Microglia proliferation underlies synaptic dysfunction in the prefrontal cortex: implications for the pathogenesis of HIV-1-associated neurocognitive and affective alterations

Abstract

Microglia, which are productively infected by HIV-1, are critical for brain development and maturation, as well as synaptic plasticity. The pathophysiology of HIV-infected microglia and their role in the pathogenesis of HIV-1-associated neurocognitive and affective alterations, however, remains understudied. Three complementary aims were undertaken to critically address this knowledge gap. First, the expression of HIV-1 mRNA in the dorsolateral prefrontal cortex of postmortem HIV-1 seropositive individuals with HAND was investigated. Utilization of immunostaining and/or RNAscope multiplex fluorescent assays revealed prominent HIV-1 mRNA in microglia of postmortem HIV-1 seropositive individuals with HAND. Second, measures of microglia proliferation and neuronal damage were evaluated in chimeric HIV (EcoHIV) rats. Eight weeks after EcoHIV inoculation, enhanced microglial proliferation was observed in the medial prefrontal cortex (mPFC) of EcoHIV rats, evidenced by an increased number of cells co-localized with both Iba1 + and Ki67 + relative to control animals. Neuronal damage in EcoHIV infected rats was evidenced by pronounced decreases in both synaptophysin and postsynaptic density protein 95 (PSD-95), markers of presynaptic and postsynaptic damage, respectively. Third, regression analyses were conducted to evaluate whether microglia proliferation mechanistically underlies neuronal damage in EcoHIV and control animals. Indeed, microglia proliferation accounted for 42-68.6% of the variance in synaptic dysfunction. Collectively, microglia proliferation induced by chronic HIV-1 viral protein exposure may underlie the profound synaptodendritic alterations in HIV-1. Understanding how microglia are involved in the pathogenesis of HAND and HIV-1-associated affective disorders affords a key target for the development of novel therapeutics.

Keywords: Dendritic Spines; EcoHIV; Proliferation.

Figure 1.

HIV-1 mRNA and Iba1, a microglial marker, in the dorsolateral PFC (dlPFC) of HIV-1 seropositive individuals with symptomatic HIV-1-associated neurocognitive disorders (HAND). (A-C) Representative images from RNAscope in situ hybridization illustrated the presence of HIV-1 mRNA (A), Iba1 mRNA (B), and their co-localization in postmortem brain tissue from HIV-1 seropositive individuals. White circles on the merged image (C) demonstrate areas of high co-localization, whereas red circles illustrate areas of microglia, but not HIV-1, mRNA. (D-F) A combined RNAscope in situ hybridization and immunohistochemistry assay were utilized to confirm these observations. Indeed, representative images illustrate the presence of HIV-1 mRNA (D), activation of microglia (E), and their co-localization (F) in the dlPFC. Observations support microglia as a cell-type that harbors HIV-1 viral proteins in the dlPFC of HIV-1 seropositive individuals with symptomatic HAND. Scale bar, 10 μm.

Figure 2.

EcoHIV-eGFP expression and its co-localization with the microglial marker Iba1 in the medial prefrontal cortex (mPFC) of F344/N rats eight weeks after inoculation with chimeric HIV (EcoHIV). (A-C) Representative images from in situ hybridization experiments. (D-F) Representative images from immunohistochemistry. Consistent with observations in HIV-1 seropositive individuals, HIV-1 mRNA (A) or EcoHIV eGFP (D) are highly co-localized with markers of Iba1 (mRNA: B; Immunohistochemistry: E). Scale bar, 10 μm.

Figure 3.

EcoHIV infection productively infected microglia, resulting in microglial activation and elevated proliferation. Brain tissue from the medial prefrontal cortex of control (A-C) and EcoHIV (D-F) rats were dual-labeled with a cell marker for microglia (i.e., Iba1) and proliferation (i.e., Ki67). White circles on images B and E demonstrate positive Ki67 signal. Quantification of the number of co-localized Iba1+ and Ki67+ cells (I) revealed a statistically significant increase in EcoHIV, relative to control, animals. Scale bar, 10 μm.

Figure 4.

Animals inoculated with EcoHIV exhibited profound synaptic dysfunction in the medial prefrontal cortex. Representative images of synaptophysin (A-B), a presynaptic vesicle protein, and PSD95 (D-E), a postsynaptic scaffolding protein, are shown for both control and EcoHIV animals. Synaptophysin and PSD95 were quantified by examining the intensity of immunohistochemical staining, whereby both synaptophysin (C) and PSD95 (F) were significantly decreased in EcoHIV relative to control animals. Scale bar, 10 μm.

Figure 5.

Regression analyses were conducted to evaluate the relationship between microglial proliferation and measures of synaptic dysfunction. (A-C) Microglial proliferation, indexed using the number of co-localized Iba1⁺ and Ki67⁺ cells, accounted for 68.6%, 55.9% and 42.0% of the variance in measure of neuronal and dendritic spine morphology, synaptophysin, and postsynaptic density protein 95, respectively. Statistical output from the regression analyses are shown in (D).