Rashmi Kumari

Proximity-based proteomics using TurboID has enabled cell-type–specific profiling without the need for cell purification, although major bottlenecks in sample lysis, biotinylated protein enrichment, digestion, and mass spectrometry (MS) parameters have limited depth of proteome coverage. Here, we systematically optimized these variables using TurboID-based labeling of BV2 microglia in vitro and brain astrocytes in vivo to define conditions that maximize proteome coverage. In microglia, the optimized protocol using 8 M urea lysis with on-bead S-Trap digestion and data-independent acquisition MS (DIA-MS) identified 4,016 proteins, double the depth of prior studies, and revealed metabolic, ribosomal, lipid-processing, autophagy, and trafficking signatures. Brain astrocyte proteomes were best recovered using SDS lysis with S-Trap digestion and DIA-MS, yielding a proteome of over 3,600 highly enriched proteins, twice the depth of prior astrocyte-TurboID studies. The expanded astrocyte proteomes captured canonical astrocyte markers as well as membrane-associated, vesicular trafficking, and presynaptic protein signatures, consistent with labeling of astrocyte–neuron interface regions, including proteins involved in receptor signaling, lipid metabolism, and plasticity at tripartite synapses, and several AD risk proteins. The increased peptide recovery following S-Trap digestion allowed the reduction of starting material to 20 µg protein for DIA-MS, and enabled multiplexed tandem mass tag (TMT-MS) proteomics using even smaller samples. When applied to synaptosomes enriched from mouse brains with neuronal TurboID labeling, our pipeline identified a synapse-specific proteome of 2,529 proteins, revealing synaptic, mitochondrial and disease-relevant signatures not detectable in prior studies. By tackling critical bottlenecks from tissue processing to MS, our optimized pipelines enable cell-type and compartment-specific proximity-labeling proteomics to obtain comprehensive biological and disease-relevant insights across various biological fields.

Inhibition of voltage-gated potassium channel Kv1.3 is a therapeutic strategy to curb microglia-mediated neuroinflammation in neurodegeneration, although the cellular and signaling mechanisms of disease-modification by Kv1.3 blockers are unclear. In this study, we delineate protective mechanisms of Kv1.3 blockade in a mouse model of Alzheimers disease (AD) pathology using comprehensive transcriptomics and proteomics profiling of brain, corresponding with neuropathological effects of two translationally relevant Kv1.3 blockers, namely small molecule PAP-1 and peptide ShK-223. Following 3 months of treatment, both molecules reduced Ab plaque burden. Single nuclear RNA seq (snRNA seq) of brain nuclei showed that PAP-1 disproportionately impacted oligodendrocytes and microglia and increased crosstalk between neurons and astrocytes with endothelial cells. In contrast, ShK-223 had pronounced effects on glutamatergic neurons and astrocytes. Both blockers increased expression of myelination genes in oligodendrocytes and synaptic genes in neurons. Neuroprotective effects of PAP-1 were further confirmed by bulk brain transcriptomics and proteomics whereby PAP-1 increased levels of synaptic, cognitive resilience and mitochondrial proteins, while decreasing glial and immune pathways including STAT1/3 phosphorylation. Using proximity labeling and co-immunoprecipitation, we found that Kv1.3 interacts with STAT1/3 in microglia. Using microglial cell lines and primary microglia, we discovered a preferential functional coupling between Kv1.3 and type 2 but not type 1 IFN signaling. Brain-level disease modification by Kv1.3 blockade was reflected in the cerebrospinal fluid (CSF) via reduced levels of neurofilament-light (NEFL) and resilience protein RPH3A, both of which are increased in human AD CSF. Together, this study demonstrates functional links between Kv1.3 channels and type 2 IFN signaling and reveals distinct cellular effects of Kv1.3 blockers in AD pathology that correspond with reduced neuropathology and neuroinflammation, augmentation of resilience and neuro-vascular pathways, along with biomarkers of therapeutic effect.