Olimpia Meucci - US grants

DESCRIPTION (provided by applicant): The present proposal stems from research conducted during previous funding periods that revealed novel and unexpected roles of the chemokine CXCL12, and its primary receptor CXCR4, on central neurons and glia. Specifically, the objectives of this proposal are to characterize the effect of CXCL12 on dendritic spines, which are critically involved in neuroprotection and synaptic plasticity, and to study the modulation of this effect by opiates and HIV proteins. The proposed studies will characterize the molecular mechanisms involved in the dendritic spine changes evoked by CXCL12; determine the effect of opiates and HIV proteins on CXCL12-induced changes; and establish the potential behavioral consequences of these alterations in the context of HIV neuropathology and drug abuse. This will be accomplished using both in vitro and in vivo approaches and various techniques of cellular/molecular neurobiology and behavior. The long-term goal of this research is to identify pharmacological targets for adjuvant neuroprotective treatments that may be used to reduce neurological problems in HIV patients, thus assisting in the clinical management of populations at high risk of neuropathology, such as intravenous drug users. The studies proposed in aim 1 will provide detailed information about the kinetics and mechanisms involved in CXCL12-induced dendritic spine changes and confirm the role of the CXCL12/CXCR4 axis in the regulation of spines in vivo (with a major focus on the prefrontal cortex and hippocampus). We expect the results to support the hypothesis that CXCL12 regulates expression of gene programs that control synapse number, which ultimately results in more spines. Overall, these experiments intend to confirm the role of CXCL12/CXCR4 in maintenance of a healthy dendritic arbor and to inform some of the analysis to be conducted in aim 2 and 3. The experiments in aim 2 are important to determine if morphine and HIV viral proteins (or cellular factors induced by these proteins) affect the CXCL12 regulation of dendritic spines. Along with the results from aim 3, these findings will indicate whether morphine and HIV can precipitate neuronal damage by inhibiting normal function of this homeostatic chemokine. Finally, studies in aim 3 will unveil subtle but important differences in behavioral performance (involving the prefrontal cortex and hippocampus) between WT and HIV-Tg rats that will help us characterize the specific deficits associated with expression of HIV viral proteins in the brain as well as impairment of CXCR4 function caused by morphine/CXCR4 antagonists.

DESCRIPTION (provided by applicant): These studies aim to assess the role of cell cycle proteins in HIV neuropathogenesis. We will focus on the activation of the key CDK/RWE2F-1 pathway in cultured neurons to identify target genes potentially involved in HIV-induced neurodegeneration. The expression of such apoptotic targets will be then analyzed in brain tissue samples from HIV demented and non-demented patients to establish their relevance in vivo. Experiments have been designed to gather fundamental information related to the coupling of chemokine receptors to neuronal cell cycle proteins, and to determine the effect of HIV envelope proteins on the Rb/E2F pathway. Aim 1 will dissect the mechanisms implicated in the regulation of Rb and E2F-1 by the chemokine receptor CXCR4 in neuronal cultures and will evaluate the contribution of non-neuronal cells to the effect of SDF-1 on neurons. Aim 2 will focus on the effect of gp120s of different nature/structure on Rb and the pathways that controls the activity of p53 and E2F-1. Aim 3 will concentrate on the expression of E2F-specific targets in tissue samples from HIV-infected patients and will determine the role of cell cycle protein alterations in neuronal degeneration. To address these issues, a well-defined primary culture system, in which pure populations of rat central neurons can be examined both in the presence and in the absence of non-neuronal cells, will be used to discern direct effects of chemokines on neurons from indirect effects mediated by the glia and other non-neuronal cells. Then, human neurons and glia will be used, to confirm the most relevant observations in a human in vitro model. Finally, examination of brain tissue samples from HIV-infected patients with and without HAD, will be included to determine the relevance of our in vitro findings to the human pathology. The effects of chemokines, namely SDF-1, and HIV envelope proteins on the expression and activity of cell cycle components involved in apoptosis and differentiation will be investigated by a multidisciplinary approach including proteomics, pharmacology, molecular biology, and novel imaging/fluorescence methods. The proposed experiments will provide novel information on the roles of chemokines and neuronal-glia interactions in the context of the neurological complications of AIDS. The proteomic approach will help us recognize differential protein profiles associated with HIV neuropathogenesis, which may lead to the identification of novel disease biomarkers.

Project Summary/Abstract HIV-associated neurocognitive disorders (HAND) persist in virally suppressed patients. HAND is a heterogeneous disease that is characterized by chronic low-level inflammation, excitotoxicity, presence of neurotoxic HIV proteins, altered APP processing, and other factors that aggravate neuronal structure and function. Opioid use is common among HIV+ patients and thought to contribute to HAND, although this remains controversial. Studies from the previous funding period suggest that morphine can augment HAND by altering APP processing through a novel, iron-dependent pathway. Toxic APP cleavage products are known to reduce dendritic spines in several brain areas, which are critical mediators of learning and memory. Surprisingly, the effects of APP cleavage products on dendritic spines of the prefrontal cortex (PFC), which is an area of critical importance to HAND, have not been studied in depth. This project will elucidate the role of altered APP processing in morphine and HIV-induced neuronal deficits in the PCF. Studies will unravel the interaction between HIV and A? proteins and determine whether morphine can contribute to alteration of APP processing and spines in HAND. Research in aim 1 will concentrate on the effect of A? oligomers on dendritic spine structure and function in PFC neurons, in the presence and absence of HIV neurotoxins or morphine. These in vitro and in vivo studies will define meaningful changes in dendritic spine density/morphology/turnover in PFC neurons, and provide a full assessment of exogenously added A? actions in this critical brain area (the role of endogenous A? will be further tested in aim 3). The main goal of aim 2 is to establish the extent to which µ-opioids can affect amyloidogenesis through modulation of neuronal iron. These mechanistic studies are based on our recent discoveries suggesting a role for iron in morphine-mediated dendritic spine reduction in cortical neurons. They are also supported by the finding that endosome deacidification leads to increase of cytosolic Fe2+ and A?. Importantly, cytosolic Fe2+ regulates APP expression whilst endolysosomal pH modulates protein degradation. In aim 3, we will employ newly generated molecular tools able to shift APP processing to the ?-cleavage pathway or prevent APP cleavage by ?-secretases. After functional testing in primary cultures, the most promising constructs will be expressed in PFC neurons of HAND animal models to determine how effectively they reduce A? levels in the presence of HIV proteins/morphine and if A? reduction contributes to recovery of PFC cognitive tasks. This proposal will shed light on iron-dependent mechanisms of accelerated cognitive decline in HIV+/opiate abusing subjects, which is becoming increasingly relevant as ART- treated patients live longer.

DESCRIPTION (provided by applicant): The goal of this multi-investigator project is to develop an animal model of HIV neuropathology that can be used to assess: 1) cognitive function, 2) neuronal and non-neuronal degeneration in the prefrontal cortex and 3) electrophysiological properties of cells and circuits in prefrontal cortical networks. With the advent of improved combination antiretroviral therapy, HIV infection has been transformed from a fatal illness to a chronic manageable condition. This trend has resulted in an increasingly large population of aging individuals with prolonged exposure to HIV neurotoxins and to HIV therapeutic interventions. While there are excellent tissue culture models for studying the impact of HIV or HIV therapy on cellular processes, the options for in vivo investigation of the effects o HIV infection or chronic antiretroviral therapy are more limited, particularly as they relate to th aging brain. The ideal model for investigating such issues would provide the opportunity to examine and correlate cognitive performance with electrophysiological indices of neural function and neuropathology across the aging continuum with respect to onset of the HIV infection and progression of ensuing disease processes. The work outlined in this proposal will focus on CNS exposure to the HIV envelope protein gp120 in adult and aged rats and its impact on 1) performance of two prefrontal cortex-dependent behavioral tasks, 2) neuronal excitability and synaptic transmission in the prefrontal cortical circuitry and 3) the degree of neurotoxic insult t neuronal and non-neuronal cells in the prefrontal cortex. The most important aspect of this investigation is the development of an animal model that will have advantages for numerous additional in vivo studies focusing on the broad array of potential agents and mechanisms associated with HIV infection and its treatment, the time course of these events, and their impact on the aging brain. In particular this model will facilitate the identification and development of new targets and new compounds for therapeutic interventions in adult and aging HIV/AIDS patients. Across all inquiries, the model will validate the findings of in vitro tissue culture studies and their relevance to normative functions in the intact central nervous system. PUBLIC HEALTH RELEVANCE: The goal of this multi-investigator project is to develop a model of HIV neuropathology that can be used to assess: 1) executive function in behaving animals, 2) neuronal and non-neuronal degeneration in the prefrontal cortex (PFC) and 3) electrophysiological properties of cells and circuits in PFC networks. Studies will be conducted in adult and aging rats. Specific experiments will focus on CNS exposure to the HIV envelope protein gp120 and characterize its impact on: 1) performance of two PFC-dependent behavioral tasks, 2) neuronal excitability and synaptic transmission in the PFC circuitry and 3) the degree of neurotoxic insult to neuronal and non-neuronal cells in the PFC. The proposed model will have advantages for numerous additional in vivo studies focusing on the broad array of potential agents and mechanisms associated with HIV infection and its treatment, the time course of these events, and their impact on the aging brain.

? DESCRIPTION (provided by applicant): This research exploits natural features of the herpesvirus transport protein US9 as a novel approach to study, and possibly modulate, lipid rafts dependent cellular processes relevant to HIV Associated Neurocognitive Disorders (HAND). As lipid rafts represent dynamic platforms for key molecular events likely involved in these conditions, our studies will capitalize on US9 properties to trace HIV-induced changes in lipid rafts, and alter Amyloid Precursor Protein (APP) processing - for both mechanistic and interventional purposes. The proposed experiments will test the hypothesis that HIV-1 gp120/tat favor transport of amyloidogenic enzymes to membrane microdomains, leading to neurotoxicity, and that these viral proteins do not affect intraneuronal distribution of US9. This work will generate a new and powerful strategy capable of studying directly the link between HIV-1 proteins induced changes of lipid rafts and amyloidogenesis, as well as possibly discovering a novel therapeutic approach to decreasing neuronal dysfunction. Briefly, we will use US9 as a molecular tracer of neuronal protein transport to study alterations induced by HIV-1 gp120 and tat in the intraneuronal distribution of lipid rafts-associated proteins that are involved in neurotoxiciy - including, but not limited to, APP processing enzymes (Aim 1). Moreover, through US9-mediated targeting of engineered enzymes altering APP processing, we will increase expression of cellular enzymes that shift APP processing toward the non-amyloidogenic pathway and test the neurotoxic effects of the HIV proteins under these conditions (Aim 2). In order to determine both in vitro and in vivo effects of the viral proteins, these aims include experiments in primary neuronal cultures and small animal models. If successful, this research will help delineate the role of lipid rafts changes in HIV-induced neuronal damage, determine the link between amyloidogenesis and neuronal survival/injury, and lead to potential applications intended to reduce local accumulation of noxious proteins at advanced stages of disease. Thus, both the technical approach and scope of this research are highly innovative and significant, and expected to exert a substantial impact on the field of HAND.

Breast cancer patients die from metastatic disease. Primary breast adenocarcinoma spreads to distant organs by shedding circulating tumor cells (CTCs) in the blood. When these cells leave the systemic circulation, they convert into Disseminated Tumor Cells (DTCs), which are the seeds of secondary tumors. Mounting evidence indicates that existing metastases can mobilize cancer cells back into the blood, thus leading to further spreading that precipitates clinical progression. Thus, strategies aimed to impair tumor seeding have the potential to arrest or significantly decelerating the extension of the disease. Chemokine receptors have been implicated in dissemination, proliferation and survival of cancer cells. Our recent work indicates that the chemokine receptor CX3CR1 is over-expressed in both primary breast tumors and metastatic lesions and plays a role in the lodging of breast cancer cells to the skeleton of animal models. Furthermore, studies form others have shown that this receptor is expressed by cells with tumor-forming abilities and can transactivate growth factor receptors regulating proliferation and survival. Thus, based on the existing literature and our preliminary data, we hypothesize that CX3CR1 is implicated in both early and late stages of metastasis and that its blockade can both deter breast cancer cells from seeding multiple organs and counter their tumor- initiating properties. To test this hypothesis, we will use a combination of CRISPR interference (CRISPRi) and a novel small-molecule antagonist to delineate the time-frame for CX3CR1 involvement in metastatic progression and the effects of pharmacologic targeting of CX3CR1 in tertiary spreading from existing metastases. Finally, we intend to gain a mechanistic understanding of CX3CR1 signaling and determine the molecular pathways associated with tumor-initiation properties activated by this receptor in breast cancer cells. This proposal is structured into three specific aims: AIM 1. To delineate the temporal involvement of CX3CR1 in metastatic progression; AIM 2. To define the effects of CX3CR1 blockade on tumor re- seeding from existing metastases; AIM 3. To elucidate the mechanistic details of CX3CR1 involvement in the metastatic behavior of breast cancer cells. This proposal will define how CX3CR1 influences breast cancer metastatic behavior, reveal important mechanistic details of its activity in cancer cells and provide pre-clinical support for the pharmacologic targeting of this receptor, which presents a high therapeutic potential from several standpoints. From a drug-safety perspective, transgenic mice knockout for CX3CR1 are viable and exhibit no impairment of their immune response under unchallenged conditions, overt behavioral abnormalities or macroscopic anatomical alterations. Thus, if successful our work should pave the way to a novel series of therapeutics and promote multifaceted strategies to counteract the metastatic progression of breast cancer.

Abstract: HIV-associated neurocognitive disorder (HAND) persists in spite of effective control of HIV replication by modern antiretroviral therapies. Adjunctive therapies treating HAND in virally suppressed patients are needed. Studies over the previous funding period suggest that cognitive impairment in HAND is correlated to decreased dendritic spine density in prefrontal cortex (PFC) neurons, which is influenced by several factors such as HIV proteins, chronic inflammation, and opioid use. Importantly, this process may be reversible. Our previous work revealed that activation of the CXCL12/CXCR4 chemokine axis increases dendritic spine density in medial PFC neurons likely leading to cognitive improvement. On the other hand, studies in multiple species - including humans - demonstrate that opioids compromise this pathway, representing a potential mechanism of accelerated HAND. With the long-term goal of introducing targeted therapeutics for HAND, this study will dissect the mechanisms whereby the CXCL12/CXCR4 axis regulates spatiotemporal expression of discrete dendritic spines in PFC neurons. Additionally, the proposed studies will determine the relationship of the spine changes with cognitive function and how opioids or HIV impact these structural and functional outcomes. Experiments will focus on the Rac1/PAK pathway, a main regulator of spine stabilization coupled to the CXCR4 receptor. We hypothesize that CXCL12/CXCR4 signaling enhances working memory and cognitive flexibility by stabilizing normally transient thin spines. Conversely, impairment of CXCR4/Rac1/PAK contributes to synaptic and cognitive deficiencies associated with HIV infection and opioid exposure. Studies in Aim 1 will dissect the signaling pathways downstream of CXCR4 that regulate dendritic spines in cortical neurons, and characterize the effect of CXCL12 on spine dynamics. The proposed experiments will establish the importance of the Rac1/PAK pathway in CXCL12-induced spine regulation and cognitive performance. Aim 2 will examine the effects of morphine on spine morphology and function, in the presence and absence of CXCL12. Although CXCL12 may work to enhance stability of dendritic spines, the development of a transient spine to one that is integrated into the neuronal circuitry is dependent on additional factors, including neuronal activity and protein synthesis. Since morphine and other µ-opioid drugs affect neuronal activity in various ways, these compounds may have additional effects on spines that are unrelated to CXCR4 inhibition. Finally, Aim 3 will define alterations of synaptodendritic architecture and Rac1/PAK pathway activity after in vivo exposure to HIV proteins and morphine, using a small animal model of HAND. These studies will also determine whether CXCL12 treatment rescues synaptic deficits and cognitive performance in a PFC-mediated behavioral task highly relevant to HAND. Behavioral studies will be linked with downstream spine and protein/gene expression analysis in the same group of animals to establish the causal link between changes in cognitive ability and altered synaptic density or connectivity.