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

<div><p>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.</p></div

Increased membrane cholesterol induces aneuploidy.

<p>(A,B) Karyotype and FISH analysis showed an increase in total aneuploidy (A) and trisomy 21 and trisomy 12 (B) in hTERT cells treated for 48 hr with 4 µg/ml cholesterol made water-soluble in a methyl-β-cyclodextrin (MβCD) complex. (C) Mouse chromosome 16 DNA probe was used to measure aneuploidy levels in mouse neuronal precursor cells (mNPC) derived from prenatal brains of wild-type mice (E17–18) and incubated with and without 4 µg/ml of WsCh for 7 days. Quantitative FISH analysis showed a 2–fold increase in trisomy 16 in WsCh-treated cells compared to controls. mNPC harbored up to 4.6% endogenous aneuploidy, as reported previously in developing mouse and human brains <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060718#pone.0060718-Brown1" target="_blank">[73]</a>.</p

Increased trisomy 21 aneuploidy in fibroblasts and in glia and neurons of Niemann-Pick C1 patients.

<p>(A,B) FISH analysis with a DNA probe for chromosome 21 (red) and chromosome 12 (green) of fibroblasts derived from NPC1 patients (NPC1-HF) showed an increase in trisomy 21 cells compared to age-matched normal human fibroblasts. (C,D) Quantitative FISH analysis with a DNA probe for chromosome 21 (red) followed by staining with NeuN antibody (green) and DAPI (blue) of resuspended cells from frontal cortices of control and NPC brains revealed significantly higher levels of trisomy 21 in NPC neurons and glia compared to controls.</p

Characteristics of the Niemann-Pick Type C (NPC) and Control Samples.

<p>yr = year; NK = not known. Human fibroblasts were purchased from Coriell and brains from NICHD Brain and Tissue Bank for Developmental Disorders. Independent T-test revealed no age difference between NPC and control brain donors (20.9±8.6 vs. 17.3±11.1, p = 0.61).</p

Role for Ca++ in lipoprotein induced chromosome mis-segregation.

<p>(A–C) Quantitative FISH analysis with a dually labeled DNA probe for chromosomes 21 and 12 showed a significant reduction in trisomy 21 (A), tetrasomy 21 and tetrasomy 12 (B) in hTERT cells pre-treated with 1.5 mM of Ca++ chelator EGTA followed by OX-LDL compared to cells treated only with OX-LDL. Cells pre-treated with 1 mM BAPTA had significantly lower levels of OX-LDL-induced trisomy 21 and trisomy 12 (C) compared to the cells grown without chelator.</p

Ethanol attenuates lipoprotein-induced chromosome mis-segregation.

<p>(A,B) Quantitative FISH analysis of hTERT cells pre-treated with 25 mM of ethanol (EtOH) for 24 hr and co-incubated with lipoproteins and EtOH for an additional 48 hr revealed a decrease in OX-LDL and LDL-induced trisomy 21 (A) and trisomy 12 (B).</p

Mitotic spindle structure disrupted by cholesterol.

<p>(A) hTERT cells were treated for 24 hr with 4 µg/ml WsCh and the structure of the mitotic spindles observed and analyzed for abnormal DNA localization, lagging chromosomes, super-numerary centrosomes, and dis-arrayed microtubules. There was a significant increase in abnormal mitotic spindle structure induce by cholesterol exposure, with dis-arrayed microtubules and mis-localized DNA being the most prominent defects. (B) Examples of normal spindles in untreated cells and abnormal spindles in cholesterol-treated cells.</p

Lipoprotein-induced aneuploidy is dependent on LDLR and independent of APP.

<p>(A) Normal human fibroblasts (NHF) developed significantly higher levels of trisomy 7 when treated with 20 µg/ml of LDL for 48 hr compared to LDL receptor deficient human fibroblasts obtained from the patient diagnosed with familial hypercholesterolemia (FHF). (B) Quantitative FISH for chromosome 16 revealed comparable levels of trisomy 16 in primary splenocytes derived from nontransgenic (NON) and APP knockout (APPKO) mice upon incubation with 20 µg/ml of OX-LDL or LDL for 48 hr, indicating that the aneugenic activity of lipoproteins is independent of a functional APP gene and of its product Aβ.</p

Lipoprotein treatment induces aneuploidy.

<p>(A) Actively growing hTERT-HME1 cells were treated with 20 µg/ml of OX-LDL, LDL or HDL for 48 hr, arrested in metaphase and Giemsa stained for karyotype analysis. All lipids induced significantly higher levels of aneuploidy compared to untreated cells, with LDL and or OX-LDL exhibiting a much stronger aneugenic effect than HDL. (B–E) FISH analysis of the same lipoprotein-treated cells showed that OX-LDL-induced trisomy 21 and trisomy 12 (B,D), and that both OX-LDL and LDL-induced tetrasomy 21 and 12 (C,E). (F) Karyotype analysis of an aliquot of the cells from the same treatment showed very few, but equal numbers of polyploid cells, indicating that the tetrasomies observed are due to chromosome mis-segregation of chromosomes 21 or 12, and not a result of chromosome duplication before cell division.</p

LDL induces trisomy 7 in human aortic smooth muscle cells.

<p>Quantitative FISH analysis of HASM cells showed an increase in trisomy 7 when incubated with 20 µg/ml of LDL, but not HDL for 48 hr.</p