Inhibition of endoplasmic reticulum associated degradation reduces endoplasmic reticulum stress and alters lysosomal morphology and distribution

Disturbances in proteostasis are observed in many neurodegenerative diseases. This leads to activation of protein quality control to restore proteostasis, with a key role for the removal of aberrant proteins by proteolysis. The unfolded protein response (UPR) is a protein quality control mechanism of the endoplasmic reticulum (ER) that is activated in several neurodegenerative diseases. Recently we showed that the major proteolytic pathway during UPR activation is via the autophagy/lysosomal system. Here we investigate UPR induction if the other major proteolytic pathway of the ER -ER associated degradation (ERAD)-is inhibited. Surprisingly, impairment of ERAD results in decreased UPR activation and protects against ER stress toxicity. Autophagy induction is not affected under these conditions, however, a striking relocalization of the lysosomes is observed. Our data suggest that a protective UPR-modulating mechanism is activated if ERAD is inhibited, which involves lysosomes. Our data provide insight in the cross-talk between proteolytic pathways involved in ER proteostasis. This has implications for neurodegenerative diseases like Alzheimer’s disease where disturbed ER proteostasis and proteolytic impairment are early phenomena in the pathology.     

Endoplasmic reticulum proteostasis impairment in aging

Perturbed neuronal proteostasis is a salient feature shared by both aging and protein misfolding disorders. The proteostasis network controls the health of the proteome by integrating pathways involved in protein synthesis, folding, trafficking, secretion, and their degradation. A reduction in the buffering capacity of the proteostasis network during aging may increase the risk to undergo neurodegeneration by enhancing the accumulation of misfolded proteins. As almost one‐third of the proteome is synthetized at the endoplasmic reticulum (ER), maintenance of its proper function is fundamental to sustain neuronal function. In fact, ER stress is a common feature of most neurodegenerative diseases. The unfolded protein response (UPR) operates as central player to maintain ER homeostasis or the induction of cell death of chronically damaged cells. Here, we discuss recent evidence placing ER stress as a driver of brain aging, and the emerging impact of neuronal UPR in controlling global proteostasis at the whole organismal level. Finally, we discuss possible therapeutic interventions to improve proteostasis and prevent pathological brain aging.     

Corrupted ER‐mitochondrial calcium homeostasis promotes the collapse of proteostasis

Aging and age‐related diseases are associated with a decline of protein homeostasis (proteostasis), but the mechanisms underlying this decline are not clear. In particular, decreased proteostasis is a widespread molecular feature of neurodegenerative diseases, such as Alzheimer's disease (AD). Familial AD is largely caused by mutations in the presenilin encoding genes; however, their role in AD is not understood. In this study, we investigate the role of presenilins in proteostasis using the model system Caenorhabditis elegans. Previously, we found that mutations in C. elegans presenilin cause elevated ER to mitochondria calcium signaling, which leads to an increase in mitochondrial generated oxidative stress. This, in turn, promotes neurodegeneration. To understand the cellular mechanisms driving neurodegeneration, using several molecular readouts of protein stability in C. elegans, we find that presenilin mutants have widespread defects in proteostasis. Markedly, we demonstrate that these defects are independent of the protease activity of presenilin and that reduction in ER to mitochondrial calcium signaling can significantly prevent the proteostasis defects observed in presenilin mutants. Furthermore, we show that supplementing presenilin mutants with antioxidants suppresses the proteostasis defects. Our findings indicate that defective ER to mitochondria calcium signaling promotes proteostatic collapse in presenilin mutants by increasing oxidative stress.     

The stress rheostat: an interplay between the unfolded protein response (UPR) and autophagy in neurodegeneration

The unfolded protein response (UPR) is a conserved adaptive reaction that increases cell survival under conditions of endoplasmic reticulum (ER) stress. The UPR controls diverse processes such as protein folding, secretion, ER biogenesis, protein quality control and macroautophagy. Occurrence of chronic ER stress has been extensively described in neurodegenerative conditions linked to protein misfolding and aggregation, including Amyotrophic lateral sclerosis, Prion-related disorders, and conditions such as Parkinson's, Huntington's, and Alzheimer's disease. Strong correlations are observed between disease progression, accumulation of protein aggregates, and induction of the UPR in animal and in vitro models of neurodegeneration. In addition, the first reports are available describing the engagement of ER stress responses in brain post-mortem samples from human patients. Despite such findings, the role of the UPR in the central nervous system has not been addressed directly and its contribution to neurodegeneration remains speculative. Recently, however, pharmacological manipulation of ER stress and autophagy - a stress pathway modulated by the UPR - using chemical chaperones and autophagy activators has shown therapeutic benefits by attenuating protein misfolding in models of neurodegenerative disease. The most recent evidence addressing the role of the UPR and ER stress in neurodegenerative disorders is reviewed here, along with therapeutic strategies to alleviate ER stress in a disease context.     

Getting the better of ER stress

Research over the past few years has highlighted the ability of the unfolded protein response (UPR) to minimize the deleterious effects of accumulated misfolded proteins under both physiological and pathological conditions. The endoplasmic reticulum (ER) adapts to endogenous and exogenous stressors by expanding its protein-folding capacity and by stimulating protective processes such as autophagy and antioxidant responses. Although it is clear that severe ER stress can elicit cell death, several recent studies have shown that low levels of ER stress may actually be beneficial to cells by eliciting an adaptive UPR that ‘preconditions’ the cell to a subsequent lethal insult; this process is called ER hormesis. The findings have important implications for the treatment of a wide variety of diseases associated with defective proteostasis, including neurodegenerative diseases, diabetes, and cancer. Here, we review the physiological and pathological functions of the ER, with a particular focus on the molecular mechanisms that lead to ER hormesis and cellular protection, and discuss the implications for disease treatment.     

Protein folding stress in neurodegenerative diseases: a glimpse into the ER

Several neurodegenerative diseases share common neuropathology, primarily featuring the presence in the brain of abnormal protein inclusions containing specific misfolded proteins. Recent evidence indicates that alteration in organelle function is a common pathological feature of protein misfolding disorders, highlighting perturbations in the homeostasis of the endoplasmic reticulum (ER). Signs of ER stress have been detected in most experimental models of neurological disorders and more recently in brain samples from human patients with neurodegenerative disease. To cope with ER stress, cells activate an integrated signaling response termed the unfolded protein response (UPR), which aims to reestablish homeostasis in part through regulation of genes involved in protein folding, quality control and degradation pathways. Here we discuss the particular mechanisms currently proposed to be involved in the generation of protein folding stress in different neurodegenerative conditions and speculate about possible therapeutic interventions.     

Preventing α-synuclein aggregation: The role of the small heat-shock molecular chaperone proteins

Protein homeostasis, or proteostasis, is the process of maintaining the conformational and functional integrity of the proteome. The failure of proteostasis can result in the accumulation of non-native proteins leading to their aggregation and deposition in cells and in tissues. The amyloid fibrillar aggregation of the protein α-synuclein into Lewy bodies and Lewy neuritis is associated with neurodegenerative diseases classified as α-synucleinopathies, which include Parkinson's disease and dementia with Lewy bodies. The small heat-shock proteins (sHsps) are molecular chaperones that are one of the cell's first lines of defence against protein aggregation. They act to stabilise partially folded protein intermediates, in an ATP-independent manner, to maintain cellular proteostasis under stress conditions. Thus, the sHsps appear ideally suited to protect against α-synuclein aggregation, yet these fail to do so in the context of the α-synucleinopathies. This review discusses how sHsps interact with α-synuclein to prevent its aggregation and, in doing so, highlights the multi-faceted nature of the mechanisms used by sHsps to prevent the fibrillar aggregation of proteins. It also examines what factors may contribute to α-synuclein escaping the sHsp chaperones in the context of the α-synucleinopathies.     

Interplay of endoplasmic reticulum stress and autophagy in neurodegenerative disorders

The common underlying feature of most neurodegenerative diseases such as Alzheimer disease (AD), prion diseases, Parkinson disease (PD), and amyotrophic lateral sclerosis (ALS) involves accumulation of misfolded proteins leading to initiation of endoplasmic reticulum (ER) stress and stimulation of the unfolded protein response (UPR). Additionally, ER stress more recently has been implicated in the pathogenesis of HIV-associated neurocognitive disorders (HAND). Autophagy plays an essential role in the clearance of aggregated toxic proteins and degradation of the damaged organelles. There is evidence that autophagy ameliorates ER stress by eliminating accumulated misfolded proteins. Both abnormal UPR and impaired autophagy have been implicated as a causative mechanism in the development of various neurodegenerative diseases. This review highlights recent advances in the field on the role of ER stress and autophagy in AD, prion diseases, PD, ALS and HAND with the involvement of key signaling pathways in these processes and implications for future development of therapeutic strategies.     

HHV-6A infection dysregulates autophagy/UPR interplay increasing beta amyloid production and tau phosphorylation in astrocytoma cells as well as in primary neurons,possible molecular mechanisms linking viral infection to Alzheimer's disease

HHV-6A and HHV-6B are neurotropic viruses able to dysregulate autophagy and activate ER stress/UPR in several cell types. The appropriate functioning of these processes is required for cell homeostasis, particularly in post-mitotic cells such as neuronal cells. Interestingly, neurodegenerative diseases such as Alzheimer's disease (AD) are often accompanied by autophagy dysregulation and abnormal UPR activation. This study demonstrated for the first time that HHV-6A infection of astrocytoma cells and primary neurons reduces autophagy, increases Aβ production and activates ER stress/UPR promoting tau protein hyper-phosphorylation. Our results support previous studies suggesting that HHV-6A infection may play a role in AD and unveil the possible underlying molecular mechanisms involved.     

Unfolded proteins and endoplasmic reticulum stress in neurodegenerative disorders

The stimuli for neuronal cell death in neurodegenerative disorders are multi-factorial and may include genetic predisposition, environmental factors, cellular stressors such as oxidative stress and free radical production, bioenergy failure, glutamate-induced excitotoxicity, neuroinflammation, disruption of Ca(2+) -regulating systems, mitochondrial dysfunction and misfolded protein accumulation. Cellular stress disrupts functioning of the endoplasmic reticulum (ER), a critical organelle for protein quality control, leading to induction of the unfolded protein response (UPR). ER stress may contribute to neurodegeneration in a range of neurodegenerative disorders. This review summarizes the molecular events occurring during ER stress and the unfolded protein response and it specifically evaluates the evidence suggesting the ER stress response plays a role in neurodegenerative disorders.     

The endoplasmic reticulum proteostasis network profoundly shapes the protein sequence space accessible to HIV envelope

The sequence space accessible to evolving proteins can be enhanced by cellular chaperones that assist biophysically defective clients in navigating complex folding landscapes. It is also possible, at least in theory, for proteostasis mechanisms that promote strict quality control to greatly constrain accessible protein sequence space. Unfortunately, most efforts to understand how proteostasis mechanisms influence evolution rely on artificial inhibition or genetic knockdown of specific chaperones. The few experiments that perturb quality control pathways also generally modulate the levels of only individual quality control factors. Here, we use chemical genetic strategies to tune proteostasis networks via natural stress response pathways that regulate the levels of entire suites of chaperones and quality control mechanisms. Specifically, we upregulate the unfolded protein response (UPR) to test the hypothesis that the host endoplasmic reticulum (ER) proteostasis network shapes the sequence space accessible to human immunodeficiency virus-1 (HIV-1) envelope (Env) protein. Elucidating factors that enhance or constrain Env sequence space is critical because Env evolves extremely rapidly, yielding HIV strains with antibody- and drug-escape mutations. We find that UPR-mediated upregulation of ER proteostasis factors, particularly those controlled by the IRE1-XBP1s UPR arm, globally reduces Env mutational tolerance. Conserved, functionally important Env regions exhibit the largest decreases in mutational tolerance upon XBP1s induction. Our data indicate that this phenomenon likely reflects strict quality control endowed by XBP1s-mediated remodeling of the ER proteostasis environment. Intriguingly, and in contrast, specific regions of Env, including regions targeted by broadly neutralizing antibodies, display enhanced mutational tolerance when XBP1s is induced, hinting at a role for host proteostasis network hijacking in potentiating antibody escape. These observations reveal a key function for proteostasis networks in decreasing instead of expanding the sequence space accessible to client proteins, while also demonstrating that the host ER proteostasis network profoundly shapes the mutational tolerance of Env in ways that could have important consequences for HIV adaptation.

The host cell’s endoplasmic reticulum proteostasis network has a profound, constraining impact on the protein sequence space accessible to HIV’s envelope protein, which is a major target of the host’s adaptive immune system; in particular, upregulation of stringent quality control pathways appears to restrict the viability of destabilizing envelope variants.     

The therapeutic effects of 4-phenylbutyric acid in maintaining proteostasis

Recently, there has been an increasing amount of literature published on the effects of 4-phenylbutyric acid (4-PBA) in various biological systems. 4-PBA is currently used clinically to treat urea cycle disorders under the trade name Buphenyl. Recent studies however have explored 4-PBA in the context of a low weight molecular weight chemical chaperone. Its properties as a chemical chaperone prevent misfolded protein aggregation and alleviate endoplasmic reticulum (ER) stress. As the ER is responsible for folding proteins targeted for use in membranes or secreted out of the cell, failure of maintaining adequate ER homeostasis may lead to protein misfolding and subsequent cell and organ pathology. Accumulation of misfolded proteins within the ER activates the unfolded protein response (UPR), a molecular repair response. The activation of the UPR aims to restore ER and cellular proteostasis by regulating the rate of synthesis of newly formed proteins as well as initiating molecular programs aimed to help fold or degrade misfolded proteins. If proteostasis is not restored, the UPR may initiate pro-apoptotic pathways. It is suggested that 4-PBA may help fold proteins in the ER, attenuating the activation of the UPR, and thus potentially alleviating various pathologies. This review discusses the biomedical research exploring the potential therapeutic effects of 4-PBA in various in vitro and in vivo model systems and clinical trials, while also commenting on the possible mechanisms of action.     

Synaptic proteostasis in Parkinson's disease

There are over 7 million people worldwide suffering from Parkinson's disease, and this number will double in the next decade. Causative mutations and risk variants in >20 genes that predominantly act at synapses have been linked to Parkinson's disease. Synaptic defects precede neuronal death. However, we are only now beginning to understand which molecular mechanisms contribute to this synaptic dysfunction. In this review, we discuss recent data demonstrating that Parkinson proteins act centrally to various protein quality control pathways at the synapse, and we argue that disturbed synaptic proteostasis is an early driver of neurodegeneration in Parkinson's disease.     

RNA metabolism: putting the brake on the UPR

The unfolded protein response (UPR) is a major signaling cascade that determines cell fate under conditions of endoplasmic reticulum (ER) stress. The kinetics and amplitude of UPR responses are tightly controlled by several feedback loops and the expression of positive and negative regulators. In this issue of EMBO Reports, the Wilkinson lab uncovers a novel function of nonsense‐mediated RNA decay (NMD) in fine‐tuning the UPR 1 . NMD is an mRNA quality control mechanism known to destabilize aberrant mRNAs that contain premature termination codons. In this work, NMD was shown to determine the threshold of stress necessary to activate the UPR, in addition to adjusting the amplitude of downstream responses and the termination phase. These effects were mapped to the control of the mRNA stability of IRE1, a major ER stress transducer. This study highlights the dynamic crosstalk between mRNA metabolism and the proteostasis network demonstrating the physiological relevance of normal mRNA regulation by the NMD pathway.     

Loss of proteostasis induced by amyloid beta peptide in brain endothelial cells

Abnormal accumulation of amyloid-β (Aβ) peptide in the brain is a pathological hallmark of Alzheimer's disease (AD). In addition to neurotoxic effects, Aβ also damages brain endothelial cells (ECs) and may thus contribute to the degeneration of cerebral vasculature, which has been proposed as an early pathogenic event in the course of AD and is able to trigger and/or potentiate the neurodegenerative process and cognitive decline. However, the mechanisms underlying Aβ-induced endothelial dysfunction are not completely understood. Here we hypothesized that Aβ impairs protein quality control mechanisms both in the secretory pathway and in the cytosol in brain ECs, leading cells to death. In rat brain RBE4 cells, we demonstrated that Aβ_1–40 induces the failure of the ER stress-adaptive unfolded protein response (UPR), deregulates the ubiquitin–proteasome system (UPS) decreasing overall proteasome activity with accumulation of ubiquitinated proteins and impairs the autophagic protein degradation pathway due to failure in the autophagic flux, which culminates in cell demise. In conclusion, Aβ deregulates proteostasis in brain ECs and, as a consequence, these cells die by apoptosis.