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5.1.1 Mitochondrial mutations
MtDNA mutations are an important cause of inherited disease. Point mutations, deletions and insertion mutations are the most common in human mitochondrial diseases and in the aging population (http://www.mitomap.org, 2010, Wallace, 2005). Though mtDNA large-scale deletions occur less frequently, these mutations contribute significantly to mitochondrial diseases, ageing and neurodegeneration. Different mtDNA deletions have been demonstrated to be important for the development of mitochondrial diseases such as Kearns–Sayre syndrome (Zeviani et al., 1988, Shoffner et al., 1989, Moraes et al., 1989), myopathies (Manfredi et al., 1997, Wong et al., 2003) progressive external ophthalmoplegia (Moraes et al., 1989, Wong et al., 2003, Johns et al., 1989), diabetes (Ballinger et al., 1992) and deafness (Ballinger et al., 1992) etc.
Pathogenic mtDNA deletions were first discovered in 1988 (Holt et al., 1988; Wallace et al., 1988), following this initial report, hundreds of point mutations, deletions, and rearrangements of mtDNA have been described in association with mitochondrial disease (http://www.mitomap. org). These diseases manifest primarily within the brain and skeletal muscle and are termed; mitochondrial encephalomyopathies (Chan, 2006)
With the exception of erythrocytes, human cells hold several copies of mtDNA per cell, termed as polyploid, forming the basis of an important aspect of mitochondrial genetics; homoplasmy and heteroplasmy. Homoplasmy is the term given when all copies of the mitochondrial genome within a cell are identical, where heteroplasmy indicates the mixing of two or more mitochondrial genotypes, which includes both wild-type and mutation containing genomes. (Chan, 2006).
Around 20% of patients suffering from mitochondrial disease harbour a single large-scale mtDNA deletion (Bender et al., 2006) where this mutation is the major cause of respiratory deficiency in a number of post-mitotic tissues including brain and muscle (Tawil and Griggs, 2002, Oldfors et al., 2006). Most reported mtDNA deletions occur within the major arc of the mitochondrial genome (Oldfors et al., 2006), although research on aged muscle, has identified unusual deletions spanning beyond the origin of light strand replication (OL) into the minor arc (Santorelli et al., 1996, Sciacco et al., 1996, Moslemi et al., 1997, Rygiel et al., 2016). Most large-scale mtDNA deletions are heteroplasmic and are prone to expansion (Rygiel et al., 2016).
5.1.2 MtDNA deletion formation
The methodology underlying the formation of mtDNA deletions remains elusive, however two hypotheses have been deeply investigated, both of which surround the mispairing or misannealing of DNA base pairs which then facilitates mtDNA deletion formation.
The first involves mtDNA replication (Haber 2000, Hanekemp and Thorsness 1999, Prithivirajsingh, 2007, Halliwell, 1992) where mtDNA deletion formation occurs via replication. In this model, the light strand remains single stranded until OL is exposed, allowing repeat sequences to attach to their complementary sites, with the remaining DNA sequence being removed by DNA ligase. The second mechanism entails mtDNA deletion formation occurring through the repair of damaged mtDNA (Arai et al., 2006, DeGrey, 1997, triggered by single-stranded mtDNA regions, produced via exonucleases at double-strand breaks (DSBs). The single strand consequently anneals with repeat sequences on the other single-stranded mtDNA or non-coding region (NCR). Following this repair, ligation and degradation of the exposed single strands initiate mtDNA deletion formation; with the generation of a mitochondrial genome harbouring a deleted segment (Krishnan et al., 2008).
5.1.3 SNpc vulnerability to mtDNA damage
MtDNA deletions in the SNpc clonally expand within individual cells, and if high levels of an individual mutation are reached (usually around 60%), this can cause a biochemical defect. SNpc neurons are highly susceptible to somatic mtDNA deletions, as a result of several factors which increase oxidative stress generated via DA metabolism, ageing and neurodegeneration, identified through respiratory chain deficiency (Halliwell, 1992, Kraystbergs et al., 2006). Furthermore, DAergic neurons exhibit pace making activity which maintains DA levels within the striatum. Alterations in the ATP levels within neurons can occur as a consequence of mtDNA mutation accumulation and also perturbed mitochondrial bioenergetics. This is triggered by oxidative stress with advancing age within SNpc neurons, where ROS production can cause double strand breaks within mtDNA and possibly contribute to the clonal expansion of damaged mtDNA (Arai et al., 2006)
5.1.4 MtDNA deletions in SNpc neurons
Initial studies within colonic stem cell correlated clonal expansion of mtDNA mutations of the mitochondrial genome to COX deficiency (Taylor et al., 2003). Following on from this, several studies have proceeded to correlate mtDNA deletion load within individual SNpc neurons with COX deficiency (Bender et al., 2006, Kraystbergs et al., 2006, Reeve et al., 2008 and Dolle et al., 2016).
Identification of a mitochondrial respiratory chain defect was carried out by histochemical analysis of both cytochrome c oxidase (COX) and succinate dehydrogenase (SDH) in frozen midbrain sections from control subjects and individuals with PD, as research has shown an age-related increase in COX-deficient cells in human brain (Cottrell et al., 2001). Bender et al., (2006)showed that COX-deficiency was observed within the SNpc of individuals with PD and ageing, in comparison to age-matched control subjects (Kraystbergs et al., 2006, Bender et al., 2006).
Further investigation into mtDNA defects within individual SNpc neurons leading to COX deficiency involved sequencing of the entire mitochondrial genome of 9 individual COX-deficient neurons from 3 individuals with PD, where no somatic mtDNA point mutations were found in 7 cells REF. The absence of pathogenic mtDNA point mutations may be indicative of the clonal expansion of deleted mtDNA molecules. DNA extraction and long range PCR analysis of 50 individual laser-micro dissected neurons from frozen SNpc of aged controls or individuals with PD, resulted in no detection of wild-type mtDNA but only deleted mtDNA, where the level of deletion was increased in aged controls and individuals with PD (Reeve et al., 2008). Moreover, within individual SNpc neurons from aged controls and individuals with PD, clonal expansion of deleted species in both normal and COX-deficient neurons were identified; where the deleted mtDNA species in individual cells were of different sizes, suggesting that the mtDNA deletion was unique to each individual neuron, compatible with the hypothesis that these were acquired mtDNA mutations. REF.
A real-time PCR assay was developed in the study by Bender et al., (2006), aimed to compare the amplification of mtDNA from the ND1 gene with amplification of mtDNA from the ND4 gene (He et al., 2002) as the major arc of the mitochondrial genome is where most deletions occur, encompassing ND4 but not ND1, and also to measure mtDNA copy number. The real-time PCR assay calculated a deletion level between 55–60% for these neurons. SNpc neurons with normal COX activity from individuals with PD or from controls were shown to harbour very high levels of deleted mtDNA; approximately 52% in individuals with PD and 43% in aged controls. The level of mtDNA deletion showed a highly significant correlation of deletion level with age. Furthermore, the level of mtDNA deletion was also significantly greater in the COX-deficient neurons than in the neurons with normal COX activity, confirming that COX deficiency is attributable to high levels of deleted mtDNA (Bender et al., 2006)
In terms of understanding the deletion formation mechanisms within SNpc neurons, Reeve et al., 2008 characterised mtDNA deletion within single SNpc neurons from 3 patient groups: controls, PD patients, and a patient with Parkinsonism due to multiple mtDNA deletions. 89 mtDNA deletions were identified, where no difference in the types of mtDNA deletions between these groups was indicative of a comparable deletion formation mechanism (Reeve et al., 2008).
Most recently, Dolle et al., 2016, investigated mtDNA deletions, with a particular interest in copy-number alterations in single neurons from the SNpc. This study showed that mtDNA copy number increases with age in these neurons, consequently maintaining the level of wild-type mtDNA population despite deletion accumulation. These findings indicate that ageing SNpc neurons can uniquely upregulate mtDNA copy number, supported by the levels of somatic mtDNA deletion in each cell. This proposes an intrinsic neuroprotective mechanism within these neurons, which allow neurons to maintain wild-type mtDNA molecules, regardless of age-dependent accumulation of somatic deletion. However, this upregulation was not observed in individuals with PD, and resulted in the depletion of the wild-type mtDNA population (Dolle et al., 2016).
5.1.5 Generating dopaminergic neurons from IPSCs
The Nobel Prize in Physiology or Medicine (2012) was awarded to Shinya Yamanaka and Sir John B. Gurdon for their ground-breaking work which allowed the transformation of almost any terminally differentiated cell to be reprogrammed to a pluripotent state. IPSCs can be directly differentiated into A9 midbrain DAergic neurons, as well as into other neuronal types, which include enteric neurons, olfactory neurons, and cortical neurons that are also affected in PD. This provides as an incomparable PD model, where pathologically studies can be conducted in live neurons (Xiao et al., 2016).
Of the neuronal types that are perturbed in PD, the midbrain DAergic neurons of SNpc are the most damaged, where a loss of these neurons are observed with the disease (Dolt et al., 2017). Until recently, several protocols have attempted to generate these DAergic neurons from pluripotent stem cells, which were unsuccessful until researchers began to delve into the biology of floor plate tissue. The floor plate of the midbrain; a glial structure crucial in the development of the nervous system within vertebrates, gives rise to nigral DAergic neurons, where the ventral neural tube degenerates in the spinal cord and hindbrain, whilst survives in the midbrain and thus generating midbrain DAergic neurons (Ono et al., 2007).
Human floor plate cells differentiated from human embryonic stem cells (hESCs) into engraftable midbrain DAergic neurons comprise of the activation of the sonic hedgehog (SHH) pathway (Fasano et al., 2010). With the addition of GSK3b inhibitor, CHIR99021 (CHIR), and the further activation of the WNT signalling pathway midbrain floor plates were created (Kriks et al., 2011) and differentiated into midbrain DAergic neurons. These neurons cultured in vitro, were found to express SNpc markers, such as GIRK2, and exhibit in vivo electrophysiological properties observed in SNpc neurons, such as slow spiking (Kriks et al., 2011). On grafting midbrain DAergic progenitors into 6-hydroxy-dopamine (6- OHDA) lesioned mice and rats, viable and functional neurons were identified, where the DA deficiency was rescued in these animals (Kriks et al., 2011).
Adjusting and optimising the various growth factors supplemented to the facilitate midbrain DAergic neuron development is critical to producing the desired neuronal cell type. Consequently, these protocols have been optimised with either one or two cell lines, where the transferring of this protocol to other cell lines, will also require further optimisation and in some cases may not be reproducible (Boulting et al., 2011, Devine et al., 2011). This can be controlled for by quantifying neuronal subtypes in differentiated neurons (Dolt et al., 2017).
5.1.6 Single transcription factor induced functional neurons
The generation of neurons from IPSCs proves a powerful tool to understand neurodegenerative mechanisms, where two major limitations have been identified. The first limitation describes characteristic differences between pluripotent cell lines (Osafune et al., 2008; Hu et al., 2009; Bock et al., 2011), where neurons derived from the same protocol from two different cell lines can display different properties (Wu et al., 2007). iPS cell lines can also exhibit changes over time in culture (Mekhoubad et al., 2012). The second limitation comes down to the time-consuming protocols, as some differentiation protocols can continue for over a month, therefore causing a prolonged time to conduct studies (Johnson et al., 2007).
Zhang et al., 2013 demonstrate that neurogenin-2 (NGN2) overexpression can rapidly convert IPScs into neuronal cells. The forced NGN2 expression converted IPSCs into neuron-like cells, in less than one week, which exhibited mature neuronal morphology in less than two weeks, quicker than any other available method for generating neurons from human ES IPSCs. Furthermore, almost 100% of surviving IPSCs were converted into neurons, indicating that forced expression of a single transcription factor; NGN2 can successfully induce neuronal differentiation with high yield. This robust method renders itself highly efficient in comparison to the Kriks protocol which comprises of the use of four transcription factors for neuronal conversion, under the SMAD (similar to the products of the drosophila MAD genes and the c. elegans SMA genes) inhibition protocol, which spans a 30 day time period (Kriks et al., 2011)
Moreover, it was shown that neuronal NGN2 cells could form synapses, and could be used successfully to observe short-term neuronal plasticity, conduct Ca2+ imaging, as well as be manipulated to represent human genetic disorders. Furthermore, under this protocol, neurons can be generated in 7 days, which permits a quick turnaround of experiments, which allows the potential to carry out mechanistic studies within neurons easily (Zhang et al., 2013).
5.1.7 IPSCs harbouring a large scale mtDNA deletion
Recently, Russell et al., (2018) delved into the methodology of clonal expansion within single, large scale mtDNA deletion IPScs reprogrammed into NGN2-IPSCs. The study described below utilised reprogrammed IPScs from Patient ‘A’; a female child with Pearson’s syndrome caused by a ~6.0 kb single, large-scale mtDNA deletion spanning m.7777- 13794 removing the following genes; COXII, ATPase 6 and 8, COXIII, ND3, ND4L, ND4, ND5 and part of ND6 and several tRNAs. Reprogramming of the deletion line A was conducted using a sendai virus (a single stranded cytoplasmic RNA vector [Tokusumi et al.,2002]) and Cyto-Tune-iPS reprogramming kit; yielding 2 clones with of < 10% and 40% mtDNA deletion respectively (Russell et al., 2018).
The long term culture of these IPScs showed a consistent increase in mtDNA deletion levels over time, where mtDNA heteroplasmy (60%) correlated with an increased respiratory deficiency (Russell et al., 2018). In this study, mitochondrial complex protein expression as well as mitochondrial function through TMRM staining was assessed between 2 cell lines that harboured varying levels of mtDNA deletion to verify the presence of a mitochondrial defect. Following on from this, IPScs were differentiated into neurons where live recordings were obtained using TMRM staining to observe differences in mitochondrial trafficking as a consequence of respiratory chain deficiency. This experiment utilised this model to compare modifications in mitochondrial trafficking in SNpc neurons from IPScs derived from mitochondrial disease patients harbouring a large scale mtDNA deletion.
5.2 Aims of this study
- Confirm the mitochondrial defect within IPScs harbouring a single, large scale mtDNA deletion via observing mitochondrial protein complex expression and alterations in mitochondrial membrane potential through TMRM staining
- Differentiate iPS cell lines harbouring two levels of large scale mtDNA deletion into dopaminergic neurons and quantify the efficiency of this differentiation
5.3 METHODS & MATERIALS
Generation of IPScs from patient fibroblasts harbouring a large scale mtDNA deletion, as well their differentiation into neurons are described in detail within (Methods and Materials, section 2.1).
5.3.1 Characterisation of mitochondrial function of low and high heteroplasmy IPSc
184.108.40.206 Cell lysis for western blot. IPScs were washed with PBS before being scraped off with cell scrapers, followed by centrifugation at 350g at 4°C for 10 minutes in ice cold PBS to pellet the cells. The supernatant was removed again and the pellet was re-suspended in cell lysis buffer (Table 6.1A), which was followed by a 30 second vortex and subsequent 10 min incubation on ice. Lysed cells were then centrifuged at 560g at 4°C for 2 minutes, where the supernatant contain cell proteins was retained eliminating the cytoskeleton of the cell, snap frozen on dry ice, and stored at -80 °C until use.
220.127.116.11 Bradford assay. Bovine serum albumin (BSA) was used to prepare a standard curve by diluting a gradient of volumes (0, 2, 5, 10, 15 and20 μl) of 1μg/μlBSA into 800 μl H20. Following this, 1μlof unknown sample was also added to 800μl H20, alongside 200μlBradford reagent. Protein molecules combining to the Coomassie blue under the acidic conditions triggers a brown to blue colour change, due a shift in absorption maximum from 465 to 595 nm, which measures amino acid residues such as arginine, lysine and histidine (Bradford, 1976).
Unknown sample protein concentrations were determined from values generated from the standard curve. The standard curve and samples were placed into an optical bottom plate and wavelengths were observed on SpectraMax M3 plate reader. Varying concentrations of BSA, created a standard curve, with concentration were plotted on the x-axis and absorbance plotted on the y-axis. A linear regression was calculated, from which the equation of the line was re-arranged to calculate the absorbance and therefore concentration of the unknown samples.
18.104.22.168 Sample preparation and gel electrophoresis. 20μg of low and high heteroplasmy IPSc lysates were loaded and separated via gel electrophoresis. Lysates were prepared with sample buffer (Table 5.1A) and 10% dithiothreitol (DTT) in 200μL PCR tubes. Proteins were denatured by heating samples at 37°C for 30 minutes (to observe Complex IV) in order for the efficient transfer of proteins through the gel matrix. This was followed by centrifugation at 14,000 RPM for 10 minutes. The SpectraTM multicolour broad range protein ladder (ThermoFisher) and protein samples were loaded into 12% Mini-PROTEAN TGX (Bio-Rad) precast gels and run for 60 minutes at 120 Volts in Tris-glycine running buffer.
22.214.171.124 Protein transfer. After 60 minutes, the gel was moved and placed in between the two ion reservoir stacks and the blotting membrane of the Trans-Blot Turbo Transfer system Transfer Pack. This membrane sandwich was then placed into a cassette and inserted into the Trans-Blot Turbo Transfer system (Bio-Rad). This turbo transfer system allowed rapid and efficient transfer at <25V for 3 minutes at 2.5 A.
126.96.36.199 Detecting mitochondrial function via immunoblotting.The membrane was transferred into blocking buffer (Table 6.1A) and placed on a roller for 60 minutes at room temperature to eliminate non-specific antibody binding. Primary antibodies (Table 5.1C) were also diluted in blocking buffer and placed on a membrane and incubated overnight at 4°C on a rotator. This membrane underwent 3x 10 minute TBST washes, which was followed by secondary antibody incubation for 60 minutes at room temperature.
188.8.131.52 Membrane development. After secondary antibody incubation, the membrane was washed 3x in TBST for 10 minutes. Image development was carried out through the AmershamTM ECLTM prime reagent kit; which comprises of luminol and peroxide solutions These solutions are utilised by the horseradish peroxidase enzyme conjugated on the secondary antibody and which catalyses these reagents to produce detectable light. The membrane was incubated in a 1:1 ratio of both solutions for 5 minutes, followed by ChemiDoc system imaging and densitometry analysis (Image Lab) respectively.
Table 5.1 Western blot reagents
5.3.2 Quantifying mtDNA deletion in neurons
184.108.40.206 Freezing cells for PCR. A pellet of 1×105 cells/mL were frozen at -80 °C until ready for DNA extraction.
220.127.116.11 DNA extraction and single cell lysis. DNA extraction from IPScs was conducted using single cell lysis buffer [50 mM Tris HCl (pH 8.5), 0.1% Tween 20, 0.2 mg/ml proteinase K (ThermoFisher, cat# 25530-049)]. Cells were incubated in buffer at 2 hours at 56 °C, followed by a 10 minute 95°C Proteinase K denaturing step, to prevent proteinase K interfering with the Taqman mastermix during the PCR reaction.
18.104.22.168 Real Time PCR primers. The real time PCR protocol was followed as described by Rygiel et al., 2015. PCR primers for these reactions and fluorogenic probes for these regions were designed by (He et al., 2002). The following primers were synthesised: ND1– forward primer (L3485-3504), ND1 reverse primer (H3532-355), ND1 probe (L3506-3529) and MT-ND1 VIC-5′-CCATCACCCTCTACATCACCGCCC-3′-MGB (np 3506-3529). ND4 – forward primer (L12087-12109), ND4 reverse primer (H12140-12170), ND4 probe (L12111-12138) and MT-ND4 FAM-5′-CCGACATCATTACCGGGTTTTCCTCTTG-3′-MGB (np 12111-12138) (Table 5.2).
22.214.171.124 Real Time PCR assay. Taqman chemistry was used to perform real-time PCR. All PCR amplification was carried out in 20μL reactions; 5μL of the standard curve and DNA samples and 15μLPCR master mix. All samples were run in triplicate in a Veriti 96 well (Applied BioSystems) PCR plate. Each plate contained serial 1:10 dilutions of a 7D1-B2M plasmid construct containing ND1 and ND4 genes in dH2O to generate a standard curve. (Rygiel et al., 2015). Plates were sealed, and vortexed to mix all the reagents with the DNA samples and spun down. mtDNA deletion levels were quantified in low and high heteroplasmy IPScs on a StepOne Plus (Applied Biosystems) real-time PCR machine, protocol as follows; 50°C for 2 minutes, 95°C for 10 minutes, 40 cycles of 95°C for 15 seconds, 60°C for 1 minute.
126.96.36.199 Real Time PCR analysis. MtDNA deletion levels were analysed where the trend line of the standard curve was assessed for efficiency of the PCR reaction and determining the level of each probe indicative of the presence of the gene. The gradient of the standard curve for every reaction needed to fall within the range of -3.25 to -3.45, with -3.33 being the optimal value. MTND1 is preserved in both WT and mutant mtDNA in this cell line , therefore acted as reference for mtDNA as, ND4 is deleted. The ratios of MT-ND1/D-Loop, MT-ND4/D-Loop and MT-ND4/MT-ND1 were calculated using standard curves. Below 10% heteroplasmy the assay was found to become less accurate due to increasing measurement error (Russell et al., 2018). The reasons for this are unclear, therefore any sample with measured heteroplasmy of 0–10% was classed as <1Ct.
Table 5.2. Real time PCR reagents for one reaction
|Reagents||Base pairs||Volume (μL)||Final concentration||Manufacturer|
|Taqman Universal PCR master mix||–||10||1x||Applied Biosystems|
|MTND1 forward primer||L3485-3504||0.6||10 µM||Eurofins MWG|
|MTND1 reverse primer||H3532-355||0.6||10 µM||Eurofins MWG|
|MTND4 forward primer||L12087-12109||0.6||10 µM||Eurofins MWG|
|MTND4 reverse primer||H12140-1217||0.6||10 µM||Eurofins MWG|
|MTND1 probe VIC||L3506-3529||0.4||5 µM||Life Technologies|
|MTND4 probe FAM||L12111-12138||0.4||5 µM||Life Technologies|
5.3.3 Neuronal imaging
188.8.131.52 Fixing cells, immunofluorescence and live imaging experiments. Conducted as highlighted in (Methods and Materials, section 2.3). Primary and secondary antibodies used in experiments are highlighted in (Table 5.3).
Table 5.3. Immunofluorescence primary and secondary antibodies
|Tyrosine Hydroxylase (TH)||1:200||DAergic neurons||Rabbit/IgG1||Sigma|
|γ-aminobutyric acid (GABA)||1:1000||Inhibitory (GABA) neurons||Rabbit||Sigma|
|Vglut||1:500||Glutamatergic neurons||IgG2a||Synaptic systems|
|MTCO1 (COX IV)||1:200||Mitochondrial mass marker||IgG2a||Abcam|
|β-tubulin||1:500||All immature neurons||Rabbit||Sigma|
|Microtubule associated Protein (MAP2)||1:200||Neuronal marker||Rabbit||Abcam|
|Alexa Fluor 405 nm||1:200||Secondary Antibody marker||ThermoFisher|
|Alexa Fluor 546 nm|
|Alexa Fluor 647 nm|
|Alexa Fluor 750 nm|
5.4.1 Understanding mitochondrial function in IPScs harbouring a large scale mtDNA deletion
Russell et al., (2018) have observed a reduction in mitochondrial oxygen comsumption within the IPSCs harbouring a highler level of mtDNA deletion. This study aimed to quantify mitochondrial protein complex expression within these two lines to confirm alterations in mitochondrial oxidative capacity in IPScs and potential their differentiated neuronal phenotype harbouring a large scale mtDNA deletion.
The large scale deletion in this line spans 6kb (m.7777- 13794) of the mitochondrial genome. Therefore, mitochondrial protein expression was assessed to detect alterations between the two IPSc lines. Lysates were obtained from low and high heteroplasmy IPScs,followed by western blot to observe protein levels of mitochondrial complex subunits. The following mitochondrial complex subunits were analysed: Complex I (NDUFB8), complex II (SDHA), complex IV (MTCO1) and complex V (ATP50) (Figure 5.1). Protein expression was semi-quantitated against alpha-tubulin loading control.
These results demonstrated that IPScs containing a higher heteroplsmy (~42% mtDNA deletion) displayed a significant decrease in Complex I and Complex IV protein expression level, , in comparison to IPScs with lower heteroplasmy (<10%) (Figure 5.1A-B). These findings demonstrate that differences in heteroplasmy cause a mitochondrial phenotype of reduced oxidative capapcity due to the low expression of the key proteins within the mitochondrial respiratory chain.
Figure 5.1. Respiratory chain protein expression in low and high heteroplasmy iPS cells. A) Western blot analyses of Complex I, II, IV and V of the respiratory chain via staining with NDUFB8, SDHA, MTCO1, and ATP50 antibodies. The downregulation of Complex 1 (NDUFB8) and Complex IV (MTCO1) can be observed within IPSc harbouring large scale mtDNA deletion (A.2 ~42% mtDNA deletion). All quantification was normalised to alpha-tubulin loading control. B) Quantification of western blot was carried on Fiji, where relative protein intensities were normalised to alpha tubulin loading control. Results demonstrate a decrease in protein level of Complex I and Complex IV, though these changes were not significant (N=2). Kruskal Wallis one way ANOVA tests were carried out with post Dunn’s test to compare the expression of each protein between complex as well as cell lines. Re-check significance
184.108.40.206 Mitochondrial membrane potential in large scale mtDNA deletion IPSCs vs Neurons
Resting mitochondria sustain ion transfer within the IMM. Protons pumped from the mitochondrial matrix into the IMS whilst electrons are transferred through the ETC, creates a mitochondrial membrane potential (ΔΨm) which is harnessed by the F1/F0 ATPase to produce ATP. The development of membrane potential reliant fluorescent stains have become a robust method to assess ΔΨm. TMRM, a fluorescent lipophilic cationic dye is efficient for this purpose (Tehrani et al., 2018).
To observe alterations in mitochondrial membrane potential between both cell lines, and to confirm whether a decrease in these values were observed in these cell lines, Russell et al., (2018) quantified the level of mitochondrial membrane potential with the IPScs. These findings demonstrated that TMRM fluorescence was slightly higher within IPScs with high heteroplasmy (~53%), in comparison to the low heteroplasmy IPSc (Figure 5.2A), although this change was not significant.
This study observed alterations in TMRM intensity in differentiated neurons (8 days maturation) of these cell lines, in order to understand whether neurons also expressed a similar mitochondrial phenotype detected in the IPSCs. Both cell lines were differentiated into neurons by expression of NGN2, followed by quantification of TMRM intensity. NGN2 neurons harbouring a higher level of deletion showed a slight decrease in TMRM intensity, suggesting a slightly perturbed mitochondrial membrane potential in comparison to the low heteroplasmy neurons (Figure 5.2B). However, these changes were not significant, consistent with results from Russell et al. in the IPSCs.
Figure 5.2. Mitochondrial membrane potential analysis in large scale deletion IPSc and neurons. A) IPSC mitochondrial inner membrane potential as visualised by TMRM fluorescence, demonstrates increased TMRM fluorescence in patient line harbouring increased (53%) mtDNA deletion (Figure taken with permission from Russell et al., 2018). B) TMRM fluorescence withinNGN2 derived neurons after 8 days maturation, where conversely a decreased TMRM fluorescence is observed within patients harbouring ~42% mtDNA deletion. Number of axonal processes analysed for low heteroplasmy neurons (N=230) and high heteroplasmy neurons (N=250). No significant difference in observed in ∆Ψm between low and high heteroplasmy IPSc and neurons.
5.4.2 Differentiation of NGN2NGN2 stem cells into dopaminergic neurons
In addition to characterising mitochondrial function within these patient cell lines, differentiated low (<10%) and high (<40%) heteroplasmy NGN2 neurons were studied to understand the effect of heteroplasmy has on mitochondrial trafficking. Following the protocol described by Russell et al., 2018, IPSCs were successfully differentiated into a heterogeneous population of neurons harbouring two levels of a large scale mtDNA deletion.
As the aim of this thesis is to understand mitochondrial trafficking within PD. The study of mitochondrial trafficking in DAergic neurons from these lines is essential. As the NGN2 neurons are a heterogeneous population of varying neuronal subtypes, efforts to drive differentiation of a DAergic-only neuronal subtype from these lines were attempted. Indeed, formation of midbrain DAergic neurons involves the addition of different growth factors and supplements at various stages of neuronal growth and differentiation (Table 5.4). Different growth factors and supplements were added throughout NGN2 neuronal development in accordance to the Krik’s protocol (Kriks et al., 2011), to support DAergic neuronal differentiation of low heteroplasmy neurons. (Figure 5.3).
The 3 main factors added to proliferation media were sonic hedgehog (SHH), Purmorphamine and ChIR99021 (CHIR). SHH is a morphogen promotes DAergic neuron formation along the dorsoventral axis, though this axis formation does not occur in cell culture. Purmorphamine acts as an SHH agonist and therefore enhances DAergic neuron formation. The addition of ChIR, allows the reprogramming of embryonic stem cells via WNT-signalling (Table 5.4). Growth factors were taken from Kriks et al., (2011) protocol. Finally, doxycycline was added to the proliferation media with and without factors to drive NGN2 expression (Figure 5.3).
Table 5.4. Supplements used in NGN2 dopaminergic differentiation
|Sonic hedgehog (shh)||100 ng /ml||Signals for DAergic neuron formation along dorsoventral axis (PM)|
|Purmorphamine||2 μM||SHH agonist (PM)|
|Chir – 99021 (CHIR)||3 μM||It allows reprogramming of ES cells. It has also shown to activate WNT-signalling and induce formation of floor and roof plate (PM)|
|DAPT||10 μM||γ-secretase inhibitor; inhibits notch signalling which increases DAergic neurons (DM)|
|Ascorbic acid||0.2 mM||Cell survival (DM)|
|Brain derived neurotrophic factor (BDNF)||20 nM||Survival and morphological differentiation of neurons (DM)|
|Fibroblast Growth factor 8 (FGF8)||100 ng/ ml||Survival of DAergic neurons, also involved in signalling via dorsoventral axis (DM)|
|Glial cell line derived neurotrophic factor (GDNF)||20 ng/ ml||Survival and morphological differentiation of neurons (DM)|
|Dibutyryl c-amp||0.5 mM||Enhances neuronal maturation (DM)|
The factors added to the differentiation media included; BDNF, GDNF, FGF8, DAPT, L-Ascorbic acid and (db)–cAMP growth factors (Table 5.4). FGF8 promotes the survival of DAergic neurons and also further signals along the dorsoventral axis in the brain. DAPT inhibits NOTCH signalling, which in turn increases DAergic neuron formation. L-Ascorbic acid further promotes cell survival. BDNF, GDNF and cAMP factors were all added to enhance neuronal maturation (Figure 5.3).
Tyrosine hydroxylase (TH) catalyses the conversion of L-tyrosine to L-3,4-dihydroxyphenylalanine (L-DOPA), using molecular oxygen (O2) As L-DOPA is a precursor for DA, TH was selected as a marker for DAergic neurons. Once NGN2 neurons had reached 7 days of maturation, differentiated neurons were fixed stained with MAP2 (Microtubule associated protein 2) to identify neuronal processes, and Hoechst to stain nuclei, followed by subsequent imaging.
Figure 5.3. Conditions for DA neuron differentiation – Supplements added to proliferation and differentiation media, when differentiating neurons into DA neurons (Protocol adapted from Kriks et al., 2011)
Neurons were grown under 4 different conditions; Condition number 1, followed the standard differentiation protocol with the addition of supplements that are known to enhance DA neuron formation (Kriks et al., 2011) during proliferation and differentiation of NGN2 neurons. Condition number 2 followed the standard protocol by Russell et al., 2018, without the addition of any factors, condition 3 does this and condition does that. Condition 3 entails a protocol that does not involve the addition of doxycycline during proliferation and all growth supplements during differentiation. Condition 4 comprises the same protocol as condition 3 thought with the addition of doxycycline.
TH staining was used to observe whether DAergic neurons were being generated by these various protocols which showed that DAergic neurons were only formed under conditions 1 and 2, but not 3 and 4 (Figure 5.4).
Furthermore, as experiments proceeded with culturing neurons under conditions 1 and 2, it was found that the addition of DAergic supplements (as observed in Condition 1) were not reproducible, due to neuronal death (data not shown). On the first day of NPC culture, DAergic growth factors; SHH, Purmorphamine and CHIR were added and maintained over the 72 hour NPC culture. Within the dish, NPCS are seeded to be densely packed, in order to inhibit their spontaneous differentiation into neurites and enhance proliferation. The addition of these factors perturbed NPC growth, disrupted their distribution and altered cell morphology. This resulted in low yield of surviving and viable NPCs primed for differentiation. Growth condition 2 (Russell et al., 2018 protocol) was then selected due to the presence of DAergic neurons after differentiation, but also due to efficiency of the neuronal differentiation compared with other conditions.
Figure 5.4. Low and high heteroplasmy DA neuron differentiation – A-B) DAergic neurons are observed in growth conditions 1 and 2 due to bright TH staining (red) and MAP2 (green) staining C) Condition 3 – indicate no TH-positive or MAP2 staining suggesting no neurons are formed D) Condition 4 shows no generation of neurons, due to cell death. E) Repeat differentiation using condition 2 of high heteroplasmy neurons exhibit TH-positive neurons indicative of DAergic subtypes Images taken at x 20 magnification.
Furthermore, to ascertain the proportion of DAergic neurons in this NGN2 heterogeneous neuronal population TH- positive neurons were counted. TH-positive neurons within this neuronal population were compared against MAP2 and Hoescht staining. Analysis revealed that low (<10%) and high (43.7%) heteroplasmy neurons showed 56.5% and 75.4% MAP2 expression and 38.3% and 52.5% TH-positive staining respectively (Figure 5.5). This result indicates that neurons harbouring a higher level of mtDNA deletion, generated more neurons in comparison to the lower heteroplasmy line, as well as exhibiting increased TH expression indicative of DAergic neurons.
Figure 5.5. TH-MAP2 expression in low and high heteroplasmy neurons. Low heteroplasmy harbour a <10 % mtDNA deletions, in comparison to high heteroplasmy neurons which demonstrates a 43.7% mtDNA deletion. Quantification of TH and MAP2 neurons indicate that in low heteroplasmy neurons 38.3% and 56.5% are TH and MAP2 positive. This is increased in high heteroplasmy neurons which express 52.5% and 75.4% TH and MAP2 positive staining.
Following this quantification, immunofluorescence was conducted on these neurons to compare the levels of other neuronal subtypes within the generated population. In addition to quantifying DAergic (TH antibody staining) expression, GABAergic (Gamma-aminobutyric acid) (GABA antibody staining) and glutamatergic (VGLUT1 antibody staining) positive neurons were also counted to understand other neuronal subtype expression within a heterogeneous neuronal population.
Quantification of 2145 low heteroplasmy neuronal cell bodies were analysed for the neurotransmitter triad: TH, GABA, and VGLUT1 staining which demonstrated that 96% of neurons expressed TH, 88% were GLUT positive and 82% were GABAergic positive (Figure 5.6). Despite a fully homogenous DAergic neuronal population could not be generated from these experiments, it is evident from that over 90% of NGN2 neurons express TH.
The aim of this experiment was to observe whether NGN2 neurons could produce DAergic neurons, and therefore as this was confirmed in the immunofluorescence studies of the low heteroplasmy cell line, it was not repeated in the high heteroplasmy cell line, as both cell lines are derived from the same parent cell line and therefore it was assumed there would not be much variation.
Figure 5.6. Quantification of neuronal differentiation. Tyrosine hydroxylase, glutamatergic and GABAergic quantification of low heteroplasmy NGN2 neurons. Co-localisation analysis measured as a percentage against total number of nuclei. Increased TH expression in NGN2 neurons (96%), followed by glutamatergic expression (88%) and GABAergic expression at (82%), where error bars represent mean +/- SEM. (N = 358 cell bodies). Images were again taken at x20 magnification.
As a large number of overlap in staining was observed, the colocalisation of these antibodies within these neurons were observed. This study counted the colocalisation of TH-GABA-GLUT, TH-GABA, TH-GLUT and GABA-GLUT staining within 499 low heteroplasmy neuronal cell bodies.
Results revealed that the colocalisation of these neurotransmitters within a heterogeneous neuronal population demonstrate large variability. The highest antibody colocalisation within these neurons were observed in the TH-GABA-GLUT cohort (Figure 5.8(, showing that 76.5% of cell bodies expressed TH-GABA-GLUT staining , followed by TH-GABA (Figure 5.9) colocalisation at 13.22% of neurons. TH-GLUT and GABA-GLUT colocalisation was counted as 0.6 % and 2.8% respectively (Figure 5.7 and 5.8). All counts were carried out as a percentage against total number of nuclei.
Therefore, once neuronal subtypes were quantified, a heterogeneous population of neurons was used in all future experiments, where differentiation of the high heteroplasmy neurons were carried out following the standard protocol where neurons harboured ~43% mtDNA deletion. From here onwards, the differentiation protocol described by Russell et al., (2018) (Condition 2) was used to generated neurons harbouring a large scale mtDNA deletion to image mitochondrial trafficking (See Chapter 6).
Figure 5.7. TH—GABA-GLUT colocalisation analyses. TH-GABA-GLUT colocalisation is 76.5%, followed by TH-GABA colocalisation at 13.22% of neurons. TH-GLUT and GABA-GLUT colocalisation is 0.6 % and 2.8% respectively. Analysis was conducted in low heteroplasmy cell bodies. (p<0.001)
Figure 5.8. Quadruple neuronal staining of low heteroplasmy neurons – Co-localisation of TH, Glutamatergic and GABAergic neurons. White arrow heads indicate colocalised staining of TH-GABA-VGLUT1 cell bodies. Images taken at x 20 magnification.
Figure 5.9. TH-GABA localisation in low heteroplasmy neurons – Co-localisation of TH and GABAergic neurons. White arrow heads indicate colocalised staining of TH and GABA positive staining. Images taken at x 20 magnification.
5.5.1 Summary of results
- Western blot analysis demonstrated that IPScs harbouring a large scale mtDNA deletion exhibit a moderate but not significant downregulation of mitochondrial complex I and IV but not complex V compared with isogenic control
- Differentiated neurons derived from low and high heteroplasmy IPScs showed a heterogeneous neuron population containing TH-expressing neurons indicate of DAergic neurons
5.5.2 Large scale mtDNA deletion IPScs exhibit dysfunctional mitochondrial phenotype
This study manipulated a induced pluripotent stem cell model harbouring a large scale mtDNA deletion (spanning 6017 base pairs from m.7777-13794, which removes COXII, ATPase 6 and 8, COXIII, ND3, ND4L, ND4, ND5 and part of ND6 and several tRNAs. Deletions are the most common mtDNA mutation type observed in substantia nigra neurons, which are affected in PD. In order to comprehend the level of respiratory chain deficiency within a cell line harbouring an mtDNA deletion for future experiments (See Chapter 7) both disease and control IPScs were analysed for OXPHOS protein expression by western blot. Furthermore, their differentiated neuronal forms
COX deficiency is observed in the SNpc is caused specifically by mtDNA deletions reaching up to 50% heteroplasmy, while point mtDNA point mutation accumulation have not been (Bender et al., 2006;Kraytsberg et al., 2006), (Reeve et al., 2009; Taylor et al., 2003). Therefore, an mtDNA deletion cell line was used in this study as opposed to inducing mitochondrial damage via a complex inhibitor i.e. Rotenone or MPTP is physiologically relevant. This cell line can provide a disease model for PD patients harbouring pathogenic mtDNA deletions, allowing insight into the workings of this disease and how mtDNA deletions can affect the severity of the disease.
Though it is largely considered that human derived IPSCs rely primarily on ATP production via glycolysis (Cherry et al., 2013), a switch from utilising glycolysis to generating ATP via OXPHOS has been reported during IPSc differentiation (Mandal et al 2011). The mtDNA deletion described in this cell line interrupts the mitochondrial complex I NADH dehydrogenase genes ND4 and ND5, which are required for translation of all products of the mitochondrial genome (encoding components of OXPHOS complexes I, III, IV, and V). Having already established a decrease in both OCR and ECAR after FCCP treatment, Russell et al., 2018 characterised this cell line with a reduced capacity to utilise oxidative respiration. This biochemical deficiency was further investigated in this study in order to demonstrate respiratory chain deficiency within the high heteroplasmy cell line (for subsequent experiments), showing that both complex I (NDUFB8) and IV (MTCO1) were down regulated determined by western blot analyses.
Interestingly, however, mitochondrial membrane potential (Δψm) analyses conducted within low and high heteroplasmy IPSCs (Russell et al., 2018) and neurons in this study did not display any significant changes between lines. TMRM fluorescence was slightly increased in higher heteroplasmy IPSCs, where the opposite was observed within the higher heteroplasmy neurons of this study. A constant Δψm is crucial in maintaining healthy mitochondria, and though perturbed Δψm in IPSCs and neurons are observed in this study, these remain insignificant. This finding could perhaps be due to the upregulation in Complex II (SDHA) observed in the high heteroplasmy cells, which compensates for the impairment of Complex I (NDUFB8) and IV (MTCO1) in the respiratory chain. This alternative mechanism means to shuttle electrons into the respiratory chain, facilitating the maintenance of the inner membrane electrical potential of mitochondria. Furthermore, upregulation is simultaneously observed within the Complex V (ATP50) subunit, indicating that Complex V could be generating more ATP to balance the electrical potential of the mitochondrial within neurons harbouring a large scale mtDNA deletion.
5.5.3 Using IPSc with a mitochondrial dysfunction as Parkinson’s disease model
Whist the generation of DAergic IPScs from PD patient lines provides a powerful tool to understand PD pathology, the traditional protocol to generate these midbrain DAergic neurons, requires a long differentiation time period. This is because it is crucial to create the correct biological milieu for these DA neurons via the addition of DAergic growth factors. Also the cell lines studied in these experiments were readily available, and were provided by Dr Oliver Russell
Several studies have utilised PD- patient IPScs, some of which now successfully demonstrate disease-specific phenotypes in differentiated neurons (Nguyne et al., 2011, Siebler et al., 2011,Cooper et al., 2012, Jiang et al., 2012, Sanchez-Danes et al., 2012), where this study attempted to observe the effect of perturbed mitochondrial function in PD. This study therefore attempted to generate DAergic IPSc-derived NGN2 neurons from mitochondrial disease patient harbouring a large scale mtDNA deletion, to elucidate whether mtDNA deletions may be crucial in DAergic neuronal loss exhibited in ageing and PD (Kraystbergs et al., 2006, Bender et al., 2006).
As the focus of this PhD project is to observe mitochondrial trafficking within PD, this study attempted to culture DAergic neurons from a large scale mtDNA deletion cell line for the first time. Initial experiments were carried out to differentiate neurons into a homogenous DA population. It was found that though DAergic neurons were not this study could not generate a homogeneous TH positive population.
Furthermore via protocols previously established, directed neuronal growth towards a DAergic subtype were conducted using morphogens such as sonic hedgehog (SHH) and fibroblast growth factor-8a (FGF8a) (Cooper et al., 2010), where further neuronal maturation was achieved through the addition of ascorbic acid, BDNF, GDNF and cAMP (Schneider et al., 2007,Chambers et al, 2009, Cooper et al., 2010). The selection of which growth factors were added at the NPC stage were trialled, with SHH, CHIR and Purmorphamine being added to aid DAergic growth during neuronal proliferation and L-ascorbic acid, FGF8, and C-AMP were added to promote DAergic neuronal survival from NPCS to neurons. The fine tuning of these supplements such as SHH, CHIR and Purmorphamine are critical in generating DAergic neurons. Studies have shown that increased CHIR expression can create hindbrain neurons, and contrastingly, little CHIR can cause neurons to undergo a rostral-directed differentiation (Kirkeby et al., 2012). The concentration of supplements used within the Kriks et al., (2001) and Hartfield et al., (2014) protocols were replicated in this study, where sole DAergic neuronal population could not be generated. Therefore, protocols that have been already optimised within a cell line, cannot always be transferred to use on another other cell line, where often further validation is required with varying concentrations of these supplements in order to combat the failure to generate a homogeneous DAergic neuronal population (Boulting et al., 2011, Devine et al., 2011).
Unfortunately, this study could not culture a homogenous DAergic population, with the addition of these factors, most likely dependant on the NGN2 differentiation protocol, which generates neurons within 8 days from NPCs. Perhaps, as many studies report that a longer time period is required to actually successfully culture these neurons, not enough time was allowed to generate DAergic neurons and therefore only created adverse effects on the cells. Rock inhibitor prevents apoptosis and in this study, even in the presence of ROCKi, not enough NPCs survived, as the DA supplements caused large amounts of cell death. As the focus of this project was to observe the impact of mtDNA deletions in mitochondrial trafficking in PD, it was decided that the heterogeneous population of neurons would be utilised in experiments, containing a largely TH-positive population harbouring a large scale mtDNA deletion.
5.5.4 NGN2 immunofluorescence colocalisation analyses
The proneural gene neurogenin 2 (NGN2), is a transcription factor crucial for neuronal differentiation but also (Fode et al., 1998), for the generation of neuronal subtype-specification within the CNS (Ma et al., 1999; Scardigli et al., 2001). Within the ventral midbrain (VM) region, NGN2 expression is crucial to mesencephalic DAergic neurons generation, showing that NGN2 plays a role in DAergic neuron development (Thompson et al., 2006). Knockout NGN2 mice have demonstrated a reduction in DA neuron number in the developing ventral midbrain in comparison to wild type postnatal mice, which suggests that, in the VM, NGN2 is involved in specific mesencephalic DAergic neuronal differentiation. As over 90% of the NGN2 neuronal population in this study stained positive for TH, it may be possible that NGN2 actually facilitates DAergic neuronal development in this cell line, without the addition of DAergic supplements (Andersson et al., 2005).
Immunofluorescent analysis of these large scale mtDNA deletion neurons revealed the co-localised staining of TH, GABA and Glutamatergic neurons. This is interesting, in regards to our understanding of the nigrostriatal pathway, where DAergic and GABAergic neurons have been known to partake in the nigrostriatal pathway (GonzaÂlez-HernaÂndez et al., 2001). Further investigation into this pathway has demonstrated that a nonDAergic nigrostriatal pathway exists within the substantia nigra reticula which utilises GABA (Fibiger et al., 1972; Maler et al., 1973; van der Kooy et al., 1981; Swanson, 1982; Gerfen et al., 1987; Hattori et al., 1991, Rodríguez & González‐Hernández, 1999).Experiments revealed that within rat nigrotectal neurons the co-expression of TH and GAD (involved in GABA synthesis, Campbell et al., 1991) were found (GonzaÂlez-HernaÂndez et al., 2001) (Figure 5.9). Therefore, take together, a particular proportion of DAergic nigrostriatal neurons express GABA and if differentiated NGN2 neurons favour developing mesencephalic neuronal subtypes, it can be hypothesised that the overlap in immunostaining could be predicted, as the neurophysiology of the nigrostriatal pathway comprises of this colocalisation.
Whilst TH and GABA have been found to co-localise in these studies, the co-expression of VGLUT1 and GABA was also observed. Abundant co-expression of VGLUT1 and GABA has been observed in cortical and hippocampal glutamatergic synapses (Kao et al., 2004; Fattorini et al., 2009; Zander et al., 2010), where electrophysiological experiments hypothesise that GABA is co-released from glutamatergic hippocampal mossy fibre terminals (MFTs) during postnatal development (Walker et al. 2001; Gutiérrez 2003; Safiulina et al., 2006). GABA functions as an inhibitory neurotransmitter and the GABAergic signalling machinery arises earlier than glutamatergic transmission; GABA synthesis occurs from glutamate via glutamate decarboxylase (GAD). During GAB synthesis, glutamate (an excitatory neurotransmitter) is converted into GABA (an inhibitory neurotransmitter) (Petroff, 2002, Schousboe, 2007). Therefore the colocalisation of GABA and VGLUT1 observed in this experiment may be causative of immature excitatory glutamatergic neurons that are still undergoing the conversion into GABAergic neurons. And thus, it can be concluded that the large overlap in TH-GLUT-GABA colocalisation observed in this study is a combination of the NGN2 preference towards mesencephalic neuronal derivation and immature GABAergic neurons.
Figure 5.9. Mesostriatal projections. There are supposedly three mesostriatal pathways. The first DAergic pathway originates from DAergic midbrain nuclei (blue), the second GABAergic pathway (red) is derived from GAD67 positive neurons in the substantia nigra pars reticula (SNR) and finally the DAergic/GABAergic pathway (green) which arises from DAergic neurons (GAD65 mRNA positive) in the substantia nigra pars compacta (SNC) and the ventral tegmental area (VTA). (Image adapted from GonzaÂlez-HernaÂndez et al., 2001)
5.6 limitations of this STUDY
- Concentrations of the growth factors added to promote DAergic neuronal growth were taken from the Kriks et al., protocol, which were reproduced in this study. It may have been that incorrect supplement concentrations were being administered to the NGN2 neurons, and therefore may have perturbed NPC differentiation. However, as this aim of this project is to observe mitochondrial movement in an IPSC model harbouring a mitochondrial defect, it was decided not to optimise this protocol due to time constraints, and continued to observe mitochondrial trafficking in a heterogeneous population of neurons, as NGN2 supports DAergic neurons.
- The overlap in the colocalisation of these neurotransmitters, firstly rendered the generation of a homogenous DAergic population a practically challenging task, from which the further quantification of the three neuronal subtype expression even more of an intricate challenge. Therefore, it was decided that both low and high heteroplasmy cell lines would be differentiated into a heterogeneous neuronal population for all future experiments.
5.7 ConclusionS & FUTURE WORK
These studies confirms decreased mitochondrial complex I and complex IV activity in the high heteroplasmy IPSc in comparison to low heteroplasmy IPScs. This model provides as a useful tool that provides insight to PD patients, since large scale mtDNA deletions are seen in affected neurons.
Future work in this study:
- Observe whether mitochondrial respiratory chain complex I and IV levels are altered within short and long term cultured neurons via western blot analyses
- Lentiviral transfection of a DA specific-GFP tag (either tyrosine hydroxylase or Dopamine transporter protein) into NGN2 IPSCs to highlight dopamine expressing neurons within a heterogeneous population of neurons during live cell image