Research Projects

This section introduces current projects of the Developmental and Cellular Biology group.

Is Parkinson’s disease a neuro-developmental disorder?

Parkinson’s disease (PD) is the second most common neurodegenerative disorder, leading to a variety of motor and non-motor symptoms. Interestingly, non-motor symptoms often appear a decade or more before the first signs of motor symptoms. Some of these non-motor symptoms are remarkably similar to those observed in cases of impaired neurogenesis and several PD-related genes have been shown to play a role in embryonic or adult neurogenesis. Indeed, animal models deficient in Nurr1, Pitx3, SNCA and PINK1 display deregulated embryonic neurogenesis and LRRK2 and VPS35 have been implicated in neuronal development-related processes such as Wnt / b-catenin signaling and neurite outgrowth. Finally, the roles of PD related genes, SNCA, LRRK2, VPS35, Parkin, PINK1 and DJ-1 have been studied in NSCs, progenitor cells and induced pluripotent stem cells, demonstrating a role for some of these genes in stem/progenitor cell proliferation and maintenance. Together, these studies strongly suggest a link between deregulated neurogenesis and the onset and progression of PD and present strong evidence that, in addition to a neurodegenerative disorder, PD can also be regarded as a developmental disorder.

Pluripotent human embryonic stem cells have been successfully generated from early stage human embryos and can differentiate into various cell types. However, to develop cellular models of human diseases, it is necessary to generate cell lines with genomes predisposed to diseases (carrying the involved mutations). The reprogramming of somatic cells to a pluripotent state (induced pluripotent stem cells, iPSCs) by overexpression of specific genes has been carried out successfully. Thereby it has also become possible to generate disease- and patient specific iPSCs.  For this method Shinya Yamanaka received the Nobel Prize in Physiology/Medicine in 2012. We use this technology to generate iPSCs from Parkinson’s disease patients for in vitro disease modeling.

We use Parkinson’s disease patients’ specific iPSCs to generate neural stem cells and dopaminergic neurons. Importantly we not only include patients with mutations in classically know Parkinson’s disease associated genes (e.g., LRRK2, VPS35, Pink1, Parkin, ATP13A2 and SNCA) but we also include patients with novel mutations as well as idiopathic patients. As controls iPSCs from healthy individuals are used. In the thereby derived neural stem cells and dopaminergic neurons we define PD-associated cellular phenotypes (e.g., neurite complexity, neuronal differentiation, cell death, stress resistance, mitochondria activity etc.). Additionally, we use these cells to establish co-culture systems mimicking the complex cellular composition in the intact brain. Finally, these cells represent excellent platforms for new genetic modifiers or small molecule drugs addressing PD-associated motor symptoms (dopaminergic neurons) and non-motor symptoms.

 

Molecular functions of Parkinson’s disease associated mutations

Among the PD associated genes LRRK2 is special because mutations have been described not only in familial forms of Parkinson’s disease but also in sporadic cases. LRRK2 is strongly expressed in neural stem cells, but yet the exact molecular function of LRRK2 in these cells remains unknown. By performing a systemic analysis of the gene expression profile of LRRK2 deficient NSCs we found that the expression of several Parkinson’s disease associated genes, like oxidation & reduction in mitochondria, are deregulated upon LRRK2 absence. Our data indeed indicate that LRRK2 regulates the level of cellular oxidative stress and thereby influences survival of neural stem cells. Furthermore, the lack of LRRK2 leads to an upregulation of neuronal differentiation inducing processes, including an upregulation of certain microRNAs like Let-7a and miR-9. On the other hand the constitutive mutant LRRK2(R1441G), known to cause Parkinson’s disease, leads to downregulation of the same activities. In agreement with the function of Let-7a and miR-9 during neuronal differentiation, LRRK2 deficient NSCs differentiate faster than wild type cells, while LRRK2(R1441G) expressing NSCs show impaired neuronal differentiation. In current projects in the lab we aim on using systems biology approaches to understand the interaction of the various Parkinson’s disease associated genes/proteins with each other. Additionally, we are very interested in the molecular and cellular connection of these genes to cell fate specification associated processes and microRNA activity regulation. We hope that a more detailed understanding of these processes will reveal new modifiers of Parkinson’s disease and will lead to the identification of new targets that can be addressed to affect onset and progression of the disease.

Similar to LRRK2 we are also investigating the molecular function of other PD associated genes (Pink1, SNCA, ATP13A2 etc.), for several of them the regulation of Mitochondria, Lysosomes and vesicle transport is of particular interest for us.

 

Somatic mutations and protein aggregate spreading  

Mutations that occur post-zygotically are called somatic mutations. This kind of mutations leads to genetic mosaicism. Since these mutations are likely to mainly occur during embryogenesis, they would be missed in ectoderm-derived neural cells, when genotyping is conducted in mesoderm-derived lymphocytes. Actually, it is estimated that each gene is likely to mutate several times post-zygotically. Interestingly, somatic mutations already previously have been associated to neurodegenerative disorders, it was even suggested that differences in Parkinson's disease phenotype in monozygotic twins with LRRK2 mutations, are caused by additional somatic mutations. The process of somatic mutation might lead to the acquisition of certain disease causing mutations only in a subset of neural cells. However, if these mutations lead to protein aggregate formation and spreading to neighbouring cells, this might be sufficient to cause PD. In this project we use induced pluripotent stem cell based in vitro disease models to address whether cellular mosaicism can be the basis for protein aggregate spreading and Parkinson’s disease phenotypes. In this project we currently focus on the genes LRRK2, VPS35 and SNCA.

Batten’s disease / Neuronal Ceroid-Lipofuscinosis

The disease Neuronal Ceroid-Lipofuscinosis (NCL) or Batten’s disease is the most prevalent pediatric hereditary neurovisceral storage disorder. In this project we aim on in vitro modelling neuronal ceroid lipofuscinoses (JNCL) in human cells. We focus on the two NCL associated genes CLN3 and CLN12. CLN12/ATP13A2 is of particular interest since mutations in this gene have been associated to Batten’s disease as well as Parkinson’s disease. Besides patient specific cells we use the CRISPR/Cas9 technology to generate human iPSCs with the disease associated mutations. Based on these iPSCs human cerebral organoids are generated for in vitro disease modeling. In the next steps these models shall be used for the identification of small molecule compounds that might become lead substances for drug treatment strategies.

 

Brain-on-a-Chip technology and Brain Organoids for advanced in vitro disease modeling

One of the main limitations in neuroscience and in the modelling of neurodegenerative diseases is the lack of advanced experimental in vitromodels that truly recapitulate the complexity of the human brain.

Therefore, we are working on the generation of brain-like organoids. Since we are particularly interested in Parkinson’s disease, we put a specific emphasis on the development of organoids resembling the human midbrain. Besides free-floating organoid cultures we make use of microfluidics technology to develop Brain-on-a-Chip solutions that allow for a high degree of automation and increased throughput.

We use these advanced in vitrodisease models to investigate the developmental contribution to Parkinson’s disease and to test novel experimental approaches to treat PD.

 

Generation of 3D human brain models. A) Stem cells in Matrigel droplets are used as the starting cell population for the generation of free floating brain organoids. B) Immunofluorescence image of a several week old brain organoid where different neural cell populations are labelled in different colours. C) Prototype of our novel microfluidics device for the parallel cultivation of 16 brain organoids. D) 3D neuronal network grown in a microfluidics device, the colours indicate different neuronal cell populations.

Watch video on Brain-on-a-Chip technology and Brain Organoids for advanced in vitrodisease modeling:

https://youtu.be/HYFnEdlPbCg

 

Automated high-throughput high-content imaging and analysis platform

The advent of genome editing tools also let us develop genetically encoded reporters in human induced pluripotent stem (iPS) cells in a straight forward manner. Combining CRISPR/Ca9 technique with pH responsive fluorescent proteins allowed us to evaluate intracellular pH and interrogate specific subcellular compartments. We stablished an automated high-throughput high-content imaging and analysis platform with our in house developed algorithm for classification of specimens in an unbiased manner. Using the cell culture automated platform at the LCSB we are currently performing a screen of natural compounds coming from Australia in collaboration with NatureBank and University of Griffith.