|dc.description.abstract||Parkinson’s disease (PD) is the second largest and progressive neurological disorder caused by selective loss of dopaminergic neurons in substantia nigra pars compacta. Familial PD can be attributed to mutations in different genes. Genetic, environmental and unknown factors may interplay and underlie idiopathic forms of PD. Research on PD is confronted by the long preclinical phase of disease, clinical and genetic heterogeneity and incomplete understanding of disease pathogenesis. It is, therefore, critical to understand the molecular basis of disease, and identify biological biomarkers to diagnose before the onset of irreversible loss of neurons. The current project focusses on identifying mutations in known PD genes to comprehend the underlying molecular mechanisms through genetic and functional studies, and identifying a panel of small molecules which can be used as “probes” to differentiate PD mutations and/or PDs and controls through multidimensional screening. All the experiments described in this project were carried out on human olfactory neurosphere derived (ONS) cells which are primary, unmodified cells previously used to model PD and to identify biologically active small molecules.
First, ONS cells from control and sporadic PD patients were screened for copy number variation in known PD genes by MLPA (multiplex ligation dependent probe amplification) which identified heterozygous deletions in the ONS cells of two sporadic PD patients i.e. 2704 (PARK2 ex 02), and 2509 (PARK2 ex 5-7). The later exhibited heterozygous deletions in the adjacent exons spanning introns 4 and 7. Whole exome sequencing for this sample did not identify additional disease causing mutations in any putative Parkinsonism-related gene.
Alternative splice variant analysis of PARK2 identified four novel alternative splice variants (SV ex 4-7, SV ex 5-7, SVex 5-8 and SVex 3-9) in 2509 (PARK2 ex 5-7) and another PD ONS cell line. Qualitative analysis of PARK2 splice variants showed more alternative variants in the disease group than in the controls. Treatment of ONS cells with mitochondrial toxins, 50 nM rotenone and 10 uM CCCP also showed changes in splicing patterns of PARK2 and SNCA. Overall, this may suggest that splicing of PARK2 and SNCA can be exacerbated on exposure to mitochondrial stressors which can be quantified further by qPCR. Only the full length transcript was observed for DJ1 in the samples tested.
PD ONS cells, exposed to 10uM of the mitochondrial uncoupler CCCP, showed increased susceptibility to cell death, as determined by number of cells surviving treatment compared to control ONS. The SNCA full length transcript level was significantly higher in the LRRK2 G2019S containing cell lines on exposure to 50nM rotenone compared to the controls and idiopathic PDs. LRRK2 containing cell lines manifested an overexpression of SYT-11, increased LysoTracker and increased LC3b intensities; this may suggest autophagosomal and lysosomal impairments that may be linked to the ATP13A2/SYT11 pathway. SYT-11 along with ATP13A2, a lysosomal type 5 P-type ATPase, regulates lysosmal function, the autophagy pathway and alpha-synuclein clearance. The altered expression of PGC-1α may suggest impaired mitochondrial biogenesis in some PD cell lines. Interestingly, cell lines carrying mutations in different regions of PARK2 showed different responses to the toxins which may be due to different pathways involved.
Seven ONS cell lines carrying mutations in different PD genes (LRRK2 G2019S, LRRK2 R1441, PINK1 G411S, PARK2 ex 5-7, PARK2 x02, KCNJ15) were prepared for high content screening of cellular, nuclear, mitochondrial, lysosomal, endosomal and autophagosomal analysis. The statistical suitability of cytological profiling was determined by subjecting previous collected data-sets to combined analyses. We observed a classification accuracy of approximately 90% using the vehicle (DMSO) treated profiles alone from PDs and controls. The lysosomal, mitochondrial and LC3b parameters were found to be the most important variables for discrimination between disease and control cell lines. Future experiments may involve the profiling of early onset cases or by challenging the ONS cells with different toxins to identify different aetiological subtypes.
In the next phase, small molecules which showed some biological activity in one patient cell line were studied in an attempt to identify probes which help to differentiate between PD and control cell lines. This could also lead to the identification of cytological signatures corresponding to the mutations they harbour, or the specific cellular pathways effected, in particular. A probe panel of small molecules was identified by comparing PD mutant ONS and controls, which showed disease or mutation specific cytological profiles. Some small molecules identified in this study have been known previously for their neuroprotective effect e.g. curcumin, Dihydroergocristine mesylate, Iso-tetrandrine, radicicol, berbamine and oxyacanthine sulfate. Little functional evidence is available to ascertain their mechanism of action. Moreover, cytological profiling can be expanded to include other cellular organelles e.g. endoplasmic reticulum and Golgi complex and the results obtained in this study can be validated by using large number of samples.
In conclusion, this study identified novel PARK2 splice variants in ONS cells, the functional evidence of which can help to comprehend how alternative splice variants can lead to neurodegeneration in PD. The functional evidence on genetic forms of PD may propose mitochondrial and endo-lysosomal impairments which require further investigation. Cytological profiling also suggests that ONS cells from PD patients reflect subtle morphological differences in various cellular organelles which can be pronounced on treatment with stressors or with small molecules which can be used as biomarkers for PD.||