Contributions of mTOR Activation-Mediated Upregulation of Synapsin

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The contributions of mTOR activation-mediated upregulation of synapsin II and neurite outgrowth to hyperalgesia in STZ-induced diabetic rats Wan-you He, Bin Zhang, Wei-cheng Zhao, Jian He, Lei Zhang, Qing-ming Xiong, Jing Wang, and Han-bing Wang ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00680 • Publication Date (Web): 20 Feb 2019 Downloaded from http://pubs.acs.org on February 21, 2019

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ACS Chemical Neuroscience

The contributions of mTOR activation-mediated upregulation of synapsin II and neurite outgrowth to hyperalgesia in STZ-induced diabetic rats Wan-you He, Bin Zhang, Wei-cheng Zhao, Jian He, Lei Zhang, Qing-ming Xiong, Jing Wang, Han-bing Wang* Department of Anesthesiology, The First People’s Hospital of Foshan, 81# North of Ling Nan Road, Foshan 528000, China

*Corresponding author: Han-bing Wang Department of Anesthesiology, The First People’s Hospital of Foshan, 81# North of Ling Nan Road, Foshan 528000, China Phone:

+0086‐180-8316-2503,

Fax:

+0086‐8316‐3871,

E-mail:

[email protected] Author Affiliations Wan-you He, MSA, Phone: +0086-8316-3871, Fax: +0086‐8316‐3871, E-mail: [email protected]

Department of Anesthesiology, The First People’s Hospital of Foshan, 81# North of Ling Nan Road, Foshan 528000, China Bin Zhang, MD, Prof. Department of Anesthesiology, The First People’s Hospital of Foshan, 81# North of Ling Nan Road, Foshan 528000, China E-mail: [email protected] Wei-cheng Zhao, MD, Prof. Department of Anesthesiology, The First People’s Hospital of Foshan, 81# North of Ling Nan Road, Foshan 528000, China E-mail: [email protected] Jian He, MSA, Department of Anesthesiology, The First People’s Hospital of Foshan, 81# North of Ling Nan Road, Foshan 528000, China

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E-mail: [email protected] Lei Zhang, MSA, Department of Anesthesiology, The First People’s Hospital of Foshan, 81# North of Ling Nan Road, Foshan 528000, China E-mail: [email protected] Qing-ming Xiong, MSA, Department of Anesthesiology, The First People’s Hospital of Foshan, 81# North of Ling Nan Road, Foshan 528000, China E-mail: [email protected] Jing Wang, MSA, Department of Anesthesiology, The First People’s Hospital of Foshan, 81# North of Ling Nan Road, Foshan 528000, China E-mail: [email protected]

Abstract


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Painful diabetic neuropathy (PDN) is among the common complications in diabetes mellitus (DM), with its underlying mechanisms largely unknown. Synapsin II is primarily expressed in the spinal dorsal horn, and its upregulation mediates a superfluous release of glutamate and a deficiency of GABAergic interneuron synaptic transmission, which is directly implicated in the facilitation of pain signals in the hyperalgesic nociceptive response. Recently, synapsin II has been revealed to be associated with the modulation of neurite outgrowth, whereas the process of this neuronal structural neuroplasticity following neuronal hyperexcitability still remains unclear. In this study, we found that under conditions of elevated glucose, TNF-α induced the activation of mTOR, mediating the upregulation of synapsin II and neurite outgrowth in dorsal horn neurons. In vivo, we demonstrated that mTOR and synapsin II were upregulated and co-expressed in the spinal dorsal horn neurons in rats with streptozotocin (STZ)-induced diabetes. Furthermore, the intrathecal administration of the mTOR inhibitor rapamycin or synapsin II shRNA significantly diminished the expression of synapsin II, effectively mitigating hyperalgesia in PDN rats. We are the first to discover that in STZ-induced diabetic rats the activation of mTOR mediates the upregulation of synapsin II and neurite outgrowth, both contributing to hyperalgesia. These findings may benefit the clinical therapy of PDN by provision of a novel target.

Keywords: Diabetes complications; hyperalgesia; synapsin II; neurite outgrowth; mTOR

1. INTRODUCTION Painful diabetic neuropathy (PDN) is among the most common complications of diabetes mellitus, affecting approximately one-third of patients1. However, the mechanisms of PDN have not been unmasked. Neuronal synaptic plasticity is widely deemed to underlie neuronal hyperexcitability, improper synaptic transmission and pain hypersensitivity2. Accumulating evidence suggests that modifications in both the synaptic structural proteins (i.e. the abnormal assembly and localization of synapsins) and neurite maladaptive outgrowth are key events in the process of synaptic plasticity and excessive



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neural excitatory transmissions in the spinal dorsal horn3. Hence, clarification of these mechanisms for neuronal synaptic plasticity is expected to benefit the understanding of the pathophysiology of hyperalgesia in PDN. The regulation of synaptogenesis and the retention as well as release of neurotransmitters during long-term potentiation (LTP) are vital mechanisms for the synaptic plasticity of dorsal horn neurons4,5. This process is controlled by the coordinated regulation of intracellular signaling, synapsin expression and synapse formation during pain hypersensitivity6,7. Synapsins, classified as a class of neuron-specific synaptic vesicle phosphoproteins, are paramount signaling components in the regulation of neuronal development and nociceptive processing5,8. Synapsin II, one of the major isoform of synapsins, was originally described in excitatory synapses in laminae I and II of the dorsal horn5. Furthermore, experimental evidence shows that the upregulation of synapsin II is directly involved in the facilitation of formalin-induced inflammatory and neuropathic pain5,8. The release of excessive glutamate and a deficiency in GABAergic interneuron synaptic transmissions have been testified to be responsible for the development of PDN9,10. Intriguingly, synapsin II is reported to exert a crucial role in the regulation of the release of glutamate and gamma-aminobutyric acid (GABA) in neuropathic pain8. Thus, we speculated that synapsin II might serve as a contributor in the nociceptive processing in PDN. Recent evidence has delineated that the mammalian target of rapamycin (mTOR) (consisting two complexes: mTORC1 and mTORC2), integrates these growth signals and determines the outcome of synaptic vesicle phosphoprotein synthesis and localization at potentiated synapses for synaptic plasticity through translation control

11,12.

Furthermore, the activation

of mTOR is essential for pain-related synaptic plasticity and behavioral hypersensitivity13-15, indicating that mTOR might mediate the upregulation of synapsin II and neurite outgrowth in PDN. Additionally, there is a paucity of evidence as to the explicit role of mTOR in neuronal branching and neurite extension associated with neuronal plasticity in the pain hypersensitivity in diabetic neuropathy. Tumor necrosis factor alpha (TNF-α), identified as a pro-inflammatory cytokine, is also reportedly involved in PDN16,17. Patients with diabetic


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neuropathy may experience more severe pain with overexpressed and intensified immunoreactivity of TNF-α17. Recently, studies have divulged that TNF-α exerts a dominant role in the modulation of excitatory synaptic transmission and spinal synaptic plasticity as well as hyperalgesia; however, the mechanism by which TNF-α induces neuronal plasticity and hyperalgesia in PDN still remains anecdotal. Notwithstanding that TNF-α could dependently or independently activate the mTOR pathway via the PI3K pathways18,19, whether TNF-α mediates the activation of mTOR in nociceptive processing still remains a puzzle. Hence, we hypothesized that TNF-α/mTOR might regulate the synapsin II and neurite outgrowth, both of which are implicated in the pathogenesis of hyperalgesia in streptozotocin (STZ)-induced diabetic rat model. Herein, we conducted the experiment to investigate the explicit effect of TNF-α/mTOR on synapsin II both in vitro and in vivo.

2. RESULTS 2.1 TNF-α Exposure Upregulated the Expression of Synapsin II and Enhanced Neurite Outgrowth under High Glucose Conditions via the mTOR Pathway in Dorsal Horn Neurons To evaluate whether the pro-inflammatory cytokine TNF-α could drive the upregulation of synapsin II and neurite outgrowth under high glucose conditions via the mTOR pathway, we assessed the expression of synapsin II by western blotting and evaluated the morphology of dorsal horn neurons by immunocytochemical analysis using the β-tubulin III (a neuron-specific antibody).

Exposure

of

cultured

dorsal

horn

neurons

to

increased

concentrations of D-glucose (30 mM) or D-mannitol (24.5 mM + 5.5 mM glucose) for 24 h failed to induce the activation of mTOR and the expression of synapsin II as well as the neuronal morphology, whereas exposure to TNF-α (10 ng/ml) or the combinatory exposure to TNF-α and high glucose activated mTOR (Fig. 1B and C, MAOVA; F (4, 15) = 3.59, p = 0.030 in p-mTOR; F (4, 15) = 4.05, p = -0.000 in p-S6K) and upregulated the expression of synapsin II (Fig. 1D, MAOVA, F (4, 15)= 3.68, p = 0.028) and increased neurite outgrowth



(Fig. 2G-I and 3G-I), notably in dorsal horn neurons exposed to the combinatory treatment of elevated concentrations of D-glucose (30 mM) with TNF-α for 7 consecutive days (Figure. 1A-D, 2G-I, and 3G-I). However, this upregulation in synapsin II expression and neurite outgrowth under a high glucose condition was blocked by rapamycin (100 nM) (Fig. 1A-D and 3G-I; Branch length, F (5, 174) = 2.61, p = 0.026; TNTN, F (5, 174) = 3.02, p = 0.012; TNBL, F (5, 174) = 2.89, p = 0.016), and the adopted drug concentration can selectively inhibit the activity of the mTOR pathway20 Moreover, the genetic inhibition of synapsin II (shNRA) also prevented neurite outgrowth in dorsal horn neurons exposed to the combination of TNF-α and high glucose (t test, t = 5.33, p = 0.003 in Fig. 1E). Notably, rapamycin or synapsin II shRNA also decreased networks, mean branch length and mean network size (branches) of neuronal clusters in dorsal horn neurons exposed to TNF-α or the combination of TNF-α and high glucose (Fig. 2A-I; networks, F (5, 102)= 2.91, p = 0.016; mean branch length, F (5, 102)= 2.77, p = 0.022; mean network size (branches), F (5, 102) = 2.59, p = 0.030). 2.2 Intrathecal Administration of mTORC1 Inhibitor Rapamycin Reduced Synapsin II Expression and Mitigated Hyperalgesia in STZ-induced Diabetic Rats To investigate whether the maladaptive overexpression of synapsin II is dependent on the activation of mTOR in the spinal cord in rats with PDN, we intrathecally delivered the mTORC1 specific inhibitor rapamycin to the spinal cord through an intrathecal catheter and evaluated the MWT with von Frey filaments. Indeed, mTOR was activated and the expression of synapsin II was upregulated in the spinal cord from rats with PDN (Fig. 4A-E). The intrathecal injection of rapamycin (10 μg once daily for 7 days) suppressed the activity of mTORC1 (p-S6K is a specific phosphorylation target of mTORC1 and represents an indicator for monitoring mTORC1 status21, diminished the expression of synapsin II and ameliorated hyperalgesia in PDN rats (ANOVA among groups, F (2,9) = 5.93 and p = 0.023 and repeated measures ANOVA in the group PDN + RAP, F (2,9) = 6.07 and p = 0.021 in Fig. 4A, F (2,17) = 5.61 and p = 0.026 in Figure. 5C, F (2,17) = 6.21 and p = 0.020 in Figure. 4D, F


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(2,17) = 5.14 and p = 0.032 in Figure. 4E), indicating the potentially pivotal role of mTOR in the modulation of synapsin expression and in the nociceptive process. Notably, the intrathecal injection of rapamycin also indirectly inhibited the phosphorylation and activation of S6K (p-s6k) in the spinal cord (Fig. 4, B and D). In vitro, we demonstrated that TNF-α induced the activation of mTOR under high-glucose conditions (Fig. 1, A and B). Importantly, studies have confirmed that TNF-α can activate mTOR/S6K through multiple signaling pathways22, and we also observed that the TNF-α expression in the spinal cord was increased in STZ-induced diabetic rats, and this effect could be reversed by intrathecal injection of rapamycin (ANOVA among groups, F (2,9) = 4.63, p = 0.041 in Fig. 4F), indicating that spinal TNF-α promotes the activation of mTOR in PDN rats. Simultaneously, intrathecal injection of rapamycin prevents the expression of TNF-α. Furthermore, our prior study demonstrated

that

mTOR

activation

facilitated

neuronal

excitatory

transmission and hyperalgesia in PDN15, supporting the hypothesis that TNF-α/mTOR mediates the overexpression of synapsin II in the process of pain hypersensitivity in PDN. 2.3 Upregulation and Co-expression of Synapsin II and mTOR in the Spinal Dorsal Horn in Rats with PDN To explore the localization of synapsin II and the contribution of mTOR in the spinal cord of PDN rat, we established an STZ-induced diabetic rat model for investigation. In this model, the expression levels of synapsin II and mTOR in the spinal cord of PDN rats were significantly upregulated versus the normal controls (Fig. 4, B, C, D and E). As illustrated, the results of immunofluorescence assay of lumbar spinal cord slices validated the elevated expression of synapsin II, which was expressed primarily in the superficial laminae of the dorsal horn (Fig. 5, H and M), also supportive of earlier studies5. In addition, 83.9% of synapsin II-positive products co-expressed with p-mTOR (Fig. 5, H and M), which is essential for synapse formation neurite outgrowth and neuronal development23,24. Synapsin II was profoundly enhanced in the lumbar spinal cord by western blot, which coincided with the activation of mTOR (Fig. 4C-E), authenticating the close correlation between the activation



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of mTOR and the maladaptive upregulation of synapsin II in the dorsal horn in rats with PDN. 2.4 Synapsin II shRNA Attenuates Hyperalgesia in STZ-induced Diabetic Rat Model To further verify that the expression of synapsin II is relevant to the hyperalgesia in PDN progression in the STZ-induced diabetic rat model, we analyzed the nociceptive behavior of naïve rats and rats with genetically inhibited synapsin II expression (using a shRNA that specifically targets rat synapsin II). The recombinant synapsin II shRNA lentiviral vectors were intrathecally injected three weeks after the STZ injection. In addition, the MWT was assessed 1, 3, 5, and 7 days after the intrathecal injection. We observed that the knockdown of synapsin II significantly increased the MWT compared to that of the naïve rats, indicating that the aberrant upregulation of spinal synapsin II was involved in the pain hypersensitivity of PDN rats (Fig. 6B, t-test, t = 4.78, p = 0.002; Fig. 6C, ANOVA, F (2, 9) = 6.07, p = 0.021, and repeated measures ANOVA in group PDN and group SHP, F (2, 15) = 4.92, p = 0.023). 2.5 Synaptic Plasticity of Dorsal Horn Neurons in Slice Cultures Prepared from PDN Rats Notwithstanding the involvement of synapsin II in the modulation of synapse formation, neurite outgrowth, neuronal excitability, and consequently, pain sensitivity thresholds, the role of synapsin II in the regulation of nociceptive processing-related neurite outgrowth and excessive synaptic transmission is still poorly understood, especially in PDN. To interrogate the precise role of the mTOR/synapsin II pathway in modulating synaptic plasticity and neuronal excitability in PDN rats, spinal cord sections were prepared and whole-cell patch-clamp recordings were conducted in lamina II neurons where ample nociceptive neurons are localized. The excitatory synaptic transmissions were tested

by

recording

spontaneous

EPSCs

(sEPSCs)

in

synapsin

II

shRNA-treated and wild-type PDN rats. As depicted, the spinal cord slices obtained from PDN rats presented with a robust increase in sEPSC frequency


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and amplitude when compared with those of the normal control (Fig. 7, ANOVA, F (3,60) = 3.46 and p = 0.022 in Fig. 7C, F (3,60) = 3.29 and p = 0.027 in Fig. 7D). These elevations in frequency and amplitude were almost abolished after superfusion of spinal cord slices or of slices from rats in the diabetic + rapamycin group (RAP group) or the diabetic + shRNA group (shRNA group) (Fig. 7). We observed that the activation of mTOR mediated the overexpression of synapsin II, contributing to neuronal hyperexcitability and excessive synaptic transmission, which are both involved in hyperalgesia.

3. DISCUSSION In the present study, we demonstrated that high glucose could facilitate the pro-inflammatory cytokine TNF-α driving the upregulation of synapsin II and neurite outgrowth via the mTOR pathway. mTORC1 inhibitor rapamycin or synapsin II shRNA reduced the synapsin II expression and mitigated hyperalgesia in STZ-induced diabetic rats. The electrophysiological asssay demenstrated that excessive excitatory synaptic transmissions of sEPSCs were approximately abolished in rats treated with rapamycin or synapsin II shRNA, which further indicated that mTOR activation-mediated upexpression of synapsin II contributed to neuronal hyperexcitability and excessive synaptic transmission, two key events involved in hyperalgesia of PDN. We are the first to discover that the activation of mTOR mediates the upregulation of synapsin II and neurite outgrowth, both contributing to hyperalgesia in STZ-induced diabetic rats. The findings might benefit to provide novel candidate targets for therapeutics of PDN. Immoderate synaptic transmissions in the spinal dorsal horn coupled with aberrant neuronal firing have been hypothesized to contribute to the development of neuropathic pain25,26 . Neurite outgrowth, including neurite extension and neuronal branching, is the structural foundation of neuronal plasticity27. Structural neuroplasticity has the noticeable property of being capable of altering the efficiency of synaptic transmissions according to the discharge patterns of neurons receiving nociceptive stimulation28-30. In consistency with the prior report, we employed an anti-β-tubulin III antibody (a microtubule element of the tubulin family), which was present almost



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exclusively in neurons to identify neurite outgrowth31. Neurite outgrowth in the adult mammalian central nervous system is severely limited, mainly due to the microenvironment at lesion sites. TNF-α could enhance neurite outgrowth in cultured adult sensory neurons through an NF-κB-dependent pathway32. Similarly, our data revealed that TNF-α remarkably enhanced neuronal neurite outgrowth, especially in dorsal horn neurons under hyperglycemic conditions, which is considered to contribute to the excessive neuronal circuitry for pain processing and the exaggerated perception of pain33,34. In hyperglycemic conditions, advanced glycation end-products (AGEs), a consequence of abnormal glucose metabolism, can activate divergent signaling pathways (i.e. the NF-κB, JAK-STAT and ERK pathways) via their ligands to promote neurite outgrowth of adult sensory neurons35,36. Moreover, TNF-α can also activate multiple intracellular signal transduction

pathways,

including

NF-κB,

JAK-STAT

and

ERK,

and

cooperatively promotes neurite outgrowth when combined with high-glucose conditions. In our experiment, however, exclusive exposure of dorsal horn neurons to hyperglycemia did not modify their morphological properties, which was probably ascribed to the hyperglycemic toxicity which also impairs neural function and dendritic and synaptic regeneration and partially neutralizes the promoting effects of AGEs in neurite outgrowth37. TNF-α can reportedly promote neurite outgrowth via a NF-κB-dependent pathway in cultured DRG neurons32. In our experiment, TNF-α and hyperglycemia synergistically induced neurite outgrowth via mTOR-dependent pathway in cultured dorsal horn neurons. Moreover, nerve growth factor (NGF) could facilitate neurite outgrowth, including neurite elongation and branching38. However, evidence indicated that NGF was decreased in the DRG and dorsal horn and contributed to concomitant allodynia in PDN39, indicating that the promotion of neurite outgrowth is independent of NGF. Neurite outgrowth is closely related to abnormal synapse formation and excessive synaptic transmission in the spinal dorsal horn, which are involved in nociception and hyperalgesia40-42. Besides, synapsin II has been associated with the regulation of synaptic transmission and neurite outgrowth23,43. In this study, we evidenced that synapsin II colocalized with β-tubulin III in dorsal horn neurons and was expressed in nascent axonal sprouting (marked by β-tubulin


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III), implicating synapsin II located in neurite outgrowth. The genetic inhibition (shRNA) of synapsin II prevented the promotion of neurite outgrowth, strongly supporting the imperative role for synapsin II in the modification of neurite outgrowth in spinal dorsal horn neurons. Given that neuropathic pain in patients is relieved by routine analgesic medications, we can exclude that abnormal structural neuroplasticity, such as excessive neurite outgrowth, can lead to axonal disconnection, exceptional neuronal excitement transmission in organs, or even the excessive amplification of cascades in the neuronal circuitry, all of which are involved in the pathophysiology of hyperalgesia and cannot be relieved by general, repeated medications that modulate neuronal functions44. Accordingly, we have found that excessive neurite outgrowth provoked a complicated, unwieldy neuronal circuitry in nociceptive neurons in spinal dorsal horn under combinatory exposure to hyperglycemia and inflammation. In addition, the inhibition of synapsin II effectively prevented improper neurite outgrowth and excessive neural excitatory transmission, relieving hyperalgesia in PDN rats. Furthermore, we have validated that the genetic inhibition of synapsin II eliminated the increase in sEPSC frequency and amplitude in dorsal horn neurons. These changes depend on glutamate release from excitatory neurotransmitters and excessive neural excitatory transmission. Our results demonstrated that synapsin II-mediated neurite outgrowth is involved in neuroplasticity and behavior hypersensitivity, which was supported by a prior report, in which synapsin II was confirmed to be primarily localized at the synapses of primary afferent neurons on the surface lamina of the dorsal horn and the overexpression of synapsin II is essential for neurite outgrowth and the facilitation of nociceptive signal transmission5. Other studies have also confirmed the involvement of synapsin II in the modulation of glutamate and GABA release in the spinal cord in the condition of nerve injury, in which the maladaptive expression of synapsin II resulted in a dysbalance between GABAergic synaptic and glutamatergic transmission, contributing to neuronal hyperexcitatory behavior8. Herein, we affirmed that intrathecal rapamycin could suppress the activity of spinal mTORC1, and assuaged mechanical hypersensitivity in PDN rats, the former of which was validated by other researches21. Additionally, our previous



research showed that intrathecal administration of rapamycin also inhibited the activation of DRG mTORC1 and the phosphorylation of Nav1.8, and prevented nociceptive information transmission of DRG in PDN15, also supported by a recent study that intrathecal rapamycin could block the activities of dorsal root. Therefore, we cannot preclude the effect of intrathecal rapamycin on the expression of CGRP and/or substance P in DRG and dorsal root in STZ-diabetic rats. Besides, we have some limitations of designating intrathecal rapamycin or vacant lentiviral vector in normal control rats, because evidence demonstrates intrathecal administration of rapamycin or empty lentiviral vector has no effect on normal behavior function45,20. mTOR-mediated translation control is indispensable for structural protein synthesis and neuronal synaptic plasticity11,13. In this study, nonetheless, we substantiated that the expression of the synaptic vesicle protein synapsin II depends on mTORC1 activity. We verified that mTOR activation promoted the upregulation of synapsin II and structural neuroplasticity (neurite outgrowth), which could be reversed by rapamycin. Furthermore, the inhibition of mTORC1 or the employment of synapsin II shRNA could downregulate the expression of synapsin II and mitigate neuronal hyperexcitability and pain hypersensitivity in STZ-induced diabetic rats. However, it is worth noting that TNF-α per se is not an immediate upstream signal of mTOR. Strong evidence suggests that TNF-α activates the mTOR/S6K pathway through PI-3K-dependent and independent mechanisms18,19, and repeated verification of this canonical pathway would not benefit research. In addition, we adopted a concentration of 100 nM rapamycin for the inhibition of mTORC1 in dorsal horn neurons and an intrathecal administration of rapamycin at 10 μg daily (for 7 consecutive days) for the inhibition of mTORC1 in the spinal cord on the grounds that the validation of these doses have been published in previous studies21,46 . Here, we disclosed that rapamycin prevented synapsin II expression (Figure. 6, F and J), indicating that intrathecal treatment did affect the dorsal horn, which was also supported by other researchers47. Intriguingly, our results also authenticated that intrathecal rapamycin could decrease the level of spinal TNF-α in STZ-induced diabetic rats, indicating that TNF-α might be also modulated by mTOR via transcriptional regulation in a positive feedback manner13. There are some limitations in the present study. A growing body of


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evidence indicated that central mechanisms exerted a crucial role in hyperalgesia of PDN, as evidenced by intrathecal delivery of inhibitors, such as shRNA or siRNA vector targeting spinal molecules effectively reversing the nociceptive behavior5,11,13,21,45. Despite the remarkable inhibitory effect of rapamycin and shRNA on synapsin II in the spinal cord, we could not exclude their effects on DRG. In addition, due to the previous report that intrathecal rapamycin or synapsin II genetic knockout alone showed no evident impacts on basal mechanical sensitivity, rapamycin-alone or shRNA-alone group was not adopted for control in our present study5,21. Moreover, we do not have the intrathecal rapamycin- or shNRA-treated normal control group with and without TNF-α treatment, on the grounds that intrathecal injection of TNF-α may reportedly affect multiple tissues including DGR, spinal dorsal root and the spine, evoking neuropathy in many sites, all of which lead to a comprehensive pain sensitization in animal behavior48-50. In such cases, the control groups would lack consistency and involve more confounders, which would not comply with our protocol of investigating single tissues in the spine. In addition, our literature retrieval found that intrathecal injection of TNF-α is also supported. Thus, we designated cell experiment on the grounds of the paucity of confounders, which may facilitate the study of TNF-α/mTOR pathway. In summary, our results demonstrated that targeting mTOR contributes to substantial analgesic relief in PDN rats, which is attributed to a decreased expression of synapsin II and neurite outgrowth in the spinal cord of diabetic rats induced by STZ. Collectively, our experiment intensely supports the vital role of synapsin II in mediating neurite outgrowth in PDN-associated hyperalgesia.

4. MATERIALS AND METHODS 4.1 Cell Culture and Stimulation Dorsal horn neurons were dissociated as described as follows51: dissected from sacrificed Sprague-Dawley rats at embryonic days 4-7, isolated in 1% Trypsin-EDTA (Invitrogen, Carlsbad, CA, USA), and thereafter cultured in EMEM (Gibco, Grand Island, NY, USA) supplemented with 2.0 mM L-glutamine, 5.5 mM glucose, 50 ng/ml nerve growth factor (NGF) (Harlan



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Bioscience, Indianapolis, IN, USA), 30 nM selenium, 10 nM hydrocortisone, 10 nM

β-estradiol,

100

μg/ml

transferrin,

and

1,000

U/ml

penicillin/streptomycin/neomycin (ABX) solution. Unless otherwise specified, all the other agents were customized from Sigma-Aldrich (St. Louis, MO, USA). The fully-grown dorsal horn neurons in the logarithmic phase were dissociated by addition of 1% trypsin in PBS solution and plated at a density of 10,000 cells per dish or 5,000 cells per collagen-coated glass slide. Subsequent to culture of 24 hr in plating medium, the neuronal medium was replaced with the desired glucose medium, and the neurons were incubated for 7 days52. Media with escalated concentrations of glucose were prepared at the final glucose concentration of 30 mM D-glucose or D-mannitol (24.5 mM + 5.5 mM D-glucose). Two days thereafter, synapsin II shRNA neurons were transduced with a recombinant pLVX-puro lentiviral vector targeting synapsin II or with a vacant vector, with transfection duration maintained for 5 consecutive days. Dorsal horn neurons were also exposed to TNF-α (Sigma, 10 ng/ml), rapamycin (100 nM) or control vehicle for 24 hr during the last day in vitro as indicated. 4.2 Morphological Analysis of Dorsal Horn Neurons The well-cultured dorsal horn neurons were randomized into six groups: normal glucose control group (Nor.Glu, D-mannitol 24.5 mM + 5.5 mM D-glucose), high glucose medium group (Hy.Glu, D-glucose 30 mM), normal glucose + TNF-α (Nor.Glu + TNF-α, TNF-α: 10 ng/ml), hyperglucose + TNF-α (Hy.Glu + TNF-α), hyperglucose + TNF-α + rapamycin (Hy.Glu + TNF-α + Rapa, rapamycin: 100 nM), hyperglucose + TNF-α + Synapsin II shRNA group (Hy.Glu + TNF-α + shRNA) at day 1 in vitro. The synapsin II shRNA was transfected by Lipofectamine 3000 reagent (Invitrogen) and the transfected dorsal horn neurons were incubated with Lipofectamine 3000 at 37°C with 5% CO2 for 7 days as indicated. 24 hours later, the neurons were analyzed for neuronal outgrowth. The neuronal morphology and neurite outgrowth of neuronal clusters were assessed by microscopy and immunocytochemical


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double staining using anti-β-tubulin III antibody and synapsin II in spinal dorsal horn neurons subsequent to combined exposure to TNF-α and hyperglycemia, respectively. The morphology of the dorsal horn neurons and neuronal complexity (number and lengths) of branches at levels of single cell and clustered neurons were quantified by NIH ImageJ to assess the size and complexity of neuronal outgrowth as previously reported53, since dorsal horn neurons are usually highly branched and the extent of the arborization of the neuronal branches (tree) is correlated with the number and distribution of nociceptive information inputs that the neuron can receive and process.

Quantitative analyses of networks (number of objects in the image that contained at least 1 junction of pixels and corresponding neurons in more than one branch,

n N

B n 0

n

 1 ), mean network size (average neuronal branching of

each neuron or the mean number of branches per network,

neuronal footprint (

I average I maxinum

1 N

N

b

n

) and

n

 x  y  s 2 , where I represents the pixel intensity in

the binarized image, s represents the calibrated length of one pixel, x represents the width of the image in pixels, and y represents the height of the image in pixels) of neuronal clusters were conducted by MINA macro of ImageJ as reported previously .18 Images from each group were randomly obtained and pre-processed to improve quality prior to binarizatoin and skeletonization for morphological analysis of networks . The original images were processed using unsharp mask, enhance local contrat (CLAHE), median of filters, binarized and skeletonized. Neuronal footprint (the area occupied by morphological structures of neurons) is calculated from the binarized image prior to skeletonization. Other descriptive parameters are calculated from the skeletonized image. The statistical analyses of the total neuronal branch tip number (TNBTN), average neuronal branching length (ANBL), and total



Page 16 of 38

neuronal branching length (TNBL) of single dorsal horn neuron were performed by GraphPad Prism 6. The projection images were traced with NIH ImageJ (available at https://imagej.net/Fiji/Downloads, and AnalyzeSkeleton plugins was download from http://imagej.net/AnalyzeSkeleton), processed using lookup tables grays, FFT bandpass filter, brightness contrast, filters unsharp mask, noise despeckle, threshold adjust, noise despeckle, binary close, removal of outliers and skeletonization. Neurite outgrowth of each prepared neuron was assessed using the NeuronJ plugin (ImageScience containing NeuronJ plugin must be added to the update list) to analyze skeleton. A series of concentric circles at 5-μm intervals were outlined around the soma, and branch crossings with each circle were automatically calculated. Images

for

neuronal

development

analysis

were

captured

by

an

Olympus-BX53 biological microscope (Olympus, Tokyo, Japan). 4.3 Animal Model of PDN

Male Sprague-Dawley rats (Laboratory Animal Center of Guangdong Province, Guangzhou, China), average weight of 180 to 220g, were housed in separate cages in standard laboratory environment, with food and water ad libitum. Diabetes mellitus was elicited by a single intraperitoneal administration of freshly dissolved STZ (60 mg/kg; Sigma) in sterile 0.1 M sodium citrate buffer (pH 4.5) and confirmed by determination of the blood glucose level with Accu-Chek test strips (Roche Diagnostics, Indianapolis, IN, USA) 3 days after the STZ injection, with all rats presenting with high levels of blood glucose (>16.7 mmol/L) recruited for the diabetic groups15. Control animals (n = 6) were injected with vehicle alone. PDN rats were validated by determination of the nociceptive thresholds by means of von Frey filaments (Stoelting, Wood Dale, IL, USA). All diabetic rats in our study successfully developed mechanical hyperalgesia approximately 21 days after STZ injection. Notwithstanding the retarded growth rate, all diabetic rats remained relatively healthy (body weight gain: 5-10 g per week in the diabetic group versus 30-40


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g per week in the non-diabetic group). The intrathecal administrations in rats were commenced 3 weeks after the STZ or vehicle treatment. All experimental protocols were approved by the Animal Care Committee of Sun Yat-Sen University and conformed to the regulations on animal care accredited by the National Institutes of Health and the institutional animal ethical committee. 4.4 Viral Vector for Synapsin II Knockdown and Validation For local (dorsal horn) knockdown of synapsin II, an HIV-1-based recombinant pLVX-puro lentiviral vector harboring a short hairpin RNA (shRNA) targeting the synapsin II or a vacant control lentiviral vector was employed in this study54 . shRNA expression was driven by the human U6 promoter (pol III) cassette with the use of the constitutively active human cytomegalovirus (CMV) promoter to encode a EGFP gene as a marker of transduction. pLVX-puro vectors were generated by the means of a Lenti-X™ HT Packaging System (Clontech) with the human embryonic kidney 293T cell line (293T). Two different shRNAs sequences (5’- CGACCAAAGGTGGTCCGCGTCTC-3’; 5’-CGGACAGCAGCTTCATTGCCAAC-3’)

were

synthesized

to

mitigate

off-target effects and both cloned into the lentiviral vector. The vector was purified using an iodixanol gradient and ultracentrifugation. The product of the gradient underwent a buffer exchange procedure to remove the iodixanol and to further concentrate/purify the virus. The final virus in PBS had a titer of 1×108 viral particles/ml. The synapsin II shRNA or vacant lentiviral vector (1: 1) was intrathecally injected into rats anesthetized with sevoflurane in oxygen three weeks after STZ administration. The rats were allowed to recover for one week for stable transgenic expression prior to biological detection. The successful shRNA knockdown of synapsin II was validated by qPCR. The enlarged lumbar spinal cords were extracted for mRNA analysis. The RNA was extracted by the means of a homogenizing kit (Tel-Test, Friendswood, Texas, USA), which isolated the RNA from DNA and protein using chloroform, with total RNA precipitated with isopropanol. Contaminated DNA was discarded with Turbo DNA-free (Life Technologies, CA, USA) using a rigorous protocol, and 5 μg of purified RNA was reverse transcribed into cDNA (Invitrogen SuperScript III First-Strand Synthesis System). For quantification of



the resulting cDNA, qPCR was conducted using a SYBR Green method55. 4.5 Intrathecal Catheter Implantation and Drug Administration For intrathecal injections, a catheter was implanted as previously described15. Cannulae were implanted while the rats were under sodium pentobarbital (60 mg/kg, i.p.). With a 1-cm midline incision in the back, the muscles were retracted to expose the L4-5 vertebrae, and polyethylene tubing (PE-10) was inserted into the subarachnoid space and advanced 1.2 cm cephalad at the level of the enlarged spinal cord lumbar segments. In addition, the catheter was secured to the paraspinal muscle of the back and then tunneled subcutaneously to exit from the dorsal neck region, where it was secured to the skin. The rats were allowed to recover for 4 to 5 days prior to the administration of STZ or vehicle. The position of the PE-10 catheter was confirmed by the intrathecal administration of 15 μL of 2% lidocaine, which induced paralysis of both hind extremities. For treatments, the drugs (10 μL) were intrathecally administered, followed by normal saline (15 mL) once daily for 7 consecutive days. Rapamycin (5 μg dissolved in 4% DMSO in saline and sonicated prior to intrathecal injection), vehicle (4% DMSO in a volume equal to that of the rapamycin solution), synapsin II shRNA or control vector were injected as appropriate. 4.6 Behavioral Measurement For the measurement of mechanical hyperalgesia, rats (6 rats per group) were randomly placed in individual plexiglas compartments for acclimation for 20 to 30 min, and the mechanical withdrawal threshold (MWT) was evaluated using von Frey filaments (Stoelting, Wood Dale, IL, USA) with buckling forces between 2.0 and 15.0 g according to the up-down method56. Mechanical hyperalgesia was defined as a >50% decrease in the MWT compared to baseline values. 4.7 Enzyme-linked Immunosorbent Assay (ELISA) Analysis


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The spinal cord was homogenized in normal saline (NS), with homogenate frozen at -20°C for 5 min before centrifugation at 4,000 rpm for 15 min. The resultant supernatant was harvested to profile the TNF-α levels with TNF-α detection kits (KeyGen Biotech Company, Nanjing, China) following the manufacturer’s instructions. The concentration of TNF-α in the spinal tissue was calculated as ng/ml of protein. 4.8 Western Blotting Analysis Dorsal horn neurons and enlarged lumbar spinal cords were harvested52. The cells or tissues were homogenized and triturated in homogenization buffer (10 mM Tris-HCl pH 7.4, 5.5 mM NaF, 1 mM sodium orthovanadate, 320 mM sucrose, 1 mM EDTA, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 1 M leupeptin, and 2 mM pepstatin A), respectively. At the end of centrifugation at 1,000 g for 20 min at 4°C to remove the nuclei and debris, the supernatant was collected and thereafter the samples were heated for 5 min at 95°C. After boiling in 5× sample buffer, the protein samples (50-μg) were electrophoresed by SDS-PAGE and transferred onto a PVDF membrane. Rinsed with PBS, the PVDF membranes were blocked with 5% non-fat dry milk for 1h at room temperature (r/t) and probed with primary antibodies targeting phospho-mTOR (p-mTOR, 1:1000 dilution, ser-2448, Abcam, Cambridge, MA, USA), mTOR (1:1000 dilution, rabbit polyclonal, Cell Signaling Technology, Danvers, MA, USA), phospho-p70S6K (p-S6K, 1:1000 dilution, thr-389, Abcam), p70S6K (1:1000 dilution, Abcam), synapsin II (1:1000 dilution, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) overnight at 4°C. Having been rinsed, the membranes were incubated with an HRP-conjugated mouse anti-GAPDH antibody (1:10,000; Hangzhou Xianzhi Biological Co. Ltd., Hangzhou, China). GAPDH was employed as the loading control. The proteins were detected with anti-rabbit or anti-mouse secondary antibodies and visualized by the chemiluminescence reagents from the Millipore ECL Kit (Millipore, NJ, USA), followed by exposure to Hyperfilm (Kodak). The densitometry of the blots was analyzed with ImageJ software, and normalized to the density of GAPDH in the equal sample.



Page 20 of 38

4.9 Immunocytochemistry Immunocytochemistry staining of dorsal horn neurons and enlarged lumbar spinal cords were implemented as previously described52. Neurons were rinsed with PBS and fixed with 4% paraformaldehyde/4% sucrose for 10 min at r/t. After washes with PBS, cells were incubated in a solution of 1% Triton X-100 in PBS for 10 min to permeabilize the membranes at r/t. After three washes in PBS to remove the detergents, endogenous peroxidase and peroxidase-like activity was blocked by the cell incubation with PBS consisting of 10% goat serum/0.2% Tween 20 for 20 min. Then, the primary antibodies (i.e., anti-synapsin II, mouse monoclonal, 1:100 dilution, Abcam; anti-β-tubulin III, rabbit monoclonal, 1:100 dilution; Elabscience Biotechnology Co. Ltd., Wuhan, China) were added to the cells for 2h at r/t. Finally, the cells were rinsed with PBS and incubated with corresponding secondary antibodies. The rats were deeply anesthetized with chloral hydrate (400 mg/ kg) and perfused with 4% paraformaldehyde for immunohistochemistry. Enlarged lumbar spinal cords were harvested, post-fixed at 4°C for 4 h, and immersed in 30% (w/v) sucrose in PBS buffer for 24 h at 4°C. Transverse spinal cord sections (at the thickness of 25 μm) were blocked with 5% v/v normal goat serum in PBS buffer for 1 h at r/t and then incubated with the following primary antibodies: anti-synapsin II (rabbit monoclonal, 1:200 dilution; Santa Cruz Biotechnology) and anti-p-mTOR (mouse monoclonal, 1:200 dilution, Abcam) for 48 h at 4°C. Following the incubation of primary antibodies, species-specific secondary antibodies (goat anti-rabbit IgG, and Cy3-conjugated goat anti-mouse IgG, Boster Biological Technology Co. Ltd., Wuhan, China ) were employed at a 1:200 dilution in PBS buffer for 4 h at r/t in the darkness, thereafter the slices were stained with DAPI (1:5000 dilution). Finally, the sections were manipulated with a routine hematoxylin and eosin staining method as follows: counterstained with hematoxylin, dehydrated, cleared and cover-mounted. The spinal

sections

were

directly

stained

with

Cy3-conjugated

and

FITC-conjugated species-specific secondary antibodies as the specificity controls. The cells and sections were observed under an Olympus-BX53


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biological microscope (Olympus), with images photographed and analyzed with a Fluoview 1000 microscope (Olympus). 4.10 Preparation of Slice Cultures At the end of the final behavioral test, a portion of enlarged lumbar spinal cord (L4-L5) was isolated from rats under sevoflurane anesthetization according to the previous report2. The spinal cord segment was stored in preoxygenated, ice-cold Krebs solution (117 mM NaCl, 3.6 mM KCl, 2.5 mM CaCl2, 1.2 mM MgCl2, 1.2 mM NaH2PO4, 25 mM NaHCO3, and 11 mM glucose) saturated with 95% O2 and 5% CO2 at 36±1°C for at least 2-3 hours prior to the experiment. The spinal segment was placed in a shallow groove formed in an agar block and glued to the bottom of the microslicer stage. Transverse sections were cut on a vibrating microslicer at a thickness of 600 μm.

4.11 Electrophysiology The whole-cell patch-clamp recordings were conducted in voltage clamp mode from lamina II neurons as previously documented2. Under a dissecting microscope with transmitted illumination, the substantia gelatinosa (SG, lamina II) was clearly visible as a relatively translucent band across the dorsal horn. Patch pipettes (5-10 MΩ) were fabricated from thin-walled borosilicate glass-capillary tubing (1.5 mm outer diameter, World Precision Instruments) and were filled with internal solution containing the following: 135 mM potassium gluconate, 5 mM KCl, 0.5 mM CaCl2, 2 mM MgCl2, 5 mM EGTA, 5 mM HEPES, 5 mM ATP-Mg. With establishment of the whole-cell configuration, neurons were voltage-clamped at a potential of -70 mV to record the spontaneous excitatory postsynaptic currents (sEPSCs). After the baseline recordings, neurons were treated with TNF-α (10 ng/ml, i.e. 0.59 nM) for 3 min, which was dilated in Kreb's solution (pH ≈ 7.2). Despite a previous study that exposure to TNF-α at the concentration of 10 ng/ml but not 1 ng/ml induced a marked increase in the sEPSCs, we did not adopt higher concentrations on the grounds that TNF-α may have neurotoxic effects at concentrations >10 ng/ml.



Membrane currents were amplified with an Axopatch 200B amplifier (Axon Instruments), with recording filtered at 2 kHz and digitized at 5 kHz. Data were acquired with pCLAMP 10.5.1 software and analyzed with a Mini Analysis program (Synaptosoft Inc., Leonia, NJ, USA). 4.12 Data Analysis and Statistics All data are represented as the means ± SEM. Data analysis and statistical comparisons were performed by means of SPSS 15.0 (SPSS Inc., Chicago, IL, USA). When normality and homogeneity of variance assumptions were eligible, value variations were assessed. The differences in the expression of spinal p-mTOR, p-S6K synapsin II, TNF-α, and MWT between groups were tested using one-way ANOVAs followed by Student-Newman-Keuls tests or the difference in between-group comparisons, such as spinal synapsin II expression, were analyzed by Student’s t-tests, with differences in p-mTOR, p-S6K, synapsin II expression and morphological analysis in dorsal horn neurons among groups compared by the multifactor analysis of variance, followed by the LSD method (Fisher's Least Significant Difference test) or by Student’s t-tests providing only two groups were analyzed. In the case of absence of homogeneity of variance by the logarithmic transformation, the equivalent non-parametric tests were adopted. For data from behavioral tests, a repeated measurement of general linear model ANOVAs was employed to compare data from various testing days. The criterion for statistical significance was set at P < 0.05.

AUTHORS’ CONTRIBUTION WYH, HBW, and BZ conceived the study and designed the experiments. WYH, QMX, and JH performed the experiments. WYH, JH and JW collected and analyzed the data. WYH and HBW drafted the manuscript. All authors have read and approved the final manuscript.

COMPETING INTERESTS


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The authors declare no competing interests.

ACKNOWLEDGMENTS This study was funded by the National Natural Science Foundation of China (No. 81771357), and the Natural Science Foundation of Guangdong Province (No. 2014A030313719 and No. 2017A030313587).The authors are sincerely grateful to Dr. Xiaoyan Yang for excellent technical support and Professor Pan Li for critical revision of the manuscript.

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Figure legends Figure 1. Effects of TNF-α and/or hyperglycemia on mTOR and synapsin II expression in spinal dorsal horn neuron cultures. (A) Representative images of mTOR, phospho-mTOR (p-mTOR), S6K, phospho-S6K (p-S6K) and synapsin II immunoreactive bands are presented in the upper left corner, with the respective loading control (GAPDH). (B-D) The relative protein expression levels of p-mTOR, p-S6K, and synapsin II in whole-cell extracts from dorsal horn cultures were analyzed by Western blotting. (E) Western blot analysis of the expression of synapsin II in whole-cell extracts from dorsal horn neurons transiently transfected with synapsin II shRNA lentiviral vector (60 nM) or negative control shRNA (1:1) on the third day of culture, with transfection duration maintained for 5 days. At the end of transfection, neurons were harvested for Western blotting analysis. Data are presented as the means ± SEM of at least five independent cell cultures. The abbreviations for groups of normal glucose control (D-mannitol 24.5 mM+5.5 mM D-glucose), normal glucose + TNF-α (10 ng/ml), hyperglucose (30 mM D-glucose), hyperglucose + TNF-α, hyperglucose + TNF-α + rapamycin (100 nM) are shown as Nor.Glu., Nor.Glu+TNF-α, Hy.Glu., Hy.Glu+TNF-α+Rapa. *P < 0.05 vs. Nor.Glu; #P < 0.05 vs. Hy.Glu; $P < 0.05 vs. Nor.Glu+TNF-α; &P < 0.05 vs. Hy.Glu+TNF-α.



Figure 2. Dorsal horn neuronal clusters and neurite outgrowth were assessed by immunocytochemistry double staining using anti-β-tubulin III antibody and anti-synapsin II antibody, respectively. (A-D) Exposure to TNF-α affects neuron morphology and promotes neurite outgrowth, especially under conditions of high glucose. (E) Rapamycin inhibits the activity of mTOR and prevents neurite outgrowth. (F) Knockdown of synapsin II inhibits neurite outgrowth. Neurons were transfected with vacant lentiviral vector or synapsin II shRNA for 5 days, followed by combined exposure to TNF-α and high glucose for 1 day. 18 imagines of each group were obtained and made quantitative analysis of neuronal clusters, including networks, mean network size and neuronal footprint by MINA macro of ImageJ. Magnification 400×; scale bar 100 μm. Figure 3. TNF-α promotes neurite outgrowth, especially in hyperglycemic conditions, and the inhibition of mTOR or synapsin II prevents neurite outgrowth in spinal dorsal horn neurons. (A-F) A representative image is shown for each neuronal morphological parameter and for neurite outgrowth stained using an anti-β-tubulin III antibody. Dorsal horn neurons were transfected with synapsin II shRNA lentiviral vector (60 nM) or negative control shRNA (1:1) on the third day of culture in high glucose medium. After transfection for 5 days, neurons were harvested for immunocytochemistry staining, and images were recorded. Magnification 200×; scale bar 50 μm. (G) Quantification of axonal length. (H–J) Quantification of TDBTN, ADBL and TDBL in dorsal horn neurons after 7 days of exposure to high glucose and (or) 1 day of TNF-α. Data are presented as the means ± SEM. 30 neurons were analyzed in each condition. *P < 0.05 vs. Nor.Glu; #P < 0.05 vs. Hy.Glu; $P < 0.05 vs. Nor.Glu+TNF-α; &P < 0.05 vs. Hy.Glu+TNF-α. Figure 4. Intrathecal administration of rapamycin inhibited the expression of spinal synapsin II and relieved hyperalgesia in STZ-induced diabetic rat model.


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(A) Effects of intrathecal rapamycin injections on MWT in PDN rats in comparison with the normal controls. The MWT in response to mechanical stimulation by von Frey filaments was measured at the indicated time points. Means ± SD, n = 6 per group. (B-E) Western blot analysis of mTOR, p-mTOR, S6K, p-S6K and synapsin II expression in the spinal cord of PDN rats. (B) The blots show the changes in protein expression levels. (C-E) Statistical analysis of the expression levels of mTOR, p-mTOR (C), p-S6K (D) and synapsin II (E) in total spinal cord extracts from different groups. (F) The levels of spinal TNF-α in PDN rats, PDN rats with rapamycin and normal control rats by ELISA. The abbreviations for groups of the normal control, pain diabetic neuropathy (PDN) + vehicle (4% DMSO), and PDN + rapamycin (10 μg) are shown as CON, PDN and PDN + RAP, respectively. Mean ± SEM, n = 4 per group. *P < 0.05 vs. CON; #P < 0.05 vs. PDN.

Figure 5. Synapsin II and p-mTOR were upregulated and co-expressed in the spinal dorsal horn of rats with painful diabetic neuropathy (A, B, E, F, I, J) Expression levels of synapsin II and p-mTOR were increased in the spinal dorsal horn of STZ-induced diabetic rats. (H-M) Representative digital images are shown of neurons double-immunolabeled with synapsin II (green) and p-mTOR (red). Synapsin II-positive staining was concentrated in the spinal dorsal horn and colocalized with p-mTOR; scale bar = 50 μm. Figure 6. Spinal inhibition of synapsin II (shRNA) expression attenuated hyperalgesia in STZ-induced diabetic rats. (A-B) Western blot analysis of the expression of synapsin II in the spinal cord of rats from the PDN model. Protein expression levels of synapsin II were reduced in synapsin II shRNA-transfected rats compared with those of the vacant lentiviral vector control. (A) Representative immunoblots of spinal synapsin II expression in the groups. (B) Statistical analysis of synapsin II expression in the different groups. n = 4 per group. &P < 0.05 vs. Control. (C)



The MWT response to mechanical stimulation by von Frey filaments was assessed. Means ± SEM values, n = 6 per group. The abbreviations for groups of the normal control, pain diabetic neuropathy (PDN) + vacant lentiviral vector, and PDN + synapsin II shRNA are shown as CON, PDN and SHP, respectively. *P < 0.05 vs. CON; #P < 0.05 vs. PDN.

Figure 7. Effects of rapamycin or synapsin II shRNA intrathecal injections on excitatory synaptic transmission, as evidenced by sEPSC amplitude and frequency in spinal lamina II neurons. (A) Patch-clamp recordings of sEPSC frequency in spinal cord slices obtained from naïve rats and diabetic rats. (B and C) Patch-clamp recordings of the amplitudes (C) and mean sEPSC (mEPSC, B) in spinal cord slices obtained from naïve rats and diabetic rats. Spinal lamina II neurons showed an increase in sEPSC and mEPSC amplitude in diabetic rats compared with those of naïve rats, but this increase was abolished in rats treated with rapamycin (10 μg per day for 7 days) or synapsin II shRNA. (D) Compared with naïve rats, the increased frequency in the diabetic rats was abolished by rapamycin and was partially reduced by etanercept. The abbreviations for the groups of normal control, diabetes, diabetes + rapamycin, and diabetes + shRNA are CON, PDN, RAP, and shRNA, respectively. *P < 0.05 vs. CON; #P < 0.05 vs. PDN.


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Contributions of mTOR Activation-Mediated Upregulation of Synapsin

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