Human synaptic plasticity gene expression profile and dendritic spine density changes in HIV-infected human CNS cells: role in HIV-associated neurocognitive disorders (HAND)

Abstract

HIV-associated neurocognitive disorders (HAND) is characterized by development of cognitive, behavioral and motor abnormalities, and occur in approximately 50% of HIV infected individuals. Our current understanding of HAND emanates mainly from HIV-1 subtype B (clade B), which is prevalent in USA and Western countries. However very little information is available on neuropathogenesis of HIV-1 subtype C (clade C) that exists in Sub-Saharan Africa and Asia. Therefore, studies to identify specific neuropathogenic mechanisms associated with HAND are worth pursuing to dissect the mechanisms underlying this modulation and to prevent HAND particularly in clade B infection. In this study, we have investigated 84 key human synaptic plasticity genes differential expression profile in clade B and clade C infected primary human astrocytes by using RT(2) Profile PCR Array human Synaptic Plasticity kit. Among these, 31 and 21 synaptic genes were significantly (≥3 fold) down-regulated and 5 genes were significantly (≥3 fold) up-regulated in clade B and clade C infected cells, respectively compared to the uninfected control astrocytes. In flow-cytometry analysis, down-regulation of postsynaptic density and dendrite spine morphology regulatory proteins (ARC, NMDAR1 and GRM1) was confirmed in both clade B and C infected primary human astrocytes and SK-N-MC neuroblastoma cells. Further, spine density and dendrite morphology changes by confocal microscopic analysis indicates significantly decreased spine density, loss of spines and decreased dendrite diameter, total dendrite and spine area in clade B infected SK-N-MC neuroblastoma cells compared to uninfected and clade C infected cells. We have also observed that, in clade B infected astrocytes, induction of apoptosis was significantly higher than in the clade C infected astrocytes. In conclusion, this study suggests that down-regulation of synaptic plasticity genes, decreased dendritic spine density and induction of apoptosis in astrocytes may contribute to the severe neuropathogenesis in clade B infection.

Figure 1. Neuronal dendrite and spine measurement by Image J analysis.

A typical dendrite segment from a pyramidal neuron is shown, and the six quantification parameters labeled as follows. 1) Total dendrite area is measured by drawing a box around the whole image; 2) dendrite diameter is obtained by drawing a line across the dendrite thickness at a place of average width; 3) spine density is the total number of spines divided by the dendrite length; 4) spine area is measured by drawing a box around the whole spine; 5) Dendrite length uses the broken line tool to measure the length and 6) Spine length. (Smith et al., 2009. Reversal of long-term dendritic spine alterations in Alzheimer disease models. Proc Natl Acad Sci U S A 106: 16877-16882. Copyright (2009) National Academy of Sciences, U.S.A.)

Figure 2. a. Scatter plot analysis of the changes in synaptic plasticity gene expression in clade B infected astrocytes.

Pair wise comparison of control primary human astrocytes (No HIV infection) and clade B infected astrocytes by scatter plot analysis. Spots associated with individual human synaptic plasticity gene were collected and converted into log10 scale. The central line indicates unchanged gene expression. The synaptic plasticity genes with expression levels higher or lower in clade B infected astrocytes than the control cells are expected to produce dots that deviate from the centerline. b. Scatter plot analysis of the changes in synaptic plasticity gene expression in clade C infected astrocytes. Pair wise comparison of control primary human astrocytes (No HIV infection) and clade C infected astrocytes by scatter plot analysis. Spots associated with individual human synaptic plasticity gene were collected and converted into log10 scale. The central line indicates unchanged gene expression. The dots are allocated to positions that are above or below than the +3 fold or −3 fold line when the differences are greater than three folds.

Figure 3. Flow-cytometry analysis of postsynaptic density and dendrite morphology regulatory proteins.

Levels of ARC, NMDAR1 and GRM1 proteins were measured in clade B and C infected astrocytes by flow-cytometry. The expression levels of these three proteins were significantly less in clade B infected astrocytes than clade C (p<0.02) infected and uninfected control cells (p<0.001).

Figure 4. Confocal Images of DiI stained SK-N-MC cells.

(4a) Control SK-N-MC cells: High number of long spines on the dendritic length, high dendrite diameter, total dendrite area and spine area. (4b) Clade B infected cells: Loss of spines on the dendrite length, decreased dendrite diameter, dendrite and spine area was observed than the control and clade C infected SK-N-MC cells. (4c) Clade C infected cells: Loss of number of spines, decreased dendrite diameter, dendrite area and spine area was observed than control cells.

Figure 5. Dendrite morphology changes in HIV-1 clade B and clade C infected SK-N-MC neuroblastoma cells.

SK-N-MC neuroblastoma cells were grown onto the glass coverslips and infected with the HIV clade B and C for 7 days. Cover slips were stained with the DiI stain and observed under confocal microscopy. Randomly selected pictures in each group of the cells were captured in confocal microscope. Image J software was used to analyze the total dendrite area (5a), spine density (5b), dendrite diameter (5c), spine area (5d), spine length (5e) and number of spines (5f).

Figure 6. Apoptosis induction in HIV infected primary human astrocytes.

Apoptosis induction was studied using double cell labeling with Annexin V-PE and 7-AAD. Seven days after infection, cells were collected and re-suspended in binding buffer containing Annexin V-PE and 7-AAD, and then processed for flow cytometry analysis. Representative flow figure for one of three experiments is shown here. In each box, cells in lower left corner (negative for 7-AAD and Annexin V-PE) are viable, cells in the upper right corner (positive for 7-AAD and Annexin V-PE) are necrotic or late apoptotic cells, while cells in the lower right corner (Annexin V-PE positive but negative to 7-AAD) are early apoptotic cells.