Project Title: Elucidating the Role of Microglial Ephrin-B1 in Microglial-Neuron Contact Dependent Inflammation and Synaptic Pruning in HIV Associated Neurocognitive Disorder (HAND)
Problem:37 million people worldwide are infected with Human Immunodeficiency Virus (HIV) . Of these 37 million, an estimated 15-55% of people living with HIV develop HIV associated neurocognitive disorder (HAND) [2, 3]. Symptomatically, this disorder manifests as cognitive impairment, motor dysfunction and speech impairments; while neuropathologically, it is defined by synaptic degeneration and glial cell activation [4-8]. HIV viral entry requires CD4 and co-receptors CXCR4 and/or CCR5 . In the CNS, microglia are the only expressors of CD4 highlighting their critical role as a reservoir for HIV and key mediator of neuroinflammation and neuronal damage . The mechanism by which this neuropathology occurs still remains unclear. Preliminary data in our lab suggest the role of cell-cell contact dependent ephrin-B1/EphB2 receptor signaling in HIV-induced neuronal damage. Additionally, our preliminary data reveals that in vivo HIVgp120tg increases total and microglial specific ephrin-B1 expression in the CNS and in vitro IFNβ, a neuroprotective treatment for HAND, relocalizes ephrin-B1 from the surface of microglia. As microglia are the key mediators of HIV induced neuronal damage, understanding how modulation of microglial ephrin-B1 influences microglial activation and neuronal health could be fundamental into unlocking the mechanism for HIV induced neuronal damage and help guide therapeutic development.
Rationale of the Approach:
Ephrin-B/EphB is a family of receptor tyrosine kinases. Ephrin-B (ligand) and EphB (receptor) signal bidirectionally, where forward signaling occurs in the receptor-expressing cell and reverse signaling in the ligand-expressing cell. Signaling cues regulate an assortment of downstream changes including filament rearrangement via RhoA/cdc42/Rac to control neuronal outgrowth, STAT3 activation, adhesion and repulsion (reviewed in ). Beyond its signaling role, ephrin-B’s have been reported to transendocytose EphB receptors on other cell types and with it engulfing parts of the surrounding cell membrane .
Overexpression of ephrin-B1/EphB2 has been implicated in the upregulation of CCL2, IL-6, TNF-α and pSTAT3 and in infiltration of immune cells in an assortment of inflammatory diseases [13-15]. In HIV, these inflammatory markers strongly correlate with neuronal damage more so than even HIV viral load and other parameters. Additionally, a lesser characterized and understood microglial function is synaptic pruning in healthy and diseased adult brains. It has been shown; however, that microglia can phagocytose stressed, yet viable neurons . Through postnatal and developmental studies, it is known that the two primary mechanisms of microglial synaptic pruning involve CX3CL1 and its receptor CX3CR1, and the complement proteins C1q/C3 and their receptors [17,18]. In this proposed study, we hypothesize that the ephrin-B/EphB system may be another integral player in mediating synaptic pruning, through its transendocytosis capability, and inflammation via microglia-neuronal contact in the context of HIV induced neuronal injury (See figure for graphical abstract).
Details of the Approach: The approach is a two-pronged approach to ascertain microglial ephrin-B1’s role in both inflammation and synaptic pruning: 1) Identify how microglial specific ephrin-B1 signaling induces an inflammatory profile Ephrin-B/EphB signaling occurs bi-directionally. To assess if EphB2 signaling to microglial ephrin-B1 generates an inflammatory microglial phenotype, we will treat primary human microglia (pHMC) with pre-clustered EphB2-Fc recombinant protein with/without ephrin-B1. We will first assess activation by examining ephrin-B1, Src and STAT3 phosphorylation. Specific cellular and/or secreted expression of inflammatory markers will be examined by nanostring glial profiling panel (RNA) and LEGENDplex inflammation panel 1 (secreted protein).
2) Determine if microglial ephrin-B1 is necessary for HIV infected microglia mediated synaptic pruning and neuronal injury. A hallmark of HIV induced neuronal damage is synaptic degeneration. To identify if ephrin-B1 is crucial for synaptic pruning, a synaptosome engulfment assay will be performed with HIV infected pHMCs, with/without ephrin-B1. HIV infected pHMC supernatants will be transferred onto iPSC-derived mixed neurons and astrocytes (iNeuron/iAstro) or HIV infected pHMCs will be co-cultured with iNeuron/iAstro to determine if microglial ephrin-B1 knockdown prevents HIV induced neurotoxicity via contact or non-contact dependent mechanisms. Synaptic health will be quantified by PSD-95/ VGluT1 (excitatory) and gephyrin/VGAT (inhibitory) staining and reduced synaptic pruning will be assessed by microglial colocalization with these synaptic markers. In addition, cFOS staining will be performed to determine neuronal activation.
How it will Affect the Broader Field:This will be the first proposed study to understand the underlying dynamics with which IFNβ may confer neuroprotection, by specifically understanding the functional role of modulating microglial ephrin-B1. This will help us understand the underlying pathophysiology of HAND more clearly and guide us in establishing more directed therapeutics for HAND, to which none exist. Furthermore, this will help describe a general mechanism for microglia’s role in regulating synapse integrity and overall inflammation during disease via glia-neuronal interactions. In addition, this mechanism could shed light on other neurodegenerative diseases which may have the ephrin-B/EphB system as an underlying cause of disease and could implicate regulators of ephrin-B/EphB, such as IFNβ, as potential therapeutics. Studies describing the regulation of ephrin-B1 are few, and presence of microglial ephrin-B1 has not been previously reported, let alone its functional role in CNS health and disease. This study seeks to explore a newly identified avenue for microglial-neuronal interaction via the ephrin-B/EphB signaling and help delineate which interactions are contact vs non-contact dependent.
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