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.

SHEP1 partners with CasL to promote marginal zone B-cell maturation.

The marginal zone is a cellular niche bordering the marginal sinus of the spleen that contains specialized B-cell and macrophage subsets poised to capture bloodborne antigens. Marginal zone B cells are retained in this niche by integrin-mediated signaling induced by G protein-coupled receptors (GPCRs) and, likely, the B-cell receptor (BCR). Sphingosine-1-phosphate (S1P) signaling via the S1P family of GPCRs is known to be essential for B-cell localization in the marginal zone, but little is known about the downstream signaling events involved. Here, we demonstrate that the adaptor protein SHEP1 is required for marginal zone B-cell maturation. SHEP1 functions in concert with the scaffolding protein CasL, because we show that SHEP1 and CasL are constitutively associated in B cells. SHEP1 association is required for the BCR or S1P receptor(s) to induce the conversion of CasL into its serine/threonine hyperphosphorylated form, which is important for lymphocyte adhesion and motility. Thus, SHEP1 orchestrates marginal zone B-cell movement and retention as a key downstream effector of the BCR and S1P receptors.

Modulation of murine complement receptor type 2 (CR2/CD21) ectodomain shedding by its cytoplasmic domain.

Ectodomain shedding is a mechanism that regulates numerous functions of cell surface proteins. The extracellular domain of the human complement receptor 2 (CR2/CD21) is released by proteolytic cleavage as a soluble protein through a variety of stimuli including the thiol antioxidants N-acetylcysteine (NAC) and glutathione (GSH), and the oxidant pervanadate (PV). In addition, PV mimics B cell antigen receptor (BCR) signaling. Here, we show that murine CD21 is shed upon those stimuli and that the cytoplasmic domain is an important modulator for CD21-shedding. B cells expressing a mutant CD21 cytoplasmic domain with only three amino acids (KHR) showed increased CD21-shedding and required lower stimuli concentrations. At lower PV concentrations, wildtype CD21 was up-regulated on the cell surface, whereas at higher PV concentrations the ectodomain was shed. These findings further indicate that GSH and NAC utilize different pathways than PV to activate CD21-shedding. Altogether, as pre-activated B cells express higher CD21 levels than resting mature B cells or fully activated and antigen-experienced B cells, we suggest CD21-shedding to be a mechanism to fine-tune B cell activation.

IFNß Protects Neurons from Damage in a Murine Model of HIV-1 Associated Brain Injury.

Infection with human immunodeficiency virus-1 (HIV-1) causes brain injury. Type I interferons (IFNα/ß) are critical mediators of any anti-viral immune response and IFNß has been implicated in the temporary control of lentiviral infection in the brain. Here we show that transgenic mice expressing HIV-1 envelope glycoprotein 120 in their central nervous system (HIVgp120tg) mount a transient IFNß response and provide evidence that IFNß confers neuronal protection against HIVgp120 toxicity. In cerebrocortical cell cultures, neuroprotection by IFNß against gp120 toxicity is dependent on IFNα receptor 1 (IFNAR1) and the ß-chemokine CCL4, as IFNAR1 deficiency and neutralizing antibodies against CCL4, respectively, abolish the neuroprotective effects. We find in vivo that IFNß mRNA is significantly increased in HIVgp120tg brains at 1.5, but not 3 or 6 months of age. However, a four-week intranasal IFNß treatment of HIVgp120tg mice starting at 3.5 months of age increases expression of CCL4 and concomitantly protects neuronal dendrites and pre-synaptic terminals in cortex and hippocampus from gp120-induced damage. Moreover, in vivo and in vitro data suggests astrocytes are a major source of IFNß-induced CCL4. Altogether, our results suggest exogenous IFNß as a neuroprotective factor that has potential to ameliorate in vivo HIVgp120-induced brain injury.

Possible tetramerisation of the proteasome maturation factor POMP/proteassemblin/hUmp1 and its subcellular localisation.

The proteasome is a multisubunit complex with a central role in non-lysosomal proteolysis and the processing of proteins for presentation by the MHC class I pathway. The 16kDa proteasome maturation protein POMP (also named proteassemblin or hUmp1) acts as a chaperone and is essential for the maturation of the 20S proteasome proteolytic core complex. However, the exact mechanism, timing and localisation of mammalian proteasome assembly remains elusive. We sought to investigate the localisation of POMP within the cell and therefore purified the protein and produced a polyclonal antibody. For immunisation, POMP was overexpressed and purified from a bacterial GST-system. Interestingly, after removal of the GST-tag, POMP was hardly detectable by Coomassie blue- and Ponceau red-staining. However, with a reverse zinc-staining, the protein could easily be visualised. POMP was gel-filtrated and eluted from a calibrated chromatography column with an apparent molecular weight of approximately 64kDa, suggesting that it forms tetramers. Moreover, localisation studies by immunofluorescence stainings and confocal microscopy revealed that POMP is present in the cytoplasm as well as in the nucleus.

Combination of methamphetamine and HIV-1 gp120 causes distinct long-term alterations of behavior, gene expression, and injury in the central nervous system.

Methamphetamine (METH) abuse is frequent in individuals infected with human immunodeficiency virus type-1 (HIV-1) and is suspected to aggravate HIV-associated neurocognitive disorders (HAND). METH is a psychostimulant that compromises several neurotransmitter systems and HIV proteins trigger neuronal injury but the combined effects of viral infection and METH abuse are incompletely understood. In this study we treated transgenic mice expressing the HIV envelope protein gp120 in the brain (HIV-1 gp120tg) at 3-4 months of age with an escalating-dose, multiple-binge METH regimen. The long-term effects were analyzed after 6-7 months of drug abstinence employing behavioral tests and analysis of neuropathology, electrophysiology and gene expression. Behavioral testing showed that both HIV-1 gp120tg and WT animals treated with METH displayed impaired learning and memory. Neuropathological analysis revealed that METH similar to HIV-1 gp120 caused a significant loss of neuronal dendrites and pre-synaptic terminals in hippocampus and cerebral cortex of WT animals. Electrophysiological studies in hippocampal slices showed that METH exposed HIV-1 gp120tg animals displayed reduced post-tetanic potentiation, whereas both gp120 expression and METH lead to reduced long-term potentiation. A quantitative reverse transcription-polymerase chain reaction array showed that gp120 expression, METH and their combination each caused a significant dysregulation of specific components of GABAergic and glutamatergic neurotransmission systems, providing a possible mechanism for synaptic dysfunction and behavioral impairment. In conclusion, both HIV-1 gp120 and METH caused lasting behavioral impairment in association with neuropathology and altered gene expression. However, combined METH exposure and HIV-1 gp120 expression resulted in the most pronounced, long lasting pre- and post-synaptic alterations coinciding with impaired learning and memory.

Ectodomain shedding and generation of two carboxy-terminal fragments of human complement receptor 2/CD21.

Numerous cell surface proteins are functionally regulated by a proteolytic cleavage event termed 'ectodomain shedding'. The complement receptor 2/CD21 extracellular domain (ectodomain) is constitutively released as a soluble form (sCD21), but its liberation can also be induced by various physiological and pharmacological stimuli. CD21-shedding modulates B cell activation, and sCD21 can activate other immune cells and allows transfer of immune complexes from marginal zone B cells to follicular dendritic cells. Deletion of the cytoplasmic domain of CD21 augments CD21-shedding, while removal of the extracellular membrane-adjacent short consensus repeat 16 abolishes shedding. Carboxy-terminal fragments (CTFs) and intracellular domains (ICDs) result from ectodomain shedding and regulated intramembrane cleavage (RIP) of CTFs, respectively. By modulating gene transcription, CTFs and ICDs can regulate cell function and homeostasis. Here, we demonstrate that two membrane-tethered CD21 CTFs of 8 and 16kDa are constitutively present in human B cells, while only the 8kDa CTF was detectable in murine B cells. Glutathione (GSH) regulates extracellular redox levels and is a known inducer of CD21-shedding. Interestingly, GSH-treatment of B lymphocytes only augmented sCD21 levels, but not CD21-CTF levels. In contrast, B cell activation led to increased CD21-CTF levels, suggesting a functional role for the CD21-CTFs in B cell activation and maintenance of B cell homeostasis.