SIV-Mediated Synaptic Dysfunction Is Associated with an Increase in Synapsin Site 1 Phosphorylation and Impaired PP2A Activity

Abstract

Although the reduction of viral loads in people with HIV undergoing combination antiretroviral therapy has mitigated AIDS-related symptoms, the prevalence of neurological impairments has remained unchanged. HIV-associated CNS dysfunction includes impairments in memory, attention, memory processing, and retrieval. Here, we show a significant site-specific increase in the phosphorylation of Syn I serine 9, site 1, in the frontal cortex lysates and synaptosome preparations of male rhesus macaques infected with simian immunodeficiency virus (SIV) but not in uninfected or SIV-infected antiretroviral therapy animals. Furthermore, we found that a lower protein phosphatase 2A (PP2A) activity, a phosphatase responsible for Syn I (S9) dephosphorylation, is primarily associated with the higher S9 phosphorylation in the frontal cortex of SIV-infected macaques. Comparison of brain sections confirmed higher Syn I (S9) in the frontal cortex and greater coexpression of Syn I and PP2A A subunit, which was observed as perinuclear aggregates in the somata of the frontal cortex of SIV-infected macaques. Synaptosomes from SIV-infected animals were physiologically tested using a synaptic vesicle endocytosis assay and FM4-64 dye showing a significantly higher baseline depolarization levels in synaptosomes of SIV⁺-infected than uninfected control or antiretroviral therapy animals. A PP2A-activating FDA-approved drug, FTY720, decreased the higher synaptosome depolarization in SIV-infected animals. Our results suggest that an impaired distribution and lower activity of serine/threonine phosphatases in the context of HIV infection may cause an indirect effect on the phosphorylation levels of essential proteins involving in synaptic transmission, supporting the occurrence of specific impairments in the synaptic activity during SIV infection.SIGNIFICANCE STATEMENT Even with antiretroviral therapy, neurocognitive deficits, including impairments in attention, memory processing, and retrieval, are still major concerns in people living with HIV. Here, we used the rhesus macaque simian immunodeficiency virus model with and without antiretroviral therapy to study the dynamics of phosphorylation of key amino acid residues of synapsin I, which critically impacts synaptic vesicle function. We found a significant increase in synapsin I phosphorylation at serine 9, which was driven by dysfunction of serine/threonine protein phosphatase 2A in the nerve terminals. Our results suggest that an impaired distribution and lower activity of serine/threonine phosphatases in the context of HIV infection may cause an indirect effect on the phosphorylation levels of essential proteins involved in synaptic transmission.

Keywords: HAND; HIV-1; neurocognition; simian immunodeficiency virus; synapsin I.

Figure 1.

Hyperphosphorylation of Syn I (S9) in the frontal cortex of SIV⁺ rhesus macaques. A, Representative Western blots of whole-tissue protein lysates from the frontal cortex of uninfected, SIV⁺, and SIV⁺ ART animals using an antibody against Syn I (S9). B, Bands were quantified and normalized against actin or GAPDH signals. There was a significant difference among groups. A higher Syn I (S9) phosphorylation was detected in the frontal cortex lysates from SIV⁺ compared with uninfected or SIV⁺ ART. No changes were observed in the expression levels of total Syn I (B, bottom). C, Immunohistochemistry of sections of uninfected, SIV⁺, and SIV⁺ ART probed with anti-Syn I (S9, green) and β III tubulin (red) antibodies. D, Quantification of experiments illustrated in C revealed a significant difference among groups. A significant increase in the intensities of the positive Syn I (S9) signals was detected. **p < 0.005 (one-way ANOVA with Tukey HSD). *p < 0.05 (one-way ANOVA with Tukey HSD). Error bars indicate SD of mean; ± SEM.

Figure 2.

Hyperphosphorylation of Syn I (S9) in parietal and occipital lobes of SIV⁺ rhesus macaques. Representative sections of parietal (A) and occipital (B) lobes of uninfected, SIV⁺, and SIV⁺ ART animals were immunostained with antibodies against β III tubulin (red) and p-Syn I (S9, green). P-Syn I (S9) signal intensities were measured and quantified. Significant increases in the immunopositive p-Syn I (S9) signals were observed in SIV⁺ sections compared with uninfected or SIV⁺ ART sections. PL, Parietal lobe; OL, occipital lobe. *p < 0.05 (one-way ANOVA with Tukey HSD). **p < 0.005 (one-way ANOVA with Tukey HSD). Error bars indicate SD of mean; ± SEM.

Figure 3.

Effect of SIV infection on Syn I phosphorylation at S62/S67, S549, and S603 sites in the frontal cortex of rhesus macaques. A, Protein lysates of the frontal cortex from uninfected, SIV⁺, and SIV⁺ ART animals were analyzed by Western blot using specific antibodies against Syn I (S62/S67, S549, and S603) sites. B, Band intensities were quantified and normalized against GAPDH and graphed as mean. No significant changes were observed among groups in the phosphorylation levels of S62/S67 and S549. However, a significant increase was detected in Syn I (S603) when frontal cortex lysates from uninfected were compared with SIV⁺ ART animals. *p < 0.05 (one-way ANOVA with Tukey HSD). Error bars indicate SD of mean; ± SEM.

Figure 4.

A significant increase in Syn I (S9) phosphorylation of synaptosome preparations from SIV⁺ rhesus macaques. A, Synaptosome preparations of uninfected, SIV⁺, and SIV⁺ ART were subjected to immunoblot analysis using antibodies recognizing the indicated phosphorylation sites of Syn I. Antibodies against GAPDH and total Syn I were used to assess protein loads. B, Band intensities from every sample were quantified and normalized against their corresponding GAPDH band, and means were graphed. A significant increase was observed among groups in Syn I (S9) (ANOVA, p < 0.005). Although no significant differences were detected among groups in S62/67, S549, and S603 phosphorylation, there was a significant increase in S549 phosphorylation between synaptosomes of SIV⁺ and SIV⁺ ART animals. *p < 0.05 (one-way ANOVA with Tukey HSD). **p < 0.005 (one-way ANOVA with Tukey HSD). Error bars indicate SD of mean; ± SEM.

Figure 5.

Assessment of the activities of CREB, PKA, CaMK I, and CaMK IV in synaptosomes of the frontal cortex of SIV⁺ rhesus macaques. A, Western blots of synaptosome preparations, which were probed with an antibody against p-CREB. No differences among groups were observed. B, Expression of PKA catalytic subunit in synaptosomes of uninfected, SIV⁺, and SIV⁺ ART animals. No differences among the groups were observed. C, Assessment of PKA activity in synaptosome preparations of the frontal cortex of rhesus macaques. D, Assessment of CaMKI and CaMKIV activities using phosphospecific antibodies in synaptosome preparations of uninfected, SIV⁺, and SIV⁺ ART rhesus macaque frontal cortex. E, No differences among the groups were observed (one-way ANOVA with Tukey HSD).

Figure 6.

Analysis of CaMKII activity compared with CaMKII total protein in synaptosomes of SIV⁺ animals. Synaptosome (A, B) and total lysates (C, D) preparations of uninfected, SIV⁺, and SIV⁺ ART rhesus macaques frontal cortex were analyzed using Western blot. The intensities of pCaMKII and CaMKII bands were quantified and graphed alone or as pCaMKII/CaMKII ratio. *p < 0.05 (one-way ANOVA with Tukey HSD).

Figure 7.

Impairments in the expression, cellular distribution, and activity of PP2A A subunit in SIV⁺ frontal cortex. A, Synaptosome preparations of the frontal cortex from uninfected, SIV⁺, and SIV⁺ ART animals were subjected to Western blot analysis with an antibody recognizing PP2A A subunit. The same blot was reprobed with an antibody against GAPDH to assess and quantify protein loads. B, To graph the means, band densities were measured and normalized against GAPDH. There was a significant difference of PP2A A subunit expression among groups. C, Representative images of the frontal cortex of uninfected, SIV⁺, and SIV⁺ ART animals, which were immunoprobed with Syn I (red) and PP2A A (green), revealed lower PP2A A-positive signals in neurons somata. Neurons coexpressing Syn I and PP2A A in sections of SIV⁺ animals exhibited accumulation of perinuclear staining (arrowhead); whereas in uninfected and SIV⁺ ART sections, the stainings were more dispersed (arrows). D, Intensities of PP2A A signals were measured and graphed. A significant decrease was observed in the signals from SIV⁺ sections. E, Equal amounts of lysates from uninfected and SIV⁺ animals were subjected to immunoprecipitation using PP2A A subunit antibody and phosphatase assay using pNPP as a substrate. A significant reduction of PP2A activity in synaptosomes of SIV⁺ animals is plotted. fostriecin (1 μm) specifically inhibited PP2A activity. ^#p < 0.05 (Student's t test). *p < 0.05 (one-way ANOVA with Tukey HSD). ***p < 0.001 (one-way ANOVA with Tukey HSD). Error bars indicate SD of mean; ± SEM; A.U., Fluorescence arbitrary units.

Figure 8.

Endocytosis assay of SVs using synaptosomes from the frontal cortex of rhesus macaques. A, Frontal cortex synaptosomes were assayed on glass microplates in the presence of 40 mm KCl and FM4–64. Brighter fluorescence signals are an indication of increased SV endocytosis, and the dye and lipid interactions thus increase in depolarization of synaptosomes. Significantly higher baseline fluorescence signals were detected in synaptosomes of SIV⁺ animals, even in the absence of KCl. Synaptosomes from SIV⁺ animals pretreated with an inhibitor of dynamin-mediated endocytosis exhibited low signals in both the presence and absence of KCl. B, Quantification of fluorescence signals using the total fluorescence signals recorded in different conditions are graphed. H.d., Hydroxy dynasore. *p < 0.05 (one-way ANOVA with Tukey HSD). **p < 0.005 (one-way ANOVA with Tukey HSD). Error bars indicate SD of mean; ± SEM.

Figure 9.

Treatment of synaptosomes with a phosphatase-activating drug (FTY720) mitigates SV endocytosis in frontal cortex synaptosomes of rhesus macaques. A, FTY720 was applied in different concentrations on frontal cortex synaptosomes in SVE assay in the presence of 40 mm KCl and FM4–64. FTY720 produced lower fluorescence signals in a concentration-dependent fashion. In SIV⁺ synaptosomes, higher concentrations of FTY720 were required to quench the signals. B, Quantification of fluorescence signals detected in A using the ratio of the total fluorescence recorded after depolarization by 40 mm KCl in different conditions. *p < 0.05 (Student's t test). **p < 0.005 (Student's t test). Error bars indicate mean ± SEM.

Figure 10.

Dynamics of the impact of SIV infection and ART regimen on Syn I phosphorylations. A, Amino acid residues studied in this work and their approximate positions in the Syn I domains (A–E). B, Observed changes in phosphorylations at multiple sites of Syn I in total lysates versus synaptosome preparations and functional impact of phosphorylation of these sites in normal physiological conditions based on Cesca et al. (2010). Thin arrows indicate insignificant trends. Thick arrows indicate significant changes. NC, No changes; AA, amino acids; NT, neurotransmitter.