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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 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
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Fig1: 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

Mentions: Synaptosome isolation About 500 mg of dorsolateral prefrontal cortex from Brodmann area 9 was dissected free while frozen. Synaptosomes were isolated at 4°C by differential centrifugation in a discontinuous sucrose gradient using a standard procedure (Dodd et al. 1981; Eshleman et al. 2001; Gray and Whittaker 1962; Mash et al. 2002; Wood et al. 1996). Specimens were thawed briefly and added to 5 mL of ice cold buffered sucrose (0.32 M sucrose, 5 mM HEPES, 25 μL protease inhibitor cocktail (Sigma Aldrich), and 50 μL phosphatase inhibitor cocktail (EMD Chemicals, Inc., Gibbstown, NJ, USA), pH 7.4). They were homogenized with eight strokes in a Potter–Elvehjem tissue grinder with 0.1 to 0.5 mm tolerance (Fisher Scientific), with intermittent immersion in an ice bath for 30 s between strokes. Homogenates were centrifuged at 1,000×g for 10 min at 4°C to yield a crude pellet (P1a) and supernatant (S1a). P1a was washed and centrifuged as above, yielding a second pellet (P1b) and supernatant (S1b). The supernatants were combined into S1, brought to a volume of 10 mL, and centrifuged at 10,000×g for 20 min at 4°C to produce the P2 pellet. The P2 pellet was resuspended in 2.5 mL buffered sucrose and layered over a discontinuous gradient composed of 2.5 mL each of buffered sucrose with concentrations of 0.8, 1.0, and 1.2 M from top to bottom. P2 was centrifuged at 150,000×g for 2 h at 4°C. The P3 synaptosome fraction was collected from the interface of the 1.0- and 1.2-M layers. P3 was washed in 0.32 M buffered sucrose at 150,000×g for 30 min at 4°C. The P3 pellet was collected and stored frozen until use. The relative enrichment of synaptic protein markers and depletion of glial markers in these synaptosome isolates was compared to the starting homogenate using Western blotting of pre- and postsynaptic protein markers, as listed in Fig. 1.Fig. 1


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

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

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
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2824116&req=5

Fig1: 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
Mentions: Synaptosome isolation About 500 mg of dorsolateral prefrontal cortex from Brodmann area 9 was dissected free while frozen. Synaptosomes were isolated at 4°C by differential centrifugation in a discontinuous sucrose gradient using a standard procedure (Dodd et al. 1981; Eshleman et al. 2001; Gray and Whittaker 1962; Mash et al. 2002; Wood et al. 1996). Specimens were thawed briefly and added to 5 mL of ice cold buffered sucrose (0.32 M sucrose, 5 mM HEPES, 25 μL protease inhibitor cocktail (Sigma Aldrich), and 50 μL phosphatase inhibitor cocktail (EMD Chemicals, Inc., Gibbstown, NJ, USA), pH 7.4). They were homogenized with eight strokes in a Potter–Elvehjem tissue grinder with 0.1 to 0.5 mm tolerance (Fisher Scientific), with intermittent immersion in an ice bath for 30 s between strokes. Homogenates were centrifuged at 1,000×g for 10 min at 4°C to yield a crude pellet (P1a) and supernatant (S1a). P1a was washed and centrifuged as above, yielding a second pellet (P1b) and supernatant (S1b). The supernatants were combined into S1, brought to a volume of 10 mL, and centrifuged at 10,000×g for 20 min at 4°C to produce the P2 pellet. The P2 pellet was resuspended in 2.5 mL buffered sucrose and layered over a discontinuous gradient composed of 2.5 mL each of buffered sucrose with concentrations of 0.8, 1.0, and 1.2 M from top to bottom. P2 was centrifuged at 150,000×g for 2 h at 4°C. The P3 synaptosome fraction was collected from the interface of the 1.0- and 1.2-M layers. P3 was washed in 0.32 M buffered sucrose at 150,000×g for 30 min at 4°C. The P3 pellet was collected and stored frozen until use. The relative enrichment of synaptic protein markers and depletion of glial markers in these synaptosome isolates was compared to the starting homogenate using Western blotting of pre- and postsynaptic protein markers, as listed in Fig. 1.Fig. 1

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