Yasuko Kitao

Transmission of cell stress from endoplasmic reticulum to mitochondria: enhanced expression of Lon protease

The rat homologue of a mitochondrial ATP-dependent protease Lon was cloned from cultured astrocytes exposed to hypoxia. Expression of Lon was enhanced in vitro by hypoxia or ER stress, and in vivo by brain ischemia. These observations suggested that changes in nuclear gene expression (Lon) triggered by ER stress had the potential to impact important mitochondrial processes such as assembly and/or degradation of cytochrome c oxidase (COX). In fact, steady-state levels of nuclear-encoded COX IV and V were reduced, and mitochondrial-encoded subunit II was rapidly degraded under ER stress. Treatment of cells with cycloheximide caused a similar imbalance in the accumulation of COX subunits, and enhanced mRNA for Lon and Yme1, the latter another mitochondrial ATP-dependent protease. Furthermore, induction of Lon or GRP75/mtHSP70 by ER stress was inhibited in PERK (−/−) cells. Transfection studies revealed that overexpression of wild-type or proteolytically inactive Lon promoted assembly of COX II into a COX I–containing complex, and partially prevented mitochondrial dysfunction caused by brefeldin A or hypoxia. These observations demonstrated that suppression of protein synthesis due to ER stress has a complex effect on the synthesis of mitochondrial-associated proteins, both COX subunits and ATP-dependent proteases and/or chaperones contributing to assembly of the COX complex.

Expression of the oxygen-regulated protein ORP150 accelerates wound healing by modulating intracellular VEGF transport

Expression of angiogenic factors such as VEGF under conditions of hypoxia or other kinds of cell stress contributes to neovascularization during wound healing. The inducible endoplasmic reticulum chaperone oxygen-regulated protein 150 (ORP150) is expressed in human wounds along with VEGF. Colocalization of these two molecules was observed in macrophages in the neovasculature, suggesting a role of ORP150 in the promotion of angiogenesis. Local administration of ORP150 sense adenovirus to wounds of diabetic mice, a treatment that efficiently targeted this gene product to the macrophages of wound beds, increased VEGF antigen in wounds and accelerated repair and neovascularization. In cultured human macrophages, inhibition of ORP150 expression caused retention of VEGF antigen within the endoplasmic reticulum (ER), while overexpression of ORP150 promoted the secretion of VEGF into hypoxic culture supernatants. Taken together, these data suggest an important role for ORP150 in the setting of impaired wound repair and identify a key, inducible chaperone-like molecule in the ER. This novel facet of the angiogenic response may be amenable to therapeutic manipulation.

Expression of the endoplasmic reticulum molecular chaperone (ORP150) rescues hippocampal neurons from glutamate toxicity.

A series of events initiated by glutamate-receptor interaction perturbs cellular homeostasis resulting in elevation of intracellular free calcium and cell death. Cells subject to such environmental change express stress proteins, which contribute importantly to maintenance of metabolic homeostasis and viability. We show that an inducible chaperone present in endoplasmic reticulum (ER), the 150-kDa oxygen-regulated protein (ORP150), is expressed both in the human brain after seizure attack and in mouse hippocampus after kainate administration. Using mice heterozygous for ORP150 deficiency, exposure to excitatory stimuli caused hippocampal neurons to display exaggerated elevation of cytosolic calcium accompanied by activation of mu-calpain and cathepsin B, as well as increased vulnerability to glutamate-induced cell death in vitro and decreased survival to kainate in vivo. In contrast, targeted neuronal overexpression of ORP150 suppressed each of these events and enhanced neuronal and animal survival in parallel with diminished seizure intensity. Studies using cultured hippocampal neurons showed that ORP150 regulates cytosolic free calcium and activation of proteolytic pathways causing cell death in neurons subject to excitatory stress. Our data underscore a possible role for ER stress in glutamate toxicity and pinpoint a key ER chaperone, ORP150, which contributes to the stress response critical for neuronal survival.

Role of Herp in the endoplasmic reticulum stress response

Application of differential display to cultured rat astrocytes allowed cloning of Herp cDNA. Although Herp was strongly induced by endoplasmic reticulum (ER) stress, it decayed rapidly consequent to proteasome‐mediated degradation. To investigate the role of this molecule in terms of the stress response, Herp knockout cells were developed using F9 embryonic carcinoma cells. F9 Herp null cells were more vulnerable to ER stress compared with F9 wild‐type cells. In the early period of ER stress (0–8 h after tunicamycin treatment), Herp null cells displayed enhanced ER stress signalling and stabilization of an endogenous ERAD substrate, compared with wild‐type cells. In the intermediate period (8–20 h after tunicamycin treatment), Herp null cells displayed reduced ER stress signalling, whereas in the late period (20–40 h after tunicamycin treatment), Herp null cells manifested irreversible cellular changes that lead to apoptotic cell death. Transfection analysis revealed that the N‐terminal region, including the ubiquitin‐like domain of Herp, was required for the survival of F9 cells under ER stress. These results indicate that Herp is a short‐lived Ub‐like protein improving the balance of folding capacity and protein loads in the ER and plays crucial roles for the ER stress resistance in F9 cells.

ORP150/HSP12A protects renal tubular epithelium from ischemia-induced cell death

The 150 kDa oxygen‐regulated protein (ORP150) is an inducible endoplasmic reticulum (ER) chaperone with cytoprotective properties in settings of cell stress, such as ischemia/reperfusion (I/R). Renal tissue from patients with acute renal failure displayed strong induction of ORP150 in tubular epithelium. In a rodent model of renal I/R injury, ORP150 was expressed in both the ischemic and contralateral kidney, principally in the thick ascending loop of Henle (TAL) and distal tubules. Cultured renal epithelial cells exposed to hypoxic or hyperosmotic conditions displayed induction of ORP150. Renal tubular epithelial cells stably transfected with ORP150 sense or antisense cDNA displayed a strong correlation between ORP150 expression and vulnerability to hypoxic/osmotic stress; higher levels of ORP150 were protective, whereas lower levels increased susceptibility to cell death. Compared with nontransgenic controls, transgenic mice overexpressing ORP150 subjected to renal I/R displayed a blunted rise of serum creatinine and blood urea nitrogen, and enhanced survival of TAL, consistent with cytoprotection. In contrast, heterozygous ORP150+/− mice, with lower levels of ORP150, showed enhanced renal injury. These data are consistent with the possibility that ORP150 exerts cytoprotective effects in renal tubular epithelia subjected to I/R injury and suggest a key role for ER stress in the renal tubular response to acute renal failure.

Topographic projections from the basal ganglia to the nucleus tegmenti pedunculopontinus pars compacta of the cat with special reference to pallidal projections

SummaryProjections from the basal ganglia to the nucleus tegmenti pedunculopontinus pars compacta (TPC) were studied by using anterograde and retrograde tracing techniques with horseradish peroxidase conjugated with wheat germ agglutinin (WGA-HRP) in the cat. Following WGA-HRP injections into the medial TPC area, a substantial number of retrogradely labeled cells were seen in the entopeduncular nucleus (EP) and medial half of the substantia nigra pars reticulata (SNr), whereas following WGA-HRP injections into the lateral TPC area, labeled cells were marked in the caudal half of the globus pallidus (GP) and lateral half of the SNr. To confirm the retrograde tracing study, WGA-HRP was injected into the EP or the caudal GP, and anterograde labeling was observed in the TPC areas. Terminal labeling was located in the medail TPC area in the EP injection case, while terminal labeling was observed in the lateral TPC area in the caudal GP injection case. Projections from the striatum to the pallidal complex (the EP and the caudal GP) were also studied autoradiographically by injecting amino acids into various parts of the caudate nucleus and the putamen. Terminal labeling was distributed over the whole extent of the EP and the rostral GP following injections into the rostral striatum (the head of the caudate nucleus or the rostral part of the putamen), while terminal labeling was distributed over the caudal GP following injections into the caudal striatum (the body of the caudate nucleus or the caudal part of the putamen). From these findings, we conclude that there exists a medio-lateral topography in the projection from the basal ganglia to the TPC: The EP receives afferent projections from the rostral striatum and projects to the medial TPC area, whereas the caudal GP receives projections from the caudal striatum and sends fibers to the lateral TPC area.

ORP150/HSP12A Regulates Purkinje Cell Survival: A Role for Endoplasmic Reticulum Stress in Cerebellar Development

The endoplasmic reticulum (ER) stress response contributes to neuronal survival in ischemia and neurodegenerative processes. ORP150 (oxygen-regulated protein 150)/HSP12A (heat shock protein 12A), a novel stress protein located in the ER, was markedly induced in Purkinje cells maximally at 4-8 d after birth, a developmental period corresponding to their vulnerability to cell death. Both terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end-labeling analysis and immunostaining using anti-activated caspase-3 antibody revealed that transgenic mice with targeted neuronal overexpression of ORP150 (Tg ORP150) displayed diminished cell death in the Purkinje cell layer and increased numbers of Purkinje cells up to 40 d after birth (p < 0.01), compared with those observed in heterozygous ORP150/HSP12A-deficient (ORP150+/-) mice and wild-type littermates (ORP150+/+). Cultured Purkinje cells from Tg ORP150 mice displayed resistance to both hypoxia- and AMPA-induced stress. Behavioral analysis, using rotor rod tasks, indicated impairment of cerebellar function in Tg ORP150 animals, consistent with the concept that enhanced survival of Purkinje cells results in dysfunction. These data suggest that ER chaperones have a pivotal role in Purkinje cell survival and death and thus may highlight the importance of ER stress in neuronal development.

ATF6alpha promotes astroglial activation and neuronal survival in a chronic mouse model of Parkinson's disease.

Accumulating evidence suggests a crucial role for the unfolded protein response (UPR) in Parkinson’s disease (PD). In this study, we investigated the relevance of the UPR in a mouse model of chronic MPTP/probenecid (MPTP/P) injection, which causes severe and persistent degeneration of dopaminergic neurons. Enhanced activation of the UPR branches, including ATF6α and PERK/eIF2α/ATF4, was observed after MPTP/P injections into mice. Deletion of the ATF6α gene accelerated neuronal degeneration and ubiquitin accumulation relatively early in the MPTP/P injection course. Surprisingly, astroglial activation was strongly suppressed, and production of the brain-derived neurotrophic factor (BDNF) and anti-oxidative genes, such as heme oxygenase-1 (HO-1) and xCT, in astrocytes were reduced in ATF6α −/− mice after MPTP/P injections. Decreased BDNF expression in ATF6α −/− mice was associated with decreased expression of GRP78, an ATF6α-dependent molecular chaperone in the ER. Decreased HO-1 and xCT levels were associated with decreased expression of the ATF4-dependent pro-apoptotic gene CHOP. Consistent with these results, administration of the UPR-activating reagent tangeretin (5,6,7,8,4′-pentamethoxyflavone; IN19) into mice enhanced the expression of UPR-target genes in both dopaminergic neurons and astrocytes, and promoted neuronal survival after MPTP/P injections. These results suggest that the UPR is activated in a mouse model of chronic MPTP/P injection, and contributes to the survival of nigrostriatal dopaminergic neurons, in part, through activated astrocytes.

Monosynaptic nigral inputs to the pedunculopontine tegmental nucleus neurons which send their axons to the medial reticular formation in the medulla oblongata. An electron microscopic study in the cat

An electron microscopic study in the cat has suggested that neurons in the substantia nigra pars reticulata (SNr) project to the medial reticular formation of the medulla oblongata (MRF) via the pedunculopontine tegmental nucleus (PPN): PPN neurons, which were labeled with a retrograde tracer (WGA-HRP; horseradish peroxidase conjugated to wheat germ agglutinin) injected into the MRF, were found to be in synaptic contact with axon terminals which were degenerated with neurotoxic agents applied into the SNr.

Deletion of SERP1/RAMP4, a Component of the Endoplasmic Reticulum (ER) Translocation Sites, Leads to ER Stress

ABSTRACT Stress-associated endoplasmic reticulum (ER) protein 1 (SERP1), also known as ribosome-associated membrane protein 4 (RAMP4), is a Sec61-associated polypeptide that is induced by ER stress. SERP1−/− mice, made by targeted gene disruption, demonstrated growth retardation, increased mortality, and impaired glucose tolerance. Consistent with high levels of SERP1 expression in pancreas, pancreatic islets from SERP1−/− mice failed to rapidly synthesize proinsulin in response to a glucose load. In addition, reduced size and enhanced ER stress were observed in the anterior pituitary of SERP1−/− mice, and growth hormone production was slowed in SERP1−/− pituitary after insulin stimulation. Experiments using pancreatic microsomes revealed aberrant association of ribosomes and the Sec61 complex and enhanced ER stress in SERP1−/− pancreas. In basal conditions, the Sec61 complex in SERP1−/− microsomes was more cofractionated with ribosomes, compared with SERP1+/+ counterparts, in high-salt conditions. In contrast, after glucose stimulation, the complex showed less cofractionation at an early phase (45 min) but more at a later phase (120 min). Although intracellular insulin/proinsulin levels were not significantly changed in both genotypes, these results suggest that subtle changes in translocation efficiency play an important role in the regulation of ER stress and rapid polypeptide synthesis.