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

Gelman BB, Nguyen TP - J Neuroimmune Pharmacol (2009)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Texas Medical Branch, 301 University Blvd, Galveston, TX 77555, USA. bgelman@utmb.edu

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.

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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
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Fig2: 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

Mentions: Immunoblotting Nine promising protein candidates identified by mass spectrometry were evaluated in individual synaptosome extracts by Western blotting. Three proteins were significantly different in one or more of the HIV-infected groups; another protein was changed marginally (Fig. 2a). Synapsin 1b concentration was sharply decreased in the HIV-positive subjects with high brain HIV-1 and LMP7. 14-3-3ζ concentration was increased by 75% in the subjects with high brain HIV-1 and LMP7 (p < 0.01). Synaptosome 14-3-3ε was increased about 2-fold in the HIV-positive subjects with high HIV-1 and LMP7, as compared to those with low HIV-1 (p < 0.05). When the protein concentrations were compared to HIV-1 concentrations in brain, CSF, and blood, Pearson correlation coefficients were positive and significant for brain and CSF HIV-1 loads (Table 3). Stathmin was marginally decreased in the high HIV-1 group (Fig. 2c) and was significantly correlated with brain HIV-1 (Table 3). Immunoblots confirmed that the IPS subunit LMP7 was increased 3-fold in synaptosome and whole cell preparations both (Figs. 1 and 2a). Concentrations of six other proteins on the original list of 31 candidates in Table 2 were not significantly altered in the immunoblots (Fig. 2c).Fig. 2


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

Gelman BB, Nguyen TP - J Neuroimmune Pharmacol (2009)

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
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Fig2: 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
Mentions: Immunoblotting Nine promising protein candidates identified by mass spectrometry were evaluated in individual synaptosome extracts by Western blotting. Three proteins were significantly different in one or more of the HIV-infected groups; another protein was changed marginally (Fig. 2a). Synapsin 1b concentration was sharply decreased in the HIV-positive subjects with high brain HIV-1 and LMP7. 14-3-3ζ concentration was increased by 75% in the subjects with high brain HIV-1 and LMP7 (p < 0.01). Synaptosome 14-3-3ε was increased about 2-fold in the HIV-positive subjects with high HIV-1 and LMP7, as compared to those with low HIV-1 (p < 0.05). When the protein concentrations were compared to HIV-1 concentrations in brain, CSF, and blood, Pearson correlation coefficients were positive and significant for brain and CSF HIV-1 loads (Table 3). Stathmin was marginally decreased in the high HIV-1 group (Fig. 2c) and was significantly correlated with brain HIV-1 (Table 3). Immunoblots confirmed that the IPS subunit LMP7 was increased 3-fold in synaptosome and whole cell preparations both (Figs. 1 and 2a). Concentrations of six other proteins on the original list of 31 candidates in Table 2 were not significantly altered in the immunoblots (Fig. 2c).Fig. 2

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Texas Medical Branch, 301 University Blvd, Galveston, TX 77555, USA. bgelman@utmb.edu

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.

Show MeSH
Related in: MedlinePlus