The program project for Neurodegenerative Disease group focuses on two leading ageing diseases, i.e. Parkinson’s disease (PD) and Alzheimer’s disease. Group projects will adopt various testing platforms that have already been established in Chang-Gung Molecular Medicine Research Center or proposed in Gerontology Research Center and will take advantage of precious clinical resources obtained from Chang-Gung Memorial Hospitals for integrative basic-clinic research. Approached by genomics, proteomics, metabolomics, molecular imaging and bioengineering, we expect to identify useful pre-clinical marker(s) or risk factor(s) in PD/AD and transform successively into translational research. The studies will cover series of experiments at molecular, cellular and systemic levels, assisted with cutting-edge biomedical techniques and advanced analytical instruments. The disease models could also bridge with Chinese Herbal ingredient group for interdisciplinary attempts to advance our current knowledge in PD and AD. The PD projects will focus on genetic mutation decoding, characterization of PD models of PINK1 and LRRK2, cell-based therapy on MPTP-induced PD model and metabolomic analyses of PD animals. The AD projects will focus on molecular mechanism ofb-amyloid (Ab) aggregation in GM1 ganglioside matrix, Ab-induced inflammation and signaling, G-CSF therapy and AV-45 imaging on Ab Tg2576 mice. The outlines of individual project are listed in below:

Project 1. Genetic diagnosis and translational research in Parkinson disease (PI: Dr. Chin-Song Lu; co-PIs: Drs. Yah-Huei Chou Wu and Tzu-Chen Yen).

The development and practical application of genetic diagnostic would be essential in post-genomic era, in particular several mutations in domestic PD patients are quite different from western society. Successively decoding the genetic mutations related to PD would facilitate the understanding of gene regulation and protein-protein interaction in disease progress. The main purpose of this project is to establish gene bank from domestic PD patients and correlate with their behavioral and structural phenotypes, assisted by molecular imaging for diagnostic standardization. To this end, we will (1) systemic screen genetic mutation in sporadic PD patients and (2) explore the unknown genetic causes in familial Parkinsonism (FPM) with the analyses of PCR, SSCP, DHPLC, qPCR, MALDI-TOF and direct sequencing. (3) Those genetic findings will correlate with clinical symptoms and brain imaging (99mTc-TRODAT, 123I-IBZM, AV-133, fMRI) database for translational research.

Project 2. An investigation on the pathogenic mechanisms of autosomal recessive PARK6 and dominant PARK8 using mouse animal models (PI: Dr. Hong-Li Wang; co-PIs: Drs. Tu-Hsueh Yeh and Yi-Hsin Weng).

The recent identification of genes linked to familiar form of Parkinson’s disease led to a better understanding of novel proteins and molecular pathways that may cause the neurodegeneration of substantia nigra (SN) dopaminergic cells and resulting PD syndrome. Clinical features of PD patients with LRRK2 or PINK1 mutation are similar to those of sporadic PD patients, suggesting that common cellular and molecular mechanisms are involved in the pathogenesis of both hereditary and sporadic PD. Therefore, elucidation of the pathogenic mechanism underlying PARK6 or PARK8 is expected to shed a light on the molecular pathogenesis of more common sporadic PD. To this end, we will (1) investigate the molecular pathogenesis of PARK6 and physiological functions of PINK1 in vivo using PINK1 knockout mice; (2) investigate cellular and molecular mechanisms of mutant LRRK2-induced neurotoxicity and degeneration of dopaminergic nigrostriatal system in mutant LRRK2 Tg mice; (3) examine, via proteomic approaches, the possible substrates of PINK1 and LRRK2 in mutant striatal tissues.

Project 3. Explore the impact of subventricular zone progenitor/stem cell proliferation and piggyBac-iPS on MPTP-induced PD animal model (PI: Dr. Jin-Chung Chen; co-PIs: Drs. Chiung-Yuan Wu and Mei-Ling Cheng).

In adult brain, subventricular zone (SVZ) contains a mass of stem/progenitor cells (NPCs), under normal physiological condition, they proliferate and migrate to olfactory bulb through rostral migratory stream. Those NPCs receive dopamine innervation from the substantia nigra and heavily expressed dopamine D3 receptors. Recent findings indicated D3 agonists could induce numbers of BrdU+ cells and offer neural protection in the striatum of MPTP-treated animals, suggests a possible link of D3 DA receptors with SVZ neurogenesis. The purpose of this project is to test if cell-based therapy would rescue DA degeneration in MPTP-induced PD animal model. To this end, we will (1) test if D3 agonist treatment would promote SVZ neurogenesis and its therapeutic potential and cellular mechanism; (2) taking advantage of advanced piggyBac-iPS technique, we will use it as alternative source and also induce specific PD patient iPS for toxicity study. (3) Considering the advanced NMR and LC/MS techniques in modern metabolomic analyses, we will monitor the oxidative and metabolic index in striatum of MPTP-induced PD animals and/or biological samples (CSF, blood or urine) from PD patients.

Project 4. Model studies of molecular mechanisms during amyloid aggregation toward molecular therapeutics for Alzheimer’s disease (PI: Dr. Chien Chou; co-PIs: Drs. Yu-Sun Chang, Jau-Song Yu, Yin Yeh [UC Davis] and Atul Parikh [UC Davis]).

The pathological hallmark in AD is the accumulation of extracellular deposits of b-amyloid (Ab) in senile plaques. Soluble Ab is non-toxic and predominantly in a-helical structure, whiles aggregated Ab fibrils enrich in b-sheet conformation, is generally believed to represent one of the earliest steps during AD progress. However, molecular mechanism by which this conversion is effected remains poorly understood. Recent finding suggests a novel form of Ab, tightly bound to GM1 ganglioside, serves as a precursor to seed the Ab fibril formation. We hypothesize that GM1 recognizing soluble Ab is linked to cholesterol enriched, raft-like micro-environment and successful therapeutics require the exogenous presentation of high affinity GM1 in raft-mimetic environment. To this end, we will (1) compare the Ab binding affinity to GM1 in cholesterol and sphingomyelin enriched and depleted bilayer configurations; (2) determine if Ab binding targets GM1 into raft-like micro-environment or conversely alters the composition of “naïve” rafts and (3) explore if exogenous presentation of liposomes (contain high-affinity GM1) can divert Ab away from the primary supported bilayers and thus provide a basis for novel therapeutic development..

Project 5. Cellular mechanism of pro-inflammatory cytokines and ß-amyloid interplay in Alzheimer’s disease (PI: Dr. Chun-Mao Yang; co-PIs: Drs. Mei-Jie Chou and Wen-Chuin Hsu).

Oxidative stress (ROS) can be sensed by the cells and trigger intracellular signaling cascades potentiating chronic inflammation. Interplay between ROS and inflammatory proteins induced by Aß peptide and cytokines leading to neurodegeneration remains largely unknown. Based on our previous studies, these inflammatory proteins may change the BBB function in various brain injuries and neuroinflammation. Therefore, we hypothesize that the Aß peptide and cytokines- initiated neurodegeneration is mediated by the generation of ROS and the induction of inflammatory proteins (MMPs, COX-2 or cPLA2) in brain cells. To test this hypothesis, this proposal will investigate the molecular mechanisms underlying Aß peptide and cytokines regulated expression of MMPs, cPLA2, or COX-2 in astrocytes, including (1) to evaluate whether the expression of inflammatory proteins induced by Aß peptide and cytokines, (2) to characterize the role of ROS in expression of inflammatory proteins induced by Aß peptide and cytokines, (3) to identify the cellular and nuclear signaling pathways by Aß peptide and cytokines and (4) to evaluate the roles of these up-regulated inflammatory proteins on astrocytic or neuronal physiological and pathological functions in vitro and in vivo studies.

Project 6. Evaluation of F18-AV-45 in neural plaques detection in Aß transgenic mice: PET study and G-CSF therapy (PI: Dr. Ho-Ling Liu; co-PIs: Drs. Shiaw-Pyng Wey, Mei-Ping Kung [U. Penn.] and Che-Kun James Shen [Academia Sinica]).

Detection the A senile plaques with F-18-AV-45 radiotracer has been recently setup as a gold standard in AD diagnostics world-wide. Application of this tracer in AD animal research would provide a sensitive indicator to semi-quantify the plaque formation hence could facilitate the drug screening process for selecting the potential therapeutic agents. In addition, imaging of A plaques in AD animal brain would form a valid and sensitive end-up assay in helping the molecular mechanism approaches. The main purpose of this project is to use molecular imaging techniques for AD transgenic study, in order to establish a testable platform for mechanistic and pharmacological examination. To this end, we will (1) apply the animal micro-PET and MRI (7T MRI in Chang-Gung Memorial Hospital Molecular Imaging Center) in Tg2576 or other AD animal models; (2) apply immunohistochemisty and fluorescence technique for Aß plaques detection for correlation with in vivobrain imaging data; (3) use F-18-AV-45 radiotracer for disease progress study in Tg2576 AD mice and (4) evaluate the effect of G-CSF therapy in Tg2576 AD mice, assisted with micro-PET imaging.

Project 7. Application of non-invasive focused ultra-sound for drug and stem delivery in treatment of PD and AD (PI: Hao-Li Liu; co-PIs: Drs. Jiann-Der Lee and Kun-Ju Lin).

Blood-brain barrier (BBB) plays a protective role in the central nervous system to prevent the movement of many substances into the brain, thus limit over 95 % of therapeutic agents entering into brain for potential neurodegenerative disorder treatment. It has been recently discovered that focused ultrasound can locally and reversibly disrupt BBB, which opens new frontier toward delivering large therapeutic agents, such as growth factors NGF, BDNF and/or GDNF for PD and AD treatment. To monitor the BBB opening process from MRI, we have been implemented contrast-enhanced T1-weighted MR imaging for preclinical animal test and show fruitful research achievement and proof the potential and value of focused ultrasound brain drug delivery approach. Hence, the aim of this project is to (1) develop a novel focused-ultrasound treatment platform for AD or PD treatment; (2) deliver therapeutic stem cells or growth factors that cannot normally penetrate through BBB to control neuronal degeneration in PD animal models and (3) via the similar application, deliver Aß antibody or G-CSF to control neuronal degeneration in AD animal models.

Heart failure (HF) is a major and increasingly common cardiovascular syndrome, and is the end result of many cardiovascular disorders. Moreover, the syndrome is one of the most common reasons for new or recurrent hospitalizations among persons over 65 years of age. To fight with this problem, one of the best ways is to identify the high risk group subject to HF by new risk stratification biomarkers far before the happening of diseases. By elucidating the relationship between biomarkers and pathogenesis in HF, the novel strategies that involve suitable medicine and rehabilitation will be developed to prevent early hemodynamic dysfunction and thrombotic events, further retarding the progression of HF.

HF is associated with neurohumoral changes as the body attempts to reverse the effect of reduced cardiac output and organ perfusion. The sympathetic and renin–angiotensin–aldosterone systems are activated in an attempt to increase myocardial contractility, heart rate and vasoconstriction, and expand extracellular fluid volume. Persistent neurohumoral excitation, however, actually results in deterioration of myocardial function with inflammatory response, end-organ damage, and skeletal muscle derangement, which lead to worsened exercise capacity. According to previous investigations, the biomarkers of HF may include various influence factors that involved inflammation, oxidative stress, extracellular matrix remodeling, neurohormones, and mycardiocyte stress/injury. To our knowledge, an integrated strategy for the analytic multimarkers in HF progression has not yet been established. This proposal will integrate the research sources in CGU oxidative stress/damage and metabolomic core laboratories to establish an excellent research platform for analyzing HF multimarkers including oxidative stress profiles, metabolomics, and mycardiocyte stress/injury.

Cardiac dysfunction is generally considered to be a critical influencing factor for the abnormal exercise response observed in HF population. However, clinical investigations have indicated weak correlation between resting ventricular function and exercise tolerance as well as failure of inotropic and vasodilatory agents to improve exercise capacity, implying that cardiac dysfunction is not the only factor contributing to progressive exercise intolerance in patients with HF, impaired pulmonary and skeletal muscle function are also thought to have a role. Recently, abnormal cerebral metabolism has been diagnosed using magnetic resonance spectroscopy in patients with advanced HF, which abnormality is chiefly attributable to cerebral hypo-perfusion. This proposal will be the first to integrate the novel bioreactance-based measurement with near-infrared spectroscopy and automatic gas analysis to identify the roles of ventilatory and cardiac-cerebral-muscle hemodynamic responses to exercise on functional impairments in patients with HF. Additionally, hemorheology, atherothrombosis, and angiogenesis modulated by erythrocyte, monocyte, and endothelial progenitor cells in patients with HF will be also determined in this investigation.

The importance of oxidative stress is increasingly emerging, with respect to a pathophysiological mechanism of the myocardium remodeling responsible for HF progression. However, the molecular mechanisms in HF progression are complex and not fully defined. Therefore, this proposal will focus on the dissection of molecular mechanisms concerning involvement of ROS in HF. Increased understanding of signal transduction mechanisms underlying oxidative stress/inflammation gene regulations will create opportunities for the development of therapeutic strategies beneficial for HF.

In the etiology of HF, coronary artery disease is frequently encountered, associated with severe or end-stage of coronary artery obstruction. In the population of aging, available conduit vessels are often not enough for a complete re-vascularization when bypass surgery is performed. Thus, it is important in the future to bio-engineer an artificial vessel, especially from an autologous source and non-invasively. Vascular progenitor cells contain a few different cell types, at least smooth muscle progenitor cells (SMPCs) and endothelial progenitor cells (EPCs). These progenitor cells have unlimited potentials in both risk stratification and tissue engineering. In this project, we will use updated technologies to find out potential gene targets for separating SMPCs from EPCs. To use these targets to identify SMPCs, development of antibodies appropriate for flow cytometer use is mandatory to positively select SMPCs from other contaminated or mixed cell populations. Finally, combined with the magnet system, we will test whether our work can separate pure SMPCs from other cell populations and specifically head for vascular conduit engineering.

Decreased exercise capacity negatively affects an individual’s ability to adequately perform activities required for normal daily life and, therefore, their independence and quality of life. Physical training can have beneficial effects on neurohumoral, inflammatory, metabolic and central hemodynamic responses, as well as on endothelial, skeletal muscle and cardiovascular function, leading to improvement in functional capacity and quality of life. However, which exercise intensity yields maximal beneficial adaptations is controversial. Aerobic interval training has been shown to rescue impaired cardiomyocyte contractility, attenuate myocardial hypertrophy, and reduce myocardial expression of atrial natriuretic peptide in animal model of post-infarction heart failure. However, underlying mechanisms of the exercise-improved regulations of cardiac hemodynamics and risk factors in patients with HF remain unclear. This proposal will further clarify how various regimens of cardiac rehabilitation affect cardiovascular hemorheological characterics and atherothrombosis/ angiogenesis-related variables in patients with HF.

In light of the above discussion, this proposal will integrate six research programs that include from bio/functional markers to clinical therapeutics in HF. By elucidating the relationship between biomarkers/physiological markers and HF pathogenesis, this proposal attempts to provide some novel strategies for developing suitable medicine/rehabilitation regimens that prevent early hemodyamic dysfunction and thrombotic events, further retarding the progression of HF. The six research programs in this proposal list as follows:

1. To establish the research platform of multi-biomarkers for analyzing oxidative stress profiles, metabolomics, and mycardiocyte stress/injury in HF progression.
2. To establish the research platform of systemic physiological markers for evaluating central/peripheral hemorheological characterics and atherothrombosis/ angiogenesis-related variables in HF progression.
3. To dissect molecular mechanisms concerning involvement of oxidative stress/inflammation inHF progression for creating opportunities for the development of therapeutic strategies beneficial for HF.
4. To develop the animal models in HF for employing novel developments of tissue engineering and cardiac rehabilitation.
5. To develop a novel technology of vascular conduit engineering for constructing an artificial vessel from an autologous source and non-invasively.

To design the “safe and effective” cardiac rehabilitation for promoting aerobic fitness and minimizing the hemodynamic limitations and vascular thrombotic events.

We will continue to validate portfolios of disease biomarkers, pathogenesis, and diagnostic criteria for metabolic syndrome and type 2 diabetes mellitus. work with animal models to illustrate the correlation and interaction of the multiple risk factors which affect metabolism and cardiovascular function during aging process. translate the research results to clinical applications.

Cognitive and behavioral indices are important indicators of mental and physical health during the aging process. Cognitive decline and related behavioral changes constitute a severe health problem for the elderly. Not only does it seriously impact their quality of, life, but for many of them it leads to dementia. Studies have shown that the differentiation of changes in cognitive function and behavior caused by diseases resulting from normal aging can help the health care professional to make a more accurate assessment and diagnosis. This then allows the health care professional to arrange for appropriate interventions to delay the onset or progress of dementia and decrease the financial burden on society. The main focus of this group project is the “cognitive and behavioral indices of aging”. Cognitive decline associated with a progressive neurological condition (e.g., Alzheimer’s disease), depression, a surgical procedure (e.g. hip fracture) or cognitive improvement as a result of an intervention (music, Tai-Chi, case management, or cognitive training) are common interests for health care providers.

1. develop cognitive and behavioral indices for the assessment, detection and diagnosis of cognitive decline in elderly patients;
2. explore the influence of aging and disease/disability on the cognitive and behavioral indices; and
3. determine the effects of dementia management and cognitive training on changes in cognitive and behavioral indices (Figure 1).

       

 

Prior accomplishment and resources

 

The PIs of our sub-projects are all well-established researchers. Dr. Yea-Ing Lotus Shyu has been collaborating with Dr. Liang from University of Michigan to conduct clinical trials on improving recovery for older patients following hip fracture that funded by National Health Research Institute since 1996 until now. She has also been collaborating with Prof. Stewart from Oregon Health Sciences University and Prof. Archbold from John A. Hartford foundation on studies related to family caregiving and management of behavioral problems for older persons with dementia. Dr. Ho-Ling Liu has been conducting studies on functional MRI and collaborating with Chicago University and NIH. Dr. Yu-Cheng Pei has collaboration with Prof. Rothwell at British Neurology Institute and Prof Ugawa at Tokyo University. Dr. Tzu-Ting Huang’s research focus in on promotion health and functional independence for older persons and has worked with Dr. Acton at the Institute of Gerontology of The University of Texas at Austin. Dr.Yi-Chen Chiu established a cognition recovery lab to study behavior problems of older persons with dementia and has collaborations with Dr. Algase and Dr. Liang from University of Michigan. Research team of Dr. Chyan-Goei Chung and Dr. Chung-Chih Lin has had rich experiences in developing health information system for older persons. They have been conducted projects on health care services for older citizen supported by Ministry of Economic Affairs and established remote monitoring and communication technology to facilitate health care services in Chang Gung Health and Culture Village. Dr. Chin-Chang Huang and Dr. Wen-Chuin Hsu establish and in charge of the Chang Gung Dementia Care Center and have been conducting studies on patients with dementia. Currently, they are conducting imaging study and competing clinical guidance related to patients with AD, DLB, and FTD. Their international research projects include studies on g-secretase inhibitor. They established the first Taiwan ADNI and recently published 2 articles on ApoE4, ApoCI, and Lrrk2. These prior accomplishments have built a good foundation for our future studies.

In addition, the Chang Gung Memorial hospital (Linkou and Taoyuan Branch), the Chang Gung Health and Culture Village and its dementia center can serve as the study sites and provide resources for this group project. In addition, the PIs of our projects have a long-term collaborative relationship with an internationally-known gerontological researcher, Dr. Jersey Liang, from the Institute of Gerontology at the University of Michigan.

 

Expected outcomes and international collaboration

 

As a result, in addition to achieve the research purposes of this group research project, collaborations with Dr. Liang for comparing population data on the cognitive and behavioral functions of the elderly in the United States and Japan can be carried out. Collaborations with the dementia research group at University of Edinburgh will be explored and conducted.

Students that participate in any of these group projects will learn how to assess and diagnose cognitive function, and how to intervene with elders that suffer from cognitive decline or from dementia. Graduate students will learn how to analyze longitudinal data, use an intelligent information system, and how to conduct a cross-national comparative analysis. Contents for courses to advance the role of health care workers by teaching them how to assess and take care of elderly patients with cognitive impairment can also be developed.