Thesis (PhD)--Stellenbosch University, 2016ENGLISH ABSTRACT : Parkinson’s disease (PD) is a neurodegenerative movement disorder characterized by the loss of
dopaminergic neurons in the substantia nigra of the midbrain. Although the aetiology of PD is still not
fully understood, it is thought to involve a combination of environmental and genetic factors. To date,
a number of PD-causing genes have been found. The PINK1 gene is of particular interest for this study,
and mutations in this gene result in autosomal recessive inheritance of early onset PD. PINK1 plays a
vital role in mitochondrial quality control and homeostasis, and in its absence it is thought to result in
an accumulation of dysfunctional mitochondria in neurons, culminating in neuronal cell death. Whilst
pharmacological and surgical interventions are available for PD, the current options exhibit adverse
side effects with long term treatment. There is a great need to develop new treatments with i. less side
effects and ii. that can simultaneously target the multiple pathways associated with this disorder. One
molecule is curcumin, the core component of the curry spice turmeric, which is well known for its
antioxidant and anti-inflammatory properties and has already been studied for its possible
neuroprotective role in Alzheimer’s disease.
The aim of the present study was to create a cellular model of PD by decreasing the expression of
PINK1 in SH-SY5Y neuroblastoma cells. Thereafter, we aimed to test the protective effects of curcumin
on this model in the presence and absence of a known stressor, paraquat. This study also aimed to detect
possible copy number variation (CNV) in PINK1 (and other PD-causing genes) in a cohort of South
African patients with PD, in order to obtain patient-derived fibroblasts to verify the results obtained
from the original cellular model.
PINK1 was knocked down using siRNA (Qiagen, USA) in SH-SY5Y neuroblastoma cells, and the
knock down was verified by quantitative real time PCR (qRTPCR) and western blotting. Thereafter,
PINK1 siRNA cells and control cells were separated into four treatment groups: i. untreated, ii. treated
with 25μM paraquat for 24hours, iii. pre-treated with 2μM curcumin for 1hour then treated with 25μM
paraquat for 24hours, or iv. treated with 2μM curcumin for 1hour, and various parameters of cellular
and mitochondrial function were measured. Cell viability was measured by an MTT assay. Western blot
analysis was performed using cleaved PARP and full-length caspase 3 markers to detect levels of
apoptosis, and LC3-II and p62 markers to detect autophagic flux. Mitochondrial respiration experiments
were completed on the Seahorse XF Analyser using the Mito Stress Test Kit and the Glycolysis Stress
Test. Flow cytometry was utilised to measure mitochondrial membrane potential (MMP) using the JC-
1 fluorochrome, and mitochondrial network was analysed by fluorescent microscopy. For CNV
detection, MLPA was performed on 210 South African PD patients and putative mutations were verified
by qRTPCR on the Lightcycler 96.
PINK1 was successfully knocked down at a gene and protein expression level. The PINK1 siRNA cells
exhibited a significant decrease in cell viability (p=0.0036), and an increase in apoptosis (p=0.0144). A
decrease in PINK1 expression also resulted in significantly decreased MMP (p=0.0008), mitochondrial
respiration (p=0.0015), ATP production (p=0.002) and glycolytic capacity (p=0.0445). No significant
changes were observed in the connectivity of the mitochondrial network, but autophagic flux was
significantly increased in the PINK1 siRNA cells, as detected by increased LC3-II levels (p=0.0152). As expected, paraquat-treated cells exhibited decreased cell viability, increased apoptosis, decreased
MMP, autophagic flux, and a more fragmented mitochondrial network. Paraquat treatment therefore
successfully acted as a stressor on the cells. Curcumin pre-treatment followed by paraquat treatment
rescued cell viability in control cells (p=0.003), and significantly decreased apoptosis in PINK1 siRNA
cells (p=0.0018). Curcumin protected mitochondrial dysfunction in PINK1 siRNA cells by increasing
MMP (p=0.0472) and maximal respiration (p=0.0014), as well as significantly increasing MMP
(p=0.0307) and maximal respiration (p=0.032) in control cells. Additionally, curcumin treatment
resulted in increased autophagic flux (p=0.0017) in stressed control cells. These results highlight a
protective effect of curcumin against paraquat and against the damaging effects on the mitochondria in
cells with decreased PINK1 expression.
Lastly, MLPA analysis did not reveal any PINK1 CNV mutations in a total of 210 South African PD
patients, and fibroblasts were therefore not obtained. A number of false positive mutations were
identified that were not verified by qRTPCR. A common polymorphism M192L resulting in a false
positive PARK2 exon 5 deletion was found in a number of patients, all of whom were of Black or Mixed
Ancestry ethnic groups. One patient was shown to harbour a heterozygous deletion in PARK2 exon 4.
In conclusion, PINK1 siRNA-mediated knock down in SH-SY5Y neuroblastoma cells can be used as
a model of PD to study aspects of mitochondrial dysfunction. Furthermore, curcumin should be
considered as a possible therapeutic target for PD, as it exhibits protective effects against paraquat at a
mitochondrial level. Given the low toxicity of curcumin, and the fact that it is already part of a dietary
regimen in most populations worldwide, further studies on elucidating its biochemical and cellular
properties are therefore warranted. The use of natural compounds such as curcumin as therapeutic
agents is currently a topical and fast-growing area of research, and holds much promise for clinical
application in various diseases including neurodegenerative disorders such as Alzheimer’s disease and
PD.AFRIKAANSE OPSOMMING : Parkinson se siekte (PD) is 'n neurodegeneratiewe beweging versteuring wat gekenmerk word deur die
verlies van dopaminergiese neurone in die brein. Hoewel die etiologie van PD nog nie ten volle verstaan
is nie, is daar denke dat dit 'n kombinasie van die omgewing en genetiese faktore behels. Tot dus ver is
daar nog net ‘n aantal gene wat PD-veroorsaak gevind. Die PINK1 geen is van besondere belang vir
hierdie studie, en mutasies in dié geen veroorsaak outosomale resessiewe oorerwing van vroeë aanvang
PD. PINK1 speel 'n belangrike rol in die mitochondriale gehaltebeheer en homeostase, en in sy
afwesigheid is dit gedink om te lei tot 'n opeenhoping van disfunksionele mitochondria in die neurone,
wat kulmineer in neuronale sel dood. Terwyl farmakologiese en chirurgiese ingrepe beskikbaar is vir
PD, die huidige opsies wys duidelike newe-effekte met lang termyn behandeling. Daar is 'n groot
behoefte om nuwe behandelings te ontwikkel met i. minder newe-effekte en ii. wat gelyktydig die
verskeie paaie wat verband hou met hierdie versteuring kan teiken. Een molekule is curcumin, die hoof
komponent van die kerrie spesery borrie, wat wel bekend is vir, sy anti-oksidant en anti-inflammatoriese
eienskappe, en is reeds bestudeer vir sy moontlike beskermende rol in Alzheimer’s se siekte.
Die doel van hierdie projek is om 'n sellulêre model van PD te skep deur die vermindering van die
uitdrukking van PINK1 in SH-SY5Y neuroblastoom selle. Ons daarop gemik om die beskermende effek
van curcumin te toets in die teenwoordigheid en afwesigheid van 'n bekende stressor, parakwat in ons
model. ‘n Additionele doelwit is om moontlike kopiegetal variasie (CNV) in die PINK1 gene (en ander
PD veroorsaakende gene) op te tel in 'n groep van die Suid-Afrikaanse pasiënte met PD. Die doel van
hierdie was om pasiënt-afgeleibare fibroblaste te kry om die resultate te verifieer vanuit die
oorspronklike model.
SH-SY5Y neuroblastoom selle was gekweek, en PINK1 is platgeslaan deur gebruik te maak van siRNA
en HiPerfect Transfectie Reagens (Qiagen, VSA). Klop van PINK1 is bevestig deur kwantitatiewe real
time PCR (qRTPCR) en westelike klad. Daarna, PINK1 siRNA selle en beheer selle was óf i. nie
behandel nie, ii. behandel met 25 um paraquat vir 24 uur per dag, iii. vooraf behandel met 2μM curcumin
vir 1 uur dan behandel met 25 um paraquat vir 24 uur per dag, of iv. behandel met 2μM curcumin vir 1
uur, en verskeie parameters van sellulêre en mitochondriale funksie is gemeet. Lewensvatbaarheid van
die selle is gemeet deur 'n MTT toets. Westerne klad analise is uitgevoer met behulp van gekleefde
PARP en vollengte caspase 3 merkers om die vlakke van apoptose te meet, en LC3-II en p62 merkers
was gebruik om autophagic vloed op te spoor. Mitochondriale respirasie eksperimente is voltooi op die
Seahorse XF Analyser met behulp van die Mito Stres Test Kit en die Glikolise Stres Toets.
Vloeisitometrie is gebruik om mitochondriale membraan potensiaal (MMP) te meet met behulp van die
JC-1 fluorochrome en die mitochondriale netwerk is geanaliseer deur fluorescent mikroskopie. Vir
CNV opsporing, was MLPA uitgevoer op 210 Suid-Afrikaanse PD pasiënte en vermeende mutasies is
bevestig deur qRTPCR op die Lightcycler 96.
PINK1 is suksesvol platgeslaan op 'n geen en proteïen uitdrukking vlak. Die PINK1 siRNA selle betoon
'n beduidende afname in lewensvatbaarheid sel (p = 0.0036), en 'n toename in apoptose (p=0.0144). 'n Afname in PINK1 uitdrukking het ook daartoe gelei na ‘n beduidende vermindering in MMP
(p=0.0008), mitochondriale respirasie (p=0.0015), ATP produksie (p=0.002) en glikolitiese kapasiteit
(p=0.0445). Geen beduidende veranderinge is waargeneem in die verbinding van die mitochondriale
netwerk nie, maar autophagic vloed het aansienlik toegeneem in die PINK1 siRNA selle, soos
waargeneem deur verhoogde vlakke in LC3-II (p=0.0152).
Soos verwag betoon, paraquat behandelde selle ‘n afname in sel lewensvatbaarheid, verhoogde
apoptose, afname in MMP, autophagic vloed, en 'n meer gefragmenteerde mitochondriale netwerk.
Parakwat behandeling het dus suksesvol opgetree as 'n stressor op die selle. Curcumin voorafbehandeling
gevolg deur paraquat behandeling het sel lewensvatbaarheid gered in beheer selle
(p=0.003), en aansienlik verminderde apoptose in PINK1 siRNA selle (p=0.0018) betoon. Curcumin
beskerm mitochondriale disfunksie deur die verhoging van MMP (p=0.0472, p=0.0307) en maksimale
respirasie (p=0.0014, p=0.032) in beide PINK1 siRNA en beheer selle. Additioneel, het curcumin
behandeling gelei tot ‘n verhoogde autophagic vloed (p=0.0017) in onderdrukte beheer selle. Hierdie
resultate beklemtoon die beskermende effek van curcumin teen parakwat en teen die skadelike resultaat
op die mitochondria in die selle met verlaagde PINK1 uitdrukking.
Laastens, MLPA ontleding het nie PINK1 CNV mutasies openbaar in 'n totaal van 210 Suid-Afrikaanse
PD pasiënte, en fibroblaste is dus nie verkry nie. 'n Aantal vals positiewe mutasies is geïdentifiseer wat
nie geverifieer is deur qRTPCR. 'n Algemene polimorfisme M192L is in 'n aantal pasiënte gevind wat
in 'n vals positiewe PARK2 ekson 5 eliminasie ontaard, waarvan almal swart of gemengde afkoms etiese
groepe is. Een pasiënt het getoon dat 'n heterosigotiese eliminasie in PARK2 ekson 4 bevind is.
Ten slotte, PINK1 siRNA-gemedieerde wat platgeslaan is in SH-SY5Y neuroblastoom selle kan gebruik
word as 'n model van PD om aspekte van mitochondriale disfunksie te bestudeer. Verder moet curcumin
beskou word as 'n moontlike terapeutiese teiken vir PD, omdat dit beskermende effekte teen parakwat
op 'n mitochondriale vlak vertoon. Gegewe die lae toksisiteit van curcumin en die feit dat dit reeds ‘n
deel vorm van die dieët in meeste populasie groepe wêreldwyd, is verdere studies op die biochemiese
en sellulêre eienskappe daarvan benodig. Die gebruik van natuurlike komposisies, soos curcumin as ‘n
terapeutiese middel is tans ‘n relevante en vinnig groeiende area van navorsing en toon baie belofte vir
kliniese toepassing in verskeie siektes soos Alzheimer’s siekte en PD