mTOR activation due to APPL1 deficiency exacerbates hyperalgesia via Rab5/Akt and AMPK signaling pathway in streptozocin-induced diabetic rats

Painful diabetic neuropathy is a common complication of diabetes mellitus with obscure underlying mechanisms. The adaptor protein APPL1 is critical in mediating the insulin sensitizing and insulin signaling. In neurons, APPL1 reportedly affects synaptic plasticity, while its role in the pathogenesis of painful diabetic neuropathy is masked. Our Western blotting revealed significantly decreased APPL1 expression in the dorsal horn in streptozocin-induced rats versus the control rats, coupled with concomitant mechanical and thermal hyperalgesia. Afterward, the determination of exact localization of APPL1 in spinal cord by immunofluorescent staining assay revealed highly expressed APPL1 in the lamina of spinal dorsal horn in control rats, with the overexpression in neurons, microglia, and underexpression in astrocytes. The APPL1 expression in laminae I and II was significantly downregulated in painful diabetic neuropathy rats. In addition, APPL1 deficiency or overexpression contributed to the increase or decrease of Map and Bassoon, respectively. The localization and immunoactivity of APPL1 and mammalian target of rapamycin (mTOR) were determined in spinal dorsal horn in painful diabetic neuropathy rats and control rats by immunohistochemistry, suggesting pronounced decrease in APPL1 expression in the superficial layer of the spinal cord in painful diabetic neuropathy rats, with p-mTOR expression markedly augmented. APPL1 knockdown by infection with lentiviral vector facilitated the activation of mTOR and abrogated mechanical withdrawal threshold values in painful diabetic neuropathy rats. Genetically overexpressed APPL1 significantly eliminated the activation of mTOR and resulted in the augmented mechanical withdrawal threshold values and thermal withdrawal latency values. Furthermore, the APPL1 levels affect phosphorylation of adenosine monophosphate-activated protein kinase (AMPK), and Akt, as well as the small GTPase, Rab5 expression in painful diabetic neuropathy rats. Our results uncovered a novel mechanism by which APPL1 deficiency facilitates the mTOR activation and thus exacerbates the hyperalgesia in streptozocin-induced diabetic rats, presumably via the regulation of Rab5/Akt and AMPK signaling pathway.