SEPT9 Upregulation in Satellite Glial Cells Associated with Diabetic Polyneuropathy in a Type 2 Diabetes-like Rat Model.

Despite the worldwide prevalence and severe complications of type 2 diabetes mellitus (T2DM), the pathophysiological mechanisms underlying the development of diabetic polyneuropathy (DPN) are poorly understood. Beyond strict control of glucose levels, clinical trials for reversing DPN have largely failed. Therefore, understanding the pathophysiological and molecular mechanisms underlying DPN is crucial. Accordingly, this study explored biochemical and neuropathological deficits in a rat model of T2DM induced through high-fat diet (HFD) feeding along with two low-dose streptozotocin (STZ) injections; the deficits were explored through serum lipid, neurobehavioral, neurophysiology, neuropathology, and immunohistochemistry examinations. Our HFD/STZ protocol induced (1) mechanical hyperalgesia and depression-like behaviors, (2) loss of intraepidermal nerve fibers (IENFs) and reduced axonal diameters in sural nerves, and (3) decreased compound muscle action potential. In addition to hyperglycemia, which was correlated with the degree of mechanical hyperalgesia and loss of IENFs, we observed that hypertriglyceridemia was the most dominant deficit in the lipid profiles of the diabetic rats. In particular, SEPT9, the fourth component of the cytoskeleton, increased in the satellite glial cells (SGCs) of the dorsal root ganglia (DRG) in the T2DM-like rats. The number of SEPT9(+) SGCs/DRG was correlated with serum glucose levels and mechanical thresholds. Our findings indicate the putative molecular mechanism underlying DPN, which presumably involves the interaction of SGCs and DRG neurons; nevertheless, further functional research is warranted to clarify the role of SEPT9 in DPN.

Exercise Normalized the Hippocampal Renin-Angiotensin System and Restored Spatial Memory Function, Neurogenesis, and Blood-Brain Barrier Permeability in the 2K1C-Hypertensive Mouse.

Hypertension is associated with blood-brain barrier alteration and brain function decline. Previously, we established the 2-kidney,1-clip (2K1C) hypertensive mice model by renin-angiotensin system (RAS) stimulating. We found that 2K1C-induced hypertension would impair hippocampus-related memory function and decrease adult hippocampal neurogenesis. Even though large studies have investigated the mechanism of hypertension affecting brain function, there remains a lack of efficient ways to halt this vicious effect. The previous study indicated that running exercise ameliorates neurogenesis and spatial memory function in aging mice. Moreover, studies showed that exercise could normalize RAS activity, which might be associated with neurogenesis impairment. Thus, we hypothesize that running exercise could ameliorate neurogenesis and spatial memory function impairment in the 2K1C-hypertension mice. In this study, we performed 2K1C surgery on eight-weeks-old C57BL/6 mice and put them on treadmill exercise one month after the surgery. The results indicate that running exercise improves the spatial memory and neurogenesis impairment of the 2K1C-mice. Moreover, running exercise normalized the activated RAS and blood-brain barrier leakage of the hippocampus, although the blood pressure was not decreased. In conclusion, running exercise could halt hypertension-induced brain impairment through RAS normalization.

Genetic loss-of-function of activating transcription factor 3 but not C-type lectin member 5A prevents diabetic peripheral neuropathy.

We investigated the mediating roles of activating transcription factor 3 (ATF3), an injury marker, or C-type lectin member 5A (CLEC5A), an inflammatory response molecule, in the induction of endoplasmic reticulum (ER) stress and neuroinflammation in diabetic peripheral neuropathy in ATF3 and CLEC5A genetic knockout (aft3-/- and clec5a-/-, respectively) mice. ATF3 was expressed intranuclearly and was upregulated in mice with diabetic peripheral neuropathy (DN) and clec5a-/- mice. The DN and clec5a-/- groups also exhibited neuropathic behavior, but not in the aft3-/- group. The upregulation profiles of cytoplasmic polyadenylation element-binding protein, a protein translation-regulating molecule, and the ER stress-related molecules of inositol-requiring enzyme 1α and phosphorylated eukaryotic initiation factor 2α in the DN and clec5a-/- groups were correlated with neuropathic behavior. Ultrastructural evidence confirmed ER stress induction and neuroinflammation, including microglial enlargement and proinflammatory cytokine release, in the DN and clec5a-/- mice. By contrast, the induction of ER stress and neuroinflammation did not occur in the aft3-/- mice. Furthermore, the mRNA of reactive oxygen species-removing enzymes such as superoxide dismutase, heme oxygenase-1, and catalase were downregulated in the DN and clec5a-/- groups but were not changed in the aft3-/- group. Taken together, the results indicate that intraneuronal ATF3, but not CLEC5A, mediates the induction of ER stress and neuroinflammation associated with diabetic neuropathy.

Roles of Neuronal Protein Kinase Cε on Endoplasmic Reticulum Stress and Autophagic Formation in Diabetic Neuropathy.

In chronic diabetic neuropathy (DN), the cellular mechanisms of neuropathic pain remain unclear. Protein kinase C epsilon (PKCε) is an intracellular signaling molecule that mediates chronic pain. This paper addresses the long-term upregulated PKCε in DN associated with endoplasmic reticulum (ER) stress and autophagic formation and correlates to chronic neuropathic pain. We found that thermal hyperalgesia and mechanical allodynia course development were associated with PKCε upregulation after DN but not skin denervation. Pathologically, PKCε upregulation was associated with the expression of inositol-requiring enzyme 1α (IRE1α; ER stress-related molecule) and ubiquitin D (UBD), which are involved in the ubiquitin-proteasome system (UPS)-mediated degradation of misfolded proteins under ER stress. Manders coefficient analyses revealed an approximately 50% colocalized ratio for IRE1α(+):PKCε(+) neurons (0.34-0.48 for M1 and 0.40-0.58 for M2 Manders coefficients). The colocalized coefficients of UBD/PKCε increased (M1: 0.33 ± 0.03 vs. 0.77 ± 0.04, p < 0.001; M2: 0.29 ± 0.05 vs. 0.78 ± 0.04; p < 0.001) in the acute DN stage. In addition, the regulatory subunit p85 of phosphoinositide 3-kinase, which is involved in regulating insulin signaling, exhibited similar expression patterns to those of IRE1α and UBD; for example, it had highly colocalized ratios to PKCε. The ultrastructural examination further confirmed that autophagic formation was associated with PKCε upregulation. Furthermore, PKCεv1-2, a PKCε specific inhibitor, reverses neuropathic pain, ER stress, and autophagic formation in DN. This finding suggests PKCε plays an upstream molecule in DN-associated neuropathic pain and neuropathology and could provide a potential therapeutic target.

Transient receptor potential vanilloid subtype 1 depletion mediates mechanical allodynia through cellular signal alterations in small-fiber neuropathy.

Transient receptor potential vanilloid subtype 1 (TRPV1) is a polymodal nociceptor that monitors noxious thermal sensations. Few studies have addressed the role of TRPV1 in mechanical allodynia in small-fiber neuropathy (SFN) caused by sensory nerve damage. Accordingly, this article reviews the putative mechanisms of TRPV1 depletion that mediates mechanical allodynia in SFN. The intraepidermal nerve fibers (IENFs) degeneration and sensory neuronal injury are the primary characteristics of SFN. Intraepidermal nerve fibers are mainly C-polymodal nociceptors and Aδ-fibers, which mediated allodynic pain after neuronal sensitization. TRPV1 depletion by highly potent neurotoxins induces the upregulation of activating transcription factor 3 and IENFs degeneration which mimics SFN. TRPV1 is predominately expressed by the peptidergic than nonpeptidergic nociceptors, and these neurochemical discrepancies provided the basis of the distinct pathways of thermal analgesia and mechanical allodynia. The depletion of peptidergic nociceptors and their IENFs cause thermal analgesia and sensitized nonpeptidergic nociceptors respond to mechanical allodynia. These distinct pathways of noxious stimuli suggested determined by the neurochemical-dependent neurotrophin cognate receptors such as TrkA and Ret receptors. The neurogenic inflammation after TRPV1 depletion also sensitized Ret receptors which results in mechanical allodynia. The activation of spinal TRPV1(+) neurons may contribute to mechanical allodynia. Also, an imbalance in adenosinergic analgesic signaling in sensory neurons such as the downregulation of prostatic acid phosphatase and adenosine A1 receptors, which colocalized with TRPV1 as a membrane microdomain also correlated with the development of mechanical allodynia. Collectively, TRPV1 depletion-induced mechanical allodynia involves a complicated cascade of cellular signaling alterations.

Activating transcription factor 3 modulates protein kinase C epsilon activation in diabetic peripheral neuropathy.

BACKGROUND: Skin denervation that develops in patients with diabetes mellitus as a neuropathic manifestation is known as diabetic peripheral neuropathy (DPN). Skin denervation is parallel to neuronal injuries that alter intracellular signaling. To date, the correlation between nerve injury and the activation of intracellular responses to neuropathic manifestations has not been elucidated; specifically, whether activating transcription factor 3 (ATF3) is responsible for neuronal injury and a critical molecule that modulates the activation of intracellular protein kinase C epsilon (p-PKCε) and pain development in DPN is a crucial question. METHODS: To address, ATF3 knockout (atf3 -/- group, C57/B6 genetic background) and wild-type mice (atf3+/+ group) received a single dose of streptozotocin (200 mg/kg) to generate a mouse model of DPN. RESULTS: Both atf3+/+ and atf3 -/- mice exhibited hyperglycemia and the same pathology of skin denervation at posttreatment month 2, but only atf3+/+ mice developed thermal hyperalgesia (P<0.001) and mechanical allodynia (P=0.002). The atf3+/+ group, but not the atf3 -/- group, had preferential ATF3 upregulation on p-PKCε(+) neurons with a ratio of 37.7%±6.1% in p-PKCε(+):ATF3(+) neurons (P<0.001). In addition, B-cell lymphoma-extra large (Bcl-XL), an antiapoptotic Bcl2 family protein, exhibited parallel patterns to p-PKCε (ie, Bcl-XL upregulation was reversed in atf3 -/- mice). These two molecules were colocalized and increased by approximately two-fold in the atf3+/+ group compared with the atf3 -/- group (30.0%±3.4% vs 13.7% ± 6.2%, P=0.003). Furthermore, linear analysis results showed that the densities of p-PKCε and Bcl-XL had a reverse linear relationship with the degrees of thermal hyperalgesia and mechanical allodynia. CONCLUSION: Collectively, this report suggested that ATF3 is a critical upstream molecule that modulates p-PKCε and Bcl-XL expression, which consequently mediated the development of neuropathic manifestation in DPN.

Treatment with methyl-ß-cyclodextrin prevents mechanical allodynia in resiniferatoxin neuropathy in a mouse model.

Specialized microdomains which have cholesterol-rich membrane regions contain transient receptor potential vanilloid subtype 1 (TRPV1) are involved in pain development. Our previous studies have demonstrated that the depletion of prostatic acid phosphatase (PAP) - a membrane-bound ectonucleotidase -- and disordered adenosine signaling reduce the antinociceptive effect. The role of membrane integrity in the PAP-mediated antinociceptive effect in small-fiber neuropathy remains unclear, especially with respect to whether TRPV1 and PAP are colocalized in the same microdomain which is responsible for PAP-mediated antinociception. Immunohistochemistry was conducted on the dorsal root ganglion to identify the membrane compositions, and pharmacological interventions were conducted using methyl-ß-cyclodextrin (MßC) - a membrane integrity disruptor that works by depleting cholesterol - in pure small-fiber neuropathy with resiniferatoxin (RTX). Immunohistochemical evidence indicated that TRPV1 and PAP were highly colocalized with flotillin 1 (66.7%±9.7%) and flotillin 2 (73.7%±6.0%), which reside in part in the microdomain. MßC mildly depleted PAP, which maintained the ability to hydrolyze phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] and delayed the development of mechanical allodynia. MßC treatment had no role in thermal transduction and neuronal injury following RTX neuropathy. In summary, this study demonstrated the following: (1) membrane cholesterol depletion preserves PAP-mediated antinociception through PI(4,5)P2 hydrolysis and (2) pain hypersensitivity that develops after TRPV1(+) neuron depletion-mediated neurodegeneration following RTX neuropathy is attributable to the downregulation of PAP analgesic signaling.

Tumor necrosis factor-α mediated pain hypersensitivity through Ret receptor in resiniferatoxin neuropathy.

Neurogenic inflammation is an onset characteristic of small fiber neuropathy (SFN), which is attributed to neuropathic manifestations. Tumor necrosis factor-α (TNFα) is a cytokine that mainly mediates neurogenic inflammation through the ligand receptor TNF receptor 1 (TNFR1), and targeting TNFα/TNFR1 signaling is a direction toward treating inflammatory diseases and injury-induced neuropathy. However, the relationships between TNFα/TNFR1 signaling and Ret signaling, which mediates pain hypersensitivity, remains elusive. This study used resiniferatoxin (RTX), an ultrapotent analog of capsaicin, to generate a mouse model of SFN, leading to marked hindpaw edema (p = 0.013) and parallel the release of TNFα (p = 0.014), which was associated with the upregulation of Ret(+) neurons (p = 0.0043) and partial depletion of TNFR1 caused by colocalization with TRPV1 depleted by RTX. Pharmacological intervention of TNFα with etanercept (Enbrel®, Wyeth), a clinical application of TNFα blockers, relieved neurogenic inflammation and caused a reduction in hindpaw thickness (p = 0.03) and TNFα releases (p = 0.01), which were determined to be associated with the normalization of mechanical allodynia (p = 0.22). The extraction of either TNFR1(+) or Ret(+) neurons from total of TNFR1(+):Ret(+) neurons indicated that TNFR1(-)/Ret(+) neurons correlated with the mechanical threshold in an antiparallel fashion (r = -0.84, p < 0.0001) but had no relationship with thermal latencies. This study confirmed that TNFα rather than TNFα mediated neuropathic manifestation through the Ret receptor, specifically mechanical allodynia in RTX neuropathy.