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Jamshed Arslan, Pharm D, PhD
Cholesterol is an essential part of animal cell membranes. Cholesterol-rich lipid rafts maintain the fluidity and protein trafficking of plasma membranes. Cellular ABCA1 protein moves cholesterol and certain fats outside of the cell. Dysregulated lipid rafts and cholesterol metabolism contribute to neural damage in Alzheimer’s disease (AD), Parkinson’s disease (PD), and prion diseases. Interestingly, prion infection and HIV infection share one common feature: reduction in ABCA1, which leads to enhanced intracellular cholesterol and overabundant, dysregulated lipid rafts. HIV is thought to use Nef protein to bring about these changes. HIV-infected subjects on anti-retroviral therapy (ART) show high levels of Nef in their blood and CSF, but this extracellular Nef (ExNef) is not high enough to explain the neural damage in HIV-associated neurocognitive disorders (HAND). A team of scientists from Australian and American medical institutes investigated if the ExNef-mediated reorganization of lipid rafts is behind the neuropathology in HIV-positive subjects. As expected, the researchers found that ExNef increased and modified the lipid rafts, and triggered a redistribution of increased amounts of AD-related proteins (APP and Tau) to the rafts. They also discovered that ExNef reduces cholesterol efflux from neural cells by reducing ABCA1.
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Dual RNAscope ISH-IHC showing formalin-fixed paraffin-embedded tissue sections of human prostate cancer were probed for ABCA1 mRNA (ACD RNAScope Probe, catalog # 432291; Fast Red chromogen, ACD catalog # 322360), right. Adjacent tissue section, left, was processed for immunohistochemistry using ABCA1 Antibody (NB400-105) followed by incubation with IgG Secondary Antibody HRP Polymer (VC003) and DAB chromogen (yellow-brown), and counterstained with hematoxylin (blue). Specific staining was localized to glandular cells.
To study the effects of ExNef on neurons, the team successfully delivered GFP-tagged Nef into the human neural cells by fluorescently-labelled extracellular vesicles produced from HEK293 cells transfected with Nef. Western blot analysis showed a decrease in total and cell surface ABCA1 that corresponded to reduced cholesterol efflux. Likewise, confocal microscopy revealed an abundance of a fluorescently-labelled raft-associated protein. Western blot and confocal microscopy also confirmed the abundance and localization of AD-related proteins, APP and Tau, to lipid rafts in ExNef treated cells. ELISA results showed tripling of Aβ42 without any abundance of PD-related protein α-synuclein. When the neural cells pre-treated with ExNef were exposed to 50 µM glutamate to study the glutamate-induced excitotoxicity (as in AD and PD), neural apoptosis was significantly increased. Increased activity of acetylcholinesterase (AChE) was also observed, indicating cholinergic hypofunction associated with cognitive decline in AD. By using a cholesterol acceptor to disrupt lipid rafts, the team was able to reverse the ExNef-induced elevation of lipid rafts, APP, Tau, and AChE.
These results suggested that lipid raft modification plays a key role in ExNef-induced neuronal impairment. Next, the team assessed the translational values of these findings in the brains of animals and humans.
IHC image of immersion fixed paraffin-embedded sections of human Alzheimer's disease brain using Goat Polyclonal Anti-Tau Antibody (AF3494). Tissue was stained with the Anti-Goat HRP-DAB Cell & Tissue Staining Kit (CTS008) (brown) and counterstained with hematoxylin (blue).
In line with the in vitro studies, the researchers found that ExNef injection into the mice reduces ABCA1 while it simultaneously enhances APP levels in the brain. They obtained post-mortem brain samples of HIV-negative (control) and ART-treated HIV-infected patients. As predicted, lower ABCA1, elevated raft marker GM1, and a trend of elevated APP and Tau were found in the brains of HIV-infected subjects with HAND.
Thus, the in vitro and in vivo findings support the idea that ExNef-mediated lipid raft modification redistributes APP and Tau to cause neuronal dysfunction.
This study provides a mechanistic explanation of HAND pathogenesis and provides a connection between HAND and AD. The findings offers a pharmacological approach to reverse neurodegeneration and neuroinflammation by opposing Nef-induced modification of lipid rafts.
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Note:
Ditiatkovski et al. (2020) used Rabbit polyclonal anti-APP (NBP1-76910)
Jamshed Arslan, Pharm D, PhD
Dr Arslan is an Assistant Professor at Salim Habib University (formerly, Barrett Hodgson University), Pakistan. His interest lies in neuropharmacology and preparing future pharmacists.
References
Ditiatkovski, M., Mukhamedova, N., Dragoljevic, D., Hoang, A., Low, H., Pushkarsky, T., Fu, Y., Carmichael, I., Hill, A. F., Murphy, A. J., Bukrinsky, M., & Sviridov, D. (2020). Modification of lipid rafts by extracellular vesicles carrying HIV-1 protein Nef induces redistribution of amyloid precursor protein and Tau, causing neuronal dysfunction. The Journal of Biological Chemistry. https://doi.org/10.1074/jbc.RA120.014642
