Dysregulated LRRK2 signaling in response to endoplasmic reticulum stress leads to dopaminergic neuron degeneration in C. elegans

Mutation of leucine-rich repeat kinase 2 (LRRK2) is the leading genetic cause of Parkinson's Disease (PD), manifested as age-dependent dopaminergic neurodegeneration, but the underlying molecular mechanisms remain unclear. Multiple roles of LRRK2 may contribute to dopaminergic neurodegeneration. Endoplasmic reticulum (ER) stress has also been linked to PD pathogenesis, but its interactive mechanism with PD genetic factors is largely unknown. Here, we used C. elegans, human neuroblastoma cells and murine cortical neurons to determine the role of LRRK2 in maintaining dopaminergic neuron viability. We found that LRRK2 acts to protect neuroblastoma cells and C. elegans dopaminergic neurons from the toxicity of 6-hydroxydopamine and/or human α-synuclein, possibly through the p38 pathway, by supporting upregulation of GRP78, a key cell survival molecule during ER stress. A pathogenic LRRK2 mutant (G2019S), however, caused chronic p38 activation that led to death of murine neurons and age-related dopaminergic-specific neurodegeneration in nematodes. These observations establish a critical functional link between LRRK2 and ER stress.     

Activation of the unfolded protein response in Parkinson's disease

Parkinson's disease (PD) is, at the neuropathological level, characterized by the accumulation of misfolded proteins. The presence of misfolded proteins can trigger a cellular stress response in the endoplasmic reticulum (ER) called the Unfolded Protein Response (UPR). The UPR has been shown to be involved in cellular models for PD. In this study, we investigated UPR activation in the substantia nigra of control and PD patients. Immunoreactivity for the UPR activation markers phosphorylated pancreatic ER kinase (pPERK) and phosphorylated eukaryotic initiation factor 2alpha (peIF2alpha) is detected in neuromelanin containing dopaminergic neurons in the substantia nigra of PD cases but not in control cases. In addition, pPERK immunoreactivity is colocalized with increased alpha-synuclein immunoreactivity in dopaminergic neurons. These data show that the UPR is activated in PD and that UPR activation is closely associated with the accumulation and aggregation of alpha-synuclein.     

ER Stress Induced by Tunicamycin Triggers α-Synuclein Oligomerization,Dopaminergic Neurons Death and Locomotor Impairment: a New Model of Parkinson’s Disease

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by progressive death of dopaminergic neurons of the substantia nigra pars compacta (SNpc), leading to the major clinical abnormalities that characterize this disease. Although PD’s etiology is unknown, α-synuclein aggregation plays a pivotal role in PD pathogenesis, which could be associated to some pathological processes such as oxidative stress, endoplasmic reticulum (ER) stress, impaired protein degradation, and mitochondrial dysfunction. Increasing experimental evidence indicates that ER stress is involved in PD, however most of the described results employed cultured cell lines and genetically modified animal models. In this study, we developed a new ER stress rat model employing the well-known ER stressor tunicamycin (Tm). To evaluate if ER stress was able to induce PD features, we performed an intranigral injection of Tm (0.1 μg/cerebral hemisphere) and animals (male Wistar rats) were analyzed 7 days post injection. The classical 6-OHDA neurotoxin model (1 μg/cerebral hemisphere) was used as an established positive control for PD. We show that Tm injection induced locomotor impairment, dopaminergic neurons death, and activation of astroglia. In addition, we observed an extensive α-synuclein oligomerization in SNpc of Tm-injected animals when compared with DMSO-injected controls. Finally, both Tm and 6-OHDA treated animals presented increased levels of ER stress markers. Taken together, these findings show for the first time that the ER stressor Tm recapitulates some of the phenotypic characteristics observed in rodent models of PD, reinforcing the concept that ER stress could be an important contributor to the pathophysiology of PD. Therefore, we propose the intranigral Tm injection as a new ER stress-based model for the study of PD in vivo.     

A new target for Parkinson’s disease: Small molecule SERCA activator CDN1163 ameliorates dyskinesia in 6-OHDA-lesioned rats

Endoplasmic reticulum (ER) stress is intimately linked to Parkinson’s disease (PD) pathophysiology. Disrupted intracellular calcium homeostasis is a major cause of the ER stress seen in dopaminergic neurons, leading to the cell death and subsequent loss of movement and coordination in patients. Dysfunctional calcium handling proteins play a major role in the promulgation of ER stress in PD. Specifically, compromised sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) has been identified as a major cause of ER stress and neuron loss in PD. We have identified a small molecule activator of SERCA that increases ER calcium content, rescues neurons from ER stress-induced cell death in vitro, and shows significant efficacy in the rat 6-hydroxydopamine (6-OHDA) model of PD. Together, these results support targeting SERCA activation as a viable strategy to develop disease-modifying therapeutics for PD.     

Neuroprotection by iron chelator against proteasome inhibitor-induced nigral degeneration

The cause of the neurodegenerative process in Parkinson's disease (PD) remains unclear, but evidence suggests that failure of the ubiquitin-proteasome system may play a major role in the pathogenesis of the disease. Iron is believed to be a key contributor to PD pathology by inducing aggregation of alpha-synuclein and by generating oxidative stress. Our present studies have shown that micro-injection of the proteasome inhibitor lactacystin into the substantia nigra (SN) of C57BL/6 mice causes significant loss of dopaminergic cells and induces intracellular inclusion body formation. We have also found that co-injection of the iron chelator desferrioxamine not only attenuates the lactacystin-induced dopamine neuron loss, but also reduces the presence of ubiquitin-positive intracellular inclusions in the SN, whereas use of iron-deficient diet has no such protective effects. These results may support that iron plays a key role in proteasome inhibitor-induced nigral pathology and that reducing iron reactivity may prevent dopaminergic neuron degeneration and reduce abnormal protein aggregation.     

The role of ubiquitin linkages on α-synuclein induced-toxicity in a Drosophila model of Parkinson's disease

Parkinson's disease (PD) is a common movement disorder marked by the loss of dopaminergic (DA) neurons in the brain stem and the presence of intraneuronal inclusions designated as Lewy bodies (LB). The cause of neurodegeneration in PD is not clear, but it has been suggested that protein misfolding and aggregation contribute significantly to the development of the disease. Misfolded and aggregated proteins are cleared by ubiquitin proteasomal system (UPS) and autophagy lysosomal pathway (ALP). Recent studies suggested that different types of ubiquitin linkages can modulate these two pathways in the process of protein degradation. In this study, we found that co-expression of ubiquitin can rescue neurons from α-syn-induced neurotoxicity in a Drosophila model of PD. This neuroprotection is dependent on the formation of lysine 48 polyubiquitin linkage which is known to target protein degradation via the proteasome. Consistent with our results that we observed in vivo , we found that ubiquitin co-expression in the cell can facilitate cellular protein degradation by the proteasome in a lysine 48 polyubiquitin-dependent manner. Taken together, these results suggest that facilitation of proteasomal protein degradation can be a potential therapeutic approach for PD.     

The relevance of iron in the pathogenesis of Parkinson's disease

Alterations of iron levels in the brain has been observed and documented in a number of neurodegenerative disorders including Parkinson's disease (PD). The elevated nigral iron levels observed in PD may reflect a dysfunction of brain iron homeostasis. Under normal physiological conditions excess iron can be sequestrated in ferritin and neuromelanin. Alternatively, the excess iron may represent a component of brain iron deposition associated with ageing. The aetiology of idiopathic PD largely remains an enigma. However, intensive investigations have provided a host of putative mechanisms that might contribute to the pathogenesis underlying the characteristic degeneration of the dopaminergic neurons in the substantia nigra (SN). The mechanisms proposed include oxidative (and nitrative) stress, inflammation, excitotoxicity, mitochondrial dysfunction, altered proteolysis and finally apoptotic induced cell death. Iron-mediated cellular destruction is mediated primarily via reactive oxygen or/and nitrogen species induced oxidative stress. Furthermore, these pathogenic mechanisms appear to be closely interlinked to the cascade of events leading to cellular death. There are conflicting reports about the stage during disease progression at which nigral iron change occurs in PD. Some have found that there are no changes in iron content SN in asymptomatic incidental Lewy body disease, suggesting it may represent a secondary event in the cascade of neuronal degeneration. In contrast, others have found an elevation of iron in SN in pre-clinical stages. These discrepancies may be attributed to the occurrence of different sub-groups of the disease. This concurs with the notion that PD represents a group of related diseases with a number of potential pathogenic pathways.     

Geraniol Protects Against the Protein and Oxidative Stress Induced by Rotenone in an In Vitro Model of Parkinson’s Disease

Dysfunction of autophagy, mitochondrial dynamics and endoplasmic reticulum (ER) stress are currently considered as major contributing factors in the pathogenesis of Parkinson’s disease (PD). Accumulation of oxidatively damaged cytoplasmic organelles and unfolded proteins in the lumen of the ER causes ER stress and it is associated with dopaminergic cell death in PD. Rotenone is a pesticide that selectively kills dopaminergic neurons by a variety of mechanism, has been implicated in PD. Geraniol (GE; 3,7-dimethylocta-trans-2,6-dien-1-ol) is an acyclic monoterpene alcohol occurring in the essential oils of several aromatic plants. In this study, we investigated the protective effect of GE on rotenone-induced mitochondrial dysfunction dependent oxidative stress leads to cell death in SK-N-SH cells. In addition, we assessed the involvement of GE on rotenone-induced dysfunction in autophagy machinery via α-synuclein accumulation induced ER stress. We found that pre-treatment of GE enhanced cell viability, ameliorated intracellular redox, preserved mitochondrial membrane potential and improves the level of mitochondrial complex-1 in rotenone treated SK-N-SH cells. Furthermore, GE diminishes autophagy flux by reduced autophagy markers, and decreases ER stress by reducing α-synuclein expression in SK-N-SH cells. Our results demonstrate that GE possess its neuroprotective effect via reduced rotenone-induced oxidative stress by enhanced antioxidant status and maintain mitochondrial function. Furthermore, GE reduced ER stress and improved autophagy flux in the neuroblastomal SK-N-SH cells. The present study could suggest that GE a novel therapeutic avenue for clinical intervention in neurodegenerative diseases especially for PD.     

Neuroinflammation and oxidative stress: Co-conspirators in the pathology of Parkinson’s disease

Parkinson’s disease (PD) is a complex disease, with genetics and environment contributing to the disease onset. Recent studies of causative PD genes have confirmed the involvement of cellular mechanisms engaged in mitochondrial and UPS dysfunction, oxidative stress and apoptosis in the progressive degeneration of the dopaminergic neurons in PD. In addition, clinical, epidemiological and experimental evidence has implicated neuroinflammation in the disease progression. This review will discuss neuroinflammation in PD, with particular focus on the genetic and toxin-based models of the disease. These studies have confirmed elevated oxidative stress and the pro-inflammatory response occurs early in the disease and these processes contribute to and/or exacerbate the nigro-striatal degeneration. In addition, the experimental models discussed here have also provided strong evidence that these pathways are an important link between the familial and sporadic causes of PD. The potential application of anti-inflammatory interventions in limiting the dopaminergic neuronal cell death in these models is discussed with evidence suggesting that the further investigation of their use as part of multi-targeted clinical trials is warranted.     

Proteasome inhibition represses unfolded protein response and Nox4, sensitizing vascular cells to endoplasmic reticulum stress-induced death

BACKGROUND: Endoplasmic reticulum (ER) stress has pathophysiological relevance in vascular diseases and merges with proteasome function. Proteasome inhibition induces cell stress and may have therapeutic implications. However, whether proteasome inhibition potentiates ER stress-induced apoptosis and the possible mechanisms involved in this process are unclear. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that proteasome inhibition with MG132, per se at non-lethal levels, sensitized vascular smooth muscle cells to caspase-3 activation and cell death during ER stress induced by tunicamycin (Tn). This effect was accompanied by suppression of both proadaptive (KDEL chaperones) and proapoptotic (CHOP/GADD153) unfolded protein response markers, although, intriguingly, the splicing of XBP1 was markedly enhanced and sustained. In parallel, proteasome inhibition completely prevented ER stress-induced increase in NADPH oxidase activity, as well as increases in Nox4 isoform and protein disulfide isomerase mRNA expression. Increased Akt phosphorylation due to proteasome inhibition partially offset the proapoptotic effect of Tn or MG132. Although proteasome inhibition enhanced oxidative stress, reactive oxygen species scavenging had no net effect on sensitization to Tn or MG132-induced cell death. CONCLUSION/RELEVANCE: These data indicate unfolded protein response-independent pathways whereby proteasome inhibition sensitizes vascular smooth muscle to ER stress-mediated cell death. This may be relevant to understand the therapeutic potential of such compounds in vascular disease associated with increased neointimal hyperplasia.     

Simultaneous inhibition of the ubiquitin-proteasome system and autophagy enhances apoptosis induced by ER stress aggravators in human pancreatic cancer cells

In contrast to normal tissue, cancer cells display profound alterations in protein synthesis and degradation. Therefore, proteins that regulate endoplasmic reticulum (ER) homeostasis are being increasingly recognized as potential therapeutic targets. The ubiquitin-proteasome system and autophagy are crucially important for proteostasis in cells. However, interactions between autophagy, the proteasome, and ER stress pathways in cancer remain largely undefined. This study demonstrated that withaferin-A (WA), the biologically active withanolide extracted from Withania somnifera, significantly increased autophagosomes, but blocked the degradation of autophagic cargo by inhibiting SNARE-mediated fusion of autophagosomes and lysosomes in human pancreatic cancer (PC) cells. WA specifically induced proteasome inhibition and promoted the accumulation of ubiquitinated proteins, which resulted in ER stress-mediated apoptosis. Meanwhile, the impaired autophagy at early stage induced by WA was likely activated in response to ER stress. Importantly, combining WA with a series of ER stress aggravators enhanced apoptosis synergistically. WA was well tolerated in mice, and displayed synergism with ER stress aggravators to inhibit tumor growth in PC xenografts. Taken together, these findings indicate that simultaneous suppression of 2 key intracellular protein degradation systems rendered PC cells vulnerable to ER stress, which may represent an avenue for new therapeutic combinations for this disease.     

Accumulation of wildtype and ALS-linked mutated VAPB impairs activity of the proteasome

Cellular homeostasis relies on a tight control of protein synthesis, folding and degradation, in which the endoplasmic reticulum (ER) quality control and the ubiquitin proteasome system (UPS) have an instrumental function. ER stress and aberrant accumulation of misfolded proteins represent a pathological signature of amyotrophic lateral sclerosis (ALS), a fatal paralytic disorder caused by the selective degeneration of motoneurons in the brain and spinal cord. Mutations in the ER-resident protein VAPB have been associated with familial forms of the disease. ALS-linked mutations cause VAPB to form cytoplasmic aggregates. We previously demonstrated that viral-mediated expression of both wildtype and mutant human VAPB (hVAPB) leads to an ER stress response that contributes to the selective death of motoneurons. However, the mechanisms behind ER stress, defective UPS and hVAPB-associated motoneuron degeneration remain elusive. Here, we show that the overexpression of wildtype and mutated hVAPB, which is found to be less stable than the wildtype protein, leads to the abnormal accumulation of ubiquitin and ubiquitin-like protein conjugates in non-human primate cells. We observed that overexpression of both forms of hVAPB elicited an ER stress response. Treatment of wildtype and mutated hVAPB expressing cells with the ER stress inhibitor salubrinal diminished the burden of ubiquitinated proteins, suggesting that ER stress contributes to the impairment of proteasome function. We also found that both wildtype and mutated hVAPB can associate with the 20S proteasome, which was found to accumulate at the ER with wildtype hVAPB or in mutant hVAPB aggregates. Our results suggest that ER stress and corruption of the proteasome function might contribute to the aberrant protein homeostasis associated with hVAPB.     

多巴胺能神经元线粒体的氧化损伤与帕金森病

帕金森病(Parkinson’s disease,PD)的一个主要病理特征就是中脑黑质多巴胺能神经元的丧失,目前研究认为该病理变化与多种因素有关,包括蛋白质异常积聚、泛素蛋白酶体系统功能异常、神经炎症、线粒体损伤和氧化应激。在帕金森病人和动物模型中,中脑黑质有着明显的氧化改变。帕金森病的遗传和环境因素均会作用于线粒体,尤其对线粒体呼吸链复合体I有着抑制作用,造成线粒体损伤,产生活性氧(ROS)。活性氧的大量产生造成脂类、蛋白质和DNA的氧化,从而加剧多巴胺能神经元的线粒体和细胞损伤。多巴胺代谢过程中会产生活性氧,该自身代谢特点决定了多巴胺能神经元存在有较高的氧化应激,易受环境因素的影响。因而,线粒体的氧化损伤在帕金森病病理发生中起着重要作用。     

Expression of mutant or cytosolic PrP in transgenic mice and cells is not associated with endoplasmic reticulum stress or proteasome dysfunction

The cellular pathways activated by mutant prion protein (PrP) in genetic prion diseases, ultimately leading to neuronal dysfunction and degeneration, are not known. Several mutant PrPs misfold in the early secretory pathway and reside longer in the endoplasmic reticulum (ER) possibly stimulating ER stress-related pathogenic mechanisms. To investigate whether mutant PrP induced maladaptive responses, we checked key elements of the unfolded protein response (UPR) in transgenic mice, primary neurons and transfected cells expressing two different mutant PrPs. Because ER stress favors the formation of untranslocated PrP that might aggregate in the cytosol and impair proteasome function, we also measured the activity of the ubiquitin proteasome system (UPS). Molecular, biochemical and immunohistochemical analyses found no increase in the expression of UPR-regulated genes, such as Grp78/Bip, CHOP/GADD153, or ER stress-dependent splicing of the mRNA encoding the X-box-binding protein 1. No alterations in UPS activity were detected in mutant mouse brains and primary neurons using the Ub(G76V)-GFP reporter and a new fluorogenic peptide for monitoring proteasomal proteolytic activity in vivo. Finally, there was no loss of proteasome function in neurons in which endogenous PrP was forced to accumulate in the cytosol by inhibiting cotranslational translocation. These results indicate that neither ER stress, nor perturbation of proteasome activity plays a major pathogenic role in prion diseases.     

Detrimental effects of proteasome inhibition activity in Drosophila melanogaster: implication of ER stress, autophagy, and apoptosis

In eukaryotes, the ubiquitin–proteasome machinery regulates a number of fundamental cellular processes through accurate and tightly controlled protein degradation pathways. We have, herein, examined the effects of proteasome functional disruption in Dmp53 ^+/+ (wild-type) and Dmp53 ^?/? Drosophila melanogaster fly strains through utilization of Bortezomib, a proteasome-specific inhibitor. We report that proteasome inhibition drastically shortens fly life-span and impairs climbing performance, while it also causes larval lethality and activates developmentally irregular cell death programs during oogenesis. Interestingly, Dmp53 gene seems to play a role in fly longevity and climbing ability. Moreover, Bortezomib proved to induce endoplasmic reticulum (ER) stress that was able to result in the engagement of unfolded protein response (UPR) signaling pathway, as respectively indicated by fly Xbp1 activation and Ref(2)P-containing protein aggregate formation. Larva salivary gland and adult brain both underwent strong ER stress in response to Bortezomib, thus underscoring the detrimental role of proteasome inhibition in larval development and brain function. We also propose that the observed upregulation of autophagy operates as a protective mechanism to “counterbalance” Bortezomib-induced systemic toxicity, which is tightly associated, besides ER stress, with activation of apoptosis, mainly mediated by functional Drice caspase and deregulated dAkt kinase. The reduced life-span of exposed to Bortezomib flies overexpressing Atg1_RNAi or Atg18_RNAi supports the protective nature of autophagy against proteasome inhibition-induced stress. Our data reveal the in vivo significance of proteasome functional integrity as a major defensive system against cellular toxicity likely occurring during critical biological processes and morphogenetic courses.     

Role of reactive oxygen species in the neurotoxicity of environmental agents implicated in Parkinson's disease

Among age-related neurodegenerative diseases, Parkinson's disease (PD) represents the best example for which oxidative stress has been strongly implicated. The etiology of PD remains unknown, yet recent epidemiological studies have linked exposure to environmental agents, including pesticides, with an increased risk of developing the disease. As a result, the environmental hypothesis of PD has developed, which speculates that chemical agents in the environment are capable of producing selective dopaminergic cell death, thus contributing to disease development. The use of environmental agents such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, rotenone, paraquat, dieldrin, and maneb in toxicant-based models of PD has become increasingly popular and provided valuable insight into the neurodegenerative process. Understanding the unique and shared mechanisms by which these environmental agents act as selective dopaminergic toxicants is critical in identifying pathways involved in PD pathogenesis. In this review, we discuss the neurotoxic properties of these compounds with specific focus on the induction of oxidative stress. We highlight landmark studies along with recent advances that support the role of reactive oxygen and reactive nitrogen species from a variety of cellular sources as potent contributors to the neurotoxicity of these environmental agents. Finally, human risk and the implications of these studies in our understanding of PD-related neurodegeneration are discussed.