New therapeutic and biomarker discovery for peripheral diabetic neuropathy: PARP inhibitor, nitrotyrosine, and tumor necrosis factor-{alpha}

Abstract

This study evaluated poly(ADP-ribose) polymerase (PARP) inhibition as a new therapeutic approach for peripheral diabetic neuropathy using clinically relevant animal model and endpoints, and nitrotyrosine (NT), TNF-alpha, and nitrite/nitrate as potential biomarkers of the disease. Control and streptozotocin-diabetic rats were maintained with or without treatment with orally active PARP inhibitor 10-(4-methyl-piperazin-1-ylmethyl)-2H-7-oxa-1,2-diaza-benzo[de]anthracen-3-one (GPI-15,427), 30 mg kg(-1) d(-1), for 10 wk after first 2 wk without treatment. Therapeutic efficacy was evaluated by poly(ADP-ribosyl)ated protein expression (Western blot analysis), motor and sensory nerve conduction velocities, and tibial nerve morphometry. Sciatic nerve and spinal cord NT, TNF-alpha, and nitrite/nitrate concentrations were measured by ELISA. NT localization in peripheral nervous system was evaluated by double-label fluorescent immunohistochemistry. A PARP inhibitor treatment counteracted diabetes-induced motor and sensory nerve conduction slowing, axonal atrophy of large myelinated fibers, and increase in sciatic nerve and spinal cord NT and TNF-alpha concentrations. Sciatic nerve NT and TNF-alpha concentrations inversely correlated with motor and sensory nerve conduction velocities and myelin thickness, whereas nitrite/nitrate concentrations were indistinguishable between control and diabetic groups. NT accumulation was identified in endothelial and Schwann cells of the peripheral nerve, neurons, astrocytes, and oligodendrocytes of the spinal cord, and neurons and glial cells of the dorsal root ganglia. The findings identify PARP as a compelling drug target for prevention and treatment of both functional and structural manifestations of peripheral diabetic neuropathy and provide rationale for detailed evaluation of NT and TNF-alpha as potential biomarkers of its presence, severity, and progression.

Figure 1

Representative Western blot analyses of sciatic nerve (panel A) and spinal cord (panel C) poly(ADP-ribosyl)ated proteins, and poly(ADP-ribosyl)ated protein content (densitometry, panels B and D), in control (C) and diabetic (D) rats maintained with or without PARP inhibitor treatment. GPI, GPI-15,427. Mean ± sem, n = 8–12 per group. **, P < 0.01 vs. control group; ##, P < 0.01 vs. untreated diabetic group.

Figure 2

Representative microphotographs of immunostaining of diabetic rat sciatic nerve for (A) NT (left); vascular endothelium (center), NT and vascular endothelium (right), and (B) NT (left); S-100 (center), NT and S-100 (right). Magnification, ×125.

Figure 3

A, Representative microphotographs of immunofluorescent staining of ventral horn motor neurons of diabetic rat spinal cord for NT (left); the DNA-binding, neuron-specific protein NeuN (center), NT and NeuN (right), Magnification, ×100. B and C, Representative microphotographs of immunofluorescent staining of white matter of diabetic rat spinal cord for (B) NT (left); the oligodendrocyte marker adenomatous polyposis coli (APC)-Ab7 (center), NT and adenomatous polyposis coli-Ab7 (right), and (C) NT (left); the astrocyte marker GFAP (center), NT and GFAP (right). Magnification, ×125.

Figure 4

Representative microphotographs of fluorescent immunostaining of diabetic rat DRG for NT (left); GS (center), NT and GS (right). Magnification, ×80.

Figure 5

NT concentrations in sciatic nerve (A) and spinal cord (B) in control (C) and diabetic (D) rats treated with and without GPI-15,427. Mean ± sem, n = 7–12 per group. * and **, P < 0.05 and <0.01 vs. controls; # and ##, P < 0.05 and <0.01 vs. untreated diabetic group.

Figure 6

TNFα concentrations in sciatic nerve (A) and spinal cord (B) in control (C) and diabetic (D) rats treated with and without GPI-15,427. Mean ± sem, n = 5–8 per group. * and **, P < 0.05 and <0.01 vs. controls; # and ##, P < 0.05 and <0.01 vs. untreated diabetic group.