Research

Broadly, we have several research projects ongoing. Some of these include:

• Kv1.3 channels as therapeutic targets for Alzheimer’s disease and ischemic stroke
• Targeting exaggerated ERK signaling in Alzheimer’s disease for disease-modification
• Understanding the role of microglial BIN1 in the pathogenesis of Alzheimer’s disease
• Cell type-specific In vivo Biotinylation Of Proteins (CIBOP) and Bio-orthogonal non-canonical amino acid tagging (BONCAT) to investigate cellular mechanisms of neurological diseases
• Plasma protein biomarker discovery for stroke using high-throughput proteomics approaches

Microglia represent the predominant myeloid cell type in the brain parenchyma. These cells take up residence in the brain during early stages of brain development and then remain long-lived residents of the brain, with slow turnover over the lifespan. Under homeostatic conditions, microglia constantly survey their environment and respond to signals from neighboring cells, including neurons and other glial cells (e.g. astrocytes). The repertoire of proteins expressed on their surface allows microglia to retain a state of readiness to rapidly respond to any perturbation or injury that occurs in the brain. In response to injury or damage, microglia undergo rapid transformations to cordon off damage, clear debris, and release factors that impact healing following injury. An important role of microglia in directly regulating synaptic maturation and maintenance, has also been recently identified. Some of these necessary functions of microglia can become dysfunctional and even detrimental, with aging and particularly following chronic damage in many neurological disorders. It is therefore not surprising that several microglial genes have been identified as genetic risk factors in neurodegenerative diseases, particularly Alzheimer’s disease. These human genetic studies, along with transcriptomic profiling studies of microglia from mice and humans, and innumerable genetic studies in animal models, implicate microglia in causal cascades of neurological diseases. Transcriptomic studies have identified immense heterogeneity in signatures of microglia in several disease models in mice, as well as in human post-mortem brain. Understanding the central pathways and molecular programs that regulate microglial functional profiles, in different contexts, can allow us to modulate microglial function to achieve disease-modification.

Like many immune cells, microglial effector functions depend on calcium flux and calcium signaling. A plethora of ion channels are expressed by microglia, some of which regulate membrane potential and calcium flux under homeostatic and in disease conditions. Ion channels such as potassium channels that are located on the cell surface fine tune calcium fluxes in immune cells such as T cells, microglia and macrophages. Microglia express voltage-gated Kv1.3, inward-rectifying Kir2.1, calcium-activated KCa3.1 and other K-ATP channels. Interestingly, pro-inflammatory activation of microglia specifically results in upregulation of Kv1.3 channels. Activated microglia surrounding amyloid ß plaques in human brain strongly upregulate this channel and activated microglia in the brains of adult 5xFAD mice (a model of AD pathology) also highly upregulate Kv1.3 channels and Kir2.1 channels. Interestingly, Kv1.3 channels are also increased in microglia and macrophages in the ischemic brain, particularly in the subacute phase (days following ischemic injury).

We have recently found that blockade of Kv1.3 channels by highly-selective peptide blockers (called ShK analogs) can inhibit neurotoxic effector functions in vitro, as well as reduce amyloid ß accumulation in the brain. We suspect this effect is due to augmented clearance of protein aggregates by microglia. Using proximity labeling methods, we have identified several novel proteins that interact with N and C termini of Kv1.3 channels in microglia, which allow functional coupling between Kv1.3 channels and immune signaling mechanisms. For example, we found that the C terminus of Kv1.3 interacts with Stat1, specifically when microglia adopt a pro-inflammatory profile. We have also shown that Kv1.3 blockade impacts Stat1 phosphorylation, as well as interferon-mediated signaling in microglia.

To further investigate the role microglia-specific Kv1.3 in AD pathogenesis, we have developed a conditional knockout (cKO) model, whereby Kv1.3 channels can be selectively deleted in microglia or other cell types of interest, and are examining how cell type-specific Kv1.3 deletion impacts pathology and behavior in AD and stroke mouse models.

Our large-scale proteomic studies of human post-mortem brains identified the ERK pathway as a central disease mechanism and a potential target in human AD. Utilizing the 5xFAD mouse model of AD, our lab has recently demonstrated that ERK activation occurs particularly in microglia, acting as a key regulator of pro-inflammatory immune responses. In vitro blockade of ERK signaling in microglia dampens the pro-inflammatory effects of interferon gamma in microglia, including several disease-associated microglial genes that are important in AD pathogenesis. Blockade of ERK signaling also reduced microglial phagocytosis of amyloid beta. To identify ERK-dependent disease mechanisms associated with pathological outcomes, we are examining the effects of systemic ERK inhibition on microglial proteomic signatures, amyloid-beta pathology, neuroinflammation, and biofluid biomarkers in 5xFAD mice. Our preliminary findings indicate that a single low-dose of a potent, selective, and brain-permeant small molecule MEK inhibitor, PD325901, was effective to suppress ERK activation in WT mice. Ongoing experiments will examine the efficacy of a long-term treatment with PD inhibitor in mouse AD models, to ameliorate neuropathology and cognitive decline in 5xFAD mice. We will also use PET to track neuroinflammation and synaptic loss in these studies and correlate how these pathological changes in the brain correlate with protein changes occurring in biofluids (mouse plasma and CSF). If successful to prevent/reverse disease progression, PD inhibitor therapy could provide clinical benefit for patients in the prodromal phase of AD. We are also conducting studies to hyper-activate or attenuate ERK signaling, specifically in microglia, as well as in neurons, using conditional genetic mouse models. Using a FRET reporter assay of ERK activity, we are examining how different receptor tyrosine kinases that signal via ERK in microglia, can alter microglial phenotypes in differential manners. This project is being conducted in close collaboration with Levi B. Wood, PhD, at Georgia Institute of Technology and Nicholas T. Seyfried, PhD, at Emory University.

Bulk proteomics has advanced our understanding of disease mechanisms involved in AD pathogenesis. However, some mechanisms exclusively occur in a specific cell type and are often restricted to a particular subcellular compartment. To address these critical specificities, our lab has employed in vivo approaches to label the proteomes of distinct cell types in the brain, to better understand cell type-specific disease mechanisms while retaining the native state of cells in the brain. Two complimentary approaches (CIBOP and BONCAT) are highlighted in Figure 5.

We are using the BONCAT approach to investigate astrocyte-specific and microglia-specific protein turnover in mouse models of AD pathology, APOE (44 vs 33) genotype, as well as ischemic brain injury. The cell type-specific in vivo biotinylation of proteins (CIBOP) method is better suited to label cytosolic proteins in brain cell types, and has been used to label the proteome of specific classes of neurons and astrocytes using proximity labeling (Figure 6) (Rayaprolu et al, Nature Communications 2022). Our very recent work on parvalbumin-interneurons-specific proteomes, in close collaboration with Matt Rowan, PhD, and his group at Emory University, revealed unique signatures of increased mitochondrial and metabolic proteins, but reduced synaptic and mTOR signaling proteins in early AD (Kumar et al, BioRxiv 2023). In line with targeting early synapse dysfunction, our ongoing project combines the neuronal-CIBOP approach with synaptosome purification, resulting in a powerful tool for resolving neuron-specific synaptic proteomes in mouse models of AD.

BIN1 represents a major genetic risk factor for late-onset AD (LOAD), second only to APOE. While much has been learned about the role of Bin1 in oligodendrocytes and neurons, the importance of microglial Bin1 in AD pathogenesis has been relatively understudied. In our studies, silencing Bin1 in microglia in vitro, as well as well conditionally deleting Bin1 in microglia in vivo, markedly altered microglial phenotypes, and dampened LPS-induced pro-inflammatory activation potentially via type I IFN signaling pathways. Our data suggest that Bin1 may regulate immune functions in microglia by interacting with Cx3cr1, Cd11c and IFN-1 and IFN-2 receptors in microglia. To examine this, we have partnered with Gopal Thinakaran, PhD, at the University of South Florida to conditionally delete Bin1 in microglia in AD mouse models and examine effects on pathology and behavior, and also use in vitro models to delineate how Bin1 regulates these immune functions of microglia. Using proximity labeling approaches, we will identify which proteins Bin1 interacts with in microglia, in different inflammatory states, and in a Bin1 isoform-dependent manner.