Diabetic peripheral neuropathy is a major debilitating late complication of diabetes, which significantly reduces the quality of life in patients. Diabetic peripheral neuropathy is associated with a wide spectrum of sensory abnormalities, where in loss of sensation or hypoalgesia to applied external stimuli is paradoxically accompanied by debilitating tonic spontaneous pain. In numerous studies on animal models of diabetic peripheral neuropathy, behavioural measurements have been largely confined to analysis of evoked withdrawal to mechanical and thermal stimuli applied to dermatomes, whereas spontaneous, on-going pain has not been widely studied. In the Streptozotocin model of type 1 diabetes, we employed the Conditioned Place Preference test to assess tonic pain. Our results indicate that both phases, that is, early evoked hypersensitivity (i.e. 5–7 weeks post-Streptozotocin) as well as late stage hypoalgesia (i.e. 17–20 weeks post-Streptozotocin) are accompanied by significant tonic pain in mice with diabetic peripheral neuropathy. We also report on the temporal relation between on-going pain and neuropathological changes in the dorsal root ganglia of mice with diabetic peripheral neuropathy up to 6 months post-Streptozotocin. Neither early hypersensitivity nor late hypoalgesia were associated with markers of cellular stress in the dorsal root ganglia. Whereas significant neutrophil infiltration was observed in the dorsal root ganglia over both early and late stages post-Streptozotocin, T-cell infiltration in the dorsal root ganglia was prominent at late stages post-Streptozotocin. Thus, longitudinal analyses reveal that similar to patients with chronic diabetic peripheral neuropathy, mice show tonic pain despite sensory loss after several months in the Streptozotocin model, which is accompanied by neuroimmune interactions in the dorsal root ganglia.

Objective: Diabetic-induced peripheral neuropathy (DPN) is a highly complex and frequent diabetic late complication, which is manifested by prolonged hyperglycemia. However, the molecular mechanisms underlying the pathophysiology of nerve damage and sensory loss remain largely unclear. Recently, alteration in metabolic flux have gained attention a basis for organ damage in diabetes; however, peripheral sensory neurons have not been adequately analyzed with respect to metabolic dysfunction. In the present study, we attempted to delineate the sequence of event occurring in alteration of metabolic pathways in relation to nerve damage and sensory loss.Methods: C57Bl6/j wild type mice were analyzed longitudinally up to 22 weeks (wks) in the streptozotocin (STZ) model of type1 diabetes. The progression of DPN was investigated by behavioral measurements of sensitivity to thermal and mechanical stimuli and quantitative morphological assessment of intraepidermal nerve fiber density. We employed a mass spectrometry-based screen to address alterations in levels of metabolites in peripheral sciatic nerve and amino acids in serum over several months post-STZ administration to elucidate metabolic dysfunction longitudinally in relation to sensory dysfunction.Results: Although hyperglycemia and body weight changes occurred early, sensory loss and reduced intraepithelial branching of nociceptive nerves was only evident at 22 wks post-STZ. The longitudinal metabolites screen in peripheral nerves demonstrated that compared to buffer-injected age-matched control mice, mice at 12 wks and 22 wks post-STZ showed an early impairment the tricaoboxylic acid (TCA cycle), which is the main pathway of carbohydrate metabolism leading to energy generation. We found that levels of citric acid, ketoglutaric acid (2 KG), succinic acid, fumaric acid and malic acid were observed to be significantly reduced in sciatic nerve at 22 wks post-STZ. In addition, we also found the increase in levels of sorbitol and L-Lactate in peripheral nerve from 12 wks post-STZ injection. Amino acid screen in serum showed that the amino acids Valine (Val), Isoleucine (Ile) and Leucine (Leu), grouped together as BCAA, increased more than 2-fold from 12 wks post-STZ. Similarly, the levels of Tyrosine (Tyr), Asparagine (Asn), Serine (Ser), Histidine (His), Alanine (Ala), and Proline (Pro) showed progressive increase with progression of diabetes. Conclusion:Our results indicate that the impaired TCA cycle metabolites in peripheral nerve is the primary cause of shunting metabolic substrate to compensatory pathways which leads to mitochondrial dysfunction and nerve damage.

Highlights d Post-translational SUMOylation critically protects sensory neuron function d SUMOylation regulates bioenergetic enzymes and controls toxic metabolites d SUMOylation functionally regulates the nociceptive ion channel TRPV1 d De-SUMOylation accelerates progression of diabetic neuropathy

Diabetic peripheral neuropathy (DPN) is one of the most common diabetic complications.Mechanisms underlying nerve damage and sensory loss following metabolic dysfunction remain large unclear. Recently, hyperglycemia-induced mitochondrial dysfunction and the generation of ROS have gained attention as possible mechanisms of organ damage in diabetes. Hypoxia-inducible factor 1 (HIF1) is a key transcription factor activated by hypoxia, hyperglycemia, nitric oxide as well as ROS, suggesting a fundamental role in DPN susceptibility. Genetically-modified mutant mice, which conditionally lack HIF1 in peripheral sensory neurons (SNS-HIF1α -/-), were analyzed longitudinally up to 6 months in the streptozotocin (STZ) model of type1 diabetes. Behavioral measurements of sensitivity to thermal and mechanical stimuli, quantitative morphological analyses of intraepidermal nerve fiber density and measurements of reactive oxygen species (ROS) in sensory neurons in vivo were undertaken over several months post-STZ injections to delineate the role of HIF1 in DPN. Longitudinal behavioral and morphological analyses at 5, 13 and 24 weeks post-STZ treatment revealed that SNS-HIF1α -/developed stronger hyperglycemia-evoked losses of peripheral nociceptive sensory axons associated with stronger losses of mechano-and heat sensation with a faster onset than HIF1α fl/fl mice. Mechanistically, these histomorphologic and behavioral differences were associated with significantly higher level of STZ-induced production of ROS in sensory neurons of SNS-HIF1α -/mice as compared with HIF1α fl/fl . Our results indicate that HIF1 is as an upstream modulator of ROS in peripheral sensory neurons and exerts a protective function in suppressing hyperglycemiainduced nerve damage by limiting ROS levels. HIF1 stabilization may be thus a new strategy target for limiting sensory loss, a debilitating late complication of diabetes. KEY MESSAGES Impaired Hypoxia-inducible factor 1α (HIF1α) signaling leads to early onset of STZinduced loss of sensation in mice  STZ-induced loss of sensation in HIF1α is associated with loss of sensory nerve fiber in skin. Activation of HIF1α signaling in diabetic mice protects the sensory neurons by limiting ROS formation generated due to mitochondrial dysfunction.

Fragment-based drug discovery (FBDD) permits efficient sampling of the vast chemical space for hit identification. Libraries are screened biophysically and fragment:protein costructures are determined by X-ray crystallography. In parallel, computational methods can derive pharmacophore models or screen virtual libraries. We screened 15 very small fragments (VSFs) (HA ≤ 11) computationally, using site identification by ligand competitive saturation (SILCS), and experimentally, by X-ray crystallography, to map potential interaction sites on the FKBP51 FK1 domain. We identified three hot spots and obtained 6 X-ray co-structures, giving a hit rate of 40%. SILCS FragMaps overlapped with X-ray structures. The compounds had millimolar affinities as determined by 15 N HSQC NMR. VSFs identified the same interactions as known FK1 binder and provide new chemical starting points. We propose a hybrid screening strategy starting with SILCS, followed by a pharmacophore-derived X-ray screen and 15 N HSQC NMR based KD determination to rapidly identify hits and their binding poses.