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

Abstract

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 also demonstrated 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.

Caspase-dependent degradation of MDMx/MDM4 cell cycle regulatory protein in amyloid β-induced neuronal damage

“…A previous study observed a small, but significant increase in MDMx mRNA in patients with AD, but this change did not associate with changes in plaque density or Braak staging [55]. Our results suggest that MDMx regulation may be occurring primarily at the protein level in the CNS, as we and others have reported previously [42, 43]. The reductions in protein expression and increases in mRNA levels suggest that a compensatory mechanism may be at play.…”

“…The mediator of MDMx degradation is dependent on the insult and the class of proteases subsequently activated by that stress, including caspases, calpains and the proteasome, as shown previously in neurons and in dividing cells [42, 43, 52]. Caspase and calpain activation are not mutually exclusive mechanisms leading to neuronal death, and our results suggest that MDMx degradation may be one point of crosstalk between these two pathways, as MDMx loss is associated with calpain-dependent neuronal damage [43, 53]. …”

“…Formalin-fixed/paraffin embedded tissue sections that are 6 μm thick were used to detect MDMx and ChAT expression using immunofluorescent detection, as described previously [43, 47]. Deparaffinization was performed using histoclear, endogenous peroxidase activity was blocked with 3% H 2 O 2 in methanol and antigen unmasking was achieved with target retrieval solution at 95°C for 1 h. Sections were blocked with 10% normal goat serum in PBS, and primary antibodies at empirically defined dilutions (ChAT: 1:50 or MDMx: 1:1,000, MAP2: 1:1,000, DAPI: 1:4,000) were incubated overnight at 4°C.…”

“…In addition, MDMx knockdown precipitates neuronal death implicating MDMx as a necessary factor for neuronal survival [42]. Finally, we have previously shown that MDMx degradation in response to calpain activation was contributing to neuronal death in an in vitro model of HIV-associated neurotoxicity [43]. …”

Measuring Inequalities in Gene Co-expression Networks of HIV-1 Infection Using the Lorenz Curve and Gini Coefficient

“…MDM4 is another representative example showing a positive and high-ranked delta Gini (value=0.36; rank=22), but a low-ranked dC (value=0.92; rank=235). This gene was recently demonstrated to be a direct calpain substrate playing roles in the HIV-induced neuronal damage [16]. The detailed values of delta Gini and dC for all comparisons of GCNs of HIV-1 infection were listed in Supplemental Table 1.…”