Brain expression of presenilins in sporadic and early-onset, familial Alzheimer's disease

BACKGROUND: Mutations in the presenilin proteins cause early-onset, familial Alzheimer's disease (FAD). MATERIALS AND METHODS: We characterized the cellular localization and endoproteolysis of presenilin 2 (PS2) and presenilin 1 (PS1) in brains from 25 individuals with presenilin-mutations causing FAD, as well as neurologically normal individuals and individuals with sporadic Alzheimer's disease (AD). RESULTS: Amino-terminal antibodies to both presenilins predominantly decorated large neurons. Regional differences between the broad distributions of the two presenilins were greatest in the cerebellum, where most Purkinje cells showed high levels of only PS2 immunoreactivity. PS2 endoproteolysis in brain yielded multiple amino-terminal fragments similar in size to the PS1 amino-terminal fragments detected in brain. In addition, two different PS2 amino-terminal antibodies also detected a prominent 42 kDa band that may represent a novel PS2 form in human brain. Similar to PS1 findings, neither amino-terminal nor antiloop PS2 antibodies revealed substantial full-length PS2 in brain. Immunocytochemical examination of brains from individuals with the N141I PS2 mutation or eight different PS1 mutations, spanning the molecule from the second transmembrane domain to the large cytoplasmic loop domain, revealed immunodecoration of no senile plaques and only neurofibrillary tangles in the M139I PS1 mutation stained with PS1 antibodies. CONCLUSIONS: Overall presenilin expression and the relative abundance of full-length and amino-terminal fragments in presenilin FAD cases were similar to control cases and sporadic AD cases. Thus, accumulation of full-length protein or other gross mismetabolism of neither PS2 nor PS1 is a consequence of the FAD mutations examined.     

The spectrum of mitochondrial DNA mutations in families with Leber hereditary optic neuroretinopathy

The mitochondrial complex I genes were sequenced in seven Leber hereditary optic neuroretinopathy (LHON) families without the ND4/11778 and ND1/3460 mutations. Four replacement mutations restricted only to LHON families were found, one in the ND1 gene at nt 4025, and three in the ND5 gene at nt 12811, 13637, and 13967. The mutations did not change evolutionarily conserved amino acids suggesting that they are not primary LHON mutations in these families. They may be considered as secondary LHON mutations serving as exacerbating factors in an appropriate genetic background. A complex III mutation, cyt b/15257, has been suggested to be one of the primary mutations causing LHON. Its presence was determined for 23 Finnish LHON families, and it was detected in two families harboring the ND4/11778 mutation. Similarly, complex IV mutation COI/7444 was screened in Finnish LHON families, and it was found in one family carrying the ND1/3460 mutation.     

Analysis of mitochondrial ND4 gene DNA sequence in Finnish families with Leber hereditary optic neuroretinopathy

A mutation in the mitochondrial DNA at nt 11,778 has recently been found in Leber hereditary optic neuroretinopathy (LHON), a maternally inherited ocular disease. The mutation is located in the ND4 gene encoding subunit 4 of the respiratory chain enzyme NADH dehydrogenase. The mutation was subsequently not found in 9 of the 20 known Finnish families with LHON, implying that there are at least two different mutations associated with the disease. Using direct sequencing of PCR-amplified mtDNA, we have now sequenced the entire ND4 region in the families without the nt 11,778 mutation to find the other mutations. No new mutations in the ND4 region were found, suggesting that the putative mtDNA mutation in these families may be in the coding regions for other subunits of NADH dehydrogenase enzyme. The sequence of ND4 gene as found to be highly homogeneous.     

A comparison of single nucleotide primer extension with mispairing PCR-RFLP in detecting a point mutation.

A recent report by Petruzzella et al. (BBRC 186, 491-497, 1992) raised a question as to whether a point mutation in the mitochondrial ND2 gene (BBRC 182, 238-246, 1992) is relevant to Alzheimer's disease. The argument was based on their inability to detect the point mutation at position 5460 in codon 331 in the DNAs extracted from 15 patients with Alzheimer's disease using mispairing PCR-RFLP. To clarify the discrepancy, we tested the DNAs reported by Petruzzella et al. for the mutation by single-nucleotide primer extension. The present work confirms our previous report and extends our finding of the point mutation in 8 of the 15 AD DNAs.     

Novel mutations in TARDBP (TDP-43) in patients with familial amyotrophic lateral sclerosis

The TAR DNA-binding protein 43 (TDP-43) has been identified as the major disease protein in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin inclusions (FTLD-U), defining a novel class of neurodegenerative conditions: the TDP-43 proteinopathies. The first pathogenic mutations in the gene encoding TDP-43 (TARDBP) were recently reported in familial and sporadic ALS patients, supporting a direct role for TDP-43 in neurodegeneration. In this study, we report the identification and functional analyses of two novel and one known mutation in TARDBP that we identified as a result of extensive mutation analyses in a cohort of 296 patients with variable neurodegenerative diseases associated with TDP-43 histopathology. Three different heterozygous missense mutations in exon 6 of TARDBP (p.M337V, p.N345K, and p.I383V) were identified in the analysis of 92 familial ALS patients (3.3%), while no mutations were detected in 24 patients with sporadic ALS or 180 patients with other TDP-43-positive neurodegenerative diseases. The presence of p.M337V, p.N345K, and p.I383V was excluded in 825 controls and 652 additional sporadic ALS patients. All three mutations affect highly conserved amino acid residues in the C-terminal part of TDP-43 known to be involved in protein-protein interactions. Biochemical analysis of TDP-43 in ALS patient cell lines revealed a substantial increase in caspase cleaved fragments, including the approximately 25 kDa fragment, compared to control cell lines. Our findings support TARDBP mutations as a cause of ALS. Based on the specific C-terminal location of the mutations and the accumulation of a smaller C-terminal fragment, we speculate that TARDBP mutations may cause a toxic gain of function through novel protein interactions or intracellular accumulation of TDP-43 fragments leading to apoptosis.     

Novel mutation at the initiation codon in the Norrie disease gene in two Japanese families

We have identified a new mutation of Norrie disease (ND) gene in two Japanese males from unrelated families; they showed typical ocular features of ND but no mental retardation or hearing impairment. A mutation was found in both patients at the initation codon of exon 2 of the ND gene (ATG to GTG), with otherwise normal nucleotide sequences. Their mothers had the normal and mutant types of the gene, which was expected for heterozygotes of the disease. The mutation of the initiation codon would cause the failure of ND gene expression or a defect in translation thereby truncating the amino terminus of ND protein. In view of the rarity and marked heterogeneity of mutations in the ND gene, the present apparently unrelated Japanese families who have lived in the same area for over two centuries presumably share the origin of the mutation.     

Mutation Screening of the <Emphasis Type="Italic">CHCHD10</Emphasis> Gene in Chinese Patients with Amyotrophic Lateral Sclerosis

Mutations in the coiled-coil-helix-coiled-coil-helix domain-containing protein 10 gene (CHCHD10), involved in mitochondrial function, have recently been reported as a causative gene of amyotrophic lateral sclerosis (ALS). The aim of this study was to obtain the mutation prevalence of CHCHD10 and the phenotypes with mutations in Chinese ALS patients. A cohort of 499 ALS patients including 487 sporadic ALS (SALS) and 12 familial ALS (FALS), from the Department of Neurology, West China Hospital of Sichuan University, were screened for mutations of all exons of the CHCHD10 gene by Sanger sequencing. Novel candidate mutations or variants were confirmed by polymerase chain reaction-restriction fragment length polymorphism in 466 healthy individuals. All patients identified with mutations of CHCHD10 gene were screened for mutations of the common ALS causative genes including C9orf72, SOD1, TARDBP, FUS, PFN1, and SQSTM1. Three heterozygous variants, including two missense mutations (c.275A?>?G (p.Y92C) and c.306G?>?C (p.Q102H)) and a synonymous change c.306G?>?A (p.Q102Q), were found in exon 3 of CHCHD10 in three alive SALS individuals (with the longest disease duration of 8.6 years), all of which were not detected in healthy controls. No mutation in CHCHD10 was identified in FALS patients. No mutation was found in the aforementioned common ALS causative genes in the patients who carried CHCHD10 mutations. The mutation frequency of CHCHD10 (0.4 %, 2/487) in a Chinese SALS population suggests CHCHD10 gene mutation appears to be an uncommon cause of ALS in Chinese populations. CHCHD10 mutations are associated with a slow progression and long disease duration.     

Novel Antibodies Reveal Inclusions Containing Non-Native SOD1 in Sporadic ALS Patients

Mutations in CuZn-superoxide dismutase (SOD1) cause amyotrophic lateral sclerosis (ALS) and are found in 6% of ALS patients. Non-native and aggregation-prone forms of mutant SOD1s are thought to trigger the disease. Two sets of novel antibodies, raised in rabbits and chicken, against peptides spaced along the human SOD1 sequence, were by enzyme-linked immunosorbent assay and an immunocapture method shown to be specific for denatured SOD1. These were used to examine SOD1 in spinal cords of ALS patients lacking mutations in the enzyme. Small granular SOD1-immunoreactive inclusions were found in spinal motoneurons of all 37 sporadic and familial ALS patients studied, but only sparsely in 3 of 28 neurodegenerative and 2 of 19 non-neurological control patients. The granular inclusions were by confocal microscopy found to partly colocalize with markers for lysosomes but not with inclusions containing TAR DNA binding protein-43, ubiquitin or markers for endoplasmic reticulum, autophagosomes or mitochondria. Granular inclusions were also found in carriers of SOD1 mutations and in spinobulbar muscular atrophy (SBMA) patients and they were the major type of inclusion detected in ALS patients homozygous for the wild type-like D90A mutation. The findings suggest that SOD1 may be involved in ALS pathogenesis in patients lacking mutations in the enzyme.     

Hyperphosphorylation as a defense mechanism to reduce TDP-43 aggregation

Several neurodegenerative diseases including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitinated inclusions (FTLD-U) are characterized by inclusion bodies formed by TDP-43 (TDP). We established cell and transgenic Drosophila models expressing TDP carboxyl terminal fragment (ND251 and ND207), which developed aggregates recapitulating important features of TDP inclusions in ALS/FTLD-U, including hyperphosphorylation at previously reported serine(403,404,409,410) residues, polyubiquitination and colocalization with optineurin. These models were used to address the pathogenic role of hyperphosphorylation in ALS/FTLD-U. We demonstrated that hyperphosphorylation and ubiquitination occurred temporally later than aggregation in cells. Expression of CK2α which phosphorylated TDP decreased the aggregation propensity of ND251 or ND207; this effect could be blocked by CK2 inhibitor DMAT. Mutation of serines(379,403,404,409,410) to alanines (S5A) to eliminate phosphorylation increased the aggregation propensity and number of aggregates of TDP, but mutation to aspartic acids (S5D) or glutamic acids (S5E) to simulate hyperphosphorylation had the opposite effect. Functionally, ND251 or ND207 aggregates decreased the number of neurites of Neuro2a cells induced by retinoic acid or number of cells by MTT assay. S5A mutation aggravated, but S5E mutation alleviated these cytotoxic effects of aggregates. Finally, ND251 or ND251S5A developed aggregates in neurons, and salivary gland of transgenic Drosophila, but ND251S5E did not. Taken together, our data indicate that hyperphosphorylation may represent a compensatory defense mechanism to stop or prevent pathogenic TDP from aggregation. Therefore, enhancement of phosphorylation may serve as an effective therapeutic strategy against ALS/FTLD-U.     

A novel G3337A mitochondrial ND1 mutation related to cardiomyopathy co-segregates with tRNALeu(CUN) A12308G and tRNAThr C15946T mutations

We describe a novel mutation in human mitochondrial NADH dehydrogenase 1 gene (ND1), a G to A transition at nucleotide position 3337, which is co-segregated with two known mutations in tRNALeu(CUN) A12308G and tRNAThr C15946T. These mutations were detected in two unrelated patients with different clinical phenotypes, exhibiting cardiomyopathy as the common symptom. The ND1 G3337A mutation that was detected was found almost homoplasmic in the two patients and it was absent in 150 individuals that were tested as control group. Mitochondrial respiratory chain complex I activity of the patients platelets was also tested and found decreased compared to those of controls. We suggest that the co-existence of mutations in tRNA and ND1 genes may act synergistically affecting the clinical phenotype. Our study highlights the enormous phenotypic diversity that exists among pathogenic mtDNA mutations and re-emphasizes the need for a more careful clinical approach.     

Changes of Activity and Isozyme Pattern of Phosphofructokinase in the Brains of Patients with Alzheimer's Disease

Abstract: A severe reduction of the in vivo cerebral glucose consumption rate is generally found in patients with Alzheimer's disease. In postmortem studies changes in the activities of key regulatory glycolytic enzymes, including 6-phosphofructokinase (PFK), have been reported in Alzheimer's disease brains, but the results obtained so far are inconsistent and controversial. We reevaluated the activity of PFK in brain tissue from clinically and neuropathologically confirmed cases of Alzheimer's disease using optimized tissue disintegration and assay methods and determined the PFK isozyme pattern. PFK activity in brains from patients with Alzheimer's disease was significantly increased in frontal and temporal cortex and unchanged in the other brain areas studied when compared with control brains. All three PFK isozymes were detected in each of the brain areas studied. In brains of Alzheimer's disease patients the level of the C-type PFK was slightly reduced at the expense of the M- and L-type subunits. The data presented do not support the results of other groups, which reported up to a 90% reduction of PFK activity in Alzheimer's disease. In contrast, the data presented clearly rule out the suggestion that changes of PFK activity might be one of the causes for the reduced glucose consumption in Alzheimer's disease brains.     

Novel mutations in an otherwise strictly conserved domain of CuZn superoxide dismutase

All mutations in the human gene for CuZn superoxide dismutase (CuZnSOD) reported to date are associated with the disease amyotrophic lateral sclerosis (ALS). These mutations, mostly of a familial nature (ALS 1, MIM 105400), span all of the coding region of this enzyme except for a highly conserved centrally located domain that includes all of exon III. We describe the identification and characterization of two mutations in this region, both found in mice. One mutation, a glutamate to lysine amino acid substitution was found in position 77 (E77K) of the strain SOD1/Ei distributed by the Jackson Laboratory. The other mutation, a lysine to glutamate substitution at position 70 (K70E) of a human transgene, was discovered in mouse line TgHS/SF-155. Enzyme activity measurements and heterodimer analysis of the CuZn SOD variant in SOD1/Ei suggest a mild loss of activity, which differs from the enzyme activity losses detected in patients with autosomal dominant ALS 1. Similarly, the presence of the mutant transgene in TgHS/SF 155 does not produce any phenotypic manifestations.     

Mutational hotspots in the mitochondrial D-loop region of cancerous and precancerous colorectal lesions in Egyptian patients

Mutations in the mitochondrial genome (mtDNA) are associated with different types of cancer, specifically colorectal cancer (CRC). However, few studies have been performed on precancerous lesions, such as ulcerative colitis (UC) lesions and adenomatous polyps (AP). The aim of this study was to identify mtDNA mutations in the cancerous and precancerous lesions of Egyptian patients. An analysis of the mutations found in six regions of the mtDNA genome (ND1, ND5, COI, tRNAser, D-loop 1, and 2) in 80 Egyptian patients (40 CRC, 20 UC, and 20 AP) was performed using polymerase chain reaction-single-strand conformational polymorphism techniques and followed up by direct sequencing. The overall incidence of mutations was 25%, 25%, and 35% in CRC, UC, and AP cases, respectively. Although there was no common mutation pattern within each group, a large number of mutations were detected in the D-loop region in all of the groups. Some mutations (e.g., T414G) were detected repeatedly in precancerous (UC and AP) and cancerous lesions. Mutations detected in patients with CRC were predominantly found in the ND1 gene (40%). Our preliminary study suggests that Egyptian patients with CRC have a large number of mtDNA mutations, especially in the D-loop region, which have not been previously reported. Mutations in the mtDNA of precancerous lesions (i.e., AP and UC) may contribute to transformation events that lead to CRC.     

ND4 mutation in transitional cell carcinoma: Does mitochondrial mutation occur before tumorigenesis?

To investigate how mitochondrial mutation occurs in cancers, we analyzed ND4 mutation in 53 transitional cell carcinomas (TCCs) of the upper urinary tract and the normal counterpart (perirenal soft tissue). Three methods, i.e., DNA sequencing, restriction fragment length polymorphism (RFLP), and denaturing high-performance liquid chromatography (DHPLC), were employed because of their different sensitive of detecting mutation. The results of sequencing and RFLP showed that ND4 mutations were only found in 24.5% (13/53) of tumor. However, 11 of these mutations could also be identified in the normal tissue by DHPLC, indicating that most mitochondrial mutations identified in tumors preexist as minor components, which are too low in quantity to be detected by less sensitive methods such as DNA sequencing. The result suggests that mtDNA mutation occurs before tumorigenesis and become apparent in cancer cells.     

A C597-->A polymorphism in the Norrie disease gene is associated with advanced retinopathy of prematurity in premature Kuwaiti infants

Retinopathy of prematurity (ROP) is a retinal vascular disease which occurs in infants with a short gestational age and low birth weight and may lead to retinal detachment and blindness. In some premature infants, ROP progresses to advanced stages despite rigorous intervention, but in the majority, it spontaneously regresses before the threshold stage. Genetic factors, e.g. mutations in the Norrie disease (ND) gene, have been implicated in determining the progression of ROP to advanced stages. We have identified a novel C597A polymorphism of the ND gene; we screened this and another mutation in the ND gene, C110G, in 210 premature Kuwaiti infants using PCR-RFLP, DNA sequence analysis and DNA enzyme immunoassay hybridization to investigate their association with advanced-stage ROP. In this cohort of premature Kuwaiti newborns, 115 of 210 babies had no eye problems and served as controls, while 95 were found to have ROP. In 71 of the 95 ROP cases, the disease spontaneously regressed at or before stage 3, while in 24 of 95 ROP cases, the disease progressed to advanced stages 4 or 5. The incidence of the AA genotype of the C597A polymorphism was considerably higher in advanced-stage ROP cases (83.3%) compared to spontaneously regressing ROP cases (0%) and the normal controls (10.4%) (p < 0.0001). For the other genotypes, no significant difference was detected between the controls and ROP cases. In the case of the C110G mutation in the ND gene, no significant differences were detected between the controls and ROP cases, and the majority of subjects had a CC genotype in all three groups.     

Nucleotide substitutions in the acetolactate synthase genes of sulfonylurea-resistant biotypes of Monochoria vaginalis (Pontederiaceae)

Some point mutations in acetolactate synthase (ALS) confer resistance to ALS-inhibiting herbicides in weeds. To clarify the evolution of the herbicide resistance of Monochoria vaginalis, a weed in rice fields in Japan, the nucleotide sequences of four genes encoding ALS were surveyed in five sulfonylurea-resistant (SU-R) and five sulfonylurea-susceptible (SU-S) biotypes. In the ALS1 gene, two SU-R biotypes showed nucleotide substitutions changing Pro197 to Ser and Leu, respectively. In a different gene, ALS3, three other SU-R biotypes showed either of the two nonsynonymous nucleotide substitutions seen in ALS1. Only two biotypes geographically located distantly from each other shared the same mutation conferring SU resistance in the same gene. These patterns of nucleotide substitutions indicate that the SU-R phenotype was acquired independently by different biotypes. Nucleotide diversity values of the genes showing SU-R mutations were higher than those of ALS2 lacking any SU-R mutation and of a putative pseudogene, ALS4. This result suggests that the maintenance of nucleotide variability within target genes provides an opportunity for the evolution of SU-R phenotypes by herbicide-driven selection for mutations conferring resistance.     

神经退化性疾病生物能量代谢和氧化应激研究进展

衰老是导致几种常见的神经系统退化性疾病的主要危险因素,包括帕金森氏病（Ｐａｒｋｉｎｓｏｎ’ｓ ｄｉｓｅａｓｅ ＰＤ）,肌萎缩性侧索硬化（Ａｍｙｏｔｒｏｐｈｉｃ ｌａｔｅｒａｌ ｓｃｌｅｒｏｓｉｓ,ＡＬＳ）,早老性痴呆（Ａｌｚｈｅｉｍｅｒ’ｓ ｄｉｓｅａｓｅ ＡＤ）和亨廷顿氏病（Ｈｕｎｔｉｎｇｔｏｎ’ｓ ｄｉｓｅａｓｅ ＨＤ）。最近研究表明,神经退化性疾病涉及到线粒体缺陷,氧化应激等因素。在脑和其它组织中,老化可导致线粒体功能的损伤和氧化损伤的增强。ＰＤ病人中,已发现线粒体复合酶体Ⅰ活性降低,氧化损伤增加和抗氧化系统活性的改变。在几例家族性ＡＬＳ病人中,也发现Ｃｕ、Ｚｎ超氧化物歧化酶（Ｃｕ,Ｚｎ ＳＯＤ）基因的突变,导致Ｃｕ、Ｚｎ超氧化物歧化酶活性减低;散发的ＡＬＳ病人氧化损伤增高。在ＨＤ病人中已发现能量代谢异常     

温州地区2型糖尿病患者线粒体DNA 3243、3316位点的突变筛查

为了解浙江省温州地区2型糖尿病病人中线粒体DNA tRNALeu (UUR)基因A3243G及NADH 脱氢酶亚单位1 (ND1)基因G3316A位点突变的发生频率, 并探讨突变与2型糖尿病主要临床指标出现的相关性。对随机收集的无血缘关系的244例温州地区2型糖尿病患者进行研究, 同时选择156例无 DM 家族史的糖耐量正常者作为对照组, 用聚合酶链反应及限制性片段长度多态性分析技术进行点突变筛选, 筛选到的异质性突变样本经T-A克隆后再作测序和变性高效液相色谱(DHPLC)确证。结果在244例的2型糖尿病患者中检出A3243G突变1例(0.410%), 156例对照者中未检出该突变, 突变发生率在两组间差异无统计学意义(P>0.05); 2型糖尿病患者中检出G3316A突变4例(1.639%), 156例对照者中检出突变2例(1. 282%), 突变发生率在两组间差异无统计学意义(P>0.05)。结果表明线粒 体tRNALeu (UUR) 基因A3243G突变在浙江温州2型糖尿病人群中发生频率低, 不是温州人群中2型糖尿病的常见病因。线粒体ND1基因G3316A突变在糖尿病人群中的发生频率也较低, 且在正常人群中也有出现, 可能仅为人群中线粒体DNA的基因多态性。