Biology subjects

Rasband, M. N.

Publications and source records attributed to Rasband, M. N..

10 recordsLinked to original sources

bioRxiv2026

Paralemmin 2 is a shared, alternatively-spliced regulator of axon initial segments and paranodal junctions in oligodendrocytes

Nervous system function depends on highly specialized axonal membrane domains. For example, axon initial segments (AIS), nodes of Ranvier, and paranodal junctions are essential for action potential initia-tion and saltatory conduction. These domains use shared molecular machinery converging on the master scaffolding protein AnkyrinG (AnkG). Using endogenous AnkG-TurboID proximity proteomics, we identify Paralemmin 2 (Palm2), a previously uncharacterized CNS protein, as a shared component of the AIS and nodes of Ranvier in neurons, and paranodes in oligodendrocytes. Palm2 localizes transiently to the AIS and nodes during development, but is found at paranodes throughout life, reflecting cell type-specific splicing of a lipid-modified domain both necessary and sufficient for AnkG interaction. Mice lacking oli-godendroglial Palm2 assemble normal paranodes during development, but aged mice lose paranode in-tegrity due to loss of paranodal AnkG and Neurofascin-155. Our findings identify Palm2 as a lipid raft-associated regulator of axonal and oligodendroglial membrane domains and reveal cell type-specific pro-tein variants as a recurring principle for specializing axonal and glial membrane architectures.

neuroscience↗

bioRxiv2025

OASIS: in vivo AAV-mediated transduction and genome editing of adult oligodendrocytes

New viral approaches have revolutionized neuroscience by precisely delivering genes in neurons; for example, to control or monitor activity in specific neuronal cell types. In contrast, the manipulation of oligodendrocytes requires the Cre-LoxP system and gene-by-gene engineering, breeding, and genotyping. Here we introduce OASIS (Oligodendrocyte AAV-CRISPR mediated Specific In vivo editing System), a versatile platform that combines SELECTIV, an AAV-receptor-based transduction strategy, with HiUGE, an NHEJ-mediated CRISPR/Cas9 knock-in approach. We show efficient and specific oligodendrocyte transduction across the brain and tagging of endogenous cytoskeletal, myelin, cell adhesion, scaffolding, and junctional proteins. OASIS enables sparse yet reliable labeling, allowing direct visualization of a proteins subcellular localization with single-cell resolution. We successfully fused the biotin-ligase TurboID with endogenous oligodendroglial Neurofascin-155, thereby achieving targeted biotinylation of the axoglial junction. OASIS is rapidly customizable for any gene-of-interest. Together, OASIS overcomes longstanding barriers in oligodendrocyte biology, providing a powerful system for precise, customizable genome editing and subcellular visualization in the adult brain.

neuroscience↗

bioRxiv2024

Diverse and Location-Specific Roles of PlexinA2, PlexinA4, and NCAM in Developing Hippocampal Mossy Fibers

Mossy fibers (MFs) originate from dentate granule cells and innervate area CA3 of the hippocampus. Upon entry of CA3, MFs partition into two prominent axon bundles, the suprapyramidal tract (SPT) and infrapyramidal tract (IPT) and form lamina specific synaptic contacts in the stratum lucidum (SL) and stratum oriens (SO), respectively. Here we employed new mouse lines to dissect the function of Sema6A and its receptors, PlexinA2 (PlxnA2) and PlxnA4, in developing MFs. In Sema6a-/- mice, MF partitioning into SPT and IPT bundles is incomplete and IPT axons in the SO are overextended, while the SPT correctly innervates the SL. Loss of neuronal Sema6a results in defective MF patterning and we show that this involves Sema6A reverse signaling. Plxna4 controls MF partitioning, SPT axon bundling and laminar targeting to the SL, as well as IPT length. Many of these defects are replicated in mice deficient for PlxnA4 GAP catalytic activity, underscoring the importance of this GAP domain. MFs are tightly fasciculated in Plxna2-/- mice and fail to separate into SPT and IPT bundles, and defects are significantly reduced in PlxnA2 GAP mutants, highlighting the involvement of GAP-independent signaling events. To further explore the molecular basis of aberrant axon fasciculation, we employed anti-PlxnA2 dependent proximity biotinylation and identified several PlxnA2-associated Ig-CAM family members. We observed a genetic interaction between Plxna2 and Ncam1, but not Plxna4 and Ncam1, for SPT and IPT formation and positioning in CA3. Together, our studies provide insights into the multifaceted and overlapping, yet distinct, functions of PlxnA family members in orchestrating specific guidance decisions in developing MFs.

developmental biology↗

bioRxiv2024

TRIM46 is not required for axon specification or axon initial segment formation in vivo

Vertebrate nervous systems use the axon initial segment (AIS) to initiate action potentials and maintain neuronal polarity. The microtubule-associated protein tripartite motif containing 46 (TRIM46) was reported to regulate axon specification, AIS assembly, and neuronal polarity through the bundling of microtubules in the proximal axon. However, these claims are based on TRIM46 knockdown in cultured neurons. To investigate TRIM46 function in vivo, we examined TRIM46 knockout mice. Contrary to previous reports, we find that TRIM46 is dispensable for AIS formation and maintenance, and axon specification. TRIM46 knockout mice are viable, have normal behavior, and have normal brain structure. Thus, TRIM46 is not required for AIS formation, axon specification, or nervous system function. We also show TRIM46 enrichment in the first [~]100 m of axon occurs independently of ankyrinG (AnkG), although AnkG is required to restrict TRIM46 only to the AIS. Our results suggest an unidentified protein may compensate for loss of TRIM46 in vivo and highlight the need for further investigation of the mechanisms by which the AIS and microtubules interact to shape neuronal structure and function. SIGNIFICANCE STATEMENTA healthy nervous system requires the polarization of neurons into structurally and functionally distinct compartments, which depends on both the axon initial segment (AIS) and the microtubule cytoskeleton. In contrast to previous reports, we show that the microtubule-associated protein TRIM46 is not required for axon specification or AIS formation in mice. Our results emphasize the need for further investigation of the mechanisms by which the AIS and microtubules interact to shape neuronal structure and function.

neuroscience↗

bioRxiv2024

Age-dependent regulation of axoglial interactions and behavior by oligodendrocyte AnkyrinG

The bipolar disorder (BD) risk gene ANK3 encodes the scaffolding protein AnkyrinG (AnkG). In neurons, AnkG regulates polarity and ion channel clustering at axon initial segments and nodes of Ranvier. Disruption of neuronal AnkG causes BD-like phenotypes in mice. During development, AnkG is also expressed at comparable levels in oligodendrocytes and facilitates the efficient assembly of paranodal junctions. However, the physiological roles of glial AnkG in the mature nervous system, and its contributions to BD-like phenotypes, remain unexplored. Here, we generated oligodendroglia-specific AnkG conditional knockout mice and observed the destabilization of axoglial interactions in aged but not young adult mice. In addition, these mice exhibited profound histological, electrophysiological, and behavioral pathophysiologies. Unbiased translatomic profiling revealed potential compensatory machineries. These results highlight the critical functions of glial AnkG in maintaining proper axoglial interactions throughout aging and suggests a previously unrecognized contribution of oligodendroglial AnkG to neuropsychiatric disorders.

neuroscience↗

bioRxiv2023

Spatially Resolved Proteomic Profiling Uncovers Structural and Functional Regulators of the Axon Initial Segment

The axon initial segment (AIS) is a specialized neuronal compartment required for action potential generation and neuronal polarity. However, understanding the mechanisms regulating AIS structure and function has been hindered by an incomplete knowledge of its molecular composition. Here, using immuno-proximity biotinylation we further define the AIS proteome and its dynamic changes during neuronal maturation. Among the many AIS proteins identified, we show that SCRIB is highly enriched in the AIS both in vitro and in vivo, and exhibits a periodic architecture like the axonal spectrin-based cytoskeleton. We found that ankyrinG interacts with and recruits SCRIB to the AIS. However, loss of SCRIB has no effect on ankyrinG. This powerful and flexible approach further defines the AIS proteome and provides a rich resource to elucidate the mechanisms regulating AIS structure and function.

neuroscience↗

bioRxiv2023

Antibody-directed extracellular proximity biotinylation reveals Contactin-1 regulates axo-axonic innervation of axon initial segments.

Axon initial segment (AIS) cell surface proteins mediate key biological processes in neurons including action potential initiation and axo-axonic synapse formation. However, few AIS cell surface proteins have been identified. Here, we used antibody-directed proximity biotinylation to define the cell surface proteins in close proximity to the AIS cell adhesion molecule Neurofascin. To determine the distributions of the identified proteins, we used CRISPR-mediated genome editing for insertion of epitope tags in the endogenous proteins. We found Contactin-1 (Cntn1) among the previously unknown AIS proteins we identified. Cntn1 is enriched at the AIS through interactions with Neurofascin and NrCAM. We further show that Cntn1 contributes to assembly of the AIS-extracellular matrix, and is required for AIS axo-axonic innervation by inhibitory basket cells in the cerebellum and inhibitory chandelier cells in the cortex.

neuroscience↗

bioRxiv2021

Ankyrin-R regulates fast-spiking interneuron excitability through perineuronal nets and Kv3.1b K+ channels

Neuronal ankyrins cluster and link membrane proteins to the actin and spectrin-based cytoskeleton. Among the three vertebrate ankyrins, little is known about neuronal Ankyrin-R (AnkR). We report AnkR is highly enriched in Pv+ fast-spiking interneurons in mouse and human. We identify AnkR-associated protein complexes including cytoskeletal proteins, cell adhesion molecules (CAMs), and perineuronal nets (PNNs). We show that loss of AnkR from forebrain interneurons reduces and disrupts PNNs, decreases anxiety-like behaviors, and changes the intrinsic excitability and firing properties of Pv+ fast-spiking interneurons. These changes are accompanied by a dramatic reduction in Kv3.1b K+ channels. We identify a novel AnkR-binding motif in Kv3.1b, and show that AnkR is both necessary and sufficient for Kv3.1b membrane localization in interneurons and at nodes of Ranvier. Thus, AnkR regulates Pv+ fast-spiking interneuron function by organizing ion channels, CAMs, and PNNs, and linking these to the underlying {beta}1 spectrin-based cytoskeleton.

neuroscience↗

bioRxiv2020

Remyelination alters the pattern of myelin in the cerebral cortex

Destruction of oligodendrocytes and myelin sheaths in cortical gray matter profoundly alters neural activity and is associated with cognitive disability in multiple sclerosis (MS). Myelin can be restored by regenerating oligodendrocytes from resident progenitors; however, it is not known whether regeneration restores the complex myelination patterns in cortical circuits. Here we performed time lapse in vivo two photon imaging in somatosensory cortex of adult mice to define the kinetics and specificity of myelin regeneration after acute oligodendrocyte ablation. These longitudinal studies revealed that the pattern of myelination in cortex changed dramatically after regeneration, as new oligodendrocytes were formed in different locations and new sheaths were often established along axon segments previously lacking myelin. Despite the dramatic increase in axonal territory available, oligodendrogenesis was persistently impaired in deeper cortical layers that experienced higher gliosis. The repeated reorganization of myelin patterns in MS may alter circuit function and contribute to cognitive decline.

neuroscience↗

bioRxiv2017

Employing single-stranded DNA donors for the high-throughput production of conditional knockout alleles in mice

The International Mouse Phenotyping Consortium is generating null allele mice for every protein-coding gene in the genome and characterizing these mice to identify gene-phenotype associations. To test the feasibility of using CRISPR/Cas9 gene editing to generate conditional knockout mice for this large-scale resource, we employed Cas9-mediated homology driven repair (HDR) with short and long single-stranded oligodeoxynucleotides (ssODNs and lssODNs). Using pairs of guides and ssODNs donating loxP sites, we obtained putative conditional allele founder mice, harboring both loxP sites, for 23 of 30 genes targeted. LoxP sites integrated in cis in at least one F0 for 18 of 23 targeted genes. However, loxP sites were mutagenized in 4 of 18 in cis lines. HDR efficiency correlated with Cas9 cutting efficiency but was not influenced by ssODN homology arm symmetry. By contrast, using pairs of guides and a single lssODN to introduce a loxP-flanked exon, conditional allele founders were generated for all 4 genes targeted. Our studies demonstrate that Cas9-mediated HDR with pairs of ssODNs can generate conditional null alleles at many loci, but reveal inefficiencies when applied at scale. In contrast, lssODNs are amenable to high-throughput production of conditional alleles when they can be employed.

genetics↗