Biology subjects

Trimmer, J.

Publications and source records attributed to Trimmer, J..

6 recordsLinked to original sources

bioRxiv2025

Kv2/Kv6.4 heteromeric potassium channels are expressed in spinal motor neurons and localized at C-bouton synapses

Voltage-gated K+ channels of the Kv2 family co-assemble with electrically silent KvS subunits in specific subpopulations of brain neurons, forming heteromeric Kv2/KvS channels with distinct functional properties. Little is known about the composition and function of Kv2 channels in spinal cord neurons, however. Here, we show that while Kv2.1 is broadly expressed in multiple classes of spinal cord neurons, the Kv6.4 "electrically-silent" subunit is specifically expressed in motor neurons. In motor neurons, we find that Kv6.4 protein is co-clustered with Kv2.1 and Kv2.2 subunits at endoplasmic reticulum-plasma membrane (ER-PM) junctions beneath C-bouton synapses. In Kv2.1 S590A mutant mice, in which Kv2.1 is unable to bind ER VAP proteins, Kv2.1 and Kv6.4 clustering at ER-PM junctions is severely reduced suggesting Kv2 channels are localized at ER-PM junctions by the same molecular mechanism in motor neurons and brain neurons. Moreover, clustering of Kv6.4, as well as the AMIGO-1 auxiliary subunit, are severely reduced in Kv2.1 knockout mice and moderately reduced in Kv2.2 knockout mice. Thus, expression and localization of Kv6.4 subunits is dependent on Kv2 subunits, likely through their co-assembly into heteromeric channels. Finally, we find that presynaptic C-boutons and postsynaptic clusters of the ER-resident sigma1-receptor are preserved in motor neurons of Kv2 knockout mice. Together, these findings identify a specific Kv2/KvS channel subtype expressed in motor neurons that localizes to C-bouton junctions where it could regulate neuronal excitability and signaling at ER-PM junctions.

neuroscience↗

bioRxiv2025

MAPT Splicing Modulators as a Therapeutic Strategy for Tauopathies

Tauopathies are neurodegenerative diseases characterized by the abnormal accumulation of microtubule-associated protein tau (MAPT) in the brain. These disorders, like frontotemporal dementia (FTD-Tau), currently lack effective therapies and can occur sporadically or be inherited when associated with MAPT gene mutations. The MAPT gene region encompassing exon 10 and adjacent introns is a hotspot for pathogenic variants, including splicing mutations that enhance exon 10 inclusion and increase 4R tau expression, and gain-of-function mutations that generate aggregation-prone mutant 4R tau protein. For these 4R-specific tauopathies, a targeted mRNA splicing approach that promotes exon 10 exclusion may offer therapeutic benefit. In this study, we discovered novel splicing modulator compounds (SMCs) that promote MAPT exon 10 exclusion, and demonstrated their efficacy in FTD patient-derived neuronal models carrying the tau-P301L gain-of-function mutation or the tau-S305N splicing mutation. Treatment with SMC reduced 4R tau expression and decreased the accumulation of hyperphosphorylated tau (pTau), oligomeric and insoluble tau, thereby rescuing tau-associated neuronal toxicity. Importantly, our lead SMC corrected the 3R/4R splice ratio in vivo and significantly reduced pTau in the brain of a gene- replacement (GR) mouse model expressing the human tau-N279K splicing mutation. These findings support the therapeutic potential of this class of small molecules and establish MAPT pre- mRNA splicing modulation as a promising strategy for the treatment of 4R tauopathies. One Sentence SummaryDiscovery of SMCs that correct MAPT splicing, reduce 4R tau, and rescue pathology in patient- derived neuronal and in vivo models of 4R tauopathies.

neuroscience↗

bioRxiv2023

Developing a Toolbox of Antibodies Validated for Array Tomography-Based Imaging of Brain Synapses

Antibody-based imaging techniques rely on reagents whose performance may be application-specific. Because commercial antibodies are validated for only a few purposes, users interested in other applications may have to perform extensive in-house antibody testing. Here we present a novel application-specific proxy screening step to efficiently identify candidate antibodies for array tomography (AT), a serial section volume microscopy technique for high-dimensional quantitative analysis of the cellular proteome. To identify antibodies suitable for AT-based analysis of synapses in mammalian brain, we introduce a heterologous cell-based assay that simulates characteristic features of AT, such as chemical fixation and resin embedding that are likely to influence antibody binding. The assay was included into an initial screening strategy to generate monoclonal antibodies that can be used for AT. This approach simplifies the screening of candidate antibodies and has high predictive value for identifying antibodies suitable for AT analyses. In addition, we have created a comprehensive database of AT-validated antibodies with a neuroscience focus and show that these antibodies have a high likelihood of success for postembedding applications in general, including immunogold electron microscopy. The generation of a large and growing toolbox of AT-compatible antibodies will further enhance the value of this imaging technique. Significance StatementArray tomography (AT) is a powerful volume microscopy technique for high-dimensional analysis of complex protein populations in cells and organelles, including synapses. AT involves the use of ultrathin serial sections embedded in resin and subjected to multiple rounds of immunofluorescence antibody (Ab) labeling and imaging. AT relies on antibody-based detection of proteins but because commercial antibodies are typically validated for other applications they often fail for AT. To identify antibodies with high probability of success in AT we developed a novel screening strategy and used this to create a comprehensive database of AT-validated antibodies for neuroscience.

neuroscience↗

bioRxiv2019

Precision calcium imaging of dense neural populations via a cell body-targeted calcium indicator

Methods for one-photon fluorescent imaging of calcium dynamics in vivo are popular due to their ability to simultaneously capture the dynamics of hundreds of neurons across large fields of view, at a low equipment complexity and cost. In contrast to two-photon methods, however, one-photon methods suffer from higher levels of crosstalk between cell bodies and the surrounding neuropil, resulting in decreased signal-to-noise and artifactual correlations of neural activity. Here, we address this problem by engineering cell body-targeted variants of the fluorescent calcium indicator GCaMP6f. We screened fusions of GCaMP6f to both natural as well as engineered peptides, and identified fusions that localized GCaMP6f to within approximately 50 microns of the cell body of neurons in live mice and larval zebrafish. One-photon imaging of soma-targeted GCaMP6f in dense neural circuits reported fewer artifactual spikes from neuropil, increased signal-to-noise ratio, and decreased artifactual correlation across neurons. Thus, soma-targeting of fluorescent calcium indicators increases neuronal signal fidelity and may facilitate even greater usage of simple, powerful, one-photon methods of population imaging of neural calcium dynamics.

neuroscience↗

bioRxiv2018

A toolbox of IgG subclass-switched recombinant monoclonal antibodies for enhanced multiplex immunolabeling of brain

Generating recombinant monoclonal antibodies (R-mAbs) from mAb-producing hybridomas offers numerous advantages that increase the effectiveness, reproducibility, and transparent reporting of research. We report here the generation of a novel resource in the form of a library of recombinant R-mAbs validated for neuroscience research. We cloned immunoglobulin G (IgG) variable domains from cryopreserved hybridoma cells and input them into an integrated pipeline for expression and validation of functional R-mAbs. To improve efficiency over standard protocols, we eliminated aberrant Sp2/0-Ag14 hybridoma-derived variable light transcripts using restriction enzyme treaent. Further, we engineered a plasmid backbone that allows for switching of the IgG subclasses without altering target binding specificity to generate R-mAbs useful in simultaneous multiplex labeling experiments not previously possible. The method was also employed to rescue IgG variable sequences and generate functional R-mAbs from a non-viable cryopreserved hybridoma. All R-mAb sequences and plasmids will be archived and disseminated from open source suppliers.

neuroscience↗

bioRxiv2018

Organizing neuronal ER-PM junctions is a conserved nonconducting function of Kv2 plasma membrane ion channels

Endoplasmic reticulum (ER) and plasma membrane (PM) form junctions crucial to ion and lipid signaling and homeostasis. The Kv2.1 ion channel is unique among PM proteins in organizing ER-PM junctions. Here, we show that this organizing function is conserved between Kv2 family members that differ in their biophysical properties, modulation and cellular expression. Manipulation of actin cytoskeleton surrounding Kv2 ER-PM junctions affects their spatial organization. Kv2-containing ER-PM junctions overlap with those formed by canonical ER-PM tethers. ER-PM junction organization by Kv2 channels is unchanged by point mutations that eliminate ion conduction, but abolished by those that eliminate PM clustering without impacting ion channel function. Kv2.2 is distinct in lacking the reversible modulation of junction organization present in Kv2.1. Brain neurons in Kv2 double knockout mice have altered ER-PM junctions, demonstrating a conserved in vivo function for Kv2 family members distinct from their canonical role as ion-conducting channels shaping neuronal excitability.

cell biology↗