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

The impact of immediate breast reconstruction on the time to delivery of adjuvant therapy: the iBRA-2 study

Background: Immediate breast reconstruction (IBR) is routinely offered to improve quality-of-life for women requiring mastectomy, but there are concerns that more complex surgery may delay adjuvant oncological treatments and compromise long-term outcomes. High-quality evidence is lacking. The iBRA-2 study aimed to investigate the impact of IBR on time to adjuvant therapy. Methods: Consecutive women undergoing mastectomy ± IBR for breast cancer July–December, 2016 were included. Patient demographics, operative, oncological and complication data were collected. Time from last definitive cancer surgery to first adjuvant treatment for patients undergoing mastectomy ± IBR were compared and risk factors associated with delays explored. Results: A total of 2540 patients were recruited from 76 centres; 1008 (39.7%) underwent IBR (implant-only [n = 675, 26.6%]; pedicled flaps [n = 105,4.1%] and free-flaps [n = 228, 8.9%]). Complications requiring re-admission or re-operation were significantly more common in patients undergoing IBR than those receiving mastectomy. Adjuvant chemotherapy or radiotherapy was required by 1235 (48.6%) patients. No clinically significant differences were seen in time to adjuvant therapy between patient groups but major complications irrespective of surgery received were significantly associated with treatment delays. Conclusions: IBR does not result in clinically significant delays to adjuvant therapy, but post-operative complications are associated with treatment delays. Strategies to minimise complications, including careful patient selection, are required to improve outcomes for patients

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

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

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

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

Sequence Similarity Comparison of AD-related proteins, between Humans and species of rodent: Sequence Similarity Scores and Sequence Identity (%) shown for each gene.

*<p>The MAPT sequence of guinea pig was predicted.</p