Pin1 levels are downregulated during ER stress in human neuroblastoma cells

Previously, we showed that pretangle neurons in Alzheimer’s disease (AD) brain display unfolded protein stress in the endoplasmic reticulum (ER). Others showed that the peptidylprolyl isomerase Pin1 protects against tangle formation by facilitating tau dephosphorylation, corroborating with the lower expression of Pin1 observed in tangle-bearing neurons. In this study, we investigated Pin1 expression under ER stress conditions. We show that in human, but not mouse neuroblastoma cells, Pin1 is downregulated in response to ER stress, in accordance with the presence of an ER stress response element in the mouse, but not the human Pin1 gene. This study creates a starting point to investigate whether modulation of the ER stress response may prevent or delay tau pathology in AD.     

Endoplasmic reticulum stress in PLP-overexpressing transgenic rats: gray matter oligodendrocytes are more vulnerable than white matter oligodendrocytes

Studies dealing with transport of proteins from the oligodendrocyte cell body to the myelin sheath reveal the presence of different transport pathways. Proteolipid protein (PLP) is synthesized at the rough endoplasmic reticulum (ER) and then processed through the Golgi apparatus and transported to the myelin membranes. Myelin basic protein (MBP) on the other hand is synthesized locally at the ends of cell processes where its messenger RNA is translated on free ribosomes. Here we show that in rats that overexpress PLP, impairment of PLP transport from the cell body to the processes interferes with the translocation of other membrane proteins such as myelin-associated glycoprotein (MAG) and myelin oligodendrocyte glycoprotein (MOG), but not with peripherally translated MBP. In addition, it also impedes the transport of non-myelin proteins, for example the amyloid precursor protein (APP). At the ultrastructural level, the ER of these metabolically disturbed oligodendrocytes revealed extreme swelling of the cisternae, and immunohistochemistry revealed intense expression of the ER chaperone molecule BiP/GRP78 and ER folding enzyme protein disulfide isomerase (PDI). These features suggest that these oligodendrocytes, which were found exclusively in gray matter areas of the spinal cord, started an unfolded protein response while suffering from ER stress. Some of these disturbed oligodendrocytes were seen to undergo programmed cell death. These results indicate that gray matter oligodendrocyte differ from white matter oligodendrocytes in their capacity to stabilize metabolic disturbances by an unfolded protein response.     

Aβ induces endoplasmic reticulum stress causing possible proteasome impairment via the endoplasmic reticulum–associated degradation pathway

Background: Accumulation of β‐amyloid is a major pathology of Alzheimer’s disease (AD). As in other neurodegenerative diseases, it is also reported that proteasome activity is deteriorated in post‐mortem brains of AD patients. However, the mechanism of proteasomal dysfunction in AD remains unexplained. There is, however, increasing reported evidence that the unfolded protein response (UPR) is involved in AD pathology. Here we show that Aβ causes not only the UPR leading to endoplasmic reticulum (ER) stress mediated cell death, but also proteasomal dysfunction in cultured cells. Methods: Mouse primary cultured neurons and other cultured cells such as HEK 293T or SH‐SY5Y were treated with Aβ or other reagents, such as thapsigargin and lactacystin, to study UPR or proteasome activity. The UPR was investigated using proteins or mRNA expression. To ascertain proteasome activity, we also recruited SH‐SY5Y cells stably transfected with GFP^u. Results: In vitro study showed that UPR, phosphorylation of eIF‐2α and BiP degradation preceded proteasome dysfunction. It is known that the UPR of ER occurs with the assistance of proteasome as ER‐associated protein degradation (ERAD). Conclusion: This evidence, taken together, suggests that Aβ may induce proteasome dysfunction by preceding the UPR through ER‐associated protein degradation.     

Identification of major S‐nitrosylated proteins in murine experimental autoimmune encephalomyelitis

Nitrosative stress has been implicated in the pathophysiology of several CNS disorders, including multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). We have recently shown that protein nitrosothiols (PrSNOs) accumulate in the brain of MS patients, and there is indirect evidence that PrSNO levels are also increased in EAE. In this study we sought to identify the major PrSNOs in the spinal cord of EAE animals prepared by active immunization of C57/BL6 mice with MOG_35–55 peptide. For this purpose, PrSNOs from control and EAE mice at various disease stages were derivatized with HPDP‐biotin, and the biotinylated proteins were isolated with streptavidin‐agarose. Proteins from total and streptavidin‐bound fractions were then analyzed by Western blotting using antibodies against the major S‐nitrosylated substrates of CNS tissue. With this approach we found that the proportion of S‐nitrosylated neurofilament proteins, NMDA receptors, α/β‐tubulin, β‐actin, and GAPDH is increased in EAE. Other potential substrates either were not S‐nitrosylated in vivo (HCN3, HSP‐72, CRMP‐2, γ‐actin, calbindin) or their S‐nitrosylation levels were unaltered in EAE (Na/K ATPase, hexokinase, glycogen phosphorylase). We also discovered that neuronal specific enolase is the major S‐nitrosylated protein in acute EAE. Given that S‐nitrosylation affects protein function, it is likely that the observed changes are significant to the pathophysiology of inflammatory demyelination. © 2009 Wiley‐Liss, Inc.     

Endoplasmic reticulum stress and induction of the unfolded protein response in human sporadic amyotrophic lateral sclerosis

The unfolded protein response (UPR) is induced at symptom onset and disease end stage in rodent models of familial amyotrophic lateral sclerosis (ALS) that express superoxide dismutase (SOD1) mutations. However, ninety percent of human ALS is sporadic and mutations in SOD1 account for only 2% of total ALS. Here we show that a full UPR, including induction of stress sensor kinases, chaperones and apoptotic mediators, is also present in spinal cords of human patients with sporadic disease. Furthermore, the UPR chaperone protein disulphide isomerase (PDI) was present in CSF and was aggregated and widely distributed throughout the motor neurons of these patients. We also show up-regulation of UPR prior to the onset of symptoms in SOD1 rodents, implying an active role in disease. This study offers new insights into pathogenesis, placing ER stress onto a generic pathophysiology for ALS.     

Derlin-1-immunopositive inclusions in patients with Alzheimer's disease

Amyloid plaques and neurofibrillary tangles are the major pathological hallmarks of Alzheimer's disease. Neurofibrillary tangles are composed of filaments and paired helical filaments containing polymerized hyperphosphorylated tau protein. Derlin proteins are a family of proteins that are conserved in all eukaryotes, in which they function in endoplasmic reticulum-associated degradation. Protein disulfide isomerase (PDI) is a member of the thioredoxin superfamily and is believed to accelerate the folding of disulfide-bonded proteins in the luminal space of the endoplasmic reticulum. In this study, we found that derlin-1 and PDI were colocalized in neurofibrillary tangles in the brain of patients with Alzheimer's disease. Derlin-1 and PDI may work as partners to avoid the accumulation of unfolded proteins in Alzheimer's disease. Furthermore, we found that derlin-1 was immunopositive for neurofibrillary tangles and upregulated in Alzheimer's disease and that derlin-1 may play an important role in endoplasmic reticulum-associated degradation during the pathogenesis of Alzheimer's disease. We hypothesize that derlin-1 was upregulated to avoid the aggregation of unfolded proteins. Despite the upregulation of derlin-1, the functions of chaperone proteins and Alzheimer tau protein were lost and these proteins were also accumulated. Finally, they were involved in neurofibrillary tangles. These results suggest that derlin-1 may be associated with endoplasmic reticulum stress in neuronal cells in Alzheimer's disease.     

Mutant SOD1 from spinal cord of G93A rats is destabilized and binds to inner mitochondrial membrane

Mutations in Cu/Zn superoxide dismutase (SOD1) cause amyotrophic lateral sclerosis (ALS). Mechanisms of mutant SOD1 toxicity are unknown, but increased SOD1 activity can boost production of reactive oxygen species (ROS) in the mitochondrial intermembrane space (IMS). Using non-reducing SDS-PAGE we found that in G93A-SOD1 rats the mutant SOD1 was prominently destabilized only in the diseased spinal cord, where this mutant enzyme was also up regulated in the IMS with increased ability to bind the inner membrane of isolated non-transgenic mitoplasts. These mitoplasts increased ROS production when exposed to mutant SOD1 from the spinal cord at the presymptomatic stage. The levels of disulfide-reduced SOD1 peaked at the end stage of the disease, whereas protein disulfide isomerase (PDI), a chaperone capable of rearranging disulfide bonds between cysteine residues of SOD1, was increased prior to the end stage. IMS binding and increased ROS production by destabilized SOD1 may contribute to mitochondrial damage in G93A-SOD1 rats.     

Activating transcription factor 6α deficiency exacerbates oligodendrocyte death and myelin damage in immune‐mediated demyelinating diseases

Endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) play a critical role in immune‐mediated demyelinating diseases, including multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE), by regulating the viability of oligodendrocytes. Our previous studies show that activation of the PERK branch of the UPR protects myelinating oligodendrocytes against ER stress in young, developing mice that express IFN‐γ, a key pro‐inflammatory cytokine in MS and EAE, in the CNS. Several studies also demonstrate that PERK activation preserves oligodendrocyte viability and function, protecting mice against EAE. While evidence suggests activation of the ATF6α branch of the UPR in oligodendrocytes under normal and disease conditions, the effects of ATF6α activation on oligodendrocytes in immune‐mediated demyelinating diseases remain unknown. Herein, we showed that ATF6α deficiency had no effect on oligodendrocytes under normal conditions. Interestingly, we showed that ATF6α deficiency exacerbated ER stressed‐induced myelinating oligodendrocyte death and subsequent myelin loss in the developing CNS of IFN‐γ‐expressing mice. Moreover, we found that ATF6α deficiency increased EAE severity and aggravated EAE‐induced oligodendrocyte loss and demyelination, without affecting inflammation. Thus, these data suggest the protective effects of ATF6α activation on oligodendrocytes in immune‐mediated demyelinating diseases.     

Identification of the protein disulfide isomerase family member PDIp in experimental Parkinson's disease and Lewy body pathology

Parkinson's disease (PD) is a slowly progressing neurodegenerative disorder with no clear etiology. Pathological hallmarks of the disease include the loss of dopaminergic neurons from the substantia nigra (SN) and the presence of Lewy bodies (LBs) (alpha-synuclein and ubiquitin-positive, eosinophilic, cytoplasmic inclusions) in many of the surviving neurons. Experimental modeling of PD neurodegeneration using the neurotoxins 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 1-methyl-4-phenyl-pyridinium (MPP(+)) has identified changes in gene expression of different endoplasmic reticulum (ER) stress proteins associated with MPTP- and PD-related neurodegeneration. We show that the protein disulfide isomerase (PDI) family member pancreatic protein disulfide isomerase (PDIp), previously considered exclusively expressed in pancreatic tissue, is uniquely upregulated among PDI family members within 24 h following exposure of retinoic acid (RA)-differentiated SH-SY5Y human neuroblastoma cells to either 1 mM MPP(+) or 10 microM of the highly specific proteasome inhibitor lactacystin. RT-PCR confirms PDIp expression in brain of post-mortem human PD subjects and immunohistochemical studies demonstrate PDIp immunoreactivity in LBs. Collectively, these findings suggest that increased PDIp expression in dopaminergic (DA) neurons might contribute to LB formation and neurodegeneration, and that this increased PDIp expression may be the result of proteasome impairment.     

TorsinA in PC12 cells: localization in the endoplasmic reticulum and response to stress

Most cases of early-onset torsion dystonia are caused by deletion of GAG in the coding region of the DYT1 gene encoding torsinA. This autosomal dominant neurologic disorder is characterized by abnormal movements, believed to originate from neuronal dysfunction in the basal ganglia of the human brain. The torsins (torsinA and torsinB) are members of the "ATPases associated with a variety of cellular activities" (AAA(+)) superfamily of proteins that mediate chaperone and other functions involved in conformational modeling of proteins, protection from stress, and targeting of proteins to cellular organelles. In this study, the intracellular localization and levels of endogenous torsin were evaluated in rat pheochromocytoma PC12 cells following differentiation and stress. TorsinA, apparent MW 37 kDa, cofractionates with markers for the microsomal/endoplasmic reticulum (ER) compartment and appears to reside primarily within the ER lumen based on protease resistance. TorsinA immunoreactivity colocalizes with the lumenal ER protein protein disulfide isomerase (PDI) and extends throughout neurites. Levels of torsinA did not increase notably in response to nerve growth factor-induced differentiation. None of the stress conditions tested, including heat shock and the unfolded protein response, affected torsinA, except for oxidative stress, which resulted in an increase in the apparent MW of torsinA and redistribution to protrusions from the cell surface. These findings are consistent with a relatively rapid covalent modification of torsinA in response to oxidative stress causing a change in state. Mutant torsinA may interfere with and/or compromise ER functions, especially in dopaminergic neurons, which have high levels of torsinA and are intrinsically vulnerable to oxidative stress.     

Role of PDI in regulating tissue factor: FVIIa activity

Cell exposed tissue factor (TF) is generally in a low procoagulant (“cryptic”) state, and requires an activation step (decryption) to exhibit its full procoagulant potential. Recent data suggest that TF decryption may be regulated by the redox environment through the oxidoreductase activity of protein disulfide isomerase (PDI). In this article we review PDI contribution to different models of TF decryption, namely the disulfide switch model and the phosphatidylserine dynamics, and hypothesize on PDI contribution to TF self-association and association with lipid domains. Experimental evidence debate the disulfide switch model of TF decryption and its regulation by PDI. More recently we showed that PDI oxidoreductase activity regulates the phosphatidylserine equilibrium at the plasma membrane. Interestingly, PDI reductase activity could maintain TF in the reduced monomeric form, while also maintaining low exposure of PS, both states correlated with low procoagulant function. In contrast, PDI inhibition or oxidants may promote the adverse effects with a net increase in coagulation. The relative contribution of disulfide isomerization and PS exposure needs to be further analyzed to understand the redox control of TF procoagulant function. For the moment however TF regulation remains cryptic.     

Transgenic mouse and cell culture models demonstrate a lack of mechanistic connection between endoplasmic reticulum stress and tau dysfunction

In vivo aggregation of tau protein is a hallmark of many neurodegenerative disorders, including Alzheimer's disease (AD). Recent evidence has also demonstrated activation of the unfolded protein response (UPR), a cellular response to endoplasmic reticulum (ER) stress, in AD, although the role of the UPR in disease pathogenesis is not known. Here, three model systems were used to determine whether a direct mechanistic link could be demonstrated between tau aggregation and the UPR. The first model system used was SH‐SY5Y cells, a neuronal cultured cell line that endogenously expresses tau. In this system, the UPR was activated using chemical stressors, tunicamycin and thapsigargin, but no changes in tau expression levels, solubility, or phosphorylation were observed. In the second model system, wild‐type 4R tau and P301L tau, a variant with increased aggregation propensity, were heterologously overexpressed in HEK 293 cells. This overexpression did not activate the UPR. The last model system examined here was the PS19 transgenic mouse model. Although PS19 mice, which express the P301S variant of tau, display severe neurodegeneration and formation of tau aggregates, brain tissue samples did not show any activation of the UPR. Taken together, the results from these three model systems suggest that a direct mechanistic link does not exist between tau aggregation and the UPR. © 2010 Wiley‐Liss, Inc.     

Endoplasmic reticulum stress features are prominent in Alzheimer disease but not in prion diseases in vivo

Prion diseases and Alzheimer disease (AD) share a variety of clinical and neuropathologic features (e.g. progressive dementia, accumulation of abnormally folded proteins in diseased tissue, and pronounced neuronal loss) as well as pathogenic mechanisms like generation of oxidative stress molecules and complement activation. Recently, it was suggested that neuronal death in AD may have its origin in the endoplasmic reticulum (ER). Cellular stress conditions can interfere with protein folding and subsequently cause accumulation of unfolded or misfolded proteins in the ER lumen. The ER responds to this by the activation of adaptive pathways, which are termed unfolded protein response (UPR). The UPR transducer PERK, which launches the most immediate response to ER stress (i.e. the transient attenuation of mRNA translation), and the downstream effector of PERK, eIF2alpha, were shown to be activated in AD. We demonstrate that neither in sporadic nor in infectiously acquired or inherited human prion diseases can the activated forms of PERK and eIF2alpha be detected, except when concomitant neurofibrillary pathology is present; whereas the distribution of phosphorylated PERK correlates with abnormally phosphorylated tau in AD. In brains of scrapie-affected mice and mice infected with sporadic or variant Creutzfeldt-Jakob disease, activated PERK is only very faintly expressed. The lack of prominent activation of the PERK-eIF2alpha pathway in prion diseases suggests that, in contrast to AD, ER stress does not play a crucial role in neuronal death in prion disorders.     

Stress protein expression in early phase spinal cord ischemia/reperfusion injury*

Spinal cord ischemia/reperfusion injury is a stress injury to the spinal cord. Our previous studies using differential proteomics identified 21 differentially expressed proteins (n > 2) in rabbits with spinal cord ischemia/reperfusion injury. Of these proteins, stress-related proteins included protein disulfide isomerase A3, stress-induced-phosphoprotein 1 and heat shock cognate protein 70. In this study, we established New Zealand rabbit models of spinal cord ischemia/reperfusion injury by abdominal aorta occlusion. Results demonstrated that hind limb function initially improved after spinal cord ischemia/reperfusion injury, but then deteriorated. The pathological morphology of the spinal cord became aggravated, but lessened 24 hours after reperfusion. However, the numbers of motor neurons and interneurons in the spinal cord gradually decreased. The expression of protein disulfide isomerase A3, stress-induced-phosphoprotein 1 and heat shock cognate protein 70 was induced by ischemia/reperfusion injury. The expression of these proteins increased within 12 hours after reperfusion, and then decreased, reached a minimum at 24 hours, but subsequently increased again to similar levels seen at 6-12 hours, showing a characterization of induction-inhibition-induction. These three proteins were expressed only in cytoplasm but not in the nuclei. Moreover, the expression was higher in interneurons than in motor neurons, and the survival rate of interneurons was greater than that of motor neurons. It is assumed that the expression of stress-related proteins exhibited a protective effect on neurons.     

Endoplasmic reticulum stress as a novel neuronal mediator in Alzheimer’s disease

Abstract

Alzheimer’s disease (AD) is one of the most common types of progressive dementias. The typical neuropathological changes in AD include extracellular senile plaques, intracellular neurofibrillary tangles, and loss of neurons. The pathogenetic mechanism of this disease is not comprehensively understood yet. Recently, endoplasmic reticulum stress (ER stress) has been considered as a potential event involved in AD development. Some AD-related factors, such as misfolded protein and Ca²⁺ depletion, could disrupt the homeostasis of ER lumen. In AD, the aggregated amyloid-beta peptide (Abeta) could induce ER stress in an assembly dependent way. The presenilin has been identified as a Ca²⁺ channel. Mutations of presenilin could change the balance of Ca²⁺ in ER lumen and thus disrupts the ER homeostasis. Furthermore, the ER stress could lead to cellular disorders like inflammation. Through activating the expression of inflammatory factors, ER stress triggers inflammatory response in AD pathology. Herein, we reviewed the recent progress of ER stress-induced unfolded protein response (UPR) and the roles of ER stress in AD pathological process.     

Overexpression of human S100B exacerbates cerebral amyloidosis and gliosis in the Tg2576 mouse model of Alzheimer's disease

Alzheimer's disease (AD) is the most common progressive dementia and is pathologically characterized by brain deposition of amyloid‐β (Aβ) peptide as senile plaques. Inflammatory and immune response pathways are chronically activated in AD patient brains at low levels, and likely play a role in disease progression. Like microglia, activated astrocytes produce numerous acute‐phase reactants and proinflammatory molecules in the AD brain. One such molecule, S100B, is highly expressed by reactive astrocytes in close vicinity of β‐amyloid deposits. We have previously shown that augmented and prolonged activation of astrocytes has a detrimental impact on neuronal survival. Furthermore, we have implicated astrocyte‐derived S100B as a candidate molecule responsible for this deleterious effect. To evaluate a putative relationship between S100B and AD pathogenesis, we crossed transgenic mice overexpressing human S100B (TghuS100B mice) with the Tg2576 mouse model of AD, and examined AD‐like pathology. Brain parenchymal and cerebral vascular β‐amyloid deposits and Aβ levels were increased in bigenic Tg2576‐huS100B mice. These effects were associated with increased cleavage of the β‐C‐terminal fragment of amyloid precursor protein (APP), elevation of the N‐terminal APP cleavage product (soluble APPβ), and activation of β‐site APP cleaving enzyme 1. In addition, double transgenic mice showed augmented reactive astrocytosis and microgliosis, high levels of S100 expression, and increased levels of proinflammatory cytokines as early as 7–9 months of age. These results provide evidence that (over)‐expression of S100B acts to accelerate AD‐like pathology, and suggest that inhibiting astrocytic activation by blocking S100B biosynthesis may be a promising therapeutic strategy to delay AD progression. © 2009 Wiley‐Liss, Inc.     

S6 kinase phosphorylated at T229 is involved in tau and actin pathologies in Alzheimer's disease

The 70‐kDa ribosomal protein S6 kinase (S6K), a serine/threonine kinase that modulates the phosphorylation of the 40S ribosomal protein S6, regulates cell cycle progression and is known as a tau kinase in Alzheimer's disease (AD). In AD brains, neurofibrillary tangles (NFTs) have been shown to be positively stained with antibodies against S6K proteins phosphorylated at T389 (pT389‐S6K) or T421/S424 (pT421/S424‐S6K) by the mammalian target of rapamycin and mitogen‐activated protein kinase pathways, respectively. However, there is little information available about S6K proteins directly phosphorylated at T229 (pT229‐S6K) by the PI3K‐PDK1 pathway. In the present study, we investigated the distribution of pT229‐S6K in post mortem human brain tissues from elderly (control) and patients with AD using immnunoblotting and immunohistochemistry. pT229‐S6K immunoreactivity was localized to small granular structures in neurons and endothelial cells in control and AD brains. In AD brains, intense pT229‐S6K immunoreactivity was detected in 16.3% of AT8‐positive NFTs, neuropil threads, and dystrophic neurites in the hippocampus and other vulnerable brain areas. In addition, Hirano bodies were also positive for pT229‐S6K but were negative for pT389‐S6K or pT421/S424‐S6K. The present results indicate that S6K phosphorylation via the PI3K‐PD1 pathway is involved in tau pathology in NFTs and abnormal neurites as well as actin pathology in Hirano bodies.