Tissue transglutaminase cross-links beclin 1 and regulates autophagy in MPP-treated human SH-SY5Y cells

Tissue transglutaminase (tTG) is a cross-linking enzyme involved in protein aggregation during Parkinson’s disease (PD) pathogenesis. Autophagy is inhibited by tTG activation via a mechanism in which cross-linking of beclin 1, an autophagy initiator at the level of the endoplasmic reticulum (ER), has been implicated. We reported increased tTG protein levels and activity at the ER in both PD brain and in a PD-mimicking cell system. Here we characterized the interaction between tTG and beclin 1 at the ER membrane and the role of tTG in reduced autophagy in an in vitro model of PD, using differentiated SH-SY5Y neurons treated with the PD-mimic MPP⁺. We found that under PD-mimicking conditions, beclin 1 and tTG partially colocalized at the ER, beclin 1 levels increased at the ER, and tTG readily cross-linked beclin 1 which was prevented by enzymatic blockade of tTG. Under these conditions, accumulation of beclin 1 at the ER was enhanced by inhibition of tTG activity. In line with these observations and the role of beclin 1 in autophagy, levels of the autophagy marker protein LC3II in MPP⁺-treated cells, were significantly increased by inhibition of tTG activity. Our data provide first evidence for a role of tTG-mediated regulation of beclin 1 and autophagy in MPP⁺-treated human SH-SY5Y cells.     

Insights into links between familial and sporadic Parkinson's disease: physical relationship between UCH-L1 variants and chaperone-mediated autophagy

Ubiquitin C-terminal hydrolase L1 (UCH-L1) is expressed abundantly in neurons and has been reported to be a major target of oxidative/carbonyl damage associated with sporadic Parkinson's disease (PD). The I93M mutation in UCH-L1 is also associated with familial PD. We recently reported that UCH-L1 physically interacts with LAMP-2A, the lysosomal receptor for chaperone-mediated autophagy (CMA), and Hsc70 and Hsp90, both of which can function as components of the CMA pathway. We found that the levels of these interactions were aberrantly increased by the I93M mutation, and that expression of I93M UCH-L1 in cells induced the CMA inhibition-associated increase in the amount of alpha-synuclein, a risk factor for PD. The interactions of UCH-L1 with LAMP-2A, Hsc70 and Hsp90 were also abnormally enhanced by carbonyl modification of UCH-L1. We propose that aberrant interactions of UCH-L1 variants with CMA machinery, at least partly, underlie the pathogenesis of I93M UCH-L1-associated PD, and possibly of sporadic PD. Our findings may provide novel insights into the links between familial and sporadic PD.     

Insights into links between familial and sporadic Parkinson's disease: Physical relationship between UCH-L1 variants and chaperone-mediated autophagy

Ubiquitin C-terminal hydrolase L1 (UCH-L1) is expressed abundantly in neurons and has been reported to be a major target of oxidative/carbonyl damage associated with sporadic Parkinson’s disease (PD). The I93M mutation in UCH-L1 is also associated with familial PD. We recently reported that UCH-L1 physically interacts with LAMP-2A, the lysosomal receptor for chaperone-mediated autophagy (CMA), and Hsc70 and Hsp90, both of which can function as components of the CMA pathway. We found that the levels of these interactions were aberrantly increased by the I93M mutation, and that expression of I93M UCH-L1 in cells induced the CMA inhibition-associated increase in the amount of α-synuclein, a risk factor for PD. The interactions of UCH-L1 with LAMP-2A, Hsc70 and Hsp90 were also abnormally enhanced by carbonyl modification of UCH-L1. We propose that aberrant interactions of UCH-L1 variants with CMA machinery, at least partly, underlie the pathogenesis of I93M UCH-L1-associated PD, and possibly of sporadic PD. Our findings may provide novel insights into the links between familial and sporadic PD.

Addendum to: Kabuta T, Furuta A, Aoki S, Furuta K, Wada K. Aberrant interaction between Parkinson disease-associated mutant UCH-L1 and the lysosomal receptor for chaperone-mediated autophagy. J Biol Chem 2008; Epub ahead of print.     

Selective Molecular Alterations in the Autophagy Pathway in Patients with Lewy Body Disease and in Models of α-Synucleinopathy

Background

Lewy body disease is a heterogeneous group of neurodegenerative disorders characterized by α-synuclein accumulation that includes dementia with Lewy bodies (DLB) and Parkinson''s Disease (PD). Recent evidence suggests that impairment of lysosomal pathways (i.e. autophagy) involved in α-synuclein clearance might play an important role. For this reason, we sought to examine the expression levels of members of the autophagy pathway in brains of patients with DLB and Alzheimer''s Disease (AD) and in α-synuclein transgenic mice.

Methodology/Principal Findings

By immunoblot analysis, compared to controls and AD, in DLB cases levels of mTor were elevated and Atg7 were reduced. Levels of other components of the autophagy pathway such as Atg5, Atg10, Atg12 and Beclin-1 were not different in DLB compared to controls. In DLB brains, mTor was more abundant in neurons displaying α-synuclein accumulation. These neurons also showed abnormal expression of lysosomal markers such as LC3, and ultrastructural analysis revealed the presence of abundant and abnormal autophagosomes. Similar alterations were observed in the brains of α-synuclein transgenic mice. Intra-cerebral infusion of rapamycin, an inhibitor of mTor, or injection of a lentiviral vector expressing Atg7 resulted in reduced accumulation of α-synuclein in transgenic mice and amelioration of associated neurodegenerative alterations.

Conclusions/Significance

This study supports the notion that defects in the autophagy pathway and more specifically in mTor and Atg7 are associated with neurodegeneration in DLB cases and α-synuclein transgenic models and supports the possibility that modulators of the autophagy pathway might have potential therapeutic effects.     

GBA deficiency promotes SNCA/α-synuclein accumulation through autophagic inhibition by inactivated PPP2A

Loss-of-function mutations in the gene encoding GBA (glucocerebrosidase, β, acid), the enzyme deficient in the lysosomal storage disorder Gaucher disease, elevate the risk of Parkinson disease (PD), which is characterized by the misprocessing of SNCA/α-synuclein. However, the mechanistic link between GBA deficiency and SNCA accumulation remains poorly understood. In this study, we found that loss of GBA function resulted in increased levels of SNCA via inhibition of the autophagic pathway in SK-N-SH neuroblastoma cells, primary rat cortical neurons, or the rat striatum. Furthermore, expression of the autophagy pathway component BECN1 was downregulated as a result of the GBA knockdown-induced decrease in glucocerebrosidase activity. Most importantly, inhibition of autophagy by loss of GBA function was associated with PPP2A (protein phosphatase 2A) inactivation via Tyr307 phosphorylation. C2-ceramide (C2), a PPP2A agonist, activated autophagy in GBA-silenced cells, while GBA knockdown-induced SNCA accumulation was reversed by C2 or rapamycin (an autophagy inducer), suggesting that PPP2A plays an important role in the GBA knockdown-mediated inhibition of autophagy. These findings demonstrate that loss of GBA function may contribute to SNCA accumulation through inhibition of autophagy via PPP2A inactivation, thereby providing a mechanistic basis for the increased PD risk associated with GBA deficiency.     

Autophagic clearance of Sin Nombre hantavirus glycoprotein Gn promotes virus replication in cells

Hantavirus glycoprotein precursor (GPC) is posttranslationally cleaved into two glycoproteins, Gn and Gc. Cells transfected with plasmids expressing either GPC or both Gn and Gc revealed that Gn is posttranslationally degraded. Treatment of cells with the autophagy inhibitors 3-methyladenine, LY-294002, or Wortmanin rescued Gn degradation, suggesting that Gn is degraded by the host autophagy machinery. Confocal microscopic imaging showed that Gn is targeted to autophagosomes for degradation by an unknown mechanism. Examination of autophagy markers LC3-I and LC3-II demonstrated that both Gn expression and Sin Nombre hantavirus (SNV) infection induce autophagy in cells. To delineate whether induction of autophagy and clearance of Gn play a role in the virus replication cycle, we downregulated autophagy genes BCLN-1 and ATG7 using small interfering RNA (siRNA) and monitored virus replication over time. These studies revealed that inhibition of host autophagy machinery inhibits Sin Nombre virus replication in cells, suggesting that autophagic clearance of Gn is required for efficient virus replication. Our studies provide mechanistic insights into viral pathogenesis and reveal that SNV exploits the host autophagy machinery to decrease the intrinsic steady-state levels of an important viral component for efficient replication in host cells.     

Paraquat, but not maneb, induces synucleinopathy and tauopathy in striata of mice through inhibition of proteasomal and autophagic pathways

SNCA and MAPT genes and environmental factors are important risk factors of Parkinson's disease [PD], the second-most common neurodegenerative disease. The agrichemicals maneb and paraquat selectively target dopaminergic neurons, leading to parkinsonism, through ill-defined mechanisms. In the current studies we have analyzed the ability of maneb and paraquat, separately and together, to induce synucleinopathy and tauopathy in wild type mice. Maneb was ineffective in increasing α-synuclein [α-Syn] or p-Tau levels. By contrast, paraquat treatment of mice resulted in robust accumulation of α-Syn and hyperphosphorylation of Tau in striata, through activation of p-GSK-3β, a major Tau kinase. Co-treatment with maneb did not enhance the effects of paraquat. Increased hyperacetylation of α-tubulin was observed in paraquat-treated mice, suggesting cytoskeleton remodeling. Paraquat, but not maneb, inhibited soluble proteasomal activity on a peptide substrate but this was not associated with a decreased expression of 26S proteasome subunits. Both paraquat and maneb treatments increased levels of the autophagy inhibitor, mammalian target of rapamycin, mTOR, suggesting impaired axonal autophagy, despite increases in certain autophagic proteins, such as beclin 1 and Agt12. Autophagic flux was also impaired, as ratios of LC3 II to LC3 I were reduced in treated animals. Increased mTOR was also observed in postmortem human PD striata, where there was a reduction in the LC3 II to LC3 I ratio. Heat shock proteins were either increased or unchanged upon paraquat-treatment suggesting that chaperone-mediated autophagy is not hampered by the agrichemicals. These studies provide novel insight into the mechanisms of action of these agrichemicals, which indicate that paraquat is much more toxic than maneb, via its inhibitory effects on proteasomes and autophagy, which lead to accumulation of α-Syn and p-Tau.     

The UCH-L1 gene encodes two opposing enzymatic activities that affect alpha-synuclein degradation and Parkinson's disease susceptibility

The assumption that each enzyme expresses a single enzymatic activity in vivo is challenged by the linkage of the neuronal enzyme ubiquitin C-terminal hydrolase-L1 (UCH-L1) to Parkinson's disease (PD). UCH-L1, especially those variants linked to higher susceptibility to PD, causes the accumulation of alpha-synuclein in cultured cells, an effect that cannot be explained by its recognized hydrolase activity. UCH-L1 is shown here to exhibit a second, dimerization-dependent, ubiquityl ligase activity. A polymorphic variant of UCH-L1 that is associated with decreased PD risk (S18Y) has reduced ligase activity but comparable hydrolase activity as the wild-type enzyme. Thus, the ligase activity as well as the hydrolase activity of UCH-L1 may play a role in proteasomal protein degradation, a critical process for neuronal health.     

Tyrosine kinase inhibition facilitates autophagic SNCA/α-synuclein clearance

The effects of ABL1/ABL inhibition on clearance of SNCA/α-synuclein were evaluated in animal models of α-synucleinopathies. Parkinson disease (PD) is a movement disorder characterized by death of dopaminergic substantia nigra (SN) neurons and brain accumulation of SNCA. The tyrosine kinase ABL1 is activated in several neurodegenerative diseases. An increase in ABL1 activity is detected in human postmortem PD brains. Lentiviral expression of SNCA in the mouse SN activates ABL1 via phosphorylation, while lentiviral Abl expression increases SNCA levels. Administration of the brain-penetrant tyrosine kinase inhibitor Nilotinib decreases Abl activity and facilitates autophagic clearance of SNCA in transgenic and lentiviral gene transfer models. Subcellular fractionation demonstrates accumulation of SNCA and hyperphosphorylated MAPT/Tau (p-MAPT) in autophagic vacuoles in SNCA-expressing brains, while Nilotinib treatment leads to protein deposition into the lysosomes, suggesting enhanced autophagic clearance. These data suggest that Nilotinib may be a therapeutic strategy to degrade SNCA in PD and other α-synucleinopathies.     

Streptococcus pneumoniae Induces Autophagy through the Inhibition of the PI3K-I/Akt/mTOR Pathway and ROS Hypergeneration in A549 Cells

The present study focused on the action mechanism of S. pneumoniae (Sp) in inducing autophagy in human alveolar epithelial cells. Sp, a gram-positive extracellular bacterium, activates autophagy with considerably increased microtuble-associated protein light chain 3 (LC3) punctation in A549 cells. The accumulation of typical autophagosomes and conjugation of LC3 to phosphatidylethanolamine were observed in Sp-infected cells as an indication of autophagy. Using the pneumolysin (PLY) mutant, we successfully demonstrated that PLY is involved in initiating autophagy without affecting the expression levels of PI3K-III and Beclin1. PLY-mediated autophagy depends on the inhibition of the phosphoinositide 3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway. Furthermore, Sp could also lead to the reactive oxygen species (ROS) hypergeneration in A549 cells. Taken together, Sp infection-induced autophagy is PLY-mediated through ROS hypergeneration and mTOR inhibition. PI3K-I and rapamycin (autophagy inducers) enhanced bacterial clearance, whereas wortmannin (autophagy inhibitor) and acetylcysteine (ROS inhibitor) reduced intracellular bacteria clearance. Thus, Sp-induced autophagy represents a host-protective mechanism, providing new insight into the pathogenesis of respiratory tract Sp infection.     

Effects of UCH-L1 on α-synuclein over-expression mouse model of Parkinson's disease

The rare inherited form of Parkinson's disease (PD), PARK5 , is caused by a missense mutation in ubiquitin carboxy-terminal hydrolase-L1 ( UCH-L1 ) gene, resulting in Ile93Met substitution in its gene product (UCH-L1^Ile93Met). PARK5 is inherited in an autosomal-dominant mode, but whether the Ile93Met mutation gives rise to a gain-of-toxic-function or loss-of-function of UCH-L1 protein remains controversial. Here, we investigated the selective vulnerabilities of dopaminergic (DA) neurons in UCH-L1-transgenic (Tg) and spontaneous UCH-L1-null gracile axonal dystrophy mice to an important PD-causing insult, abnormal accumulation of α-synuclein (αSyn). Immunohistochemistry of midbrain sections of a patient with sporadic PD showed αSyn- and UCH-L1-double-positive Lewy bodies in nigral DA neurons, suggesting physical and/or functional interaction between the two proteins in human PD brain. Recombinant adeno-associated viral vector-mediated over-expression of αSyn for 4 weeks significantly enhanced the loss of nigral DA cell bodies in UCH-L1^Ile93Met-Tg mice, but had weak effects in age-matched UCH-L1^wild-type-Tg mice and non-Tg littermates. In contrast, the extent of αSyn-induced DA cell loss in gracile axonal dystrophy mice was not significantly different from wild-type littermates at 13-weeks post-injection. Our results support the hypothesis that PARK5 is caused by a gain-of-toxic-function of UCH-L1^Ile93Met mutant, and suggest that regulation of UCH-L1 in nigral DA cells could be a future target for treatment of PD.     

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.     

Mitochondria drive autophagy pathology via microtubule disassembly

Neurons are exquisitely dependent on quality control systems to maintain a healthy intracellular environment. A permanent assessment of protein and organelle “quality” allows a coordinated action between repair and clearance of damage proteins and dysfunctional organelles. Impairments in the intracellular clearance mechanisms in long-lived postmitotic cells, like neurons, result in the progressive accumulation of damaged organelles and aggregates of aberrant proteins. Using cells bearing Parkinson disease (PD) patients’ mitochondria, we demonstrated that aberrant accumulation of autophagosomes in PD, commonly interpreted as an abnormal induction of autophagy, is instead due to defective autophagic clearance. This defect is a consequence of alterations in the microtubule network driven by mitochondrial dysfunction that hinder mitochondria and autophagosome trafficking. We uncover mitochondria and microtubule-directed traffic as main players in the regulation of autophagy in PD.     

Aberrant interaction between Parkinson disease-associated mutant UCH-L1 and the lysosomal receptor for chaperone-mediated autophagy

Parkinson disease (PD) is the most common neurodegenerative movement disorder. An increase in the amount of alpha-synuclein protein could constitute a cause of PD. Alpha-synuclein is degraded at least partly by chaperone-mediated autophagy (CMA). The I93M mutation in ubiquitin C-terminal hydrolase L1 (UCH-L1) is associated with familial PD. However, the relationship between alpha-synuclein and UCH-L1 in the pathogenesis of PD has remained largely unclear. In this study, we found that UCH-L1 physically interacts with LAMP-2A, the lysosomal receptor for CMA, and Hsc70 and Hsp90, which can function as components of the CMA pathway. These interactions were abnormally enhanced by the I93M mutation and were independent of the monoubiquitin binding of UCH-L1. In a cell-free system, UCH-L1 directly interacted with the cytosolic region of LAMP-2A. Expression of I93M UCH-L1 in cells induced the CMA inhibition-associated increase in the amount of alpha-synuclein. Our findings may provide novel insights into the molecular links between alpha-synuclein and UCH-L1 and suggest that aberrant interaction of mutant UCH-L1 with CMA machinery, at least partly, underlies the pathogenesis of PD associated with I93M UCH-L1.     

Autophagy Activation Is Involved in 3,4-Methylenedioxymethamphetamine (‘Ecstasy’)—Induced Neurotoxicity in Cultured Cortical Neurons

Autophagic (type II) cell death, characterized by the massive accumulation of autophagic vacuoles in the cytoplasm of cells, has been suggested to play pathogenetic roles in cerebral ischemia, brain trauma, and neurodegenerative disorders. 3,4-Methylenedioxymethamphetamine (MDMA or ecstasy) is an illicit drug causing long-term neurotoxicity in the brain. Apoptotic (type I) and necrotic (type III) cell death have been implicated in MDMA-induced neurotoxicity, while the role of autophagy in MDMA-elicited neurotoxicity has not been investigated. The present study aimed to evaluate the occurrence and contribution of autophagy to neurotoxicity in cultured rat cortical neurons challenged with MDMA. Autophagy activation was monitored by expression of microtubule-associated protein 1 light chain 3 (LC3; an autophagic marker) using immunofluorescence and western blot analysis. Here, we demonstrate that MDMA exposure induced monodansylcadaverine (MDC)- and LC3B-densely stained autophagosome formation and increased conversion of LC3B-I to LC3B-II, coinciding with the neurodegenerative phase of MDMA challenge. Autophagy inhibitor 3-methyladenine (3-MA) pretreatment significantly attenuated MDMA-induced autophagosome accumulation, LC3B-II expression, and ameliorated MDMA-triggered neurite damage and neuronal death. In contrast, enhanced autophagy flux by rapamycin or impaired autophagosome clearance by bafilomycin A1 led to more autophagosome accumulation in neurons and aggravated neurite degeneration, indicating that excessive autophagosome accumulation contributes to MDMA-induced neurotoxicity. Furthermore, MDMA induced phosphorylation of AMP-activated protein kinase (AMPK) and its downstream unc-51-like kinase 1 (ULK1), suggesting the AMPK/ULK1 signaling pathway might be involved in MDMA-induced autophagy activation.     

SGPL1 (sphingosine phosphate lyase 1) modulates neuronal autophagy via phosphatidylethanolamine production

Macroautophagy/autophagy defects have been identified as critical factors underlying the pathogenesis of neurodegenerative diseases. The roles of the bioactive signaling lipid sphingosine-1-phosphate (S1P) and its catabolic enzyme SGPL1/SPL (sphingosine phosphate lyase 1) in autophagy are increasingly recognized. Here we provide in vitro and in vivo evidence for a previously unidentified route through which SGPL1 modulates autophagy in neurons. SGPL1 cleaves S1P into ethanolamine phosphate, which is directed toward the synthesis of phosphatidylethanolamine (PE) that anchors LC3-I to phagophore membranes in the form of LC3-II. In the brains of SGPL1^fl/fl/Nes mice with developmental neural specific SGPL1 ablation, we observed significantly reduced PE levels. Accordingly, alterations in basal and stimulated autophagy involving decreased conversion of LC3-I to LC3-II and increased BECN1/Beclin-1 and SQSTM1/p62 levels were apparent. Alterations were also noticed in downstream events of the autophagic-lysosomal pathway such as increased levels of lysosomal markers and aggregate-prone proteins such as APP (amyloid β [A4] precursor protein) and SNCA/α-synuclein. In vivo profound deficits in cognitive skills were observed. Genetic and pharmacological inhibition of SGPL1 in cultured neurons promoted these alterations, whereas addition of PE was sufficient to restore LC3-I to LC3-II conversion, and control levels of SQSTM1, APP and SNCA. Electron and immunofluorescence microscopy showed accumulation of unclosed phagophore-like structures, reduction of autolysosomes and altered distribution of LC3 in SGPL1^fl/fl/Nes brains. Experiments using EGFP-mRFP-LC3 provided further support for blockage of the autophagic flux at initiation stages upon SGPL1 deficiency due to PE paucity. These results emphasize a formerly overlooked direct role of SGPL1 in neuronal autophagy and assume significance in the context that autophagy modulators hold an enormous therapeutic potential in the treatment of neurodegenerative diseases.     

Resveratrol-activated AMPK/SIRT1/autophagy in cellular models of Parkinson's disease

Excessive misfolded proteins and/or dysfunctional mitochondria, which may cause energy deficiency, have been implicated in the etiopathogenesis of Parkinson's disease (PD). Enhanced clearance of misfolded proteins or injured mitochondria via autophagy has been reported to have neuroprotective roles in PD models. The fact that resveratrol is a known compound with multiple beneficial effects similar to those associated with energy metabolism led us to explore whether neuroprotective effects of resveratrol are related to its role in autophagy regulation. We tested whether modulation of mammalian silent information regulator 2 (SIRT1) and/or metabolic energy sensor AMP-activated protein kinase (AMPK) are involved in autophagy induction by resveratrol, leading to neuronal survival. Our results showed that resveratrol protected against rotenone-induced apoptosis in SH-SY5Y cells and enhanced degradation of α-synucleins in α-synuclein-expressing PC12 cell lines via autophagy induction. We found that suppression of AMPK and/or SIRT1 caused decrease of protein level of LC3-II, indicating that AMPK and/or SIRT1 are required in resveratrol-mediated autophagy induction. Moreover, suppression of AMPK caused inhibition of SIRT1 activity and attenuated protective effects of resveratrol on rotenone-induced apoptosis, further suggesting that AMPK-SIRT1-autophagy pathway plays an important role in the neuroprotection by resveratrol on PD cellular models.     

Stearoyl-CoA Desaturase 1 Activity Is Required for Autophagosome Formation

Autophagy is one of the major degradation pathways for cytoplasmic components. The autophagic isolation membrane is a unique membrane whose content of unsaturated fatty acids is very high. However, the molecular mechanisms underlying formation of this membrane, including the roles of unsaturated fatty acids, remain to be elucidated. From a chemical library consisting of structurally diverse compounds, we screened for novel inhibitors of starvation-induced autophagy by measuring LC3 puncta formation in mouse embryonic fibroblasts stably expressing GFP-LC3. One of the inhibitors we identified, 2,5-pyridinedicarboxamide, N2,N5-bis[5-[(dimethylamino)carbonyl]-4-methyl-2-thiazolyl], has a molecular structure similar to that of a known stearoyl-CoA desaturase (SCD) 1 inhibitor. To determine whether SCD1 inhibition influences autophagy, we examined the effects of the SCD1 inhibitor 28c. This compound strongly inhibited starvation-induced autophagy, as determined by LC3 puncta formation, immunoblot analyses of LC3, electron microscopic observations, and p62/SQSTM1 accumulation. Overexpression of SCD1 or supplementation with oleic acid, which is a catalytic product of SCD1 abolished the inhibition of autophagy by 28c. Furthermore, 28c suppressed starvation-induced autophagy without affecting mammalian target of rapamycin activity, and also inhibited rapamycin-induced autophagy. In addition to inhibiting formation of LC3 puncta, 28c also inhibited formation of ULK1, WIPI1, Atg16L, and p62/SQSTM1 puncta. These results suggest that SCD1 activity is required for the earliest step of autophagosome formation.     

Impairment of chaperone-mediated autophagy induces dopaminergic neurodegeneration in rats

Chaperone-mediated autophagy (CMA) involves the selective lysosomal degradation of cytosolic proteins such as SNCA (synuclein α), a protein strongly implicated in Parkinson disease (PD) pathogenesis. However, the physiological role of CMA and the consequences of CMA failure in the living brain remain elusive. Here we show that CMA inhibition in the adult rat substantia nigra via adeno-associated virus-mediated delivery of short hairpin RNAs targeting the LAMP2A receptor, involved in CMA's rate limiting step, was accompanied by intracellular accumulation of SNCA-positive puncta, which were also positive for UBIQUITIN, and in accumulation of autophagic vacuoles within LAMP2A-deficient nigral neurons. Strikingly, LAMP2A downregulation resulted in progressive loss of nigral dopaminergic neurons, severe reduction in striatal dopamine levels/terminals, increased astro- and microgliosis and relevant motor deficits. Thus, this study highlights for the first time the importance of the CMA pathway in the dopaminergic system and suggests that CMA impairment may underlie PD pathogenesis.