Early in the HIV pandemic, it became evident that people living with HIV (PLWH) develop a wide range of neurological and neurocognitive complications. Even after the introduction of combination antiretroviral therapy (cART), which dramatically improved survival of PLWH, the overall number of people living with some form of HIV-associated neurocognitive disorders (HAND) seemed to remain unchanged, although the incidence of dementia declined and questions about the incidence and diagnosis of the mildest form of HAND arose. To better understand this complex disease, several transcriptomic analyses have been conducted in autopsy samples, as well as in non-human primates and small animal rodent models. However, genetic studies in the HIV field have mostly focused on the genetic makeup of the immune system. Much less is known about the genetic underpinnings of HAND. Here, we provide a summary of reported transcriptomic and epigenetic changes in HAND, as well as some of the potential genetic underpinnings that have been linked to HAND, and discuss future directions with hurdles to overcome and angles that remain to be explored.

Background

The chemokine receptor CXCR4 (CD184) and its natural ligand CXCL12 contribute to many physiological processes, including decisions about cell death and survival in the central nervous system. In addition, CXCR4 is a co-receptor for human immunodeficiency virus (HIV)-1 and mediates the neurotoxicity of the viral envelope protein gp120. However, we previously observed that CXCL12 also causes toxicity in cerebrocortical neurons but the cellular mechanism remained incompletely defined.

Methods

Primary neuronal-glial cerebrocortical cell cultures from rat were exposed to a neurotoxicity-inducing CXCL12 concentration for different times and the activity of the stress-associated mitogen-activated protein kinase p38 (p38 MAPK) was assessed using an in vitro kinase assay. Neurotoxicity of CXCL12 and cellular localization of p38 MAPK was analyzed by immunofluorescence microscopy. Pharmacological inhibition of NMDA-type glutamate receptor-gated ion channels (NMDAR) of L-type Ca²⁺ channels was employed during 12- and 24-h exposure to neurotoxic amounts of CXCL12 to study the effects on active p38 MAPK and neuronal survival by Western blotting and microscopy, respectively. Neurotoxicity of CXCL12 was also assessed during pharmacological inhibition of p38 MAPK.

Results

Here, we show that a neurotoxic amount of CXCL12 triggers a significant increase of endogenous p38 MAPK activity in cerebrocortical cells. Immunofluorescence and Western blotting experiments with mixed neuronal-glial and neuron-depleted glial cerebrocortical cells revealed that the majority of active/phosphorylated p38 MAPK was located in neurons. Blockade of NMDAR-gated ion channels or L-type Ca²⁺ channels both abrogated an increase of active p38 MAPK and toxicity of CXCL12 in cerebrocortical neurons. Inhibition of L-type Ca²⁺ channels with nimodipine kept the active kinase at levels not significantly different from baseline while blocking NMDAR with MK-801 strongly reduced phosphorylated p38 MAPK below baseline. Finally, we confirmed that directly blocking p38 MAPK also abrogated neurotoxicity of CXCL12.

Conclusions

Our findings link CXCL12-induced neuronal death to the regulation of NMDAR-gated ion channels and L-type Ca²⁺ channels upstream of p38 MAPK activation.

People living with HIV (PLWH) continue to develop HIV-associated neurocognitive disorders despite combination anti-retroviral therapy. Lipocalin-2 (LCN2) is an acute phase protein that has been implicated in neurodegeneration and is upregulated in a transgenic mouse model of HIV-associated brain injury. Here we show that LCN2 is significantly upregulated in neocortex of a subset of HIV-infected individuals with brain pathology and correlates with viral load in CSF and pro-viral DNA in neocortex. However, the question if LCN2 contributes to HIV-associated neurotoxicity or is part of a protective host response required further investigation. We found that the knockout of LCN2 in transgenic mice expressing HIVgp120 in the brain (HIVgp120tg) abrogates behavioral impairment, ameliorates neuronal damage, and reduces microglial activation in association with an increase of the neuroprotective CCR5 ligand CCL4. In vitro experiments show that LCN2 neurotoxicity also depends on microglia and p38 MAPK activity. Genetic ablation of CCR5 in LCN2-deficient HIVgp120tg mice restores neuropathology, suggesting that LCN2 overrides neuroprotection mediated by CCR5 and its chemokine ligands. RNA expression of 168 genes involved in neurotransmission reveals that neuronal injury and protection are each associated with genotype- and sex-specific patterns affecting common neural gene networks. In conclusion, our study identifies LCN2 as a novel factor in HIV-associated brain injury involving CCR5, p38 MAPK and microglia. Furthermore, the mechanistic interaction between LCN2 and CCR5 may serve as a diagnostic and therapeutic target in HIV patients at risk of developing brain pathology and neurocognitive impairment.

Macrophages (MΦ) infected with human immunodeficiency virus (HIV)-1 or activated by its envelope protein gp120 exert neurotoxicity. We found previously that signaling via p38 mitogen-activated protein kinase (p38 MAPK) is essential to the neurotoxicity of HIVgp120-stimulated MΦ. However, the associated downstream pathways remained elusive. Here we show that cysteinyl-leukotrienes (CysLT) released by HIV-infected or HIVgp120 stimulated MΦ downstream of p38 MAPK critically contribute to neurotoxicity. SiRNA-mediated or pharmacological inhibition of p38 MAPK deprives MΦ of CysLT synthase (LTC4S) and, pharmacological inhibition of the cysteinyl-leukotriene receptor 1 (CYSLTR1) protects cerebrocortical neurons against toxicity of both gp120-stimulated and HIV-infected MΦ. Components of the CysLT pathway are differentially regulated in brains of HIV-infected individuals and a transgenic mouse model of NeuroHIV (HIVgp120tg). Moreover, genetic ablation of LTC4S or CysLTR1 prevents neuronal damage and impairment of spatial memory in HIVgp120tg mice. Altogether, our findings suggest a novel critical role for cysteinyl-leukotrienes in HIV-associated brain injury.

Synapse dysfunction and loss are central features of neurodegenerative diseases, caused in part by the accumulation of protein oligomers. Amyloid-β, tau, prion, and α-synuclein oligomers bind to the cellular prion protein (PrP^C), resulting in the activation of macromolecular complexes and signaling at the post-synapse, yet the early signaling events are unclear. Here we sought to determine the early transcript and protein alterations in the hippocampus during the pre-clinical stages of prion disease. We used a transcriptomic approach focused on the early-stage, prion-infected hippocampus of male wild-type mice, and identify immediate early genes, including the synaptic activity response gene, Arc/Arg3.1, as significantly upregulated. In a longitudinal study of male, prion-infected mice, Arc/Arg-3.1 protein was increased early (40% of the incubation period), and by mid-disease (pre-clinical), phosphorylated AMPA receptors (pGluA1-S845) were increased and metabotropic glutamate receptors (mGluR5 dimers) were markedly reduced in the hippocampus. Notably, sporadic Creutzfeldt-Jakob disease (sCJD) post-mortem cortical samples also showed low levels of mGluR5 dimers. Together, these findings suggest that prions trigger an early Arc response, followed by an increase in phosphorylated GluA1 and a reduction in mGluR5 receptors.

Endolysosomal defects in neurons are central to the pathogenesis of prion and other neurodegenerative disorders. In prion disease, prion oligomers traffic through the multivesicular body (MVB) and are routed for degradation in lysosomes or for release in exosomes, yet how prions impact proteostatic pathways is unclear. We found that prion-affected human and mouse brain showed a marked reduction in Hrs and STAM1 (ESCRT-0), which route ubiquitinated membrane proteins from early endosomes into MVBs. To determine how the reduction in ESCRT-0 impacts prion conversion and cellular toxicity in vivo, we prion-challenged conditional knockout mice (male and female) having Hrs deleted from neurons, astrocytes, or microglia. The neuronal, but not astrocytic or microglial, Hrs-depleted mice showed a shortened survival and an acceleration in synaptic derangements, including an accumulation of ubiquitinated proteins, deregulation of phosphorylated AMPA and metabotropic glutamate receptors, and profoundly altered synaptic structure, all of which occurred later in the prion-infected control mice. Finally, we found that neuronal Hrs (nHrs) depletion increased surface levels of the cellular prion protein, PrP^C, which may contribute to the rapidly advancing disease through neurotoxic signaling. Taken together, the reduced Hrs in the prion-affected brain hampers ubiquitinated protein clearance at the synapse, exacerbates postsynaptic glutamate receptor deregulation, and accelerates neurodegeneration.SIGNIFICANCE STATEMENT Prion diseases are rapidly progressive neurodegenerative disorders characterized by prion aggregate spread through the central nervous system. Early disease features include ubiquitinated protein accumulation and synapse loss. Here, we investigate how prion aggregates alter ubiquitinated protein clearance pathways (ESCRT) in mouse and human prion-infected brain, discovering a marked reduction in Hrs. Using a prion-infection mouse model with neuronal Hrs (nHrs) depleted, we show that low neuronal Hrs is detrimental and markedly shortens survival time while accelerating synaptic derangements, including ubiquitinated protein accumulation, indicating that Hrs loss exacerbates prion disease progression. Additionally, Hrs depletion increases the surface distribution of prion protein (PrP^C), linked to aggregate-induced neurotoxic signaling, suggesting that Hrs loss in prion disease accelerates disease through enhancing PrP^C-mediated neurotoxic signaling.