HIV-1 Tg rats as a model of neuroAIDS
A thorough neurobiological investigation of the neurobiology of HIV-induced neuronal dysfunction and its evolving phenotype in the context of viral suppression has been limited by the lack of validated small animal models to probe the effects of concomitant low level expression of multiple HIV-1 products in disease-relevant cells in the CNS. The HIV-1 Tg rats are constitutive, making them a model of persistent exposure to viral proteins, although lack of induciblity of the HIV-1 provirus can be seen as a limitation. Here we show that HIV-1 Tg rats express viral proteins in microglia and astrocytes, display astrogliosis and microgliosis, and have gene expression changes consistent with human neuroAIDS. Collectively, these results support that HIV-1 Tg rats are a valid animal model of neuroAIDS.
Supporting the functional significance of the gene expression alterations reported here, HIV-1 Tg rats showed working memory impairments in spontaneous alternation behavior (SAB) in the T-Maze, a paradigm sensitive to prefrontal cortex and hippocampal function. Rats were tested in the dark in the T-Maze with no visual cues provided to minimize the influence of cataracts in HIV-1 Tg rats, as done by others [
33]. In this regard it should be noted that spontaneous alternation behavior (SAB) has been used in aged rats with visual impairments [
34] and to compare anophthalmic mutant mice to wild-type controls [
35]. It has been previously shown that HIV transgenic rats exhibit impairment in a spatial reversal learning task [
36] without any deficit in spatial memory or motor activity [
37]. Our results extend these studies by showing that HIV rats also exhibit impairment in a working memory task. The deficits in the reversal learning task and the working memory task may both be explained by an impairment in the ability of HIV rats to shift attention to a new target, a decreased working memory capacity and/or an increase in perseveration.
Gene expression changes in HIV-1 Tg rats
Gene expression and pathway analysis with the GSEA algorithm show evidence of neuroinflammatory processes and gliosis and synaptodendritic injury that together with the impairment in working memory are reminiscent of HAND in humans. In particular, the GSEA-based pathway analysis that we employed suggests key dysregulation in IFN regulated pathways, including ISG-15, which exerts antiviral activities through members of the Endosomal sorting complex required for transport (ESCRT) proteins [
38]. ISG-15 was proposed to be a predictive biomarker for HAND [
39,
40]. In particular, ISG-15 was shown to correlate with neuropathology and viral load and ISG-15 CSF levels may be predictive of future neurocognitive impairments [
38]. Several other inflammation-related genes induced in the hippocampus of HIV-1 Tg rats were previously associated with neuroAIDS in humans including IGF binding proteins [
41], prostaglandin D2 (PGD2) synthase [
42], and the pro-alpha2 chain of type I collagen, Col1A2 [
43]. Prostaglandins have been implicated in inflammation-induced working memory deficits [
44] and imaging evidence supports that brain arachidonic acid metabolism in HIV-1 transgenic rats is upregulated [
45]. PGD2 has been implicated in the induction of astrogliosis and demyelination [
42,
46] and has been associated with immune activation, astrogliosis, microgliosis, and neuroAIDS in humans [
42]. Col1A2 has been associated with neuroAIDS in humans [
43]. Collagen induction in the CNS has been also observed in brain injury, Alzheimer’s disease, temporal lobe epilepsy, and multiple sclerosis plaques [
47‐
49].
GSEA analysis showed downregulation of genesets related to neuronal function, neuronal trophism, synaptic plasticity, as well as genes and pathways associated with degenerative diseases such as Huntington’s disease and Parkinson’s disease, which include genes involved in protein misfolding and mitochondrial function. Decreased expression of synaptic plasticity and axon guidance genes was also observed in patients with HIVE in the The National NeuroAIDS Tissue Consortium Brain Gene Array [
50] and in another study [
43], consistent with the notion that HAND in the cART setting correlates with synaptodendritic injury [
17,
18]. Dysregulations of genes related to Parkinson’s disease and Huntington’s disease and mitochondrial function, is consistent with previous studies suggesting mitochondrial dysfunction and oxidative stress as potential contributors to HAND [
51].
Signaling systems involved in neuronal trophism and synaptic maturation and plasticity that were found to be differentially regulated in HIV-1 Tg rats involve IGF, ErbB and netrin, all of which have been shown to exert trophic actions on dendritic spines [
52‐
55], suggesting that dysregulation of these pathways can contribute to the synaptodendritic injury seen in neuroAIDS [
17,
18].
IGF signaling has been implicated in synaptic trophism, depression and Parkinson’s disease [
55‐
57]. GSEA highlighted differential expression of IGF signaling genes including increased Igfbp2 and Igfbp6 expression, which were also found to be increased in the CSF of HIV-1 patients [
41]. The IGFBPs have high affinities for the IGFs [
58,
59] and their increased expression may result in reduced trophic support as well as inflammatory processes [
60‐
62]. The IGF activated gene TDAG51/PHLDA1 [
63], which is expressed in both neurons and glial cells [
64], is implicated in insulin signaling [
65] and has been shown to have differential effects on susceptibility to apoptosis [
66‐
68].
ErbB2/B4 receptors and their ligand neuregulin-1 (NRG1) are encoded by candidate susceptibility genes for schizophrenia [
69]. Functional NRG1 receptors consist of ErbB4 homodimers or heterodimers between ErbB2, ErbB3 and ErbB4 since NRG1 binds ErbB3 and ErbB4, and ErbB2 and ErbB4 have intrinsic tyrosine kinase activity [
69]. Mice that lack the ErbBs with intrinsic tyrosine kinase activity (ErbB2 and ErbB4) in the CNS have reduced dendritic spine density and behavioral abnormalities [
53], possibly implicating this ErbB signaling in synaptodendritic injury. Netrins are secreted mediators that exert multiple trophic actions including axon guidance [
70], synaptic plasticity [
71], prevention of apoptosis [
72,
73], adult neurogenesis including in the course of CNS regeneration [
74‐
76], which is believed to be impaired in neuroAIDS [
77], and synaptic trophism [
54].
The GSEA analysis highlights a substantial downregulation of intracellular signaling pathways relevant to trophic support such as the PI3K-mTOR signaling pathway, which mediates a vast array of trophic signals [
78]; the LKB1 pathway, which is key to neuronal survival following mitochondrial insults [
79] and cross-talks with the PI3K-mTOR pathway; and the ILK pathway, which is involved in mediating trophic signals of the extracellular matrix and trophic factors as well as in the anti-apoptotic effect of the PI3K pathway [
80]. Recent studies have indicated impaired processing and transport of neurotrophic factors in HIV-1 neuropathogenesis [
81‐
83]. The PI3K-mTOR signaling pathway is involved in synaptic plasticity [
84‐
86] and was shown to be dysregulated in the frontal cortex of patients with neuroAIDS [
43]. Downregulated genesets also contain numerous MAP kinases including ERK1 (MAPK3), which was previously implicated in the neurological actions of HIV-1 products [
87].
The GSEA analysis also points to coordinated mitochondria and peroxisome dysregulations as well as dysregulation of phospholipid metabolism. Peroxisomes and mitochondria exhibit a functional interplay in fatty acid processing and intermediate metabolism [
88,
89]. It could be envisioned that if peroxisomal metabolism is slowed, critical metabolic intermediates (e.g., acetyl-CoA) may not be adequately supplied to mitochondria and, conversely, disruption of mitochondrial metabolism can similarly affect peroxisomal function. Peroxisome dysfunction may also contribute to increased lipid peroxidation and cellular aging in HIV-1 [
90,
91]. In apparent agreement with the dysregulation of these pathways, evidence suggestive of altered phospholipid metabolism has been reported in HIV-1 patients [
92,
93] and peroxisome dysregulation can be indicative of altered brain oxidative balance associated with HIV-1 [
94].
Two cellular targets of HIV-1, Fbxw11 and nucleophosmin/B23, were also differentially regulated in the hippocampus of HIV-1 Tg rats. Fbxw11 is the gene coding for βTrCP, a cellular ubiquitin ligase that was found to be bound by the HIV-1 Vpu viral protein [
95]. Nucleophosmin/B23, encoded by the Npm1 gene, is a nuclear chaperone implicated in the nuclear transport of Tat [
96].
Potential therapeutic targets for neuroAIDS suggested by gene expression analysis of HIV-1 Tg rats
Gene expression analysis can be a useful tool both to gain insights in the pathogenesis and for the identification of potential new therapeutic targets by identifying the signaling pathways that have the potential to modify the disease pathophysiology [
97‐
99]. This is especially needed in neuroAIDS as no targeted therapy other than cART is currently recommended for the management of HAND [
100,
101]. Adjunctive therapies for HAND explored to date include the low-affinity antagonist of the NMDA type glutamate receptor memantine, the calcium channel blocker nimodipine, and the monoamine oxidase B inhibitor selegiline, in addition to antioxidants and anti-inflammatory drugs such as minocycline. Memantine and nimodipine are intended to protect from excitotoxic neuronal damage associated with excessive glutamate release [
102,
103]. Selegiline is a drug used for the treatment of early-stage Parkinson’s disease, depression and dementia [
104,
105].
Our gene expression results showing decreased expression of Parkinson's disease-related genesets lend support for the use of selegiline in HAND and potentially for other therapeutics used in Parkinson's disease such as methylphenidate, a stimulant that reduces reuptake of dopamine and norepinephrine that has proven beneficial in lowering fatigue scores in HIV patients [
106]. The present results of increased PGD2 synthase (Ptgds), is in keeping with previous studies that showed elevated PGD2 levels in HIV-1-positive patients [
42]. A potential role of prostaglandins in HIV-1 neuropathogenesis is also indicated by increased expression of the prostaglandin synthetic enzyme COX-2, which is also a characteristic of other degenerative conditions such as Alzheimer's disease and amyotrophic lateral sclerosis [
107,
108]. Dysregulation of prostaglandin synthesis in HAND lends support to the use of inhibitors of cyclooxygenase-2 (COX-2) as well as newer compounds targeting the PGD2 receptor. In this regard, a COX-2 inhibitor has shown promise in downregulating immune activation and improving T cell function in HIV-1 patients [
109].
The present gene expression results as well as the previous literature also point to a role in HAND of several signaling systems with the potential to provide trophic support to synapses and reverse the synaptodendritic injury associated with HAND [
17,
18]. Among them are IGF, ErbB and netrin signaling and the PI3K-mTOR signal transduction pathway, which mediates trophic and plastic actions of various signaling systems.
IGF-1 therapy has shown potential in models of both Parkinson’s disease [
110,
111] and Alzheimer's disease [
112,
113]. These effects are at least in part mediated by recruitment of PI3K [
111,
114]. Additionally, chronic treatment with IGF-1 was protective
in vitro against gp120-mediated neuronal damage and was synergistic with erythropoietin (EPO), at least in part, through cooperative activation of PI3K [
115]. GLP-1 and GLP-1 analogs, which activate partially overlapping signal transduction pathways as insulin and IGF-1 [
114], have also shown beneficial effects in preclinical models of neurodegenerative disorders [
116‐
119]. There is currently great interest in developing drugs to modulate NRG1
–ErbB4 and netrin signalling [
120,
121]. Netrin agonists may be potentially beneficial in neuroAIDS both by exerting neurotrophic actions and by reducing inflammation [
120]. The PI3K pathway is itself a potential target for reversal of the synaptodendritic injury associated with HAND. However, as PI3K appears to play a role in the regulation of the HIV-1 LTR promoter and virus latency [
122,
123], activation of PI3K in the cells that harbor the provirus may increase levels of expression of HIV-1 products, potentially leading to detrimental effects on cognition.