|Ph.D Student||Bergman Oded|
|Subject||Impaired Assembly of Complex l of the Respiratory Chain as|
a Possible Cause for Mitochondrial Dysfunction in
|Department||Department of Medicine||Supervisor||Professor Emeritus Dorit Ben-Shachar|
|Full Thesis text|
Mitochondrial respiratory chain impairments including complex I (CoI) deficits have been observed in schizophrenia (SZ). Our group has previously shown abnormalities in CoI nuclear encoded subunits NDUFV1, NDUFV2 and NDUFS1 in SZ patients. Interestingly, these subunits are labile, meaning that during CoI homeostasis newly imported subunits interchange existing ones in the holo-complex. We hypothesize that defects in holo-CoI homeostasis play a crucial role in mitochondrial dysfunctions in SZ.
We studied Epstein-Barr virus transformed lymphocytes from SZ patients and healthy subjects. Reduced CoI-driven respiration has been observed in SZ-permeabilized cells, indicating a deficit specific to CoI. Isolated holo-CoI activity was also reduced in patients, indicating the impairment is in CoI per-se. CoI synthesis rate was decreased in patients, with no change in its mitochondrial DNA encoded subunits. Although the level of CoI labile subunits did not differ in the isolated complex between patients and controls, they showed reduced variability suggesting lower turnover in patients. In addition, cytosolic and mitochondrial levels were altered in SZ. This discrepancy between altered level in cytosolic and mitochondria, with no change in the isolated complex, also suggests altered turnover rate of these labile subunits. Mitochondrial cAMP, which inhibits the degradation of these subunits, showed higher levels in SZ, further supporting a lower turnover rate. As the later may stem from impaired import, we studied this process focusing on NDUFV2. Our results show impaired NDUFV2 mitochondrial import in which the origin of protein and mitochondria contributed to the import deficits in an additive manner. The translation machinery of both cohorts was artificial, indicating that the impaired import stems from the NDUFV2 transcript. Sanger sequencing showed no difference between SZ and controls. However, sequence quality was consistently reduced in SZ with an apparent mixture of PCR products, 2 associated with lower PCR products level. We analyzed the sequence by additional approaches and found no difference.
RNA modifications can interfere with RT enzymes and hamper cDNA synthesis. We hypothesized that transcript modification can cause the abnormalities observed in the RT-PCR products. Based on the prior sequencing reactions, we identified a suspected modification site around 359bp downstream to the ATG. Utilizing RT reactions at high and low dNTPs concentration followed by PCR (RTL-P), we validated the presence of the modification, in both cohorts. Notably, in the control samples, modification level showed a strong and significant correlation with NDUFV2 cytosolic pre-protein level, suggesting that the modification has a functional significance in translation. Such a correlation was absent in the SZ cell lines, suggesting either the involvement of additional factors in the translation or that a deviation from an optimal level of modification can impair translation. We believe mRNA post-transcriptional modifications may represent a global phenomenon in SZ.
The current study proposes CoI and its labile subunits as important players in mitochondrial dysfunction, further supporting mitochondria as a pathological factor in SZ. The findings on CoI opens the road for future research that may identify new targets for intervention in SZ and lead to better understanding of the pathophysiology of the disorder.