Investigating the Molecular and Cellular Events Causing Alzheimer's Disease Using Zebrafish
We have two Honours project positions open in our molecular biology laboratory each year. The projects are designed to include both molecular work (e.g. PCR; western blotting; gene design and construction; CRISPR Cas9 endogenous gene editing) and work with zebrafish (e.g. egg injection for forced gene expression, gene knockout, or transgenesis; in situ transcript hybridsation; breeding and genotyping). Our laboratory philosophy regarding Honours projects is that these should be technically very feasible to allow students the satisfaction of achieving real results while also producing data that can be incorporated into scientific publications.
Since our work is rapidly evolving we prefer not to give specific project descriptions months in advance since these may not be valid when Honours study actually begins. We are happy to talk to students about possible projects at any time but decisions on actual projects are made in consultation with our Honours students in late January of each year. If you wish to apply for Honours work in our laboratory then simply write down "Alzheimer's Disease Genetics Laboratory" in your list of preferences. Please email Michael Lardelli to arrange a time to talk about Honours projects in our laboratory.
Master and Ph.D. Projects
We welcome Master and Ph.D. students who wish to investigate the molecular events underlying Alzheimer's disease using the zebrafish as a genetic model. Projects can be based in molecular biology, bioinformatics, or a combination of these areas. Please email Michael Lardelli to discuss this further.
Research Projects in Alzheimer's Disease Genetics
Our laboratory is currently involved in a number of projects investigating the molecular changes that cause Alzheimer's disease. Our main focus is on the function of the PRESENILIN genes (PSEN1 and PSEN2) that are the major loci for mutations causing Familial Alzheimer's Disease (FAD). Over 160 mutations causing FAD are known in the human PSEN1 gene. Nearly all are mis-sense mutations that alter the protein coding sequence rather than non-sense mutations that truncate the protein. This led to the belief that truncated PRESENILIN proteins have no function. However, the unique analytical opportunities offered by the zebrafish model allowed us to show this is false. Instead, truncated PRESENILIN proteins can have potent dominant effects, (see Nornes et al. 2008; Newman et al. 2014; Moussavi Nik et al. 2015).
We are currently using gene editing technologies such as TALENs and the CRISPR Cas9 system to introduce FAD mutations into zebrafish. We are then analysing the cellular stresses that these mutations cause by examining the brains of young zebrafish using deep sequencing of the brain transcriptome followed by gene regulatory network analysis. Our first papers on these analyses have now been submitted. A very important aspect of our work is that we are analysing the dominant FAD mutations in the heterozygous state that they occur in humans. (We do this because our previous published work indicates that e.g. mutant PRESENILIN alleles dominantly interact with non-mutant PRESENILIN alleles to produce the disease phenotype - see below.) We are also able to minimise "gene expression noise" in our analyses by reducing both genetic and environmental variation. To do this we use inbred strains of fish and we analyse the progeny of pairs of mated fish that are raised together under identical conditions - i.e. in the same tank. Our analyses also include considerable experimental replication - multiple heterozygous mutant brains are compared to multiple non-mutant brains - and all the brains are from siblings produced from a pair of mated fish and raised together. In order to identify the initial, causative stresses that produce Alzheimer's disease we analyse brains taken from the fish when they are recently sexually mature - i.e. at a time before pathology would be advanced in humans.
Since we work at the forefront of Alzheimer's disease research using zebrafish, we have developed assays for Alzheimer's disease-relevant cellular activities. For example, we have developed the first in vivo assay for the PRESENLINs' "gamma-secretase" enzyme activity that is required to produce a small protein fragment named Amyloid-beta (thought by many to act in a toxic fashion in Alzheimer's disease brains - see Wilson and Lardelli 2013). Using this unique assay we discovered that the effects of different PRESENILIN truncations on APP cleavage and Notch signalling are not equal. Also, truncated PRESENILIN proteins apparently exert their dominant effects through interaction with normal, full-length PRESENILIN proteins (Newman et al. 2014). This has implications for our understanding of diseases other than FAD that are also caused by mutations in the PRESENILIN genes (e.g. Frontotemporal Dementia and the inherited skin condition Familial Acne Inversa).
We have used our gamma-secretase assay to study the action of a particular truncated form of the PSEN2 protein named "PS2V" that is seen in the brains of people with late onset (>65 years) Alzheimer's disease. When expressed in zebrafish, a PS2V-like molecule interacts with the full-length zebrafish Psen1 protein to increase gamma-secretase activity (Newman et al. 2014). Interestingly, during evolution PS2V has been lost from mice and rats but not from most other mammals or even zebrafish. This is one more indication that mice and rats may be poor choices for modeling Alzheimer's disease. We recently published a paper analyzing PS2V in the guinea pig and showing that this rodent has the potential to be a superior Alzheimer's disease model (see Sharman et al. 2013). Our discoveries regarding PS2V have implications for understanding the cause(s) of the sporadic (non-inherited) form of Alzheimer's disease that constitutes 98% of all Alzheimer's disease cases.
The Lardelli laboratory collaborates with other Alzheimer's disease research laboratories in Australia and we also assist a number of other laboratories in Adelaide to use zebrafish in their research.