Impaired Protein Degradation Research Articles

Autophagy stimulus affects different kinase pathways and promotes HuR protein activation and SQSTM1/p62 protein synthesis in ARPE‐19 cells

PurposeAge‐related macular degeneration (AMD) pathogenesis is characterized by protein degradation impairment in retinal pigment epithelial (RPE) cells. We previously found that the expression of autophagy receptor SQSTM1/p62 is positively regulated by the RNA‐binding HuR/ELAV protein. We investigated the effects of AICAR (autophagy inducer, 5‐aminoimidazole‐4‐ carboxamide‐1‐β‐D‐ribofuranoside) and MG‐132 (proteasome inhibitor) co‐treatment on HuR activation, p62 expression, and the kinases potentially involved.MethodsARPE‐19 cells were treated with MG‐132 (1 µM) and/or AICAR (2 mM) for increasing times (up to 2 h) and subjected to cell fractionation. SQSTM1/p62 mRNA and protein levels were measured by qRT‐PCR and Western blotting, respectively. HuR protein levels and its phosphorylated status were evaluated by Western blotting. The effects of puromycin (1 mM, protein synthesis inhibitor) and various kinase inhibitors were also tested.ResultsAICAR+MG‐132 co‐treatment for 2 h induces HuR protein up‐regulation, its cytoplasmic translocation and phosphorylation, as well as increased expression of p62 protein, being the latter one blunted by puromycin. AICAR+MG‐132 co‐treatment affects various kinases with differential outcomes.ConclusionsAICAR+MG‐132 co‐treatment leads to HuR activation and p62 protein translation. Different protein kinase pathways are likely involved in these events.

Histone deacetylase 6 delays motor neuron degeneration by ameliorating the autophagic flux defect in a transgenic mouse model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by the selective loss of motor neurons. Abnormal protein aggregation and impaired protein degradation are believed to contribute to the pathogenesis of this disease. Our previous studies showed that an autophagic flux defect is involved in motor neuron degeneration in the SOD1(G93A) mouse model of ALS. Histone deacetylase 6 (HDAC6) is a class II deacetylase that promotes autophagy by inducing the fusion of autophagosomes to lysosomes. In the present study, we showed that HDAC6 expression was decreased at the onset of disease and became extremely low at the late stage in ALS mice. Using lentivirus-HDAC6 gene injection, we found that HDAC6 overexpression prolonged the lifespan and delayed the motor neuron degeneration in ALS mice. Moreover, HDAC6 induced the formation of autolysosomes and accelerated the degradation of SOD1 protein aggregates in the motor neurons of ALS mice. Collectively, our results indicate that HDAC6 has neuroprotective effects in an animal model of ALS by improving the autophagic flux in motor neurons, and autophagosome-lysosome fusion might be a therapeutic target for ALS.

The transition zone protein Rpgrip1l regulates proteasomal activity at the primary cilium.

Mutations in RPGRIP1L result in severe human diseases called ciliopathies. To unravel the molecular function of RPGRIP1L, we analyzed Rpgrip1l(-/-) mouse embryos, which display a ciliopathy phenotype and die, at the latest, around birth. In these embryos, cilia-mediated signaling was severely disturbed. Defects in Shh signaling suggested that the Rpgrip1l deficiency causes an impairment of protein degradation and protein processing. Indeed, we detected a cilia-dependent decreased proteasomal activity in the absence of Rpgrip1l. We found different proteasomal components localized to cilia and identified Psmd2, a component of the regulatory proteasomal 19S subunit, as an interaction partner for Rpgrip1l. Quantifications of proteasomal substrates demonstrated that Rpgrip1l regulates proteasomal activity specifically at the basal body. Our study suggests that Rpgrip1l controls ciliary signaling by regulating the activity of the ciliary proteasome via Psmd2.

Modeling non-hereditary mechanisms of Alzheimer disease during apoptosis in yeast.

Impaired protein degradation and mitochondrial dysfunction are believed to contribute to neurodegenerative disorders, including Alzheimer disease (AD). In patients suffering from non-hereditary AD, UBB+1, the frameshift variant of ubiquitin B, accumulated in neurons affected by neurofibrillary tangles, which is a pathological hallmark. We established a yeast model expressing high levels of UBB+1, and could demonstrate that UBB+1 interfered with both the ubiquitin-proteasome system (UPS) and mitochondrial function. More precisely, UBB+1 promoted the mitochondrion-localized production of the basic amino acids arginine, ornithine, and lysine, which we identified as the decisive toxic event culminating in apoptosis. Inducing the UPS activity at mitochondria prevented the lethal basic amino acid accumulation and avoided UBB+1-triggered cell loss. The arginine/ornithine metabolism is altered in brains of AD patients, and VMS1, the mitochondrion-specific UPS component, co-existed with UBB+1 in neurofibrillary tangles. Therefore, our data suggest that aberrant basic amino acid synthesis is a crucial link between UPS dysfunction and mitochondrial damage during AD progression.

A start codon CMT1X mutation associated with transient encephalomyelitis causes complete loss of Cx32.

X-linked Charcot-Marie-Tooth disease (CMTX1) results from numerous mutations in the GJB1 gene encoding the gap junction protein connexin32 (Cx32) and is one of the commonest forms of inherited neuropathy. Owing to the expression of Cx32 not only in Schwann cells but also in oligodendrocytes, a subset of CMT1X patients develops central nervous system (CNS) clinical manifestations in addition to peripheral neuropathy. While most GJB1 mutations appear to cause peripheral neuropathy through loss of Cx32 function, the cellular mechanisms underlying the CNS manifestations remain controversial. A novel start codon GJB1 mutation (p.Met1Ile) has been found in a CMT1X patient presenting with recurrent episodes of transient encephalomyelitis without apparent signs of peripheral neuropathy. In order to clarify the functional consequences of this mutation, we examined the cellular expression of two different constructs cloned from genomic DNA including the mutated start codon. None of the cloned constructs resulted in detectable expression of Cx32 by immunocytochemistry or immunoblot, although mRNA was produced at normal levels. Furthermore, co-expression with the other major oligodendrocyte connexin, Cx47, had no negative effect on GJ formation by Cx47. Finally, lysosomal and proteasomal inhibition in cells expressing the start codon mutant constructs failed to recover any detection of Cx32 as a result of impaired protein degradation. Our results indicate that the Cx32 start codon mutation is equivalent to a complete loss of the protein with failure of translation, although transcription is not impaired. Thus, complete loss of Cx32 function is sufficient to produce CNS dysfunction with clinical manifestations.

Ubiquitin-dependent proteolysis in yeast cells expressing neurotoxic proteins.

Critically impaired protein degradation is discussed to contribute to neurodegenerative disorders, including Parkinson's, Huntington's, Alzheimer's, and motor neuron diseases. Misfolded, aggregated, or surplus proteins are efficiently degraded via distinct protein degradation pathways, including the ubiquitin-proteasome system, autophagy, and vesicular trafficking. These pathways are regulated by covalent modification of target proteins with the small protein ubiquitin and are evolutionary highly conserved from humans to yeast. The yeast Saccharomyces cerevisiae is an established model for deciphering mechanisms of protein degradation, and for the elucidation of pathways underlying programmed cell death. The expression of human neurotoxic proteins triggers cell death in yeast, with neurotoxic protein-specific differences. Therefore, yeast cell death models are suitable for analyzing the role of protein degradation pathways in modulating cell death upon expression of disease-causing proteins. This review summarizes which protein degradation pathways are affected in these yeast models, and how they are involved in the execution of cell death. I will discuss to which extent this mimics the situation in other neurotoxic models, and how this may contribute to a better understanding of human disorders.

The cleavage product of amyloid-β protein precursor sAβPPα modulates BAG3-dependent aggresome formation and enhances cellular proteasomal activity.

Alzheimer's disease (AD) is the major age-associated form of dementia characterized by gradual cognitive decline. Aberrant cleavage of the amyloid-β protein precursor (AβPP) is thought to play an important role in the pathology of this disease. Two principal AβPP processing pathways exist: amyloidogenic cleavage of AβPP resulting in production of the soluble N-terminal fragment sAβPPβ, amyloid-β (Aβ), which accumulates in AD brain, and the AβPP intracellular domain (AICD) sAβPPα, p3 and AICD are generated in the non-amyloidogenic pathway. Prevalence of amyloidogenic versus non-amyloidogenic processing leads to depletion of sAβPPα and an increase in Aβ. Although sAβPPα is a well-accepted neurotrophic protein, molecular effects of this fragment remains unknown. Different studies reported impaired protein degradation pathways in AD brain, pointing to a role of disturbed proteasomal activity in the pathogenesis of this disease. Here we studied the possible role of sAβPPα in Bag3-mediated selective macroautophagy and proteasomal degradation. Employing human IMR90 cells, HEK 293 cells, and primary neurons, we demonstrate that sAβPPα prevents the proteotoxic stress-induced increase of Bag3 at the protein and at the mRNA level indicating a transcriptional regulation. Intriguingly, p62 and LC3, two other key players of autophagy, were not affected. Moreover, the formation and the accumulation of disease-related protein aggregates were significantly reduced by sAβPPα. Interestingly, there was a significant increase of proteasomal activity by sAβPPα as demonstrated by using various proteasome substrates. Our findings demonstrate that sAβPPα modulates Bag3 expression, aggresome formation, and proteasomal activity, thereby providing first evidence for a function of sAβPPα in the regulation of proteostasis.

Attenuation of endoplasmic reticulum stress as a treatment strategy against ischemia/reperfusion injury.

Attenuation of endoplasmic reticulum stress as a treatment strategy against ischemia/reperfusion injury.

The importance of uromodulin as regulator of salt reabsorption along the thick ascending limb.

Uromodulin (UMOD), also named Tamm Horsfall protein, is the most abundant protein secreted in the urine under normal conditions. It was purified the first time in 1950 and since then considerable efforts have highlighted its importance in human pathophysiology. However, its precise biological functions still remain elusive. The clinical interest in UMOD derives from the evidence that UMOD genetic mutations result in tubulointerstitial nephropathies currently known as UMODassociated kidney disease (UAKD), rare genetic disorders characterized by hyperuricaemia, gout and a progressive decline of renal function [1]. In addition, UMOD has been proposed to modulate water and electrolyte homeostasis by acting on the main transporters expressed along the thick ascending limb (TAL) and the early distal convoluted tubule. The precursor undergoes extensive post-translational modifications through the endoplasmic reticulum (ER) and the Golgi apparatus, and ultimately it is targeted to the apical membrane [2]. From the luminal site, it is cleaved by an unknown protease and then released into the tubular fluid. The exact mechanism linking UMOD mutations to renal concentrating defect has not been fully characterized. It has been suggested that the filamentous gel-like structure of the extracellular domain may serve as a barrier to water permeability [3]. In vitro and in vivo studies have recently demonstrated that UMOD modulates the function of the Na-K-2Cl (NKCC2) co-transporter [4] and the renal outer medullary potassium (ROMK) channel [5] (Figure 1). UMOD-deficient mice showed normal electrolyte balance at basal and urine concentrating defect after water deprivation [6]. Impaired urine concentration was coupled with a compensatory up-regulation of distal Na transporters, including the Na-Cl co-transporter (NCC), suggesting indirectly an impaired function of the TAL. Immunostaining analysis revealed the absence of any difference in NKCC2 protein abundance on the apical membrane between knockout (KO) and wild-type (WT) mice, but an increased sub-apical immunoreactivity, with overall increased NKCC2 protein abundance compared with WT [4]. It is possible that an impaired protein degradation, in the absence of UMOD, resulted in NKCC2 accumulation. However, the same study demonstrated that phospho-NKCC2 levels, a marker of NKCC2 activity, were lower in KO than WT mice, and intraperitoneal injection of frusemide resulted in attenuated natriuretic and cloruretic responses, further supporting the hypothesis of reduced NKCC2 activity in the absence of UMOD. Consistent with these findings, in vitro NKCC2 phosphorylation was enhanced in the presence of UMOD, indicating that also in cultured cells UMOD promoted NKCC2 activity. This hypothesis has been further corroborated by recent observations linking salt-sensitive hypertension with a genetic UMOD variant leading to an increased UMOD synthesis and secretion in humans [7]. Transgenic mice expressing this genetic variant resembled human features, showing salt-sensitive hypertension. This finding correlated with the up-regulation of NKCC2, with increased protein phosphorylation via the STE20/SPS1-related proline/alanine-rich kinase (SPAK) and the down-regulation of the negative regulator kidney-specific KS-SPAK. Besides NKCC2, UMOD has been shown to modulate also the activity of ROMK. Renigunta et al. [5] have shown that UMOD co-localized with ROMK in a protein lysate from mice, while in oocytes, co-expression of UMOD and ROMK resulted in increased current amplitude, associated with an increased surface ROMK abundance. Patients suffering from UAKD commonly do manifest a defect in urine concentrating ability even before the decline of the GFR [8]. The mechanism by which UMOD mutations lead to urine concentrating defect in humans remains to be better elucidated. In this issue of NDT, Labriola et al. [9] show original data exploring the tubular function of a patient suffering UAKD during the early phase of the disease.

ESCRT-0 Protein Hepatocyte Growth Factor-regulated Tyrosine Kinase Substrate (Hrs) Is Targeted to Endosomes Independently of Signal-transducing Adaptor Molecule (STAM) and the Complex Formation with STAM Promotes Its Endosomal Dissociation

The ESCRT-0 complex, consisting of the hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) and the signal-transducing adaptor molecule (STAM) proteins, recognizes ubiquitylated cargo during the initial step of endosomal sorting. The endosomal accumulation of overexpressed Hrs has been reported previously to be associated with endosome enlargement. In this study, we have found that co-expressing exogenous STAM1 in Hrs-overexpressing cells leads to a diffuse localization for a large part of the Hrs accumulated on endosomes and a recovery of the impaired cargo protein degradation process, thus suggesting that exogenous STAM abrogates the abnormalities of the Hrs-positive endosomes. A fluorescently labeled Hrs, introduced into the cells by membrane permeabilization, exhibited endosomal localization in the absence of STAM1 and gradually dissociated from the endosomes upon the sequential addition of recombinant STAM1. Furthermore, when microinjected into cells, the fluorescently labeled Hrs also showed endosomal accumulation; however, ESCRT-0 complexes formed prior to the microinjection did not. Analysis of the state of the complex in HeLa cells using blue-native PAGE revealed that the membrane-associated Hrs exists partly as a monomer and not only in the STAM1-bound form. Thus, our data suggest that the membrane binding and dissociation cycle of the ESCRT-0 proteins on the endosomal membrane is a critical step during the cargo sorting process.

Proteasome inhibition and oxidative reactions disrupt cellular homeostasis during heme stress.

Dual control of cellular heme levels by extracellular scavenger proteins and degradation by heme oxygenases is essential in diseases associated with increased heme release. During severe hemolysis or rhabdomyolysis, uncontrolled heme exposure can cause acute kidney injury and endothelial cell damage. The toxicity of heme was primarily attributed to its pro-oxidant effects; however additional mechanisms of heme toxicity have not been studied systematically. In addition to redox reactivity, heme may adversely alter cellular functions by binding to essential proteins and impairing their function. We studied inducible heme oxygenase (Hmox1)-deficient mouse embryo fibroblast cell lines as a model to systematically explore adaptive and disruptive responses that were triggered by intracellular heme levels exceeding the homeostatic range. We extensively characterized the proteome phenotype of the cellular heme stress responses by quantitative mass spectrometry of stable isotope-labeled cells that covered more than 2000 individual proteins. The most significant signals specific to heme toxicity were consistent with oxidative stress and impaired protein degradation by the proteasome. This ultimately led to an activation of the response to unfolded proteins. These observations were explained mechanistically by demonstrating binding of heme to the proteasome that was linked to impaired proteasome function. Oxidative heme reactions and proteasome inhibition could be differentiated as synergistic activities of the porphyrin. Based on the present data a novel model of cellular heme toxicity is proposed, whereby proteasome inhibition by heme sustains a cycle of oxidative stress, protein modification, accumulation of damaged proteins and cell death.

Abstract 1325: Profilin-1 overexpression downregulates phosphorylation of p27kip1 leading to its nuclear accumulation in breast cancer cells

Abstract Profilin-1 is a ubiquitously expressed actin-binding protein that is downregulated in human breast cancer. Overexpression of profilin-1 in human breast cancer cells suppresses their tumorigenic potential. We had previously shown that profilin-1 overexpression elevates the expression level of p27kip1 leading to cell-cycle arrest in G1 phase. In the present work, we explore the underlying mechanism. Our studies showed that profilin-1-induced accumulation of p27kip1 in breast cancer cells occurs most dramatically in the nuclear compartment and this is due to impaired protein degradation. Consistent with these observations, we found that phosphorylation of p27kip1 at T187 (a prerequisite for p27kip1 degradation by SCF-Skp2 complex in the nucleus) and S10 (a post-translational event that promotes nuclear export of p27kip1) residues are both significantly inhibited by profilin-1 overexpression. Elevating profilin-1 level suppresses AKT activation, and hyper-activating AKT through overexpression of a constitutively active mutant relieves profilin-1-induced inhibition of T187 phosphorylation and elevation of p27kip1. These data demonstrate that profilin-1 can control the cellular level of p27kip1 in breast cancer cells through affecting its post-translational modification in an AKT-dependent pathway. These findings may provide mechanistic insights underlying the tumor-suppressive action of profilin-1. Citation Format: Chang Jiang, William Veon, Partha Roy. Profilin-1 overexpression downregulates phosphorylation of p27kip1 leading to its nuclear accumulation in breast cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1325. doi:10.1158/1538-7445.AM2014-1325

Autophagy stimulus promotes HuR protein phosphorylation and SQSTM1/p62 protein up‐regulation in ARPE‐19 cells

AbstractPurpose Age‐related macular degeneration (AMD) pathogenesis involves impaired protein degradation in retinal pigment epithelial (RPE) cells. Our recent observation reveals that the expression of autophagy receptor SQSTM1/p62 is positively regulated by HuR protein (mRNA‐stabilizing human Embryonic Lethal Abnormal Vision protein) (Viiri et al. 8(7):e69563, 2013, PLoS One). In this study, we investigated the effects of AICAR (autophagy inducer, 5‐aminoimidazole‐4‐carboxamide‐1‐β‐D‐ribofuranoside) with/without MG‐132 (proteasome inhibitor) on HuR post‐translational activation and p62 protein levels in ARPE‐19 cells.Methods ARPE‐19 cells were treated with MG‐132 (1µM) and/or AICAR (2mM) for increasing times (from 15 min to 2 h) and subjected to cell fractionation. HuR and SQSTM1/p62 protein levels were measured by Western blotting. HuR phosphorylation in threonine residues was evaluated by Western blotting following immunoprecipitation.Results AICAR+MG‐132 co‐treatment induces early translocation of HuR protein from the nucleus to the cytoplasm (already after 15 min). In this latter cellular fraction, after 2 h an increase of HuR protein phosphorylation (+94% vs control) and an up‐regulation of p62 protein levels are observed (+85% vs control).Conclusion In the cytoplasm of ARPE‐19 cells AICAR+MG‐132 co‐treatment leads to HuR protein activation, which in turn may determine the increased p62 protein expression.

Selective increase of in vivo firing frequencies in DA SN neurons after proteasome inhibition in the ventral midbrain.

The impairment of protein degradation via the ubiquitin-proteasome system (UPS) is present in sporadic Parkinson's disease (PD), and might play a key role in selective degeneration of vulnerable dopamine (DA) neurons in the substantia nigra pars compacta (SN). Further evidence for a causal role of dysfunctional UPS in familial PD comes from mutations in parkin, which results in a loss of function of an E3-ubiquitin-ligase. In a mouse model, genetic inactivation of an essential component of the 26S proteasome lead to widespread neuronal degeneration including DA midbrain neurons and the formation of alpha-synuclein-positive inclusion bodies, another hallmark of PD. Studies using pharmacological UPS inhibition in vivo had more mixed results, varying from extensive degeneration to no loss of DA SN neurons. However, it is currently unknown whether UPS impairment will affect the neurophysiological functions of DA midbrain neurons. To answer this question, we infused a selective proteasome inhibitor into the ventral midbrain in vivo and recorded single DA midbrain neurons 2 weeks after the proteasome challenge. We found a selective increase in the mean in vivo firing frequencies of identified DA SN neurons in anesthetized mice, while those in the ventral tegmental area (VTA) were unaffected. Our results demonstrate that a single-hit UPS inhibition is sufficient to induce a stable and selective hyperexcitability phenotype in surviving DA SN neurons in vivo. This might imply that UPS dysfunction sensitizes DA SN neurons by enhancing 'stressful pacemaking'.

Hepcidin knockout mice fed with iron-rich diet develop chronic liver injury and liver fibrosis due to lysosomal iron overload

Hepcidin is the central regulator of iron homeostasis and altered hepcidin signalling results in both hereditary and acquired iron overload. While the association between iron overload and development of end-stage liver disease is well established, the underlying mechanisms are largely unknown. To improve that, we analysed hepcidin knockout (KO) mice as a model of iron overload-associated liver disease. Hepcidin wild type (WT) and KO mice fed with 3% carbonyl iron-containing diet starting at one month of age were compared to age-matched animals kept on standard chow. Liver histology and serum parameters were used to assess the extent of liver injury and fibrosis. Iron distribution was determined by subcellular fractionation and electron microscopy. Among mice kept on iron-rich diet, 6 months old hepcidin KO mice (vs. WT) displayed profound hepatic iron overload (3,186 ± 411 vs. 1,045 ± 159 μg/mg tissue, p<0.005), elevated liver enzymes (ALT: KO 128 ± 6, WT 56 ± 5 IU/L, p<0.05), mild hepatic inflammation and hepatocellular apoptosis. Twelve, but not six months old KO mice fed with iron-rich diet developed moderate liver fibrosis. The liver injury was accompanied by a marked lysosomal iron overload and lysosomal fragility with release of cathepsin B into the cytoplasm. Increased p62 levels and autofluorescent iron complexes suggested impaired protein degradation. As a mechanism leading to lysosomal iron overload, the autophagy (lysosomal influx) was increased. Hepcidin KO mice represent a novel model of iron overload-related liver diseases and implicate lysosomal injury as a crucial event in iron toxicity.

MTOR-independent, autophagic enhancer trehalose prolongs motor neuron survival and ameliorates the autophagic flux defect in a mouse model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disorder caused by selective motor neuron degeneration. Abnormal protein aggregation and impaired protein degradation pathways may contribute to the disease pathogenesis. Although it has been reported that autophagy is altered in patients and animal model of ALS, little is known about the role of autophagy in motor neuron degeneration in this disease. Our previous study shows that rapamycin, an MTOR-dependent autophagic activator, accelerates disease progression in the SOD1G93A mouse model of ALS. In the present report, we have assessed the role of the MTOR-independent autophagic pathway in ALS by determining the effect of the MTOR-independent autophagic inducer trehalose on disease onset and progression, and on motor neuron degeneration in SOD1G93A mice. We have found that trehalose significantly delays disease onset prolongs life span, and reduces motor neuron loss in the spinal cord of SOD1G93A mice. Most importantly, we have documented that trehalose decreases SOD1 and SQSTM1/p62 aggregation, reduces ubiquitinated protein accumulation, and improves autophagic flux in the motor neurons of SOD1G93A mice. Moreover, we have demonstrated that trehalose can reduce skeletal muscle denervation, protect mitochondria, and inhibit the proapoptotic pathway in SOD1G93A mice. Collectively, our study indicated that the MTOR-independent autophagic inducer trehalose is neuroprotective in the ALS model and autophagosome-lysosome fusion is a possible therapeutic target for the treatment of ALS.

De Novo Prion Aggregates Trigger Autophagy in Skeletal Muscle

In certain sporadic, familial, and infectious prion diseases, the prion protein misfolds and aggregates in skeletal muscle in addition to the brain and spinal cord. In myocytes, prion aggregates accumulate intracellularly, yet little is known about clearance pathways. Here we investigated the clearance of prion aggregates in muscle of transgenic mice that develop prion disease de novo. In addition to neurodegeneration, aged mice developed a degenerative myopathy, with scattered myocytes containing ubiquitinated, intracellular prion inclusions that were adjacent to myocytes lacking inclusions. Myocytes also showed elevated levels of the endoplasmic reticulum chaperone Grp78/BiP, suggestive of impaired protein degradation and endoplasmic reticulum stress. Additionally, autophagy was induced, as indicated by increased levels of beclin-1 and LC3-II. In C2C12 myoblasts, inhibition of autophagosome maturation or lysosomal degradation led to enhanced prion aggregation, consistent with a role for autophagy in prion aggregate clearance. Taken together, these findings suggest that the induction of autophagy may be a central strategy for prion aggregate clearance in myocytes. IMPORTANCE In prion diseases, the prion protein misfolds and aggregates in the central nervous system and sometimes in other organs, including muscle, yet the cellular pathways of prion aggregate clearance are unclear. Here we investigated the clearance of prion aggregates in the muscle of a transgenic mouse model that develops profound muscle degeneration. We found that endoplasmic reticulum stress pathways were activated and that autophagy was induced. Blocking of autophagic degradation in cell culture models led to an accumulation of aggregated prion protein. Collectively, these findings suggest that autophagy has an instrumental role in prion protein clearance.

Up-regulation of Synaptotagmin IV within amyloid plaque-associated dystrophic neurons in Tg2576 mouse model of Alzheimer’s disease

AimTo investigate the involvement of the vesicular membrane trafficking regulator Synaptotagmin IV (Syt IV) in Alzheimer’s disease pathogenesis and to define the cell types containing increased levels of Syt IV in the β-amyloid plaque vicinity.MethodsSyt IV protein levels in wild type (WT) and Tg2576 mice cortex were determined by Western blot analysis and immunohistochemistry. Co-localization studies using double immunofluorescence staining for Syt IV and markers for astrocytes (glial fibrillary acidic protein), microglia (major histocompatibility complex class II), neurons (neuronal specific nuclear protein), and neurites (neurofilaments) were performed in WT and Tg2576 mouse cerebral cortex.ResultsWestern blot analysis showed higher Syt IV levels in Tg2576 mice cortex than in WT cortex. Syt IV was found only in neurons. In plaque vicinity, Syt IV was up-regulated in dystrophic neurons. The Syt IV signal was not up-regulated in the neurons of Tg2576 mice cortex without plaques (resembling the pre-symptomatic conditions).ConclusionsSyt IV up-regulation within dystrophic neurons probably reflects disrupted vesicular transport or/and impaired protein degradation occurring in Alzheimer’s disease and is probably a consequence but not the cause of neuronal degeneration. Hence, Syt IV up-regulation and/or its accumulation in dystrophic neurons may have adverse effects on the survival of the affected neuron.

AICAR promotes cleansing of MG‐132‐induced protein aggregates in ARPE‐19 cells

AbstractPurpose Age‐related macular degeneration pathogenesis involves impaired protein degradation in retinal pigment epithelial (RPE) cells. The ubiquitin‐proteasome system and the autophagy pathway are major proteolytic processes in eukaryotic cells. SQSTM1/p62 has been shown as a key player linking the proteasomal and lysosomal clearance systems. The present study investigated the effects of AICAR (AICA ribonucleotide, 5‐aminoimidazole‐4‐carboxamide‐1‐β‐D‐ribofuranoside) with/without MG‐132 (proteasome inhibitor) on autophagy regulation in RPE cells.Methods ARPE‐19 cells were treated with MG‐132 (5µM) and/or AICAR (2mM). p62 and MAP1LC3A/LC3 (LC3II) were analyzed by Western blotting. LC3 lipidation was used to study autophagic flux. Transmission electron microscopy was used to detect protein aggregates and autophagosomes. pDendra2‐hLC3 construct was used to detect macroautophagy in confocal microscopy analysis.Results AICAR+MG‐132 co‐treatment induces autophagy clearance of p62 and increased LC3 lipidation. AICAR is able to completely abolish the MG‐132‐induced protein aggregation after 24 h treatment; at the same time, the co‐treatment increases the number of autophagic vesicles. Cells treated with MG‐132+AICAR exhibit a strong reduction of the perinuclear aggregates containing both LC3II and p62 proteins.Conclusion Autophagy is emerging as a novel target of therapies aimed to counteract protein aggregation and improve cell viability. In this way, our findings indicate that AICAR could be useful in the acceleration of protein clearance in RPE cells.

ELAVL1/HuR‐mediated accumulation of SQSTM1/p62 during proteasomal inhibition in human ARPE‐19 cells

AbstractPurpose Impaired protein degradation in retinal pigment epithelial (RPE) cells contributes to age‐related macular degeneration (AMD) pathogenesis. SQSTM1/p62 protein is involved in autophagy and proteasome‐mediated proteolysis, and expressed strongly under toxic stimuli. ELAVL1/HuR is a RNA‐binding protein which, in response to various stressors, regulates gene expression, finally affecting key cellular functions. Main aims of our study were to investigate whether p62 mRNA would be a target of HuR protein, and whether a link between these two proteins exists in RPE cells. Finally, the effects of proteasome inhibition and autophagy induction on p62 and HuR levels in RPE cells were also evaluated.Methods AlphaScreen technology and immunoprecipitation coupled with real‐time qPCR were used to study the binding between HuR protein and p62 mRNA. ARPE‐19 cells were treated with MG‐132 (proteasome inhibitor; 5µM) and/or AICAR (AICA ribonucleotide, 5‐aminoimidazole‐4‐carboxamide‐1‐β‐D‐ribofuranoside; 2mM). P62 and HuR levels were analyzed by real‐time qPCR and Western blotting. HuR silenced ARPE‐19 cells and negative control cells were also employed.Results MG‐132 treatment up‐regulates HuR, which binds p62 mRNA thus contributing, at post‐transcriptional level, to the increase of p62 protein level in ARPE‐19 cells. The addition of AICAR promoted cleansing by autophagy of p62, but not HuR.Conclusion p62, whose mRNA represents a novel target of HuR, is positively regulated as a cellular response to proteasome inhibition. P62 is degraded by autophagy‐mediated pathway, while HuR through proteasome. These findings may be relevant for AMD.