Diabetic Sensory Neurons Research Articles

Novologue Therapy Requires Heat Shock Protein 70 and Thioredoxin-Interacting Protein to Improve Mitochondrial Bioenergetics and Decrease Mitophagy in Diabetic Sensory Neurons.

Diabetic peripheral neuropathy (DPN) is a complication of diabetes whose pathophysiology is linked to altered mitochondrial bioenergetics (mtBE). KU-596 is a small molecule neurotherapeutic that reverses symptoms of DPN, improves sensory neuron mtBE, and decreases the pro-oxidant protein, thioredoxin-interacting protein (Txnip) in a heat shock protein 70 (Hsp70)-dependent manner. However, the mechanism by which KU-596 improves mtBE and the role of Txnip in drug efficacy remains unknown. Mitophagy is a quality-control mechanism that selectively targets damaged mitochondria for degradation. The goal of this study was to determine if KU-596 therapy improved DPN, mtBE, and mitophagy in an Hsp70- and Txnip-dependent manner. Mito-QC (MQC) mice express a mitochondrially targeted mCherry-GFP fusion protein that enables visualizing mitophagy. Diabetic MQC, MQC × Hsp70 knockout (KO), and MQC × Txnip KO mice developed sensory and nerve conduction dysfunctions consistent with the onset of DPN. KU-596 therapy improved these measures, and this was dependent on Hsp70 but not Txnip. In MQC mice, diabetes decreased mtBE and increased mitophagy and KU-596 treatment reversed these effects. In contrast, KU-596 was unable to improve mtBE and decrease mitophagy in MQC × Hsp70 and MQC × Txnip KO mice. These data suggest that Txnip is not necessary for the development of the sensory symptoms and mitochondrial dysfunction induced by diabetes. KU-596 therapy may improve mitochondrial tolerance to diabetic stress to decrease mitophagic clearance in an Hsp70- and Txnip-dependent manner.

KU-596 decreases mitochondrial superoxide and improves bioenergetics following downregulation of manganese superoxide dismutase in diabetic sensory neurons

Neuronal mitochondrial dysfunction and oxidative stress are key pathophysiologic mechanisms of diabetic peripheral neuropathy (DPN). KU-596 is a small molecule modulator of heat shock protein 90 (Hsp90) that can reverse clinically relevant measures of DPN in diabetic animal models. Mechanistically, drug efficacy requires Hsp70 and correlates with improving mitochondrial maximal respiratory capacity (MRC) and decreasing oxidative stress in diabetic sensory neurons. The goal of this study was to determine if ex vivo treatment of diabetic neurons with KU-596 improves MRC by decreasing glucose-induced oxidative stress in an Hsp70-dependent manner. Sensory neurons were isolated from non-diabetic or diabetic mice wild type (WT) or Hsp70 knockout (Hsp70 KO) mice and treated with KU-596 in the presence of low or high glucose concentrations. In diabetic WT and Hsp70 KO neurons, hyperglycemia significantly increased superoxide levels, but KU-596 only decreased superoxide in WT neurons. Similarly, KU-596 significantly improved MRC in diabetic WT neurons maintained in high glucose but did not improve MRC in diabetic Hsp70 KO neurons under the same conditions. Since manganese superoxide dismutase (MnSOD) is the main mechanism to detoxify mitochondrial superoxide radicals, the cause and effect relationship between improved respiration and decreased oxidative stress was examined after knocking down MnSOD. Downregulating MnSOD in diabetic WT neurons increased hyperglycemia-induced superoxide levels, which was still significantly decreased by KU-596. However, KU-596 did not improve MRC following MnSOD knockdown. These data suggest that the ability of KU-596 to improve MRC is not necessarily dependent on decreasing mitochondrial superoxide in a MnSOD-dependent manner.

Diabetic neuropathy and the sensory neuron: New aspects of pathogenesis and their treatment implications.

Diabetic polyneuropathy (DPN) continues to be generally considered as a “microvascular” complication of diabetes mellitus alongside nephropathy and retinopathy. The microvascular hypothesis, however, might be tempered by the concept that diabetes directly targets dorsal root ganglion sensory neurons. This neuron‐specific concept, supported by accumulating evidence, might account for important features of DPN, such as its early sensory neuron degeneration. Diabetic sensory neurons develop neuronal atrophy alongside a series of messenger ribonucleic acid (RNA) changes related to declines in structural proteins, increases in heat shock protein, increases in the receptor for advanced glycation end‐products, declines in growth factor signaling and other changes. Insulin is recognized as a potent neurotrophic factor, and insulin ligation enhances neurite outgrowth through activation of the phosphoinositide 3‐kinase–protein kinase B pathway within sensory neurons and attenuates phenotypic features of experimental DPN. Several interventions, including glucagon‐like peptide‐1 agonism, and phosphatase and tensin homolog inhibition to activate growth signals in sensory neurons, or heat shock protein overexpression, prevent or reverse neuropathic abnormalities in experimental DPN. Diabetic sensory neurons show a unique pattern of microRNA alterations, a key element of messenger RNA silencing. For example, let‐7i is widely expressed in sensory neurons, supports their growth and is depleted in experimental DPN; its replenishment improves features of DPN models. Finally, impairment of pre‐messenger RNA splicing in diabetic sensory neurons including abnormal nuclear RNA metabolism and structure with loss of survival motor neuron protein, a neuron survival molecule, and overexpression of CWC22, a splicing factor, offer further novel insights. The present review addresses these new aspects of DPN sensory neurodegeneration.

Diabetic polyneuropathy, sensory neurons, nuclear structure and spliceosome alterations: a role for CWC22.

ABSTRACTUnique deficits in the function of adult sensory neurons as part of their early neurodegeneration might account for progressive polyneuropathy during chronic diabetes mellitus. Here, we provide structural and functional evidence for aberrant pre-mRNA splicing in a chronic type 1 model of experimental diabetic polyneuropathy (DPN). Cajal bodies (CBs), unique nuclear substructures involved in RNA splicing, increased in number in diabetic sensory neurons, but their expected colocalization with survival motor neuron (SMN) proteins was reduced – a mislocalization described in motor neurons of spinal muscular atrophy. Small nuclear ribonucleoprotein particles (snRNPs), also participants in the spliceosome, had abnormal multiple nuclear foci unassociated with CBs, and their associated snRNAs were reduced. CWC22, a key spliceosome protein, was aberrantly upregulated in diabetic dorsal root ganglia (DRG), and impaired neuronal function. CWC22 attenuated sensory neuron plasticity, with knockdown in vitro enhancing their neurite outgrowth. Further, axonal delivery of CWC22 siRNA unilaterally to locally knock down the aberrant protein in diabetic nerves improved aspects of sensory function in diabetic mice. Collectively, our findings identify subtle but significant alterations in spliceosome structure and function, including dysregulated CBs and CWC22 overexpression, in diabetic sensory neurons that offer new ideas regarding diabetic sensory neurodegeneration in polyneuropathy.

Erratum to: Adenosine Monophosphate-Activated Protein Kinase Abates Hyperglycaemia-Induced Neuronal Injury in Experimental Models of Diabetic Neuropathy: Effects on Mitochondrial Biogenesis, Autophagy and Neuroinflammation.

Impaired adenosine monophosphate kinase (AMPK) signalling under hyperglycaemic conditions is known to cause mitochondrial dysfunction in diabetic sensory neurons. Facilitation of AMPK signalling is previously reported to ameliorate inflammation and induce autophagic response in various complications related to diabetes. The present study assesses the role of AMPK activation on mitochondrial biogenesis, autophagy and neuroinflammation in experimental diabetic neuropathy (DN) using an AMPK activator (A769662). A769662 (15 and 30 mg/kg, i.p) was administered to Sprague–Dawley rats (250–270 g) for 2 weeks after 6 weeks of streptozotocin (STZ) injection (55 mg/kg, i.p.). Behavioural parameters (mechanical/thermal hyperalgesia) and functional characteristics (motor/sensory nerve conduction velocities (MNCV and SNCV) and sciatic nerve blood flow (NBF)) were assessed. For in vitro studies, Neuro2a (N2A) cells were incubated with 25 mM glucose to simulate high glucose condition and then studied for mitochondrial dysfunction and protein expression changes. STZ administration resulted in significant hyperglycaemia (>250 mg/dl) in rats. A769662 treatment significantly improved mechanical/thermal hyperalgesia threshold and enhanced MNCV, SNCV and NBF in diabetic animals. A769662 exposure normalised the mitochondrial superoxide production, membrane depolarisation and markedly increased neurite outgrowth of N2A cells. Further, AMPK activation also abolished the NF-κB-mediated neuroinflammation. A769662 treatment increased Thr-172 phosphorylation of AMPK results in stimulated PGC-1α-directed mitochondrial biogenesis and autophagy induction. Our study supports that compromised AMPK signalling in hyperglycaemic conditions causes defective mitochondrial biogenesis ultimately leading to neuronal dysfunction and associated deficits in DN and activation of AMPK can be developed as an attractive therapeutic strategy for the management of DN.

Adenosine Monophosphate-Activated Protein Kinase Abates Hyperglycaemia-Induced Neuronal Injury in Experimental Models of Diabetic Neuropathy: Effects on Mitochondrial Biogenesis, Autophagy and Neuroinflammation.

Impaired adenosine monophosphate kinase (AMPK) signalling under hyperglycaemic conditions is known to cause mitochondrial dysfunction in diabetic sensory neurons. Facilitation of AMPK signalling is previously reported to ameliorate inflammation and induce autophagic response in various complications related to diabetes. The present study assesses the role of AMPK activation on mitochondrial biogenesis, autophagy and neuroinflammation in experimental diabetic neuropathy (DN) using an AMPK activator (A769662). A769662 (15 and 30mg/kg, i.p) was administered to Sprague-Dawley rats (250-270g) for 2weeks after 6weeks of streptozotocin (STZ) injection (55mg/kg, i.p.). Behavioural parameters (mechanical/thermal hyperalgesia) and functional characteristics (motor/sensory nerve conduction velocities (MNCV and SNCV) and sciatic nerve blood flow (NBF)) were assessed. For in vitro studies, Neuro2a (N2A) cells were incubated with 25mM glucose to simulate high glucose condition and then studied for mitochondrial dysfunction and protein expression changes. STZ administration resulted in significant hyperglycaemia (>250mg/dl) in rats. A769662 treatment significantly improved mechanical/thermal hyperalgesia threshold and enhanced MNCV, SNCV and NBF in diabetic animals. A769662 exposure normalised the mitochondrial superoxide production, membrane depolarisation and markedly increased neurite outgrowth of N2A cells. Further, AMPK activation also abolished the NF-κB-mediated neuroinflammation. A769662 treatment increased Thr-172 phosphorylation of AMPK results in stimulated PGC-1α-directed mitochondrial biogenesis and autophagy induction. Our study supports that compromised AMPK signalling in hyperglycaemic conditions causes defective mitochondrial biogenesis ultimately leading to neuronal dysfunction and associated deficits in DN and activation of AMPK can be developed as an attractive therapeutic strategy for the management of DN.

Diabetic-induced increased sodium channel activity attenuated by tetracaine in sensory neurons in vitro

The present study was aimed to explore correlation between the altered pain perception and Na+ channel activity in diabetic animals as well as the effect of tetracaine on sensory neurons of diabetic rat. In streptozotocin-induced diabetic rats behavioral nociceptive parameters were assessed. The Na+ current (INa) was obtained using whole-cell voltage-clamp configuration in dorsal root ganglion (DRG) neurons isolated from diabetic rat (in vitro). In addition, the effects of tetracaine on altered Na+ channel activity associated with diabetes in small DRG neurons were evaluated. After induction of diabetes mechanical allodynia, thermal hyperalgesia and Na+ channel activity were altered significantly in 4th and 6th week in relation to the control. Altered pain parameters were in correlation with increased INa in time-dependent manner. In comparison to age-matched control (−1.10±0.20nA) the INa was found to be −2.49±0.21nA at 4th week and −3.71±0.28nA at 6th week. The increased activity of Na+ channels was blocked by tetracaine even in diabetic condition. The depression of the INa on tetracaine exposure was not sensitive to the voltage or time. The conductance curve shifted towards right around −8.0mV. The alterations in neuropathic pain associated with diabetes and Na+ channel activity has been clearly correlated in time-dependent manner. The INa density was increased significantly with the progression of neuropathic pain. Local anesthetic, tetracaine potentially blocked the Na+ channel activity in diabetic sensory neurons.

Glucagon-Like Peptide 1, Insulin, Sensory Neurons, and Diabetic Neuropathy

Like insulin, glucagon-like peptide 1 (GLP-1) may have direct trophic actions on the nervous system, but its potential role in supporting diabetic sensory neurons is uncertain. We identified wide expression of GLP-1 receptors on dorsal root ganglia sensory neurons of diabetic and nondiabetic mice. Exendin-4, a GLP-1 agonist, increased neurite outgrowth of adult sensory neurons in vitro. To determine the effects ofexendin-4 in comparison with continuous low- or high-dose insulin in vivo, we evaluated parallel cohorts of type 1 (streptozotocin-induced) and type 2 (db/db) mice of 2 months' diabetes duration with established neuropathy during an additional month of treatment. High-dose insulin alone reversed hyperglycemia in type 1 diabetic mice, partly reversed thermal sensory loss, improved epidermal innervation but failed to reverse electrophysiological abnormalities. Exendin-4 improved both sensory electrophysiology and behavioral sensory loss. Low-dose insulin was ineffective. In type 2 diabetes, hyperglycemia was uncorrected, and neither insulin nor exendin-4 reversed sensory electrophysiology, sensory behavior, or loss of epidermal axons. However, exendin-4 alone improved motor electrophysiology. Receptor for advanced glycosylated end products and nuclear factor-κB neuronal expression were not significantly altered by diabetes or treatment. Taken together, these results suggest that although GLP-1 agonists and insulin alone are insufficient to reverse all features of diabetic neuropathy, in combination, they might benefit some aspects of established diabetic neuropathy.

Effect of protein kinase C?? inhibitor on Ca2+ homeostasis in diabetic sensory neurons

To elucidate the direct effect of selective protein kinase Cbeta inhibitor LY333531 on diabetic sensory neurons, we examined intracellular Ca(2+) concentration in isolated rat dorsal root ganglion neurons using the fluorescent Ca(2+) indicator fura-2. The duration of calcium transients induced by high (50 mM) extracellular K in small diabetic dorsal root ganglion neurons was significantly prolonged compared with that in control neurons. This prolonged intracellular Ca concentration elevation in diabetic neurons was normalized rapidly and reversibly by LY333531 in a dose-dependent manner, and the effect of LY333531 was completely abolished by pretreating the neurons with mitochondrial calcium uniporter inhibitor, Ruthenium 360. These results suggest that LY333531 has an ameliorating effect on calcium homeostasis of diabetic sensory neurons via mitochondrial calcium buffering.

Sensory neurons with activated caspase-3 survive long-term experimental diabetes.

Long-term experimental diabetes may best model the prominent and irreversible sensory deficits of chronic human diabetic polyneuropathy. Whereas irretrievable loss of sensory neurons, if present, would be an unfortunate feature of the disease, systematic unbiased counting has indicated that sensory neurons survive long-term experimental diabetes. In this study, we examined whether incipient cell loss from apoptosis in chronic experimental diabetes might nonetheless be in process, or whether neurons somehow adapt to their chronic insults. We examined sensory neurons in L4 and L5 dorsal root ganglia of long-term experimental streptozotocin-induced diabetic rats using transferase-mediated dUTP nick-end labeling (TUNEL), 4',6-diamidino-2-phenylindole (DAPI) staining of nuclear morphology, and electron microscopic appraisal of cell morphology. None provided any evidence for ongoing apoptosis. Despite this confirmation that sensory neurons survive, neurons had elevated expression of activated caspase-3 in unique patterns that included their nuclei, cytoplasm, and proximal axonal segments. Bcl-2 expression, a marker of antiapoptosis signaling, was observed in similar numbers of diabetic and nondiabetic neurons. In contrast, diabetic sensory neurons had elevated expression of the DNA repair enzyme poly(ADP-ribose) polymerase (PARP) in their nuclei, cytoplasm, and proximal axonal segments not overlapping with caspase-3 localization. Diabetic sensory neurons also had an apparent rise in cytoplasmic labeling of nitrotyrosine, a marker of peroxynitrite toxicity reported to activate PARP.