Synaptic proteins linked to HIV-1 infection and immunoproteasome induction: proteomic analysis of human synaptosomes

Abstract

Infection of the central nervous system with human immunodeficiency virus type 1 (HIV-1) can produce morphological changes in the neocortical synaptodendritic arbor that are correlated with neurocognitive impairment. To determine whether HIV-1 infection influences the protein composition of human synapses, a proteomic study of isolated nerve endings was undertaken. Synaptosomes from frontal neocortex were isolated using isopyknic centrifugation from 19 human brain specimens. Purity and enrichment were assessed by measuring pre- and postsynaptic protein markers. Two-dimensional polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry was used to screen for proteins differentially expressed in HIV/AIDS. The concentrations of 31 candidate protein spots were potentially abnormal in HIV-infected decedents with HIV encephalitis and/or increased expression of immunoproteasome subunits. Immunoblots showed that the concentration of some of them was related to HIV-1 infection of the brain and immunoproteasome (IPS) induction. Synapsin 1b and stathmin were inversely related to brain HIV-1 load; 14-3-3zeta and 14-4-4epsilon proteins were higher in subjects with HIV-1 loads. Perturbed synaptosome proteins were linked with IPS subunit composition, and 14-3-3zeta was histologically colocalized with IPS subunits in stained neocortical neurons. Proteomics illustrates that certain human proteins within the synaptic compartment are involved with changes in the synaptodendritic arbor and neurocognitive impairment in HIV-1-infected people.

Fig. 1

Immunoblots show that human cerebrocortical synaptosome preparations from frontal neocortex specimens of eight human subjects are enriched with synaptic marker proteins. a The immunoproteasome subunit LMP7 was increased in synaptosomes from the HIV-positive subjects. Pre- (b) and postsynaptic (c) marker proteins were enriched in synaptosomes by up to 7-fold when compared to unfractionated homogenates. d Marker proteins for non-neural cells, including mononuclear phagocytes (CD68) and astrocytes (GFAP) were sharply depleted in these synaptosome isolates. See Table 1 for added description of the proteins

Fig. 2

Immunoblots of synaptosomes prepared from frontal neocortex from all 19 human subjects. Comparison groups are according to status of HIV-1 infection, neocortical HIV-1 RNA load (low and high), and neocortical immunoproteasome subunit expression (low and high). a Subjects with high HIV-1 loads had significantly more synaptosome LMP7 as compared to the other groups. b Synapsin 1b concentration was decreased in subjects with high HIV-1 load and abundant LMP7; 14-3-3 zeta and 14-3-3 epsilon were increased with high HIV-1 and LMP7. c The decreased stathmin in subjects with high HIV-1 and LMP7 was marginal (p < 0.1). Concentrations of HIV-1 RNA in brain cortex in the three respective groups (log₁₀ copies per gram of wet weight) were 0 ± 0, 2.77 ± 1.50, and 4.81 ± 0.48 (t test: p = 0.0000055, low versus high). The values for the IPS subunit LMP7 concentration in the three groups were 1.00 ± 0.79, 2.02 ± 1.02, and 4.03 ± 2.15 in relative density units (t test: p = 0.0315, low versus high). Six other candidate protein species tested were not different statistically. IPS neocortical immunoproteasome concentration. Mean ± one standard deviation. *p < 0.05; **p < 0.01

Fig. 3

Confocal immunofluorescence microscopy for 14-3-3ζ (red) and the immunoproteasome subunit marker LMP2 (green) in a subject with HIV encephalitis and high concentrations of these proteins in the synaptosome preparation. Punctate deposits of both proteins outline the distinct shape of a pyramidal neocortical neuron and surrounding neuropil. The perikaryon and neuropil both contain deposits of these proteins, which morphologically resemble synaptic densities. Arrows in the large bottom panel denote foci where the antigens are colocalized in the merged image. Attempts to localize synapsin 1 were not successful technically because its concentration was sharply decreased in HIV encephalitis. Scale bar = 10 μm