Efficacy of therapy by MK-28 PERK activation in the Huntington's disease R6/2 mouse model.

There is currently no disease-modifying therapy for Huntington's disease (HD). We recently described a small molecule, MK-28, which restored homeostasis in HD models by specifically activating PKR-like ER kinase (PERK). This activation boosts the unfolded protein response (UPR), thereby reducing endoplasmic reticulum (ER) stress, a central cytotoxic mechanism in HD and other neurodegenerative diseases. Here, we have tested the long-term effects of MK-28 in HD model mice. R6/2 CAG (160) mice were treated by lifetime intraperitoneal injections 3 times a week. CatWalk measurements of motor function showed strong improvement compared to untreated mice after only two weeks of MK-28 treatment and continued with time, most significantly at 1 â€‹mg/kg MK-28, approaching WT values. Seven weeks treatment significantly improved paw grip strength. Body weight recovered and glucose levels, which are elevated in HD mice, were significantly reduced. Treatment with another PERK activator, CCT020312 at 1 â€‹mg/kg, also caused amelioration, consistent with PERK activation. Lifespan, measured in more resilient R6/2 CAG (120) mice with daily IP injection, was much extended by 16 days (20%) with 0.3 â€‹mg/kg MK-28, and by 38 days (46%) with 1 â€‹mg/kg MK-28. No toxicity, measured by weight, blood glucose levels and blood liver function markers, was detectable in WT mice treated for 6 weeks with 6 â€‹mg/kg MK-28. Boosting of PERK activity by long-term treatment with MK-28 could be a safe and promising therapeutic approach for HD.

COP I and II dependent trafficking controls ER-associated degradation in mammalian cells.

Misfolded proteins and components of the endoplasmic reticulum (ER) quality control and ER associated degradation (ERAD) machineries concentrate in mammalian cells in the pericentriolar ER-derived quality control compartment (ERQC), suggesting it as a staging ground for ERAD. By tracking the chaperone calreticulin and an ERAD substrate, we have now determined that the trafficking to the ERQC is reversible and recycling back to the ER is slower than the movement in the ER periphery. The dynamics suggest vesicular trafficking rather than diffusion. Indeed, using dominant negative mutants of ARF1 and Sar1 or the drugs Brefeldin A and H89, we observed that COPI inhibition causes accumulation in the ERQC and increases ERAD, whereas COPII inhibition has the opposite effect. Our results suggest that targeting of misfolded proteins to ERAD involves COPII-dependent transport to the ERQC and that they can be retrieved to the peripheral ER in a COPI-dependent manner.

The Sigma-1 receptor is an ER-localized type II membrane protein.

The Sigma-1 receptor (S1R) is a transmembrane protein with important roles in cellular homeostasis in normal physiology and in disease. Especially in neurodegenerative diseases, S1R activation has been shown to provide neuroprotection by modulating calcium signaling, mitochondrial function and reducing endoplasmic reticulum (ER) stress. S1R missense mutations are one of the causes of the neurodegenerative Amyotrophic Lateral Sclerosis and distal hereditary motor neuronopathies. Although the S1R has been studied intensively, basic aspects remain controversial, such as S1R topology and whether it reaches the plasma membrane. To address these questions, we have undertaken several approaches. C-terminal tagging with a small biotin-acceptor peptide and BirA biotinylation in cells suggested a type II membrane orientation (cytosolic N-terminus). However, N-terminal tagging gave an equal probability for both possible orientations. This might explain conflicting reports in the literature, as tags may affect the protein topology. Therefore, we studied untagged S1R using a protease protection assay and a glycosylation mapping approach, introducing N-glycosylation sites. Both methods provided unambiguous results showing that the S1R is a type II membrane protein with a short cytosolic N-terminal tail. Assessments of glycan processing, surface fluorescence-activated cell sorting, and cell surface biotinylation indicated ER retention, with insignificant exit to the plasma membrane, in the absence or presence of S1R agonists or of ER stress. These findings may have important implications for S1R-based therapeutic approaches.

Pridopidine reduces mutant huntingtin-induced endoplasmic reticulum stress by modulation of the Sigma-1 receptor.

The endoplasmic reticulum (ER)-localized Sigma-1 receptor (S1R) is neuroprotective in models of neurodegenerative diseases, among them Huntington disease (HD). Recent clinical trials in HD patients and preclinical studies in cellular and mouse HD models suggest a therapeutic potential for the high-affinity S1R agonist pridopidine. However, the molecular mechanisms of the cytoprotective effect are unclear. We have previously reported strong induction of ER stress by toxic mutant huntingtin (mHtt) oligomers, which is reduced upon sequestration of these mHtt oligomers into large aggregates. Here, we show that pridopidine significantly ameliorates mHtt-induced ER stress in cellular HD models, starting at low nanomolar concentrations. Pridopidine reduced the levels of markers of the three branches of the unfolded protein response (UPR), showing the strongest effects on the PKR-like endoplasmic reticulum kinase (PERK) branch. The effect is S1R-dependent, as it is abolished in cells expressing mHtt in which the S1R was deleted using CRISPR/Cas9 technology. mHtt increased the level of the detergent-insoluble fraction of S1R, suggesting a compensatory cellular mechanism that responds to increased ER stress. Pridopidine further enhanced the levels of insoluble S1R, suggesting the stabilization of activated S1R oligomers. These S1R oligomeric species appeared in ER-localized patches, and not in the mitochondria-associated membranes nor the ER-derived quality control compartment. The colocalization of S1R with the chaperone BiP was significantly reduced by mHtt, and pridopidine restored this colocalization to normal, unstressed levels. Pridopidine increased toxic oligomeric mHtt recruitment into less toxic large sodium dodecyl sulfate-insoluble aggregates, suggesting that this in turn reduces ER stress and cytotoxicity.

PERK Pathway and Neurodegenerative Disease: To Inhibit or to Activate?

With the extension of life span in recent decades, there is an increasing burden of late-onset neurodegenerative diseases, for which effective treatments are lacking. Neurodegenerative diseases include the widespread Alzheimer's disease (AD) and Parkinson's disease (PD), the less frequent Huntington's disease (HD) and Amyotrophic Lateral Sclerosis (ALS) and also rare early-onset diseases linked to mutations that cause protein aggregation or loss of function in genes that maintain protein homeostasis. The difficulties in applying gene therapy approaches to tackle these diseases is drawing increasing attention to strategies that aim to inhibit cellular toxicity and restore homeostasis by intervening in cellular pathways. These include the unfolded protein response (UPR), activated in response to endoplasmic reticulum (ER) stress, a cellular affliction that is shared by these diseases. Special focus is turned to the PKR-like ER kinase (PERK) pathway of the UPR as a target for intervention. However, the complexity of the pathway and its ability to promote cell survival or death, depending on ER stress resolution, has led to some confusion in conflicting studies. Both inhibition and activation of the PERK pathway have been reported to be beneficial in disease models, although there are also some reports where they are counterproductive. Although with the current knowledge a definitive answer cannot be given on whether it is better to activate or to inhibit the pathway, the most encouraging strategies appear to rely on boosting some steps without compromising downstream recovery.

Oxidoreductases in Glycoprotein Glycosylation, Folding, and ERAD.

N-linked glycosylation and sugar chain processing, as well as disulfide bond formation, are among the most common post-translational protein modifications taking place in the endoplasmic reticulum (ER). They are essential modifications that are required for membrane and secretory proteins to achieve their correct folding and native structure. Several oxidoreductases responsible for disulfide bond formation, isomerization, and reduction have been shown to form stable, functional complexes with enzymes and chaperones that are involved in the initial addition of an N-glycan and in folding and quality control of the glycoproteins. Some of these oxidoreductases are selenoproteins. Recent studies also implicate glycan machinery-oxidoreductase complexes in the recognition and processing of misfolded glycoproteins and their reduction and targeting to ER-associated degradation. This review focuses on the intriguing cooperation between the glycoprotein-specific cell machineries and ER oxidoreductases, and highlights open questions regarding the functions of many members of this large family.

Insulin-like growth factor 2 (IGF2) protects against Huntington's disease through the extracellular disposal of protein aggregates.

Impaired neuronal proteostasis is a salient feature of many neurodegenerative diseases, highlighting alterations in the function of the endoplasmic reticulum (ER). We previously reported that targeting the transcription factor XBP1, a key mediator of the ER stress response, delays disease progression and reduces protein aggregation in various models of neurodegeneration. To identify disease modifier genes that may explain the neuroprotective effects of XBP1 deficiency, we performed gene expression profiling of brain cortex and striatum of these animals and uncovered insulin-like growth factor 2 (Igf2) as the major upregulated gene. Here, we studied the impact of IGF2 signaling on protein aggregation in models of Huntington's disease (HD) as proof of concept. Cell culture studies revealed that IGF2 treatment decreases the load of intracellular aggregates of mutant huntingtin and a polyglutamine peptide. These results were validated using induced pluripotent stem cells (iPSC)-derived medium spiny neurons from HD patients and spinocerebellar ataxia cases. The reduction in the levels of mutant huntingtin was associated with a decrease in the half-life of the intracellular protein. The decrease in the levels of abnormal protein aggregation triggered by IGF2 was independent of the activity of autophagy and the proteasome pathways, the two main routes for mutant huntingtin clearance. Conversely, IGF2 signaling enhanced the secretion of soluble mutant huntingtin species through exosomes and microvesicles involving changes in actin dynamics. Administration of IGF2 into the brain of HD mice using gene therapy led to a significant decrease in the levels of mutant huntingtin in three different animal models. Moreover, analysis of human postmortem brain tissue and blood samples from HD patients showed a reduction in IGF2 level. This study identifies IGF2 as a relevant factor deregulated in HD, operating as a disease modifier that buffers the accumulation of abnormal protein species.

A novel specific PERK activator reduces toxicity and extends survival in Huntington's disease models.

One of the pathways of the unfolded protein response, initiated by PKR-like endoplasmic reticulum kinase (PERK), is key to neuronal homeostasis in neurodegenerative diseases. PERK pathway activation is usually accomplished by inhibiting eIF2α-P dephosphorylation, after its phosphorylation by PERK. Less tried is an approach involving direct PERK activation without compromising long-term recovery of eIF2α function by dephosphorylation. Here we show major improvement in cellular (STHdhQ111/111) and mouse (R6/2) Huntington's disease (HD) models using a potent small molecule PERK activator that we developed, MK-28. MK-28 showed PERK selectivity in vitro on a 391-kinase panel and rescued cells (but not PERK-/- cells) from ER stress-induced apoptosis. Cells were also rescued by the commercial PERK activator CCT020312 but MK-28 was significantly more potent. Computational docking suggested MK-28 interaction with the PERK activation loop. MK-28 exhibited remarkable pharmacokinetic properties and high BBB penetration in mice. Transient subcutaneous delivery of MK-28 significantly improved motor and executive functions and delayed death onset in R6/2 mice, showing no toxicity. Therefore, PERK activation can treat a most aggressive HD model, suggesting a possible approach for HD therapy and worth exploring for other neurodegenerative disorders.

Letting go of O-glycans.

The generation of free N-glycans, or unconjugated oligosaccharides derived from N-linked glycoproteins, is well understood, but whether a similar fate awaits O-linked glycoprotein carbohydrates was unknown. Hirayama et al. now reveal, by using only mannose as an energy source, the generation of free O-glycans in Saccharomyces cerevisiae, in the lumen of a secretory compartment, possibly the vacuole. These findings uncover the presence of a possible regulated degradation pathway for O-mannosylated glycoproteins.

Compartmentalization and Selective Tagging for Disposal of Misfolded Glycoproteins.

The ability of mammalian cells to correctly identify and degrade misfolded secretory proteins, most of them bearing N-glycans, is crucial for their correct function and survival. An inefficient disposal mechanism results in the accumulation of misfolded proteins and consequent endoplasmic reticulum (ER) stress. N-glycan processing creates a code that reveals the folding status of each molecule, enabling continued folding attempts or targeting of the doomed glycoprotein for disposal. We review here the main steps involved in the accurate processing of unfolded glycoproteins. We highlight recent data suggesting that the processing is not stochastic, but that there is selective accelerated glycan trimming on misfolded glycoprotein molecules.

Protein Misfolding and ER Stress in Huntington's Disease.

Increasing evidence in recent years indicates that protein misfolding and aggregation, leading to ER stress, are central factors of pathogenicity in neurodegenerative diseases. This is particularly true in Huntington's disease (HD), where in contrast with other disorders, the cause is monogenic. Mutant huntingtin interferes with many cellular processes, but the fact that modulation of ER stress and of the unfolded response pathways reduces the toxicity, places these mechanisms at the core and gives hope for potential therapeutic approaches. There is currently no effective treatment for HD and it has a fatal outcome a few years after the start of symptoms of cognitive and motor impairment. Here we will discuss recent findings that shed light on the mechanisms of protein misfolding and aggregation that give origin to ER stress in neurodegenerative diseases, focusing on Huntington's disease, on the cellular response and on how to use this knowledge for possible therapeutic strategies.

Mannosidase activity of EDEM1 and EDEM2 depends on an unfolded state of their glycoprotein substrates.

Extensive mannose trimming of nascent glycoprotein N-glycans signals their targeting to endoplasmic reticulum-associated degradation (ERAD). ER mannosidase I (ERManI) and the EDEM protein family participate in this process. However, whether the EDEMs are truly mannosidases can be addressed only by measuring mannosidase activity in vitro. Here, we reveal EDEM1 and EDEM2 mannosidase activities in vitro. Whereas ERManI significantly trims free N-glycans, activity of the EDEMs is modest on free oligosaccharides and on glycoproteins. However, mannosidase activity of ERManI and the EDEMs is significantly higher on a denatured glycoprotein. The EDEMs associate with oxidoreductases, protein disulfide isomerase, and especially TXNDC11, enhancing mannosidase activity on glycoproteins but not on free N-glycans. The finding that substrate unfolded status increases mannosidase activity solves an important conundrum, as current models suggest general slow mannose trimming. As we show, misfolded or unfolded glycoproteins are subject to differentially faster trimming (and targeting to ERAD) than well-folded ones.

[2-3H]Mannose-labeling and Analysis of N-linked Oligosaccharides.

Modifications of N-linked oligosaccharides of glycoproteins soon after their biosynthesis correlate to glycoprotein folding status. These alterations can be detected in a sensitive way by pulse-chase analysis of [2-3H]mannose-labeled glycoproteins, with enzymatic removal of labeled N-glycans, separation according to size by HPLC and radioactive detection in a scintillation counter.

Common fixation-permeabilization methods cause artifactual localization of a type II transmembrane protein.

We found that a localization artifact can arise from common immunofluorescence methods. Specifically, cell fixation and permeabilization can cause mislocalization of a type II membrane-bound protein, ER mannosidase I, from its native localization in vesicles to the Golgi complex. Live cell microscopy and interestingly also mild cell fixation with paraformaldehyde without membrane permeabilization do not present this artifact.

Mannosidase IA is in Quality Control Vesicles and Participates in Glycoprotein Targeting to ERAD.

Endoplasmic reticulum-associated degradation (ERAD) of a misfolded glycoprotein in mammalian cells requires the removal of 3-4 alpha 1,2 linked mannose residues from its N-glycans. The trimming and recognition processes are ascribed to ER Mannosidase I, the ER-degradation enhancing mannosidase-like proteins (EDEMs), and the lectins OS-9 and XTP3-B, all residing in the ER, the ER-derived quality control compartment (ERQC), or quality control vesicles (QCVs). Folded glycoproteins with untrimmed glycans are transported from the ER to the Golgi complex, where they are substrates of other alpha 1,2 mannosidases, IA, IB, and IC. The apparent redundancy of these enzymes has been puzzling for many years. We have now determined that, surprisingly, mannosidase IA is not located in the Golgi but resides in QCVs. We had recently described this type of vesicles, which carry ER α1,2 mannosidase I (ERManI). We show that the overexpression of alpha class I α1,2 mannosidase IA (ManIA) significantly enhances the degradation of ERAD substrates and its knockdown stabilizes it. Our results indicate that ManIA trims mannose residues from Man9GlcNAc2 down to Man5GlcNAc2, acting in parallel with ERManI and the EDEMs, and targeting misfolded glycoproteins to ERAD.

Protein aggregation and ER stress.

Protein aggregation is a common feature of the protein misfolding or conformational diseases, among them most of the neurodegenerative diseases. These disorders are a major scourge, with scarce if any effective therapies at present. Recent research has identified ER stress as a major mechanism implicated in cytotoxicity in these diseases. Whether amyloid-ß or tau in Alzheimer's, α-synuclein in Parkinson's, huntingtin in Huntington's disease or other aggregation-prone proteins in many other neurodegenerative diseases, there is a shared pathway of oligomerization and aggregation into amyloid fibrils. There is increasing evidence in recent years that the toxic species, and those that evoke ER stress, are the intermediate oligomeric forms and not the final amyloid aggregates. This review focuses on recent findings on the mechanisms and importance of the development of ER stress upon protein aggregation, especially in neurodegenerative diseases, and possible therapeutic approaches that are being examined. This article is part of a Special Issue entitled SI:ER stress.

Non-invasive diagnosis of liver fibrosis and cirrhosis.

The evaluation and follow up of liver fibrosis and cirrhosis have been traditionally performed by liver biopsy. However, during the last 20 years, it has become evident that this "gold-standard" is imperfect; even according to its proponents, it is only "the best" among available methods. Attempts at uncovering non-invasive diagnostic tools have yielded multiple scores, formulae, and imaging modalities. All are better tolerated, safer, more acceptable to the patient, and can be repeated essentially as often as required. Most are much less expensive than liver biopsy. Consequently, their use is growing, and in some countries the number of biopsies performed, at least for routine evaluation of hepatitis B and C, has declined sharply. However, the accuracy and diagnostic value of most, if not all, of these methods remains controversial. In this review for the practicing physician, we analyze established and novel biomarkers and physical techniques. We may be witnessing in recent years the beginning of the end of the first phase for the development of non-invasive markers. Early evidence suggests that they might be at least as good as liver biopsy. Novel experimental markers and imaging techniques could produce a dramatic change in diagnosis in the near future.

Mammalian ER mannosidase I resides in quality control vesicles, where it encounters its glycoprotein substrates.

Endoplasmic reticulum α1,2 mannosidase I (ERManI), a central component of ER quality control and ER-associated degradation (ERAD), acts as a timer enzyme, modifying N-linked sugar chains of glycoproteins with time. This process halts glycoprotein folding attempts when necessary and targets terminally misfolded glycoproteins to ERAD. Despite the importance of ERManI in maintenance of glycoprotein quality control, fundamental questions regarding this enzyme remain controversial. One such question is the subcellular localization of ERManI, which has been suggested to localize to the ER membrane, the ER-derived quality control compartment (ERQC), and, surprisingly, recently to the Golgi apparatus. To try to clarify this controversy, we applied a series of approaches that indicate that ERManI is located, at the steady state, in quality control vesicles (QCVs) to which ERAD substrates are transported and in which they interact with the enzyme. Both endogenous and exogenously expressed ERManI migrate at an ER-like density on iodixanol gradients, suggesting that the QCVs are derived from the ER. The QCVs are highly mobile, displaying dynamics that are dependent on microtubules and COP-II but not on COP-I vesicle machinery. Under ER stress conditions, the QCVs converge in a juxtanuclear region, at the ERQC, as previously reported. Our results also suggest that ERManI is turned over by an active autophagic process. Of importance, we found that membrane disturbance, as is common in immunofluorescence methods, leads to an artificial appearance of ERManI in a Golgi pattern.

Glycan regulation of ER-associated degradation through compartmentalization.

The internal environment of the eukaryotic cell is divided by membranes into various organelles, containing diverse functional subcompartments, which allow complex cellular life. The quality control of newly made secretory proteins relies on the ability of the endoplasmic reticulum (ER) to segregate and compartmentalize molecules at different folding states. These folding states are communicated by N-glycans present on most secretory proteins. In ER-associated degradation (ERAD), protein molecules that have been identified as terminally misfolded are sent for degradation at the cytosolic proteasomes after being dislocated from the ER to the cytosol. This review will focus on how misfolded glycoprotein molecules are segregated from their properly folded counterparts and targeted to ERAD. The pathway involves compartmentalization, which is intimately linked to differential N-glycan processing. Recent data suggests that these processes are very dynamic, and include transient assembly of ERAD machinery complexes.

ER stress-induced eIF2-alpha phosphorylation underlies sensitivity of striatal neurons to pathogenic huntingtin.

A hallmark of Huntington's disease is the pronounced sensitivity of striatal neurons to polyglutamine-expanded huntingtin expression. Here we show that cultured striatal cells and murine brain striatum have remarkably low levels of phosphorylation of translation initiation factor eIF2α, a stress-induced process that interferes with general protein synthesis and also induces differential translation of pro-apoptotic factors. EIF2α phosphorylation was elevated in a striatal cell line stably expressing pathogenic huntingtin, as well as in brain sections of Huntington's disease model mice. Pathogenic huntingtin caused endoplasmic reticulum (ER) stress and increased eIF2α phosphorylation by increasing the activity of PKR-like ER-localized eIF2α kinase (PERK). Importantly, striatal neurons exhibited special sensitivity to ER stress-inducing agents, which was potentiated by pathogenic huntingtin. We could strongly reduce huntingtin toxicity by inhibiting PERK. Therefore, alteration of protein homeostasis and eIF2α phosphorylation status by pathogenic huntingtin appears to be an important cause of striatal cell death. A dephosphorylated state of eIF2α has been linked to cognition, which suggests that the effect of pathogenic huntingtin might also be a source of the early cognitive impairment seen in patients.