Italo Mocchetti - US grants

Nerve growth factor (NGF) plays an important role in neuronal trophism of the central nervous system. Since NGF has been shown to influence the survival of cholinergic neurons in the basal forebrain, a potential use of NGF has been suggested for the therapy of neurodegenerative diseases. However, NGF is known to poorly cross the blood brain barrier, therefore, the direct use of NGF as a therapeutic tool is limited. Since NGF is already produced in the brain, the use of pharmacological agents that enhance NGF biosynthesis becomes a crucial alternative. To this end, it is essential to learn whether NGF biosynthesis can be pharmacological regulated in the brain. It has been shown that different pharmacological stimuli that activate cAMP formation, including beta-adrenergic receptor (BAR) simulation, induce synthesis and release of NGF from C6 rat glioma cells or normal astrocytes in culture. These findings suggest that noradrenaline can regulate NGF biosynthesis in astrocytes. However, before inferring that these results reflect a mechanism of physiological significance, it is necessary to investigate whether BAR stimulation enhances NGF biosynthesis in the brain. This hypothesis will be tested in this proposal by measuring changes in the biosynthesis of NGF in different brain regions of rats, after acute and chronic (2 weeks, 1 month) administration of clenbuterol, a lipophilic BAR agonist. Moreover, other specific alpha- and beta-noradrenergic receptor agonists and antagonists will be used to characterize the specificity of clenbuterol's effect. NGF biosynthesis will be estimated by measure NGF mRNA content, by a quantitative Northern blot analysis, and NGF content, by a two site enzyme immunoassay (ELISA). 21 day old, young adult (3 months) and aged (2 year old) rats will be used to establish whether BAR stimulation enhances NGF biosynthesis in the brain throughout life. These studies will be combined with those aimed to establish whether NGF, pharmacological induced, exerts a physiological effect on the forebrain cholinergic system. To this end, after the pharmacological treatments, choline acetyltransferase (ChAT) activity and immunohistochemistry will be determined, together with NGF receptor (mRNA and protein), in rats of different ages. Finally, C6 rat glioma cells will be used to investigate whether other signal transduction mechanisms (protein kinase C activation and steroids) are operative in the regulation of NGF biosynthesis. It is the ultimate goal of this project to supply significant new information concerning mechanisms regulating NGF biosynthesis which could be used to gain further knowledge on the physiological importance of NGF in the brain and to develop new therapeutic tolls for the treatment of neuronal degeneration.

Italo Mocchetti: IBN-9905113

Lay Summary. The brain contains high levels of gangliosides, natural compounds that are important constituents of the cell membranes. However, the physiological role of gangliosides in the brain is not fully understood. Experimental data have shown that GM1, the prototype of gangliosides found in the brain, promotes neuronal differentiation, maturation and growth. The overall goal of this proposal is to examine the molecular mechanisms by which gangliosides influence structure and function of neurons. The properties of GM1 demonstrate striking similarities to neurotrophic factors, especially the neurotrophins, endogenous substances that are required for an appropriate brain development and function. This proposal main working hypothesis is that gangliosides enhance neuronal function by interacting with the neurotrophins and in particular their receptors. Using cell lines which express each of the neurotrophin receptors, this proposal will investigate: 1) which neurotrophin receptor subtype is activated, 2) which portion of the ganglioside molecule is involved in this activation, and 3) which region of the receptor is activated by gangliosides. This proposal will provide new discoveries in the field of ganglioside biology and brain function.

DESCRIPTION (provided by applicant): Neurodegeneration is very often seen in patients with human immunodeficiency virus type I (HIV-1). The HIV-1 envelope protein gp120 has been implicated in the neurotoxicity caused by HIV-1, however, little is known about the molecular mechanism(s) of gp120 neurotoxicity. We have used, as an experimental model to study these mechanisms, primary culture of cerebellar granule cell neurons obtained from 8-day-old rats. These neurons undergo cell death when exposed to glutamate or other neurotoxic agents. gp120-induced a time-and concentration-dependent neurotoxicity as determined by in situ TUNEL and MTT assay. In fact, exposure of these cultures to 200 pM-20 nM of gp120 for 24 hr induced apoptosis in at least 80 percent of neurons. Moreover, the morphological appearance of these neurons was altered by gp120 such that the majority of these neurons showed short neuronal processes (both neurite and dendrites). Thus, gp120 induces a form of neuronal atrophy, which mimics that observed in young patients infected with the HIV. Neurotrophic factors have been shown to prevent apoptosis and neuronal atrophy caused by neurotoxic agents. In particular, brain derived neurotrophic factor (BDNF) has been shown to prevent glutamate toxicity in granule cells as well as in other neuronal populations. Thus, the first hypothesis that we will test is whether BDNF could limit gp120 neurotoxicity. Preliminary data in support of this hypothesis have been obtained by showing that exposure of cerebellar granule cells to BDNF prevented gp120 neurotoxicity. These findings give exciting hints to the potential mechanisms of gp120 toxicity in vivo and allowed us to formulate our overall hypotheses that gp120 may reduce synthesis and release of BDNF and therefore renders neurons more vulnerable to glutamate. As preliminary data to support this hypothesis, we show that gp120 decreases BDNF synthesis in cerebellar granule cells. However, it remains to establish whether such mechanism is operative in vivo as well. RNase protection assay, two-site immunoassay and trkB tyrosine phosphorylation will be used to document BDNF biosynthesis/activity in cerebellar granule cells in response to various concentrations of gp120. We will also use an in vivo model to provide direct evidence of the relevance of our in vitro findings. Gp120 will be injected in selected areas of the brain in postnatal and adult rats, as well as in wild type and BDNF heterozygous null (+/-) mice to further establish a direct relationship between availability of BDNF and gp120 neuronal toxicity. These studies are aimed at obtaining more information on the potential role of the neurotrophins in limiting gp120 toxicity and consequently on neuronal plasticity. Results from these studies will provide i) new insights into the molecular mechanisms whereby HIV causes neuronal degeneration and ii) the significance of endogenous neurotrophins in the development of new therapeutic approach for HIV-1 related neurodegenerative diseases.

DESCRIPTION (provided by applicant): Patients with Acquired Immune Deficiency Syndrome-related Dementia (AIDSD) exhibit a broad spectrum of motor impairments and cognitive deficits, which follow or parallel cellular loss, atrophy and morphological changes in their brains. The viral envelope glycoprotein gp120 has been suggested to be a causal agent of neuronal loss. However, little is known about the molecular and cellular mechanisms of this effect. The aim of the present study is to test the novel and challenging but timely hypothesis that gp120, released from microglia or astrocytes, causes apoptotic cell death by being internalized by neurons and retrogradely transported to neuronal cell bodies. We plan to test this hypothesis by using two strains of gp120, IIIB and BaL, which bind to CXCR4 and CCR5 chemokine receptors, respectively. Gp120 will be acutely injected into two well-established neuronal pathways of the rat brain, the nigrostriatal and septohippocampal pathways. In particular, we will inject gp120 into the caudate nucleus or fimbria and examine gp120 immunoreactivity in the substantia nigra and septal nuclei, respectively. These studies will be followed by analyses of cell types transporting gp120 by using antibodies against specific neuronal and non-neuronal markers. In addition, we will examine whether neurons positive for gp120 show signs of apoptosis by histologically measuring active caspase-3,-8 and -9. Moreover, we will investigate the hypothesis that gp120 may enter the central nervous system by axonal retrograde transport from peripheral nerves. The rat sciatic nerve will be exposed to gp120 and gp120 immunoreactivity will be determined in dorsal root ganglia and motor neurons of lumbar spinal cord. The retrograde transport hypothesis will be further tested by ligating the sciatic nerve to block axonal transport and examining gp120 accumulation. Overall, the results of the present study will provide a major break-through on the mechanisms whereby gp120 causes neuronal cell death and the related morphological alterations that may account for the motor and cognitive deficits found in AIDSD.

[unreadable] DESCRIPTION (provided by applicant): The mechanism(s) responsible for the therapeutic effect of antidepressant drugs is not completely understood. One current view regarding the pathology of depression proposes that this mental illness results from dysfunction of synaptic connections, perhaps due to the loss of synaptic contacts. Conversely, recent experimental data have suggested that the therapeutic action of antidepressants may involve their ability to increase neurogenesis and synaptic strength. These properties may result more from the induction of neurotrophic factor synthesis than activation of monoamine receptors. This suggestion is supported by preliminary data showing that antidepressants increase the synthesis of basic fibroblast growth factor (FGF2), a neurotrophic factor that exerts neurotrophic activity on catecholaminergic neurons and promotes neurogenesis in the adult brain. These experimental data give rise to a novel and challenging but timely hypotheses that depression is characterized by lack of trophic support and that antidepressants restore synaptic plasticity in adult brain by inducing the synthesis of FGF2. As an experimental tool to test these hypotheses, we propose to use a rat model of depression termed chronic, unpredictable mild stress (CMS), by which rats develop anhedonia, one of the core symptoms of major depression. In particular, we plan to investigate whether CMS down-regulates FGF2 expression (mRNA and protein) in the brain and whether antidepressants such as desipramine and fluoxetine will reverse this down-regulation. Moreover, since CMS evokes apoptotic cell death in the frontal cortex, we propose to test the neuroprotective activity of antidepressants in this animal model of depression by examining the ability of antidepressants and FGF2 to limit apoptotic cell death. Apoptosis will be determined by measuring caspase- 3 activation and in situ terminal deoxynucleotidyl transferase-mediated biotinylated DTP nick end labeling (TUNEL) staining in several brain areas of CMS rats. Overall, the results of the present study will provide a major break-through on the mechanisms whereby antidepressants affect neuronal plasticity in mature rats. Data from this proposal may help elucidate the therapeutic efficacy of antidepressants in affective disorders. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Patients with the human immunodeficiency virus type 1 (HIV-1) develop in the late phase of infection a complex of neurological signs termed Acquired Immune Deficiency Syndrome-related Dementia (AIDSD). These patients exhibit a broad spectrum of motor impairments and cognitive deficits, which follow or parallel cellular loss, atrophy and morphological changes in their brains. The viral envelope glycoprotein gp120 has been suggested to be one of the causal agents of neuronal loss observed in these patients. However, little is known about the molecular and cellular mechanism(s) of the neurotoxic effect of gp120. Data from a previous proposal have established that gp120 causes neuronal cell death by activating an apoptotic pathway through the chemokine receptor CXCR4. Interestingly, the neurotrophin brain-derived neurotrophic factor (BDNF) prevents gp120-mediated neurotoxicity in vitro by decreasing CXCR4 levels. These data lead to the overall hypothesis that BDNF may reduce gp120 toxicity also in vivo. We propose to study the molecular and cellular mechanisms whereby BDNF blocks gp120 neurotoxicity and the involvement of CXCR4. In particular, we plan to examine the role of CXCR4 in gp120-mediated toxicity in vivo by injecting gp120IIIB into the lateral ventricle of rats and comparing its toxic activity with that of gpBal, a strain of gp120 that binds to the chemokine receptor CCR5. We propose to analyze apoptosis by immunohistochemical and biochemical assessment of activated caspase-3 and caspase-9, two proteases that play a crucial role in the apoptotic pathway. These studies will be accompanied by experiments aimed at establishing the molecular and cellular mechanisms whereby BDNF regulates CXCR4 expression. We propose to use BDNF heterozygous (+/-) mice and cerebellar granule cells in culture. In these studies, we will test the hypothesis that BDNF is neuroprotective against gp120 because of its ability to reduce the expression of CXCR4 or increase its internalization. CXCR4 expression will be determined by histological and biochemical detection of CXCR4 immunoreactivity and mRNA, and CXCR4 internalization will be monitored by quantifying beta-arrestin- mediated endocytosis. Moreover, we propose to establish whether BDNF, infused in the lateral ventricle or by recombinant adeno-associated virus, impairs the ability of gp120 to induce apoptosis. Overall, the results of the present study 1) will provide a major break-through on the mechanisms whereby gp120 causes neuronal cell death and the related morphological alterations that may account for the motor and cognitive deficits found in human AIDSD, and 2) will explore the significance of endogenous and exogenous BDNF in the development of a new therapeutic approach for HIV-1-related neurodegenerative diseases.

[unreadable] DESCRIPTION (provided by applicant): Neuronal apoptosis plays an essential role in normal brain development and neurodegeneration. Results of recent studies indicate that after brain injury this type of neuronal death preferentially proceeds through the intrinsic pathway of caspase activation. The release of mitochondrial cytochrome c (Cyto-c) to the cytoplasm plays a central role in this pathway. Cyto-c release is mediated by pro-apoptotic members of Bcl-2 family, which are induced in injured neurons in response to DNA damage. On the other hand, other reports indicate that the release of Cyto-c from mitochondria is not sufficient for the induction of the intrinsic apoptotic pathway. Despite of Cyto-c release, progression of this pathway is inhibited by the extracellular signal-regulated protein kinases Erk1/2 able to block activation of caspase-9 and promote cell survival by direct phosphorylation of procaspase-9. A pathway, which restrains this protective mechanism in apoptotic neurons, remains unknown. In this application we propose a possible link between DNA damage, the release of Cyto-c from mitochondria, dephosphorylation and inactivation of pro-survival Erk1/2, activation of caspases, and neuronal death. Our working hypothesis is that the progression of DNA damage-induced apoptosis in neurons depends on p53-mediated activation of specific dual specificity phosphatases (DUSPs) able to promote processing of procaspase-9 through selective Erk1/2 inactivation. If our hypothesis is correct, inhibition of DUSP activity after neuronal injury must be neuroprotective. To address the hypothesis, we propose 2 related Specific Aims: (1) To examine neuronal expression of DUSP gene family members in response to DNA damage in vitro and after brain trauma in vivo and (2) To assess a role for inducible DUSPs in inhibition of Erk1/2, caspase activation, and neuronal death. Results of this study may have fundamental impact on identifying novel treatment strategies for a wide variety of acute and chronic neurodegenerative conditions where the contribution of DNA damage and p53-dependent apoptosis to neuronal loss is highly significant. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Human Immunodeficiency virus -1 (HIV)-associated dementia (HAD) is characterized by a severe loss of neurons and fibers throughout the brain. It is estimated that a third of HIV-infected individuals in the USA are intravenous opiate drug abusers. It is believed that heroin, morphine and other intravenous opiate drugs may exacerbate the neurotoxic effect of HIV. However, little is known about the molecular and cellular mechanisms of this effect despite the enormous expansion of knowledge regarding the complex factors that determine slow but progressive neuronal death in these patients. Opiates, and in particular morphine, have been shown to cause abnormalities in the functioning of immune cells including microglia and to render these cells more susceptible to HIV infection. However, experimental data have shown that morphine synergizes with HIV viral proteins in causing apoptosis even in the absence of microglia. Thus, alteration of immune/glial cells alone cannot explain loss of neurons in HAD. Chronic opiates alter the expression of chemokine receptors CCR5 and CXCR4, known to mediate the neurotoxic effect of HIV. Thus, opiates may use this cellular mechanism to amplify HIV neurotoxic property. The HIV protein gp120 reduces the levels of brain-derived neurotrophic factor BDNF (BDNF) in rat brain. BDNF is a natural modulator of chemokine receptor expression. Therefore, we propose the hypothesis that HAD derives from a multiple step interaction between opiates and HIV that causes a profound alteration of endogenous cellular mechanisms of neuroprotection and plasticity. To test this hypothesis we will first use rodents (in vitro and in vivo studies) to test the role of morphine and two strains of HIV envelop proteins gp120 on chemokine and chemokine receptors and neuronal survival. Moreover, we propose a series of comprehensive studies in human subjects aimed at establishing the role of BDNF and BDNF polymorphism on the development of HAD. Such studies include BDNF analysis of post-mortem brains of HAD subjects with a history of heroin abuse, as well as examination of a cohort of HIV infected subjects (from the Women's Interagency HIV Study cohort) for BDNF polymorphisms. We believe that lack of BDNF is a risk factor for HAD. The long term aim of this application is to establish a new mechanism of HIV opiates interaction that explains why neurons of drug abusers are more vulnerable to apoptosis than non abusers. This mechanism includes reduction of trophic support and enhancement of pro-apoptotic chemokine receptors in neurons. Proving such mechanism will help develop effective drug therapy to reverse or delay HAD. PUBLIC HEALTH RELEVANCE: At least a third of subjects infected with Human Immunodeficiency Virus-1 are drug abusers. The combination of infection, morphine, heroin addition (or other drugs) is believed to be toxic to the brain. This application will study the mechanisms as well as the genetic abnormalities that may be risk factors for developing brain damage in these subjects.

[unreadable] DESCRIPTION (provided by applicant): A subset of patients infected with human immunodeficiency virus-1 (HIV) develop Acquired Immunodeficiency Syndrome Dementia Complex (ADC) or HIV-associated dementia (HAD), a disorder characterized by motor impairments and cognitive dysfunctions. A severe loss of neurons and fibers throughout the brain is seen in these patients. Despite the enormous expansion of knowledge regarding the complex factors that determine slow but progressive neuronal death in HAD, no effective drug therapy to reverse or delay this damage has yet emerged. Thus, the discovery of new compounds that reduce neuronal degeneration in HAD is crucial. Neurotrophic factors are polypeptides that exhibit a broad spectrum of biological actions, including neuronal protection and restoration of synaptic contacts. Brain-derived neurotrophic factor (BDNF) is a prototypical trophic factor that reduces neuronal apoptosis evoked by the HIV envelope protein gp120, used to generate an experimental model of HAD. Thus, BDNF could be an ideal therapy for HAD patients to minimize neuronal damage. Unfortunately, BDNF administered systemically does not reach the injured neurons. Recently, we have discovered that LIGA20, a semi-synthetic sphingolipid that enters the brain after oral administration, has a neurotrophic profile similar to BDNF. The neuroprotective property of LIGA20 in vitro is related to its ability to activate TrkB, the BDNF receptor that modulates the neuroprotective property of this neurotrophic factor. These preliminary data lead to the hypothesis that LIGA20 may have a neuroprotective property similar to BDNF. The overall goal of this proposal is to establish the pharmacological and neuroprotective profile of LIGA20 in vivo. In particular, we propose to test the hypothesis that LIGA20 prevents apoptosis and neuronal loss in acute and subchronic animal models of HAD. These include: acute intrastriatal injection of gp120 in mice and mice injected with HIV-infected human monocytes-derived macrophages. Neuronal apoptosis, loss of dopamine and inflammatory responses will be determined by histological and biochemical means. The data obtained in this proposal could have important therapeutic implications because LIGA20 may be used instead of BDNF to prevent neuronal cell death in HAD individuals. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Axonal injury is seen in subjects with Human Immunodeficiency Virus type 1 (HIV) associated dementia (HAD). The lack of documented evidence of direct infection of neurons by HIV has prompted studies to reveal possible mechanisms for HIV-mediated axonal damage. However, the nature and the site of initial injury to the neuron are unknown; in particular it is unclear if the process starts in the axons itself or in the neuronal cell body. Several lines of independent investigations have shown that the viral envelope protein gp120 promotes neuronal degeneration. The neurotoxic effect of gp120 occurs through chemokine receptors. Preliminary evidence has shown that the chemokine receptor CXCR4 promotes endocytosis of T-tropic gp120 into neurons. Endocytosis of gp120 is followed by axonal transport through microtubules. The length of axons and survival rate of neurons that do not internalize gp120 is significantly greater when compared to those accumulating gp120. Moreover, agents that block axonal transport are neuroprotective against gp120 even in the presence of cytokines, suggesting that the toxic effect of gp120 may not necessarily require an inflammatory response. Most importantly, gp120 endocytosis and transport precede synaptic simplification and caspase-3 activation and therefore neuronal cell death, suggesting that accumulation and axonal transport of gp120 are crucial to its toxicity. These data lead to a new challenging hypothesis that gp120 binding to CXCR4 receptor initiates a series of events that triggers direct axonal toxicity. However, little is known about the cellular and molecular mechanisms of this effect. Internalized gp120 could cause axonal damage and neuronal apoptosis by affecting axonal proteins or impairing the axonal transport of important molecules that are necessary for neuronal survival and maintenance. The experiments planned in this project will examine whether gp120 affects selected regulatory kinases of axonal transport, or microtubule-associated protein such as Tau, or other cytoskeleton proteins that when impaired, promote the dying-back pattern of degeneration. These experiments will be accompanied by histological analysis of gp120 immunoreactivity within endosomes or other cellular organelles, as well as by biochemical analyses of pro-inflammatory cytokines. The ability of gp120 to induce a rapid axonal/dendritic degeneration raises the hypothesis that this protein is sufficient to initiate an irreversible neurodegenerative process even in the absence of other endogenous neurotoxins or other patho-physiological insults. If proven, our hypothesis that gp120 injures neurons by being internalized and transported to their cell bodies will lead to a major discovery in this field. Overall, we expect to provide new significant data that help in the design of adjunct therapies against synaptic simplifications caused by HIV.

DESCRIPTION (provided by applicant): Human Immunodeficiency Virus-1 (HIV) infection of the central nervous system may cause a neurological syndrome termed HIV-associated neurocognitive disorders (HAND). HAND includes minor neurocognitive disorders and a more severe form of motor and cognitive impairments termed HIV associated dementia (HAD). Although treatment with highly active antiretroviral agents decreases the load of HIV in the brain, the prevalence of HAND is actually increased due to longer life. Therefore, adjunctive and combined therapies must be developed to prevent and perhaps reverse the neurologic deficits observed in these individuals. These subjects exhibit synaptic simplification and neuronal apoptosis. However, the molecular mechanisms leading to synaptic pathology are unknown. Key to developing effective therapies is a better understanding of the molecular and cellular mechanisms by which the virus causes these neuropathological features. HIV and its viral envelope protein gp120 promote axonal retraction and dendritic simplification. These effects are reproduced by the proneurotrophin brain-derived neurotrophic factor (proBDNF) and are mediated by the p75 neurotrophin receptor (p75NTR), a member of the tumor necrosis factor family. Thus, we have generated the hypothesis that proBDNF facilitates HIV neurotoxicity by promoting the activation of p75NTR. We have obtained preliminary data in support to this hypothesis. Indeed, we have shown that brains of HAD subjects exhibit a higher amount of proBDNF than non-HAD subjects. Moreover, in vitro data have determined that HIV and gp120-mediated synaptodendritic simplification is blocked by p75NTR antagonists. Thus, this proposal will test the novel hypothesis that HIV injures neurons by a gp120-mediated mechanism that involves the release of proBDNF, which in turn promotes axonal/dendritic degeneration via a p75NTR-mediated mechanism. Experiments will be carried out to establish the role of p75NTR in HIV/gp120 mediated neurotoxicity, and the cellular mechanisms whereby HIV/gp120 promotes proBDNF accumulation. These include testing the activity of enzymes involved in processing proBDNF to mature BDNF. These experiments will be accompanied by studies examining proteolytic enzymes involved in proBDNF processing in postmortem human brain as well as in animal models of HAD. Studies are also planned to establish the relationship (if any) between proBDNF and microglia activation and inflammation. We expect to provide new significant data on the role of p75NTR in HIV neurotoxicity. These data will help in the design of p75NTR antagonists as an adjunct therapy against synaptic simplification caused by HIV.

? DESCRIPTION (provided by applicant): One challenge in Human Immunodeficiency Virus (HIV) research involves the discovery and characterization of compounds that prevent or limit the neurodegeneration that follows HIV infection of the brain. The chemokine CCL5 inhibits entry of M-tropic HIV strains into macrophages/microglia by affecting the binding of the envelop protein gp120 to the co-receptor CCR5. Interestingly, CCL5 also prevents neuronal cell death mediated by the T-tropic gp120 and the viral protein Tat, which have no affinity for CCR5. Therefore, the mechanism of action of this chemokine remains to be fully characterized. Recent studies and our preliminary data have shown that CCL5 activates a G protein coupled-receptor 75 (GPR75) which encodes for a 540 amino-acid orphan receptor of the Gq¿ family. This receptor is more abundant in the brain than in the immune organs. Moreover, CCL5 activates various pro-survival signaling molecules, including inositol triphosphate, phosphatidylinositol 3-kinase and its downstream targets protein kinase B and extracellular signal-regulated kinases, in SH-SY5Y cells. These cells do not express CCR5, CCR3 and CCR1, receptors known to bind to CCL5. Moreover, CCL4, CCL7 and CCL3, other chemokines that bind to CCR5, CCR3 and CCR1, failed to activate these signaling molecules in these cells. Therefore, GPR75 could be the missing link to explain the neuroprotective activity of CCL5. In this exploratory proposal we will test the innovative hypothesis that GPR75 activation by CCL5 promotes neuroprotection against viral proteins (e.g. gp120 and Tat). To test this hypothesis, we propose to identify and characterize CCL5 binding activity and affinity for GPR75 by Scatchard analysis, cross-linking, receptor internalization, mass spectrometry and surface plasmon resonance (Biacore) studies (AIM 1) in both human as well as rodent neurons. These experiments will be accompanied by studies examining the role of GPR75 in CCL5-mediated neuroprotection against T-tropic gp120 and Tat in neurons in vitro (AIM 2). We expect to provide new significant data on the ability of CCL5 to bind to GPR75 and its role in preventing the neurotoxicity of HIV proteins. These data will help in the design of small molecule GPR75 agonists as an adjunct therapy against synaptic simplification caused by HIV.

Abstract Opioids, such as heroin and morphine, have been shown to cause abnormalities in the functioning of immune cells and to render these cells more ?susceptible? to human immunodeficiency virus-1 (HIV) infection. In addition, heroin, morphine, and other intravenous opiate drugs may accelerate/amplify the neurotoxic mechanisms of HIV that lead to the development of HIV associated neurocognitive disorders (HAND), even in the presence of antiretroviral therapy. However, HIV positive opiate users undergo cycles of withdrawal, which could modify several cellular mechanisms that negatively impact synaptic repair. Few studies have addressed the neurotoxic effects of opioid withdrawal in conjunction with HIV and their mechanisms. Our preliminary data indicate that these mechanisms may include the proliferation of pro-inflammatory (e.g. M1-like) microglia. Therefore, we propose to test the hypothesis that opiate withdrawal, through proliferation of reactive microglia, augments the neurotoxic property of the HIV protein gp120. To test this hypothesis, we will examine whether morphine withdrawal accelerates neuronal degeneration observed in gp120 transgenic mice and whether methadone reverses this effect. Other experiments will determine the cellular and molecular mechanisms whereby withdrawal is neurotoxic. To this end, we will examine the hypothesis that morphine withdrawal promotes microglia proliferation by increasing colony-stimulating factor 1, a chemokine that regulates the proliferation of adult microglia. The long-term goal of this proposal is to establish whether the neurotoxic effects of HIV could be managed in opioid abusers by reducing the cycles of opioid withdrawal. These studies are of high significance since they could elucidate a novel mechanism contributing to HAND in drug abusers. A better understanding of these mechanisms may lead to the development of novel and more effective drug therapies to delay HAND.

Abstract Synaptodendritic neuronal injury is emerging as an important mediator of cognitive deficits in HIV-positive individuals. Nevertheless, the molecular and cellular mechanisms of how HIV causes axonal pruning are still far from established. This precludes effective treatment of the neurological complications. Recent findings report that the HIV soluble envelope protein gp120 is endocytosed into neurons and binds to tubulin ?-3 (TUBB3), a major component of neuronal microtubules (MTs), which are essential for neuronal function. Neurons that endocytose gp120 exhibit neurite retraction and activation of caspase-3, raising the hypothesis that the endocytic process and the binding to TUBB3 are crucial for gp120-mediated neuronal injury. In this exploratory proposal, we will test this hypothesis by examining gp120-neurotoxicity in the presence of compounds that displace gp120 from binding to TUBB3 (AIM 1). Gp120 internalization is CXCR4 receptor mediated. To discriminate between chemokine receptor signaling and binding to MTs as a mechanism of neurotoxicity, experiments in AIM 2 will test whether the delivery of gp120 inside neurons by mesoporous silica nanoparticles is neurotoxic. The characterization of how viral proteins interact with the neuronal cytoskeleton and negatively affect mature synapses will provide considerable insights toward understanding the mechanism underlying HIV-associated neurocognitive disorders. If successful, this proposal will establish that gp120, after its internalization, injures neurons by binding to and damaging neuronal MTs. This is a novel mechanism of gp120 neurotoxicity that, if proven, will help in the design of compounds that will inhibit the neurotoxic effect of gp120.

Abstract Human immunodeficiency virus-1 (HIV) infection of the central nervous system damages synapses and promotes neuronal injury that culminates in HIV-associated neurocognitive disorders (HAND). How HIV damages synapses is still under investigation. Viral proteins, including the envelope protein gp120, have emerged as leading candidates to explain HIV-mediated neurotoxicity, though the mechanisms remain unclear. The balance between neuronal survival and damage is predominantly governed by neurotrophic factors, and in particular, brain-derived neurotrophic factor (BDNF) and its precursor proBDNF. proBDNF, when bound to the neurotrophin receptor p75 (p75NTR) activates a pro-apoptotic signal. We have shown that brains of HAND subjects, as well as neurons exposed to gp120, exhibit a significant increase of proBDNF, which correlates with a decreased expression of furin, a key enzyme in the processing of proBDNF. The removal of one allele of p75NTR, the receptor for proBDNF, rescues the loss of synapses seen in gp120 transgenic mice. Therefore, we hypothesize that HIV damages synapses through the ability of gp120 to increase proBDNF and therefore activating p75NTR. This is an important line of research because synaptic degeneration dysfunction has been linked to numerous neurodegenerative diseases but only preliminarily to HAND. The molecular and cellular mechanisms of how gp120 causes impairs/damages synapses remain under investigation. This application proposes a comprehensive set of experiments to test the main hypothesis. In particular (AIM 1), we will test the hypothesis that gp120 reduces furin levels by directly binding to this endoprotease. We will utilize (AIM 2) p75NTR-/- neurons and p75NTR antagonists to examine the mechanisms and signaling of gp120 neurotoxicity. We will perform behavioral studies for memory function (AIM 3) in gp120 transgenic (gp120tg) mice intercrossed with p75NTR null mice to investigate whether the removal of one allele for p75NTR rescues the memory impairment observed in gp120tg mice. Finally, (AIM 4) we will use human samples including the cerebrospinal fluid (CSF) to determine whether the levels of proBDNF are altered in different subgroups of HAND subjects. Levels of gp120 will also be measured in the CSF. These experiments might establish a correlation between levels of proBDNF, gp120 and neurocognitive impairment. We expect to provide new significant data on the role of p75NTR in HIV-mediated synaptic simplification.

Project Summary/Abstract This is a new application for an Institutional Training Grant in the basic and clinical aspects of Neurological Disorders caused by HIV, better known as HIV-associated neurocognitive disorders (HAND). The Training Program in NeuroHIV (TPNH) is requesting support for 4 advanced predoctoral students who will be trained in research on how HIV causes neural injury by faculty actively participating in the Neuroscience and Immunology programs at Georgetown University. The purpose of this training program is to prepare scientists to investigate molecular and cellular mechanisms of neurodegenerative processes as well as the immunological responses triggered by HIV. Our goal is to train researchers on the basic science aspect of developing neurotherapeutics and/or treatment strategies to reduce the neuronal and glial impairments that result from HIV infection of the brain. An experienced and well-funded group of 22 faculty members with a wide range of research interests and expertise in CNS function, neurodegeneration, virology and immunology will participate in the Ph.D. training program. Most students will enter graduate school through the Georgetown University Interdisciplinary Program in Neuroscience (IPN). In the first year they will take course work and rotate through the laboratories of potential mentors. In the second year, those interested in the TPNH will take specifically relevant courses dealing with neurodegenerative disorders, immunology, and immunopathogenesis of HIV infection, as well as extended integrative reasoning and statistical literacy training, and professional development training (i.e., grant writing and presentation skills). In the second year of graduate school, students will formally apply to the TPNH with the outline of a thesis research proposal approved by a potential mentor or co-mentors from the IPN/Immunology Training Faculty. Thesis research will start after the completion of the Ph.D. program?s qualifying exam. In their third and fourth years, trainees will participate in training sessions with clinician-scientists to provide the necessary experience in basic and clinical science that is key to the understanding of HAND from bench to therapeutics. Trainees will report results from their research in yearly student seminars, presentations at national meetings, and as publications in peer-reviewed journals. With respect to public health, this program will create a core of neuroscientists trained for research and/or management of research programs, through which new and more effective treatments for HAND and related neuro-immuno-degenerative disorders will be developed.