Usp9X Regulates Cell Death in Malignant Peripheral Nerve Sheath Tumors.

Malignant peripheral nerve sheath tumors (MPNSTs) are the leading cause of death in neurofibromatosis type 1 (NF1) patients. Current treatment modalities have been largely unsuccessful in improving MPNST patient survival, making the identification of new therapeutic targets urgent. In this study, we found that interference with Usp9X, a deubiquitinating enzyme which is overexpressed in nervous system tumors, or Mcl-1, an anti-apoptotic member of the Bcl-2 family whose degradation is regulated by Usp9X, causes rapid death in human MPNST cell lines. Although both Usp9X and Mcl-1 knockdown elicited some features of apoptosis, broad spectrum caspase inhibition was ineffective in preventing knockdown-induced MPNST cell death suggesting that caspase-independent death pathways were also activated. Ultrastructural examination of MPNST cells following either Usp9X interference or pharmacological inhibition showed extensive cytoplasmic vacuolization and swelling of endoplasmic reticulum (ER) and mitochondria most consistent with paraptotic cell death. Finally, the Usp9X pharmacological inhibitor WP1130 significantly reduced human MPNST growth and induced tumor cell death in an in vivo xenograft model. In total, these findings indicate that Usp9X and Mcl-1 play significant roles in maintaining human MPNST cell viability and that pharmacological inhibition of Usp9X deubiquitinase activity could be a therapeutic target for MPNST treatment.

The volatile anesthetic methoxyflurane protects motoneurons against excitotoxicity in an in vitro model of rat spinal cord injury.

Neuroprotection of the spinal cord during the early phase of injury is an important goal to determine a favorable outcome by prevention of delayed pathological events, including excitotoxicity, which otherwise extend the primary damage and amplify the often irreversible loss of motor function. While intensive care and neurosurgical intervention are important treatments, effective neuroprotection requires further experimental studies focused to target vulnerable neurons, particularly motoneurons. The present investigation examined whether the volatile general anesthetic methoxyflurane might protect spinal locomotor networks from kainate-evoked excitotoxicity using an in vitro rat spinal cord preparation as a model. The protocols involved 1h excitotoxic stimulation on day 1 followed by electrophysiological and immunohistochemical testing on day 2. A single administration of methoxyflurane applied together with kainate (1h), or 30 or even 60 min later prevented any depression of spinal reflexes, loss of motoneuron excitability, and histological damage. Methoxyflurane per se temporarily decreased synaptic transmission and motoneuron excitability, effects readily reversible on washout. Spinal locomotor activity recorded as alternating electrical discharges from lumbar motor pools was fully preserved on the second day after application of methoxyflurane together with (or after) kainate. These data suggest that a volatile general anesthetic could provide strong electrophysiological and histological neuroprotection that enabled expression of locomotor network activity 1 day after the excitotoxic challenge. It is hypothesized that the benefits of early neurosurgery for acute spinal cord injury (SCI) might be enhanced if, in addition to injury decompression and stabilization, the protective role of general anesthesia is exploited.

Mechanisms underlying cell death in ischemia-like damage to the rat spinal cord in vitro.

New spinal cord injury (SCI) cases are frequently due to non-traumatic causes, including vascular disorders. To develop mechanism-based neuroprotective strategies for acute SCI requires full understanding of the early pathophysiological changes to prevent disability and paralysis. The aim of our study was to identify the molecular and cellular mechanisms of cell death triggered by a pathological medium (PM) mimicking ischemia in the rat spinal cord in vitro. We previously showed that extracellular Mg(2+) (1 mM) worsened PM-induced damage and inhibited locomotor function. The present study indicated that 1 h of PM+Mg(2+) application induced delayed pyknosis chiefly in the spinal white matter via overactivation of poly (ADP-ribose) polymerase 1 (PARP1), suggesting cell death mediated by the process of parthanatos that was largely suppressed by pharmacological block of PARP-1. Gray matter damage was less intense and concentrated in dorsal horn neurons and motoneurons that became immunoreactive for the mitochondrial apoptosis-inducing factor (the intracellular effector of parthanatos) translocated into the nucleus to induce chromatin condensation and DNA fragmentation. Immunoreactivity to TRPM ion channels believed to be involved in ischemic brain damage was also investigated. TRPM2 channel expression was enhanced 24 h later in dorsal horn and motoneurons, whereas TRPM7 channel expression concomitantly decreased. Conversely, TRPM7 expression was found earlier (3 h) in white matter cells, whereas TRPM2 remained undetectable. Simulating acute ischemic-like damage in vitro in the presence of Mg(2+) showed how, during the first 24 h, this divalent cation unveiled differential vulnerability of white matter cells and motoneurons, with distinct changes in their TRPM expression.