Date of Award
Doctor of Philosophy (PhD)
Although combined antiretroviral therapy (cART) effectively suppresses HIV-1 infection and increases the lifespan of people living with HIV, up to 50% of HIVinfected individuals are experiencing HIV-associated neurocognitive disorder (HAND), with clinical symptoms ranging from mild cognitive impairment to severe dementia, for which no effective treatment is currently available. Synaptodendritic impairments, such as decreased dendritic spines and synaptic loss, that occur in various brain regions including the prefrontal cortex and hippocampus are key pathological features of HAND that correlates closely with neurocognitive impairments. The development of synaptodendritic impairment in HAND in the post-cART era is complicated and not fully understood; However, chronic presence of viral proteins, such as gp120 and Tat, may disrupt neuronal homeostasis in the CNS could lead to synaptodendritic impairments and functional alterations of neurons. An important mechanism that is disrupted in HAND is endolysosome function. Endolysosome dysfunction is seen in a variety of neurodegenerative disorders and plays a crucial role in disrupting the homeostasis of the cell. We have shown that gp120 and Tat as well as certain antiretroviral (ART) drugs de-acidify endolysosomes, induce enlargement of endolysosomes, and disrupt the function of endolysosomes. Endolysosomes are important in the maintenance and xviii neuronal homeostasis of neurons as neurons are long-lived post-mitotic cells that are extremely polarized cells with extensive processes that require constant membrane trafficking to establish and maintain axonal and somatodendritic plasma membrane domains. HIV-1 viral protein induced endolysosome dysfunction could contribute to the development of synaptodendritic impairments in HAND. Thus, preventing endolysosome dysfunction represents a promising therapeutic strategy. It is well known that estrogen exerts enhancing effects on cognition and synaptic function and 17β-estradiol (17βE2), the primary estrogen in the blood, has been recognized as a potential therapeutic agent against HIV-associated neurocognitive disorders. Previous studies have shown that 17βE2 suppresses HIV-1 replication, protects against HIV-1 protein-induced neuronal cell death, and promotes synapse formation. However, feminizing effects and other side effects such as cancer, thromboembolism, coronary heart disease, and stroke may limit the use of 17βE2 in the larger general population. Our studies focused on 17α-estradiol (17αE2), a natural non-feminizing estrogen that has neuroprotective effects and is the predominant form of estrogen in the brain. In the prefrontal cortex and the hippocampus, regions that facilitate learning and memory, membrane-bound ER are present and found in dendritic spines while nuclear estrogen receptors (ER) are found to a lesser extent. The presence of extranuclear membrane-bound estrogen receptors in neurons has been implicated in estrogen’s enhancing effect on cognition and synaptic xix function. ERα has been detected in hippocampal neurons and located on endolysosomes such that ERα was thought to be degraded. However, as a soluble cytosolic protein, ERα most likely resides on the cytosolic side of the endolysosome membranes rather than in the lumen where it might be degraded. Rather, the endolysosome localization of ERα may alter endolysosome function directly. We have shown that gp120 and Tat induced neurotoxic effects and contributed to the development of HAND, at least in part, by disrupting the function of the endolysosomes. We hypothesize that 17αE2 protects against gp120-and Tat-induced endolysosome dysfunction and synaptodendritic injury via ERα present on endolysosomes. The present studies were conducted in primary cultured hippocampal neurons and a hippocampal cell line. HIV-1 transgenic (Tg) rats that model HAND were treated with 17αE2. We used a range of different methods to test our hypothesis. Methods used to determine the effect of 17αE2 on HIV-1 protein-induced endolysosome dysfunction and synaptodendritic injury include, ratio-metric endolysosome pH measurement, immunostaining for assessing morphology and function and endolysosomes, live-cell imaging and immunostaining for quantification of changes in dendritic spines, and Golgi-Cox staining for quantification of alteration in dendritic spines in hippocampus of HIV-1 Tg rats. Methods used to assess the involvement of ERα include ERα knockdown with siRNA approach and overexpressing the ERα mutant (C451A) that lacks endolysosome localization. xx We found that treating hippocampal neurons with either gp120 or Tat enlarged endolysosomes, elevated endolysosome pH, lowered the percentage of cathepsin D positive endolysosomes, and decreased dendritic spine density. 17αE2 not only prevented gp120-and Tat-induced endolysosome de-acidification and enlargement, but also prevented gp120-and Tat-induced decreases in dendritic spine density. ERα knockdown blocks the protective effects of 17αE2 against gp120-and Tat-induced endolysosome dysfunction and reduction in dendritic spines. Over-expressing the ERα mutant (C451A) that lacks endolysosome localization blocks the protective effects of 17αE2 against gp120- induced endolysosome dysfunction and reduction in dendritic spines. Furthermore, 17αE2 treatment attenuates the development of endolysosome enlargement and reduction in dendritic spines in HIV-1 Tg rats. Our findings support our hypothesis that 17αE2 protects against gp120-and Tatinduced endolysosome dysfunction and synaptodendritic injury via endolysosome localization of ERα. Our findings suggest that 17αE2 represents a promising therapeutic agent that may slow or prevent the progression of HAND.
Miller, Nicole, "Endolysosome Involvement In The Protective Effects Of 17ơ-Estradiol Against HIV-1 Protein-Induced Neuronal Injury" (2021). Theses and Dissertations. 4089.
Available for download on Saturday, August 26, 2023