Rab7 Silencing Prevents μ-Opioid Receptor Lysosomal Targeting and Rescues Opioid Responsiveness to Strengthen Diabetic Neuropathic Pain Therapy

Abstract

Painful diabetic neuropathy is poorly controlled by analgesics and requires high doses of opioids, triggering side effects and reducing patient quality of life. This study investigated whether enhanced Rab7-mediated lysosomal targeting of peripheral sensory neuron μ-opioid receptors (MORs) is responsible for diminished opioid responsiveness in rats with streptozotocin-induced diabetes. In diabetic animals, significantly impaired peripheral opioid analgesia was associated with a loss in sensory neuron MOR and a reduction in functional MOR G-protein-coupling. In control animals, MORs were retained mainly on the neuronal cell membrane. In contrast, in diabetic rats, they were colocalized with upregulated Rab7 in LampI-positive perinuclear lysosome compartments. Silencing endogenous Rab7 with intrathecal Rab7-siRNA or, indirectly, by reversing nerve growth factor deprivation in peripheral sensory neurons not only prevented MOR targeting to lysosomes, restoring their plasma membrane density, but also rescued opioid responsiveness toward better pain relief. These findings elucidate in vivo the mechanisms by which enhanced Rab7 lysosomal targeting of MORs leads to a loss in opioid antinociception in diabetic neuropathic pain. This is in contrast to peripheral sensory neuron MOR upregulation and antinociception in inflammatory pain, and provides intriguing evidence that regulation of opioid responsiveness varies as a function of pain pathogenesis.

Evidence for MOR on cell membrane, sarcoplasmatic reticulum and mitochondria in left ventricular myocardium in rats

“…Western blot experiments were performed as three independent experiments in duplicate. The integrated optical density (IOD) was calculated by use of Java Image processing and analysis software (Image J, NIH) as described previously [39].…”

Cellular localization and adaptive changes of the cardiac delta opioid receptor system in an experimental model of heart failure in rats

“…After incubation with the secondary antibody (peroxidase-conjugated anti-rabbit, 1:20.000, Jackson ImmunoResearch for DOR and peroxidase-conjugated anti-mouse, 1:7.500, Abcam for PENK) for 2 h at room temperature, reactive bands were visualized after incubation of the membrane in ECL solutions (Amersham) for 1 min and by immediate exposure to autoradiographic film for 5-20 min. The integrated optical density (OID) was calculated using Java Image processing and analysis software (Image J, NIH) as described previously [30].…”

“…Finally, the tissues were washed in PBS, mounted on vectashield (Vector Laboratories), and viewed under a Zeiss LSM 510 laser scanning microscope (Carl Zeiss, Göttingen, Germany). The integrated optical density (OID) was calculated by use of Java Image processing and analysis software (Image J, NIH) as described previously [30].…”

Upregulation of the kappa opioidergic system in left ventricular rat myocardium in response to volume overload

“…Finally, the tissue sections were washed in PBS, mounted on vectashield (Vector Laboratories) and viewed under a Zeiss LSM 510 laser-scanning microscope (Carl Zeiss, Göttingen, Germany). The integrated optical density (IOD) was calculated by use of Java Image processing and analysis software (Image J, NIH) as described previously [23].…”

“…Western blot experiments were performed as three independent experiments in duplicate. The integrated optical density (IOD) was calculated by use of Java Image processing and analysis software (Image J, NIH) as described previously [23].…”