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Researchers Identify Key Mechanisms Underlying HIV-Associated Cognitive Disorders - HIV/Brain Damaged reduced by Rapamycin in Mice, in Vitro - new study
 
 
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"HIV-associated neurocognitive disorder (HAND) occurrence is increasing in aging HIV patients"
 
from Jules: while everyone is focused on a hope for a cure many HIV+ older than 50 are increasingly suffering with cognitive impairment & neurologic damage.
 
"rapamycin reduced the incidence of neurodegeneration in the mice and in cell models.....These in vivo results show that Tat-mediated alterations in autophagy lead to cell death and that this effect is reversed with Rapam......Our studies confirm Tat causes neurodegeneration in vivo and in vitro, both of which are reversed by Rapam treatment."
 
What is autophagy?
 
http://www.news-medical.net/health/Autophagy-What-is-Autophagy.aspx......http://en.wikipedia.org/wiki/Autophagy
 
"Autophagy is a normal physiological process in the body that deals with destruction of cells in the body. It maintains homeostasis or normal functioning by protein degradation and turnover of the destroyed cell organelles for new cell formation. During cellular stress the process of Autophagy is upscaled and increased.Oct 7, 2014"
 
"the combined impact of proteins, such as Tat, Nef, and gp120, on neuronal autophagy is likely to exacerbate neurodegeneration.....The present studies follow up on a recent report that showed Tat affects the neuronal lysosome pathway and accumulation of autophagy proteins"
 
"Currently >30 million people live with HIV worldwide. Modern treatment regimens result in HIV suppression and immune recovery; however, the prevalence of HIV-associated neurocognitive disorders (HAND) and neurodegeneration has remained the same or increased, in particular among people over the age of 50. In the United States, the aging population represents one of the fastest growing groups with HIV"
 
"Our current data may partially explain mechanisms that lead to HAND; Tat, in concert with other factors, may initially alter neuronal autophagy in a deleterious manner, while other processes or chronic exposure to Tat and overuse of the autophagy system may result in lower autophagy levels and concomitant neurodegeneration in late stages of HIV infection. Therapies that increase LAMP2A function through blocking interactions with Tat or increasing LAMP2A expression and increasing autophagy efficiency may be useful in combatting HAND."
 
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Researchers Identify Key Mechanisms Underlying HIV-Associated Cognitive
 
Disorders - full text below
 
press release
 
http://ucsdnews.ucsd.edu/pressrelease/researchers_identify_key_mechanisms_underlying_hiv_associated_cognitive_dis
 
While antiretroviral therapies have significantly improved and extended the lives of many HIV patients, another insidious and little discussed threat looms for aging sufferers – HIV-associated neurocognitive disorders (HAND). The disorders, which strike more often in HIV patients over age 50, can result in cognitive impairment, mild to severe, making everyday tasks a challenge.
 
But new findings, published today by researchers at the University of California, San Diego School of Medicine, open the door to the development of new therapies to block or decrease cognitive decline due to HIV-associated neurocognitive disorders (HAND), estimated to affect 10 to 50 percent of aging HIV sufferers to some degree.
 
The study is published in the Feb. 4 issue of the Journal of Neuroscience. Eliezer Masliah, MD, a professor of neurosciences and pathology, is senior author; Jerel Adam Fields, PhD, a postdoctoral researcher in Masliah's lab, is first author.
 
"Most people know HIV affects the immune system's ability to fight disease, but they may not be aware that HIV gets into the brain and can damage brain cells," said Masliah, an investigator with the HIV Neurobehaviorial Research Center at UC San Diego.
 
There are several types of HAND, the most common being Mild Neurocognitive Disorder (MND). "Most of the cases we see are mild to moderate," said Masliah. But even mild cognitive problems can interfere with everyday functioning and reduce quality of life, he added, noting that sufferers may have difficulty with daily activities like balancing a checkbook or driving directions.
 
In their study, the researchers sought to understand the mechanisms by which HIV damages brain cells. They focused on the HIV tat protein's role in a critical disposal process, known as autophagy, in neurons. "Neurons produce a lot of proteins as part of their normal functions, some of which are damaged and need to be cleared away," said Masliah. "Autophagy acts like a garbage disposal and removes and destroys the damaged proteins."
 
Masliah and colleagues found that HIV tat "hijacks" the disposal process by interfering with key pathways. "HIV tat is secreted from infected cells in the brain, and subsequently enters neurons where it binds to a protein that is important for multiple autophagy pathways," explained Fields. "This binding disrupts the neuronal autophagy process, resulting in the accumulation of damaged proteins and death of the neuron. Overtime, this may lead to impaired cognitive abilities."
 
To counteract this disruption, Fields said the team conducted mouse studies using the cancer drug rapamycin, which has been reported to promote autophagy in other cell types. "By speeding up neuronal autophagy, we hoped to override the disruptive effects of HIV tat on the process," he said.
 
The experiments produced positive results. "We found that rapamycin reduced the incidence of neurodegeneration in the mice and in cell models," said Fields. While the feasibility of rapamycin as a neurological treatment in humans is currently inconclusive, Fields said the study's results are exciting because they prove, in principle, that enhancing autophagy reduces tat-induced neurodegeneration.
 
"By understanding the molecular underpinnings of how HIV proteins kill nerve cells, we can design drugs that will block this process," said Masliah. Co-authors include Wilmar Dumaop, UCSD Department of Pathology; Simona Elueteri, Sofia Campos, Elisabeth Serger, Margarita Trejo, Kori Kosberg, Anthony Adame, Brian Spencer and Edward Rockenstein, UCSD Department of Neurosciences; and Johnny J. He, departments of Cell Biology and Immunology, University of North Texas Health Science Center.
 
Funding for this research came, in part, from the National Institute of Aging (grant AG043384), the National Institute of Mental Health (grants MH062962, MH5974 and MH83506) and the National Institute for Neurological Disorders and Stroke (grant 1F32NS083426-01).
 
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The Journal of Neuroscience, 4 February 2015
 
HIV-1 Tat Alters Neuronal Autophagy by Modulating Autophagosome Fusion to the Lysosome: Implications for HIV-Associated Neurocognitive Disorders
 
Abstract
 
Antiretroviral therapy has increased the life span of HIV+ individuals; however, HIV-associated neurocognitive disorder (HAND) occurrence is increasing in aging HIV patients. Previous studies suggest HIV infection alters autophagy function in the aging CNS and HIV-1 proteins affect autophagy in monocyte-derived cells. Despite these findings, the mechanisms leading to dysregulated autophagy in the CNS remain unclear. Here we sought to determine how HIV Tat dysregulates autophagy in neurons. Tat caused a dose-dependent decrease in autophagosome markers, microtubule-associated protein-1 light chain ß II (LC3II), and sequestosome 1(SQSTM1), in a membrane-enriched fraction, suggesting Tat increases autophagic degradation. Bafilomycin A1 increased autophagosome number, LC3II, and SQSTM1 accumulation; Tat cotreatment diminished this effect. Tat had no effect when 3-methyladenine or knockdown of beclin 1 blocked early stages of autophagy. Tat increased numbers of LC3 puncta and resulted in the formation of abnormal autophagosomes in vitro. Likewise, in vivo studies in GFAP-Tat tg mice showed increased autophagosome accumulation in neurons, altered LC3II levels, and neurodegeneration. These effects were reversed by rapamycin treatment. Tat colocalized with autophagosome and lysosomal markers and enhanced the colocalization of autophagosome with lysosome markers. Furthermore, co-IP studies showed that Tat interacts with lysosomal-associated membrane protein 2A (LAMP2A) in vitro and in vivo, and LAMP2A overexpression reduces Tat-induced neurotoxicity. Hence, Tat protein may induce autophagosome and lysosome fusion through interaction with LAMP2A leading to abnormal neuronal autophagy function and dysregulated degradation of critical intracellular components. Therapies targeting Tat-mediated autophagy alterations may decrease neurodegeneration in aging patients with HAND.
 
Introduction
 
Currently >30 million people live with HIV worldwide. Modern treatment regimens result in HIV suppression and immune recovery; however, the prevalence of HIV-associated neurocognitive disorders (HAND) and neurodegeneration (Budka et al., 1987; Wiley and Achim, 1994; Gendelman et al., 1997; Cherner et al., 2007; Heaton et al., 2010) has remained the same or increased (Joska et al., 2010; Heaton et al., 2011), in particular among people over the age of 50. In the United States, the aging population represents one of the fastest growing groups with HIV (Scott et al., 2011).
 
Mechanisms of neurodegeneration causing HAND are not completely understood; however, recent studies have shown that HIV proteins interfere with clearance pathways such as macroautophagy (Alirezaei et al., 2008a, b; Zhou et al., 2011), a pathway necessary for recycling proteins or defective and older intracellular organelles (Cuervo, 2004). Macroautophagy (hereafter, autophagy) involves nucleation, initiation, elongation, and termination. Initially, autophagy-related proteins form a phagophore, which develops into the autophagosome, a double-membrane sac that delivers cytoplasmic material to the lysosomal compartment for degradation (Codogno et al., 2012). MAP1 light chain 3 (LC3I), one of the core autophagy proteins, is cleaved and conjugated into the membrane (LC3II) during autophagosomal formation. LC3II interaction with SQSTM1 mediates selective recruitment of ubiquitylated proteins to the autophagosome (Pankiv et al., 2007). Both commonly used at markers of autophagy, LC3II levels are affected by autophagy initiation as well as degradation, whereas SQSTM1 levels are inversely proportional to autophagy activity (Shvets et al., 2008). Alterations in autophagy pathways have been described in AD (Nixon et al., 2005; Pickford et al., 2008), PD (Cuervo et al., 2004; Crews et al., 2010), and other CNS disorders (Cuervo, 2004). Similarly, neurodegeneration patterns during HIV infection have been linked to defects in autophagy (Alirezaei et al., 2008a, b; Zhou et al., 2011; Fields et al., 2013). Recently, HIV gp120 and Nef have been shown to affect autophagy, respectively (Kyei et al., 2009; Fields et al., 2013). HIV-1 Tat suppresses autophagy function in macrophages (Li et al., 2011) and bystander monocytes (Van Grol et al., 2010). Tat treatment caused altered endolysosome morphology and function leading to neurotoxic effects (Hui et al., 2012). Therefore, we aimed to investigate the molecular targets of the autophagy pathway in neurons that are affected by Tat.
 
In these studies we found that in in vitro or in GFAP-Tat tg mice Tat induces abnormal neuronal autophagosome formation, and associates with lysosome-associated membrane protein 2A (LAMP2A). Tat reversed Bafilomycin A1 (BafA1)-mediated block of degradation of autophagy markers and induced colocalization of autophagosome and lysosome markers. Last, Tat induced neurotoxicity in vitro and neurodegeneration in vivo, and Rapamycin (Rapam) reversed these effects. These findings suggest that Tat directly alters lysosome fusion to autophagosomes, possibly through interaction with LAMP2A. This mechanism could contribute, in concert with other Tat actions, HIV proteins, or inflammatory factors, to neurodegeneration in HAND and reduced neuronal autophagy in aged HIV patients.
 
Discussion
 
The present study shows that HIV-1 Tat increases degradation of autophagic markers, and disrupts molecular inhibition of autophagy in neuronal cells. We found that Tat overcomes the actions of BafA1 on LC3II and SQSTM1 accumulation. Tat alters autophagosome morphology and quantity in neurons, though this effect was prevented when initial stages of autophagy were inhibited with shBECN1. Tat colocalizes with autophagy marker LC3 and lysosomal protease, CTSD, and induces LC3 and CTSD colocalization. Ultrastructure of Tat-treated neuronal cells and GFAP-Tat tg mice shows robust changes in autophagosome morphology and quantity. Moreover, Tat associates with neuronal LAMP2A in vitro and in brain homogenates from a GFAP-Tat tg mouse model of HAND. Importantly, Rapam or Torin 1 treatment and LAMP2A overexpression reduces Tat neurotoxic effects. Last, Rapam reversed the autophagy alterations and ameliorated the neurodegenerative and inflammatory phenotype in the GFAP-Tat tg mouse model. Our data suggest that Tat may facilitate abnormal autophagolysosome formation through interaction with lysosomal LAMP2A, leading to neurodegeneration.
 
The present findings are consistent with previous studies in which HIV proteins are secreted from infected cells and subsequently enter bystander cells (Benelli et al., 2000; Kandanearatchi et al., 2005; Kaul and Lipton, 2006; Alirezaei et al., 2007). HIV proteins affect cell processes through interaction with caspase machinery or NMDA receptors and thereby induce apoptosis, regulate calcium levels, alter glutamate excitotoxicity, and induce chemotaxis in bystander cells (Bonavia et al., 2001; Singh et al., 2005; Hargus and Thayer, 2013; Dalvi et al., 2014; Darbinian et al., 2014). Nef, a viral replication facilitator, prevents the destruction of HIV components by autophagolysosomes by inhibiting autophagic maturation in the host cells (Kyei et al., 2009). Gp120 was reported to increase autophagy in uninfected T-cells (Espert et al., 2007) as well as in LV-infected SK N SH cells (Zhou et al., 2011). Interestingly, in vitro experiments using HIV-infected microglial supernatant or gp120-transgenic mice showed a decrease in autophagy through possible decreases in Beclin levels and activity (Alirezaei et al., 2008a; Fields et al., 2013). Given that multiple HIV factors are expressed during HIVE, the combined impact of proteins, such as Tat, Nef, and gp120, on neuronal autophagy is likely to exacerbate neurodegeneration. Further investigations must be conducted to detect the singular as well as the aggregate effects of these proteins. The present studies follow up on a recent report that showed Tat affects the neuronal lysosome pathway and accumulation of autophagy proteins (Hui et al., 2012). Hui et al. (2012) found that Tat associates with lysosomes and reduces LC3II levels in neurons. LC3II levels are not sufficient to determine autophagy flux due to the fact that LC3II levels result from equilibrium of LC3II formation and degradation, and often must be analyzed in context with autophagosome presence and other markers. For example, BafA1 and Chloro disrupt autophagy by blocking acidification of lysosomes, and subsequent fusion of lysosomes with autophagosomes (Amadoro et al., 2014), resulting in accumulation of LC3II and SQSTM1. However, Tat reduced SQSTM1 and LC3II levels suggesting increased autophagic degradation. In contrast to the Hui et al. (2012) studies, and in support of the notion that Tat facilitates autophagosome and lysosome fusion, Tat is able to counter BafA1 and Chloro-mediated block in LC3II and SQSTM1 degradation. Tat may overcome this effect by promoting fusion of autophagosomes with lysosomes more rapidly than BafA1 or Chloro can block lysosome acidification (Fig. 13), leading to aberrant degradation of important neuronal organelles.
 
Autophagosome number is altered in the brains of HIV-infected persons (Alirezaei et al., 2008a); however, the nature of the autophagy pathway is such that a snapshot of protein levels is not sufficient to specify a pathologic mechanism.
 
Our laboratory recently reported autophagy machinery levels are altered in postmortem brains of HIV patients (Zhou et al., 2011), and more recently that "young" (<50) HIV patients express high levels of autophagy machinery while levels are low in brains from "aged" (>50) patients (Fields et al., 2013). The aged and young distinctions are made in the context of HIV infection, consistent with other reports that suggest HIV patients over 50 are more susceptible to cognitive impairments (Wendelken and Valcour, 2012). This may be explained by our results showing that Tat increases GFP-LC3 autophagosomes present in neuronal cells and primary neurons; however, BafA1 had a similar effect on GFP-LC3 puncta. BafA1 increases GFP-LC3 puncta by inhibiting degradation of LC3II, but Tat may counteract this, and induce LC3II degradation by facilitating autophagolysosome formation. Accordingly, knockdown of BECN1 prevents Tat-induced GFP-LC3 puncta formation, suggesting macroautophagy is necessary for these effects. This may also explain how Tat increases the number of autophagosome-like structures per cell as imaged by electron microscopy. Cargo-filled autophagosomes fuse with acidic lysosomes through interaction with LAMP2A (Bandyopadhyay and Cuervo, 2007; Zhang and Cuervo, 2008). Interestingly, Tat colocalizes with LC3 and CTSD, indicating association with both autophagosomes and lysosomes. Moreover, Tat enhances LC3 and CTSD double immunolabeling indicating increased autophagosome fusion with lysosomes. LAMP2A is a target of pathologic proteins such as α-syn, and this association may contribute to Parkinson's disease (Cuervo et al., 2004). Furthermore, LAMP2A is necessary for efficient macroautophagy and chaperone-mediated autophagy (CMA) progression (González-Polo et al., 2005; Zhang and Cuervo, 2008). Indeed, LAMP2A levels are reduced with age in some organs, and it has been suggested that restoring levels may reverse some aging effects (Saftig and Eskelinen, 2008; Saftig et al., 2008; Zhang and Cuervo, 2008). Importantly, Tat associates with LAMP2A in a way that may facilitate autophagolysosome formation and thereby increase LC3II degradation, which explains how Tat increases LC3+ puncta while decreasing LC3II protein levels in neurons. Accordingly, increased GFP-LC3 vesicles in Tat-treated cells may signify active autophagolysosomes, but GFP-LC3 puncta in BafA1-treated cells may represent accumulated LC3II+ autophagosomes. Conversely, LAMP2A is important for CMA (Cuervo et al., 2004; Bandyopadhyay and Cuervo, 2007; Zhang and Cuervo, 2008; Alvarez-Erviti et al., 2010). Therefore, Tat may interfere with CMA and thereby indirectly increase macroautophagy function as a feedback mechanism to maintain recycling of neuronal components. The fact that Rapam or Torin 1 treatment, and overexpression of LAMP2A, reduced Tat-mediated neurotoxicity suggests that Tat promotes deleterious and dysregulated autophagic degradation. This effect, along with other deleterious Tat mechanisms, may result in the observed neurodegeneration.
 
The inducible GFAP-Tat tg mouse model provides a unique method for studying the role of Tat in neuropathogenesis. The model is associated with neurodegeneration and neurotoxicity similar to that seen in brains of HIV patients (Kim et al., 2003). In our studies, increased LC3 puncta coincided with 2 weeks of Tat expression. This corroborates data in our cell models by showing that in vivo Tat expression affects neurons over an extended period. In vitro, upon Tat treatment we found decreased LC3II and SQSTM1 levels while LC3+ puncta were increased, suggesting an increase in autophagic flux. Similarly, in GFAP-Tat tg mice after 2 weeks of Tat expression we found increased LC3+ puncta in neurons and increased neurodegeneration. This mechanism may explain differences in the brain; autophagy protein levels increased in brains of young and decreased in brains of aged HIV patients (Fields et al., 2013). Tat appears to be closely associated with the autophagy machinery in neurons from the GFAP-Tat tg model, suggesting a similar mechanism as seen in our cell models. Our studies confirm Tat causes neurodegeneration in vivo and in vitro, both of which are reversed by Rapam treatment. Paradoxically, Rapam and Tat both increase autophagic flux, but Rapam reverses Tat-induced neurodegeneration. This effect may be due to robust induction of neuronal macroautophagy by Rapam that overcomes the multiple insults Tat causes in neurons. Another possibility is that mTor inhibition ameliorates Tat-induced neurotoxicity through a pathway other than autophagy induction; future studies will focus on determining this mechanism. Tat-induced autophagy alterations may occur at the autophagolysosome formation stage in a way that leads to neurodegeneration through aberrant degradation. These data may suggest Tat plays a specific role, along with other pathogenic mechanisms, promoting deleterious neuronal autophagy alterations that are rectified through normal macroautophagy induction or increased LAMP2A, which may sequester Tat. Nonetheless, it is clear that our results do not provide conclusive evidence that LAMP2A is involved in Tat-mediated effects on autophagy. Future investigations will focus on how Tat and LAMP2A interactions, and which portions, affect autophagolysosome formation in neurons. Moreover, astrocyte LC3+ puncta are also increased, suggesting Tat affects glial autophagy. Increased astrocyte activation and LC3 signal are both reversed following Rapam treatment, and this may contribute to reduced neurotoxicity in vivo by rectifying astrocyte autophagy dysfunction. Further studies are necessary to fully understand the role of Tat on astrocyte autophagy as well as the mechanism at play in neurons.
 
Our current data may partially explain mechanisms that lead to HAND; Tat, in concert with other factors, may initially alter neuronal autophagy in a deleterious manner, while other processes or chronic exposure to Tat and overuse of the autophagy system may result in lower autophagy levels and concomitant neurodegeneration in late stages of HIV infection. Therapies that increase LAMP2A function through blocking interactions with Tat or increasing LAMP2A expression and increasing autophagy efficiency may be useful in combatting HAND.
 
 
 
 
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