HERV-K (HML-2) Envelope Protein Induces Mitochondrial Depolarization and Neurotoxicity via Endolysosome Iron Dyshomeostasis.

Human endogenous retroviruses (HERVs) are associated with the pathogenesis of amyotrophic lateral sclerosis (ALS); a disease characterized by motor neuron degeneration and cell death. The HERV-K subtype HML-2 envelope protein (HERV-K Env) is expressed in the brain, spinal cord, and cerebrospinal fluid of people living with ALS and through CD98 receptor-linked interactions causes neurodegeneration. HERV-K Env-induced increases in oxidative stress are implicated in the pathogenesis of ALS, and ferrous iron (Fe2+) generates reactive oxygen species (ROS). Endolysosome stores of Fe2+ are central to iron trafficking and endolysosome deacidification releases Fe2+ into the cytoplasm. Because HERV-K Env is an arginine-rich protein that is likely endocytosed and arginine is a pH-elevating amino acid, it is important to determine HERV-K Env effects on endolysosome pH and whether HERV-K Env-induced neurotoxicity is downstream of Fe2+ released from endolysosomes. Here, we showed using SH-SY5Y human neuroblastoma cells and primary cultures of human cortical neurons (HCNs, information on age and sex was not available) that HERV-K Env (1) is endocytosed via CD98 receptors, (2) concentration dependently deacidified endolysosomes, (3) decreased endolysosome Fe2+ concentrations, (4) increased cytosolic and mitochondrial Fe2+ and ROS levels, (5) depolarized mitochondrial membrane potential, and (6) induced cell death, effects blocked by an antibody against the CD98 receptor and by the endolysosome iron chelator deferoxamine. Thus, HERV-K Env-induced increases in cytosolic and mitochondrial Fe2+ and ROS as well as cell death appear to be mechanistically caused by HERV-K Env endocytosis, endolysosome deacidification, and endolysosome Fe2+ efflux into the cytoplasm.

Neurotoxic properties of the Zika virus envelope protein.

Prenatal Zika virus (ZIKV) infection is a serious global concern as it can lead to brain injury and many serious birth defects, collectively known as congenital Zika syndrome. Brain injury likely results from viral mediated toxicity in neural progenitor cells. Additionally, postnatal ZIKV infections have been linked to neurological complications, yet the mechanisms driving these manifestations are not well understood. Existing data suggest that the ZIKV envelope protein can persist in the central nervous system for extended periods of time, but it is unknown if this protein can independently contribute to neuronal toxicity. Here we find that the ZIKV envelope protein is neurotoxic, leading to overexpression of poly adenosine diphosphate -ribose polymerase 1, which can induce parthanatos. Together, these data suggest that neuronal toxicity resulting from the envelope protein may contribute to the pathogenesis of post-natal ZIKV-related neurologic complications.

Detection of SARS-CoV-2 Nucleocapsid and Microvascular Disease in the Brain: A Case Report.

BACKGROUND AND OBJECTIVES: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection can cause a wide range of neurologic complications; however, its neuropenetrance during the acute phase of the illness is unknown. METHODS: Extracellular vesicles were isolated from brain biopsy tissue from a patient undergoing epilepsy surgery using ultracentrifugation and analyzed by Western blot and qPCR for the presence of virus protein and RNA, respectively. Biopsy tissue was assessed by immunohistochemistry for the presence of microvascular damage and compared with 3 other non-COVID surgical epilepsy brain tissues. RESULTS: We demonstrate the presence of viral nucleocapsid protein in extracellular vesicles and microvascular disease in the brain of a patient undergoing epilepsy surgery shortly after SARS-CoV-2 infection. Endothelial cell activation was indicated by increased levels of platelet endothelial cell adhesion molecule-1 and was associated with fibrinogen leakage and immune cell infiltration in the biopsy tissue as compared with control non-COVID surgical epilepsy brain tissues. DISCUSSION: Despite the lack of evidence of viral replication within the brain, the presence of the nucleocapsid protein was associated with disease-specific endothelial cell activation, fibrinogen leakage, and immune cell infiltration.

Human Endogenous Retrovirus K Envelope in Spinal Fluid of Amyotrophic Lateral Sclerosis Is Toxic.

OBJECTIVE: Human endogenous retroviruses have been implicated in neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). Expression of human endogenous retrovirus K (HERV-K) subtype HML-2 envelope (Env) in human neuronal cultures and in transgenic mice results in neurotoxicity and neurodegeneration, and mice expressing HML-2 Env display behavioral and neuromuscular characteristics resembling ALS. This study aims to characterize the neurotoxic properties of HML-2 Env. METHODS: Env neurotoxicity was detected in ALS cerebrospinal fluid and confirmed using recombinant Env protein in a cell-based assay and a mouse model. The mechanism of neurotoxicity was assessed with immunoprecipitation followed by mass spectrometry and Western blot, and by screening a panel of inhibitors. RESULTS: We observed that recombinant HML-2 Env protein caused neurotoxicity resulting in neuronal cell death, retraction of neurites, and decreased neuronal electrical activity. Injection of the Env protein into the brains of mice also resulted in neuronal cell death. HML-2 Env protein was also found in the cerebrospinal fluid of patients with sporadic ALS. The neurotoxic properties of the Env and the cerebrospinal fluid could be rescued with the anti-Env antibody. The Env was found to bind to CD98HC complexed to ß1 integrin on the neuronal cell surface. Using a panel of compounds to screen for their ability to block Env-induced neurotoxicity, we found that several compounds were protective and are linked to the ß1 integrin pathway. INTERPRETATION: HERV-K Env is released extracellularly in ALS and causes neurotoxicity via a novel mechanism. Present results pave the way for new treatment strategies in sporadic ALS. ANN NEUROL 2022;92:545-561.

Fluconazole Is Neuroprotective via Interactions with the IGF-1 Receptor.

There is a continuing unmet medical need to develop neuroprotective strategies to treat neurodegenerative disorders. To address this need, we screened over 2000 compounds for potential neuroprotective activity in a model of oxidative stress and found that numerous antifungal agents were neuroprotective. Of the identified compounds, fluconazole was further characterized. Fluconazole was able to prevent neurite retraction and cell death in in vitro and in vivo models of toxicity. Fluconazole protected neurons in a concentration-dependent manner and exhibited efficacy against several toxic agents, including 3-nitropropionic acid, N-methyl D-aspartate, 6-hydroxydopamine, and the HIV proteins Tat and gp120. In vivo studies indicated that systemically administered fluconazole was neuroprotective in animals treated with 3-nitropropionic acid and prevented gp120-mediated neuronal loss. In addition to neuroprotection, fluconazole also induced proliferation of neural progenitor cells in vitro and in vivo. Fluconazole mediates these effects through upregulation and signaling via the insulin growth factor-1 receptor which results in decreased cAMP production and increased phosphorylation of Akt. Blockade of the insulin growth factor-1 receptor signaling with the selective inhibitor AG1024 abrogated the effects of fluconazole. Our studies suggest that fluconazole may be an attractive candidate for treatment of neurodegenerative diseases due to its protective properties against several categories of neuronal insults and its ability to spur neural progenitor cell proliferation.

Therapeutic candidates for the Zika virus identified by a high-throughput screen for Zika protease inhibitors.

When Zika virus emerged as a public health emergency there were no drugs or vaccines approved for its prevention or treatment. We used a high-throughput screen for Zika virus protease inhibitors to identify several inhibitors of Zika virus infection. We expressed the NS2B-NS3 Zika virus protease and conducted a biochemical screen for small-molecule inhibitors. A quantitative structure-activity relationship model was employed to virtually screen ∼138,000 compounds, which increased the identification of active compounds, while decreasing screening time and resources. Candidate inhibitors were validated in several viral infection assays. Small molecules with favorable clinical profiles, especially the five-lipoxygenase-activating protein inhibitor, MK-591, inhibited the Zika virus protease and infection in neural stem cells. Members of the tetracycline family of antibiotics were more potent inhibitors of Zika virus infection than the protease, suggesting they may have multiple mechanisms of action. The most potent tetracycline, methacycline, reduced the amount of Zika virus present in the brain and the severity of Zika virus-induced motor deficits in an immunocompetent mouse model. As Food and Drug Administration-approved drugs, the tetracyclines could be quickly translated to the clinic. The compounds identified through our screening paradigm have the potential to be used as prophylactics for patients traveling to endemic regions or for the treatment of the neurological complications of Zika virus infection.

A Strategy to Identify Compounds that Affect Cell Growth and Survival in Cultured Mammalian Cells at Low-to-Moderate Throughput.

Cytotoxicity is a critical parameter that needs to be quantified when studying drugs that may have therapeutic benefits. Because of this, many drug screening assays utilize cytotoxicity as one of the critical characteristics to be profiled for individual compounds. Cells in culture are a useful model to assess cytotoxicity before proceeding to follow up on promising lead compounds in more costly and labor-intensive animal models. We describe a strategy to identify compounds that affect cell growth in a tdTomato expressing human neural stem cells (NSC) line. The strategy uses two complementary assays to assess cell number. One assay works via the reduction of 3-(4,5-dimethylthizol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to formazan as a proxy for cell number and the other directly counts the tdTomato expressing NSCs. The two assays can be performed simultaneously in a single experiment and are not labor intensive, rapid, and inexpensive. The strategy described in this demonstration tested 57 compounds in an exploratory primary screen for toxicity in a 96-well plate format. Three of the hits were characterized further in a six-point dose response using the same assay set-up as the primary screen. In addition to providing excellent corroboration for toxicity, comparison of results from the two assays may be effective in identifying compounds affecting other aspects of cell growth.

Protease-activated receptor-1 activation by granzyme B causes neurotoxicity that is augmented by interleukin-1ß.

BACKGROUND: The cause of neurodegeneration in progressive forms of multiple sclerosis is unknown. We investigated the impact of specific neuroinflammatory markers on human neurons to identify potential therapeutic targets for neuroprotection against chronic inflammation. METHODS: Surface immunocytochemistry directly visualized protease-activated receptor-1 (PAR1) and interleukin-1 (IL-1) receptors on neurons in human postmortem cortex in patients with and without neuroinflammatory lesions. Viability of cultured neurons was determined after exposure to cerebrospinal fluid from patients with progressive multiple sclerosis or purified granzyme B and IL-1ß. Inhibitors of PAR1 activation and of PAR1-associated second messenger signaling were used to elucidate a mechanism of neurotoxicity. RESULTS: Immunohistochemistry of human post-mortem brain tissue demonstrated cells expressing higher amounts of PAR1 near and within subcortical lesions in patients with multiple sclerosis compared to control tissue. Human cerebrospinal fluid samples containing granzyme B and IL-1ß were toxic to human neuronal cultures. Granzyme B was neurotoxic through activation of PAR1 and subsequently the phospholipase Cß-IP3 second messenger system. Inhibition of PAR1 or IP3 prevented granzyme B toxicity. IL-1ß enhanced granzyme B-mediated neurotoxicity by increasing PAR1 expression. CONCLUSIONS: Neurons within the inflamed central nervous system are imperiled because they express more PAR1 and are exposed to a neurotoxic combination of both granzyme B and IL-1ß. The effects of these inflammatory mediators may be a contributing factor in the progressive brain atrophy associated with neuroinflammatory diseases. Knowledge of how exposure to IL-1ß and granzyme B act synergistically to cause neuronal death yields potential novel neuroprotective treatments for neuroinflammatory diseases.

Interaction of paroxetine with mitochondrial proteins mediates neuroprotection.

There are severe neurological complications that arise from HIV infection, ranging from peripheral sensory neuropathy to cognitive decline and dementia for which no specific treatments are available. The HIV proteins secreted from infected macrophages, gp120 and Tat, are neurotoxic. The goal of this study was to screen, identify and develop neuroprotective compounds relevant to HIV-associated neurocognitive disorders (HAND). We screened more than 2000 compounds that included FDA approved drugs for protective efficacy against oxidative stress-mediated neurodegeneration and identified selective serotonin reuptake inhibitors (SSRIs) as potential neuroprotectants. Numerous SSRIs were then extensively evaluated as protectants against neurotoxicity as measured by changes in neuronal cell death, mitochondrial potential, and axodendritic degeneration elicited by HIV Tat and gp120 and other mitochondrial toxins. While many SSRIs demonstrated neuroprotective actions, paroxetine was potently neuroprotective (100 nM potency) against these toxins in vitro and in vivo following systemic administration in a gp120 neurotoxicity model. Interestingly, the inhibition of serotonin reuptake by paroxetine was not required for neuroprotection, since depletion of the serotonin transporter had no effect on its neuroprotective properties. We determined that paroxetine interacts selectively and preferentially with brain mitochondrial proteins and blocks calcium-dependent swelling but had less effect on liver mitochondria. Additionally, paroxetine induced proliferation of neural progenitor cells in vitro and in vivo in gp120 transgenic animals. Therefore, SSRIs such as paroxetine may provide a novel adjunctive neuroprotective and neuroregenerative therapy to treat HIV-infected individuals.

Compounds with species and cell type specific toxicity identified in a 2000 compound drug screen of neural stem cells and rat mixed cortical neurons.

Human primary neural tissue is a vital component for the quick and simple determination of chemical compound neurotoxicity in vitro. In particular, such tissue would be ideal for high-throughput screens that can be used to identify novel neurotoxic or neurotherapeutic compounds. We have previously established a high-throughput screening platform using human induced pluripotent stem cell (iPSC)-derived neural stem cells (NSCs) and neurons. In this study, we conducted a 2000 compound screen with human NSCs and rat cortical cells to identify compounds that are selectively toxic to each group. Approximately 100 of the tested compounds showed specific toxicity to human NSCs. A secondary screen of a small subset of compounds from the primary screen on human iPSCs, NSC-derived neurons, and fetal astrocytes validated the results from >80% of these compounds with some showing cell specific toxicity. Amongst those compounds were several cardiac glycosides, all of which were selectively toxic to the human cells. As the screen was able to reliably identify neurotoxicants, many with species and cell-type specificity, this study demonstrates the feasibility of this NSC-driven platform for higher-throughput neurotoxicity screens.

Combination fluconazole/paroxetine treatment is neuroprotective despite ongoing neuroinflammation and viral replication in an SIV model of HIV neurological disease.

Effective combined antiretroviral therapy (cART) in HIV-infected patients has made HIV a treatable infection; however, debilitating HIV-associated neurocognitive disorders (HAND) can still affect approximately 50% of HIV-infected individuals even under cART. While cART has greatly reduced the prevalence of the most severe form of HAND, milder forms have increased in prevalence, leaving the total proportion of HIV-infected individuals suffering from HAND relatively unchanged. In this study, an in vitro drug screen identified fluconazole and paroxetine as protective compounds against HIV gp120 and Tat neurotoxicity. Using an accelerated, consistent SIV/macaque model of HIV-associated CNS disease, we tested the in vivo neuroprotective capabilities of combination fluconazole/paroxetine (FluPar) treatment. FluPar treatment protected macaques from SIV-induced neurodegeneration, as measured by neurofilament light chain in the CSF, APP accumulation in axons, and CaMKIIα in the frontal cortex, but did not significantly reduce markers of neuroinflammation or plasma or CNS viral loads. Since HIV and SIV neurodegeneration is often attributed to accompanying neuroinflammation, this study provides proof of concept that neuroprotection can be achieved even in the face of ongoing neuroinflammation and viral replication.

PACAP27 is protective against tat-induced neurotoxicity.

Human immunodeficiency virus type-1 (HIV) infection of the central nervous system promotes neuronal injury and apoptosis that culminate in HIV-associated neurocognitive disorders (HAND). Viral proteins, such as transactivator of transcription (Tat), have emerged as leading candidates to explain HIV-mediated neurotoxicity, though the mechanism remains unclear. To determine the effects of Tat, rat cortical neurons were exposed to nanomolar concentrations of Tat for various time points. Within a few hours, Tat induced the production of reactive oxygen species (ROS), and other indices of mitochondrial destabilization. In addition, we observed a significant induction of DNA double-strand breaks (DSBs) by Tat. We next investigated the neuroprotective activity of the pituitary adenylate cyclase-activating polypeptide 27 (PACAP27) against these cardinal features of Tat-induced neurodegeneration. PACAP27 (100 nM) inhibited all Tat-mediated toxic effects including DNA DSBs. Importantly, PACAP27 prevented the induction of neuronal loss induced by Tat. The neuroprotective effect of PACAP27 is correlated with its ability to release the anti-apoptotic chemokine CCL5. Our data support a mechanism of Tat neurotoxicity in which Tat induces mitochondrial destabilization, thus increasing the release of ROS, which causes DNA DSBs leading to cell death. PACAP27, through CCL5, mitigates the effects of Tat-induced neuronal dysfunction, suggesting that PACAP27 could be a new strategy for an adjunct therapy against HIV-associated neurocognitive disorders.

Neurotrophin strategies for neuroprotection: are they sufficient?

As people are living longer, the prevalance of neurodegenerative diseases continues to rise resulting in huge socio-economic consequences. Despite major advancements in studying the pathophysiology of these diseases and a large number of clinical trials currently there is no effective treatment for these illnesses. All neuroprotective strategies have either failed or have shown only a minimal effect. There has been a major shift in recent years exploring the potential of neuroregenerative approaches. While the concept of using neurotropins for therapeutic purposes has been in existence for many years, new modes of delivery and expression of this family of molecules makes this approach now feasilble. Further neurotropin mimetics and receptor agonists are also being developed. The use of small molecules to induce the expression of neurotropins including repurposing of FDA approved drugs for this approach is another strategy being pursued. In the review we examine these new developments and discuss the potential for such approaches in the context of the pathophysiology of neurodegenerative diseases.

Functional screening assays with neurons generated from pluripotent stem cell-derived neural stem cells.

Rapid and effective drug discovery for neurodegenerative disease is currently impeded by an inability to source primary neural cells for high-throughput and phenotypic screens. This limitation can be addressed through the use of pluripotent stem cells (PSCs), which can be derived from patient-specific samples and differentiated to neural cells for use in identifying novel compounds for the treatment of neurodegenerative diseases. We have developed an efficient protocol to culture pure populations of neurons, as confirmed by gene expression analysis, in the 96-well format necessary for screens. These differentiated neurons were subjected to viability assays to illustrate their potential in future high-throughput screens. We have also shown that organelles such as nuclei and mitochondria could be live-labeled and visualized through fluorescence, suggesting that we should be able to monitor subcellular phenotypic changes. Neurons derived from a green fluorescent protein-expressing reporter line of PSCs were live-imaged to assess markers of neuronal maturation such as neurite length and co-cultured with astrocytes to demonstrate further maturation. These studies confirm that PSC-derived neurons can be used effectively in viability and functional assays and pave the way for high-throughput screens on neurons derived from patients with neurodegenerative disorders.

Catechins protect neurons against mitochondrial toxins and HIV proteins via activation of the BDNF pathway.

Currently, there is no effective treatment for neurological complications of infection with the human immunodeficiency virus that persists despite the use of combination antiretroviral therapy. A medium throughput assay was developed for screening neuroprotective compounds using primary mixed neuronal cells and mitochondrial toxin 3-nitropropionic acid. Using this assay, a library of 2,000 compounds was screened. Out of 256 compounds that showed variable degrees of neuroprotection, nine were related to epicatechin, a monomeric flavonoid found in cocoa and green tea leaves that readily crosses the blood-brain barrier. Hence, catechin, epicatechin, and the related compound, epigallocatechin gallate (EGCG) were further screened for their neuroprotective properties against HIV proteins Tat and gp120, and compared to those of resveratrol. Epicatechin and EGCG targets the brain-derived neurotrophic factor (BDNF) and its precursor proBDNF signaling pathways, normalizing both Tat-mediated increases in proapoptotic proBDNF and concomitant Tat-mediated decreases in the mature BDNF protein in hippocampal neurons. Epicatechin and epigallocatechin gallate were more potent than catechin or resveratrol as neuroprotectants. Due to its simpler structure and more efficient blood-brain barrier penetration properties, epicatechin might be the best therapeutic candidate for neurodegenerative diseases including HIV-associated neurocognitive disorders where oxidative stress is an important pathophysiological mechanism.

The human immunodeficiency virus coat protein gp120 promotes forward trafficking and surface clustering of NMDA receptors in membrane microdomains.

Infection by the human immunodeficiency virus (HIV) can result in debilitating neurological syndromes collectively known as HIV-associated neurocognitive disorders. Although the HIV coat protein gp120 has been identified as a potent neurotoxin that enhances NMDA receptor function, the exact mechanisms for this effect are not known. Here we provide evidence that gp120 activates two separate signaling pathways that converge to enhance NMDA-evoked calcium flux by clustering NMDA receptors in modified membrane microdomains. gp120 enlarged and stabilized the structure of lipid microdomains on dendrites by mechanisms that involved a redox-regulated translocation of a sphingomyelin hydrolase (neutral sphingomyelinase-2) to the plasma membrane. A concurrent pathway was activated that accelerated the forward traffic of NMDA receptors by a PKA-dependent phosphorylation of the NR1 C-terminal serine 897 (masks an ER retention signal), followed by a PKC-dependent phosphorylation of serine 896 (important for surface expression). NMDA receptors were preferentially targeted to synapses and clustered in modified membrane microdomains. In these conditions, NMDA receptors were unable to laterally disperse and did not internalize, even in response to strong agonist induction. Focal NMDA-evoked calcium bursts were enhanced by threefold in these regions. Inhibiting membrane modification or NR1 phosphorylation prevented gp120 from accelerating the surface localization of NMDA receptors. Disrupting the structure of membrane microdomains after gp120 treatments restored the ability of NMDA receptors to disperse and internalize. These findings demonstrate that gp120 contributes to synaptic dysfunction in the setting of HIV infection by interfering with NMDA receptor trafficking.

Inhibition of Tat-mediated HIV-1 replication and neurotoxicity by novel GSK3-beta inhibitors.

The HIV-1 protein Tat is a critical regulator of viral transcription and has also been implicated as a mediator of HIV-1 induced neurotoxicity. Here using a high throughput screening assay, we identified the GSK-3 inhibitor 6BIO, as a Tat-dependent HIV-1 transcriptional inhibitor. Its ability to inhibit HIV-1 transcription was confirmed in TZM-bl cells, with an IC(50) of 40nM. Through screening 6BIO derivatives, we identified 6BIOder, which has a lower IC(50) of 4nM in primary macrophages and 0.5nM in astrocytes infected with HIV-1. 6BIOder displayed an IC(50) value of 0.03nM through in vitro GSK-3ß kinase inhibition assays. Finally, we demonstrated 6BIO and 6BIOder have neuroprotective effects on Tat induced cell death in rat mixed hippocampal cultures. Therefore 6BIO and its derivatives are unique compounds which, due to their complex mechanisms of action, are able to inhibit HIV-1 transcription as well as to protect against Tat induced neurotoxicity.

GPI-1046 increases presenilin-1 expression and restores NMDA channel activity.

BACKGROUND: Previously we showed that 6-hydroxydopamine lesions of the substantia nigra eliminate corticostriatal LTP and that the neuroimmunolophilin ligand (NIL), GPI-1046, restores LTP. METHODS: We used cDNA microarrays to determine what mRNAs may be over- or under-expressed in response to lesioning and/or GPI-1046 treatment. Patch clamp recordings were performed to investigate changes in NMDA channel function before and after treatments. RESULTS: We found that 51 gene products were differentially expressed. Among these we found that GPI-1046 treatment up-regulated presenilin-1 (PS-1) mRNA abundance. This finding was confirmed using QPCR. PS-1 protein was also shown to be over-expressed in the striatum of lesioned/GPI-1046-treated rats. As PS-1 has been implicated in controlling NMDA-receptor function and LTP is reduced by lesioning we assayed NMDA mediated synaptic activity in striatal brain slices. The lesion-induced reduction of dopaminergic innervation was accompanied by the near complete loss of NDMA receptor-mediated synaptic transmission between the cortex and striatum. GPI-1046 treatment of the lesioned rats restored NMDA-mediated synaptic transmission but not the dopaminergic innervation. Restoration of NDMA channel function was apparently specific as the sodium channel current density was also reduced due to lesioning but GPI-1046 did not reverse this effect. We also found that restoration of NMDA receptor function was also not associated with either an increase in NMDA receptor mRNA or protein expression. CONCLUSION: As it has been previously shown that PS-1 is critical for normal NMDA receptor function, our data suggest that the improvement of excitatory neurotransmission occurs through the GPI-1046-induced up-regulation of PS-1.

Neuroprotective and antiretroviral effects of the immunophilin ligand GPI 1046.

HIV infection results in a neurodegenerative disorder for which currently there is no effective therapy available. Currently, available antiretroviral therapy has no impact on the production of early regulatory HIV proteins once the virus is integrated. Of these proteins, Tat was shown to be toxic to neurons. We, thus, used an in vitro neuronal culture system to determine if immunophilin ligands could protect against Tat-induced neurotoxicity. We found that GPI 1046 had potent neuroprotective effects in this model. The compound was able to protect the neurons even though it only partially obliterated Tat-induced oxidative stress in neurons, suggesting that other mechanisms may be important in mediating its neuroprotective effect. Furthermore, GPI 1046 showed inhibition of HIV replication and Tat-mediated long terminal repeat (LTR) activation suggesting that this class of compounds may be worthy of further exploration as a potential treatment for HIV dementia.