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.


Introduction
Patients with diabetes often experience chronic diabetic neuropathy (DN), which presents as sensory neuropathy symptoms, including pain, tingling, numbness, and skin denervation in the limbs.Skin denervation may induce nerve sensitization [1], which consequently results in chronic pain through the activation of protein kinase C epsilon (PKCε) [2,3].PKCε is an important intracellular signaling molecule in primary afferent nociceptors [2,4,5].For example, PKCε upregulation paralleled the skin denervation-associated neuropathic pain [2], suggesting that PKCε in nociceptors has a role in regulating neuropathic pain.However, the function of PKCε has remained controversial because of its protective role in acute stress [6] and its modulatory role in chronic diabetes-related metabolic disorders [2,7].
Endoplasmic reticulum (ER) stress is induced under hyperglycemia and is also a protective mechanism for eliminating misfolded proteins in DN [8].There are several enzymes involved in the precise regulation of protein folding and the degradation of misfolded proteins under ER-associated degradation (ERAD) [9,10] and the ubiquitin-proteasome system (UPS) [11].The precise regulation requires additional molecular modulation; for example, protein degradation is regulated by PKCε activation, which utilizes UPS in downstream signaling cascades that trigger ubiquitination [12] and the subsequent degradation of PKCε [13,14].Moreover, the activation of ubiquitination through the upregulation of ubiquitin D (UBD) further regulates the signaling pathway of inositol-requiring enzyme 1α (IRE1α) under ER stress [15].The subdomain of ER is involved in phagophore formation, an initial step in autophagy [16].Pathologically, autophagy is critical in Fig. 1 Diagram of regulating endoplasmic reticulum (ER) stress and autophagy by the neuronal ε isoform of protein kinase C (PKCε).PKCε was upregulated under hyperglycemia and subsequentially led to the induction of ER stress and autophagy, which mediated diabetic neuropathy (DN).The pathology of ER stress includes the upregulation of inositol-requiring enzyme 1α (IRE1α) and ubiquitin D (UBD).Upregulated PKCε-mediated ER stress, autophagy, and DN were reversed by PKCεv1-2, a PKCε specific inhibitor diabetes-associated metabolic diseases [17].However, the molecular significance of PKCε, ER stress, and autophagic formation in the cellular pathophysiology of nociceptive receptors remains unknown; for example, whether PKCε is activated under hyperglycemia and correlated with the neuropathic pain in DN, which involved ER stress and autophagic formation.
This study investigated the duration of chronic neuropathic pain in DN and examined the coexpression profiles of PKCε with IRE1α, UBD, and p85 of phosphoinositide 3-kinase (PI3Kp85), which are involved in ER stress, dysfunctional protein elimination, and insulin signaling regulation, respectively.The current study also provided ultrastructural evidence of autophagic formation in DN.The inhibition of PKCε with a PKCε-specific inhibitor, namely PKCεv1-2, reversed neuropathic activity, expression of proteins involved in ER stress, and autophagic formation.This finding suggested that PKCε serves as an upstream modulator of ER stress and autophagy in small nociceptors, which mediates DN (Fig. 1).

Streptozotocin-induced DN and Animal Groups
We established a DN mouse model by administering a single dose of streptozotocin (STZ; 200 mg/kg, Sigma, St. Louis, MO, USA) to 8-week-old C57/B6 mice through intraperitoneal injection.Briefly, their blood glucose levels were examined weekly by using a commercially available glucometer (Accu-Chek Go, Roche Diagnostics GmbH, Mannheim, Germany), and the mice exhibiting hyperglycemia (glucose level > 400 mg/dL) were included in the following experiments (DN group).The mice that exhibited mild-to-moderate hyperglycemia (glucose level < 400 mg/ dL) were assigned to the non-DN (nDN) group.The mice that received an equal volume of citrate buffer served as the control (citrate group).Experiments were conducted at different time points on the DN mice in the following subgroups: posttreatment month 1 (DNm1), posttreatment month 2 (DNm2), and posttreatment month 5 (DNm5), and the blood glucose level were measured before sacrificed to ensure the animal in each group met the hyperglycemia for DN (glucose level > 400 mg/dL) and hypoglycemia (glucose level < 400 mg/dL) for nDN criteria.To eliminate the bias of age factors, the citrate group included each corresponding time of experimental tissues.The mice were housed in plastic cages under a 12-h light-dark cycle with ad libitum access to food and water.Without glycemic control, the survival of DN mice was limited after 5 months of STZ administration.Therefore, the endpoint of this study was set at DNm5, and all procedures were coded and blinded and minimized animal suffering by ethical guidelines.

Neuropathic Pain Evaluation
The activity and appearance of the mice were checked before the evaluation, which consisted of tests of thermal (hot plate test) and mechanical (von Frey monofilament test) responses.
Hot Plate test.The mice were placed on a 52 °C hot plate (IITC, Woodland Hills, CA, USA) enclosed in a Plexiglas cage.The withdrawal latency of the hindpaw to thermal stimulation was determined to an accuracy of 0.1 s.Each test session comprised three trials at 30-min intervals, and the withdrawal criteria included shaking, licking, or jumping from the hot plate.The mean latency was expressed as the threshold of each animal to thermal stimulation.
von Frey Monofilament test.The changes in the mechanical threshold of each group were assessed using the upand-down method with different calibers of von Frey monofilaments (Somedic Sales AB, Hörby, Sweden) in accordance with our established protocol [18].In brief, a series of monofilaments were applied to the plantar region of the hindpaw.If paw withdrawal occurred, a monofilament of a smaller caliber was applied; however, if the paw was not withdrawn, a monofilament of a larger caliber was applied.Four additional stimuli with monofilaments of various calibers were applied on the basis of the preceding responses, and the mechanical thresholds were calculated using a formula [8].

Evaluation and Quantitation of Protein Gene Product 9.5(+) Intraepidermal Nerve Fibers
We revealed intraepidermal nerve fibers (IENFs) by using a pan-axonal marker, protein gene product (PGP)9.5, in immunohistochemical studies.Briefly, the mice received intracardiac perfusion with 0.1 M phosphate buffer (PB), followed by 4% paraformaldehyde in 0.1 M PB.After perfusion, the footpad skin was postfixed for another 6 h and stored in PB.For cryosection, the footpad skin was cryoprotected with 30% sucrose in PB overnight and sectioned perpendicular to the epidermis (30-µm thickness) on an HM440E sliding microtome (Microm, Walldorf, Germany).Footpad sections were incubated with anti-PGP 9.5 (1:1000, UltraClone, Isle of Wight, UK) antiserum following a regular immunostaining protocol, and the reaction product was visualized using 3,3ʹ-diaminobenzidine (Sigma).For quantitation, PGP9.5(+)IENFs were counted, and the quantified criteria were conducted following established criteria in a coded manner [8,19].IENF density was defined as the lead citrate and were then observed and photographed using an electron microscope (Hitachi, Tokyo, Japan).

Pharmacological Intervention: PKCε Inhibitor Administration
To examine the role of PKCε in DN-related neuropathology and neuropathic pain, PKCε activity was inhibited using a pharmacological, PKCε-specific inhibitor, PKCεv1-2 (Calbiochem, La Jolla, CA, USA; Stock: 2 mg/ml).Briefly, the drugs were freshly prepared with normal saline and delivered through a lumbar puncture (1 µg/5 µL) [20] using a Hamilton microsyringe (Hamilton, Reno, NV, USA) [18].To investigate the pharmacological effects of the PKCεv1-2 inhibitor, two administration protocols were performed: (1) on the next day after STZ treatment, administration every 2 days for 4 weeks (DNw4; mPKCεI group; cumulative dose: 15 µg/mouse) and (2) administration of normal saline by using the same protocol to serve as a negative control (vePKCεI group).After treatment, the mice were housed in plastic cages under a 12-h light-dark cycle with ad libitum access to water and food.Changes in neuropathic pain were assessed at week 1 (DNw1), week 2 (DNw2), week 3 (DNw3), and DNw4 after PKCεv1-2 inhibitor administration.

Statistical Analysis
To minimize individual variation, each group had 5-8 animals.All data are expressed as the mean ± standard derivation of the mean.One-way repeated-measures analysis of variance (ANOVA) followed by Tukey's post hoc test was performed, and p < 0.05 was considered statistically significant.
To avoid bias in neuronal density measurements, only neurons with a clear nuclear profile were counted.The neuronal areas were measured using ImageJ version 1.44d.

Autophagic Formation in STZ-induced DN and PKCε Inhibition Reversed Autophagic Formation
Since ER stress initiates autophagic formation [14], we further performed ultrastructural examinations of DRGs to confirm whether PKCε blockade could reverse the autophagic formation in DN (Fig. 8).The rough ER (rER) comprised stacks of flattened membrane-bound cisternae with a lucent lumen.Double-membrane mitochondria were observed in the citrate group (Fig. 8A and inset in 8A1).By contrast, numerous swollen vesicles represented the degradation of rER and the mitochondria (Fig. 8B), which were designated as the early autophagosome (As in Fig. 8B1-8B3) and late autolysosome stages of autophagy (Aly in Fig. 8B1-8B3) in the DN mice.The amorphous masses in the swollen vesicles implied the accumulation of misfolded or unfolded proteins; additionally, we observed some autophagosomes with a double-limiting membrane [21] within the DRG soma (As in Fig. 8B1-8B3).In contrast, the mPKCεI mice exhibited stacks of flattened membrane-bound cisternae and normal mitochondrial appearance, similar to the citrate mice (Fig. 8C, 8C1, and 8C2), indicating the inhibition of PKCε activity reversed autophagic formation.

Discussion
This study demonstrated that progressive chronic neuropathic pain in DN parallels PKCε expression but not IENF density.This study further highlights PKCε is an upstream molecule that regulates the expression of (1) IRE1α, an ER stress-related molecule, (2) UBD, a regulator of misfolded protein degradation and (3) PI3Kp85, an insulin signaling molecule; all these molecules correlate with the levels of neuropathic pain in DN, as demonstrated by pharmacological inhibition of PKCε.In addition, this study also revealed that ER stress and autophagic formation in the DRG of DN were alleviated by pharmacological PKCε blockade.

PKCε Activation and ER Stress Response for Neuropathic Pain
It is important to note that challenges, such as diabetic hyperglycemia, can impair the ER intrinsic pathway for protein folding and quality control, accumulating protein aggregates.ER stress and autophagy functions are similar, namely, to eliminate unfolded and misfolded proteins [17,[24][25][26].Therefore, they maintain the physiological homeostasis of the intracellular environment.Notably, ER stress and autophagy occur in several metabolic disorders, particularly neurological disorders [27][28][29].ER stress eliminates misfolded proteins by triggering an adaptive signaling cascade that is required to activate the ERAD [9,10] and UPS [11] systems.The current study found that PKCε was upregulated in small nociceptors under hyperglycemia and was highly colocalized with IRE1α and UBD; these molecules are involved in the ERAD and UPS systems, respectively.The expression of PKCε also linear to IRE1α, UBD, and PI3Kp85, suggesting that PKCε modulates ER stress through several downstream signaling cascades.
Although the pathology of ER stress could be diminished by targeting a specific ER stress signal pathway via various inhibitors, they still exhibit limited physiological effects, neuropathy [22].However, some patients with diabetes with relatively few IENFs experience no pain in quantitative sensory testing [23], and our previous report demonstrated that diabetic mice with low IENF density had no neuropathic pain [2,8].These findings suggest that additional potential biomarkers in the DRG neurons are required for evaluating neuropathic pain in DN.PKCε has been demonstrated to be an intracellular signaling messenger for long-lasting hyperalgesic priming and chronic pain [3,5].However, the pathophysiology of neuropathic pain in DN differs from hyperalgesic priming caused by peripheral local damage.The pathology of DN involves both central sensitization and peripheral IENFs degeneration; in particular, central mice appeared as a flattened membrane-bound cisternae with a lucent lumen.The double membranous structure of mitochondria was also observed.By contrast, the DN mice (B1-B3) had numerous vacuoles that were filled with amorphous or granular substances referred to as an autophagosome (As), the early autophagic vacuoles, and autolysosome (Aly), the late autophagic vacuoles.Some autophagosomes had a double-limiting membrane (arrowheads in B1-B3).(C1, C2) mPKCεI mice exhibited flattened membrane-bound cisternae of rER and normal mitochondria appearance.Bar, 250 nm such as having a poor effect in alleviating neuropathic pain in DN [30].Accordingly, targeting upstream modulators of ER stress is an additional therapeutic approach [31].In the current study, the PKCε inhibitor reversed the ER stress pathology and neuropathic pain behaviors without affecting blood glucose levels.These findings suggest that PKCε is an upstream modulator of ER stress, autophagy, and neuropathic pain.Therefore, the inactivation of PKCε might be a potential therapeutic approach.

PKCε Activation Mediates Autophagy
Pathologically, autophagy plays a housekeeping role by removing misfolded proteins and eliminating intracellular pathogens, suggesting that it is a critical process in the pathogenesis of human diseases such as DM-associated metabolic diseases [17].Previous reports have suggested that the downregulation of the PKCε signal is associated with decreased autophagic formation, which is also demonstrated by the increased anti-apoptotic effect for cancer cell lines [32,33].However, the role of autophagy in DN is ambiguous [34,35]; for example, an ultrastructural study demonstrated that autophagic adaptation was enhanced in the peripheral nerve fibers of patients with diabetes [36].However, other studies have observed reduced autophagic pathology in diabetic animals [35,37].Although previous studies have focused on exploring autophagic pathology, upstream modulation remains unknown.This report demonstrated that PKCε upregulation was parallel to autophagic pathology, as phagophores and autophagosomes were observed in the DN mice.PKCε inhibition reversed autophagy formation and neuropathic pain manifestations.Based on our findings, there seems to be an association between the formation of autophagy and the development of neuropathic pain.
Additionally, PKCε may be an upstream modulator for ER stress and autophagy.Notably, this report does not address the causal relationship between ER stress, autophagic pathology, and the development of neuropathic pain in DN.Therefore, this issue needs further investigation.Overall, this study is the first report to provide comprehensive evidence of molecular and pathological roles of PKCε, ER stress, autophagic formation, and insulin signaling in DRG neurons.The study suggests that PKCε is an effective therapeutic target in DN.