Upcoming and recent PhD dissertations from Molecular Neurology Research Program.
Time: 21 September, 2018 13:00
Place: Biomedicum Helsinki 1, Lecture Hall 1, Haartmaninkatu 8, 00290 Helsinki
Custos: Professor Timo Otonkoski
Pancreatic beta-cell dysfunction is the ultimate cause behind all forms of diabetes. Decades of research with different animal and cellular models have expanded the knowledge on the heterogeneous molecular mechanisms causing the disease. However, they present important limitations that may significantly affect the way these findings can be translated into new approaches to combat diabetes in humans. Rodent pancreatic islet development and physiology display species-specific particularities when compared to human. Similarly, rodent and human insulinoma cell lines are a convenient research tool but do not recapitulate faithfully the functionality of adult human beta-cells. To validate if the findings obtained with these models extrapolate to humans, diabetes researchers have traditionally used cadaveric donor human islets. Primary islets are scarce, highly variable in their composition and functionality and difficult to manipulate for certain experiments.
As an alternative, human pluripotent stem cells (hPSC) constitute a renewable source of beta-cells. Stem cell-derived beta-cells can be generated by directed differentiation and used as a model to study pancreatic beta-cell development and disease in vitro. They can also be transplanted into immunocompromised mice, generating humanized models where in vivo beta-cell function can be closely evaluated in a systemic context.
The goal of this thesis work was to demonstrate the use of human pluripotent stem cells as a tool to investigate monogenic diabetes disease mechanisms. For this purpose, improved hPSC differentiation protocols to the beta-cell lineage were generated utilizing 3D suspension culture approaches. Transplantation procedures were devised to create humanized mouse models that allow proper evaluation of beta-cell function in vivo. Novel CRISPR-Cas9-based techniques were established and utilized to edit the genome of hPSC and control gene transcription. Precise genome editing made possible the generation of isogenic, mutation-corrected patient-derived induced PSC, enabling the disease modeling of monogenic diabetes cases.
Using these approaches, an activating mutation in STAT3 gene was found to cause neonatal diabetes by inducing pancreas endocrinogenesis prematurely, via direct induction of master endocrine transcription factor NEUROG3. In a similar way, INS gene mutations causing proinsulin misfolding were found to impair developing beta-cell proliferation due to increased endoplasmic reticulum stress. Taken together, this thesis work highlights the versatility of hPSC combined with genome editing and transplantation as a useful approach to better elucidate and understand human diabetes.
Time: 24 August, 2018 12:00
Place: Biomedicum Helsinki 1, Lecture Hall 1, Haartmaninkatu 8, 00290 Helsinki
Custos: Professor Timo Otonkoski
Somatic cells can be reprogrammed to pluripotent state by ectopic expression of a defined set of transcription factors. These induced pluripotent stem cells (iPSC) hold great potential for biomedical applications, such as disease modelling, drug discovery and cell therapies. The derivation of iPSCs is a complex multistep process that can commonly result in inefficient or incomplete conversion of the cells. The reprogramming efficiency and the quality of the reprogrammed cells can be affected by various components of the reprogramming method, including reprogramming vectors, starting cell populations and the choice of reprogramming factors. The aim of this thesis was to explore novel approaches for improving the pluripotent reprogramming outcome.
The particular aims of this thesis were to investigate the use of recombinant Adeno-associated virus (rAAV) as a gene transfer vector for cellular reprogramming, characterization of the effects of old donor age and long term passaging on the reprogramming of fibroblasts, and development of reprogramming methods based on CRISPR/Cas9-mediated activation of endogenous reprogramming factors.
In this study, rAAV-mediated transduction of mouse embryonic fibroblasts with OCT4, SOX2, KLF4 and C-MYC was found to successfully induce reprogramming to pluripotency. Unlike initially expected, the AAV vectors were integrated with high efficiency into the host genome during the reprogramming process, resulting in all analyzed iPSCs containing vector integrations.
Both donor age and the culture time of the donor fibroblasts correlated with reduction in pluripotent reprogramming efficiency. This effect was found to be associated with upregulation of cellular P21 expression and reduction in cell proliferation. Downregulation of P21 expression by siRNA treatment was able to promote reprogramming of late passage senescent fibroblasts.
By optimizing the reprogramming factor guide composition, CRISPR/Cas9-mediated gene activation (CRISPRa) could be used to derive iPSCs reprogrammed fully by targeted activation of endogenous genes. The efficient reprogramming of somatic cells by CRISPRa was found to be dependent on the inclusion of additional guides targeting an embryonic genome activation enriched Alu-motif. Due to the direct targeting of endogenous loci and the high multiplexing capacity of CRISPRa, the reprogramming approach has a high potential for mediating comprehensive and specific reprogramming.
Overall, this thesis provides a number of novel tools and insights into the pluripotent reprogramming process. The results of this work can be used to develop more robust reprogramming methods and to improve the quality of reprogrammed cells.
Time: 07 October, 2017 12:00
Place: Biomedicum Helsinki, Lecture Hall 2, Haartmaninkatu 8, 00290 Helsinki
Defects of mitochondrial DNA (mtDNA) replication underlie common metabolic disorders. Despite mtDNA is degraded and synthesised in all cells containing mitochondria, mtDNA replication stress typically causes generation of mtDNA deletions or depletion of mtDNA copy number in muscle and brain, which manifest as mitochondrial myopathy (MM) or neurodegeneration, respectively. MtDNA replication defects, however, do not affect highly proliferative tissues, such as blood or intestine, despite their reliance on robust mtDNA replication to sustain high rates of proliferation. The mechanisms behind the tissue-specific manifestations of mtDNA replication defects remain unknown.
In this thesis, we aimed to identify the metabolic response pathways for mtDNA replication stress caused by a dominant Twinkle mtDNA helicase (TWNK) mutation leading to adult-onset MM. The study revealed that MM induces a metabolic stress response in muscle which we found to be orchestrated by one master regulator, mechanistic target of rapamycin complex I (mTORC1). The mTORC1-mediated stress response appeared to promote disease progression, and an mTORC1 inhibitor, rapamycin, remarkably improved the mitochondrial muscle disease. It ameliorated the typical hallmarks of MM: the number of ragged red fibers (RRFs) and the amount of mtDNA deletions were reduced after rapamycin treatment.
In the second part of this thesis, we studied the transcription regulation of mtDNA replication machinery. We identified a complex regulatory locus for DNA polymerase gamma (POLG) by in silico predictions, which were verified in vivo. The regulatory non-coding locus drives POLG expression specifically in the sensory interneurons of the spinal cord and oculomotor nucleus, which we found to degenerate in POLG patients. The death of these neurons might be the underlying cause of sensory neuropathy and progressive external ophthalmoplegia (PEO), which are typical clinical findings in POLG disorders. The identified regulatory locus is the first non-coding locus for a mitochondrial disease gene and offers the first candidate region for pathogenic non-coding mutations.
In conclusion, our work has identified novel contributors in the tissue-specific manifestations of mitochondrial diseases and offers multiple novel treatment targets for mitochondrial disorders, which currently lack effective treatment options.
Time: 22 September, 2017 12:00
Place: Haartman Institute, Lecture Hall 1, Haartmaninkatu 3, 00290 Helsinki
Opponent: Professor Rita Horvath, Newcastle University, United Kingdom
Mitochondrial diseases are inheritable diseases, where the function of the ATP
(adenosine triphosphate) producing organelle of the cell, is compromised. This
leads to a wide variety of phenotypes, known to arise from defects in over 200
genes. Typically manifesting organs are brain, heart, muscle, liver, endocrine
organs and sense organs. Mitochondrial disorders are often progressive, they
can manifest in multiple organs in one person and due to inheritance, other
family members might also be affected. Careful clinical assessment with
investigation of family history helps to predict the cause, but other diagnostic
assessments play a central role in diagnosis.
Elevation of blood or cerebrospinal fluid lactate has traditionally been an
indicator of mitochondrial dysfunction, but this biomarker lacks sensitivity and
specificity, and more accurate biomarkers are required. Muscle biopsy sample
is the gold standard of mitochondrial disease diagnosis. Cytochrome c oxidase
(COX) negative, succinate dehydrogenase (SDH) positive fibers and raggedred
fibers (RRFs) are hallmarks of mitochondrial dysfunction. Reduced
respiratory chain enzyme activity in tissue verifies diagnosis. Solid diagnosis
requires genetic evidence, but the expanding number of disease-causing
genes makes it difficult to choose which genes to sequence.
We report here a novel diagnostic serum biomarker, fibroblast growth factor
21 (FGF21), which is more sensitive and specific to muscle-manifesting
mitochondrial disorders than any of the conventional biomarkers used before.
It correlates with COX-negative muscle fibers and is most likely produced and
secreted by them. We also studied another recently discovered serum
biomarker, growth differentiation factor 15 (GDF15). We report that both
FGF21 and GDF15 correctly distinguish mitochondrial myopathies from nonmitochondrial
myopathies and controls, making them the most accurate
biomarkers for mitochondrial myopathies to date. The trigger for induction of
these biomarkers seems to be upstream of respiratory chain defect, most likely
initiates from the mitochondrial translational machinery.
In another study, we used next generation sequencing to search for a
pathogenic mutation in a patient with fatal infantile Alpers
hepatoencephalopathy. We identified two compound heterozygous mutations
in a novel disease gene, FARS2. This gene encodes for a protein,
mitochondrial phenylalanyl-tRNA synthetase (mtPheRS), responsible for the
charging of mitochondrial phenylalanyl-tRNA with its cognate amino acid.
Structural prediction of the mutated proteins together with functional studies in
E. coli showing decreased activity of mutant mtPheRS, verified the diagnosis.
Our results strongly support the use of FGF21 and GDF15 as first line
diagnostic assessments. Rapid progression to next-generation sequencing is
advised if both of these biomarkers are elevated, with positive predictive value
being 95%. This would reduce the need for invasive diagnostic tests.