Adaptive preconditioning in neurological diseases - therapeutic insights from proteostatic perturbations.

In neurological disorders, both acute and chronic neural stress can disrupt cellular proteostasis, resulting in the generation of pathological protein. However in most cases, neurons adapt to these proteostatic perturbations by activating a range of cellular protective and repair responses, thus maintaining cell function. These interconnected adaptive mechanisms comprise a 'proteostasis network' and include the unfolded protein response, the ubiquitin proteasome system and autophagy. Interestingly, several recent studies have shown that these adaptive responses can be stimulated by preconditioning treatments, which confer resistance to a subsequent toxic challenge - the phenomenon known as hormesis. In this review we discuss the impact of adaptive stress responses stimulated in diverse human neuropathologies including Parkinson׳s disease, Wolfram syndrome, brain ischemia, and brain cancer. Further, we examine how these responses and the molecular pathways they recruit might be exploited for therapeutic gain. This article is part of a Special Issue entitled SI:ER stress.

Mutations in the core and NS5A region of hepatitis C virus genotype 1b and correlation with response to pegylated-interferon-alpha 2b and ribavirin combination therapy.

Mutations in two regions of hepatitis C virus (HCV) have been implicated in influencing response to interferon (IFN) therapy. Substitutions in the NS5A region of HCV have been associated with response to IFN therapy, and this region has been known as the IFN sensitivity-determining region (ISDR). The mutations in the core region of HCV have also been reported to predict IFN response. The aim of this study was to investigate whether amino acid substitutions in the core region and ISDR among patients with HCV genotype 1b affect the response to IFN therapy. A total of 213 patients who completed IFN treatment were randomly selected. All patients received pegylated-IFN-alpha 2b once each week, plus oral ribavirin daily for 48 weeks. Of the 213 patients, 117 (54.9%) showed early virologic response (EVR), with HCV-negativity, at 12 weeks. Factors related to EVR on multivariate analysis were non-Gln70 and Leu91 in the core region, and ISDR mutant-type. One hundred and two (47.9%) showed a sustained virologic response (SVR). SVR occurred more frequently in patients without Gln70 (55.4%) than in those with Gln70 (21.3%) (P < 0.0001). SVR was achieved in 43.6% of patients with wild-type ISDR and 62.5% of patients with mutant-type (P = 0.0227). Of the 34 patients who simultaneously had non-Gln70 and mutant-type ISDR, 26 (76.5%) achieved SVR. Factors related to SVR on multivariate analysis were non-Gln70 and ISDR mutant-type. In conclusion, amino acid substitutions in the core region and ISDR were useful for predicting the response to IFN in patients with HCV genotype 1b.

AATF mediates an antiapoptotic effect of the unfolded protein response through transcriptional regulation of AKT1.

Endoplasmic reticulum (ER) stress-mediated cell death has an important role in the pathogenesis of chronic diseases, including diabetes and neurodegeneration. Although proapoptotic programs activated by ER stress have been extensively studied, identification and characterization of antiapoptotic programs that counteract ER stress are currently incomplete. Through the gene expression profiling of beta-cells lacking Wolfram syndrome 1 gene (WFS1), a causative gene for Wolfram syndrome, we discovered a novel antiapoptotic gene of the unfolded protein response (UPR), apoptosis antagonizing transcription factor (AATF). Here, we study the regulation of AATF, identify its target genes, and determine the basis for its antiapoptotic activities in response to ER stress. We show that AATF is induced by ER stress through the PERK-eIF2alpha pathway and transcriptionally activates the v-akt murine thymoma viral oncogene homolog 1 (AKT1) gene through signal transducer and activator of transcription 3 (Stat3), which sustains Akt1 activation and promotes cell survival. Ectopic expression of AATF or a constitutively active form of AKT1 confers on cells resistance to ER stress-mediated cell death, whereas RNAi-mediated knockdown of AATF or AKT1 renders cells sensitive to ER stress. We also discovered a positive crosstalk between the AATF and WFS1 signaling pathways. Thus, WFS1 deficiency or AATF deficiency mediates a self-perpetuating cycle of cell death. Our results reveal a novel antiapoptotic program relevant to the treatment of diseases caused by ER stress-mediated cell death.

Amyloid beta induces neuronal cell death through ROS-mediated ASK1 activation.

Amyloid beta (Abeta) is a main component of senile plaques in Alzheimer's disease and induces neuronal cell death. Reactive oxygen species (ROS), nitric oxide and endoplasmic reticulum (ER) stress have been implicated in Abeta-induced neurotoxicity. We have reported that apoptosis signal-regulating kinase 1 (ASK1) is required for ROS- and ER stress-induced JNK activation and apoptosis. Here we show the involvement of ASK1 in Abeta-induced neuronal cell death. Abeta activated ASK1 mainly through production of ROS but not through ER stress in cultured neuronal cells. Importantly, ASK1-/- neurons were defective in Abeta-induced JNK activation and cell death. These results indicate that ROS-mediated ASK1 activation is a key mechanism for Abeta-induced neurotoxicity, which plays a central role in Alzheimer's disease.

Neuroleptic malignant syndrome with prolonged catatonia in a dopa-responsive dystonia patient.

The authors describe a patient with dopa-responsive dystonia who developed neuroleptic malignant syndrome with prolonged catatonia following treatment with neuroleptic agents. Use of these agents probably expanded the patient's neuronal dysfunction beyond the nigrostriatal system to involve multiple dopaminergic systems. Electroconvulsive treatment alleviated the prolonged catatonia.

Delayed delivery of AAV-GDNF prevents nigral neurodegeneration and promotes functional recovery in a rat model of Parkinson's disease.

Glial cell line-derived neurotrophic factor (GDNF) is a strong candidate agent in the neuroprotective treatment of Parkinson's disease (PD). We investigated whether adeno-associated viral (AAV) vector-mediated delivery of a GDNF gene in a delayed manner could prevent progressive degeneration of dopaminergic (DA) neurons, while preserving a functional nigrostriatal pathway. Four weeks after a unilateral intrastriatal injection of 6-hydroxydopamine (6-OHDA), rats received injection of AAV vectors expressing GDNF tagged with FLAG peptide (AAV-GDNFflag) or beta-galactosidase (AAV-LacZ) into the lesioned striatum. Immunostaining for FLAG demonstrated retrograde transport of GDNFflag to the substantia nigra (SN). The density of tyrosine hydroxylase (TH)-positive DA fibers in the striatum and the number of TH-positive or cholera toxin subunit B (CTB, neuronal tracer)-labeled neurons in the SN were significantly greater in the AAV-GDNFflag group than in the AAV-LacZ group. Dopamine levels and those of its metabolites in the striatum were remarkably higher in the AAV-GDNFflag group compared with the control group. Consistent with anatomical and biochemical changes, significant behavioral recovery was observed from 4-20 weeks following AAV-GDNFflag injection. These data indicate that a delayed delivery of GDNF gene using AAV vector is efficacious even 4 weeks after the onset of progressive degeneration in a rat model of PD.

Gastrin and Helicobacter pylori in low-grade MALT lymphoma patients.

BACKGROUND: This study of patients with Helicobacter pylori infection and low-grade MALT lymphoma aimed to investigate: 1) the effect of H. pylori eradication therapy on the serum gastrin level, 2) whether changes of the serum gastrin level after therapy could predict the prognosis of patients with this tumour, and 3) the relationship between the gastric H. pylori load, the serum gastrin level and the status of MALT lymphoma. METHODS: Thirteen patients with documented low-grade MALT lymphoma and H. pylori infection were enrolled and received H. pylori eradication therapy as the sole initial treatment. The presence of H. pylori, the serum gastrin level, the endoscopic findings, the pathologic features of the biopsies and resected specimens, and the endoscopic ultrasonography findings were evaluated before and after therapy. Follow-up was carried out every 3-6 months. RESULTS: H. pylori eradication was eventually achieved in all 13 patients. The pretreatment fasting serum gastrin level decreased from 177.1 +/- 107.4 pg/ml to 129.2 +/- 78.1, 96.4 +/- 66.6 and 80.1 +/- 42.7 pg/ml after 0-3, 3-6 and 6-9 months, respectively (all P < 0.05). Successful eradication of H. pylori was followed by a decrease of the fasting serum gastrin level and complete regression of initial low-grade MALT lymphoma was observed in all patients. However, two patients subsequently developed recurrent high-grade MALT lymphoma or high-grade lymphoma. In one of them, the serum gastrin level rose again above the pretreatment value. In the other, however, the fasting gastrin level fell throughout the study period. The median fasting serum gastrin level before H. pylori eradication therapy was higher in the patients with tumours of the gastric body (203.4 +/- 108.9 pg/ml) than in those with tumours of the antrum and angulus (89.3 +/- 28.0 pg/ml) (P = 0.06). CONCLUSIONS: Hypergastrinaemia may be associated with an increased risk of gastric MALT lymphoma.

Inhibition of CHOP translation by a peptide encoded by an open reading frame localized in the chop 5'UTR.

Chop is a ubiquitously expressed mammalian gene encoding a small nuclear protein related to the CCAAT/enhancer-binding protein (C/EBP) family of transcription factors. CHOP protein plays an important role in various cellular processes such as growth, differentiation and programmed cell death. CHOP expression is strongly increased in response to a large variety of stresses including perturbation of the endoplasmic reticulum function, DNA damage and nutrient deprivation. Multiple mechanisms including transcriptional and post-transcriptional controls are involved in the regulation of CHOP expression. We show here that the 5'UTR of the Chop transcript plays an important role in controlling the synthesis of CHOP protein. In particular, the 5'UTR contains a conserved uORF which encodes a 31 amino acid peptide that inhibits the expression of the downstream ORF. Mutational analysis of the 5' leader region and peptide coding sequences suggests that the peptide itself inhibits expression of the downstream ORF. Such results suggest a role for uORF in limiting ribosomal access to downstream initiation sites. With respect to the importance of CHOP protein in the regulation of cellular functions, the mechanisms that regulate its basal level are of considerable interest.

Triple transduction with adeno-associated virus vectors expressing tyrosine hydroxylase, aromatic-L-amino-acid decarboxylase, and GTP cyclohydrolase I for gene therapy of Parkinson's disease.

Parkinson's disease (PD), a neurological disease suited to gene therapy, is biochemically characterized by a severe decrease in the dopamine content of the striatum. One current strategy for gene therapy of PD involves local production of dopamine in the striatum achieved by inducing the expression of enzymes involved in the biosynthetic pathway for dopamine. We previously showed that the coexpression of tyrosine hydroxylase (TH) and aromatic-L-amino-acid decarboxylase (AADC), using two separate adeno-associated virus (AAV) vectors, resulted in more effective dopamine production and more remarkable behavioral recovery in 6-hydroxydopamine-lesioned parkinsonian rats, compared with the expression of TH alone. Not only levels of TH and AADC but also levels of tetrahydrobiopterin (BH4), a cofactor of TH, and GTP cyclohydrolase I (GCH), a rate-limiting enzymes for BH4 biosynthesis, are reduced in parkinsonian striatum. In the present study, we investigated whether transduction with separate AAV vectors expressing TH, AADC, and GCH was effective for gene therapy of PD. In vitro experiments showed that triple transduction with AAV-TH, AAV-AADC, and AAV-GCH resulted in greater dopamine production than double transduction with AAV-TH and AAV-AADC in 293 cells. Furthermore, triple transduction enhanced BH4 and dopamine production in denervated striatum of parkinsonian rats and improved the rotational behavior of the rats more efficiently than did double transduction. Behavioral recovery persisted for at least 12 months after stereotaxic intrastriatal injection. These results suggest that GCH, in addition to TH and AADC, is important for effective gene therapy of PD.

Male sterility and enhanced radiation sensitivity in TLS(-/-) mice.

TLS (also known as FUS) is an RNA-binding protein that contributes the N-terminal half of fusion oncoproteins implicated in the development of human liposarcomas and leukemias. Here we report that male mice homozygous for an induced mutation in TLS are sterile with a marked increase in the number of unpaired and mispaired chromosomal axes in pre-meiotic spermatocytes. Nuclear extracts from TLS(-/-) testes lack an activity capable of promoting pairing between homologous DNA sequences in vitro, and TLS(-/-) mice and embryonic fibroblasts exhibit increased sensitivity to ionizing irradiation. These results are consistent with a role for TLS in homologous DNA pairing and recombination.

Acute myeloid leukemia possessing jumping translocation is related to highly elevated levels of EAT/mcl-1, a Bcl-2 related gene with anti-apoptotic functions.

Jumping translocations (JTs) are unbalanced chromosomal translocations in which an identical chromosomal region is translocated to the telomeric region of different chromosomes. JTs are rare in hematological malignancies where they are second translocations and may be an indicator of poor prognosis. We report a case of acute myeloid leukemia with t(16;21) and a JT in which the long arm of chromosome 1 distal to q21 is translocated to the terminal region of chromosome 10. The leukemic cells exhibit high expression of EAT/mcl1, an anti-apoptotic Bcl-2 related gene. Since EAT/mcl1 is mapped to 1q21 near the breakpoint in the JTs, high level expression of EAT/mcl1 may be associated with the poor prognosis of leukemia with JTs.

High frequency of inactivation of the imprinted H19 gene in "sporadic" hepatoblastoma.

Embryonal tumors such as Wilms' tumor (WT), embryonal rhabdomyosarcoma (eRMS) and hepatoblastoma have been thought to have a common pathogenetic mechanism. H19 was found to be inactivated in WT and eRMS either by loss of heterozygosity or by hypermethylation of the maternal allele. We show here that the expression of the H19 gene is inactivated by maternal allelic loss or hypermethylation in 7 out of 8 "sporadic" hepatoblastomas. Furthermore, we analyzed expression of the IGF2 gene. Loss of imprinting of the IGF2 gene was detected and linked to inactivation of the H19 gene in 2 hepatoblastomas. However, 2 sporadic cases demonstrated monoallelic expression of the IGF2 gene with inactivation of the H19 gene. Our results suggest that H19 may play a role as a common imprinted tumor suppressor gene in "sporadic" hepatoblastomas but may at times work independently of IGF2 expression.

The genomic breakpoint and chimeric transcripts in the EWSR1-ETV4/E1AF gene fusion in Ewing sarcoma.

Chromosome translocation creates a fusion between the EWSR1 gene and an ETS family gene. The fusion between these two genes is a characteristic feature of Ewing sarcoma. We previously identified a fourth translocation, t(17;22)(q12;q12), in genomic DNA isolated from cells of patients affected with Ewing sarcoma. The discovery of this translocation suggested that there might be a novel EWSR1-ETV4 fusion gene. In the present study, we determined the genomic breakpoint and characterized the chimeric transcript of the EWSR1-ETV4 fusion gene in two t(17;22) Ewing sarcomas. Reverse transcriptase-PCR assay showed an in-frame fusion between the 5'-terminal region of EWSR1 and the 3' end of ETV4 (alias E1AF, PEA3); the chimeric transcript could thus serve as a template for expression of a protein composed of the N-terminal portion of EWSR1 fused to the DNA-binding domain of ETV4. Long PCR and sequence analysis of genomic DNA revealed that either exon 8 or intron 7 of EWSR1 is fused to the same intron of ETV4 in both tumors. Several palindromic oligomer sequences were found close to the breakpoints in both genes. The 159-bp Alu-like sequence was repeated in the breakpoint region of the ETV4 gene. These observations suggest a mechanism of EWSR1-ETV4 gene fusion.

Molecular analysis of Ewing's sarcoma: another fusion gene, EWS-E1AF, available for diagnosis.

Ewing's sarcoma, one of the most malignant tumors of children and young adults, expresses specific chimeric genes, e.g. EWS-FLI-1, EWS-ERG, EWS-ETV1 and EWS-FEV. In this paper, we extensively characterized a new fusion gene, EWS-EIAF by means of whole cDNA sequencing, RNA blot analysis, DNA blot analysis and chromosomal analysis, and showed it to be available for the diagnosis of Ewing's sarcoma and to participate in the oncogenesis of Ewing's sarcoma. Furthermore, we conducted a genetic analysis of Ewing family tumors in conjunction with immunohistochemical analysis and ultrastructural analysis. Our results demonstrate some limitations of both genetic analysis and histopathological analysis, and establish the relationship between neurogenic phenotypes and chimera genes.

A long-term follow-up study of interferon treatment for chronic hepatitis C in Japanese patients with congenital bleeding disorders.

Twenty-one HIV negative Japanese patients with chronic hepatitis C who had congenital bleeding disorders, 15 hemophilia A, 3 hemophilia B, 1 von Willebrand's disease, 1 afibrinogenemia and 1 thrombasthenia, were treated with 9 million units 3 times a week of natural interferon (IFN)-alpha for 6 months. They were followed, biochemically and virologically, for at least 18 months after therapy discontinuation to evaluate the long-term results. Liver biopsy, hepatitis C virus (HCV) genotyping and quantification of viral load by polymerase chain reaction (PCR) were performed to identify the predictors of a favorable response to IFN treatment. One male patient with hemophilia A dropped out because of general fatigue and was excluded from evaluation. Ten (50.0%) patients continued to be HCV RNA negative in serum together with normal ALT levels throughout the study. Subtype 1b and a high level of viremia significantly associated with an unfavorable outcome on the response to IFN although liver histology was not definitive for predicting the response. We concluded that a 6-month treatment with high doses of natural IFN-alpha was effective in inducing a long-term response without relapse of viremia in 50% of chronic hepatitis C patients with congenital bleeding disorders and that HCV subtype and pretreatment level of viremia were useful predictors of the response to IFN in treating such patients.

Serum levels of soluble intercellular adhesion molecule-1 and soluble vascular cell adhesion molecule-1 in liver disease, and their changes by treatment with interferon.

Serum levels of soluble intercellular adhesion molecule-1 (sICAM-1) and soluble vascular cell adhesion molecule-1 (sVCAM-1) were measured by an enzyme-linked immunosorbent assay in patients with chronic hepatitis (n = 57), liver cirrhosis (n = 19) and hepatocellular carcinoma (n = 33). Serum levels of sICAM-1 and sVCAM-1 were significantly higher in liver disease than those in controls (P < 0.0001 and P < 0.0005, respectively). A total of 22 patients with chronic hepatitis C were treated with interferon. Pretreatment levels of sICAM-1 and sVCAM-1 were not significantly different between complete responders and non-responders. In complete responders, serum sICAM-1 and sVCAM-1 levels 1 year after interferon treatment significantly decreased compared to the pretreatment levels (P < 0.005 and P < 0.05, respectively). Post-treatment levels of sICAM-1 and sVCAM-1 in complete responders were also significantly lower than those in non-responders (P < 0.005 and P < 0.05, respectively). This suggests that monitoring soluble adhesion molecules might be useful in the follow-up of patients with liver disease.

Idiopathic adulthood ductopenia.

In 1981, a 26 year old man occasionally demonstrated elevated serum transaminase concentrations. He had no history of medication, or a personal or family history of jaundice, except for prolonged physiological jaundice as a neonate. Serum hepatitis B surface antigen, hepatitis C virus antibody and anti-mitochondrial antibody were absent. A wedge biopsy specimen revealed ductular proliferation, mild inflammation of the portal area and disappearance of bile ducts from 80% of the portal tracts. Serial sections demonstrated a vanishing bile duct. Endoscopic retrograde choledochopancreatography, portography and arteriography demonstrated no abnormalities. In 1994, the patient died of hepatic failure following a 12 year observation period. He was subsequently diagnosed with idiopathic adulthood ductopenia on the basis of the criteria proposed by Ludwig.