-
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 -
Channels (Austin, Tex.) Dec 2020Voltage gated sodium channels (Na) play a crucial role in action potential initiation and propagation. Although the discovery of Na channels dates back more than... (Review)
Review
Voltage gated sodium channels (Na) play a crucial role in action potential initiation and propagation. Although the discovery of Na channels dates back more than 65 years, and great advances in understanding their localization, biophysical properties, and links to disease have been made, there are still many questions to be answered regarding the cellular and molecular mechanisms involved in Na channel trafficking, localization and regulation. This review summarizes the different trafficking mechanisms underlying the polarized Na channel localization in neurons, with an emphasis on the axon initial segment (AIS), as well as discussing the latest advances regarding how neurons regulate their excitability by modifying AIS length and location. The importance of Na channel localization is emphasized by the relationship between mutations, impaired trafficking and disease. While this review focuses on Na1.6, other Na isoforms are also discussed.
Topics: Action Potentials; Animals; Axon Initial Segment; Female; Humans; NAV1.6 Voltage-Gated Sodium Channel; Neurons; Protein Transport; Voltage-Gated Sodium Channels
PubMed: 31841065
DOI: 10.1080/19336950.2019.1700082 -
Neurology(R) Neuroimmunology &... May 2023Autoantibodies to leucine-rich glioma inactivated protein 1 (LGI1) cause an autoimmune limbic encephalitis with frequent focal seizures and anterograde memory...
BACKGROUND AND OBJECTIVES
Autoantibodies to leucine-rich glioma inactivated protein 1 (LGI1) cause an autoimmune limbic encephalitis with frequent focal seizures and anterograde memory dysfunction. LGI1 is a neuronal secreted linker protein with 2 functional domains: the leucine-rich repeat (LRR) and epitempin (EPTP) regions. LGI1 autoantibodies are known to interfere with presynaptic function and neuronal excitability; however, their epitope-specific mechanisms are incompletely understood.
METHODS
We used patient-derived monoclonal autoantibodies (mAbs), which target either LRR or EPTP domains of LGI1 to investigate long-term antibody-induced alteration of neuronal function. LRR- and EPTP-specific effects were evaluated by patch-clamp recordings in cultured hippocampal neurons and compared with biophysical neuron modeling. K1.1 channel clustering at the axon initial segment (AIS) was quantified by immunocytochemistry and structured illumination microscopy techniques.
RESULTS
Both EPTP and LRR domain-specific mAbs decreased the latency of first somatic action potential firing. However, only the LRR-specific mAbs increased the number of action potential firing together with enhanced initial instantaneous frequency and promoted spike-frequency adaptation, which were less pronounced after the EPTP mAb. This also led to an effective reduction in the slope of ramp-like depolarization in the subthreshold response, suggesting K1 channel dysfunction. A biophysical model of a hippocampal neuron corroborated experimental results and suggests that an isolated reduction of the conductance of K1-mediated K currents largely accounts for the antibody-induced alterations in the initial firing phase and spike-frequency adaptation. Furthermore, K1.1 channel density was spatially redistributed from the distal toward the proximal site of AIS under LRR mAb treatment and, to a lesser extant, under EPTP mAb.
DISCUSSION
These findings indicate an epitope-specific pathophysiology of LGI1 autoantibodies. The pronounced neuronal hyperexcitability and SFA together with dropped slope of ramp-like depolarization after LRR-targeted interference suggest disruption of LGI1-dependent clustering of K channel complexes. Moreover, considering the effective triggering of action potentials at the distal AIS, the altered spatial distribution of K1.1 channel density may contribute to these effects through impairing neuronal control of action potential initiation and synaptic integration.
Topics: Humans; Antibodies, Monoclonal; Autoantibodies; Epitopes; Intracellular Signaling Peptides and Proteins; Leucine; Nerve Tissue Proteins; Neurons
PubMed: 37028941
DOI: 10.1212/NXI.0000000000200096 -
Translational Vision Science &... Apr 2023Axons depend on long-range transport of proteins and organelles which increases susceptibility to metabolic stress in disease. The axon initial segment (AIS) is...
PURPOSE
Axons depend on long-range transport of proteins and organelles which increases susceptibility to metabolic stress in disease. The axon initial segment (AIS) is particularly vulnerable due to the high bioenergetic demand of action potential generation. Here, we prepared retinal ganglion cells derived from human embryonic stem cells (hRGCs) to probe how axonal stress alters AIS morphology.
METHODS
hRGCs were cultured on coverslips or microfluidic platforms. We assayed AIS specification and morphology by immunolabeling against ankyrin G (ankG), an axon-specific protein, and postsynaptic density 95 (PSD-95), a dendrite-specific protein. Using microfluidic platforms that enable fluidic isolation, we added colchicine to the axon compartment to lesion axons. We verified axonopathy by measuring the anterograde axon transport of cholera toxin subunit B and immunolabeling against cleaved caspase 3 (CC3) and phosphorylated neurofilament H (SMI-34). We determined the influence of axon injury on AIS morphology by immunolabeling samples against ankG and measuring AIS distance from soma and length.
RESULTS
Based on measurements of ankG and PSD-95 immunolabeling, microfluidic platforms promote the formation and separation of distinct somatic-dendritic versus axonal compartments in hRGCs compared to coverslip cultures. Chemical lesioning of axons by colchicine reduced hRGC anterograde axon transport, increased varicosity density, and enhanced expression of CC3 and SMI-34. Interestingly, we found that colchicine selectively affected hRGCs with axon-carrying dendrites by reducing AIS distance from somas and increasing length, thus suggesting reduced capacity to maintain excitability.
CONCLUSIONS
Thus, microfluidic platforms promote polarized hRGCs that enable modeling of axonopathy.
TRANSLATIONAL RELEVANCE
Microfluidic platforms may be used to assay compartmentalized degeneration that occurs during glaucoma.
Topics: Humans; Retinal Ganglion Cells; Microfluidics; Axons; Action Potentials
PubMed: 37010860
DOI: 10.1167/tvst.12.4.1 -
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 -
Molecular Neurobiology Dec 2021The axon initial segment (AIS) is essential for maintaining neuronal polarity, modulating protein transport into the axon, and action potential generation. These...
The axon initial segment (AIS) is essential for maintaining neuronal polarity, modulating protein transport into the axon, and action potential generation. These functions are supported by a distinctive actin and microtubule cytoskeleton that controls axonal trafficking and maintains a high density of voltage-gated ion channels linked by scaffold proteins to the AIS cytoskeleton. However, our knowledge of the mechanisms and proteins involved in AIS cytoskeleton regulation to maintain or modulate AIS structure is limited. In this context, formins play a significant role in the modulation of actin and microtubules. We show that pharmacological inhibition of formins modifies AIS actin and microtubule characteristics in cultured hippocampal neurons, reducing F-actin density and decreasing microtubule acetylation. Moreover, formin inhibition diminishes sodium channels, ankyrinG and βIV-spectrin AIS density, and AIS length, in cultured neurons and brain slices, accompanied by decreased neuronal excitability. We show that genetic downregulation of the mDia1 formin by interference RNAs also decreases AIS protein density and shortens AIS length. The ankyrinG decrease and AIS shortening observed in pharmacologically inhibited neurons and neuron-expressing mDia1 shRNAs were impaired by HDAC6 downregulation or EB1-GFP expression, known to increase microtubule acetylation or stability. However, actin stabilization only partially prevented AIS shortening without affecting AIS protein density loss. These results suggest that mDia1 maintain AIS composition and length contributing to the stability of AIS microtubules.
Topics: Animals; Axon Initial Segment; Axons; Cells, Cultured; Cytoskeleton; Formins; Hippocampus; Mice; Microtubules; Neurons
PubMed: 34458961
DOI: 10.1007/s12035-021-02531-6 -
Advanced Science (Weinheim,... Oct 2023In tauopathy conditions, such as Alzheimer's disease (AD), highly soluble and natively unfolded tau polymerizes into an insoluble filament; however, the mechanistic...
In tauopathy conditions, such as Alzheimer's disease (AD), highly soluble and natively unfolded tau polymerizes into an insoluble filament; however, the mechanistic details of this process remain unclear. In the brains of AD patients, only a minor segment of tau forms β-helix-stacked protofilaments, while its flanking regions form disordered fuzzy coats. Here, it is demonstrated that the tau AD nucleation core (tau-AC) sufficiently induced self-aggregation and recruited full-length tau to filaments. Unexpectedly, phospho-mimetic forms of tau-AC (at Ser324 or Ser356) show markedly reduced oligomerization and seeding propensities. Biophysical analysis reveal that the N-terminus of tau-AC facilitates the fibrillization kinetics as a nucleation motif, which becomes sterically shielded through phosphorylation-induced conformational changes in tau-AC. Tau-AC oligomers are efficiently internalized into cells via endocytosis and induced endogenous tau aggregation. In primary hippocampal neurons, tau-AC impaired axon initial segment plasticity upon chronic depolarization and is mislocalized to the somatodendritic compartments. Furthermore, it is observed significantly impaired memory retrieval in mice intrahippocampally injected with tau-AC fibrils, which corresponds to the neuropathological staining and neuronal loss in the brain. These findings identify tau-AC species as a key neuropathological driver in AD, suggesting novel strategies for therapeutic intervention.
Topics: Mice; Humans; Animals; Alzheimer Disease; tau Proteins; Brain; Neurons; Phosphorylation
PubMed: 37594721
DOI: 10.1002/advs.202302035 -
ELife Dec 2021Inhibitory neurons in mammalian cortex exhibit diverse physiological, morphological, molecular, and connectivity signatures. While considerable work has measured the...
Inhibitory neurons in mammalian cortex exhibit diverse physiological, morphological, molecular, and connectivity signatures. While considerable work has measured the average connectivity of several interneuron classes, there remains a fundamental lack of understanding of the connectivity distribution of distinct inhibitory cell types with synaptic resolution, how it relates to properties of target cells, and how it affects function. Here, we used large-scale electron microscopy and functional imaging to address these questions for chandelier cells in layer 2/3 of the mouse visual cortex. With dense reconstructions from electron microscopy, we mapped the complete chandelier input onto 153 pyramidal neurons. We found that synapse number is highly variable across the population and is correlated with several structural features of the target neuron. This variability in the number of axo-axonic ChC synapses is higher than the variability seen in perisomatic inhibition. Biophysical simulations show that the observed pattern of axo-axonic inhibition is particularly effective in controlling excitatory output when excitation and inhibition are co-active. Finally, we measured chandelier cell activity in awake animals using a cell-type-specific calcium imaging approach and saw highly correlated activity across chandelier cells. In the same experiments, in vivo chandelier population activity correlated with pupil dilation, a proxy for arousal. Together, these results suggest that chandelier cells provide a circuit-wide signal whose strength is adjusted relative to the properties of target neurons.
Topics: Animals; Female; Male; Mice; Microscopy, Electron, Transmission; Pyramidal Cells; Synapses; Visual Cortex
PubMed: 34851292
DOI: 10.7554/eLife.73783 -
The Journal of Neuroscience : the... Jul 2022Brain enriched voltage-gated sodium channel (VGSC) Na1.2 and Na1.6 are critical for electrical signaling in the CNS. Previous studies have extensively characterized...
Brain enriched voltage-gated sodium channel (VGSC) Na1.2 and Na1.6 are critical for electrical signaling in the CNS. Previous studies have extensively characterized cell-type-specific expression and electrophysiological properties of these two VGSCs and how their differences contribute to fine-tuning of neuronal excitability. However, because of a lack of reliable labeling and imaging methods, the subcellular localization and dynamics of these homologous Na1.2 and Na1.6 channels remain understudied. To overcome this challenge, we combined genome editing, super-resolution, and live-cell single-molecule imaging to probe subcellular composition, relative abundances, and trafficking dynamics of Na1.2 and Na1.6 in cultured mouse and rat neurons and in male and female mouse brain. We discovered a previously uncharacterized trafficking pathway that targets Na1.2 to the distal axon of unmyelinated neurons. This pathway uses distinct signals residing in the intracellular loop 1 between transmembrane domain I and II to suppress the retention of Na1.2 in the axon initial segment and facilitate its membrane loading at the distal axon. As mouse pyramidal neurons undergo myelination, Na1.2 is gradually excluded from the distal axon as Na1.6 becomes the dominant VGSC in the axon initial segment and nodes of Ranvier. In addition, we revealed exquisite developmental regulation of Na1.2 and Na1.6 localizations in the axon initial segment and dendrites, clarifying the molecular identity of sodium channels in these subcellular compartments. Together, these results unveiled compartment-specific localizations and trafficking mechanisms for VGSCs, which could be regulated separately to modulate membrane excitability in the brain. Direct observation of endogenous voltage-gated sodium channels reveals a previously uncharacterized distal axon targeting mechanism and the molecular identity of sodium channels in distinct subcellular compartments.
PubMed: 35672149
DOI: 10.1523/JNEUROSCI.0086-22.2022 -
Cells Aug 2020By binding to actin filaments, non-muscle myosin II (NMII) generates actomyosin networks that hold unique contractile properties. Their dynamic nature is essential for... (Review)
Review
By binding to actin filaments, non-muscle myosin II (NMII) generates actomyosin networks that hold unique contractile properties. Their dynamic nature is essential for neuronal biology including the establishment of polarity, growth cone formation and motility, axon growth during development (and axon regeneration in the adult), radial and longitudinal axonal tension, and synapse formation and function. In this review, we discuss the current knowledge on the spatial distribution and function of the actomyosin cytoskeleton in different axonal compartments. We highlight some of the apparent contradictions and open questions in the field, including the role of NMII in the regulation of axon growth and regeneration, the possibility that NMII structural arrangement along the axon shaft may control both radial and longitudinal contractility, and the mechanism and functional purpose underlying NMII enrichment in the axon initial segment. With the advances in live cell imaging and super resolution microscopy, it is expected that in the near future the spatial distribution of NMII in the axon, and the mechanisms by which it participates in axonal biology will be further untangled.
Topics: Axons; Growth Cones; Humans
PubMed: 32858875
DOI: 10.3390/cells9091961