Team:ESBS-Strasbourg/proteolux/application/neuro

• Alzeimer disease
• Occurrence and costs
• Genetics
• Pathology
• The Tau protein
• Calpains
• Caspases
• Utilization of the ProteoLux® Pro system

Alzeimer disease

The Alzheimer disease (AD) is a common form of dementia. This neurodegenerative and terminal disease was first described by German neuropathologist Alois Alzheimer in 1906 and was named after him. Its most prevalent form occurs by person over 65 years and it is responsible for ca. 60 percent of the 24 million dementia cases [54].

Characteristic for the disease is a deterioration of cognitive capacities, which follows by a decrease of regular activities and apparent behavioral disorders.

Alzheimer occurs nearly exclusively by people of high age. A rising prevalent can be observed in countries with an aging population. By people under 65 of age ca. 2% are affected, by 70 year old people it is nearly 3%, by the 75 years old 6% and by the 85 year old 20% of the people are affected [54].

In 2007, 29 million people worldwide were affected by the AD. A computer simulation based on the population date of the United Nations showed that 106 million people will be affected by AD in 2050 [55].

Dementias like Alzheimer are one of the most costly diseases in the United States and Europe and can cause massive problems today and in the future. The costs are direct medical costs such as nursing home care or nonmedical cost such as day care with a loss of productivity of the caregiver. The worldwide costs of dementia have been estimated to 160 billion dollar per year. They will dramatically increase in coming years. [56]

There exists a genetic component which causes AD. Five to ten percent of the affected persons show a familiar accumulation or FAD (familial Alzheimer Disease) which results from mutations of the presenilin 1 gene on the chromosome 14, on the presenilin 2 gene on chromosome 1 or the APP (Amyloid-Precursor-Protein) gene on chromosome 21 [57].

The Down syndrome with tripled chromosome 21 also heightens the risk of developing dementia like Alzheimer, but a diagnosis is often difficult due to already affected cognitive capacities of these patients.

Also a mutation variant of the SORL1 gene is known as a risk factor which heightens change of becoming affected by the Alzheimer disease. [58]

By patients affected senile plaques and fibril accretion are formed. The protein accretions of the plaques consist mostly of beta amyloid peptide. The intracellular located neurofibrils consist of Tau protein. This protein aggregates to fibrils when it is hyper phosphorylated. It is not yet clear if this phosphorylation is of secondary nature or if its triggers the disease. [59]
During the cause of disease the brain mass decreases due to a dyeing of neurons which is called a Brain atrophy. Additionally the neurotransmitter acetylcholine is not produced in sufficient quantity. The reason for this diminished production is an under expression of the enzyme cholinacetyltranferase which catalyzes the reaction of acetyl CoA and choline which leads to a general decrease of congenital capacities. [59]

The precursor of the Aβ peptide is the Amyloid precursor protein (APP), which is an integral membrane protein. Most of the protein protrudes out of the cell into the extracellular matrix, while just a small part is in the intracellular space of the cell. It is a type one trans membrane protein by which the amino terminus is outside and the carboxyl terminus is inside the cell. [59]

APP is cleaved by certain different secretases (alpha-, beta-, and gamma-secretase) which can result in a release of the Aβ peptide out of the precursor protein. There are basically two different mechanism existing. [59]

1. The amyloidogenic mechanism : APP is first cleaved by the beta secretase and then by the gamma secretase. This incision within the transmembrane domain leads to a release of the Aβ peptide.
2. The non amyloidogenic mechanism: APP is cleaved by an alpha- secretase. The incision takes place within a part of the APP which contains Aβ. This prevents from the generation of the Aβ peptide. The result is a release of a relatively big part of the protein which further mechanism is not clear yet.

Figure 1 | The two different mechanisms of APP cleavage
By the non amyloidogenic mechanism APP is cleaved by an alpha secretase (ADAM 9,10 or 17) with the extracellular domain. In the amyloidogenic mechanism APP is first cleaved by a beta secretase (BACE1) and followed by a second incision of a gamma secretase (PEN-2, APH-1 and Nicastrin) which releases the Aβ peptide

Both pathways can take place parallel in neuron cells. The length of the Aβ peptides can vary. The main type is the Aβ 40 peptide consisting of 40 amino acids, Aβ 42 peptide being represented in a smaller part. The length of the peptide is significant as the longer peptide with 42 amino acids has a far higher tendency for aggregation as the smaller peptide. [59]

Another factor for the Alzheimer disease is a dysfunction of mitochondrion. A blockade of the respiratory chain at the complex IV leads to a high production of radicals which can damage the cells. If this blockade is a result of the high Aβ production or if Aβ is produced as an antioxidant to deal with the high oxidative stress is not yet clear.

The function of the tau protein is to sustain the stability of the cytoskeleton and the proteins which are attached by the microtubules. The Tau protein is regulated by the MAPT gene where a mutation can lead to several diseases like pick diseases or corticobasal degeneration. In the Alzheimer disease accretion of Tau protein leads to senile plaques. In theory it is believed that these plaques disrupt the calcium homeostasis of the cells leading to an apoptosis. [60]

Posttranslational alterations on the MAPT gene on chromosome 17 result in nine isoforms of the gene in the human central nervous system. By the construction of filament structures single Tau proteins are joined over their repeating part. The amino terminal end is then cleaved. A hyper phosphorylation of paired helical Tau filaments leads to a misfolded not functional protein which is unable to interact with microtubules. As a result the accretion of these neurofibrillary lesions can be observed in the brains of Alzheimer patients [61].

As demonstrated previously, the Tau protein and its fragments play a very important role in the Alzheimer disease. Indeed, it has been shown that truncated Tau protein was present in Alzheimer disease and that the cleavage of tau is an early event of the Alzheimer disease development. The cleavage of Tau into fragments is done by several proteases such as caspases and calpains. However the real implication of the Tau protein as well as its level of degradation and aggregation is not fully understood yet. Some studies have been conducted on the Tau fragments, the proteases causing those fragments and the aggregation of those resulting fragments. So far, even though those aggregates have been shown to be involved in Alzheimer diseases, there has not been any clear and conclusive result allowing the establishment of the importance and the role of those aggregates.

The ProteoLux® Pro system can be used to help finding a new therapeutical target against the calpains as the research in this domain is quite advanced and the use of a tightly controlled system can improve the existing knowledge. On the other hand, the ProteoLux® system can be used in fundamental research to gather datas on the caspases and their real involvement in the Alzheimer disease. It has been clearly shown that caspases might have a role in the degradation of Tau protein into fragments. However there is no clear result pointing out their exact implication and the importance of this implication compared to the calpains. Therefore more datas are first required before going more into details with the caspase research as pharmacological target. [62]

Calpains are nonlysosomal, neutral cysteine proteases activated by calcium. There are two types of calpains expressed in the brain, the most characterized among the calpains. They are the µ-calpain and the m-calpain. They are presented as a heterodimer form. Until now the difference in activity between those two forms of calpains is not known, because they also have the same substrate. Normal transient activation of calpains in a normal functioning cell is involved in cell signaling, synaptic plasticity, and protein turnover. In contrast, sustained activation of calpains contributes to both chronic neurodegeneration associated with Parkinson’s disease, Alzheimer’s disease, and Huntington’s disease.

Several calpains inhibitors already exist for the study of the calpains activity, however they do not allow a tight control of the calpain concentration or activity in the cell. That is where the ProteoLux® Pro system comes into action.

As said previously, the caspases’ role in the Alzheimer disease has not been clearly defined yet. The ProteoLux® Pro system could be a major tool in the study of this caspase system and the discovery of new leading knowledge on the caspases. All the new knowledge acquired could be further developed to reach a level such as the calpains’ stage and then be used as a pharmacological target.

It is clearly visible that the characterization and a better comprehension of the Tau and Tau related aggregates are needed. A clear and complete understanding of those mechanisms could help to understand the disease process and could lead to new pharmacological targets. The idea is to use the ProteoLux® Pro system in order to study the mechanism of Tau fragmentation. The ProteoLux® Pro system can be implemented in cells culture from Alzheimer patients. Each protease involved in the Tau fragmentation could be studied in different cell cultures using the ProteoLux® Pro system. The protease would be fused to a Phytochrome Interacting Factor (PIF) and a degradation tag (Lambda-O in N-terminal and DAS in C-terminal). After implementation of the ClpXP system in the cell culture, the level of the targeted protease could be adjusted to a certain concentration using a succession of activating and inactivating pulses. Indeed the modeling has shown that the ProteoLux® Pro system can adjust the protein concentration in the cell. This allows a precise study of the targeted protease.

The utilization of the ProteoLux® Pro will without any doubt bring new insights in the mechanisms of the Alzheimer disease factors and development. It will also lead to new breakthrough in the creation of new pharmacological targets and substances. Indeed being able to control the level or even the formation of Tau aggregates might lead to cure the Alzheimer disease.