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Proteomic profiling of brain cell types using isolation-based strategies pose limitations in resolving cellular phenotypes representative of their native state. We describe a mouse line for cell type-specific expression of biotin ligase TurboID, for in vivo biotinylation of proteins. Using adenoviral and transgenic approaches to label neurons, we show robust protein biotinylation in neuronal soma and axons throughout the brain, allowing quantitation of over 2000 neuron-derived proteins spanning synaptic proteins, transporters, ion channels and disease-relevant druggable targets. Next, we contrast Camk2a-neuron and Aldh1l1-astrocyte proteomes and identify brain region-specific proteomic differences within both cell types, some of which might potentially underlie the selective vulnerability to neurological diseases. Leveraging the cellular specificity of proteomic labeling, we apply an antibody-based approach to uncover differences in neuron and astrocyte-derived signaling phospho-proteins and cytokines. This approach will facilitate the characterization of cell-type specific proteomes in a diverse number of tissues under both physiological and pathological states. Current isolation-based approaches for cell type-specific proteomics pose several challenges. Here, the authors present an approach for in vivo cell type-specific protein labeling to characterize proteomic differences between neurons and astrocytes in their native state in adult mouse brain.

The repeated failures of amyloid-targeting therapies have challenged our narrow understanding of Alzheimer’s disease (AD) pathogenesis and inspired wide-ranging investigations into the underlying mechanisms of disease. Increasing evidence indicates that AD develops from an intricate web of biochemical and cellular processes that extend far beyond amyloid and tau accumulation. This growing recognition surrounding the diversity of AD pathophysiology underscores the need for holistic systems-based approaches to explore AD pathogenesis. Here we describe how network-based proteomics has emerged as a powerful tool and how its application to the AD brain has provided an informative framework for the complex protein pathophysiology underlying the disease. Furthermore, we outline how the AD brain network proteome can be leveraged to advance additional scientific and translational efforts, including the discovery of novel protein biomarkers of disease.

In Alzheimer’s disease (AD), a multitude of genetic risk factors and early biomarkers are known. Nevertheless, the causal factors responsible for initiating cognitive decline in AD remain controversial. Toxic plaques and tangles correlate with progressive neuropathology, yet disruptions in circuit activity emerge before their deposition in AD models and patients. Parvalbumin (PV) interneurons are potential candidates for dysregulating cortical excitability as they display altered action potential (AP) firing before neighboring excitatory neurons in prodromal AD. Here, we report a novel mechanism responsible for PV hypoexcitability in young adult familial AD mice. We found that biophysical modulation of K v 3 channels, but not changes in their mRNA or protein expression, were responsible for dampened excitability in young 5xFAD mice. These K + conductances could efficiently regulate near-threshold AP firing, resulting in gamma-frequency-specific network hyperexcitability. Thus, biophysical ion channel alterations alone may reshape cortical network activity prior to changes in their expression levels. Our findings demonstrate an opportunity to design a novel class of targeted therapies to ameliorate cortical circuit hyperexcitability in early AD.

Mitochondria influence cellular function through both cell-autonomous and non-cell autonomous mechanisms, such as production of paracrine and endocrine factors. Here, we demonstrate that mitochondrial regulation of the secretome is more extensive than previously appreciated, as both genetic and pharmacological disruption of the electron transport chain caused upregulation of the Alzheimer’s disease risk factor apolipoprotein E (APOE) and other secretome components. Indirect disruption of the electron transport chain by gene editing of SLC25A mitochondrial membrane transporters as well as direct genetic and pharmacological disruption of either complexes I, III, or the copper-containing complex IV of the electron transport chain elicited upregulation of APOE transcript, protein, and secretion, up to 49-fold. These APOE phenotypes were robustly expressed in diverse cell types and iPSC-derived human astrocytes as part of an inflammatory gene expression program. Moreover, age- and genotype-dependent decline in brain levels of respiratory complex I preceded an increase in APOE in the 5xFAD mouse model. We propose that mitochondria act as novel upstream regulators of APOE-dependent cellular processes in health and disease.

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.

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.

Background Exposure to repetitive head impacts (RHI) is associated with increased risk for chronic traumatic encephalopathy (CTE), a neurodegenerative tauopathy, and other neuropathological changes. Biological drivers of RHI-related neurodegeneration are not well understood. We interrogated the plasma proteome in aging adults with prior RHI compared to healthy controls (CTL) and individuals with Alzheimer’s disease (AD), including a subset characterized neuropathologically at autopsy. Methods Proximity extension assay (Olink Explore®) quantified 2,779 plasma proteins in 22 RHI patients (all AD-biomarker negative), 39 biomarker-confirmed AD, and 44 CTL. A subset of participants went to autopsy ( N  = 16) allowing for comparisons of the antemortem plasma proteome between autopsy-confirmed CTE + ( N  = 7) and CTE- ( N  = 9). Differential abundance and co-expression network analyses identified plasma proteomic signatures of RHI, which were functionally annotated using gene ontology and cell type enrichment analysis. Nonparametric correlations examined plasma proteomic associations with orthogonally-measured plasma biomarkers, global cognitive function, and semi-quantitative ratings of neuropathology burden at autopsy. Results Differential abundance analysis revealed 434 increased (vs. 6 decreased) proteins in RHI vs. CTL and 193 increased (vs. 14 decreased) in RHI vs. AD. Network analysis identified 9 protein co-expression modules (M1-M9), of which 7 were elevated in RHI compared to AD or CTL. Modules with increased abundance in RHI were enriched for mitochondrial/metabolic, cell division, and immunovascular (e.g., cell adhesion, TNF-signaling) processes. RHI-related modules exhibited strong and selective correlations with immunoassay-based plasma IL-6 in RHI cases, including the M2 TNF-signaling/cell adhesion module which harbored proteins that strongly tracked with cognitive function. RHI-related plasma protein signatures were similar in the subset of participants with autopsy-confirmed CTE, including immune and metabolic modules that positively correlated with medial temporal lobe tau and TDP-43 burden. Conclusions Molecular pathways in plasma most consistently implicated in RHI were tied to immune response, mitochondrial function, and cell metabolism. RHI-related proteomic signatures tracked with antemortem cognitive severity and postmortem neuropathological burden, providing converging evidence for their role in disease progression. Differentially abundant proteins and co-expression modules in RHI may inform mechanisms linking RHI to increased dementia risk, thus guiding diagnostic biomarker and therapeutic development for at-risk populations.

The autosomal dominant spinocerebellar ataxias (SCAs) are a diverse group of neurological disorders anchored by the phenotypes of motor incoordination and cerebellar atrophy. Disease heterogeneity is appreciated through varying comorbidities: dysarthria, dysphagia, oculomotor and/or retinal abnormalities, motor neuron pathology, epilepsy, cognitive impairment, autonomic dysfunction, and psychiatric manifestations. Our study focuses on SCA13, which is caused by several allelic variants in the voltage-gated potassium channel KCNC3 (Kv3.3). We detail the clinical phenotype of four SCA13 kindreds that confirm causation of the KCNC3R423H allele. The heralding features demonstrate congenital onset with non-progressive, neurodevelopmental cerebellar hypoplasia and lifetime improvement in motor and cognitive function that implicate compensatory neural mechanisms. Targeted expression of human KCNC3R423H in Drosophila triggers aberrant wing veins, maldeveloped eyes, and fused ommatidia consistent with the neurodevelopmental presentation of patients. Furthermore, human KCNC3R423H expression in mammalian cells results in altered glycosylation and aberrant retention of the channel in anterograde and/or endosomal vesicles. Confirmation of the absence of plasma membrane targeting was based on the loss of current conductance in cells expressing the mutant channel. Mechanistically, genetic studies in Drosophila, along with cellular and biophysical studies in mammalian systems, demonstrate the dominant negative effect exerted by the mutant on the wild-type (WT) protein, which explains dominant inheritance. We demonstrate that ocular co-expression of KCNC3R423H with Drosophila epidermal growth factor receptor (dEgfr) results in striking rescue of the eye phenotype, whereas KCNC3R423H expression in mammalian cells results in aberrant intracellular retention of human epidermal growth factor receptor (EGFR). Together, these results indicate that the neurodevelopmental consequences of KCNC3R423H may be mediated through indirect effects on EGFR signaling in the developing cerebellum. Our results therefore confirm the KCNC3R423H allele as causative for SCA13, through a dominant negative effect on KCNC3WT and links with EGFR that account for dominant inheritance, congenital onset, and disease pathology.

Mutations in MATR3 have been associated with amyotrophic lateral sclerosis (ALS) as well as a form of distal myopathy termed vocal cord pharyngeal distal myopathy (VCPDM). To begin to understand how mutations in MATR3 may cause disease, here we provide initial characterization of transgenic (Tg) mice expressing human wild-type (WT) MATR3 (MATR3 WT ) and ALS-mutant F115C MATR3 (MATR3 F115C ) proteins under the control of the mouse prion promoter (MoPrP). For each construct, we established multiple independent lines of mice that stably transmitted the transgene. Unexpectedly, for all stably-transmitting lines examined, MATR3 transgenic mRNA expression was more robust in muscle, with minimal expression in spinal cord. The levels of transgenic mRNA in muscle did not differ between mice from our lead MATR3 F115C line and lead MATR3 WT line, but mice from the lead MATR3 F115C line had significantly higher levels of MATR3 protein in muscle over the lead MATR3 WT line. Mice from the three independent, established lines of MATR3 F115C mice developed weakness in both fore- and hind-limbs as early as < 1 months of age; whereas, MATR3 WT mice aged to > 20 months were not overtly distinguishable from non-transgenic (NT) littermates based on basic motor phenotype. Muscle of both MATR3 WT and MATR3 F115C mice showed vacuoles by 2 months of age which worsened by ~ 10 months, but vacuolation was noticeably more severe in MATR3 F115C mice. Overall, our results indicate that increasing the levels of MATR3 in muscle can cause pathologic changes associated with myopathy, with MATR3 F115C expression causing overt muscle atrophy and a profound motor phenotype. The findings suggest that analysis of muscle pathology in individuals harboring ALS-linked MATR3 mutations should be routinely considered.

INTRODUCTION Factors underlying discordant visual and quantitative amyloid beta–positron emission tomography (Aβ‐PET) results and their clinical implications are not well understood. METHODS Participants from the 1Florida Alzheimer's Disease Research Center (1FLADRC) underwent Aβ‐PET, blood draw, brain magnetic resonance imaging (MRI), and neuropsychological testing. We evaluated differences in demographics, apolipoprotein E (APOE) status, biomarkers, and cognition among older adults with concordant and discordant visual‐quantitative Aβ‐PET. Discordance was defined as positive visual read (V) of Aβ‐PET with below‐threshold Centiloid quantification (Q; CL <25; V+/Q–) or negative visual read with CL ≥25 (V–/Q+). RESULTS We studied 386 participants (mean age ± SD: 70.7 ± 7.8, 55.2% female, 44.6% Hispanic White). Compared to V+/Q–, V–/Q+ had a higher frequency of APOE ε4 carriers (40%). Black/African American participants were overrepresented in V–/Q+ (40.9%). Both discordant groups had higher plasma phosphorylated tau 217 (p‐tau217) and glial fibrillary acidic protein (GFAP) than V–/Q– but lower than V+/Q+. Discordant groups had greater gray matter volume and better cognitive performance than V+/Q+. DISCUSSION Discordant Aβ‐PET findings likely hold clinical significance and may reflect early stages of neuropathological progression. Highlights Groups with concordant/discordant visual‐quantitative amyloid beta–positron emission tomography (Aβ‐PET) results were compared. Visual–/quant+ were more likely than visual+/quant– to be apolipoprotein E (APOE) ε4 carriers and Black/African American. Discordant groups had higher plasma phosphorylated tau 217 (p‐tau217) and glial fibrillary acidic protein (GFAP) than concordant negative. Discordant groups had less atrophy and better cognition than concordant positive. Centiloid quantification should supplement visual reads in clinical settings.