Accumulation of p62 in degenerated spinal cord under chronic mechanical compression

Intracellular accumulation of altered proteins, including p62 and ubiquitinated proteins, is the basis of most neurodegenerative disorders. The relationship among the accumulation of altered proteins, autophagy, and spinal cord dysfunction by cervical spondylotic myelopathy has not been clarified. We examined the expression of p62 and autophagy markers in the chronically compressed spinal cord of tiptoe-walking Yoshimura mice. In addition, we examined the expression and roles of p62 and autophagy in hypoxic neuronal cells. Western blot analysis showed the accumulation of p62, ubiquitinated proteins, and microtubule-associated protein 1 light chain 3 (LC3), an autophagic marker, in the compressed spinal cord. Immunohistochemical examinations showed that p62 accumulated in neurons, axons, astrocytes, and oligodendrocytes. Electron microscopy showed the expression of autophagy markers, including autolysosomes and autophagic vesicles, in the compressed spinal cord. These findings suggest the presence of p62 and autophagy in the degenerated compressed spinal cord. Hypoxic stress increased the expression of p62, ubiquitinated proteins, and LC3-II in neuronal cells. In addition, LC3 turnover assay and GFP-LC3 cleavage assay showed that hypoxic stress increased autophagy flux in neuronal cells. These findings suggest that hypoxic stress induces accumulation of p62 and autophagy in neuronal cells. The forced expression of p62 decreased the number of neuronal cells under hypoxic stress. These findings suggest that p62 accumulation under hypoxic stress promotes neuronal cell death. Treatment with 3-methyladenine, an autophagy inhibitor decreased the number of neuronal cells, whereas lithium chloride, an autophagy inducer increased the number of cells under hypoxic stress. These findings suggest that autophagy promotes neuronal cell survival under hypoxic stress. Our findings suggest that pharmacological inducers of autophagy may be useful for treating cervical spondylotic myelopathy patients.     

Accumulation of p62 in degenerated spinal cord under chronic mechanical compression: functional analysis of p62 and autophagy in hypoxic neuronal cells

Intracellular accumulation of altered proteins, including p62 and ubiquitinated proteins, is the basis of most neurodegenerative disorders. The relationship among the accumulation of altered proteins, autophagy, and spinal cord dysfunction by cervical spondylotic myelopathy has not been clarified. We examined the expression of p62 and autophagy markers in the chronically compressed spinal cord of tiptoe-walking Yoshimura mice. In addition, we examined the expression and roles of p62 and autophagy in hypoxic neuronal cells. Western blot analysis showed the accumulation of p62, ubiquitinated proteins, and microtubule-associated protein 1 light chain 3 (LC3), an autophagic marker, in the compressed spinal cord. Immunohistochemical examinations showed that p62 accumulated in neurons, axons, astrocytes, and oligodendrocytes. Electron microscopy showed the expression of autophagy markers, including autolysosomes and autophagic vesicles, in the compressed spinal cord. These findings suggest the presence of p62 and autophagy in the degenerated compressed spinal cord. Hypoxic stress increased the expression of p62, ubiquitinated proteins, and LC3-II in neuronal cells. In addition, LC3 turnover assay and GFP-LC3 cleavage assay showed that hypoxic stress increased autophagy flux in neuronal cells. These findings suggest that hypoxic stress induces accumulation of p62 and autophagy in neuronal cells. The forced expression of p62 decreased the number of neuronal cells under hypoxic stress. These findings suggest that p62 accumulation under hypoxic stress promotes neuronal cell death. Treatment with 3-methyladenine, an autophagy inhibitor decreased the number of neuronal cells, whereas lithium chloride, an autophagy inducer increased the number of cells under hypoxic stress. These findings suggest that autophagy promotes neuronal cell survival under hypoxic stress. Our findings suggest that pharmacological inducers of autophagy may be useful for treating cervical spondylotic myelopathy patients.     

Suberoylanilide Hydroxamic Acid Triggers Autophagy by Influencing the mTOR Pathway in the Spinal Dorsal Horn in a Rat Neuropathic Pain Model

Histone acetylation levels can be upregulated by treating cells with histone deacetylase inhibitors (HDACIs), which can induce autophagy. Autophagy flux in the spinal cord of rats following the left fifth lumber spinal nerve ligation (SNL) is involved in the progression of neuropathic pain. Suberoylanilide hydroxamic acid (SAHA), one of the HDACIs can interfere with the epigenetic process of histone acetylation, which has been shown to ease neuropathic pain. Recent research suggest that SAHA can stimulate autophagy via the mammalian target of rapamycin (mTOR) pathway in some types of cancer cells. However, little is known about the role of SAHA and autophagy in neuropathic pain after nerve injury. In the present study, we aim to investigate autophagy flux and the role of the mTOR pathway on spinal cells autophagy activation in neuropathic pain induced by SNL in rats that received SAHA treatment. Autophagy-related proteins and mTOR or its active form were assessed by using western blot, immunohistochemistry, double immunofluorescence staining and transmission electron microscopy (TEM). We found that SAHA decreased the paw mechanical withdrawal threshold (PMWT) of the lower compared with SNL. Autophagy flux was mainly disrupted in the astrocytes and neuronal cells of the spinal cord dorsal horn on postsurgical day 28 and was reversed by daily intrathecal injection of SAHA (n?=?100 nmol/day or n?=?200 nmol/day). SAHA also decreased mTOR and phosphorylated mTOR (p-mTOR) expression, especially p-mTOR expression in astrocytes and neuronal cells of the spinal dorsal horn. These results suggest that SAHA attenuates neuropathic pain and contributes to autophagy flux in astrocytes and neuronal cells of the spinal dorsal horn via the mTOR signaling pathway.

     

The protective role of metformin in autophagic status in peripheral blood mononuclear cells of type 2 diabetic patients

Autophagy plays an important role in the pathophysiology of type 2 diabetes (T2D). Metformin is the most common antidiabetic drug. The main objective of this study was to explore the molecular mechanism of metformin in starvation‐induced autophagy in peripheral blood mononuclear cells (PBMCs) of type 2 diabetic patients. PBMCs were isolated from 10 diabetic patients and 7 non‐diabetic healthy volunteers. The autophagic puncta and markers were measured with the help of monodansylcadaverine staining and western blot. Additionally, transmission electron microscopy was also performed. No significant changes were observed in the initial autophagy marker protein levels in PBMCs of T2D after metformin treatment though diabetic PBMCs showed a high level of phospho‐mammalian target of rapamycin, p62 and reduced expression of phospho‐AMP‐activated protein kinase and lysosomal membrane‐associated protein 2, indicating a defect in autophagy. Also, induction of autophagy by tunicamycin resulted in apoptosis in diabetic PBMCs as observed by caspase‐3 cleavage and reduced expression of Bcl2. Inhibition of autophagy by bafilomycin rendered consistent expression of p62 indicating a defect in the final process of autophagy. Further, electron microscopic studies also confirmed massive vacuole overload and a sign of apoptotic cell death in PBMCs of diabetic patients, whereas metformin treatment reduced the number of autophagic vacuoles perhaps by lysosomal fusion. Thus, our results indicate that defective autophagy in T2D is associated with the fusion process of lysosomes which could be overcome by metformin.     

p62 filaments capture and present ubiquitinated cargos for autophagy

The removal of misfolded, ubiquitinated proteins is an essential part of the protein quality control. The ubiquitin‐proteasome system (UPS) and autophagy are two interconnected pathways that mediate the degradation of such proteins. During autophagy, ubiquitinated proteins are clustered in a p62‐dependent manner and are subsequently engulfed by autophagosomes. However, the nature of the protein substrates targeted for autophagy is unclear. Here, we developed a reconstituted system using purified components and show that p62 and ubiquitinated proteins spontaneously coalesce into larger clusters. Efficient cluster formation requires substrates modified with at least two ubiquitin chains longer than three moieties and is based on p62 filaments cross‐linked by the substrates. The reaction is inhibited by free ubiquitin, K48‐, and K63‐linked ubiquitin chains, as well as by the autophagosomal marker LC3B, suggesting a tight cross talk with general proteostasis and autophagosome formation. Our study provides mechanistic insights on how substrates are channeled into autophagy.     

miR-32-5p-mediated Dusp5 downregulation contributes to neuropathic pain

Previous studies have demonstrated that microRNAs (miRNAs) play important roles in the pathogenesis of neuropathic pain. In the present study, we found that miR-32-5p was significantly upregulated in rats after spinal nerve ligation (SNL), specifically in the spinal microglia of rats with SNL. Functional assays showed that knockdown of miR-32-5p greatly suppressed mechanical allodynia and heat hyperalgesia, and decreased inflammatory cytokine (IL-1β, TNF-α and IL-6) protein expression in rats after SNL. Similarly, miR-32-5p knockdown alleviated cytokine production in lipopolysaccharide (LPS)-treated spinal microglial cells, whereas its overexpression had the opposite effect. Mechanistic investigations revealed Dual-specificity phosphatase 5 (Dusp5) as a direct target of miR-32-5p, which is involved in the miR-32-5p-mediated effects on neuropathic pain and neuroinflammation. We demonstrated for the first time that miR-32-5p promotes neuroinflammation and neuropathic pain development through regulation of Dusp5. Our findings highlight a novel contribution of miR-32-5p to the process of neuropathic pain, and suggest possibilities for the development of novel therapeutic options for neuropathic pain.     

Galectin-3 Inhibition Is Associated with Neuropathic Pain Attenuation after Peripheral Nerve Injury

Neuropathic pain remains a prevalent and persistent clinical problem because it is often poorly responsive to the currently used analgesics. It is very urgent to develop novel drugs to alleviate neuropathic pain. Galectin-3 (gal3) is a multifunctional protein belonging to the carbohydrate-ligand lectin family, which is expressed by different cells. Emerging studies showed that gal3 elicits a pro-inflammatory response by recruiting and activating lymphocytes, macrophages and microglia. In the study we investigated whether gal3 inhibition could suppress neuroinflammation and alleviate neuropathic pain following peripheral nerve injury. We found that L5 spinal nerve ligation (SNL) increases the expression of gal3 in dorsal root ganglions at the mRNA and protein level. Intrathecal administration of modified citrus pectin (MCP), a gal3 inhibitor, reduces gal3 expression in dorsal root ganglions. MCP treatment also inhibits SNL-induced gal3 expression in primary rat microglia. SNL results in an increased activation of autophagy that contributes to microglial activation and subsequent inflammatory response. Intrathecal administration of MCP significantly suppresses SNL-induced autophagy activation. MCP also inhibits lipopolysaccharide (LPS)-induced autophagy in cultured microglia in vitro. MCP further decreases LPS-induced expression of proinflammatory mediators including IL-1β, TNF-α and IL-6 by regulating autophagy. Intrathecal administration of MCP results in adecreased mechanical and cold hypersensitivity following SNL. These results demonstrated that gal3 inhibition is associated with the suppression of SNL-induced inflammatory process andneurophathic pain attenuation.     

Proteotoxic Stress Induces Phosphorylation of p62/SQSTM1 by ULK1 to Regulate Selective Autophagic Clearance of Protein Aggregates

Disruption of proteostasis, or protein homeostasis, is often associated with aberrant accumulation of misfolded proteins or protein aggregates. Autophagy offers protection to cells by removing toxic protein aggregates and injured organelles in response to proteotoxic stress. However, the exact mechanism whereby autophagy recognizes and degrades misfolded or aggregated proteins has yet to be elucidated. Mounting evidence demonstrates the selectivity of autophagy, which is mediated through autophagy receptor proteins (e.g. p62/SQSTM1) linking autophagy cargos and autophagosomes. Here we report that proteotoxic stress imposed by the proteasome inhibition or expression of polyglutamine expanded huntingtin (polyQ-Htt) induces p62 phosphorylation at its ubiquitin-association (UBA) domain that regulates its binding to ubiquitinated proteins. We find that autophagy-related kinase ULK1 phosphorylates p62 at a novel phosphorylation site S409 in UBA domain. Interestingly, phosphorylation of p62 by ULK1 does not occur upon nutrient starvation, in spite of its role in canonical autophagy signaling. ULK1 also phosphorylates S405, while S409 phosphorylation critically regulates S405 phosphorylation. We find that S409 phosphorylation destabilizes the UBA dimer interface, and increases binding affinity of p62 to ubiquitin. Furthermore, lack of S409 phosphorylation causes accumulation of p62, aberrant localization of autophagy proteins and inhibition of the clearance of ubiquitinated proteins or polyQ-Htt. Therefore, our data provide mechanistic insights into the regulation of selective autophagy by ULK1 and p62 upon proteotoxic stress. Our study suggests a potential novel drug target in developing autophagy-based therapeutics for the treatment of proteinopathies including Huntington’s disease.     

Regulation of Autophagy and Ubiquitinated Protein Accumulation by bFGF Promotes Functional Recovery and Neural Protection in a Rat Model of Spinal Cord Injury

The role of autophagy in the recovery of spinal cord injury remains controversial; in particular, the mechanism of autophagy regulated degradation of ubiquitinated proteins has not been discussed to date. In this study, we investigated the protective role of basic fibroblast growth factor (bFGF) both in vivo and in vitro and demonstrated that excessive autophagy and ubiquitinated protein accumulation is involved in the rat model of trauma. bFGF administration improved recovery and increased the survival of neurons in spinal cord lesions in the rat model. The protective effect of bFGF is related to the inhibition of autophagic protein LC3II levels; bFGF treatment also enhances clearance of ubiquitinated proteins by p62, which also increases the survival of neuronal PC-12 cells. The activation of the downstream signals of the PI3K/Akt/mTOR pathway by bFGF treatment was detected both in vivo and in vitro. Combination therapy including the autophagy activator rapamycin partially abolished the protective effect of bFGF. The present study illustrates that the role of bFGF in SCI recovery is related to the inhibition of excessive autophagy and enhancement of ubiquitinated protein clearance via the activation of PI3K/Akt/mTOR signaling. Overall, our study suggests a new trend for bFGF drug development for central nervous system injuries and sheds light on protein signaling involved in bFGF action.     

Dynamics of the degradation of ubiquitinated proteins by proteasomes and autophagy: association with sequestosome 1/p62

Proteotoxicity resulting from accumulation of damaged/unwanted proteins contributes prominently to cellular aging and neurodegeneration. Proteasomal removal of these proteins upon covalent polyubiquitination is highly regulated. Recent reports proposed a role for autophagy in clearance of diffuse ubiquitinated proteins delivered by p62/SQSTM1. Here, we compared the turnover dynamics of endogenous ubiquitinated proteins by proteasomes and autophagy by assessing the effect of their inhibitors. Autophagy inhibitors bafilomycin A1, ammonium chloride, and 3-methyladenine failed to increase ubiquitinated protein levels. The proteasome inhibitor epoxomicin raised ubiquitinated protein levels at least 3-fold higher than the lysosomotropic agent chloroquine. These trends were observed in SK-N-SH cells under serum or serum-free conditions and in WT or Atg5(-/-) mouse embryonic fibroblasts (MEFs). Notably, chloroquine considerably inhibited proteasomes in SK-N-SH cells and MEFs. In these cells, elevation of p62/SQSTM1 was greater upon proteasome inhibition than with all autophagy inhibitors tested and was reduced in Atg5(-/-) MEFs. With epoxomicin, soluble p62/SQSTM1 associated with proteasomes and p62/SQSTM1 aggregates contained inactive proteasomes, ubiquitinated proteins, and autophagosomes. Prolonged autophagy inhibition (96 h) failed to elevate ubiquitinated proteins in rat cortical neurons, although epoxomicin did. Moreover, prolonged autophagy inhibition in cortical neurons markedly increased p62/SQSTM1, supporting its degradation mainly by autophagy and not by proteasomes. In conclusion, we clearly demonstrate that pharmacologic or genetic inhibition of autophagy fails to elevate ubiquitinated proteins unless the proteasome is affected. We also provide strong evidence that p62/SQSTM1 associates with proteasomes and that autophagy degrades p62/SQSTM1. Overall, the function of p62/SQSTM1 in the proteasomal pathway and autophagy requires further elucidation.     

Crosstalk between Spinal Astrocytes and Neurons in Nerve Injury-Induced Neuropathic Pain

Emerging research implicates the participation of spinal dorsal horn (SDH) neurons and astrocytes in nerve injury-induced neuropathic pain. However, the crosstalk between spinal astrocytes and neurons in neuropathic pain is not clear. Using a lumbar 5 (L5) spinal nerve ligation (SNL) pain model, we testified our hypothesis that SDH neurons and astrocytes reciprocally regulate each other to maintain the persistent neuropathic pain states. Glial fibrillary acidic protein (GFAP) was used as the astrocytic specific marker and Fos, protein of the protooncogene c-fos, was used as a marker for activated neurons. SNL induced a significant mechanical allodynia as well as activated SDH neurons indicated by the Fos expression at the early phase and activated astrocytes with the increased expression of GFAP during the late phase of pain, respectively. Intrathecal administration of c-fos antisense oligodeoxynucleotides (ASO) or astroglial toxin L-α-aminoadipate (L-AA) reversed the mechanical allodynia, respectively. Immunofluorescent histochemistry revealed that intrathecal administration of c-fos ASO significantly suppressed activation of not only neurons but also astrocytes induced by SNL. Meanwhile, L-AA shortened the duration of neuronal activation by SNL. Our data offers evidence that neuronal and astrocytic activations are closely related with the maintenance of neuropathic pain through a reciprocal “crosstalk”. The current study suggests that neuronal and non-neuronal elements should be taken integrally into consideration for nociceptive transmission, and that the intervention of such interaction may offer some novel pain therapeutic strategies.     

Altered neuropathic pain behaviour in a rat model of depression is associated with changes in inflammatory gene expression in the amygdala

The association between chronic pain and depression is widely recognized, the comorbidity of which leads to a heavier disease burden, increased disability and poor treatment response. This study examined nociceptive responding to mechanical and thermal stimuli prior to and following L5‐L6 spinal nerve ligation (SNL), a model of neuropathic pain, in the olfactory bulbectomized (OB) rat model of depression. Associated changes in the expression of genes encoding for markers of glial activation and cytokines were subsequently examined in the amygdala, a key brain region for the modulation of emotion and pain. The OB rats exhibited mechanical and cold allodynia, but not heat hyperalgesia, when compared with sham‐operated counterparts. Spinal nerve ligation induced characteristic mechanical and cold allodynia in the ipsilateral hindpaw of both sham and OB rats. The OB rats exhibited a reduced latency and number of responses to an innocuous cold stimulus following SNL, an effect positively correlated with interleukin (IL)‐6 and IL‐10 mRNA expression in the amygdala, respectively. Spinal nerve ligation reduced IL‐6 and increased IL‐10 expression in the amygdala of sham rats. The expression of CD11b (cluster of differentiation molecule 11b) and GFAP (glial fibrillary acidic protein), indicative of microglial and astrocyte activation, and IL‐1β in the amygdala was enhanced in OB animals when compared with sham counterparts, an effect not observed following SNL. This study shows that neuropathic pain‐related responding to an innocuous cold stimulus is altered in an animal model of depression, effects accompanied by changes in the expression of neuroinflammatory genes in the amygdala .     

p62 aggregates mediated Caspase 8 activation is responsible for progression of ovarian cancer

Increasing evidence suggests that p62/SQSTM1 functions as a signalling centre in cancer. However, the role of p62 in tumour development depends on the interacting factors it recruits and its precise regulatory mechanism remains unclear. In this study, we investigated the pro‐death signalling recruitment of p62 with the goal of improving anti‐tumour drug effects in ovarian cancer treatment. We found that p62 with Caspase 8 high expression is correlated with longer survival time compared with cases of low Caspase 8 expression in ovarian cancer. In vivo experiments suggested that insoluble p62 and ubiquitinated protein accumulation induced by autophagy impairment promoted the activation of Caspase 8 and increased cell sensitivity to cisplatin. Furthermore, p62 functional domain UBA and LIR mutants regulated autophagic flux and attenuated Caspase 8 activation, which indicates that autophagic degradation is involved in p62‐mediated activation of Caspase 8 in ovarian cancer cells. Collectively, our study demonstrates that p62 promotes Caspase 8 activation through autophagy flux blockage with cisplatin treatment. We have provided evidence that autophagy induction followed by its blockade increases cell sensitivity to chemotherapy which is dependent on p62‐Caspase 8 mediated apoptosis signalling. p62 exhibits pro‐death functions through its interaction with Caspase 8. p62 and Caspase 8 may become novel prognostic biomarkers and oncotargets for ovarian cancer treatment.     

p62 links the autophagy pathway and the ubiqutin–proteasome system upon ubiquitinated protein degradation

The ubiquitin–proteasome system (UPS) and autophagy are two distinct and interacting proteolytic systems. They play critical roles in cell survival under normal conditions and during stress. An increasing body of evidence indicates that ubiquitinated cargoes are important markers of degradation. p62, a classical receptor of autophagy, is a multifunctional protein located throughout the cell and involved in many signal transduction pathways, including the Keap1–Nrf2 pathway. It is involved in the proteasomal degradation of ubiquitinated proteins. When the cellular p62 level is manipulated, the quantity and location pattern of ubiquitinated proteins change with a considerable impact on cell survival. Altered p62 levels can even lead to some diseases. The proteotoxic stress imposed by proteasome inhibition can activate autophagy through p62 phosphorylation. A deficiency in autophagy may compromise the ubiquitin–proteasome system, since overabundant p62 delays delivery of the proteasomal substrate to the proteasome despite proteasomal catalytic activity being unchanged. In addition, p62 and the proteasome can modulate the activity of HDAC6 deacetylase, thus influencing the autophagic degradation.     

Knockdown of Linc00052 alleviated spinal nerve ligation-triggered neuropathic pain through regulating miR-448 and JAK1

The dysfunction of the nervous system contributes to neuropathic pain. Long noncoding RNAs are reported to participate in neuropathic pain. Recently, Linc00052 is implicated to be closely associated with multiple diseases. Nevertheless, the mechanisms of Linc00052 remain barely explored in neuropathic pain development. Currently, spinal nerve ligation (SNL) triggered neuropathic pain was employed in our investigation. Here, we assessed the function of Linc00052 in SNL rat models. Interestingly, we reported Linc00052 was significantly elevated in SNL rats. Loss of Linc00052 could reduce neuropathic pain progression via regulating the behaviors of neuropathic pain. Additionally, knockdown of Linc00052 repressed the processes of neuroinflammation. Interleukin (IL)-6 and tumor necrosis factor α level were inhibited while IL-10 was induced by the silence of Linc00052. Moreover, we predicted miR-448 can serve as a target of Linc00052. miR-448 exerts a crucial power in several diseases. Currently, we exhibited miR-448 was remarkably downregulated in SNL rats. RNA immunoprecipitation experiments validated the association between miR-448 and Linc00052. Inhibition of Linc00052 could reverse the roles of miR-448 on neuropathic pain development. Furthermore, Janus kinase 1 (JAK1) was displayed as the putative target of miR-448 in the present investigation. It was showed that JAK1 was induced in SNL rats. Loss of miR-448 could dramatically induce the expression of JAK1, which was rescued by knockdown of Linc00052. Taken these together, our study implied that Linc00052 functioned as a novel target of neuropathic pain via sponging miR-448 and regulating JAK1.     

Phasing out the bad—How SQSTM1/p62 sequesters ubiquitinated proteins for degradation by autophagy

The degradation of misfolded, ubiquitinated proteins is essential for cellular homeostasis. These proteins are primarily degraded by the ubiquitin-proteasome system (UPS) and macroautophagy/autophagy serves as a backup mechanism when the UPS is overloaded. How autophagy and the UPS are coordinated is not fully understood. During the autophagy of misfolded, ubiquitinated proteins, referred to as aggrephagy, substrate proteins are clustered into larger structures in a SQSTM1/p62-dependent manner before they are sequestered by phagophores, the precursors to autophagosomes. We have recently shown that SQSTM1/p62 and ubiquitinated proteins spontaneously phase separate into micrometer-sized clusters in vitro. This enabled us to characterize the properties of the ubiquitin-positive substrates that are necessary for the SQSTM1/p62-mediated cluster formation. Our results suggest that aggrephagy is triggered by the accumulation of substrates with multiple ubiquitin chains and that the process can be inhibited by active proteasomes.     

NBR1 co-operates with p62 in selective autophagy of ubiquitinated targets

Selective degradation of intracellular targets, such as misfolded proteins and damaged organelles, is an important homeostatic function that autophagy has acquired in addition to its more general role in restoring the nutrient balance during stress and starvation. Although the exact mechanism underlying selection of autophagic substrates is not known, ubiquitination is a candidate signal for autophagic degradation of misfolded and aggregated proteins. p62/SQSTM1 was the first protein shown to bind both target-associated ubiquitin (Ub) and LC3 conjugated to the phagophore membrane, thereby effectively acting as an autophagic receptor for ubiquitinated targets. Importantly, p62 not only mediates selective degradation but also promotes aggregation of ubiquitinated proteins that can be harmful in some cell types. Is p62 the only autophagic receptor for selective autophagy? Looking for proteins that interact with ATG8 family proteins, we identified NBR1 (neighbor of BRCA1 gene 1) as an additional LC3- and Ub-binding protein. NBR1 is degraded by autophagy depending on its LC3-interacting region (LIR) but does not strictly require p62 for this process. Like p62, NBR1 accumulates and aggregates when autophagy is inhibited and is a part of pathological inclusions. We propose that NBR1 together with p62 promotes autophagic degradation of ubiquitinated targets and simultaneously regulates their aggregation when autophagy becomes limited.     

The deubiquitinating enzyme USP36 controls selective autophagy activation by ubiquitinated proteins

Initially described as a nonspecific degradation process induced upon starvation, autophagy is now known also to be involved in the degradation of specific ubiquitinated substrates such as mitochondria, bacteria and aggregated proteins, ensuring crucial functions in cell physiology and immunity. We report here that the deubiquitinating enzyme USP36 controls selective autophagy activation in Drosophila and in human cells. We show that dUsp36 loss of function autonomously inhibits cell growth while activating autophagy. Despite the phenotypic similarity, dUSP36 is not part of the TOR signaling pathway. Autophagy induced by dUsp36 loss of function depends on p62/SQSTM1, an adaptor for delivering cargo marked by polyubiquitin to autophagosomes. Consistent with p62 requirement, dUsp36 mutant cells display nuclear aggregates of ubiquitinated proteins, including Histone H2B, and cytoplasmic ubiquitinated proteins; the latter are eliminated by autophagy. Importantly, USP36 function in p62-dependent selective autophagy is conserved in human cells. Our work identifies a novel, crucial role for a deubiquitinating enzyme in selective autophagy.     

Inhibiting astrocytic activation: a novel analgesic mechanism of ketamine at the spinal level?

Although ketamine is widely used as an analgesic agent and has an anti-allodynic effect on neuropathic pain, the underlying analgesic mechanisms are not fully explained by the modern 'neuronal-based' theories. As emerging studies have focused on the critical role of spinal astrocytes in the pathological pain states, we have hypothesized that there exist some 'astrocytes-related' mechanisms in the analgesic function of ketamine. In the present study, using the spinal nerve ligation (SNL) pain model, we investigated the anti-nociceptive effects of intraperitoneal or intrathecal ketamine on SNL-induced neuropathic pain response, meanwhile, we investigated the astrocytic activation after ketamine administration on SNL rats. Behavioral data showed that either intraperitoneal or intrathecal ketamine inhibited SNL-induced allodynia, however, immunohistochemistry showed that SNL induced astrocytic activation was suppressed by intrathecal but not intraperitoneal ketamine. Using quantitative Western blot analysis, our report showed that intrathecal ketamine down-regulated glial fibrillary acidic protein expression, suggesting inhibition of SNL-induced astrocytic activation, which wasn't influenced by intraperitoneal administration. We conclude that intraperitoneal ketamine could alleviate SNL-induced neuropathic pain via the classical 'neuronal-based' mechanisms, but in addition, 'astrocytes-related' mechanisms were also important underlying the anti-allodynic effect of intrathecal ketamine.     

BDNF Contributes to Spinal Long-Term Potentiation and Mechanical Hypersensitivity Via Fyn-Mediated Phosphorylation of NMDA Receptor GluN2B Subunit at Tyrosine 1472 in Rats Following Spinal Nerve Ligation

Previously we have demonstrated that brain-derived neurotrophic factor (BDNF) contributes to spinal long-term potentiation (LTP) and pain hypersensitivity through activation of GluN2B-containing N-methyl-d-aspartate (GluN2B-NMDA) receptors in rats following spinal nerve ligation (SNL). However, the molecular mechanisms by which BDNF impacts upon GluN2B-NMDA receptors and spinal LTP still remain unclear. In this study, we first documented that Fyn kinase-mediated phosphorylation of GluN2B subunit at tyrosine 1472 (pGluN2B^Y1472) was involved in BDNF-induced spinal LTP and pain hypersensitivity in intact rats. Second, we revealed a co-localization of Fyn and GluN2B-NMDA receptor in cultured dorsal horn neurons, implying that Fyn is a possible intermediate kinase linking BDNF/TrkB signaling with GluN2B-NMDA receptors in the spinal dorsal horn. Furthermore, we discovered that both SNL surgery and intrathecal active Fyn could induce an increased expression of dorsal horn pGluN2B^Y1472, as well as pain hypersensitivity in response to von Frey filaments stimuli; and more importantly, all these actions were effectively abrogated by pre-treatment with either PP2 or ifenprodil to respectively inhibit Fyn kinase and GluN2B-NMDA receptors activity. Moreover, we found that intrathecal administration of BDNF scavenger TrkB-Fc prior to SNL surgery, could prevent the nerve injury-induced increase of both pFyn^Y420 and pGluN2B^Y1472 expression, and also inhibit the mechanical allodynia in neuropathic rats. Collectively, these results suggest that Fyn kinase-mediated pGluN2B^Y1472 is critical for BDNF-induced spinal LTP and pain hypersensitivity in SNL rats. Therefore, the BDNF-Fyn-GluN2B signaling cascade in the spinal dorsal horn may constitute a key mechanism underlying central sensitization and neuropathic pain development after peripheral nerve injury.