Stem cell models of C9orf72-linked Frontotemporal Dementia

The GGGGCC repeat expansion in C9orf72 is the most common genetic cause of frontotemporal dementia and amyotrophic lateral sclerosis (ALS). Expanded repeat-associated toxicity from either RNA foci or dipeptide protein repeats (DPRs) as well as a loss of C9orf72 function due to haploinsufficiency, have all been described as potential pathogenic mechanisms in C9orf72-linked FTD/ALS (C9-FTD/ALS). Moreover, the C9orf72 protein has been suggested to play an important role in autophagy and lysosomal trafficking. The work presented in this thesis includes the investigation of FTD-related phenotypes and C9orf72 haploinsufficiency in C9orf72 patient iPSC-derived cortical neurons (iPSC-CNs), and the generation and study of C9orf72 knockout iPSC-CNs. C9orf72 patient iPSC-CNs exhibited: (i) sense RNA foci, (ii) poly-GP and poly-GR, (iii) C9orf72 promoter hypermethylation, (iv) reduction in C9orf72 total mRNA levels and (v) increased expression of intron1-retaining transcript, compared to control iPSC-CNs, with considerable variability between C9orf72 lines. No reduction in C9orf72 protein levels was detected in C9orf72 iPSC-CNs at 150 and 260 days in vitro. CRISPR/Cas9-mediated knockout of C9orf72 resulted in significant downregulation of autophagy-related genes ULK1, LC3B and LAMP1 in C9orf72-/- iPSC-CNs compared to isogenic C9orf72+/- and/or WT iPSC-CNs, indicative of altered basal autophagy. Moreover, preliminary findings suggest that induction of mitophagy is impaired in C9orf72-/- iPSC-CNs. Future work will be required to replicate this finding and further examine the potential role of C9orf72 in autophagy and mitophagy in iPSC-CNs and how its loss may contribute to C9-FTD/ALS

APPLYING INTEGRATED, SPATIAL TOOLS IN THE FRAMEWORK OF EU COHESION POLICY (2014-2020).

The need for a thorough comprehension and integration into the planning practice of the spatial features of European policies has recently emerged and has been reinforced. The recognition in the Treaty of Lisbon of the territorial cohesion as being the third target of the Policy, combined with the aims of the financial and social cohesion has further strengthened its spatial dimension. One of the results of the renovation during the 2014-2020 period, was the prediction and encouragement of the use of an integrated and place-based approach along with the promotion of spatial governance. The present paper investigates the degree of integration of the spatial dimensions of the Cohesion Policy in member states during that period. What is pursued is an evaluation of the way and the degree of adaptation of the place-based approach to the frame of the policy of Integrated Territorial Development. Thus, the spatial dimension of the development planning of each country could be assessed within this context. The key questions focus on functionality and potential scope, the integration approach, and the utilization of the territorial tools of spatial development. The research is based on a comparative analysis of Member States key policy documents

Public- Private Partnerships in Southeastern Europe: The case of Croatia

This paper aims to define Public Private Partnerships (PPPs) that are considered as a major approach in delivering public infrastructure projects in recent years. The paper analyses PPPs in Southeastern Europe, particularly in Croatia, describing the projects that have been achieved and the efforts of governments to promote the PPPs as alternative means of funding and bearing into consideration the formulation of the theoretical framework of PPP. The paper concludes that although the need for infrastructural projects in Croatia is huge, however the number of projects proposed are confined, according the Agency for PPPs for the approved PPP project

Public- Private Partnerships in Southeastern Europe: The case of Croatia

This paper aims to define Public Private Partnerships (PPPs) that are considered as a major approach in delivering public infrastructure projects in recent years. The paper analyses PPPs in Southeastern Europe, particularly in Croatia, describing the projects that have been achieved and the efforts of governments to promote the PPPs as alternative means of funding and bearing into consideration the formulation of the theoretical framework of PPP. The paper concludes that although the need for infrastructural projects in Croatia is huge, however the number of projects proposed are confined, according the Agency for PPPs for the approved PPP project

Mutations in valosin-containing protein (VCP) decrease ADP/ATP translocation across the mitochondrial membrane and impair energy metabolism in human neurons

Mutations in the gene encoding valosin-containing protein (VCP) lead to multisystem proteinopathies including frontotemporal dementia. We have previously shown that patient-derived VCP mutant fibroblasts exhibit lower mitochondrial membrane potential, uncoupled respiration, and reduced ATP levels. This study addresses the underlying basis for mitochondrial uncoupling using VCP knockdown neuroblastoma cell lines, induced pluripotent stem cells (iPSCs), and iPSC-derived cortical neurons from patients with pathogenic mutations in VCP. Using fluorescent live cell imaging and respiration analysis we demonstrate a VCP mutation/knockdown-induced dysregulation in the adenine nucleotide translocase, which results in a slower rate of ADP or ATP translocation across the mitochondrial membranes. This deregulation can explain the mitochondrial uncoupling and lower ATP levels in VCP mutation-bearing neurons via reduced ADP availability for ATP synthesis. This study provides evidence for a role of adenine nucleotide translocase in the mechanism underlying altered mitochondrial function in VCP-related degeneration, and this new insight may inform efforts to better understand and manage neurodegenerative disease and other proteinopathies

Developmental regulation of tau splicing is disrupted in stem cell-derived neurons from frontotemporal dementia patients with the 10 + 16 splice-site mutation in MAPT

The alternative splicing of the tau gene, MAPT, generates six protein isoforms in the adult human CNS. Tau splicing is developmentally regulated and dysregulated in disease. Mutations in MAPT that alter tau splicing cause frontotemporal dementia (FTD) with tau pathology, providing evidence for a causal link between altered tau splicing and disease. The use of induced pluripotent stem cell (iPSC) derived neurons has revolutionized the way we model neurological disease in vitro. However, as most tau mutations are located within or around the alternatively spliced exon 10, it is important that iPSC-neurons splice tau appropriately in order to be used as disease models. To address this issue, we analysed the expression, and splicing of tau in iPSC-derived cortical neurons from control patients and FTD patients with the 10+16 intronic mutation in MAPT. We show that control neurons only express the fetal tau isoform (0N3R), even at extended time points of 100 days in vitro. Neurons from FTD patients with the 10+16 mutation in MAPT express both 0N3R and 0N4R tau isoforms, demonstrating that this mutation overrides the developmental regulation of exon 10 inclusion in our in vitro model. Further, at extended time-points of 365 days in vitro, we observe a switch in tau splicing to include six tau isoforms as seen the adult human CNS. Our results demonstrate the importance of neuronal maturity for use in in vitro modeling and provide a system that will be important for understanding the functional consequences of altered tau splicing

Genetic and phenotypic characterization of complex hereditary spastic paraplegia

The hereditary spastic paraplegias are a heterogeneous group of degenerative disorders that are clinically classified as either pure with predominant lower limb spasticity, or complex where spastic paraplegia is complicated with additional neurological features, and are inherited in autosomal dominant, autosomal recessive or X-linked patterns. Genetic defects have been identified in over 40 different genes, with more than 70 loci in total. Complex recessive spastic paraplegias have in the past been frequently associated with mutations in SPG11 (spatacsin), ZFYVE26/SPG15, SPG7 (paraplegin) and a handful of other rare genes, but many cases remain genetically undefined. The overlap with other neurodegenerative disorders has been implied in a small number of reports, but not in larger disease series. This deficiency has been largely due to the lack of suitable high throughput techniques to investigate the genetic basis of disease, but the recent availability of next generation sequencing can facilitate the identification of disease- causing mutations even in extremely heterogeneous disorders. We investigated a series of 97 index cases with complex spastic paraplegia referred to a tertiary referral neurology centre in London for diagnosis or management. The mean age of onset was 16 years (range 3 to 39). The SPG11 gene was first analysed, revealing homozygous or compound heterozygous mutations in 30/97 (30.9%) of probands, the largest SPG11 series reported to date, and by far the most common cause of complex spastic paraplegia in the UK, with severe and progressive clinical features and other neurological manifestations, linked with magnetic resonance imaging defects. Given the high frequency of SPG11 mutations, we studied the autophagic response to starvation in eight affected SPG11 cases and control fibroblast cell lines, but in our restricted study we did not observe correlations between disease status and autophagic or lysosomal markers. In the remaining cases, next generation sequencing was carried out revealing variants in a number of other known complex spastic paraplegia genes, including five in SPG7 (5/97), four in FA2H (also known as SPG35) (4/97) and two in ZFYVE26/SPG15. Variants were identified in genes usually associated with pure spastic paraplegia and also in the Parkinson’s disease-associated gene ATP13A2, neuronal ceroid lipofuscinosis gene TPP1 and the hereditary motor and sensory neuropathy DNMT1 gene, highlighting the genetic heterogeneity of spastic paraplegia. No plausible genetic cause was identified in 51% of probands, likely indicating the existence of as yet unidentified genes

Progressive Motor Neuron Pathology and the Role of Astrocytes in a Human Stem Cell Model of VCP-Related ALS.

Motor neurons (MNs) and astrocytes (ACs) are implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS), but their interaction and the sequence of molecular events leading to MN death remain unresolved. Here, we optimized directed differentiation of induced pluripotent stem cells (iPSCs) into highly enriched (> 85%) functional populations of spinal cord MNs and ACs. We identify significantly increased cytoplasmic TDP-43 and ER stress as primary pathogenic events in patient-specific valosin-containing protein (VCP)-mutant MNs, with secondary mitochondrial dysfunction and oxidative stress. Cumulatively, these cellular stresses result in synaptic pathology and cell death in VCP-mutant MNs. We additionally identify a cell-autonomous VCP-mutant AC survival phenotype, which is not attributable to the same molecular pathology occurring in VCP-mutant MNs. Finally, through iterative co-culture experiments, we uncover non-cell-autonomous effects of VCP-mutant ACs on both control and mutant MNs. This work elucidates molecular events and cellular interplay that could guide future therapeutic strategies in ALS

Creation of an Open-Access, Mutation-Defined Fibroblast Resource for Neurological Disease Research

Our understanding of the molecular mechanisms of many neurological disorders has been greatly enhanced by the discovery of mutations in genes linked to familial forms of these diseases. These have facilitated the generation of cell and animal models that can be used to understand the underlying molecular pathology. Recently, there has been a surge of interest in the use of patient-derived cells, due to the development of induced pluripotent stem cells and their subsequent differentiation into neurons and glia. Access to patient cell lines carrying the relevant mutations is a limiting factor for many centres wishing to pursue this research. We have therefore generated an open-access collection of fibroblast lines from patients carrying mutations linked to neurological disease. These cell lines have been deposited in the National Institute for Neurological Disorders and Stroke (NINDS) Repository at the Coriell Institute for Medical Research and can be requested by any research group for use in in vitro disease modelling. There are currently 71 mutation-defined cell lines available for request from a wide range of neurological disorders and this collection will be continually expanded. This represents a significant resource that will advance the use of patient cells as disease models by the scientific community

iPSC-derived neuronal models of PANK2-associated neurodegeneration reveal mitochondrial dysfunction contributing to early disease

Mutations in PANK2 lead to neurodegeneration with brain iron accumulation. PANK2 has a role in the biosynthesis of coenzyme A (CoA) from dietary vitamin B5, but the neuropathological mechanism and reasons for iron accumulation remain unknown. In this study, atypical patient-derived fibroblasts were reprogrammed into induced pluripotent stem cells (iPSCs) and subsequently differentiated into cortical neuronal cells for studying disease mechanisms in human neurons. We observed no changes in PANK2 expression between control and patient cells, but a reduction in protein levels was apparent in patient cells. CoA homeostasis and cellular iron handling were normal, mitochondrial function was affected; displaying activated NADH-related and inhibited FADH-related respiration, resulting in increased mitochondrial membrane potential. This led to increased reactive oxygen species generation and lipid peroxidation in patient-derived neurons. These data suggest that mitochondrial deficiency is an early feature of the disease process and can be explained by altered NADH/FADH substrate supply to oxidative phosphorylation. Intriguingly, iron chelation appeared to exacerbate the mitochondrial phenotype in both control and patient neuronal cells. This raises caution for the use iron chelation therapy in general when iron accumulation is absent