Unfolded Protein Response Activation Markers Research Articles

Investigating mechanisms of sporadic progressive supranuclear palsy in autopsy‐validated human iPSC‐derived midbrain organoids and human brain tissue

AbstractBackgroundProgressive supranuclear palsy (PSP) is the most common primary tauopathy, with a constellation of pathological features including 4R‐tau positive neurofibrillary tangles and tufted astrocytes. Most PSP cases are sporadic and associated with common structural variation in the 17q21.31 MAPT locus as well as other risk loci, including EIF2AK3 which is critical for the unfolded protein response (UPR). Despite these known genetic risk associations, mechanisms underlying disease pathogenesis are unclear. To investigate candidate mechanisms, there is a critical need for model systems that better recapitulate the cellular complexity of the human brain. Human induced pluripotent stem cell (hiPSC) patient‐derived organoid models have emerged as powerful tools to study molecular and cellular changes in a disease‐relevant genomic context.MethodFibroblasts grown from sporadic PSP patient skin cells were cultured in vitro and reprogrammed into hiPSCs. HiPSC‐derived midbrain organoids were generated in suspension spinner flasks through pharmacological directed differentiation, processed and screened for patterning using qRT‐PCR, immunohistochemistry (IHC), and quantitative immunoblot. Total tau, tau isoform, hyperphosphorylated tau (p‐tau), and UPR activation markers were assessed in organoids and human brain tissue. Single‐nucleus RNA sequencing (snRNA‐seq) was performed on the subthalamic nucleus and adjacent structures in autopsy‐validated PSP brain tissue and clinically normal controls.ResultPSP and control organoids displayed a cytoarchitectural and gene expression pattern consistent with the developing neuroectoderm and midbrain. We observed disease‐relevant differences between PSP and control organoids, including increased high molecular weight p‐tau, a higher ratio of p‐tau:total tau, and increased levels of 4R‐tau in the PSP organoids. Human PSP brain snRNA‐seq data showed dysregulation of the UPR in astrocytes and neuronal subpopulations. We also observed different UPR activation levels in PSP organoids. These findings were validated by neurohistological characterization of the UPR in autopsy brain tissue.ConclusionSingle nucleus transcriptomic and neurohistological data reveals dysregulation of the UPR in key cell types affected in PSP. PSP patient‐derived organoids develop a characteristic midbrain cellular composition and recapitulate key disease‐relevant features, including elevation of toxic tau proteoforms and UPR dysregulation. This sporadic PSP organoid model will provide insight into cell‐type specific drivers of neurodegeneration that underlie sporadic tauopathy.

Involvement of the unfolded protein response in the protective effects of growth hormone releasing hormone antagonists in the lungs.

Growth hormone releasing hormone (GHRH) antagonists enhance endothelial barrier function and counteract the LPS-induced lung endothelial hyperpermeability, the cardinal feature of the acute respiratory distress syndrome (ARDS). The unfolded protein response (UPR) is a multifaceted molecular mechanism, strongly involved in tissue defense against injury. The current study introduces the induction of UPR by GHRH antagonists, since those peptides induced several UPR activation markers, including the inositol-requiring enzyme-1α (IRE1α), the protein kinase RNA-like ER kinase (PERK), and the activating transcription factor 6 (ATF6). On the other hand, the GHRH agonist MR-409 exerted the opposite effects. Furthermore, GHRH antagonists counteracted the kifunensine (UPR suppressor)-induced lung endothelial barrier dysfunction. Our observations suggest that UPR mediates, at least in part, the protective effects of GHRH antagonists in the lung microvasculature. To the best of our knowledge; this is the first study to provide experimental evidence in support of the hypothesis that UPR induction is a novel mechanism by which GHRH antagonists oppose severe human disease, including ARDS.

Unfolded protein response activation in C9orf72 frontotemporal dementia is associated with dipeptide pathology and granulovacuolar degeneration in granule cells.

A repeat expansion in the C9orf72 gene is the most prevalent genetic cause of frontotemporal dementia (C9‐FTD). Several studies have indicated the involvement of the unfolded protein response (UPR) in C9‐FTD. In human neuropathology, UPR markers are strongly associated with granulovacuolar degeneration (GVD). In this study, we aim to assess the presence of UPR markers together with the presence of dipeptide pathology and GVD in post mortem brain tissue from C9‐FTD cases and neurologically healthy controls. Using immunohistochemistry we assessed the presence of phosphorylated PERK, IRE1α and eIF2α in the frontal cortex, hippocampus and cerebellum of C9‐FTD (n = 18) and control (n = 9) cases. The presence of UPR activation markers was compared with the occurrence of pTDP‐43, p62 and dipeptide repeat (DPR) proteins (poly(GA), ‐(GR) & ‐(GP)) as well as casein kinase 1 delta (CK1δ), a marker for GVD. Increased presence of UPR markers was observed in the hippocampus and cerebellum in C9‐FTD compared to control cases. In the hippocampus, overall levels of pPERK and peIF2α were higher in C9‐FTD, including in granule cells of the dentate gyrus (DG). UPR markers were also observed in granule cells of the cerebellum in C9‐FTD. In addition, increased levels of CK1δ were observed in granule cells in the DG of the hippocampus and granular layer of the cerebellum in C9‐FTD. Double‐labelling experiments indicate a strong association between UPR markers and the presence of dipeptide pathology as well as GVD. We conclude that UPR markers are increased in C9‐FTD and that their presence is associated with dipeptide pathology and GVD. Increased presence of UPR markers and CK1δ in granule cells in the cerebellum and hippocampus could be a unique feature of C9‐FTD.

Proteostasis in epicardial versus subcutaneous adipose tissue in heart failure subjects with and without diabetes

BackgroundCardiovascular diseases (CVDs) are leading cause of death and primary cause of morbidity and mortality in diabetic population. Epicardial adipose tissue (EAT) covers the heart's surface and is a source of biomolecules regulating heart and blood vessel physiology. The protective activation of the unfolded protein response (UPR) and autophagy allows the cardiomyocyte reticular network to restore energy and/or nutrient homeostasis and to avoid cell death. However, an excessive or prolonged UPR activation can trigger cell death. UPR activation is an early event of diabetic cardiomyopathies and deregulated autophagy is associated with CVDs. ResultsAn upregulation of UPR markers (glucose-regulated protein 78 KDa, glucose-regulated protein 94 KDa, inositol-requiring enzyme 1α, protein kinase RNA-like ER kinase and CCAAT/−enhancer-binding protein homologous protein (CHOP) gene) in EAT compared to subcutaneous adipose tissue (SAT), was observed as well as the UPR-related apoptosis marker caspase-4/procaspase-4 ratio but not in CHOP protein levels. Additionally, levels of ubiquitin and ubiquitinated proteins were decreased in EAT. Moreover, upregulation of autophagy markers (5′ adenosine monophosphate-activated protein kinase, mechanistic target of rapamycin, Beclin 1, microtubule-associated protein light chain 3-II, lysosome-associated membrane protein 2, and PTEN-induced putative kinase 1) was observed, as well as an increase in the apoptotic Bim but not the ratio between Bim and the anti-apoptotic Bcl-2 in EAT. Diabetic patients show alterations in UPR activation markers but not in autophagy or apoptosis markers. ConclusionUPR and autophagy are increased in EAT compared to SAT, opening doors to the identification of early biomarkers for cardiomyopathies and novel therapeutic targets.

Activation of the unfolded protein response and granulovacuolar degeneration are not common features of human prion pathology.

Human prion diseases are fatal neurodegenerative disorders with a genetic, sporadic or infectiously acquired aetiology. Neuropathologically, human prion diseases are characterized by deposition of misfolded prion protein and neuronal loss. In post-mortem brain tissue from patients with other neurodegenerative diseases characterized by protein misfolding, including Alzheimer’s disease (AD) and frontotemporal lobar degeneration with tau pathology (FTLD-tau), increased activation of the unfolded protein response (UPR) has been observed. The UPR is a cellular stress response that copes with the presence of misfolded proteins. Recent studies have indicated that UPR activation is also involved in experimental models of prion disease and have suggested intervention in the UPR as a therapeutic strategy. On the other hand, it was previously shown that the active form of the UPR stress sensor dsRNA-activated protein kinase-like ER kinase (PERK) is not increased in post-mortem brain tissue samples from human prion disease cases. In the present study, we assessed the active form of another UPR stress sensor, inositol-requiring enzyme 1α (IRE1α), in human post-mortem frontal cortex of a large cohort of sporadic, inherited and acquired prion disease patients (n = 47) and non-neurological controls. Immunoreactivity for phosphorylated IRE1α was not increased in prion disease cases compared with non-neurological controls. In addition, immunoreactivity for phosphorylated PERK was unaltered in human prion disease cases included in the current cohort. Moreover, no difference in the extent of granulovacuolar degeneration, a pathological feature associated with the presence of UPR activation markers, was detected. Our data indicate that, in contrast to AD and primary tauopathies, activation of the UPR is not a common feature of human prion pathology.Electronic supplementary materialThe online version of this article (doi:10.1186/s40478-016-0383-7) contains supplementary material, which is available to authorized users.

The unfolded protein response is associated with early tau pathology in the hippocampus of tauopathies

The unfolded protein response (UPR) is a stress response activated upon disturbed homeostasis in the endoplasmic reticulum (ER). Previously, we reported that the activation of the UPR closely correlates with the presence of phosphorylated tau (p-tau) in Alzheimer's disease (AD). As well as increased presence of intracellular p-tau, AD brains are characterized by extracellular deposits of β amyloid (Aβ). Recent in vitro studies have shown that Aβ can induce ER stress and activation of the UPR. The aim of the present study is to investigate UPR activation in sporadic tauopathies like progressive supranuclear palsy (PSP) and Pick's disease (PiD), and familial cases with frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) which carry mutations in the gene encoding for tau (MAPT). The presence of phosphorylated pancreatic ER kinase (pPERK) and phosphorylated inositol requiring enzyme 1α (pIRE1), which are indicative of an activated UPR, was assessed by immunohistochemistry in cases neuropathologically defined as frontotemporal lobar degeneration with tau pathology (FTLD-tau). Increased presence of UPR activation markers pPERK and pIRE1 was observed in neurons and glia in FTLD-tau cases, in contrast to FTLD subtypes negative for tau pathology or in non-neurological controls. pPERK and pIRE1 were also prominently present in relatively young carriers of MAPT mutation. A strong association between the presence of UPR activation markers and p-tau was observed in the hippocampus of FTLD-tau cases. Double immunohistochemical staining on FTLD-tau cases revealed that UPR activation is predominantly observed in neurons that show diffuse staining of p-tau. These data demonstrate that UPR activation is intimately connected with the accumulation and aggregation of p-tau, and occurs independently from Aβ deposits. Our findings provide new pathological insight into the close association between p-tau and UPR activation in tauopathies.

Endoplasmic reticulum stress: a new playER in tauopathies

The accumulation of unfolded or misfolded proteins in the lumen of the endoplasmic reticulum (ER) activates the unfolded protein response (UPR), which involves a set of protein signalling pathways and transcription factors that re-establish homeostasis and normal ER function, adapting cells to ER stress. If this adaptive response is insufficient, the UPR triggers an apoptotic program to eliminate irreversibly damaged cells. Recent observations suggest that ER stress plays an important role in the pathogenesis of various neurodegenerative disorders such as Alzheimer's disease, which is characterized by the deposition of amyloid-beta (Aβ) and hyperphosphorylated tau in susceptible brain regions. Moreover, several studies demonstrate that Aβ induces UPR activation, which in turn promotes tau phosphorylation. In the study by Nijholt and colleagues, reported in the current issue of The Journal of Pathology, the association between UPR activation and tau pathology was investigated in the brain of patients diagnosed with sporadic or familial tauopathies in which Abeta deposits are absent. The authors described that increased levels of UPR activation markers are predominantly observed in neurons within the hippocampus, being correlated with early tau phosphorylation. These findings suggest that UPR activation, which occurs in an Abeta-independent manner, is an early event during tau pathology and point to a functional crosstalk between these molecular mechanisms in tauopathies. A better understanding of UPR activation in tauopathies can thus contribute to the design of new therapeutic strategies with the purpose of promoting neuronal cell survival in these disorders.

Activation and clinical significance of the unfolded protein response in breast cancer

Introduction:The tumour microenvironment is hypoglycaemic, hypoxic and acidotic. This activates a stress signalling pathway: the unfolded protein response (UPR). The UPR is cytoprotective if the stressor is mild, but may initiate apoptosis if severe.Activation of the UPR in breast carcinoma is induced by microenvironmental stress such as glucose and oxygen deprivation, but may also be linked to oestrogen stimulation. It may be clinically significant as it may alter chemosensitivity to doxorubicin.Methods:395 human breast adenocarcinomas were immunohistochemically stained for UPR activation markers (glucose-regulated protein (GRP-78 and XBP-1). A model of UPR activation in vitro by glucose deprivation of T47D breast cancer cells was developed to determine how the UPR affects cellular sensitivity to doxorubicin and 5-fluorouracil. Cytotoxicity was assessed using a colorimetric cytotoxicity assay (MTT). The effect of oestrogen stimulation and tamoxifen exposure on UPR activation by T47D cells was determined by western blotting measurement of the key UPR protein, GRP-78.Results:Expression of GRP78 and XBP-1 was demonstrated in 76% and 90% of the breast cancers, respectively, and correlated with oestrogen receptor positivity (P=0.045 and 0.017, respectively). In vitro UPR activation induced resistance to both doxorubicin and 5-flurouracil, (P<0.05). Oestrogen stimulation induced GRP78 and XBP1 over-expression on western blotting. Tamoxifen did not block this response and may induce UPR activation in its own right.Conclusions:The UPR is activated in the majority of breast cancers and confers resistance to chemotherapy. In vitro oestrogen stimulates UPR induction. UPR activation may contribute to breast cancer chemoresistance and interact with oestrogen response elements.

The Unfolded Protein Response Is Activated in Pretangle Neurons in Alzheimer's Disease Hippocampus

Accumulation of misfolded proteins in the endoplasmic reticulum triggers a cellular stress response called the unfolded protein response (UPR) that protects the cell against the toxic buildup of misfolded proteins. Previously, we reported that UPR activation is increased in Alzheimer's disease (AD) patients. How the UPR relates to the pathological hallmarks of AD is still elusive. In the present study, the involvement of UPR activation in neurofibrillary degeneration in AD was investigated. Immunoreactivity for the phosphorylated UPR activation markers pancreatic ER kinase (pPERK), eukaryotic initiation factor 2alpha, and inositol-requiring enzyme 1alpha was observed in hippocampal neurons associated with granulovacuolar degeneration. The percentage of pPERK-immunoreactive neurons was increased in AD cases compared with nondemented control cases and with the Braak stage for neurofibrillary changes. Although absent from neurofibrillary tangles, pPERK immunoreactivity was most abundant in neurons with diffuse localization of phosphorylated tau protein. Additional analyses showed that pPERK immunoreactivity was associated with ubiquitin and the ubiquitin binding protein p62. A strong co-occurrence of immunoreactivity for both pPERK and glycogen synthase kinase 3beta in neurons was also observed. Together, these data indicate that UPR activation in AD neurons occurs at an early stage of neurofibrillary degeneration and suggest that the prolonged activation of the UPR is involved in both tau phosphorylation and neurodegeneration in AD pathogenesis.

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 2α (peIF2α) 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 α-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 α-synuclein.