The Guinea Pig as a model for sporadic Alzheimer's Disease (AD): the impact of cholesterol intake on expression of AD-related genes

Extent: 12p.We investigated the guinea pig, Cavia porcellus, as a model for Alzheimer’s disease (AD), both in terms of the conservation of genes involved in AD and the regulatory responses of these to a known AD risk factor - high cholesterol intake. Unlike rats and mice, guinea pigs possess an Aβ peptide sequence identical to human Aβ. Consistent with the commonality between cardiovascular and AD risk factors in humans, we saw that a high cholesterol diet leads to up-regulation of BACE1 (β-secretase) transcription and down-regulation of ADAM10 (α-secretase) transcription which should increase release of Aβ from APP. Significantly, guinea pigs possess isoforms of AD-related genes found in humans but not present in mice or rats. For example, we discovered that the truncated PS2V isoform of human PSEN2, that is found at raised levels in AD brains and that increases γ-secretase activity and Aβ synthesis, is not uniquely human or aberrant as previously believed. We show that PS2V formation is up-regulated by hypoxia and a high-cholesterol diet while, consistent with observations in humans, Aβ concentrations are raised in some brain regions but not others. Also like humans, but unlike mice, the guinea pig gene encoding tau, MAPT, encodes isoforms with both three and four microtubule binding domains, and cholesterol alters the ratio of these isoforms. We conclude that AD-related genes are highly conserved and more similar to human than the rat or mouse. Guinea pigs represent a superior rodent model for analysis of the impact of dietary factors such as cholesterol on the regulation of AD-related genes.Mathew J. Sharman, Seyyed H. Moussavi Nik, Mengqi M. Chen, Daniel Ong, Linda Wijaya, Simon M. Laws, Kevin Taddei, Morgan Newman, Michael Lardelli, Ralph N. Martins, Giuseppe Verdil

Hypoxia alters expression of Zebrafish Microtubule-associated protein Tau (mapta, maptb) gene transcripts

Background: Microtubule-associated protein tau (MAPT) is abundant in neurons and functions in assembly and stabilization of microtubules to maintain cytoskeletal structure. Human MAPT transcripts undergo alternative splicing to produce 3R and 4R isoforms normally present at approximately equal levels in the adult brain. Imbalance of the 3R-4R isoform ratio can affect microtubule binding and assembly and may promote tau hyperphosphorylation and neurofibrillary tangle formation as seen in neurodegenerative diseases such as frontotemporal dementia (FTD) and Alzheimer\u27s disease (AD). Conditions involving hypoxia such as cerebral ischemia and stroke can promote similar tau pathology but whether hypoxic conditions cause changes in MAPT isoform formation has not been widely explored. We previously identified two paralogues (co-orthologues) of MAPT in zebrafish, mapta and maptb. Results: In this study we assess the splicing of transcripts of these genes in adult zebrafish brain under hypoxic conditions. We find hypoxia causes increases in particular mapta and maptb transcript isoforms, particularly the 6R and 4R isoforms of mapta and maptb respectively. Expression of the zebrafish orthologue of human TRA2B, tra2b, that encodes a protein binding to MAPT transcripts and regulating splicing, was reduced under hypoxic conditions, similar to observations in AD brain. Conclusion: Overall, our findings indicate that hypoxia can alter splicing of zebrafish MAPT co-orthologues promoting formation of longer transcripts and possibly generating Mapt proteins more prone to hyperphosphorylation. This supports the use of zebrafish to provide insight into the mechanisms regulating MAPT transcript splicing under conditions that promote neuronal dysfunction and degeneration

Increased Aβ1-40 levels in the CNS of cholesterol fed guinea pigs.

<p><b>(A)</b> CSF Aβ1-40 levels (pg/mL) in the cholesterol and control fed diet groups following 12 weeks of feeding. Value is significantly increased over those animals fed the control diet (<i>p</i> = 0.011, <i>t</i> = 2.896, <i>d.f.</i> = 14). <b>(B)</b> Cerebral Aβ1-40 levels (nmol/g wet tissue) in frontal cortex and cerebellum homogenates from animals fed for 12 weeks on a high cholesterol or control diet. Increases are observed in animals fed cholesterol diet in the frontal cortex (<i>p</i> = 0.04, <i>t</i> = 2.204, <i>d.f.</i> = 14) but not in the cerebellum (<i>p</i> = 0.501, <i>t</i> = 0.684, <i>d.f.</i> = 14, ns). Values mean ± SEM.</p

Forward and reverse primers used for the qRT-PCR of <i>ADAM10</i>, <i>BACE1</i>, <i>PSEN2</i>, <i>PS2V</i> and <i>MAPT</i> transcripts.

<p>Note that guinea pig exon designations are according to the cognate exons in human since annotation of the guinea pig genome sequence is currently rudimentary.</p

Amino acid residue sequence alignment of human PSEN1 and that predicted for guinea pig, rat and mouse.

<p>Residues that are conserved in human and guinea pig but not in the rat, mouse or both are shaded in blue. Rodent residues not conserved in humans are shaded in black. Residues known to be mutated in FAD in human PSEN1 are shown in red text. Only one residue is conserved in guniea pigs (but not mice and/or rats) that is mutated in FAD (S212Y).</p

Increased <i>BACE1</i> RNA and reduced <i>ADAM10</i> RNA expression levels in brain tissue from guinea pigs fed a high cholesterol diet.

<p>Quantitative PCR analysis analysis for (A) ADAM10 and (B) BACE1 expression on total RNA extracted from the frontal cortex and cerebellum of guinea pigs fed the control or cholesterol diets. Data is represented as relative expression to RPS16. Compared to animals fed the control diet, ADAM10 expression is significantly decreased in the frontal cortex (<i>p</i><0.0001, <i>t</i> = 7.735, <i>d.f.</i> = 14) and cerebellum (<i>p</i><0.0001, <i>t</i> = 6.30, <i>d.f.</i> = 14) from animals fed cholesterol. In contrast BACE1 levels are significantly increased in the frontal cortex (<i>p</i><0.0001, <i>t</i> = 8.196, <i>d.f.</i> = 14) and cerebellum (<i>p</i><0.0001, <i>t</i> = 8.196, <i>d.f.</i> = 14). Values represent ± SEM.</p

Amino acid residue sequence alignment of Aβ in humans and that predicted for guinea pig, rat and mouse.

<p>Black shading indicates identical residues. Red box represents residues from mouse and rat Aβ that differ from those in human and guinea pig Aβ sequences.</p

Formation of the PS2V Transcript.

<p><b>A)</b> Presenilin structure in lipid bilayers: Arrowhead indicates boundary between protein sequences derived from exon 4 and 5. Dashed line indicates sequence from exon 5. Arrow indicates endoproteolysis site. Filled circle indicates γ-secretase catalytic site. <b>B)</b> PS2V forms when HMGA1a is expressed and binds to exon 5 (lighter shading) of <i>PSEN2</i> RNA causing ligation of exon 4 to exon 6 and ORF termination. <b>C)</b> Nucleotide sequence alignment of the 3′ end of exon 5 in human <i>PSEN2</i> RNA (with corresponding encoded residues) and the cognate exon of other species. Red boxes enclose sequences aligned with the HMGA1a-binding sites in human <i>PSEN2</i> RNA. <b>D)</b> mRNA from guinea brains exposed to control media or to media containing NaN₃ followed by RT-PCR analysis using primers amplifying cDNA spanning exons 3 to 7 of <i>Psen2</i>. In untreated samples a prominent ∼420 bp band is observed. In NaN₃ treated samples an additional ∼350 bp band is evident representing the cDNA fragment predicted from exclusion of the exon 5 sequence (PS2V). <b>E)</b> qPCR using a primer spanning the exon 4/6 junction PS2V cDNA showed up-regulation of PS2V mRNA in samples treated with NaN₃.</p

Total <i>MAPT</i> and <i>MAPT</i>3R transcripts are up-regulated under cholesterol fed conditions.

<p>Quantitative PCR analysis shows that, in comparison to animals fed a control diet, guinea pigs fed a cholesterol rich diet showed a significant increase in (A) total <i>MAPT</i> (<i>p</i> = 0.031, <i>t</i> = 3.560, <i>d.f.</i> = 14) and (B) <i>MAPT3</i>R (<i>p</i><0.0001, <i>t</i> = 6.468, <i>d.f.</i> = 14) transcripts but (C) no change was observed in <i>MAPT4R</i> transcripts (<i>p</i> = 0.1320, <i>t</i> = 1.60, <i>d.f.</i> = 14, ns). An increased 3R/4R ratio was observed (<i>p</i> = 0.0007, <i>t</i> = 4.326, <i>d.f.</i> = 14). Data is represented as fold change from control fed animals. Transcript levels were normalised against RPS16. Data represents ± SEM.</p

Mapt isoforms in guinea pig brain.

<p><b>A)</b> Schematic diagram of the alternative splicing pattern of human <i>Mapt</i>. Six Mapt isoforms (0N3R, 1N3R, 2N3R, 0N4R, 1N4R, 2N4R) are generated from alternative splicing of exon2, 3 and 10 of the solo Mapt gene. The alternative splicing of exon2 or/and 3 (green boxes) yields <i>Mapt</i> isoforms with 0, 1 or 2 inserts of 29 amino acid residues in the N-termini; whereas, alternative splicing of exon 10 (purple) generates isoforms with either 3 or 4 tubulin-binding repeats in the C-termini. To analyse whether this splicing pattern is conserved in guinea pig, two primer pairs, GTau0F/4R and GTau10F/14R were designed, targeting the corresponding region of the human exon2/3 and tubulin-binding repeats domains respectively in guinea pig Mapt. <b>B)</b> RT-PCR <i>Mapt</i>, using primer pairs GTau10F/14R.c DNA was isolated from a brain sample from guinea pig fed normal chow diet Two bands representing 3R and 4R Mapt were detected. <b>C)</b> RT-PCR of Guinea pig <i>Mapt</i>, using primer pairs GTau0F/4R. A single band representing 1N <i>Mapt</i> was detected.</p