Raúl Martín Guadaño

In previous research performed from our group, fibroblasts from patients with known parkin mutations have been shown to have mitochondrial dysfunction with ATP deficient production linked to CI. ATP assays assessed the proliferative and cytotoxicity response of cells, from PD patient tissue, to drug exposure or other biological compounds and/or conditions. Therefore, ATP assays are valuable, sensitive and effective method to investigate the underlying suspected energetic mechanisms involved in PD (Mortiboys et al., 2008, 2010b).

ATP linked-complex I production is facilitated by changing the media growth conditions, since fibroblasts in culture produce their ATP through glycolysis. When media is changed from glucose to galactose, fibroblasts rely on the mitochondrial oxidative phosphorylation (OXPHOS) pathway to produce their energy. One of the patients had to be removed from the overall analysis (P3). ATP assays were performed according to the availability of the tissue. Current results show an ATP reduction of ~41.5% when comparing the group of controls with the patients with

174 al., 2008). This demonstrated an overall decrease of ATP production linked to a similar defect amongst the five parkin-mutant fibroblasts cell lines reported (Mortiboys et al., 2008).

Although fibroblasts are a reliable, robust and useful model of research to study PD in patient tissue, PD is a disease that primarily affects the CNS. In vivo models, such as rodents, have been useful to investigate disease mechanisms, assess toxin exposure (i.e., rotenone, paraquat, herbicides) and to test newer treatments. They offer a great advantage by assessing their effect in motor symptoms and the SN after inducing neurotoxicity and genetic manipulation. Rodent models have shown Dopaminergic neuronal loss. Moreover, they have shown to be useful to study non-motor PD symptoms (i.e. sleep, cognition). However, they do not fully and consistently recapitulate human PD (Blesa et al., 2016; Campos et al., 2013). An alternative approach will be to study TIGAR by reprogramming fibroblasts from patients with PD-related mutations, via induced pluripotent stem cells (iPSC), direct lineage of induced neural stem (iNS), induced neural precursor (iNP), induced neurons (iN), or dopaminergic neurons (iDA). iPSC with PD-related mutations can be differentiated into neurons, which have shown the pathological mechanisms already described in PD (Playne and Connor, 2017; Xu et al., 2017). Direct reprogramming from somatic cells to a specific type of neurons offers a new alternative method for cell modelling avoiding pluripotent state. The direct conversion offers many advantages: functional neurons can be obtained faster (within weeks); the risk of having pluripotent cells left is avoided; it promises to be an efficient and feasible model. However, there are also many disadvantages: less efficiency with scarce or no Tyrosine Hydroxylase positive (TH+) cells, it could still produce longer culture

175 periods for maturation and immature and non-functional neurons, cells are post mitotic and with limited availability. These new methods are still under optimisation, for which different reprogramming protocols have been assessed. Indeed, the effect of TIGAR have been shown to be cell dependent, with complex and somehow contrary and arbitrary functions depending on the metabolic context of a specific cellular type (Bensaad et al., 2006, 2009; Madan et al., 2012). For this reason, any observations in parkin- or PINK1-mutant fibroblasts would need to be confirmed in further cellular, neuronal model systems or in PD brain tissue.

3.5.3 Tigar RNAi mediated knockdown

Initial transfections using siRNAs (Santa Cruz) against TIGAR were unsuccessful in achieving a satisfactory knockdown in controls. Several potential reasons should be considered for this. For instance, particular cell types can be more difficult to transfect, and this is well established for fibroblasts. Also, the growth and quality variability (i.e., cell confluency, density, passage, etc.) amongst the different fibroblast cell lines used, could affect the transfection process. However, even when transfections assays were performed at the same time with the same methodology, variation cannot be avoided due to cell culture behaviour. This variation in the results could also be caused by the fact that the reference genes are not stably expressed. Also, the factor of the transfection done under serum free media for six hours, might affect the expression of the genes when serum is added hours after the transfection. In Figure 3.10 b-c, the knockdown effect was seen to be reliable between β-actin and GAPDH, whereas at 24 hours (Figure 3.10 a) the knockdown effect showed the highest inconsistency of them all.

176 The siRNAs against TIGAR we used were also only targeting the 3’UTR region of the gene (Figure 3.2). Furthermore, it has been reported that siRNA mediated gene knockdown is generally less effective when targeting only the 3’ UTR. A better silencing effect is achieved when targeting the interior region of the mRNA, we therefore used a new SMARTpool of 4 siRNA against TIGAR (Dharmacon) avoiding 50 nucleotides (nt) upstream and downstream region and within the exon 4, 5 and 6 regions of the gene. Successful knockdowns of ~70% were achieved consistently across the different experiments within different cell lines (Figure 3.12), therefore we decided to assess its effect in the mitochondrial function in one LRRK2- patient, which displayed a mitochondrial defect previously reported and was available. We wanted to see if the effect of TIGAR knockdown was robust and could be applied in PD tissue regardless the genotype.