and neurodevelopmental disorders (NDDs), and most, including the recurrent 15q13.3 microdeletion disorder, have unknown disease mechanisms. We used a heterozygous 15q13.3 microdeletion mouse model and patient iPSC-derived neurons to reveal developmental defects in neuronal maturation and network activity. To identify the underlying molecular dysfunction, we developed a neuron-specific proximity-labeling proteomics (BioID2) pipeline, combined with patient mutations, to target the 15q13.3 CNV genetic driver OTUD7A. OTUD7A is an emerging independent NDD risk gene with no known function in the brain, but has putative deubiquitinase function. The OTUD7A protein-protein interaction network included synaptic, axonal, and cytoskeletal proteins and was enriched for ASD and epilepsy risk genes (Ank3, Ank2, SPTAN1, SPTBN1). The interactions between OTUD7A and Ankyrin-G (Ank3) and Ankyrin-B (Ank2) were disrupted by an epilepsy-associated OTUD7A L233F variant. Further investigation of Ankyrin-G in mouse and human 15q13.3 microdeletion and OTUD7A models revealed protein instability, increased polyubiquitination, and decreased levels in the axon initial segment, while structured illumination microscopy identified reduced Ankyrin-G nanodomains in dendritic spines. Functional analysis of human 15q13.3 microdeletion and OTUD7A models revealed shared and distinct impairments to axonal growth and intrinsic excitability. Importantly, restoring OTUD7A or Ankyrin-G expression in 15q13.3 microdeletion neurons led to a reversal of abnormalities. These data reveal a critical OTUD7A-Ankyrin pathway in neuronal development, which is impaired in the 15q13.3 microdeletion syndrome, leading to neuronal dysfunction. Furthermore, our study highlights the utility of targeting CNV genes using cell type-specific proteomics to identify shared and unexplored disease mechanisms across NDDs.
Topics: Humans; Mice; Animals; Ankyrins; DNA Copy Number Variations; Epilepsy; Neurons
PubMed: 36604605
DOI: 10.1038/s41380-022-01937-5 -
Communications Biology Jan 2023Fast synaptic inhibition is dependent on targeting specific GABAR subtypes to dendritic and axon initial segment (AIS) synapses. Synaptic GABARs are typically assembled...
Fast synaptic inhibition is dependent on targeting specific GABAR subtypes to dendritic and axon initial segment (AIS) synapses. Synaptic GABARs are typically assembled from α1-3, β and γ subunits. Here, we isolate distinct GABARs from the brain and interrogate their composition using quantitative proteomics. We show that α2-containing receptors co-assemble with α1 subunits, whereas α1 receptors can form GABARs with α1 as the sole α subunit. We demonstrate that α1 and α2 subunit-containing receptors co-purify with distinct spectrin isoforms; cytoskeletal proteins that link transmembrane proteins to the cytoskeleton. β2-spectrin was preferentially associated with α1-containing GABARs at dendritic synapses, while β4-spectrin was associated with α2-containing GABARs at AIS synapses. Ablating β2-spectrin expression reduced dendritic and AIS synapses containing α1 but increased the number of synapses containing α2, which altered phasic inhibition. Thus, we demonstrate a role for spectrins in the synapse-specific targeting of GABARs, determining the efficacy of fast neuronal inhibition.
Topics: Receptors, GABA-A; Spectrin; Synapses; Membrane Proteins; gamma-Aminobutyric Acid
PubMed: 36604600
DOI: 10.1038/s42003-022-04381-x -
Cells Dec 2022Mitochondrial Ca efflux by NCLX is a critical rate-limiting step in mitochondria signaling. We previously showed that NCLX is phosphorylated at a putative Casein Kinase...
Mitochondrial Ca efflux by NCLX is a critical rate-limiting step in mitochondria signaling. We previously showed that NCLX is phosphorylated at a putative Casein Kinase 2 (CKII) site, the serine 271 (S271). Here, we asked if NCLX is regulated by CKII and interrogated the physiological implications of this control. We found that CKII inhibitors down-regulated NCLX-dependent Ca transport activity in SH-SY5Y neuronal cells and primary hippocampal neurons. Furthermore, we show that the CKII phosphomimetic mutants on NCLX inhibited (S271A) and constitutively activated (S271D) NCLX transport, respectively, rendering it insensitive to CKII inhibition. These phosphomimetic NCLX mutations also control the allosteric regulation of NCLX by mitochondrial membrane potential (ΔΨm). Since the omnipresent CKII is necessary for modulating the plasticity of the axon initial segment (AIS), we interrogated, in hippocampal neurons, if NCLX is required for this process. Similarly to WT neurons, NCLX-KO neurons can exhibit homeostatic plasticity following M-channel block. However, while WT neurons utilize a CKII-sensitive distal relocation of AIS Na and Kv7 channels to decrease their intrinsic excitability, we did not observe such translocation in NCLX-KO neurons. Thus, our results indicate that NCLX is regulated by CKII and is a crucial link between CKII signaling and fast neuronal plasticity.
Topics: Humans; Axon Initial Segment; Casein Kinase II; Homeostasis; Mitochondria; Neuroblastoma; Neuronal Plasticity
PubMed: 36552754
DOI: 10.3390/cells11243990 -
Brain Communications 2022Grey matter damage has been established as a key contributor to disability progression in multiple sclerosis. Aside from neuronal loss and axonal transections, which...
Grey matter damage has been established as a key contributor to disability progression in multiple sclerosis. Aside from neuronal loss and axonal transections, which predominate in cortical demyelinated lesions, synaptic alterations have been detected in both demyelinated plaques and normal-appearing grey matter, resulting in functional neuronal damage. The axon initial segment is a key element of neuronal function, responsible for action potential initiation and maintenance of neuronal polarity. Despite several reports of profound axon initial segment alterations in different pathological models, among which experimental auto-immune encephalomyelitis, whether the axon initial segment is affected in multiple sclerosis is still unknown. Using immunohistochemistry, we analysed axon initial segments from control and multiple sclerosis tissue, focusing on layer 5/6 pyramidal neurons in the neocortex and Purkinje cells in the cerebellum and performed analysis on the parameters known to control neuronal excitability, i.e. axon initial segment length and position. We found that the axon initial segment length was increased only in pyramidal neurons of inactive demyelinated lesions, compared with normal appearing grey matter tissue. In contrast, in both cell types, the axon initial segment position was altered, with an increased soma-axon initial segment gap, in both active and inactive demyelinated lesions. In addition, using a computational model, we show that this increased gap between soma and axon initial segment might increase neuronal excitability. Taken together, these results show, for the first time, changes of axon initial segments in multiple sclerosis, in active as well as inactive grey matter lesions in both neocortex and cerebellum, which might alter neuronal function.
PubMed: 36451656
DOI: 10.1093/braincomms/fcac284 -
International Journal of Molecular... Nov 2022The diagnosis of chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) is based on a combination of clinical, electrodiagnostic and laboratory features. The... (Review)
Review
The diagnosis of chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) is based on a combination of clinical, electrodiagnostic and laboratory features. The different entities of the disease include chronic immune sensory polyradiculopathy (CISP) and autoimmune nodopathies. It is debatable whether CIDP occurring in the course of other conditions, i.e., monoclonal IgG or IgA gammopathy, should be treated as a separate disease entity from idiopathic CIDP. This study aims to evaluate the molecular differences of the nodes of Ranvier and the initial axon segment (AIS) and juxtaparanode region (JXP) as the potential cause of phenotypic variation of CIDP while also seeking new pathomechanisms since JXP is sequestered behind the paranode and autoantibodies may not access the site easily. The authors initially present the structure of the different parts of the neuron and its functional significance, then discuss the problem of whether damage to the juxtaparanodal region, Schwann cells and axons could cause CIDP or if these damages should be separated as separate disease entities. In particular, AIS's importance for modulating neural excitability and carrying out transport along the axon is highlighted. The disclosure of specific pathomechanisms, including novel target antigens, in the heterogeneous CIDP syndrome is important for diagnosing and treating these patients.
Topics: Humans; Polyradiculoneuropathy, Chronic Inflammatory Demyelinating; Axons; Autoantibodies; Syndrome
PubMed: 36362407
DOI: 10.3390/ijms232113621