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Apomorphine Treatment in Alzheimer Mice Promoting Amyloid-b Degradation

1. Why did they examine the levels of amyloid beta-protein, p-tau, and P53? Why evaluate the activity of proteasome and IDE?

2. Why did they choose to use this particular triple transgenic AD mouse model (3xTg-AD)?

3. Why do you think they chose Apomorphine for this study? Think about the role of dopamine in AD and see if you can find any studies that offer insight into this.

Amyloid beta-protein is a protein that accumulates in the brains of Alzheimer?s patients as senile plaque and causes neuronal degradation. P-tau is a protein that forms neurofibrillary tangles, another hallmark of Alzheimer?s disease, when hyperphosphorylated. P53 is a tumor suppressor protein that works to maintain the normal progression of the cell cycle; it also has roles in causing apoptosis, which is the function that was studied in this experiment. Levels of each of these proteins were examined in the rats studied to observe the effects of the apomorphine (APO) treatment on them. Decreased levels of these proteins would show that APO can keep at bay the physiological changes that lead to neurodegeneration in Alzheimer?s patients. Evaluating proteasome and insulin-degrading enzyme was done for the same purpose: higher levels of activity in these enzymes would indicate that they are breaking down amyloid-beta protein faster and therefore are producing a beneficial effect in the organism being studied.

The particular triple transgenic AD mouse model chosen for this study, 3x-Tg-AD, has two familial Alzheimer?s Disease-related mutations which result in the mice developing cognitive deficiencies early in life as well as significant amyloid-beta 42 aggregations that, when removed, allow for improved memory function. In this way, these mice are good models for studying the pathological effects of amyloid-beta protein when it exists in high levels within neurons, and therefore they are excellent test subjects for developing new therapies to treat Alzheimer?s disease. The third mutation they possess, a mutation in the p-tau protein, contributes to the neurodegradation observed in these mice and to their pathology.

APO, a ?non-specific dopamine agonist,? may have been chosen for use in this study because, as a dopamine agonist, it can activate dopamine receptors just as dopamine itself can, which, in the case of Alzheimer?s disease, may be necessary because dopamine may not be as available as necessary for proper functioning. Dopaminergic neurons do extend to the hippocampus, a brain structure we have learned is important in memory and learning, an a paucity of dopamine in this area could be part of the reason why Alzheimer?s patients develop dementia: there is not enough dopamine available to activate the hippocampus properly, impeding the formation of new memories and hindering the recall of old ones. I was able to find a study about APO and its effects upon MAO-A and MAO-B, and it discusses in the abstract the fact that APO acts to inhibit MAO, which would cause less degradation of available dopamine within the brain which could lead to improved memory and cognitive function in Alzheimer?s patients, among other things. APO, it seems, is on the whole a beneficial drug that could be put to good use as a treatment for patients with Alzheimer?s disease.

I think the researchers make a fairly compelling case that Apomorphine could be a novel treatment for Alzheimer?s disease, especially since current drugs merely treat some symptoms and do nothing to slow or halt the disease?s progression as it appears the Apomorphine may be able to do. AD is characterized by a progressive deficit in a host of functions due to neural degeneration. The degeneration in AD patients is thought to primarily be caused by plaques and tangles in the brain. The tangles consist mostly of the ptau protein, which works to stabilize microtubules in neurons, that has been hyperphosphorylated and so, has clumped together in a tangle. Likewise, the plaques are made up of amyloid B-protein. They also examined P53, a protein that helps to regulate the cell cycle and is known to be involved in apoptosis associated with the disease. These various proteins were examined as a gauge of how severe the AD in the model mice was and provide a quantifiable measure to see if Apomorphine is an effective treatment through reducing their concentration. Proteasome and IDE activity were also measured, both of which work to degrade AB among their other functions, meaning the researchers could determine if the Apomorphine was effective at encouraging AB degradation via activating the degradative enzymes.

The 3xTg-AD model mouse was used in the study since it develops cognitive deficiencies early in life (about 4 months) due to two separate mutations in familial AD related genes that cause the accumulation of AB as well as a mutation in the ptau protein. Since these mice have both increased AB accumulation and ptau accumulation, they have both the tangles and the plaques of humans with AD, so they should exhibit similar physiological changes as those observed in humans.

Dopamine is thought to be involved in a number of the symptoms associated with AD, since the dopaminergic (and other diffuse modulatory systems like the other catecholamines or serotonin) are involved in areas such as mood and the development of psychosis. I found a study that looked at two existing AD treatments, donepezil and galantamine both of which are inhibitors of Ach that can help mitigate some of the cognitive decline in AD. The study found that these Ach inhibitors also stimulated dopamine release, and the researchers speculate that the increased DA release may help explain those drugs ability to treat the non-cognitive symptoms of AD. Thus, Apomorphine, as a potent, non-selective DA agonist was examined in the current study in order to determine if stimulating DA release is a viable treatment for AD, and it appears that, at least in mice, it is indeed an effective treatment since there was improved memory in accordance with decreased AB (particularly, intraneurally) and ptau levels following Apomorphine administration.
http://www.ncbi.nlm.nih.gov/pubmed/15322245

The researchers examined the levels of AB protein and P-tau because they are the hallmark of Alzheimer?s disease. Aggregation of AB protein and tau tangles is what causes neurons to die by generating cell stress response and inflammatory responses within the brain. P53 plays an integral role in the stability and survival of the cell and organism as a whole. It plays an important part in apoptosis, which can be generated by the heat-shock response from AB aggregates. Proteasomes and IDE have the ability to degrade intracellular AB, P53, and P-tau, so measuring the level of these proteins is integral in measuring the effect of Apomorphine treatment.

3XTg-AD mice was selected because they are bred with two FAD mutations and a tau gene mutation. Onset of Alzheimer?s is early in these mice, we can see AB aggregation and cognitive decline in about 4 month. Actively removing these plaques lead to direct improvement in their cognitive function. Overall, they serve as a good model for measuring the effectiveness of APO injection.

Although Alzheimer?s patients are mostly well-known for their dysfunction in cholinergic neurons. However, the role of other neurotransmitters including dopamine is also important in leading to dementia. A study done by Martorana et. al performed studies measuring the cholinergic activity before and after injection of L-Dopa in healthy and AD patients, and found that there was improvement of cholinergic function in AD patients after the injection, but no improvements in healthy individuals. This shows that dopaminergic neurons influences the function of cholinergic neurons, and a decline in dopaminergic neurons plays a part in the cognitive decline we see in AD patients.

http://www.nature.com/npp/journal/v34/n10/full/npp200960a.html

The functions of AB protein, p-tau, and p53 are made clear by Drew, Clementine, and Nick. As we discussed in class today, tau protein is important in stabilizing the microtubules of the neurons. When p-tau overcompensates, it forms neurofibrillary tangles throughout the brain, causing problems and symptoms of Alzheimer's when in conjunction with AB protein problems. AB protein causes plaque build up in the neuron, which causes the neuron to malfunction. Lastly, p53 is very important in suppressing tumors and apoptosis. These are clearly related in the death and functions of a cell, and when they function improperly, they can lead to AD. Once these levels are measured throughout the cell, they have a basis for testing. Then apomorphine can be applied to see if they have an effect on the levels of these proteins. Proteasomes are important in oxidative stress, which is related to the proteins listed above. IDE is an insulin degrading enzyme and can degrade AB, implicating its involvement in AD. These enzymes have the ability to degrade AB protein, p-tau, and p53, meaning that higher levels of these enzymes will show lower levels of these proteins. They were included in the experiment to see if apomorphine increased the levels of proteasomes and IDE and therefore decreased the proteins that are closely associated with AD.

Taken from the paper itself "The triple transgenic AD mouse model (3xTg-AD) has 2 familial AD-related gene mutations (APPKM670/671NL/PS1M146V) and a tau gene mutation (TauP301L)." This means that the mice develop AD very early on in their life due to the plaque build up along with the neurofibrillary tangles due to the p-tau mutation. These are the same causes of AD in humans as well, making the comparison between the two, on a neurological basis, very similar. They also were able to remove the plaque and improve the memory of these mice. These mice then became very good test subjects because they respond the same as humans and have easily alterable cognitive abilities.

Apomorphine is a non-selective dopamine agonist, meaning it can act in the place of dopamine, and as the others said, dopamine is obviously important in mood and behavior. This would show its effects in people with AD who clearly have problems with these aspects. One other study being done is the effectiveness of huperzine A on memory and cognition of those with Alzheimers. Huperzine A is an acetylcholinesterase inhibitor (also stimulating dopamine release throughout brain), as well as donepezil and galantamine. Huperzine A is a naturally occurring substance found in a fern moss. As of now, this has shown that people with AD have had memory and cognitive improvement after taking this drug, showing that it may be an effective treatment for those with severe symptoms.

http://www.ncbi.nlm.nih.gov/pubmed/8701750

To reiterate, Amyloid-Beta (AB) is a peptide that create amyloid plaques via aggregation into smaller peptides that join to become plaque. These plaques inhibit normal brain function in AD patients. P-tau is a protein that produces large strings that aid in the stabilization of microtubules. In AD patients, these proteins float no longer bind to the microtube and instead aggregate and float as tangles in the cytosol of the axon. Finally P53 is a tumor suppressor protein that helps in the regulation of the cell cycle. As a tumor suppressor, it plays a role in apoptosis. These three proteins were chosen for observation because they are closely related to AD. Both plaques and tangles are found in Alzheimer?s lesions, and P53 plays a role in the cell death of neurons associated with the disease. Proteasome and insulin degrading enzyme are related to the degradation of the previous proteins. Thus, an increase in Proteasome and IDE expression suggests a decrease in AB, p-tau and P53 presence.

The type of mouse was chosen for having two types of Familial Alzheimer Disease (FAD) related gene mutations and a mutation in the p-tau gene (thus the triple transgenic title). The FAD related genes increase and hasten the symptoms of Alzheimer?s, specifically generating AB plaque buildup. This early expression made them convenient test subjects. The p-tau mutation increased tangle production by generating defective p-tau. The mirrored symptoms to those observed in AD patients as well as the short time frame for onset made these mice the right choice.

The choice of APO was to stimulate dopaminergic systems. Dopamine systems are associated with mood, attention, learning and working memory, to name a few. Working memory is specifically needed to hold information while processing other information. This is seen as disrupted in AD patients through the loss of the ability to process their environment. An increase in Dopamine could influence this system. This article also shows the role of AB and dopamine as precursors to apoptosis in human neurons and the use of antioxidants to inhibit apoptosis.

http://informahealthcare.com/doi/abs/10.1080/107157602100006445

Like other people have said, We know that beta secretase created 4 kd AB42 can oligomerize to form the amyloid plaques that are a hallmark of azheimers disease. Because these monomers are a precursor for plaque formation, observing a reduction in total amyloid beta protein would be beneficial. The same goes for phosphorylated tau protein, which is another hallmark of alzheimers due to microtubule breakdown and neurofibrillary tangle formation assosciated with tau hyperphosphorylation. As for p53, a reduction would show an anti-apoptotic effect, showing that not only does the drug reduce levels of protein, but it also reduces the neurotoxic effects of p-tau and AB. as for the proteasome and IDE, this protein activity would support that the breakdown of AB is occuring through natural protein degradation and not some protein degrading effect of apomorphine. Essentially the increase in protease activity is a desired effect.

the 3 times transgenic rat seems like a novel animal for studying alzheimers, especially familial alzheimers. I think that this model is beneficial because it contains three distinct mutations that contribute to alzheimers. The APP mutation will lead to an increase in AB and therefore increase neurotoxic plaque formation. The Tau mutation will increase Tau hyperphosphorylation and therefore increase microtubule degradation and tangle formation. the Ps1 mutation is a gamma secretase mutation, which will contribute to alzheimers by reducing the level of normal APP cleavage, allowing for a generalized increase in AB (pathalogical cleavage). Therefore, a beneficial effect seen in these mice will be more attributable to a real life scenario and would suggest an overall beneficial effect on multiple causes of alzheimers pathology over a beneficial effect on a specific mutation like overactive BACE1.

A lot of studies I read correlated alzheimers with a loss of dopamine, including a reduction in dopamine-beta-hydroxylase activity. Another study showed that using cholinergic drugs for alzheimers also increases dopamine release indirectly, with some of the succes of cholinergic drugs like donepezil being attributed to the increase in dopaminergic systems, not just acetylcholine. There seems to be an interplay between the cholinergic and dopaminergic systems that is mutually beneficial and seemingly clinically applicable. Therefore, the dopamine agonist apomorphine can utilise this combinatorially beneficcial effect as a mechanism for alzheimers treatment. Perhaps the next avenue of research involves harmonious combinatorial drug treatments involving cholinergic and dopaminergic increases.