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The Role of Protein Kinase R-like ER Kinase (PERK) in HIV-Associated Neurocognitive Disorders (HAND)
HIV-Associated Neurocognitive Disorders (HAND) are caused by the infection of HIV in the Central Nervous System (CNS). HIV (Human Immunodeficiency Virus) is a retrovirus that spreads due to the transmission of certain bodily fluids such as semen, blood, and breast milk. The virus’ high mortality rate is due to a host of reasons, chiefly because it has a prolonged lysogenic cycle, attacks the host immune system, and has a high mutation rate. In other words, HIV genes are able to lie dormant within host cells because the virus integrates its own RNA genes into the host DNA genome through a process called reverse transcription. In addition, by infecting helper T cells and macrophages, HIV renders the host organism more susceptible to minor infections in a condition termed AIDS (Acquired Immunodeficiency Syndrome). Furthermore, typical antiretroviral therapies are ineffective because HIV does not contain any genetic proofreading mechanisms, allowing the virus to constantly mutate into new strains that can bypass the host’s immunological memory.10 (Castano et al. 2017)
Recently, the development of a new treatment called combined anti-retroviral therapy (cART) in 1996 has drastically extended the lives of HIV+ patients. However, though patient life expectancies were extended, the quality of life for the survivors significantly deteriorated. This was due to a host of newly discovered HIV-influenced neurodegenerative symptoms that had previously not been noted because very few HIV+ patients survived long enough for neurodegeneration to occur. The most severe symptoms for HIV-associated neurocognitive disorders (HAND) include behavioral change, cognitive impairment, memory loss, and motor dysfunction so daily functions like driving and memorizing dates and locations become impossible to accomplish for HAND patients. Examples of HIV-associated neurocognitive disorders (HAND) include HIV-associated dementia (HAD), mild neurocognitive disorder (MND), and asymptomatic neurocognitive impairment (ANI). The milder forms of HAND, ANI and MND have increased prevalence since 1996 because cART is relatively ineffective in destroying the small amounts of erroneous cellular mRNA produced by ANI and MND. In contrast, the most extreme form of HAND, HAD, has reduced prevalence because the symptoms of severe neurodegeneration can be generally contained if cART is used in combination with the right protease inhibitors (PI) and integrase strand transfer inhibitors (INSTI)3. It is important to study the causes of HIV-associated neurocognitive disorders (HAND) not only because it impacts 30-60% of all HIV-infected patients2, but also because it lends a new perspective on other neurodegenerative diseases (Alzheimer’s, Parkinson’s) previously hypothesized to be caused by age-related elements as opposed to virus-induced factors. Since it is a new area of research, the specific causes of HAND are unknown and the problem is compounded by the fact that there could be several factors that contribute to development of HAND.6 (Vartak-Sharma et al. 2017)
One of the known contributing factors to HAND is the HIV TAT protein (Trans-Activator of Transcription). TAT is a master regulatory viral protein that takes advantage of the host’s cellular mechanisms to stimulate transcription for itself and other vital HIV genes, creating a positive feedback loop that doesn’t stop until the newly assembled virus lyses out of its host. Previous studies by Dr. Jordan-Sciutto indicated TAT can disrupt neuronal and glial cells and damage dopamine-rich regions of the CNS. HIV is able to enter the CNS and travel to the brain by lying hidden in infected monocytes (a type of leukocyte) that move through the blood-brain barrier. Once inside the brain, the HIV genes remains dormant in the monocytes until the monocytes mature into macrophages at which point a signal is given and HIV breaks into the lytic cycle. This causes all the HIV infected cells, including astrophytes (a type of glial cell), to stimulate the release of inflammatory proteins, cytokine signaling, and viral proteins that result in neuron inflammation and neurotoxicity.5
The TAT protein and other viral proteins also induce the inflammatory response and the Integrated Stress Response (ISR) in nearby uninfected cells, which if unregulated, can cause neuron toxicity. The ISR is responsible for regulating calcium ions, endoplasmic reticulum genes, protein synthesis, protein folding, protein degradation and the endogenous antioxidant response. Known causes of ISR include oxidative stress, hypoxia, toxins, nutrient deprivation, and viral infection. The ISR is meant to help promote cellular survival in short term cases, therefore the response is an evolutionarily conserved process in almost all human cells, however prolonged or faulty activation of the response can mean cell apoptosis. The master regulator protein of ISR, called BiP/GRP78 (BiP), stands for immunoglobulin heavy chain-binding protein/glucose-regulated protein 78. The BiP protein, and “ER-resident chaperone” activates three receptor proteins, PERK (PKR-like ER kinase), ATF6 (activating transcription factor 6), and IRE1a (inositol-requiring enzyme 1a).2 These receptor proteins are present in almost all human cells with metabolic functions, however only neuronal cells are affected by HAND because current treatments for HIV are not effective at destroying the virus in the CNS. If BiP is not working, it could lead to translation attenuation, transactivation of ER chaperone gene promoters, and ER-mediated protein degradation. BiP, once activated, goes to the Golgi body, and then the endoplasmic reticulum where it induces the ER stress response element (ESRE) and transcribes genes for the Unfolded Protein Response (UPR). The UPR is a pathway activated in the lumen of the endoplasmic reticulum and is responsible for the degradation of proteins that have been misfolded. The prolonged presence of misfolded proteins or over-degradation of normal proteins can induce the neuronal toxicity responsible for HAND.2 (Akay et al. 2012)
One of these misfolded proteins are called AB plaques, which unlike other misfolded proteins remain undetected by neuronal cells. It is still not known the reason why the accumulation of AB plaques are not regulated by any cellular pathway. It is hypothesized that AB plaques are actually supposed to prevent toxicity, so the body response categorizes toxicity as a more important factor than maintaining the homeostasis of AB plaques. However, the oligomerization (clumping) of AB plaques ironically causes toxicity. AB plaques are created when a B-site amyloid precursor protein cleaving enzyme-1 (BACE1) causes the cleavage of amyloid precursor protein (APP), which results in the formation of amyloid-B (AB) peptides. These AB peptides oligomerize to form extracellular plaques (AB plaques). In vitro studies by Dr. Jordan-Sciutto produced results that show by inhibiting BACE1 through the N-methyl-D-aspartate receptor (NMDAR), neurotoxicity is decreased via decreased level of APP. In the same study, anti-retroviral therapy (ART) was seen to be effective in inhibiting the development of extracellular plaques in HAND patients but not in Alzheimer’s, which makes sense because ART is designed to limit viral infections only.1 (Jordan-Sciutto et al. 2018)
To summarize thus far, increased stress levels (e.g. viral infection) causes the faulty activation of the ISR (Integrated Stress Response), which through BiP and the three receptor proteins (PERK, ATF6b, IRE1a) induce the malfunction of the UPR (Unfolded Protein Response) allowing misfolded proteins (AB plaques) to accumulate and oligomerize (clump together), resulting in the inflammatory neurotoxic response that is a common symptom in HAND.1,2
Finally, the grant proposal written by Dr. Jordan-Sciutto noticed similar symptoms between Alzheimer’s disease (AD) and HAND, suggesting a possible link between the two. Further analysis found the oligomerization (or accumulative clumping) of Amyloid-B (AB) plaques through cleavage of the Amyloid Precursor Protein (APP) by B-site Amyloid precursor protein Cleavage Enzyme 1 (BACE1)1 happens in both HAND and Alzheimer’s disease (AD). Faulty UPR activation is also present in both HAND and AD; although current research on AD believes the activation of the PERK protein receptor is a primary factor, yet HAND data analysis shows PERK does not seem to be correlated with any neural toxicity. By studying the similarities and differences in the age-related (AD) versus virus-induced (HAND) neurodegenerative diseases, effective drugs and treatments can be developed to delay or inhibit neurotoxic effects.1 (Jordan-Sciutto, et al. 2018)
The lab used a process called immunoblotting and immunofluorescence to allow visual and photographic evidence. Immunofluorescence was used to mark and track specific cells by marking cellular antigens with special antibodies that have a fluorescent dye. Immunoblotting was used to separate protein supernatants. Green live cell imaging is a technique used to see cells under digital fluorescent microscopes. The process is similar to gel electropheoresis, using proteins instead of genetic material. Materials used include protein assay dye (to mark and see proteins), PVDF membrane (used for western immunoblotting), and prestained molecular weight ladder (used to compare unknown materials in the gel).1,2,3
Tissue samples and Antibody Marking:
Tissue sections were obtained from the National NeuroAIDS Tissue Consortium (NNTC). In all experiments, the tissue samples were from the mid-frontal cortical autopsy tissue. Data about the age, sex, mental status, and post-mortem interval of each tissue was provided. Antibodies from various animals such as horses, mice, rabbits, and goats were mainly used for marking proteins. Mouse and rat neurons were prepared and used for the experiments. Antibodies for BACE1 and APP were used to mark and track levels of the proteins. HIV/MDM supernatants were also used to differentiate between healthy and carefully controlled HIV infected cells.1,2,3
The lab used GraphPad Prism 3.02 software for data analysis. All results must be deemed statistically relevant. For example, controlled activation of the PERK protein increased neurotoxicity but not enough to be statistically relvant.1,3
I will be studying the role PERK protein plays in the UPR and ISR pathway that cause HAND. However, the likelihood of PERK itself inducing HAND rather than simply being part of a long transduction pathway is relatively small. Previous studies show that the ATF6 controls the transcription factor for the master regulator of ISR, the immunoglobulin heavy chain-binding protein/glucose-related protein 78 (BiP/GRP78 or BiP for short). This not only enables a positive feedback loop of ATF6 itself, but activates the entire Integrated Stress Response since BiP also activates PERK and IRE1a. Thus, a faulty ATF6 protein is more likely to cause HAND than the PERK protein. Nonetheless, it is important to study the role PERK plays in UPR and ISR because it is a shared response in Alzheimer’s disease.
- Jordan-Sciutto, Kelly, et al. “BACE1 Mediates HIV-Associated and Excitotoxic Neuronal Damage Through an APP-Dependent Mechanism.” Journal of Neuroscience, Society for Neuroscience, 2 May 2018, www.jneurosci.org/content/38/18/4288.
- Akay, C, et al. “Activation Status of Integrated Stress Response Pathways in Neurones and Astrocytes of HIV-Associated Neurocognitive Disorders (HAND) Cortex.” Neuropathology and Applied Neurobiology, U.S. National Library of Medicine, Apr. 2012, www.ncbi.nlm.nih.gov/pmc/articles/PMC3708539/.
- Jordan-Sciutto, Kelly, et al. “Differential Effects of Antiretroviral Drugs on Neurons In Vitro: Roles for Oxidative Stress and Integrated Stress Response.” Journal of Neuroimmune Pharmacology : the Official Journal of the Society on NeuroImmune Pharmacology, U.S. National Library of Medicine, Mar. 2018, www.ncbi.nlm.nih.gov/pubmed/28861811.
- “Potential Diagnostic and Prognostic Tools for HIV-Associated Neurocognitive Disorders.” ScienceDaily, ScienceDaily, 20 July 2017, www.sciencedaily.com/releases/2017/07/170720113652.htm.
- “Antidepressant May Improve Cognitive Symptoms in People with HIV.” ScienceDaily, ScienceDaily, 25 Feb. 2016, www.sciencedaily.com/releases/2016/02/160225153620.htm.
- “HIV, Tat and Dopamine Transmission.” Neurobiology of Disease, Academic Press, 27 Apr. 2017, reader.elsevier.com/reader/sd/pii/S096999611730089X?token=1846B1D472146A7712AFDF5FC3A6324137FD2D3417E093B8CAA2365209F945BDAF2333BC95DA9DA22B1DDD9938DD32D6.
- Vartak-Sharma, Neha, et al. “Astrocyte Elevated Gene-1 (AEG-1) and the A(E)Ging HIV/AIDS-HAND.” Progress in Neurobiology, U.S. National Library of Medicine, Oct. 2017, www.ncbi.nlm.nih.gov/pubmed/27090750.
- Clifford, David B, and Beau M Ances. “HIV-Associated Neurocognitive Disorder.” The Lancet. Infectious Diseases, U.S. National Library of Medicine, Nov. 2013, www.ncbi.nlm.nih.gov/pmc/articles/PMC4108270/.
- Saye, Koo, et al. “Controversies in HIV-associated neurocognitive disorders.” The Lancet. Infectious Diseases, U.S. National Library of Medicine, Nov. 2014, https://www.thelancet.com/journals/laneur/article/PIIS1474-4422(14)70137-1/
- Nightingale, Sam, et al. “Controversies in HIV-Associated Neurocognitive Disorders.” The Lancet. Neurology, U.S. National Library of Medicine, Nov. 2014, www.ncbi.nlm.nih.gov/pmc/articles/PMC4313542/.
- Castano, Victor M., et al. “The Emergence and Evolution of the Research Fronts in HIV/AIDS Research.” PloS One, Public Library of Science, 25 May 2017, www.ncbi.nlm.nih.gov/pmc/articles/PMC5444800/.
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