A systems view of G protein-coupled receptor (GPCR) signaling in its native environment is central to the development of GPCR therapeutics with fewer side effects. Using the kappa opioid receptor (KOR) as a model, we employed high-throughput phosphoproteomics to investigate signaling induced by structurally diverse agonists in five mouse brain regions. Quantification of 50,000 different phosphosites provided a systems view of KOR in vivo signaling, revealing novel mechanisms of drug action. Thus, we discovered enrichment of the mechanistic target of rapamycin (mTOR) pathway by U-50,488H, an agonist causing aversion, which is a typical KOR-mediated side effect. Consequently, mTOR inhibition during KOR activation abolished aversion while preserving beneficial antinociceptive and anticonvulsant effects. Our results establish high-throughput phosphoproteomics as a general strategy to investigate GPCR in vivo signaling, enabling prediction and modulation of behavioral outcomes.
Mitochondrial dysfunction and oxidative stress are strongly implicated in neurodegenerative diseases and epilepsy. Strikingly, neurodegenerative diseases show regional specificity in vulnerability and follow distinct patterns of neuronal loss. A challenge is to understand, why mitochondria fail in particular brain regions under specific pathological conditions. A potential explanation could be provided by regional or cellular specificity of mitochondrial function. We applied high-resolution respirometry to analyze the integrated Complex I- and II (CI and CII)-linked respiration, the activity of Complex IV, and the combined CI&II-linked oxidative phosphorylation (OXPHOS)- and electron-transfer system (ETS)-capacity in microsamples obtained from distinct regions of the mouse brain. We compared different approaches to assess mitochondrial density and suggest flux control ratios as a valid method to normalize respiration to mitochondrial density. This approach revealed significant differences of CI- and CII-linked OXPHOS capacity and coupling control between motor cortex, striatum, hippocampus and pons of naïve mice. CI-linked respiration was highest in motor cortex, while CII-linked respiration predominated in the striatum. To investigate if this method could also determine differences in normal and disease states within the same brain region, we compared hippocampal homogenates in a chronic epilepsy model. Three weeks after stereotaxic injection of kainate, there was a down-regulation of CI- and upregulation of CII-linked respiration in the resulting epileptic ipsilateral hippocampus compared to the contralateral one. In summary, respirometric OXPHOS analysis provides a very sensitive diagnostic approach using small amounts of distinct brain tissues. In a single assay, information is obtained on numerous OXPHOS parameters as indicators of tissue-specific mitochondrial performance.
Focal epilepsy represents one of the most common chronic CNS diseases. The high incidence of drug resistance, devastating comorbidities, and insufficient responsiveness to surgery pose unmet medical challenges. In the quest of novel, disease‐modifying treatment strategies of neuropeptides represent promising candidates. Here, we provide the “proof of concept” that gene therapy by adeno‐associated virus (AAV) vector transduction of preprodynorphin into the epileptogenic focus of well‐accepted mouse and rat models for temporal lobe epilepsy leads to suppression of seizures over months. The debilitating long‐term decline of spatial learning and memory is prevented. In human hippocampal slices obtained from epilepsy surgery, dynorphins suppressed seizure‐like activity, suggestive of a high potential for clinical translation. AAV‐delivered preprodynorphin expression is focally and neuronally restricted and release is dependent on high‐frequency stimulation, as it occurs at the onset of seizures. The novel format of “release on demand” dynorphin delivery is viewed as a key to prevent habituation and to minimize the risk of adverse effects, leading to long‐term suppression of seizures and of their devastating sequel.
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