The release of calcium from the endoplasmic reticulum induced by amyloid-beta and prion peptides activates the mitochondrial apoptotic pathway

In this study, we analyzed whether ER Ca²⁺ release, induced by amyloid-β (Aβ) and prion (PrP) peptides activates the mitochondrial-mediated apoptotic pathway. In cortical neurons, addition of the synthetic Aβ1–40 or PrP106–126 peptides depletes ER Ca²⁺ content, leading to cytosolic Ca²⁺ overload. The Ca²⁺ released through ryanodine (RyR) and inositol 1,4,5-trisphosphate (IP₃R) receptors was shown to be involved in the loss of mitochondrial membrane potential, Bax translocation to mitochondria and apoptotic death. Our data further demonstrate that Ca²⁺ released from the ER leads to the depletion of endogenous GSH levels and accumulation of reactive oxygen species, which were also involved in the depolarization of the mitochondrial membrane. These results illustrate that the early Aβ- and PrP -induced perturbation of ER Ca²⁺ homeostasis affects mitochondrial function, activating the mitochondrial-mediated apoptotic pathway and help to clarify the mechanism implicated in neuronal death that occurs in AD and PrD.     

Molecular evidence of founder effects of fatal familial insomnia through SNP haplotypes around the D178N mutation

This work presents a detailed investigation of the genomic region surrounding the PRNP gene in a sample of patients diagnosed with fatal familial insomnia (FFI) from several European countries, notably Spain. The main focus of the study was to explore the origins of the chromosomes carrying the D178N mutation by designing a single-nucleotide polymorphism (SNP) haplotype around the PRNP gene. Haplotypes were constructed by genotyping six SNPs (rs2756271, rs13040327, rs6037932, rs13045348, rs6116474, and rs6116475) in 25 FFI patients from all over Spain. To augment the geographical scope of our study, 13 further FFI cases from Germany (9) and Italy (4) were also examined. Genotyping of SNPs in conjunction with the analysis of genealogical data for a group of FFI patients revealed the existence of two distinct haplotypes potentially associated with the D178N mutation. Of them, GCATTA-M proved to be the common haplotype of Spanish patients, whereas ACATTA-M was typical of the German cases. It is interesting to note that both haplotypes were identified in the Italian samples: GCATTA-M in a family from the Tuscany region and ACATTA-M in a family from the Veneto region. Our findings suggest the occurrence of two independent D178N-129M mutational events in Europe, preserved and transmitted from one generation to the next until nowadays. Likewise, results based on the analysis of SNP data indicate that previous hypotheses postulating that the D178N mutation had independent origins for each family and that its global distribution was determined by recurrent mutational events must be regarded with caution. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Polymorphisms of the prion protein gene (PRNP) in a Korean population

Human prion protein gene (PRNP) has been considered to be involved in the susceptibility of humans to prion diseases. Polymorphisms of methionine (Met)/valine (Val) at codon 129 and of glutamic acid (Glu)/lysine (Lys) at codon 219 are thought to play an important role in susceptibility to sporadic, iatrogenic and variant Creutzfeldt–Jakob disease (CJD). Although the genotype distribution of polymorphisms in PRNP open reading frame (ORF) has been reported in many European populations, among Asian groups, it has been reported only in the Japanese population. We examined the PRNP polymorphisms in 529 healthy Koreans. We observed that genotype frequencies at codon 129 was 94.33% Met/Met, 5.48% Met/Val, and 0.19% Val/Val with an allele frequency of 0.971:0.029 Met:Val, and that genotype frequencies at codon 219 was 92.06% Glu/Glu, 7.94% Glu/Lys, and 0% Lys/Lys with an allele frequency of 0.96:0.04 Glu:Lys. The frequencies of the Glu/Glu genotype (²=10.075, P=0.0015) and of the Glu allele (²=9.486, P=0.0021) at codon 219 were significantly higher in the Korean population than the Japanese population. In addition, the genotype frequency of heterozygotes (12.7%) at codons 129 or/and 219 was significantly lower in Koreans than in people from Great Britain (²=89.52, P<0.0001). The deletion rate of one octarepeat (R2 deletion) was 0.38%, with 99.62% undeleted homozygotes and 0% deleted homozygote. To our knowledge, the R2 octarepeat deletion has never been found in people from countries other than Korea. The data of PRNP polymorphism at codon 219 suggest that Koreans may be more sensitive to sporadic CJD than the Japanese population.     

Cellular prion protein neuroprotective function: implications in prion diseases

Prion protein can display two conformations: a normal cellular conformation (PrP) and a pathological conformation associated with prion diseases (PrP^Sc). Three complementary strategies are used by researchers investigating how PrP is involved in the pathogenesis of prion diseases: elucidation of the normal function of PrP, determination of how PrP^Sc is toxic to neurons, and unraveling the mechanism for the conversion of PrP to PrP^Sc. We review the normal function of PrP as an antioxidant and an antiapoptotic protein in vivo and in vitro. This review also addresses contrasting evidence that PrP is cytotoxic. Finally, we discuss the implication of the neuroprotective role of PrP in prion diseases.     

Inactivation of amyloid-enhancing factor (AEF): study on experimental murine AA amyloidosis

It is known that amyloid-enhancing factor (AEF) shortens the preamyloid phase in experimentally induced AA amyloidosis in mice. Because it is reported that AEF serves as both a nidus and a template for amyloid formation, AA amyloidosis may have transmissibility by a prion-like mechanism. It has been shown that amyloid fibrils also have AEF activity, and amyloid fibrils with AEF activity were named fibril-amyloid enhancing factor (F-AEF). In this study, we investigated methods to inactivate the AEF activity. AEF was extracted from the thyroid gland obtained at autopsy of a patient with AA amyloidosis. Before injection into mice, AEF was treated with several methods for inactivation. Of all the tested treatments, 1 N NaOH, 0.1 N NaOH, and autoclaving consistently demonstrated complete inactivation of AEF. Heat treatment led to incomplete inactivation, but 0.01 N NaOH, 0.001 N NaOH, pepsin, trypsin, pronase, and proteinase K treatment had no effect on AEF activity. By analysis with transmission electron microscopy, the AEF preparation contains amyloid fibrils, and a change of ultrastructure was shown after 1 N NaOH, 0.1 N NaOH, and autoclaving treatment. Furthermore, immunoblotting of AEF with antihuman AA antibody revealed that the protein band was scarcely found after autoclaving, 1 N NaOH, and 0.1 N NaOH treatment. Our results suggest that, similar to Creutzfeldt–Jakob disease (CJD), amyloidosis may require chemical or autoclaving decontamination.     

以原核细胞表达的人PrP蛋白为抗原制备抗人朊蛋？…

目的 制备针对朊蛋白的特异性抗体。方法 以初步纯化的原核细胞表达的ＧＳＴ－ＰｒＰ融合蛋白免疫家兔,制备朊蛋白特异的抗体。结果 以ＧＳＴ－ＰｒＰ－ｓ为抗原,用酶联免疫吸附试验（ＥＬＩＳＡ）检测免疫家兔血清中的朊病毒抗体效价最高可达至１２８０００。Ｗｅｓｔｅｒｎ ｂｏｌｔ检测显示所制备的抗体不仅可以与体外表达的全长及不同Ｃ－末端缺失的人朊蛋白反应,而且能够识别人,鼠脑组织中内源性朊蛋白。结论 利用ＧＳ     

Role of the AMPK pathway in promoting autophagic flux via modulating mitochondrial dynamics in neurodegenerative diseases: Insight into prion diseases

Neurons are highly energy demanding cells dependent on the mitochondrial oxidative phosphorylation system. Mitochondria generate energy via respiratory complexes that constitute the electron transport chain. Adenosine triphosphate depletion or glucose starvation act as a trigger for the activation of adenosine monophosphate-activated protein kinase (AMPK). AMPK is an evolutionarily conserved protein that plays an important role in cell survival and organismal longevity through modulation of energy homeostasis and autophagy. Several studies suggest that AMPK activation may improve energy metabolism and protein clearance in the brains of patients with vascular injury or neurodegenerative disease. Mild mitochondrial dysfunction leads to activated AMPK signaling, but severe endoplasmic reticulum stress and mitochondrial dysfunction may lead to a shift from autophagy towards apoptosis and perturbed AMPK signaling. Hence, controlling mitochondrial dynamics and autophagic flux via AMPK activation might be a useful therapeutic strategy in neurodegenerative diseases to reinstate energy homeostasis and degrade misfolded proteins. In this review article, we discuss briefly the role of AMPK signaling in energy homeostasis, the structure of AMPK, activation mechanisms of AMPK, regulation of AMPK, the role of AMPK in autophagy, the role of AMPK in neurodegenerative diseases, and finally the role of autophagic flux in prion diseases.