Perturbation of synapsins homeostasis through HIV-1 Tat-mediated suppression of BAG3 in primary neuronal cells

Abstract

HIV-1 Tat is known to be released by HIV infected non-neuronal cells in the brain, and after entering neurons, compromises brain homeostasis by impairing pro-survival pathways, thus contributing to the development of HIV-associated CNS disorders commonly observed in individuals living with HIV. Here, we demonstrate that synapsins, phosphoproteins that are predominantly expressed in neuronal cells and play a vital role in modulating neurotransmitter release at the pre-synaptic terminal, and neuronal differentiation become targets for Tat through autophagy and protein quality control pathways. We demonstrate that the presence of Tat in neurons results in downregulation of BAG3, a co-chaperone for heat shock proteins (Hsp70/Hsc70) that is implicated in protein quality control (PQC) processes by eliminating mis-folded and damaged proteins, and selective macroautophagy. Our results show that treatment of cells with Tat or suppression of BAG3 expression by siRNA in neuronal cells disturbs subcellular distribution of synapsins and synaptotagmin 1 (Syt1) leading to their accumulation in the neuronal soma and along axons in a punctate pattern, rather than being properly distributed at axon-terminals. Further, our results revealed that synapsins partially lost their stability and their removal via lysosomal autophagy was noticeably impaired in cells with low levels of BAG3. The observed impairment of lysosomal autophagy, under this condition, is likely caused by cells losing their ability to process LC3-I to LC3-II, in part due to a decrease in the ATG5 levels upon BAG3 knockdown. These observations ascribe a new function for BAG3 in controlling synaptic communications and illuminate a new downstream target for Tat to elicit its pathogenic effect in impacting neuronal cell function and behavior.

Fig. 1. HIV-1 Tat promotes alterations in synaptic vesicle proteins distribution and homeostasis.

a Single neuron images showing that Tat overexpression affects neuronal processes and synaptic vesicle distribution as stained with Tubb3 and synapsins (Syn), respectively. Synapsins accumulate close to soma upon Tat expression, compared to the control where synapsins are primarily observed in neuronal processes. b Synapsin distribution in neurons in a population wide image shows the disruption of synaptic vesicle network during Tat expression. c A second synaptic vesicle marker, synaptotagmin 1 (Syt1), confirms the aggregation of synaptic vesicle proteins in neurons upon Tat expression as compared to the Ad-Null transduction. d In addition to distribution, total synapsin proteins levels are altered in the soluble and insoluble fractions of neuronal lysate upon Tat expression. e Synaptotagmin 1 exhibits more accumulation in the insoluble fraction compared to soluble fraction (~70%) in neurons where Tat is expressed. f Dose response of soluble recombinant Tat (rTat) protein on the distribution of synapsins indicates the increased impairment at higher doses of Tat. Syt1, Syn, and Tubb3 stand for synaptotagmin 1, synapsins, and β3-tubulin, respectively

Fig. 2. HIV-1 Tat decreases BAG3 protein and mRNA.

a Tat expression reduced BAG3 protein in rat primary hippocampal and cortical neurons, R1 and R2 indicate two replicates shown on this gel. b, c Hippocampal and cortical neurons expressing Tat show time-dependent reduction in BAG3 (VCP was probed as a control). d BAG3 level drops in the brains of Tat-transgenic mice under Dox-induced Tat expression. e Tat expression results in a decreased level of BAG3 protein even during adenoviral overexpression of BAG3, indicating a post-transcriptional reduction. f Inhibition of the proteasome using MG132 does not restore Tat-induced BAG3 reduction. g Inhibition of the lysosome using Baf A1 does not block Tat-induced BAG3 decrease. h Neuronal BAG3 mRNA is decreased by Tat expression. i qRT-PCR measurement of BAG3 mRNA levels under control and Tat expression when transcription is inhibited by Actinomycin D. j Recombinant Tat decreases BAG3 mRNA levels. Quantifications show mean ± SD with n ≥ 3. P-values calculated using t-test where *P ≤ 0.01, **P ≤ 0.005, ***P ≤ 0.001

Fig. 3. BAG3 KD leads to the accumulation and aggregation of synaptic vesicle proteins.

a BAG3 co-localizes with Syt1 in the control and oxidative-stress induced neurons. b SVs probed with Syn and Syt1 indicate the accumulation of synaptic vesicles along the major neuronal processes and soma under BAG3 KD. c BAG3 KD under normal and H₂O₂-induced oxidative stress conditions results in accumulation of Syn in the insoluble fraction of neuronal protein lysate. d BAG3 KD under normal and stress conditions results in accumulation of Syt1 in the insoluble fraction of neuronal protein lysate. e Neuronal culture in a microfluidic device allows the assessment of neuronal processes separate from the soma. BAG3 KD using Ad-siBAG3 transduction leads to the increased formation and accumulation of syn-positive aggregates along processes. f BAG3 KD impairs the distribution of synapsins in the neurons as compared to the control

Fig. 4. BAG3 KD impairs lysosomal autophagy and client protein ubiquitination under oxidative stress.

a Under control and oxidative stress conditions, BAG3 KD leads to decreased level of LC3-II. b Co-immunoprecipitation with BAG3 and LC3 confirms the interaction of BAG3 and LC3. c Under control and oxidative stress conditions, BAG3 KD leads to reduced levels of ATG5, a protein essential for LC3-I to LC3-II conversion. d Co-immunoprecipitation with ATG5 antibody and probing for BAG3 confirms the interaction of BAG3 and ATG5. e BAG3 KD in neurons leads to inhibition of ubiquitination under oxidative stress and inhibition of proteasome or lysosome. The bracket shows the higher molecular weight region at which ubiquitination is impaired under BAG3 KD. f ATG5 co-localizes with Hsc70 in neurons. g BAG3 co-localizes with UB in neurons. h ATG5 and BAG3 are suppressed under Tat expression over a range of 2–4 days post-transduction. i ATG5 mRNA level is not decreased by Tat expression compared to control

Fig. 5. BAG3 interacts with synaptic vesicle proteins and synapsins likely via the WW domain.

a Co-immunoprecipitation with Syn antibody and probing for BAG3 confirms the interaction of BAG3 with synapsins in rat primary cortical and hippocampal neurons under normal and oxidative stress conditions. b Co-immunoprecipitation with BAG3 antibody and probing for synaptotagmin 1 confirms the interaction of BAG3 and Syt1. c BAG3 co localizes with Syt1 in neurons. d, e Expression of Flag-BAG3 (1–300) in neurons results in the disruption of neuronal synapsins distribution similar to BAG3 KD and Tat expression

Fig. 6. BAG3-mediated homeostasis of synaptic vesicle proteins turnover disrupted by HIV-1 Tat.

BAG3 is essential for the formation of LC3-II via regulating ATG5 protein. BAG3 is involved in the homeostasis and turnover of synapsins through lysosmal autophagy and vesicle axonal transport