FY2020 Annual Report

Brain Mechanisms for Behaviour Unit
Professor Gordon Arbuthnott

Unit members in zoom format as it became the norm during the remarkable year of 2020. (From top to bottom) Gordon Arbuthnot, Hiroko Chinone; Marianela Garcia Munoz; Teresa Hernandez Flores; Esther Lai; Junghyun Jo; Hoang Dai Tran; Jin Seok Bang; Yoko Nakano; Ai Takahashi; Thato Mokhothu; Anai Escheverria Oviedo and Charlotte Denman.

Abstract

This has been a year of big changes in which the main work of the unit is now on human organoids.  Towards year end, only Teresa and Esther were working on slices of mouse brain while the major work was on human brain organoids, 3D cultures developed from human pluripotent stem cells.  These organoids allow us access to human neurons cultured to mimic parts of brain. We have both midbrain-like organoids and striatum-like ones, with which to explore the effects of gene manipulations that mimic Parkinson’s disease.

1. Staff

  • Dr. Marianela Garcia Munoz, Staff Scientist
  • Dr. Esther Lai, Staff Scientist
  • Dr. Junghyun Jo, Staff Scientist
  • Dr. Teresa Hernandez Flores, Postdoctoral Scholar
  • Dr. Hoang Dai Tran, Postdoctoral Scholar
  • Dr. Jin Seok Bang, Postdoctoral Scholar
  • Yoko Nakano, Technician
  • Ai Takahashi, Technician
  • Charlotte Denman, Graduate Student
  • Thato Mokohothu, Graduate Student (Rotation)
  • Isabel Anai Escheverria Oviedo, Graduate Student (Rotation)
  • Hiroko Chinone, Research Unit Administrator

2. Collaborations

2.1 Development of the alpha-synuclein aggregates in Parkinsonian brain organoids

  • Description: Electron microscopic studies on human brain organiods
  • Type of collaboration: Joint research
  • Researchers:
    • Professor Mark Ellisman, National Centerfor Microscopy and Imaging Research Group, University California San Diego
    • Dr. Daniela Boassa, National Centerfor Microscopy and Imaging Research Group, University California San Diego

2.2  Organoid models of human diseases

  • Description: Development of 2D and 3D human pluripotent stem cell (hiPSC) differentiation platform for human disease modeling.
  • Type of collaboration: Joint funded research
  • Researchers:
    • Professor Dong Ryul Lee, CHA University, South Korea

3. Activities and Findings

3.1 Rotation students, changes in staff and a year of pandemic isolation

I usually start this section by looking back to the rotation students that passed through the laboratory that year. With no exceptions the graduate students share a delightful taste of wonder before moving along to other Units. This year it was Thato’s, Anai’s and Charlotte’s turn, thank you for your enthusiasm.

In comparison to previous years, a recurring departure of unit’s members left a foreseeable sense of renewal. Yoko moved to join the Terenzio Unit and was substituted by Ai, who a few months later, moved to explore different adventures in OIST Imaging Section.

At the end of last year, it became evident that to protect the health of my lungs, I had to work from home. As a result, Teresa departed without the anticipated celebration we had for all those who finished their postdoctoral experience with us; instead of the planned fanfare, her departure turned out to be a sombre distant goodbye via email. Continuing with the Unit’s decamp, Marianela now only works part time and Esther has only a few months left with us before she moves to deliver her expert support in another Unit within OIST.

Remaining in the field of exploring the many experimental questions related to neurodegeneration, is Charlotte who will develop her PhD project in the Optical Neuroimaging Unit under Professor Bernd Kuhn’s direction. She will use midbrain organoids developed from hiPSC to determine the influence of microglia in neurodegeneration. The Unit’s expert team of Jo, Dai, and Jin Seok will guide Charlotte’s desire to use organoids as her experimental model. 

3.2  Human neuron organoids

Dr. Jo, who is passionate about advancing stem cell technology using hiPSCs for Parkinson’s disease, was integrated with the unit as a part of a general interest of the University in the development of research using this new technical advance.  Dr. Jo obtained his PhD from CHA University in South Korea, known for its world-class reputation in the development of technology related to stem cells, the human genome and reproductive medicine. His postdoctoral training was in the Genome Institute of Singapore (GIS) under Prof. Huck-Hui Ng. Within that group, Junghyun Jo developed a protocol to generate human midbrain-like organoids (hMLOs) from hPSCs to recapitulate features of human midbrain by the 3D culture system (Junghyun Jo, et al. 2016. Midbrain-like Organoids from Human Pluripotent Stem Cells Contain Functional Dopaminergic and Neuromelanin-Producing Neurons. Cell Stem Cell 19(2): 248-257.) Before joining OIST, during his postdoctoral experience, Junghyun Jo had already generated organoids -deficient in the gene encoding the lysosomal enzyme, glucocerebrosidase (GBA1) displayed some similarities with Parkinsonian brains.

As Junghyun Jo joined the Unit, we applied for a big USA grant from Aligning Science Across Parkinson’s, a consortium using the Michel J. Fox organization to arrange funding for Parkinson’s disease. Although we did not get the money, the grant was shortlisted, an honor in such a competitive venue. As a result, we initiated a collaboration with Mark Ellisman and the National Center for Microscopy and Imaging Research group in the University of California San Diego that now, when the end to the pandemic is in sight, we will rekindle.  It is imperative, in the near future, to proceed by looking for the site of development of the alpha-synuclein aggregates that are part of the Lewy Bodies, a pathological marker in Parkinson’s disease brains.

Junghyun Jo redoubled his efforts this year when I left him in charge of developing the project while I was protecting my health. He has already applied for several other sources of money and received a joint grant with CHA University that provided the Unit with another postdoctoral worker Dr. Jin Seok Bang.  He could not arrive before November due to the imposed travel restrictions despite the paperwork being ready since in February. For now Jin Seok replaced Teresa in the unit from January. Importantly, a publication including Dr Jo’s work in Singapore was published this year (Kwak TH, et al, 2020. Generation of homogeneous midbrain organoids with in vivo-like cellular composition facilitates neurotoxin-based Parkinson's disease modeling." Stem Cells 38 (6): 727-740).  He also has a joint Kakenhi grant about to start, and a POC opportunity that might start in December next year.

Of the three rotation students this year, Thato worked with Teresa on dopamine release (Figure 2), Anai worked on the organoids (Figure 3), and Charlotte is still with us beginning her Ph.D.

 Figure 1. Electron microscopy section obtained from an OIST-generated human organoid, sent to the UCSD National Center for Microscopy and Imaging Research group for processing. It shows a synaptic bouton in the center of a field with space surrounding it.  Extracellular space is rare in brain but present in organoids. This collaboration waiting to be rekindled as soon as possible.                                                                           

Figure 2. Release of dopamine in slices of mouse brain in vitro.
Left Red-shifted dopamine sensor (RdLight1) expressed in 15µm diameter mice striatal neurons.
Right The change of fluorescence over the field of the injection site was plotted as the change of fluorescence/baseline with a time resolution of 10 frames per sec.

Figure 3: Top row, control organoids from hiPSC; lower row organoids devoid of the GAB1 gene that makes a glucocerebrosidase that consequently express markers for Parkinson’s disease. Columns: left, lower power fluorescent images (scale bar is 100µm). Staining: DAPI (4′,6-diamidino-2 -phenylindole dihydrochloride) a blue fluorescent dye that binds to DNA; alpha synuclein accumulations are shown in red. As expected, alpha synuclein was more common in the genetically modified organoid; S100 B is a calcium binding protein used as indicator of astrocytes in the central nervous system. Middle column higher power micrographs (scale bar 20µm) show leucine repeat kinase (LRRK2), a protein overexpressed in Parkinson’s disease and the astrocyte marker S100B. The color results from the coincident fluorescence of red (LRRK2) and cyan (S100B) dyes. Right column shows cyan fluorescence from MAP2, a microtubular associated protein that marks neurons and the LRRK2 fluorescence (red). These fluorescence results indicate that LRRK2 is present in astrocytes in organoids but not in neurons.

Meanwhile, Esther took on responsibility of examining the electrophysiology in the organoid cells, a job by the way, that I was hoping to do myself before isolation.  She already obtained some records, but some of the neurons are exhibiting immature phenotypes. In conclusion, there is more work to do to make a convincing story out of that part of our research.  Meanwhile, the work she did in the Tokyo Medical and Dental University before joining OIST, is published in iScience.  Publication of her work from her time in the unit is in revision.

 

4. Publications

4.1 Journals

  1. Violeta G. Lopez-Huerta, Jai A. Denton, Yoko Nakano, Omar Jaidar, Marianela Garcia-Munoz, Gordon W. Arbuthnott, Striatal bilateral control of skilled forelimb movement, Cell Reports 34 1-13, doi.org/10.1016/j.celrep.2020.108651 (2021)
     
  2. Bianca Sieveritz, Gordon W. Arbuthnott, Prelimbic cortical targets of ventromedial thalamic projections include inhibitory interneurons and corticostriatal pyramidal neurons in the rat, Brain Structure and Function 225 2057-2076, doi.org/10.1007/s00429-020-02109-3 (2020)

  3. Tae Hwan Kwak, Ji Hyun Kang, Sai Hali. Jonghun Kim, Kee-Pyo Kim, Chanhyeok Park3 | Ju-Hyun Lee, Ha Kyun Ryu, Ji Eun Na, Junghyun Jo, Hyunsoo Shawn Je, Huck-Hui Ng, Jeongwoo Kwon, Nam-Hyung Kim, Kwon Ho Hong, Woong Sun, Chi Hye Chung, Im Joo Rhyu, Dong Wook Han, Generation of homogeneous midbrain organoids with in vivo-like cellular composition facilitates neurotoxin-based Parkinson's disease modelling, Stem Cells 38, 727-740. DOI:10.1002/stem.3163 (2020)

  4. Kunihiko Yamashiro, Kei Hori, Esther S.K. Lai, Naofumi Uesaka, Masanobu Kano, Mikio Hoshino, AUTS2 Governs Cerebellar Development, Purkinje Cell Maturation, Motor Function and Social Communication, iScience 23 (12) 1-18 doi/10.1016/j.isci.2020.101820 (2020)

  5. Gordon Arbuthnott, An Introspective Approach: A Lifetime of Parkinson’s Disease Research and Not Much to Show for It Yet?, Cells 10, 513, 1-10  doi.org/10.3390/cells10030513 (2021)

4.2 Books and other one-time publications

Nothing to report

4.3 Oral and Poster Presentations

  1. Violeta Giselle Lopez Huerta, Marianel Garcia Munoz & Gordon William Arbuthnott, It takes two to tango and two striata to reach, FENS virtual Forum, July 11-15 (2020)
     
  2. Hernandez-Flores T., Nakano Y., Garcia-Munoz M., Arbuthnott G.W., Direct measurement of striatal cholinergic transmission with a specific genetically encoded fluorescent acetylcholine indicator in mice, FENS virtual Forum, July 11-15 (2020)
     

5. Intellectual Property Rights and Other Specific Achievements

Nothing to report

6. Meetings and Events

Nothing to report

7. Other

Nothing to report.