Background Proteomic characterization of microglia provides the most proximate assessment of functionally relevant molecular mechanisms of neuroinflammation. However, microglial proteomics studies have been limited by low cellular yield and contamination by non-microglial proteins using existing enrichment strategies. Methods We coupled magnetic-activated cell sorting (MACS) and fluorescence activated cell sorting (FACS) of microglia with tandem mass tag-mass spectrometry (TMT-MS) to obtain a highly-pure microglial proteome and identified a core set of highly-abundant microglial proteins in adult mouse brain. We interrogated existing human proteomic data for Alzheimer’s disease (AD) relevance of highly-abundant microglial proteins and performed immuno-histochemical and in-vitro validation studies. Results Quantitative multiplexed proteomics by TMT-MS of CD11b + MACS-enriched (N = 5 mice) and FACS-isolated (N = 5 mice), from adult wild-type mice, identified 1791 proteins. A total of 203 proteins were highly abundant in both datasets, representing a core-set of highly abundant microglial proteins. In addition, we found 953 differentially enriched proteins comparing MACS and FACS-based approaches, indicating significant differences between both strategies. The FACS-isolated microglia proteome was enriched with cytosolic, endoplasmic reticulum, and ribosomal proteins involved in protein metabolism and immune system functions, as well as an abundance of canonical microglial proteins. Conversely, the MACS-enriched microglia proteome was enriched with mitochondrial and synaptic proteins and higher abundance of neuronal, oligodendrocytic and astrocytic proteins. From the 203 consensus microglial proteins with high abundance in both datasets, we confirmed microglial expression of moesin (Msn) in wild-type and 5xFAD mouse brains as well as in human AD brains. Msn expression is nearly exclusively found in microglia that surround Aβ plaques in 5xFAD brains. In in-vitro primary microglial studies, Msn silencing by siRNA decreased Aβ phagocytosis and increased lipopolysaccharide-induced production of the pro-inflammatory cytokine, tumor necrosis factor (TNF). In network analysis of human brain proteomic data, Msn was a hub protein of an inflammatory co-expression module positively associated with AD neuropathological features and cognitive dysfunction. Conclusions Using FACS coupled with TMT-MS as the method of choice for microglial proteomics, we define a core set of highly-abundant adult microglial proteins. Among these, we validate Msn as highly-abundant in plaque-associated microglia with relevance to human AD.

equal contribution as first authors. Levi B. Wood and Srikant Rangaraju equal contribution as senior authors. Srikant Rangaraju lead contact.Abbreviations: 5xFAD, refers to Tg(APPSwFlLon,PSEN1*M146L*L286V)6799Vas/Mmjax strain on C57BL6/J background; AD, Alzheimer's disease; Aβ, amyloid beta; DAM, diseaseassociated microglia; ERK, extracellular signal-regulated kinase; GO, gene ontology; IFNγ, interferon gamma; JNK, c-Jun N-terminal kinase; KEGG, Kyoto Encyclopedia of Genes and Genomes; LPS, lipopolysaccharide; MAGMA, multi-marker analysis of GenoMic annotation; MAPK, mitogen-activated protein kinase; NFκB, nuclear factor kappa-light-chain-enhancer of activated B cells; PANTHER, protein analysis through evolutionary relationships; RTK, receptor tyrosine kinase; TMT, tandem mass tag.

Background The BIN1 locus contains the second-most significant genetic risk factor for late-onset Alzheimer’s disease. BIN1 undergoes alternate splicing to generate tissue- and cell-type-specific BIN1 isoforms, which regulate membrane dynamics in a range of crucial cellular processes. Whilst the expression of BIN1 in the brain has been characterized in neurons and oligodendrocytes in detail, information regarding microglial BIN1 expression is mainly limited to large-scale transcriptomic and proteomic data. Notably, BIN1 protein expression and its functional roles in microglia, a cell type most relevant to Alzheimer’s disease, have not been examined in depth. Methods Microglial BIN1 expression was analyzed by immunostaining mouse and human brain, as well as by immunoblot and RT-PCR assays of isolated microglia or human iPSC-derived microglial cells. Bin1 expression was ablated by siRNA knockdown in primary microglial cultures in vitro and Cre-lox mediated conditional deletion in adult mouse brain microglia in vivo. Regulation of neuroinflammatory microglial signatures by BIN1 in vitro and in vivo was characterized using NanoString gene panels and flow cytometry methods. The transcriptome data was explored by in silico pathway analysis and validated by complementary molecular approaches. Results Here, we characterized microglial BIN1 expression in vitro and in vivo and ascertained microglia expressed BIN1 isoforms. By silencing Bin1 expression in primary microglial cultures, we demonstrate that BIN1 regulates the activation of proinflammatory and disease-associated responses in microglia as measured by gene expression and cytokine production. Our transcriptomic profiling revealed key homeostatic and lipopolysaccharide (LPS)-induced inflammatory response pathways, as well as transcription factors PU.1 and IRF1 that are regulated by BIN1. Microglia-specific Bin1 conditional knockout in vivo revealed novel roles of BIN1 in regulating the expression of disease-associated genes while counteracting CX3CR1 signaling. The consensus from in vitro and in vivo findings showed that loss of Bin1 impaired the ability of microglia to mount type 1 interferon responses to proinflammatory challenge, particularly the upregulation of a critical type 1 immune response gene, Ifitm3. Conclusions Our convergent findings provide novel insights into microglial BIN1 function and demonstrate an essential role of microglial BIN1 in regulating brain inflammatory response and microglial phenotypic changes. Moreover, for the first time, our study shows a regulatory relationship between Bin1 and Ifitm3, two Alzheimer’s disease-related genes in microglia. The requirement for BIN1 to regulate Ifitm3 upregulation during inflammation has important implications for inflammatory responses during the pathogenesis and progression of many neurodegenerative diseases. Graphical Abstract

Kv1.3 potassium channels, expressed by proinflammatory central nervous system mononuclear phagocytes (CNS-MPs), are promising therapeutic targets for modulating neuroinflammation in Alzheimer’s disease (AD). The molecular characteristics of Kv1.3-high CNS-MPs and their cellular origin from microglia or CNS-infiltrating monocytes are unclear. While Kv1.3 blockade reduces amyloid beta (Aβ) burden in mouse models, the downstream immune effects on molecular profiles of CNS-MPs remain unknown. We show that functional Kv1.3 channels are selectively expressed by a subset of CD11b+CD45+ CNS-MPs acutely isolated from an Aβ mouse model (5xFAD) as well as fresh postmortem human AD brain. Transcriptomic profiling of purified CD11b+Kv1.3+ CNS-MPs, CD11b+CD45int Kv1.3neg microglia, and peripheral monocytes from 5xFAD mice revealed that Kv1.3-high CNS-MPs highly express canonical microglial markers (Tmem119, P2ry12) and are distinct from peripheral Ly6chigh/Ly6clow monocytes. Unlike homeostatic microglia, Kv1.3-high CNS-MPs express relatively lower levels of homeostatic genes, higher levels of CD11c, and increased levels of glutamatergic transcripts, potentially representing phagocytic uptake of neuronal elements. Using irradiation bone marrow CD45.1/CD45.2 chimerism in 5xFAD mice, we show that Kv1.3+ CNS-MPs originate from microglia and not blood-derived monocytes. We show that Kv1.3 channels regulate membrane potential and early signaling events in microglia. Finally, in vivo blockade of Kv1.3 channels in 5xFAD mice by ShK-223 reduced Aβ burden, increased CD11c+ CNS-MPs, and expression of phagocytic genes while suppressing proinflammatory genes (IL1b). Our results confirm the microglial origin and identify unique molecular features of Kv1.3-expressing CNS-MPs. In addition, we provide evidence for CNS immunomodulation by Kv1.3 blockers in AD mouse models resulting in a prophagocytic phenotype.

Objective: Magnetic resonance imaging (MRI)-guided laser interstitial thermal therapy (MRg-LITT) is an alternative to open epilepsy surgery. We assess safety and effectiveness of MRg-LITT for extratemporal lobe epilepsy (ETLE) in patients who are considered less favorable for open resection. Methods: We retrospectively reviewed sequential cases of patients with focal ETLE who underwent MRg-LITT between 2012 and 2019. Epileptogenic zones were determined from standard clinical and imaging data ± stereoelectroencephalography (SEEG). Standard stereotactic techniques, MRI thermometry, and a commercial laser thermal therapy system were used for ablations. Anatomic MRI was used to calculate ablation volumes. Clinical outcomes were determined longitudinally. Results: Thirty-five patients with mean epilepsy duration of 21.3 ± 12.2 years underwent MRg-LITT for focal ETLE at a mean age 36.4 ± 12.7 years. A mean 2.59 ± 1.45 trajectories per patient were used to obtain ablation volumes of 8.8 ± 7.5 cm 3. Mean follow-up was 27.3 ± 19.5 months. Of 32 patients with >12 months of follow-up, 17 (53%) achieved good outcomes (Engel class I + II) of whom 14 (44%) were Engel class I. Subgroup analysis revealed better outcomes for patients with lesional ETLE than for those who were nonlesional, multifocal, or who had failed prior interventions (P = .02). Of 13 patients showing favorable seizure-onset patterns (localized low voltage fast activity or rhythmic spiking on SEEG) prior to ablation, 9 (69%) achieved good outcomes, whereas only 3 of 11 (27%) who show other slower onset patterns achieved good outcomes. Minor adverse events included six patients with transient sensorimotor neurologic deficits and four patients with asymptomatic hemorrhages along the fiber tract. Major adverse events included one patient with a brain abscess that required stereotactic drainage and one patient with persistent hypothalamic obesity. Three deaths-two seizure-associated and one suicide-were unrelated to surgical procedures.