HIV-associated neurocognitive disorder (HAND), characterized by a wide spectrum of behavioral, cognitive, and motor dysfunctions, continues to affect approximately 50 % of HIV(+) patients despite the success of combination antiretroviral drug therapy (cART) in the periphery. Of note, potential toxicity of antiretroviral drugs in the central nervous system (CNS) remains remarkably underexplored and may contribute to the persistence of HAND in the cART era. Previous studies have shown antiretrovirals (ARVs) to be neurotoxic in the peripheral nervous system in vivo and in peripheral neurons in vitro. Alterations in lipid and protein metabolism, mitochondrial damage, and oxidative stress all play a role in peripheral ARV neurotoxicity. We hypothesized that ARVs also induce cellular stresses in the CNS, ultimately leading to neuronal damage and contributing to the changing clinical and pathological picture seen in HIV-positive patients in the cART era. In this report, we show that ARVs are neurotoxic in the CNS in both pigtail macaques and rats in vivo. Furthermore, in vitro, ARVs lead to accumulation of reactive oxygen species (ROS), and ultimately induction of neuronal damage and death. Whereas ARVs alone caused some activation of the endogenous antioxidant response in vitro, augmentation of this response by a fumaric acid ester, monomethyl fumarate (MMF), blocked ARV-induced ROS generation, and neuronal damage/death. These findings implicate oxidative stress as a contributor to the underlying mechanisms of ARV-induced neurotoxicity and will provide an access point for adjunctive therapies to complement ARV therapy and reduce neurotoxicity in this patient population.

Although the specific mechanism of neuronal damage in human immunodeficiency virus (HIV) -associated dementia is not known, a prominent role for NMDA receptor (NMDAR)-induced excitotoxicity has been demonstrated in neurons exposed to HIV-infected/activated macrophages. We hypothesized NMDAR-mediated activation of the calcium-dependent protease, calpain, would contribute to cell death by induction of cyclin-dependent kinase 5 (CDK5) activity. Using an in vitro model of HIV neurotoxicity, in which primary rat cortical cultures are exposed to supernatants from primary human HIV-infected macrophages, we have observed increased calpain-dependent cleavage of the CDK5 regulatory subunit, p35, to the constitutively active isoform, p25. Formation of p25 is dependent upon NMDAR activation and calpain activity and is coincident with increased CDK5 activity in this model. Further, inhibition of CDK5 by roscovitine provided neuroprotection in our in vitro model. Consistent with our observations in vitro, we have observed a significant increase in calpain activity and p25 levels in midfrontal cortex of patients infected with HIV, particularly those with HIV-associated cognitive impairment. Taken together, our data suggest calpain activation of CDK5, a pathway activated in HIVinfected individuals, can mediate neuronal damage and death in a model of HIV-induced neurotoxicity. Keywords: calpain, cyclin-dependent kinase 5, cell cycle, dementia, encephalitis, neurodegeneration, NMDA receptor. Before the widespread use of highly active antiretroviral therapy (HAART), approximately 20% of patients infected with human immunodeficiency virus (HIV) developed HIVassociated dementia (HAD) (Kaul et al. 2001;Garden 2002;Gonzalez-Scarano and Martin-Garcia 2005). In the post-HAART era, the incidence of HAD has declined to approximately 8%; however, the prevalence has increased. In addition, an increasing number (40%) of HIV-infected patients on HAART are developing a constellation of less severe neurologic symptoms referred to as minor cognitive motor disorder (MCMD) (Janssen et al. 1989;Sacktor et al. 2002;McArthur et al. 2003). Pathologic studies of the brains of patients with HAD suggest an inflammatory mechanism in the progression of this disease, as evidenced by astrogliosis, microgliosis, and perivascular macrophage infiltration (Kaul et al. 2001;Garden 2002;Ghorpade et al. 2003). Although neuronal, dendritic, and synaptic loss are features of HAD, there is little evidence of direct HIV infection of neurons (Shi et al. 1996;Corasaniti et al. 2001). Instead, neuronal dys- Abbreviations used: CDK5, cyclin-dependent kinase 5; DIV, days in vitro; DTT, dithiothreitol; ERK1/2, extracellular signal-regulated kinase 1 and 2; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HAART, highly active antiretroviral therapy; HAD, HIV-associated dementia; HIV MDM, HIV-infected, monocyte-derived macrophage; HIV, human immunodeficiency virus; HIVE, human immunodeficiency virus encephalitis; LR, linear range; MAP2, microtubule-associated protein 2...

The prevalence of HIV‐associated neurocognitive impairment (NCI), which includes HIV‐associated dementia (HAD) and minor cognitive and motor disorder (MCMD), has been increasing. HIV‐infected and/or activated macrophages/microglia in the brain initiate the neurodegeneration seen in HIV‐associated NCI via soluble neurotoxic mediators, including reactive oxygen species, viral proteins and excitotoxins. Neurotoxic factors released by macrophages/microglia injure neurones directly and alter astrocytic homeostatic functions, which can lead to excitotoxicity and oxidative stress‐mediated neuronal injury. Often, cells respond to oxidative stress by initiating the endoplasmic reticulum (ER) stress response. Thus, we hypothesize that ER stress response is activated in HIV‐infected cortex. We used immunofluorescence and immunoblotting to assess expression patterns of the ER stress proteins, BiP and ATF6, in HIV‐positive cortical autopsy tissue. Additionally, we performed immunofluorescence using cell type‐specific markers to examine BiP staining in different cell types, including neurones, astrocytes and macrophages/microglia. We observed a significant increase in BiP expression by both immunoblotting and immunofluorescence in HIV‐positive cortex compared with control tissue. Additionally, phenotypic analysis of immunofluorescence showed cell type‐specific increases in BiP levels in neurones and astrocytes. Further, ATF‐6β, an ER stress response initiator, is up‐regulated in the same patient group, as assessed by immunoblotting. These results suggest that ER stress response is activated in HIV‐infected cortex. Moreover, data presented here indicate for the first time that numbers of macrophages/microglia increase in brains of MCMD patients, as has been observed in HAD.

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Previous work demonstrated that hyperthermia (43°C for 2 h) results in delayed, apoptotic-like death in striatal neuronal cultures. We investigated early changes in mitochondrial function induced by this heat stress. Partial depolarization of the mitochondrial membrane potential (ΔΨ(m)) began about 1 h after the onset of hyperthermia and increased as the stress continued. When the heat stress ended, there was a partial recovery of ΔΨ(m), followed hours later by a progressive, irreversible depolarization of ΔΨ(m). During the heat stress, O(2) consumption initially increased but after 20-30 min began a progressive, irreversible decline to about one-half the initial rate by the end of the stress. The percentage of oligomycin-insensitive respiration increased during the heat stress, suggesting an increased mitochondrial leak conductance. Analysis using inhibitors and substrates for specific respiratory chain complexes indicated hyperthermia-induced dysfunction at or upstream of complex I. ATP levels remained near normal for ∼4 h after the heat stress. Mitochondrial movement along neurites was markedly slowed during and just after the heat stress. The early, persisting mitochondrial dysfunction described here likely contributes to the later (>10 h) caspase activation and neuronal death produced by this heat stress. Consistent with this idea, proton carrier-induced ΔΨ(m) depolarizations comparable in duration to those produced by the heat stress also reduced neuronal viability. Post-stress ΔΨ(m) depolarization and/or delayed neuronal death were modestly reduced/postponed by nicotinamide adenine dinucleotide, a calpain inhibitor, and increased expression of Bcl-xL.