Research Approaches


 

A combination of models systems ensures the quickness, in vivo testing, and patient-relevance.

The Zhang lab seeks to address two big questions:

  • What is the molecular mechanism underlying neurodegeneration?

  • How different cellular organelles/pathways connect functionally?

To address these questions, we cannot perform experiments directly on human and thus have to rely on model systems. The figure on the right summarizes the strengths of different models used in neurodegeneration studies. The advantage of the Zhang lab is to combine the power of Drosophila (fruit fly), human cell lines, mouse, and patient induced pluripotent stem cells (iPSCs): first using Drosophila and human cell lines to quickly identify potential disease mechanisms, and then verifying these mechanisms in mouse and iPSC-derived neurons, as well as patient postmortem tissues. With this strategy, we ensure the quickness, in vivo testing, and patient-relevance of our studies.

 

Drosophila: Fast and in Vivo

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Left: A normal Drosophila eye; Middle: A c9ALS/FTD Drosophila eye; Right: An eye of a c9ALS/FTD fly expressing a genetic suppressor of neurodegeneration.

As a model system, Drosophila has several major advantages. Firstly, it provides powerful genetic tools. Secondly, the generation time of Drosophila is only around two weeks, allowing complicated genetic manipulation. Furthermore, it is relatively inexpensive and easy to handle, allowing large-scale genetic screens. In addition, Drosophila has a complicated nervous system and 70% of their genes conserved in human, allowing mechanistic studies in Drosophila to be translated to human.

A commonly used phenotypic readout to study neurodegeneration in Drosophila, especially in a large-scale screen, is the external morphology of the eye. The Drosophila compound eye consists of around 800 single eyes, a.k.a. ommatidia, organized in a hexagonal pattern. Each ommatidium contains eight photoreceptor neurons surrounded by glia. Using this system, one can easily screen genetic modifiers or drugs that modulate eye degeneration. In addition to the visual system, the Drosophila motor system is also widely used to study synaptic functions and behavior.

Limitations:

One limitation of Drosophila as a model system to study neurodegenerative diseases is that it is less relevant to human, compared to mammalian model systems. In addition, many cell biology tools (e.g. antibodies) are lacking in Drosophila, making the cell biology studies sometimes difficult.

Zhang K, Coyne AC, Lloyd TE. Drosophila Melanogaster models of amyotrophic lateral sclerosis with defects in RNA metabolism. Review. Brain Research Reviews. 2018 Aug 15;1693(Pt A):109-120.

Further readings:

Ugur B, Chen K, Bellen HJ (2016) Drosophila tools and assays for the study of human diseases. Disease Models & Mechanisms 9:235-244. [Abstract]

Fly Pushing: The Theory and Practice of Drosophila Genetics. By Ralph J. Greenspan, The Neurosciences Institute, San Diego [Book Store]

 

Human Immortalized Cell Lines: Fast and Human-relevant

A second model system used in the Zhang lab is human immortalized cell lines. Given their ease and quickness to handle, they are the best model system to study the molecular and cellular mechanisms of diseases. Furthermore, they are also a great system to study the fundamental cell biology questions that are implicated in diseases, e.g. how cells respond to stress.

Limitations:

A major limitation of cell lines is that they are not in vivo. Furthermore, the immortalized cells are not neurons or glia and may not reflect what happens in these nerve cells.

 

Patient iPSC-derived Neurons: Best Patient-relevance

In the past decades, none of the neurodegenerative drugs identified in rodents have been successfully translated to human. Thus, a more patient-relevant system is required to test drugs for neurodegenerative diseases. The recently developed iPSC technology allows us to generate neurons directly from patient cells, providing a model system with the best patient-relevance. We use the iPSC-derived neurons to verify our findings from Drosophila and cell lines. We perform immunofluorescent staining, Western Blot, and cell death assays on these neurons.

Limitations:

iPSC experiments are expensive and labor-intensive. Also, they are not in vivo.

 

Mouse: in Vivo and Mammalian

Before translating our studies to clinics, it is critical to test our findings (e.g. a drug) in a mammalian model in vivo to fully assess their effects in live mammals. Thus, we use mouse models as the last step to verify our findings from Drosophila, cell lines, and iPSC-derived neurons.

Limitations:

Mouse experiments are labor-intensive and time-consuming. Also, mice are NOT human.

Currently, the Zhang lab only does limited mouse experiments. For most of our mouse work, we collaborate with Dr. Leonard Petrucelli who generated several c9ALS/FTD mouse models.

Further readings:

Chew J, Gendron TF, Prudencio M, Sasaguri H, Zhang YJ, Castanedes-Casey M, Lee CW, Jansen-West K, Kurti A, Murray ME, Bieniek KF, Bauer PO, Whitelaw EC, Rousseau L, Stankowski JN, Stetler C, Daughrity LM, Perkerson EA, Desaro P, Johnston A, Overstreet K, Edbauer D, Rademakers R, Boylan KB, Dickson DW, Fryer JD, Petrucelli L. Neurodegeneration. C9ORF72 repeat expansions in mice cause TDP-43 pathology, neuronal loss, and behavioral deficits. (2015) Science. 2015 Jun 5;348(6239):1151-4. [Abstract]

Chew J, Cook C, Gendron TF, Jansen-West K, Del Rosso G, Daughrity LM, Castanedes-Casey M, Kurti A, Stankowski JN, Disney MD, Rothstein JD, Dickson DW, Fryer JD, Zhang YJ, Petrucelli L. Aberrant deposition of stress granule-resident proteins linked to C9orf72-associated TDP-43 proteinopathy. Molecular Neurodegeneration. 2019 Feb 15;14(1):9. [Abstract]

Zhang YJ, Guo L, Gonzales PK, Gendron TF, Wu Y, Jansen-West K, O'Raw AD, Pickles SR, Prudencio M, Carlomagno Y, Gachechiladze MA, Ludwig C, Tian R, Chew J, DeTure M, Lin WL, Tong J, Daughrity LM, Yue M, Song Y, Andersen JW, Castanedes-Casey M, Kurti A, Datta A, Antognetti G, McCampbell A, Rademakers R, Oskarsson B, Dickson DW, Kampmann M, Ward ME, Fryer JD, Link CD, Shorter J, Petrucelli L. Heterochromatin anomalies and double-stranded RNA accumulation underlie C9orf72 poly(PR) toxicity. Science. 2019 Feb 15;363(6428). [Abstract]