Cagla Akay

Activation of cyclin‐dependent kinase 5 by calpains contributes to human immunodeficiency virus‐induced neurotoxicity

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 HIV‐infected individuals, can mediate neuronal damage and death in a model of HIV‐induced neurotoxicity.

Expression of the endoplasmic reticulum stress response marker, BiP, in the central nervous system of HIV‐positive individuals

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.

Parallel high throughput neuronal toxicity assays demonstrate uncoupling between loss of mitochondrial membrane potential and neuronal damage in a model of HIV-induced neurodegeneration

Neurocognitive deficits seen in HIV-associated neurocognitive disorders (HANDs) are attributed to the release of soluble factors from CNS-resident, HIV-infected and/or activated macrophages and microglia. To study HIV-associated neurotoxicity, we used our in vitro model in which primary rat neuronal/glial cultures are treated with supernatants from cultured human monocyte-derived macrophages, infected with a CNS-isolated HIV-1 strain (HIV-MDM). We found that neuronal damage, detected as a loss of microtubule-associated protein-2 (MAP2), begins as early as 2h and is preceded by a loss of mitochondrial membrane potential (Δψ(m)). Interestingly, inhibitors of calpains, but not inhibitors of caspases, blocked MAP2 loss, however neither type of inhibitor prevented the loss of Δψ(m). To facilitate throughput for these studies, we refined a MAP2 cell-based-ELISA whose data closely compare with our standardized method of hand counting neurons. In addition, we developed a tetramethyl rhodamine methyl ester (TMRM)-based multi-well fluorescent plate assay for the evaluation of whole culture Δψ(m). Together, these findings indicate that calpain activation and loss of Δψ(m) may be parallel pathways to death in HIV-MDM-treated neurons and also demonstrate the validity of plate assays for assessing multiple experimental parameters as is useful for screening neurotherapeutics for neuronal damage and death.

Activation status of integrated stress response pathways in neurones and astrocytes of HIV-associated neurocognitive disorders (HAND) cortex

C. Akay, K. A. Lindl, N. Shyam, B. Nabet, Y. Goenaga‐Vazquez, J. Ruzbarsky, Y. Wang, D. L. Kolson and K. L. Jordan‐Sciutto (2012) Neuropathology and Applied Neurobiology38, 175–200

Site-specific hyperphosphorylation of pRb in HIV-induced neurotoxicity ☆

HIV-Associated Neurocognitive Disorder (HAND) remains a serious complication of HIV infection, despite combined Anti-Retroviral Therapy (cART). Neuronal dysfunction and death are attributed to soluble factors released from activated and/or HIV-infected macrophages. Most of these factors affect the cell cycle machinery, determining cellular outcomes even in the absence of cell division. One of the earliest events in cell cycle activation is hyperphosphorylation of the retinoblastoma protein, pRb (ppRb). We and others have previously shown increased ppRb expression in the CNS of patients with HIV encephalitis (HIVE) and in neurons in an in vitro model of HIV-induced neurodegeneration. However, trophic factors also lead to an increase in neuronal ppRb with an absence of cell death, suggesting that, depending on the stimulus, hyperphosphorylation of pRb can have different outcomes on neuronal fate. pRb has multiple serines and threonines targeted for phosphorylation by distinct kinases, and we hypothesized that different stimuli may target separate sites for phosphorylation. Thus, to determine whether pRb is differentially phosphorylated in response to different stimuli and whether any of these sites is preferentially phosphorylated in association with HIV-induced neurotoxicity, we treated primary rat mixed cortical cultures with trophic factors, BDNF or RANTES, or with the neurotoxic factor, N-methyl-d-aspartate (NMDA), or with supernatants containing factors secreted by HIV-infected monocyte-derived macrophages (HIV-MDM), our in vitro model of HIV-induced neurodegeneration. We found that, while BDNF and RANTES phosphorylated serine807/811 and serine608 in vitro, treatment with HIV-MDM did not, even though these trophic factors are components of HIV-MDM. Rather, HIV-MDM targets a specific phosphorylation site, serine795, of pRb for phosphorylation in vitro and this ppRb isoform is also increased in HIV-infected brains in vivo. Further, overexpression of a nonphosphorylatable pRb (ppRb S795A) attenuated HIV-MDM-induced neurotoxicity. These findings indicate that HIV-infection in the brain is associated with site-specific hyperphosphorylation of pRb at serine795, which is not induced by other tested stimuli, and that this phosphorylation contributes to neuronal death in this disease, demonstrating that specific pRb sites are differentially targeted and may have diverse impacts on the viability of post-mitotic neurons.

E2F1 localizes predominantly to neuronal cytoplasm and fails to induce expression of its transcriptional targets in human immunodeficiency virus-induced neuronal damage

As human immunodeficiency virus (HIV) does not induce neuronal damage by direct infection, the mechanisms of neuronal damage or loss in HIV-associated dementia (HAD) remain unclear. We have shown previously that immunoreactivity of transcription factor, E2F1, increases in neurons, localizing predominantly to the cytoplasm, in HIV-associated pathologies. Here we confirm that E2F1 localization is predominantly cytoplasmic in primary postmitotic neurons in vitro and cortical neurons in vivo. To determine whether E2F1 contributes to neuronal death in HAD via transactivation of target promoters, we assessed the mRNA and protein levels of several classical E2F1 transcriptional targets implicated in cell cycle progression and apoptosis in an in vitro model of HIV-induced neurotoxicity and in cortical autopsy tissue from patients infected with HIV. By Q-PCR, we show that mRNA levels of E2F1 transcriptional targets implicated in cell cycle progression (E2F1, Cyclin A, proliferating cell nuclear antigen (PCNA), and dyhydrofolate reductase (DHFR)) and apoptosis (caspases 3, 8, 9 and p19(ARF)) remain unchanged in an in vitro model of HIV-induced neurotoxicity. Further, we show that protein levels of p19(ARF), Cyclin A, and PCNA are not altered in vitro or in the cortex of patients with HAD. We propose that the predominantly cytoplasmic localization of E2F1 in neurons may account for the lack of E2F1 target transactivation in neurons responding to HIV-induced neurotoxicity.

Calpain-mediated degradation of MDMx/MDM4 contributes to HIV-induced neuronal damage☆

Neuronal damage in HIV-associated Neurocognitive Disorders (HAND) has been linked to inflammation induced by soluble factors released by HIV-infected, and non-infected, activated macrophages/microglia (HIV M/M) in the brain. It has been suggested that aberrant neuronal cell cycle activation determines cell fate in response to these toxic factors. We have previously shown increased expression of cell cycle proteins such as E2F1 and phosphorylated pRb in HAND midfrontal cortex in vivo and in primary neurons exposed to HIV M/M supernatants in vitro. In addition, we have previously shown that MDMx (also referred to as MDM4), a negative regulator of E2F1, was decreased in the brain in a primate model of HIV-induced CNS neurodegeneration. Thus, we hypothesized that MDMx provides indirect neuroprotection from HIV-induced neurodegeneration in our in vitro model. In this report, we found significant reductions in MDMx protein levels in the mid-frontal cortex of patients with HAND. In addition, treatment of primary rat neuroglial cultures with HIV M/M led to NMDA receptor- and calpain-dependent degradation of MDMx and decreased neuronal survival, while overexpression of MDMx conferred partial protection from HIV M/M toxicity in vitro. Further, our results demonstrate that MDMx is a novel and direct calpain substrate. Finally, blocking MDMx activity led to neuronal death in vitro in the absence of toxic stimulus, which was reversed by calpain inhibition. Overall, our results indicate that MDMx plays a pro-survival role in neurons, and that strategies to stabilize and/or induce MDMx can provide neuroprotection in HAND and in other neurodegenerative diseases where calpain activation contributes to neuropathogenesis.

Persistence of HIV-Associated Neurocognitive Disorders in the Era of Antiretroviral Therapy

HIV-Associated Neurocognitive Disorders (HAND) is a serious menifestation of HIV in‐ fection in the central nervous system (CNS), and encompassess a wide spectrum of cog‐ nitive, behavioral, and motor deficits [1-3]. While the implementation of combination antiretroviral therapy (ART) has dramatically increased the life expectancy and led to significant improvements in the clinical presentation and progression of HAND, an esti‐ mated 50% of HIV-infected patientscontinue to suffer from implications of HAND in the ART era, with as much as 20% of these exhibiting symptoms of HIV-associated dementia (HAD), the most severe form of HAND [3-6]. The brain regions affected in patients with HAND has changed in the ART era in parallel to the changes observed in the clinical picture; a more subtle and insidious cortical damage mainly in the hippocampus and the temporal cortex is observed, in contrast to the overt subcortical damage seen before ART [7-10]. The underlying causes of these changes are not fully elucidated; however, exami‐ nation of the post-mortem tissue reveals the persistence of synaptic and dendritic dam‐ age in the affected brain regions [3, 11]. Emerging evidence suggests that controlling viral replication in the periphery or in the CNS may not be sufficient to control the un‐ derlying neuropathological processes that culminate in the development of HAND. The impact of HAND on the quality of life, adherence to drug regimens, and co-morbidities especially in an aging HIV population with decreased cognitive reserves, is of major con‐ cern. In this chapter, we will examine the major factors that continue to impact the CNS of HIV-infected individuals, and introduce new challenges in the successful treatment of HAND.

Antiretroviral Therapy and HIV-Associated Neurocognitive Disorders

Human immunodeficiency virus (HIV)-Associated Neurocognitive Disorders (HAND) define a wide spectrum of behavioral, cognitive and motor dysfunctions(Heaton, Clifford et al. ; Neuenburg, Brodt et al. 2002; McArthur 2004; Lindl, Marks et al. 2010). The initial criteria put forth by the American Association of Neurology described two levels of neurocognitive impairment in HIV (+) patients: HIV-associated dementia (HAD) and minor motor cognitive disorder (MCMD)(1996). However, the emergence of more subtle forms of cognitive impairment after the implementation of combination antiretroviral therapy (cART) in 1996 necessitated a revised classification, which now includes, in addition to HAD, asymptomatic neurocognitive impairment (ANI), and mild neurocognitive disorder (MND), while MCMD is no longer included in the classification (Antinori, Arendt et al. 2007). While the incidence of HAD has declined in the cART era, from 10-15% to approximately 2%; ANI and MND have become more prevalent (Heaton, Clifford et al. 2010). Overall, HAND continues to persist, affecting approximately 50% of HIV (+) patients in the cART era (Power, Boisse et al. 2009; Heaton, Clifford et al. 2010). The underlying neuropathology suggests a change from overt dementia to a more insidious neurocognitive impairment (Kusdra, McGuire et al. 2002; Anthony and Bell 2008; Everall, Vaida et al. 2009). Various factors, such as age-related vascular and metabolic changes and substance and alcohol abuse, the emergence of resistant virus species, persistent viral DNA in the central nervous system (CNS) despite successful plasma viral control, limited access of cART into the CNS, poor adherence to drug regimen, and possible cART-related neurotoxicities are suggested to contribute to these changes observed in the clinical and neuropathological presentation in the HAND brain. With the increased life expectancy, the ever-expanding repertoire of antiretroviral drugs (ARVs) and the patient-tailored, dynamic combination ARV regimens, the contribution of cART to the persistence of HAND is an urgent question that needs to be addressed to more successfully predict and prevent CNS related morbidity and mortality among HIV-infected patients.

The Stress Response in Injured Afferents of the Capsular Ligament Depends on Tensile Loading Applied to the Facet Joint

Chronic neck pain from whiplash affects over 10 million people annually in the United States [1]. Many studies have indentified the facet joint as a possible source of neck pain due to its innervation by nociceptive fibers [2]. In particular, in vivo studies have reported that distraction of the facet joint and its capsule produces both persistent firing of capsule pain fibers and persistent behavioral hypersensitivity and pain symptoms (i.e. mechanical allodynia) [2,3].Copyright