BLT Stages

Abstract Bachelor Project 2019-2020:  Ex-vivo study of regeneration of skeletal muscle due to satellite cells

The research discussed in this paper took place at the Institut Pasteur de Lille in France. In this research, the main topic is the satellite cell in muscle fibers. This project takes a closer look to the role of the biological clock and how it affects muscle stem cell activation ex-vivo. For this research, two groups of mice were used. Those following a normal day- and night cycle and another group in which they induced a jetlag effect. From these mice, muscle fibers were isolated, cultured and the muscle stem cells on these fibers were stained through primary and secondary antibodies. In this way the proteins, which are expressed by these satellite cells, are investigated. Images of these satellite cells are recovered through a spinning disk microscopy. Next to this research, the expression of genes from the biological clock pathway in skeletal muscles are measured through qPCR. There is a significant difference in expression of genes in jetlag mice compared to the control mice. The clock-genes Rev-erba and Bmal1 are less expressed in jetlag mice than in control mice. In quiescent satellite cells, the fluorescence intensity of Pax7 is less in Jet-lag mice then in control mice. In 48h ex-vivo activated cultured satellite cells, there is no significant difference between jetlag and control mice. It is clear that the clock genes are less expressed in jetlag mice than in mice who follow a normal day- and night cycle. Also, the Pax7 intensity in quiescent satellite cells is less in jetlag mice than in normal mice. In 48h ex-vivo activated cells, there is no visible difference. The Jetlag-effect has a big influence on the biological clock in mice. It has also an effect on the intensity of the Pax7 trancription factor in satellite cells, which has influence on the regeneration of skeletal muscle.

 

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 Amph^5E3, 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 Amph^5E3 deletion does affect the GaQ sequence.

 

Samenvatting eindwerk 2014-2015: Interaction between the Alzheimer’s disease genes Fak and Tau in Drosophila

In an effort to understand the underlying pathology of AD, research teams all over the world are trying to uncover the genetic and molecular mechanisms of the disease . In the follow-up of a genome-wide meta-analysis for AD, scientists at the Institut Pasteur de Lille did a genetic screen with the model organism Drosophila melanogaster and uncovered five positive genes that modify Tau neurotoxicity. Among these genes, strong interaction is observed between Tau and the fly ortholog of the focal adhesion gene PTK2B, called Fak. The interaction is assessed with the use of the small rough eye phenotype and the wing blister phenotype in Drosophila. Using the Gal4-UAS system, Tau2N4R is expressed in the eyes and wings with different Fak backgrounds. In the eyes, Fak behaved as a consistent suppressor of Tau toxicity. In the wings the proportion of Tau-induced wing blisters is reduced upon RNAi-mediated knockdown of Fak. Accordingly, Fak loss-of-function mutations suppressed Tau toxicity in the posterior part of the wing. In the present work, quantification of the wing sizes of Tau0N4R and phosphodeficient Tau^AP expression in wild type and Fak mutant backgrounds reveals that loss of Fak restores the wing size of Tau0N4R but not Tau^AP expressing wings. Rather opposite results are obtained with expression of Tau0N4R in the same Fak mutations in the notal bristles, where the Fak loss-of-function mutations seem to slightly enhance the toxicity of Tau. Using the same bristle phenotype with Tau2N4R, results show no clear interaction with Fak. To check if Fak is able to phosphorylate Tau, protein extracts of fly heads of are made for Western blotting. Using different antibodies against phosphorylated and non- phosphorylated forms of the Tau protein, no difference is observed suggesting that Fak is not a Tau kinase. Together, the results of this study confirm the genetic interaction between Fak and Tau in independent readouts in the wings but not clearly in the notal bristles. Interestingly, this effect appears not be mediated by a direct role of Fak on Tau-phosphorylation.