Biology subjects

Newman, M.

Publications and source records attributed to Newman, M..

4 recordsLinked to original sources

bioRxiv2018

The zebrafish orthologue of familial Alzheimer’s disease gene PRESENILIN 2 is required for normal adult melanotic skin pigmentation

Alzheimers disease is the most common form of age-related dementia. At least 15 mutations in the human gene PRESENILIN 2 (PSEN2) have been found to cause familial Alzheimers disease (fAD). Zebrafish possess an orthologous gene, psen2, and present opportunities for investigation of PRESENILIN function related to Alzheimers disease. The most prevalent and best characterized fAD mutation in PSEN2 is N141I. The equivalent codon in zebrafish psen2 is N140. We used genome editing technology in zebrafish to target generation of mutations to the N140 codon. We isolated two mutations: psen2N140fs, (hereafter \"N140fs\"), causing truncation of the coding sequence, and psen2T141_L142delinsMISLISV, (hereafter \"T141_L142delinsMISLISV\"), that deletes the two codons immediately downstream of N140 and replaces them with seven codons coding for amino acid residues MISLISV. Thus, like almost every fAD mutation in the PRESENILIN genes, this latter mutation does not truncate the genes open reading frame. Both mutations are homozygous viable although N140fs transcripts are subject to nonsense-mediated decay and lack any possibility of coding for an active {gamma}-secretase enzyme. N140fs homozygous larvae initially show grossly normal melanotic skin pigmentation but subsequently lose this as they grow while retaining pigmentation in the retinal pigmented epithelium. T141_L142delinsMISLISV homozygotes retain faint skin melanotic pigmentation as adults, most likely indicating that the protein encoded by this allele retains weak {gamma}-secretase activity. Null mutations in the human PRESENILIN genes do not cause Alzheimers disease so these two mutations may be useful for future investigation of the differential effects of null and fAD-like PRESENILIN mutations on brain aging.\n\nFinancial Disclosure StatementThis research was supported by grants from the National Health and Medical Research Council of Australia, GNT1061006 and GNT1126422, and by funds from the School of Biological Sciences of the University of Adelaide. HJ is supported by an Adelaide Scholarship International from the University of Adelaide.\n\nConflict of Interest StatementThe authors declare no conflict of interest.

genetics↗

bioRxiv2018

Ratiometric assays of autophagic flux in zebrafish for analysis of familial Alzheimer’s disease-like mutations

Protein aggregates such as those formed in neurodegenerative diseases can be degraded via autophagy. To assess changes in autophagic flux in zebrafish models of familial Alzheimers disease (fAD) mutations, we first developed a transgene, polyQ80-GFP-v2A-GFP, expressing equimolar amounts of aggregating polyQ80-GFP and a free GFP internal control in zebrafish embryos and larvae. This assay detects changes in autophagic flux by comparing the relative strength of polyQ80-GFP and free GFP moiety signals on western immunoblots probed with an antibody detecting GFP. However, the assays application is limited by the toxicity of polyQ80-GFP, and because aggregation of this protein may, itself, induce autophagy. To overcome these issues, we subsequently developed a similar ratiometric assay where expression of a GFP-Lc3a-GFP transgene generates initially equimolar amounts of GFP-Lc3a (directed to autophagic degradation) and a free GFP internal control. The sensitivity of this latter assay is reduced by a cellular protease activity that separates Lc3a from GFP-Lc3a, thus contributing to the apparent free GFP signal and somewhat masking decreases in autophagic flux. Nevertheless, the assay demonstrates significantly decreased autophagic flux in zebrafish lacking presenilin2 gene activity supporting that the Presenilin2 protein, like human PRESENILIN1, plays a role(s) in autophagy. Zebrafish heterozygous for a typical fAD-like, reading-frame-preserving mutation in psen1 show decreased autophagic flux consistent with observations in mammalian systems. Unexpectedly, a zebrafish model of the only confirmed reading-frame-truncating fAD mutation in a human PRESENILIN gene, the K115Efs mutation of human PSEN2, shows possibly increased autophagic flux in young zebrafish (larvae).

molecular biology↗

bioRxiv2018

Accelerated brain aging towards transcriptional inversion in a zebrafish model of familial Alzheimer’s disease

Alzheimers disease (AD) develops silently over decades. We cannot easily access and analyse pre-symptomatic brains, so the earliest molecular changes that initiate AD remain unclear. Previously, we demonstrated that the genes mutated in early-onset, dominantly-inherited familial forms of AD (fAD) are evolving particularly rapidly in mice and rats. Fortunately, some non-mammalian vertebrates such as the zebrafish preserve fAD-relevant transcript isoforms of the PRESENILIN (PSEN1 and PSEN2) genes that these rodents have lost. Zebrafish are powerful vertebrate genetic models for many human diseases, but no genetic model of fAD in zebrafish currently exists. We edited the zebrafish genome to model the unique, protein-truncating fAD mutation of human PSEN2, K115fs. Analysing the brain transcriptome and proteome of young (6-month-old) and aged, infertile (24-month-old) wild type and heterozygous fAD-like mutant female sibling zebrafish supports accelerated brain aging and increased glucocorticoid signalling in young fAD-like fish, leading to a transcriptional inversion into glucocorticoid resistance and vast changes in biological pathways in aged, infertile fAD-like fish. Notably, one of these changes involving microglia-associated immune responses regulated by the ETS transcription factor family is preserved between our zebrafish fAD model and human early-onset AD. Importantly, these changes occur before obvious histopathology and likely in the absence of A{beta}. Our results support the contributions of early metabolic and oxidative stresses to immune and stress responses favouring AD pathogenesis and highlight the value of our fAD-like zebrafish genetic model for elucidating early changes in the brain that promote AD pathogenesis. The success of our approach has important implications for future modelling of AD.

bioinformatics↗

bioRxiv2018

HMGA1 zebrafish co-orthologue hmga1b can modulate p53-dependent cellular responses but is unable to control the alternative splicing of psen1

The HIGH MOBILITY GROUP AT-HOOK 1 (HMGA1) family of chromatin-binding proteins plays important roles in cellular responses to low oxygen. HMGA1 proteins regulate gene activity both in the nucleus and within mitochondria. They are expressed mainly during embryogenesis and their upregulation in cancerous cells indicates poor prognosis. The human HMGA1a isoform is upregulated under hypoxia via oxidative stress-dependent signalling and can then bind nascent transcripts of the familial Alzheimers disease gene PSEN2 to regulate alternative splicing to produce the truncated PSEN2 protein isoform PS2V. Zebrafish where hmga1a expression is induced by hypoxia to control splicing of the psen1 gene to produce the PS2V-equivalent isoform PS1IV. Zebrafish possess a second gene with apparent HMGA1 orthology, hmga1b. Here we investigate the predicted structure of Hmga1b protein and demonstrate it to be co-orthologous to human HMGA1 and most similar in structure to human isoform HMGA1c. We show that forced over-expression of either hmga1a or hmga1b mRNA can suppress the action of the cytotoxin hydroxyurea in stimulating cell death and transcription of the genes mdm2 and cdkn1a that, in humans, are controlled by p53. Our experimental data support an important role for HMGA1 proteins in modulation of p53-dependent responses and illuminate the evolutionary subfunctionalisation.

developmental biology↗