Nucleus Accumbens AMPA Receptors Are Necessary for Morphine-Withdrawal-Induced Negative-Affective States in Rats.

UNLABELLED: Dependence is a hallmark feature of opiate addiction and is defined by the emergence of somatic and affective withdrawal signs. The nucleus accumbens (NAc) integrates dopaminergic and glutamatergic inputs to mediate rewarding and aversive properties of opiates. Evidence suggests that AMPA glutamate-receptor-dependent synaptic plasticity within the NAc underlies aspects of addiction. However, the degree to which NAc AMPA receptors (AMPARs) contribute to somatic and affective signs of opiate withdrawal is not fully understood. Here, we show that microinjection of the AMPAR antagonist NBQX into the NAc shell of morphine-dependent rats prevented naloxone-induced conditioned place aversions and decreases in sensitivity to brain stimulation reward, but had no effect on somatic withdrawal signs. Using a protein cross-linking approach, we found that the surface/intracellular ratio of NAc GluA1, but not GluA2, increased with morphine treatment, suggesting postsynaptic insertion of GluA2-lacking AMPARs. Consistent with this, 1-naphthylacetyl spermine trihydrochloride (NASPM), an antagonist of GluA2-lacking AMPARs, attenuated naloxone-induced decreases in sensitivity to brain stimulation reward. Naloxone decreased the surface/intracellular ratio and synaptosomal membrane levels of NAc GluA1 in morphine-dependent rats, suggesting a compensatory removal of AMPARs from synaptic zones. Together, these findings indicate that chronic morphine increases synaptic availability of GluA1-containing AMPARs in the NAc, which is necessary for triggering negative-affective states in response to naloxone. This is broadly consistent with the hypothesis that activation of NAc neurons produces acute aversive states and raises the possibility that inhibiting AMPA transmission selectively in the NAc may have therapeutic value in the treatment of addiction. SIGNIFICANCE STATEMENT: Morphine dependence and withdrawal result in profound negative-affective states that play a major role in the maintenance of addiction. However, the underlying neurobiological mechanisms are not fully understood. We use a rat model of morphine dependence to show that GluA1 subunits of AMPA glutamate receptors in the nucleus accumbens (NAc), a brain region critical for modulating affective states, are necessary for aversive effects of morphine withdrawal. Using biochemical methods in NAc tissue, we show that morphine dependence increases cell surface expression of GluA1, suggesting that neurons in this area are primed for increased AMPA receptor activation upon withdrawal. This work is important because it suggests that targeting AMPA receptor trafficking and activation could provide novel targets for addiction treatment.

C-RAF mutations confer resistance to RAF inhibitors.

Melanomas that contain B-RAF(V600E) mutations respond transiently to RAF and MEK inhibitors; however, resistance to these agents remains a formidable challenge. Although B- or C-RAF dysregulation represents prominent resistance mechanisms, resistance-associated point mutations in RAF oncoproteins are surprisingly rare. To gain insights herein, we conducted random mutagenesis screens to identify B- or C-RAF mutations that confer resistance to RAF inhibitors. Whereas bona fide B-RAF(V600E) resistance alleles were rarely observed, we identified multiple C-RAF mutations that produced biochemical and pharmacologic resistance. Potent C-RAF resistance alleles localized to a 14-3-3 consensus binding site or a separate site within the P loop. These mutations elicited paradoxical upregulation of RAF kinase activity in a dimerization-dependent manner following exposure to RAF inhibitors. Knowledge of resistance-associated C-RAF mutations may enhance biochemical understanding of RAF-dependent signaling, anticipate clinical resistance to novel RAF inhibitors, and guide the design of "next-generation" inhibitors for deployment in RAF- or RAS-driven malignancies.

Early measures of cognitive function predict survival in patients with newly diagnosed glioblastoma.

Cognitive dysfunction is a common manifestation of primary brain tumors. We evaluated the association between early cognitive dysfunction and prognosis in a cohort of patients with newly diagnosed glioblastoma. Ninety-one patients who completed neuropsychological assessment after tumor resection but before further treatment were identified in the MD Anderson Neuropsychology database. The relationship between performance on cognitive testing and survival was evaluated using not only Cox proportional hazards models that included clinical factors such as age and KPS but also the Kaplan-Meier method. Median survival time from surgery was 20.7 months. Rates of impairment on cognitive testing ranged from 7.1% for Similarities, to 60.0% for Hopkins Verbal Learning Test-Revised Total Recall. As continuous variables, the Clinical Trial Battery Composite, Trail Making Test Part B, and Controlled Oral Word Association test were associated with survival. Impairment on the Trail Making Test Part B, Controlled Oral Word Association, Similarities, and Digit Span were associated with mortality. Kaplan-Meier analysis demonstrated the survival impact of these tests on the group as a whole and in select patient subgroups defined by classification by the Radiation Therapy Oncology Group (RTOG) Recursive Partitioning Analysis (RPA). Cognitive impairment as measured by specific neuropsychological tests is independently associated with poor prognosis in patients with newly diagnosed glioblastoma, and this effect remains significant even within patient subgroups defined by RTOG RPA class. Executive function and attention are the cognitive domains most closely associated with prognosis in this analysis.

MEK1 mutations confer resistance to MEK and B-RAF inhibition.

Genetic alterations that activate the mitogen-activated protein kinase (MAP kinase) pathway occur commonly in cancer. For example, the majority of melanomas harbor mutations in the BRAF oncogene, which are predicted to confer enhanced sensitivity to pharmacologic MAP kinase inhibition (e.g., RAF or MEK inhibitors). We investigated the clinical relevance of MEK dependency in melanoma by massively parallel sequencing of resistant clones generated from a MEK1 random mutagenesis screen in vitro, as well as tumors obtained from relapsed patients following treatment with AZD6244, an allosteric MEK inhibitor. Most mutations conferring resistance to MEK inhibition in vitro populated the allosteric drug binding pocket or alpha-helix C and showed robust ( approximately 100-fold) resistance to allosteric MEK inhibition. Other mutations affected MEK1 codons located within or abutting the N-terminal negative regulatory helix (helix A), which also undergo gain-of-function germline mutations in cardio-facio-cutaneous (CFC) syndrome. One such mutation, MEK1(P124L), was identified in a resistant metastatic focus that emerged in a melanoma patient treated with AZD6244. Both MEK1(P124L) and MEK1(Q56P), which disrupts helix A, conferred cross-resistance to PLX4720, a selective B-RAF inhibitor. However, exposing BRAF-mutant melanoma cells to AZD6244 and PLX4720 in combination prevented emergence of resistant clones. These results affirm the importance of MEK dependency in BRAF-mutant melanoma and suggest novel mechanisms of resistance to MEK and B-RAF inhibitors that may have important clinical implications.

Anatomically dissociable effects of dopamine D1 receptor agonists on reward and relief of withdrawal in morphine-dependent rats.

RATIONALE: Chronic opiate administration induces neuroadaptations within the nucleus accumbens (NAc) and ventral tegmental area (VTA) that can contribute to dependence. We have shown that morphine dependence shifts the behavioral consequences of D1 dopamine (DA) receptor signaling: systemic administration of a D1 receptor agonist is rewarding and blocks naloxone-precipitated withdrawal signs in morphine-dependent rats, but has minimal effects in nondependent rats. These data suggest that D1 receptors acquire the ability to regulate reward and withdrawal in morphine-dependent rats. The brain regions involved in these effects are not known. OBJECTIVE: Studies were designed to test the hypothesis that the nucleus accumbens shell (NASh) and the ventral tegmental area (VTA) are important sites for mediating the behavioral effects of D1 receptor activation in morphine-dependent rats. MATERIALS AND METHODS: The effects of microinjecting the D1 receptor agonist SKF 82958 into the NASh or the VTA on place conditioning and somatic withdrawal signs were studied in morphine-dependent and nondependent rats. RESULTS: Intra-NASh microinjection of SKF 82958 (1 microg/side) established conditioned place preferences in morphine-dependent but not nondependent rats, but had no effect on naloxone-induced place aversions or somatic withdrawal signs. Intra-VTA microinjection of SKF 82958 (2 microg) did not establish place preferences under any conditions, but blocked naloxone-induced place aversions without effects on somatic withdrawal signs. CONCLUSIONS: There is an anatomical dissociation between D1 receptor-mediated reward and relief of withdrawal in morphine-dependent rats. When combined, the individual effects of D1 receptor activation in the NASh and VTA on the affective signs of precipitated morphine withdrawal resemble those seen with systemic administration.