Is the time dimension of the cell cycle re-entry in AD regulated by centromere cohesion dynamics?

Chromosomal involvement is a legitimate, yet not well understood, feature of Alzheimer disease (AD). Firstly, AD affects more women than men. Secondly, the amyloid-β protein precursor genetic mutations, responsible for a cohort of familial AD cases, reside on chromosome 21, the same chromosome responsible for the developmental disorder Down's syndrome. Thirdly, lymphocytes from AD patients display a novel chromosomal phenotype, namely premature centromere separation (PCS). Other documented morphological phenomena associated with AD include the occurrence of micronuclei, aneuploidy, binucleation, telomere instability, and cell cycle re-entry protein expression. Based on these events, here we present a novel hypothesis that the time dimension of cell cycle re-entry in AD is highly regulated by centromere cohesion dynamics. In view of the fact that neurons can re-enter the cell division cycle, our hypothesis predicts that alterations in the signaling pathway leading to premature cell death in neurons is a consequence of altered regulation of the separation of centromeres as a function of time. It is well known that centromeres in the metaphase anaphase transition separate in a non-random, sequential order. This sequence has been shown to be deregulated in aging cells, various tumors, syndromes of chromosome instability, following certain chemical inductions, as well as in AD. Over time, premature chromosome separation is both a result of, and a driving force behind, further cohesion impairment, activation of cyclin dependent kinases, and mitotic catastrophe–a vicious circle resulting in cellular degeneration and death.     

A mechanism of x chromosome aneuploidy in lymphocytes of aging women.

One and sometimes both X chromosomes in cultured lyphocytes of women donors showed division of the centromere when the centromeres of other chromosomes were entire. This premature centromere division (PCD) was associated with evidence of non-disjunction of the X chromosome. On average, 2% of metaphases from 32 women donors showed PCD, but the incidence was 4 times greater in women over 59 years of age than in women under 40 years. Increased X chromosome aneuploidy was associated with the higher frequency of PCD in cultured lymphocytes from older women. PCD of the X chromosome is considered to be the mechanism of non-disjunction causing the previously described aneuploidy in cultured lymphocytes of aging women.     

Dkk-3 is elevated in CSF and plasma of Alzheimer's disease patients

Biomarkers in CSF can offer improved diagnostic accuracy for Alzheimer's disease (AD). The present study investigated whether the glycoprotein and putative tumor suppressor Dickkopf homolog 3 (Dkk-3) is secreted into CSF and evaluated its applicability as a diagnostic marker for AD. Using our highly specific immunoenzymometric assay, Dkk-3 levels were measured in plasma and/or CSF of patients suffering from depression, mild cognitive impairment (MCI), or AD and compared with healthy subjects. Dkk-3 identity was verified by western blot and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS)/MS. High concentrations of Dkk-3 were detected in CSF compared with plasma (28.2 ± 1.3 vs. 1.22 ± 0.04 nmol/L, respectively). Consistently Dkk-3 expression was demonstrated in neurons of the cortex and epithelial cells of the choroid plexus, the major source of CSF. Significantly increased Dkk-3 levels in plasma and CSF were observed for AD patients compared with healthy subjects but not patients suffering from MCI or depression. In summary, our data indicate that elevated Dkk-3 levels are specifically associated with AD and might serve as a potential non-invasive AD biomarker in plasma.     

Premature centromere division (PCD): a dominantly inherited cytogenetic anomaly

Summary We describe a family with an increased frequency of cells with premature centromere division (PCD) of all chromosomes in four phenotypically normal individuals. This familial PCD phenomenon is apparently different from the well-described PCD of the X chromosome and from the centromere splitting in cells of patients with Roberts syndrome. Implications for genetic counseling are discussed.     

Premature centromere division dominantly inherited in a subfertile family

An increased frequency of mitoses showing premature centromere division (PCD) in every chromosome was found in lymphocyte cultures from four members of a subfertile family. These cells were observed in both the presence and absence of colchicine. Cultured fibroblasts from the proband showed only normal diploid metaphases. PCD cells seemed to have a shorter cell cycle. The anomaly was transmitted in a way compatible with autosomal dominant inheritance in this family.     

Chromosome abnormalities in tuberous sclerosis

Summary In fibroblasts cultured from biopsies of the skin lesions of six patients with tuberous sclerosis (TS) there was a variable but consistent degree of karyotypic variation. Premature centromere disjunction (PCD) of all or part of the chromosomes, micronuclei, an increased incidence of breaks, dicentric chromosomes and the presence of polyploid metaphases were found in all cultures. The PCD was of the type encountered in Roberts syndrome and its frequency varied from 8% to 30%. In metaphases with PCD of one and of two chromosomes, the chromosome involved were identified, and chromosome 3 was involved 21 times among 59 chromosomes with PCD. Chromosome 3 tends to be preferentially involved in dicentric formation. In lymphocyte cultures from the same patients there were no metaphases with PCD, but there was a slight increase of breaks and the presence of dicentric chromosomes, also involving chromosome 3. Polyploid metaphases were increased in some of the cases. Karyotypic variation can be considered a cellular phenotypic characteristic of TS in fibroblasts cultured from the skin lesions, and its type indicates disturbances in the mechanics of centromere division and of chromosome distribution at cell division.     

Mitotic Spindle Defects and Chromosome Mis-Segregation Induced by LDL/Cholesterol—Implications for Niemann-Pick C1, Alzheimer’s Disease,and Atherosclerosis

Elevated low-density lipoprotein (LDL)-cholesterol is a risk factor for both Alzheimer’s disease (AD) and Atherosclerosis (CVD), suggesting a common lipid-sensitive step in their pathogenesis. Previous results show that AD and CVD also share a cell cycle defect: chromosome instability and up to 30% aneuploidy–in neurons and other cells in AD and in smooth muscle cells in atherosclerotic plaques in CVD. Indeed, specific degeneration of aneuploid neurons accounts for 90% of neuronal loss in AD brain, indicating that aneuploidy underlies AD neurodegeneration. Cell/mouse models of AD develop similar aneuploidy through amyloid-beta (Aß) inhibition of specific microtubule motors and consequent disruption of mitotic spindles. Here we tested the hypothesis that, like upregulated Aß, elevated LDL/cholesterol and altered intracellular cholesterol homeostasis also causes chromosomal instability. Specifically we found that: 1) high dietary cholesterol induces aneuploidy in mice, satisfying the hypothesis’ first prediction, 2) Niemann-Pick C1 patients accumulate aneuploid fibroblasts, neurons, and glia, demonstrating a similar aneugenic effect of intracellular cholesterol accumulation in humans 3) oxidized LDL, LDL, and cholesterol, but not high-density lipoprotein (HDL), induce chromosome mis-segregation and aneuploidy in cultured cells, including neuronal precursors, indicating that LDL/cholesterol directly affects the cell cycle, 4) LDL-induced aneuploidy requires the LDL receptor, but not Aß, showing that LDL works differently than Aß, with the same end result, 5) cholesterol treatment disrupts the structure of the mitotic spindle, providing a cell biological mechanism for its aneugenic activity, and 6) ethanol or calcium chelation attenuates lipoprotein-induced chromosome mis-segregation, providing molecular insights into cholesterol’s aneugenic mechanism, specifically through its rigidifying effect on the cell membrane, and potentially explaining why ethanol consumption reduces the risk of developing atherosclerosis or AD. These results suggest a novel, cell cycle mechanism by which aberrant cholesterol homeostasis promotes neurodegeneration and atherosclerosis by disrupting chromosome segregation and potentially other aspects of microtubule physiology.     

Chloroplast DNA content in Dinophysis (Dinophyceae) from different cell cycle stages is consistent with kleptoplasty

Kleptoplasty is the retention of plastids obtained from ingested algal prey, which can remain temporarily functional and be used for photosynthesis by the predator. With a new approach based on cell cycle analysis, we have addressed the question of whether the toxic, bloom-forming dinoflagellate Dinophysis norvegica practice kleptoplasty or if they replicate their own plastid DNA. Dividing (G2) and non-dividing (G1) D. norvegica cells from a natural population were physically separated with a flow cytometer based on their DNA content. Average numbers of nuclear and plastid rDNA copies were quantified with real-time PCR both in the G1 and G2 group. Cells from the G1 group contained 5800 ± 340 copies of nuclear rDNA and 1300 ± 200 copies of plastid rDNA; cells from the G2 group contained 9700 ± 58 copies of nuclear rDNA and 1400 ± 220 copies of plastid rDNA (mean ± SD, n  = 3). The ratio G2/G1 in average rDNA copies per cell was 1.67 for nuclear DNA and 1.07 for plastid DNA. These ratios show that plastid acquisition in D. norvegica is either uncoupled with the cell cycle, or plastids accumulate rapidly in the beginning of the cell cycle owing to feeding, as would be expected in a protist with kleptoplastic behaviour but not in a protist with own plastid replication. In addition, flow cytometry measurements on cells from the same population used for real-time PCR showed that when kept without plastidic prey, live Dinophysis cells lost on average 36% of their plastid phycoerythrin fluorescence in 24 h. Together these findings strongly suggest that D. norvegica does not possess the ability for plastid replication.     

The Bacillus subtilis SftA (YtpS) and SpoIIIE DNA translocases play distinct roles in growing cells to ensure faithful chromosome partitioning

In several bacterial species, the faithful completion of chromosome partitioning is known to be promoted by a conserved family of DNA translocases that includes Escherichia coli FtsK and Bacillus subtilis SpoIIIE. FtsK localizes at nascent division sites during every cell cycle and stimulates chromosome decatenation and the resolution of chromosome dimers formed by recA -dependent homologous recombination. In contrast, SpoIIIE localizes at sites where cells have divided and trapped chromosomal DNA in the membrane, which happens during spore development and under some conditions when DNA replication is perturbed. SpoIIIE completes chromosome segregation post-septationally by translocating trapped DNA across the membrane. Unlike E. coli , B. subtilis contains a second uncharacterized FtsK/SpoIIIE-like protein, SftA (formerly YtpS). We report that SftA plays a role similar to FtsK during each cell cycle but cannot substitute for SpoIIIE in rescuing trapped chromosomes. SftA colocalizes with FtsZ at nascent division sites but not with SpoIIIE at sites of chromosome trapping. SftA mutants divide over unsegregated chromosomes more frequently than wild-type unless recA is inactivated, suggesting that SftA, like FtsK, stimulates chromosome dimer resolution. Having two FtsK/SpoIIIE paralogues is not conserved among endospore-forming bacteria, but is highly conserved within several groups of soil- and plant-associated bacteria.     

Total aneuploidy and age-related sex chromosome aneuploidy in cultured lymphocytes of normal men and women

Summary In PHA-cultured lymphocytes, about 8% of metaphases from 32 women were aneuploid compared to 4% of metaphases from 35 men. A significant part of this aneuploidy was characterized by sex chromosome involvement: in women, the loss or gain of X chromosomes; in men, the gain of X chromosomes and the loss or gain of Y chromosomes. The incidence of this aneuploidy was positively age-related for both sexes. Premature division of the X-chromosome centromere was closely associated with X-chromosome aneuploidy in women and men, and appeared to be the mechanism of nondisjunction causing this aneuploidy. Premature centromere division (PCD) indicated a dysfunction of the X-chromosome centromere with aging, and this dysfunction was the basic cause of age-related aneuploidy. A similar mechanism of nondisjunction may operate for the Y chromosome of men, but could not be clearly demonstrated because of the low incidence of Y-chromosome aneuploidy.The balance of the aneuploidy was characterized by chromosome loss and the involvement of all chromosome groups. It was consistent with chromosome loss from metaphase cells damaged during preparation for cytogenetic examination.     

Rate of spontaneous chromosome aberration occurrence in cultured Chinese hamster cells

The spontaneous chromosome mutation rate was studied in cultured aneuploid Chinese hamster cells (clone 237(1)) using the method of slowing down the rate of cell division in a limiting medium containing 0.1% of serum. It was shown that during one cell generation (which lasted 14 days in limiting medium) the accumulation of chromosome aberrations with time took place. The data obtained are in keeping with the assumption of a linear dependence of this accumulation on time. The spontaneous chromosome rearrangement rate was 1.2 X 10(-2) mutations per cell per 24 hours. Proceeding from this value the spontaneous chromosome aberration rate in cells with a normal duration of the cell cycle was 0.6 X 10(-2) per cell per generation.     

神经祖细胞与纤维蛋白支撑物移植对老年痴呆模型鼠的记忆认知功能改善及其机理探究

目的探讨阿尔茨海默病（AD）模型鼠双侧海马区移植含多因子孵育的神经祖细胞（NPCs）后记忆认知功能改善情况及NPCs移植后迁移定位和分化能力。 方法取胎龄10?d的C57BL/6J孕鼠,分离得胎鼠NPCs,NPCs体外分化及鉴定,AD模型鼠分3组：NPCs+因子组、因子组及PBS组,对照组为同月龄C57BL/6J小鼠;Morris水迷宫及新物体识别实验检测AD模型鼠移植NPCs细胞前,及移植1至6个月后记忆行为变化情况;通过免疫荧光,免疫组化和Western-blot检测海马区移植的NPCs向神经元和胆碱能神经元分化及迁移能力。组间比较采用F检验。 结果Morris水迷宫实验中,NPCs+因子组找到平台前的逃避潜伏期时间（14.12±7.45）s要明显低于注射PBS的AD模型鼠组[（39.65±4.64）?s,F = 2.578,P = 0.0094],因子组时间（15.68±5.34）s同样低于PBS组[（39.65±4.64）s,F?= 1.324,P = 0.0016],24 h撤去平台后,NPCs+因子组逃避潜伏期时间（15.12±3.52）s仍低于PBS组[（37.17±2.18）?s,F = 2.598,P = 0.0003],因子组时间（16.62±3.23）s同样低于PBS组[（37.17±2.18）s,F = 2.186,P = 0.0004）];新物体识别实验中,各实验组对新物体探究时间占总探究时间百分比结果中,NPCs+因子组（68.46±2.4）%要高于PBS组[（54.47±4.79）%,F =3.983,P = 0.018],因子组（65.20±1.03）%同样高于PBS组[（54.47±4.79）%,F = 21.63,P = 0.042];实验结果表明,通过移植细胞与因子AD模型鼠的记忆认知功能在早期均得到改善,随着时间的增长,移植NPCs组的记忆改善情况持续时间更长久;Western blot结果显示AD模型鼠海马区胆碱能神经元与正常C57BL/6J鼠相比表达减少,移植NPCs后,AD模型鼠脑内胆碱能神经元增多;免疫荧光与免疫组化结果显示,移植的NPCs在AD模型鼠脑内移植区存活,并向胆碱能神经元分化。 结论AD模型鼠双侧海马区移植的含多因子孵育的NPCs,通过分化成功能性的胆碱能神经元来改善AD鼠的记忆认知功能。     

Different involvement of the mitochondrial, plastidial and cytosolic ascorbate–glutathione redox enzymes in heat shock responses

Plant survival under heat stress requires the activation of proper defence mechanisms to avoid the impairment of metabolic functions. Heat stress leads to the overproduction of reactive oxygen species (ROS) in the cell. In plants, the ascorbate (ASC)-GSH cycle plays a pivotal role in controlling ROS levels and cellular redox homeostasis. Ascorbate peroxidase (APX) is the enzyme of this cycle mainly involved in ROS detoxification. In this study, the ASC-GSH cycle enzymes were analysed in the cytosol, mitochondria and plastids of tobacco Bright Yellow-2 cultured cells. The cells were also subjected to two different heat shocks (HSs; 35 or 55°C for 10 min) and the cell compartments were isolated in both conditions. The results reported here indicate that moderate HS (35°C) does not affect cell viability, whereas cell exposure to 55°C HS induces programmed cell death (PCD). In relation to ASC-GSH cycle, the three analysed compartments have specific enzymatic profiles that are diversely altered by the HS treatments. The cytosol contains the highest activity of all ASC-GSH cycle enzymes and the data reported here suggest that it acts as a redox buffer for the whole cells. In particular, the cytosolic APX seems to be the most versatile enzyme, being its activity enhanced after moderate HS and reduced during PCD induction, whereas the other APX isoenzymes are only affected in the cells undergoing PCD. The relevance of the changes in the different ASC-GSH cycle isoenzymes in allowing cell survival or promoting PCD is discussed.     

The grasshopper X chromosome

The X chromosome can be identified with the light microscope throughout all stages of the gonial cell cycle (including interphase) in the grasshopper Brachystola magna. At gonial mitotic stages the X chromosome gives the appearance of being undercondensed or negatively heteropycnotic. At interphase the X projects out from the body of the nucleus. — Examination with the electron microscope reveals that the X is compartmentalized at least two gonial cell cycles prior to the entry of the cells into meiotic prophase. The membrane layers that envelope the X chromatin at interphase remain associated with the X chromosome throughout gonial mitotic stages providing the ultrastructural basis for the apparent negative heteropycnosis observed with the light microscope. — The X chromosome is inactive in RNA synthesis during gonial mitotic stages but is hyperactive in RNA synthesis when compared to autosomes at gonial interphase. — X chromosome condensation which reaches its maximum at premieotic interphase is initiated at or prior to the pre-pentultimate gonial division.     

Increased sister chromatid exchanges in human cell lines characterized by monosomy X or structural abnormalities of the X chromosome

Summary In the present investigation we test the hypothesis that deficiencies in the X chromosome affect sister chromatid exchange (SCE) frequencies in human fibroblast cell lines. Our results show increased mean SCE frequencies in cell lines with abnormalities of the X chromosome: 45,X; 46,X,del(X) (q13), 46,X,del(X)(p11), and 46,X,i(Xq); control cell lines were 46,XX. In only one abnormal line [46,X,del(X)(p11)] was the increase not significant after correcting for multiple comparisons. If SCE formation is replication-dependent, the increased SCE frequencies might merely reflect the prolonged cell cycle we reported previously in cell lines with X chromosome abnormalities (Simpson and LeBeau 1981). Other explanations for differences between cell lines are possible, e.g., that deleted loci on the X chromosome affect cellular uptake of bromodeoxyuridine (BrDU). However, specific mechanisms were not explored directly.     

Evidence for the repeated primary non-disjunction of chromosome 21 as a result of premature centromere division (PCD)

Summary A clinically normal 28-year-old woman had three conceptuses with trisomy 21 and one normal child. She showed minimal cytogenetic evidence of mosaicism: 4% of her blood cells and 6% of skin fibroblasts had trisomy 21. Also, 7% of her blood cells showed aneuploidy of the X chromosome which was associated with premature centromere division (PCD, X); 6% of fibroblasts showed trisomy 18, 10% of fibroblasts showed PCD,21, and 1% PCD, 18. It is unlikely that this woman is a constitutional mosaic for trisomies X, 18, and 21, all at low levels. We suggest that she has a predisposition to irregular centromere separation and that chromosomes X, 18, and 21 are most susceptible to its action.     

Spontaneous and induced aneuploidy in peripheral blood lymphocytes of patients with Alzheimer’s disease

This study was aimed at assessing whether peripheral blood lymphocytes of patients with Alzheimer’s disease (AD) show significant levels of aneuploidy and high percentages of cytogenetic events in vitro, indicating a predisposition to aneuploidy spontaneously, or after chemical treatment in vitro. A group of affected individuals and a group of unaffected, age-, sex- and smoking-habit-matched controls were identified. Lymphocytes were cultured for analysis of the following cytogenetic parameters: premature centromere division (PCD), satellite associations of acrocentric chromosomes (SA) and micronuclei (MN). In a subset of subjects, the fluorescence in situ hybridization (FISH) technique was combined with the MN assay, by means of a pancentromeric DNA probe for the detection of the presence of centric material. To evaluate the sensitivity to aneuploidogenic agents, in vitro treatment of lymphocytes of affected individuals was performed by adding griseofulvin, a chemical whose supposed target is microtubule-associated protein(s). Both the spontaneous frequency of MN and the frequency of PCD was significantly higher in patient cells than in controls. Furthermore, after application of the FISH technique, we found that the majority of MN were composed of whole chromosomes (because of the phenomenon of chromosome loss). Metaphase analysis for the detection of associative events between satellite regions of acrocentric chromosomes showed no differences between the two groups under study. Analysis of sensitivity to the aneuploidogen griseofulvin showed that the patient group was characterized by lower levels of MN induction compared with controls. Our data confirm that peripheral blood lymphocytes of AD patients are prone to undergo aneuploidy spontaneously in vitro and support the hypothesis that microtubule impairment might be associated with the disease. Received: 13 January 1997 / Accepted: 30 July 1997     

Regulation of Neuronal Cell Cycle and Apoptosis by MicroRNA 34a

The cell cycle of neurons remains suppressed to maintain the state of differentiation and aberrant cell cycle reentry results in loss of neurons, which is a feature in neurodegenerative disorders like Alzheimer''s disease (AD). Present studies revealed that the expression of microRNA 34a (miR-34a) needs to be optimal in neurons, as an aberrant increase or decrease in its expression causes apoptosis. miR-34a keeps the neuronal cell cycle under check by preventing the expression of cyclin D1 and promotes cell cycle arrest. Neurotoxic amyloid β_1–42 peptide (Aβ₄₂) treatment of cortical neurons suppressed miR-34a, resulting in unscheduled cell cycle reentry, which resulted in apoptosis. The repression of miR-34a was a result of degradation of TAp73, which was mediated by aberrant activation of the MEK extracellular signal-regulated kinase (ERK) pathway by Aβ₄₂. A significant decrease in miR-34a and TAp73 was observed in the cortex of a transgenic (Tg) mouse model of AD, which correlated well with cell cycle reentry observed in the neurons of these animals. Importantly, the overexpression of TAp73α and miR-34a reversed cell cycle-related neuronal apoptosis (CRNA). These studies provide novel insights into how modulation of neuronal cell cycle machinery may lead to neurodegeneration and may contribute to the understanding of disorders like AD.     

Immunocytochemical Detection of Anti-Hippocampal Antibodies in Alzheimer's Disease and Normal Cerebrospinal Fluid

Immunocytochemical staining was performed to investigate the presence of anti-hippocampal antibodies in cerebrospinal fluid (CSF) from patients with probable Alzheimer's disease (AD) (n = 19), aged normal controls (n = 9), and young normal controls (n = 10). Marked staining of neurons in the granule cell layer of the dentate gyrus and in pyramidal neurons in CA1-3 of the rat hippocampus was observed in 5 AD CSF samples (26%), 1 aged control sample (11%), and 1 young control sample (10%). These differences were not statistically significant. One of the immunoreactive AD CSF specimens also contained high concentrations of C5b-9, the membrane attack complex. The infrequent occurrence of anti-hippocampal antibodies in AD CSF, and the detection of similar immunoreactivity in control CSF specimens, suggest that these antibodies are unlikely to play a role in the neurodegenerative process in most individuals with AD. However, elevated C5b-9 concentration in an AD CSF specimen with marked immunoreactivity to hippocampal neurons suggests the possibility that anti-neuronal antibodies may contribute to complement activation in some AD patients.