Antiretrovirals, Methamphetamine, and HIV-1 Envelope Protein gp120 Compromise Neuronal Energy Homeostasis in Association with Various Degrees of Synaptic and Neuritic Damage.

HIV-1 infection frequently causes HIV-associated neurocognitive disorders (HAND) despite combination antiretroviral therapy (cART). Evidence is accumulating that components of cART can themselves be neurotoxic upon long-term exposure. In addition, abuse of psychostimulants, such as methamphetamine, seems to aggravate HAND and compromise antiretroviral therapy. However, the combined effect of virus and recreational and therapeutic drugs on the brain is poorly understood. Therefore, we exposed mixed neuronal-glial cerebrocortical cells to antiretrovirals (ARVs) (zidovudine [AZT], nevirapine [NVP], saquinavir [SQV], and 118-D-24) of four different pharmacological categories and to methamphetamine and, in some experiments, the HIV-1 gp120 protein for 24 h and 7 days. Subsequently, we assessed neuronal injury by fluorescence microscopy, using specific markers for neuronal dendrites and presynaptic terminals. We also analyzed the disturbance of neuronal ATP levels and assessed the involvement of autophagy by using immunofluorescence and Western blotting. ARVs caused alterations of neurites and presynaptic terminals primarily during the 7-day incubation and depending on the specific compounds and their combinations with and without methamphetamine. Similarly, the loss of neuronal ATP was context specific for each of the drugs or combinations thereof, with and without methamphetamine or viral gp120. Loss of ATP was associated with activation of AMP-activated protein kinase (AMPK) and autophagy, which, however, failed to restore normal levels of neuronal ATP. In contrast, boosting autophagy with rapamycin prevented the long-term drop of ATP during exposure to cART in combination with methamphetamine or gp120. Our findings indicate that the overall positive effect of cART on HIV infection is accompanied by detectable neurotoxicity, which in turn may be aggravated by methamphetamine.

CCR5 knockout prevents neuronal injury and behavioral impairment induced in a transgenic mouse model by a CXCR4-using HIV-1 glycoprotein 120.

The innate immune system has been implicated in several neurodegenerative diseases, including HIV-1-associated dementia. In this study, we show that genetic ablation of CCR5 prevents microglial activation and neuronal damage in a transgenic model of HIV-associated brain injury induced by a CXCR4-using viral envelope gp120. The CCR5 knockout (KO) also rescues spatial learning and memory in gp120-transgenic mice. However, the CCR5KO does not abrogate astrocytosis, indicating it can occur independently from neuronal injury and behavioral impairment. To characterize further the neuroprotective effect of CCR5 deficiency we performed a genome-wide gene expression analysis of brains from HIVgp120tg mice expressing or lacking CCR5 and nontransgenic controls. A comparison with a human brain microarray study reveals that brains of HIVgp120tg mice and HIV patients with neurocognitive impairment share numerous differentially regulated genes. Furthermore, brains of CCR5 wild-type and CCR5KO gp120tg mice express markers of an innate immune response. One of the most significantly upregulated factors is the acute phase protein lipocalin-2 (LCN2). Using cerebrocortical cell cultures, we find that LCN2 is neurotoxic in a CCR5-dependent fashion, whereas inhibition of CCR5 alone is not sufficient to abrogate neurotoxicity of a CXCR4-using gp120. However, the combination of pharmacologic CCR5 blockade and LCN2 protects neurons from toxicity of a CXCR4-using gp120, thus recapitulating the finding in CCR5-deficient gp120tg mouse brain. Our study provides evidence for an indirect pathologic role of CCR5 and a novel protective effect of LCN2 in combination with inhibition of CCR5 in HIV-associated brain injury.

Dantrolene enhances antisense-mediated exon skipping in human and mouse models of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) causes profound and progressive muscle weakness and loss, resulting in early death. DMD is usually caused by frameshifting deletions in the gene DMD, which leads to absence of dystrophin protein. Dystrophin binds to F-actin and components of the dystrophin-associated glycoprotein complex and protects the sarcolemma from contraction-induced injury. Antisense oligonucleotide-mediated exon skipping is a promising therapeutic approach aimed at restoring the DMD reading frame and allowing expression of an intact dystrophin glycoprotein complex. To date, low levels of dystrophin protein have been produced in humans by this method. We performed a small-molecule screen to identify existing drugs that enhance antisense-directed exon skipping. We found that dantrolene, currently used to treat malignant hyperthermia, potentiates antisense oligomer-guided exon skipping to increase exon skipping to restore the mRNA reading frame, the sarcolemmal dystrophin protein, and the dystrophin glycoprotein complex in skeletal muscles of mdx mice when delivered intramuscularly or intravenously. Further, dantrolene synergized with multiple weekly injections of antisense to increase muscle strength and reduce serum creatine kinase in mdx mice. Dantrolene similarly promoted antisense-mediated exon skipping in reprogrammed myotubes from DMD patients. Ryanodine and Rycal S107, which, like dantrolene, targets the ryanodine receptor, also promoted antisense-driven exon skipping, implicating the ryanodine receptor as the critical molecular target.

Genetic knockouts suggest a critical role for HIV co-receptors in models of HIV gp120-induced brain injury.

Infection with HIV-1 frequently affects the brain and causes NeuroAIDS prior to the development of overt AIDS. The HIV-1 envelope protein gp120 interacts with host CD4 and chemokine co-receptors to initiate infection of macrophages and lymphocytes. In addition, the virus or fragments of it, such as gp120, cause macrophages to produce neurotoxins and trigger neuronal injury and apoptosis. Moreover, the two major HIV co-receptors, the chemokine receptors CCR5 and CXCR4, serve numerous physiological functions and are widely expressed beyond immune cells, including cells in the brain. Therefore, HIV co-receptors are poised to play a direct and indirect part in the development of NeuroAIDS. Although rodents are not permissive to infection with wild type HIV-1, viral co-receptors - more than CD4 - are highly conserved between species, suggesting the animals can be suitable models for mechanistic studies addressing effects of receptor-ligand interaction other than infection. Of note, transgenic mice expressing HIV gp120 in the brain share several pathological hallmarks with NeuroAIDS brains. Against this background, we will discuss recently completed or initiated, ongoing studies that utilize HIV co-receptor knockout and viral gp120-transgenic mice as models for in vitro and in vivo experimentation in order to address the potential roles of HIV gp120 and its co-receptors in the development of NeuroAIDS.

Activation of p38 MAPK is required in monocytic and neuronal cells for HIV glycoprotein 120-induced neurotoxicity.

HIV-1 envelope protein gp120 has been implicated in neurotoxin production by monocytic cells (i.e., macrophages and microglia), as well as in the pathogenesis of HIV-1-associated neurocognitive disorders. We previously showed in cerebrocortical cell cultures from rodents containing microglia, astrocytes, and neurons that overall inhibition of p38 MAPK signaling abrogated the neurotoxic effect of HIV-1 gp120. However, the time course of p38 MAPK activation and the contribution of this kinase in the various cell types remained unknown. In this study, we found that active p38 MAPK is required in monocytic lineage cells (i.e., macrophages and microglia) and neuronal cells for HIV gp120-induced neurotoxicity to occur. In cerebrocortical cell cultures, HIV-1 gp120 stimulated a time-dependent overall increase in active p38 MAPK, and the activated kinase was primarily detected in microglia and neurons. Interestingly, increased activation of p38 MAPK and neuronal death in response to gp120 were prevented by prior depletion of microglia or the presence of CCR5 ligand CCL4 or p38 MAPK inhibitors. In human monocytic THP-1 cells and primary monocyte-derived macrophages, HIV gp120-stimulated production of neurotoxins was abrogated by prior introduction into the cells of a dominant-negative p38 MAPK mutant or p38 MAPK small interfering RNA. In addition, the neurotoxic effects of cell-free supernatants from gp120-stimulated monocytic THP-1 cells were prevented in microglia-depleted cerebrocortical cells pretreated with a pharmacological inhibitor of p38 MAPK. Thus, p38 MAPK signaling was critical, upon exposure to HIV gp120, for the neurotoxic phenotype of monocytic cells and subsequent toxin-initiated neuronal apoptosis.

Alteration of Methamphetamine-induced stereotypic behaviour in transgenic mice expressing HIV-1 envelope protein gp120.

The use of drugs for recreational purposes, in particular Methamphetamine, is associated with an increased risk of infection with human immunodeficiency virus (HIV)-1. HIV-1 infection in turn can lead to HIV-associated neurological disorders (HAND) that range from mild cognitive and motor impairment to HIV-associated dementia (HAD). Interestingly, post mortem brain specimens from HAD patients and transgenic (tg) mice expressing the viral envelope protein gp120 in the central nervous system display similar neuropathological signs. In HIV patients, the use of Methamphetamine appears to aggravate neurocognitive alterations. In the present study, we injected HIV/gp120tg mice and non-transgenic littermate control animals with Methamphetamine dissolved in Saline or Saline vehicle and assessed locomotion and stereotyped behaviour. We found that HIVgp120-transgenic mice differ significantly from non-transgenic controls in certain domains of their behavioural response to Methamphetamine. Thus this experimental model system may be useful to further study the mechanistic interaction of both the viral envelope protein and the psychostimulant drug in behavioural alterations and neurodegenerative disease.