Team:Worldshaper-Shanghai/Human Practices

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Integrated Human practice

1.Public Engagement

1）The results of the questionnaire show that the public's understanding of Alzheimer's disease is not comprehensive. Due to the lack of knowledge about the characteristics and pathogenesis of Alzheimer's disease, disease prevention and treatment are hindered. Most of them believe that the current AD drugs are ineffective, which means that patients and their families lose hope for the treatment of Alzheimer's disease. We believe that we have the obligation to popularize basic AD knowledge and let everyone have a deeper understanding of the disease. As a result, We have integrated this knowledge into popular science materials and speech content.
2）Besides, we also know that caregivers of patients also face heavy physical and psychological pressure. Through the survey, we plan to carry out activities with high acceptance and recognition, such as popularizing mindfulness meditation, so as to truly and effectively solve the anxiety of caregivers of Alzheimer's disease. At present, one of the members of Worldshaper-Shanghai has also set up a WeChat official account called “HippoComPass” to give scientific advice on caring for patients and meditation practice courses to reduce stress.
3）In addition, about a quarter of respondent have not heard of sythetic biology and 30% of them have not heard of iGEM. Therefore, we believed the results from this young and popular field will catch their sight and incorporated this part into public participation activities.

2.Impact on the project

1）Professor Zhao told us that when we transfer full-length app to drosophila, we need to confirm the expression of APP gene in specific tissues or organs, and also need to demonstrate how we obtained positive transgenic fruit flies. According to the professor's suggestions, we focused on collecting evidence in this regard. For example, we used GAL4 / UAS system to ensure the expression of APP in special tissues and organs. Our plasmid also contains red eye gene. The success of transformation can be confirmed by the change of eye color of White eyed drosophila (the eye color of transgenic drosophila changes from yellow to orange, depending on the expression of red eye gene).
2）According to the suggestion of experts, we chose the behaviors that similar to that of patients with Alzheimer's disease as the test object, such as mobility, learning and memory, and cognitive abilities.
3）The preclinical research and evaluation of new drugs are very complex. We realize that our drug screening model can only be used as one of the tools in the process of drug research and development. But even so, a successful Drosophila model, because of its high efficiency and economic advantages, can play a vital role in the early screening stage of new drugs. This conclusion led us to further communicate with experts and enterprises engaged in animal model research and application.

3. Breakthrough in human practice work

This year, due to the particularity of our project, we encountered many problems at the beginning of human practice. For example, we were unable to obtain the first-hand information of patients and online statistics of the disease; There were also many difficulties in finding relevant doctors, experts and drug research and manufacturers. This led us to rethink our approach to gathering HP-related resources and achieved a breakthrough. Now, we have written the experience into a document and uploaded it to the contribution page.

Human Practice

1. Introductin and Motivation

The ultimate goal of this project is to explore the application potential of Drosophila drug screening model for Alzheimer's disease in the field of scientific research and drug development as far as possible, and gradually broaden horizons. Despite many obstacles, we have done our best to interact and communicate with the public, doctors, professionals and public welfare organizations.

2.Questionnaire survey

2.1 public survey

Respondents Total: 800
Gender: 65% female, 32.5% male, 2.5% yet to be determined
Age: Below 18: 31.25%, 18~30: 13.75%, 31~55: 4-%, Above 55: 15%
Educational background: Junior high school or below: 8.75%, High school: 28.75%, Associate Degree: 15%, Bachelor: 30%, Master degree: 10%, PhD: 7.5%

The first part of our investigation is to know the AD around us. Is there any AD around friends or family, and which stage have they reached? 30% of our respondent have AD patient around them, and 45.83% of them have arrived intermediate stage. 25% of patients have reached late stage. (Figures below show the results of our first part investigation.) This data was actually beyond our expectation. Alzheimer’s Diseases has been so closed to us, and a quarter of these patients have reached late stage, which means they are too hard to save.

Then, we survey the respondents’ degree of understanding toward formation of AD. As we expected, most respondents (56.25%) know part of pathology of AD. Rest of respondents mostly, 36.25% of total, know nothing about pathology of AD. Only some specialized person understands perfectly. This reflects that many audience do not know about the pathology of AD, and this can be our following direction of working.
For the people who know about AD, genetic cause was the first factor to be believed through the results. Psychological condition and living environment are the next, and so on. It can be predicted that people may be more willing to accept genetic cure and treatment.
Eventually, we asked respondents for their realization about AD hypothesis. Again, 54.9% people know nothing about pathogenic hypothesis. Cholinergic hypothesis of age-related cognitive dysfunction is the most known hypothesis through our investigation. However, there are only small part of people have heard some hypothesis. The real causes is still developing.

We investigated for their attitude toward treatment of AD in the future. Fortunately, even though people held no brief for the existing drugs, most of them still believe targeted drugs will be developed in the future.

However, not too much people learned about drug screening, this results case a doubt on the reliability of their advices above, but admittedly, their supporting to medicine gave us power to develop new efficient drugs.

2.2 Views of Alzheimer's Caregivers

Our team members has conducted a series of in-depth investigations and studies on the current situation of Alzheimer's caregivers and their anxiety issues.
Respondents Total: 123
Source of feedback: 66% of the answers received came from Jiangsu, Zhejiang and Shanghai. Because the overall economic strength of Jiangsu, Zhejiang and Shanghai is in the forefront in China, Alzheimer's patients also receive relatively good treatment. The following will show the survey results from the four aspects of the questionnaire.

Based on the data, it is not difficult to see that most people have a medium understanding of AD, and more than 90% of the people have heard of and better understand AD. Only 2.4% had never heard of it at all. It shows that the overall popularity of the disease is high.
26% of people have close contact with patients, but the proportion of direct caregivers is very small. Therefore, there are not many respondents with personal care experience. However, compared with the total number of Alzheimer's patients who account for only 13% of the total number of elderly people in China and the incidence rate is less than 4%, it is normal for us to get such results in a short time.

Since there are few direct caregivers, we only summarized some useful information.
In terms of psychological distress, the overall response was very consistent, and there was no unfair psychology due to self sacrifice, indicating that the caregivers voluntarily invested time and energy in the care of patients, showing an optimistic willingness to take care of patients. In the care process, the caregivers will be physically overworked, resulting in discomfort such as headache, sleep disorder and so on. At the same time, the problems of caregivers in life are also very consistent with expectations, and they have some problems in economic and social aspects. However, the caregivers who filled out the questionnaire did not indicate the occurrence of more serious long-term diseases, such as arthritis.
Different from the choice of caregivers, about one third of indirect caregivers believe that long-term high-intensity care will lead to grievances caused by self sacrifice, and about one quarter believe that care will lead to serious chronic diseases. Indirect caregivers are able to empathize with caregivers, which shows that people generally have strong empathy and compassion. Therefore, if the troubles of caregivers are amplified and publicized, many enthusiastic people who are willing to help should emerge in the society.
About how caregivers adjust their living conditions and psychological discomfort, more than 50% of the effective responses mentioned some ways to reduce psychological stress, such as exercise, yoga, listening to music, etc. Nearly 30% of the respondents said they didn't understand this and couldn't come up with some adjustment methods. In addition, as one of the most authoritative and effective methods for caregivers to relieve stress, mindfulness therapy, only 24.8% of respondents said they had heard of it.

3. Doctor Interviews

Dr. Ruibin Guo,General Hospital of Eastern Theater command.
Dr. Xiang Xu, Neurology Department of Shanghai Huadong Hospital

3.1 Diagnosis and treatment of Alzheimer's disease

Combined with the data of doctors, 300-600 patients with Alzheimer's disease are treated in the dementia clinic every year. Dr. Guo told us that the incidence of AD increases with age, and reports constantly refer that about 30% of people over 80 years old may develop AD. Nowadays, there is a growing awareness of AD, and many people will come to the clinic as soon as they notice that their memory is impaired. Some people even have symptoms that are not particularly obvious, and then only through some very specialized assessments can they be evaluated to see if they have AD or if the symptoms may have other underlying causes. In general, AD is an irreversible process, patients receiving treatment are in the early to mid-stage, and late stage, according to Dr.Xu.
Dr. Guo said that during treatment, doctors will choose drugs according to symptoms. Amlexan is mainly for mild to moderate cognitive impairment, and memantine is for moderate to severe cognitive impairment.
Dr. Xu also pointed out that the role of current ad drugs is basically to slow down the process of the disease. When choosing AD drugs, we should consider three aspects: the first is the safety of drugs, which can not have very serious side effects. The second is the effectiveness of drugs. The effect of treatment depends on whether the patient's cognitive level is restored. Only when the patient's cognitive level is improved can it be regarded as effective. The third is to consider the price of drugs, which is a key factor, because AD treatment is a long-term challenge. If the drugs are too expensive, patients and their families will face a heavy financial burden.
For patient's motivation for treatment, doctors thought that the motivation of AD patients for treatment depends mainly on the patient's education level and the family's care. If the patient is more educated and has a higher demand for his own quality of life, and if the family is more concerned, generally this part of patients will be more active in treatment and have better compliance.

3.2 Views on existing drugs in treating Alzheimer’s disease

The mainstream drugs for AD nowadays include two main categories, one is cholinesterase inhibitors, such as that donepezil and carbadine, and the other is NMBA receptor antagonists, such as memantine.
Dr. Guo said, one option that is currently effective is acetylcholinesterase inhibitors: e.g., Amlexan, Meperidine. When the acetylcholinesterase activity is abnormal, it will lead to an increase in some products, which will cause some memory impairment, and now a large part of the drugs used are acetylcholinesterase inhibitors to inhibit the activity of this enzyme, so as to protect the function of neurons and achieve the purpose of maintaining neuron counts, cognitive function, and memory. There is an ameliorative effect, but no blocking effect on the progression of the disease. It stems from an AD cholinergic damage hypothesis.
As for the efficacy of ad drugs, Dr. Xu said that the efficacy of drugs is not very significant, but they can not be used. Because the current drugs are effective and statistically effective compared with placebo, but they may not feel obvious curative effect for each patient. They will feel that their disease is getting worse every year, but if they do not use these drugs, the disease may get worse more obviously. Therefore, my doctor believes that drugs can delay the progress of the disease.

4. Interviews with experts and professionals

Prof. Xiaofeng Zhao, Hangzhou Normal University
Dr. You Yin, Shanghai changzheng hospital - The Second Military Medical University
Cheng Yang, Laos ElementoPharma Co., Ltd

4.1 Problems and technical difficulties at present

“Alzheimer's disease is a very hot disease. Between 200 and 300 global drug discoveries have been made since 2000, all for this one disease, but so far they have largely failed or had little effect.” Said Dr. Yin. “We can see that Alzheimer's disease is very important because for each drug development, several or even several billion dollars are spent. With so many failures, drug companies continue to invest in drug discovery.”
Dr. Yin listed several hypotheses about the pathogenesis of Alzheimer's disease, such as the effect of amyloid beta protein, the destruction of cytoskeleton by Tau protein, activation of microglia by inflammatory mechanisms, loss of cholinergic energy, and so on.
Most drugs target the amyloid beta hypothesis, including a variety of ideas, such as inhibiting preproduction of the APP base protein. In recent years, two new drugs have a big impact, one is Mannita from China, and the other is a newly approved drug jointly developed by Eisai and Biogen in Japan, which is the monoclonal antibody of a-β.
Dr. Yin gives three reasons why scientists think that the results are bad. First is because the pathogenesis is unclear. The second reason is that it intervenes too late.Patients undergoing drug development have already developed extensive pathological changes, and it is difficult to reverse their pathological changes if you go to drug intervention at this time. So, there's a lot of drug development going on right now that looks at earlier pathogens.The third is the lack of precise biomarker indicators, many patients in clinical trials are diagnosed incorrectly.
Prof. Zhao said another point of view. Most current investigations of AD, including amyloid-beta, tau, neurofibrillary tangle, and inflammation, target the disease's progress. In other words, they focus on the late stage, such as how drugs clear amyloid-beta deposits and inhibit tau hyperphosphorylation after the onset of old age. We should pay more attention to the cause mechanism, such as screening and intervention for key nodes at an early age. By detecting changes and clues of disease in its juvenile stage, we can make a more profound effect on the disease course in the late stage.
The existing drugs only relieve symptoms. However, AD is a systemic disease and involves all aspects of life, so we need to look at the problem from a macroscopic perspective before finding an entry point for research. A single drug target is not as effective as multiple drugs in combination.

4.2 Existing animal models and drosophila models

Dr. Yin said that drosophila are usually used as model animals in the early stages of research, and the later the model animals should be more like humans, such as apes, but apes are too expensive, so dogs or mice are probably the next best choices.
Through our review, we learnt that if we want to use transgenic animals to make models, generally mouse models, the most common genes are PS1 and PS2, the APP precursor gene, and the gene that promotes Tau protein production. If we use mice, a pair generally costs one thousand two thousand RMB, and not ready to use at once. The life span of mice in nature is more than 18 months. Even if the transformation gene speeds up the progress of its disease, it will take at least 12 months until we do the experiment. The cost during the process is very high, and the mice may die at any time. Zebrafish and drosophila are very low level creatures, which have the advantage of being cheap and able to replicate and reproduce in large numbers.
The drosophila modaels also have a downside. According to Dr. Yin , there are two ways to verify a model's success. The first is to identify behavioral symptoms consistent with those of people with Alzheimer's, and the second is to identify pathological changes consistent with people: an increase in age spots and tangles. Drosophila are too small and have relatively simple behaviors, which makes testing these two points difficult.If we can solve this problem, then the drosophila model could save a lot of money for Alzheimer's research.
While we transfer the full-length APP gene into fruit flies, Prof. Zhao suggests we control the main short peptide presence after APP transfer and hydrolysis by using western blot to examine types of short peptide fragments (either short peptides inhibit hydrolysis and make fewer deposits or promote hydrolysis and lead to cell death) to improve the accuracy of drug screening. At the same time, he suggests we observe the changes in specificity such as those in nerve cells and filial cells after the entry and expression of the APP gene into drosophila. By paying attention to the differences between the AD model and other animal models, we can determine the characteristics of our model and evaluate the fit between the drug and the model to increase precise drug screening efficiency.
Due to the easy trial breeding conditions, low cost, large quantity, and complete genome and nervous system information, the AD fruit fly drug screening model has practical application prospects for the amyloid-beta hypothesis. Prof. Zhao proposed three areas for us to continue researching: 1. By what means do we detect changes in the nervous system; 2. To what extent behavioral performances represent the regression of human AD; 3. Setting positive and negative controls and removing variables caused by injection with empty vector control.
Mr.Yang from Laos ElementoPharma Co., Ltd indicated that technical difficulties include the lack of understanding of the molecular mechanisms of AD; the absence of emphases on empirical evidence and clinical data rather than molecular traces; the failure to recognize the probability that AD retains heterogeneity, which corresponds to a need of sub-grouping, or differentiating, AD to more precisely identify how differing mechanisms result in various forms of AD; and the incapability of replicating a majority of experiments targeting AD published in renowned journals for efficacy verification as well as drug mass manufacturing purposes.
Similarly, if our drosophila model does not mimic AD’s pathogenesis, the implication of our work may be limited. Nevertheless, even if our model merely falsifies a signaling pathway’s role in AD progression, it succeeds in saving the time of future scientists experimenting with AD as they can intentionally avoid attempting in this direction. The meaning of constructing a model, in its essence, is to stably reflect the disease’s real-life pathogenesis, despite the perplexity of the human condition, in vivo. Refining our model may require intensive literature perusal on how to construct a model that mimics how AD progresses in humans as accurately as possible.

5.Conclusion

1)Alzheimer’s Diseases has been so closed to us, and most patients are diagnosed when the disease has developed to the middle or late stage. This kind of disease also has a negative impact on patients and their families. Caregivers have anxiety because of many direct and indirect reasons, which also brings negative effects and burdens on caregivers in physiology, psychology, economy and life.
2)At present, there is no effective drug for the treatment of Alzheimer's disease. The current AD drugs used in clinic are mainly to delay the development of the disease. Both patients and doctors hope to develop effective drugs.
3)At present, the main difficulty in the development of ad drugs is not clear about its pathogenesis. Three steps are required to understand Alzheimer's disease. The utmost priority is to examine the pathogenesis, which yields a precise, targeted understanding of signaling transduction mechanisms. Proceeding with the identification of pathways is to condense and limit the research scope within a few driver genes. Eventually, genetic engineering shall be implemented to upregulate or downregulate driver gene expression to achieve therapeutic effects.
4)Conventional drug development process includes drug target finding, in vitro high-throughput screening, animal test and clinical test. This process is usually time-consuming, especially in the validation of animal models. If the large-scale screening stage is carried out with high-level animal models, it will be costly and time-consuming.
5)Transgenic model animals are an important tool for drug screening. There are two methods to verify the success of animal models for AD drug screening: the first is to ensure that transgenic model animals show behavioral symptoms consistent with Alzheimer's disease patients, and the second is that transgenic model animals show pathological changes consistent with humans, such as amyloid plaques and neurotangles.
6）Drosophila drug screening model has the advantages of simple culture conditions, low cost, large quantity and complete information of genome and nervous system. The successful drosophila model will has the prospect of practical application.