Research Highlights 2017

Biomedical Research and Findings

APP is one of the most important proteins in relation to Alzheimer’s disease (AD). APP is cleaved by a protein (or protease) called beta-secretase, and then by the protein complex called gamma-secretase, releasing the toxic Aβ peptides. Amyloid plaques, one of the two major hallmarks of AD, are composed of aggregated Aβ peptides. APP cleavages or APP maturation occurs while APP is trafficking inside the cell, mainly from the plasma membrane to late endosomes. These multi-step biochemical reactions affect a range of biological processes such as protein maturation and protein trafficking and involve various cellular compartments. A large part of the work carried out by the Fisher scientists is related to various aspects of APP biology. Other aspects of the work, more recently initiated, involved the characterization at the molecular level of nerve cells that are vulnerable (cells that disappear first in the disease) or resistant (cells that are very similar to the vulnerable cells but that seem to be more resistant to the disease state). Nerve cells are crucial, but other cells are also important, we call them accessory cells because of their role in supporting nerve cells. A class of these cells called microglia is also at the center of the recent research happening at the Fisher Center. Finally, the Fisher scientists initiated a novel line of research using a unique type of cells derived from humans and that can be grown in culture to study the importance of various genes and pathways.


1) Screening chemical derivatives of Gleevec and mechanisms of action

Fisher scientists continued their effort to synthesize derivatives (chemically altered forms) of Gleevec and screen the derivatives for more potent beta-amyloid lowering activity and the ability to accumulate in the brain, two weaknesses that Gleevec has. Fisher scientists have now carried out mechanistic and in vivo studies with Gleevec analogs that were described in the prior progress report, as well as long term studies. Gleevec was previously shown to reduce Aβ production in cellular assays by independently inhibiting both β-secretase and gamma-secretase protease-cleavages of APP and its main metabolite. We have performed in vivo efficacy studies with two compounds that are now clearly accumulating in the brain. They also showed promising effects of one of the compounds on Aβ42 levels in mice when the compound was administered orally for 5 days, and on amyloid plaques for the second compound when given in drinking water for over 3 months. We have confirmed that the new Gleevec analogs function similarly to the parent compound.

The Fisher scientists have also focused on new analogs of DV2-103, which is a kinase inactive analog of a potent dual inhibitor with a different mechanism, targeting Abl/Src kinases. While the new compound strongly reduces Aβ40.it does not show any change in Aβ42 level in cellular assay. The kinase inactive analog DV2-103, on the other hand, reduces both Aβ40 and Aβ42 levels at a relatively higher concentration. Because, it functions similarly to Gleevec with the added advantage of accumulating significantly in mouse brain, we prepared over 80 new analogs of DV2-103 and evaluated them in cells. We identified compounds that strongly reduce Aβproductions and are greater than DV2-103.

A manuscript is in preparation for one aspect of these studies and should be submitted for publication within 3 months.

As reported last year, independently of this drug based effort, a mutation (A673T) that seems to protect elderly people from developing AD was discovered by geneticists. Remarkably, the cellular mechanisms underlying the effect of this mutation has also been identified, indicating that drugs targeting this pathway might provide protection against the development of AD. In a study centered on Gleevec action and its derivatives, the Fisher scientists discovered that Gleevec and a related compound mimic the effects of the protective mutation A673T. This also signifies that a cellular model treated with these drugs could serve as a model for the development of more effective drugs to fight AD. This study was published in February 2017 in the Proceedings of the National Academy of Sciences.


2) GSAP-dependent trafficking is essential for Aβ (Abeta) peptide accumulation

A few years ago, we identified a protein that we called GSAP (for gamma-secretase activating protein). This work was previously published in the journal Nature (He et al., 2010). While we are working on studying how GSAP regulates gamma-secretase, we are also conducting a novel line of research consisting of studying an earlier role of GSAP on protein trafficking, including APP trafficking.

Fisher scientists investigated the possibility that an early trafficking step, involving GSAP, could be relevant for APP maturation and therefore for AD. Our latest studies, using a complex set of imaging technologies coupled to biochemical experiments, demonstrate that the GSAP indeed regulates APP intracellular trafficking. Our biochemical evidence show that GSAP associates APP and seems to control APP distribution inside the cells. Indeed it was found that decreasing the amount of GSAP decreases membrane APP level. Using the state-of-the-art TIRF microscopy, the Fisher scientists found that GSAP knockdown lowers the total numbers and the membrane dwell time of surface APP vesicles, indicating a role for GSAP in the membrane retention of APP vesicles. These data therefore define a new role of GSAP on amyloidogenic processing mechanism that involves crosstalk between APP trafficking machinery and Aβ production. A manuscript is in preparation for one aspect of this study.


3) Regulation of Abeta peptide by autophagy using various chemical and biological tools

On a study previously published in the Journal of Federation of American Societies for Experimental Biology (FASEB)(Tian et al., 2011), the scientists from the Fisher Center succeeded in accelerating the breakdown of beta-amyloid. The cellular process involved is called autophagy, a system responsible for removing debris from the cells, including unwanted materials such as the protein aggregates that are hallmarks of Alzheimer’s disease. The scientists discovered that a compound called SMER28 lowers the level of beta-amyloid found in nerve cells by stimulating autophagy.

Along those lines, and following up on even earlier work from the Fisher Center (Flajolet et al., 2007), Fisher scientists have identified an unexpected way for neurons to regulate beta-amyloid degradation and metabolism. As we mentioned above, Aβ originates from sequential cleavage of the Amyloid Precursor Protein (APP). The APP first cleavage is by BACE and yields βCTF. In turn, βCTF is cleaved by Presenilin 1 (PS1) to produce Aβ. The Fisher scientists showed recently that PS1, in addition to synthesizing Aβ, can also decrease Aβ levels by directing βCTF degradation through autophagy, by facilitating the fusion inside the cells of two important cellular components, the lysosomes and the autophagosomes. This fusion event is necessary to “clean” the content of autophagosomes, the main function of which is to remove debris from the cells. This study was published in July 2017 in the Proceedings of the National Academy of Sciences.

Furthermore, the scientists identified that the phosphorylation of PS1 by the protein kinase CK1 at a specific position participate in the degradation of intracellular material. Lack of phosphorylation on Presenilin 1 causes accumulation of partially fused autophagosomes-lysosomes in mouse brain and reduced autophagic flux, therefore debris (including various forms of APP, some of which can become toxic) are more likely to accumulate inside the cells. In summary, the disturbance in this cleaning process called autophagy leads to decreased βCTF degradation and results in accumulation of toxic Aβ peptide in the brain. This study was published in July 2017 in the Proceedings of the National Academy of Sciences.


4) Understanding the vulnerability of some specific neurons to disappear early on in the disease

An important concept has been emerging in the field of Alzheimer’s disease within the last few years, the notion of selective neuronal vulnerability. This concept implicates that specific types of nerve cells (neurons) will be more susceptible to a pathological process, they will be affected and disappear sooner than others and thus be called resistant neurons. In the case of AD, it is well known based on human post mortem studies that specific neuronal types disappear before others, while some other types seem to be resistant to the disease process. The regions of higher vulnerability have been relatively well mapped in humans. A brain region called the entorhinal cortex is the region of the brain that is the most vulnerable to degeneration, where cell death happens very early on, but it is not known why. Understanding why some cells are vulnerable and others are resistant to the disease process will certainly bring new clues on the underlying causes of the disease and help design entirely new therapeutic strategies. To pursue their effort to decipher selective neuronal vulnerability in AD, Fisher scientists are using a unique set of tools and technologies that they developed over the years. Because the vulnerable neurons of interest are intermixed with other cells, it is crucial to have a way to sort the various types of neurons. The tools developed by the scientists over the years allow precisely that. They discovered dozens of new genes that are linked to the vulnerability of EC neurons. Among these, Fisher scientists described the discovery of ADV1 (for AD vulnerability 1), a gene which is mutated in very rare families that have high incidence of AD, and which seems to regulate how excitable an EC neuron is, ADV2 and ADV3 that regulate the levels of many proteins involved in the vulnerability of neurons, and ADV4 a « splice factor » that clips out of the Tau protein (the main constituent of the AD-defining neurofibrillary tangles) a fragment that can increase its propensity to aggregate, and which seems to be most active in EC neurons. These four genes, and the other ones that Fisher scientists can now connect to vulnerability are all candidate genes that could serve as therapeutic targets for the treatment of AD-related neurodegeneration, but for all of them they are now trying to prove that they are indeed involved at different steps of AD development. This work has been recently submitted for publication to the journal Nature.

A major obstacle in developing drugs against AD neurodegeneration is that the field is lacking a good in vitro platform to screen, in an efficient high-throughput manner, different drug candidates, and to test the involvement of genes in the disease process. The in vitro screening platforms that currently exist are generic neurons, which do not have the same molecular content as the most AD-vulnerable neurons, and that potentially do not activate the same disease mechanisms when they form AD-related pathology. Findings obtained in these generic neurons will probably not be relevant for the study of the disease. Fisher scientists thus decided to take advantage of their study of the most AD-vulnerable neurons – EC neurons, in order to learn how to make them from human induced pluripotent stem cells (iPSCs). Namely they are using a combination of molecular profiles of EC neurons at different stages of their development, and a large compendium of human publicly available databases, in order to predict the proteins that can drive differentiation of stem cells to an EC neuron identity. They are currently establishing a list of candidate proteins, and they will start introducing these proteins into stem cells, analyzing if the stem cells take on EC molecular and functional characteristics. Once they obtain EC neurons, it will be an extremely powerful tool for the study of the disease and the generation of therapeutic drugs.


5) Regulatory role of microglia cells in the context of Alzheimer’s disease

Microglia cells are a sub-group of the larger family of cells called glial cells. They function, in the central nervous system, as macrophages or scavengers. These cells are mobile, and gets activated very quickly as a response to various stimuli that typically indicate a problem. Following their activation is response to an insult, the cells are then moving to the problematic zone where they will engulf and digest anything that is detected as being problematic, such as synapses, amyloid plaques, neuron debris, etc. Microglia contributes to AD pathogenesis and seems to have various roles, but the mechanisms by which microglia become dysfunctional in AD remain obscure. The Fisher scientists studied how microglial Presenilin 1 (the main active component of gamma-secretase) specifically modulates microglial development in AD. They have shown that they can modulate AD-like hallmarks by manipulating in vivo the activity of Presenilin 1. They have also shown using multiple state-of-the-art molecular techniques that Presenilin 1 plays a crucial role during microglial development as well as for synaptic transmission. All together, these results demonstrate that Presenilin 1 connects microglial development and function to AD. This work has been recently submitted for publication to the journal Cell.