Institut Pasteur de Lille
Alzheimer’s disease is a neurodegenerative illness affecting millions of people worldwide. BIN1 is the second most associated risk factor for this disease, however its contribution to AD pathogenesis is not well understood.
The aim of the research is to examine neuronal defects caused by BIN1 in the photoreceptors of Drosophila melanogaster. The functional conservation between human BIN1 and its Drosophila orthologue Amphiphysin is also assessed.
The rhabdomeres of transgenic BIN1 and Amphiphysin overexpressing Drosophila flies are examined. Rhabdomeres are the light sensitive part of the retina. First cornea neutralisation is done, a quick method to visualize rhabdomeres in the facet eye. Next immunofluorescence is performed on dissected pupal retinas. For the final and more detailed visualisation, transmission electron microscopy is used.
BIN1 and Amphiphysin overexpression cause defects in the photoreceptors of the Drosophila melanogaster eye during development. The defects in BIN1 and Amphiphysin overexpressing flies are similar. Transmission electron microscopy also shows defects caused by BIN1 in the photoreceptors of adult retinas.
BIN1 and Amphiphysin cause similar defects in photoreceptors. This confirms that human BIN1 and Drosophila Amphiphysin have an evolutionary conserved function.
With the world population growing older, age-related diseases are becoming more and more the highest cause of death. In particular, Alzheimer’s disease (AD) a neurodegenerative disease, affects 50 to 75% of people with dementia, the general term of the syndrome. Most people know this disease by its typical symptoms in behavior and memory loss. These symptoms are caused by still incompletely discovered molecular pathways taking place in the brain. Two abnormal structures are found in the brain of people with AD. The first ones are deposits of amyloid beta (Ab) peptides which build up between the nerve cells of the brain. The second ones are tangles of Tau protein. In AD brains Tau protein is abnormally hyperphosphorylated and accumulated into bundles of filaments inside the nerve cells of the brain. These observations are believed to be the key to developing AD but it is unknown by which molecular pathway these are formed.
AD can be distinguished in two forms: a rare familial form called early-onset AD (EOAD) and a more sporadic form called late-onset (LOAD). Over the past years, genetic analyses were only performed on EOAD cases but with the development of genome-wide association studies (GWAS), research has shifted to the more sporadic form. By performing these studies several additional risk loci were discovered. The most recent and largest GWAS, led by team 3 of Unit 1167 at Institut Pasteur de Lille, discovered nineteen additional loci associated with AD. It is now a matter of determining the link between the physiological pathway of AD and the genes within these loci.
The gene showing the strongest association for developing AD discovered in this GWAS was bridging integrator 1 (BIN1). Team 3 decided to use Drosophila melanogaster as a model organism to perform experiments associated with BIN1 in an AD relevant background. One of the planned experiments is associated with the ortholog gene of BIN1 in Drosophila, Amphiphysin (Amph). In published experiments with Amph-null mutant flies, Amph26 and Amph5E3, the larvae appeared slower and adult flies were basically flightless (Leventis et al., 2001; Zelhof et al., 2001). The main aim is to assess if BIN1 expression in Drosophila can save the locomotor behavior of Amph deficient larvae. Lines have been created to perform these experiments but they first need to be genotyped to check if they have the Amph-null mutation. In this report the reported Amph null alleles are molecularly mapped in more detail to assess whether the flanking genes are affected or not. These experiments are performed using the polymerase chain reaction (PCR) technique and analyzing the PCR products by agarose gel electrophoresis.
Previous results of the lab have shown overexpression of BIN1 isoforms in the eye of Drosophila caused rhabdomere damage and that this would be specific to isoform 1 (Abdelfettah, Dourlen & Dermaut, 2014). However, it is also possible that isoform 1 is more expressed or accumulated compared to the other readouts. To conclude between the two hypotheses, levels of BIN1 isoforms in the adult eye are assessed by Western blot analysis.
Together, the results in this project show that the rhabdomere damage certainly is caused by BIN1 isoform 1, the neuronal isoform, which puts things in an interesting AD-context. The results also disagree the findings of Leventis et al. (2001) as the Amph5E3 deletion does affect the GaQ sequence.
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