טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentIfhar Lee
SubjectMetabolism of Heme Compound in Schizophrenia
DepartmentDepartment of Medicine
Supervisor Professor Emeritus Dorit Ben-Shachar
Full Thesis textFull thesis text - English Version


Abstract

Background: Heme was found to be involved in neuropsychiatric disorders. Previous research suggests that heme oxygenase-1 (HO1), the heme catabolic rate- limiting enzyme, is overexpressed in the substantia nigra of Parkinson’s patients and the cerebral cortex of Alzheimer’s patients. Furthermore, decrease in heme is a hallmark of neuroporphyria, which often exhibits psychotic symptoms similar to schizophrenia (SZ). Previous data demonstrated that mitochondrial Complex I (Co-I) deficiency is involved in the pathophysiology of SZ. Since heme synthesis involves the mitochondria, we hypothesized that alterations in Co-I may affect heme metabolism and vice versa.  In this study, we seek to investigate heme metabolism in SZ and the involvement of mitochondrial Co-I in heme metabolism.


Method: Heme levels were fluorescently measured in cell lysate and isolated mitochondria of EBV transformed lymphocytes (lymphoblasts) derived from SZ patients and healthy subjects. HO1, heme synthesis rate limiting enzyme Aminolevulinic acid synthase-1 (ALAS1) and phosphorylation of eukaryotic initiation factor 2-alpha (peif2a/eif2a), a downstream target of heme, were analyzed by immunoblotting and qRT-PCR. In healthy derived lymphoblasts Co-I was inhibited by applying rotenone (10,25,50nM) for 24h, 48h, and 72h. Heme levels, mRNA and protein levels of HO1 and peif2a/eif2a were assessed. Furthermore, four brain regions of Poly-IC rats, a validated animal model for SZ, were analyzed for mitochondrial related proteins, and for variations in HO1 and peif2a/eif2a.


Results:  Heme levels were significantly decreased in SZ derived EBV lymphoblasts, while no difference was observed in their mitochondria. Phosphorylation of eif2a (peif2a/eif2a), was significantly increased in SZ derived cells, suggesting that deficits in heme may have functional consequences. ALAS1 mRNA and protein levels did not differ among the two cohorts, while HO1 mRNA and protein levels were significantly elevated in SZ cells, suggesting increased heme catabolism in SZ cells.  Co-I inhibition exhibited a significant increase (24h) and a later decrease (48h) in heme levels, with no difference at 72h. A significantly elevated HO1 protein level was observed at all time points, while mRNA was significantly elevated at 24h and 48h, with slighter elevation at 72h. Phosphorylation of eif2a significantly decreased at 24h and increased at 72h. Lastly, in the medial pre-frontal (mPFC) of Poly I:C treated offspring protein levels of Co-I subunits NDUFV1 and NDUFV2, as well as mitochondrial pro-fission protein FIS1 and pro-fusion protein OPA1 were decreased. HO1 protein significantly increased in the mPFC and ventral hippocampus of Poly I:C offspring. peIF2α/eIF2α increased in the hippocampus, while decreased in the mPFC of Poly I:C offspring.


Discussion: The current study demonstrates alterations in heme metabolism in SZ derived cells, namely decreased cellular heme and increased HO1 and eif2a phosphorylation levels in lymphoblasts. Since some of these changes are evident in rotenone treated healthy-derived cells, and alterations in Co-I subunits were witnessed alongside increased HO1 levels and altered eif2a phosphorylation in the brains of Poly I:C treated rats, Co-I dysfunction can possibly be associated with these alterations. Our findings open the road for future research on the involvement of heme in the pathophysiology of SZ.