Dissertation Details

Dissertation Code :  IDpq 3398631

Dissertation Title   :  Down-regulation and alternative splicing of methionine synthase as an adaptive response to oxidative stress in aging and neurological disorders

About This Dissertation

155 pages.
Ph.D. dissertation.

The folate and cobalamin-dependent enzyme methionine synthase converts homocysteine to methionine, but oxidation of its cobalamin cofactor halts activity, leading to increased levels of homocysteine. Homocysteine can be converted to cystathionine through the transsulfuration pathway, via the enzyme cystathionine ²-synthase, resulting in glutathione synthesis. The enzymes methionine synthase and cystathionine ²-synthase flank homocysteine, and their relative enzymatic activities determine the proportion of homocysteine that can enter the methylation or transsulfuration pathways. Prevailing redox conditions may adjust this flux by modulating the enzyme via multiple mechanisms.

Glutathione is the principal intracellular antioxidant, and adequate levels of its reduced form are essential for survival in an oxidative metabolic environment. The thiol-containing amino acid cysteine is rate-limiting for glutathione synthesis and can be provided through either cellular uptake or conversion through transsulfuration.

Methionine synthase possesses a linker domain termed 'cap' that is responsible for covering the susceptible cobalamin from oxidation, and loss of this domain may lead to enzyme inactivation. Oxidative stress, associated with lower glutathione levels, is an important contributor to neurodevelopmental and neurodegenerative disorders such as autism, Alzheimer's disease, and schizophrenia.

I used qRT-PCR to evaluate the level of methionine synthase mRNA in post-mortem human cortex and found a significant decrease with age, from 28 weeks of fetal gestation to greater than 80 years. Domain-specific PCR showed that the cap/cobalamin ratio is significantly decreased in subjects over the age of 60, implying age-dependent alternative splicing of the cap domain. Further studies revealed the deletion of exons 19 and 20 of the cap domain, which would reduce the domain size and possibly favor inactivation of MS, leading to increased flux of homocysteine into the transsulfuration pathway and glutathione formation.

A comparison study between autistic subjects and age-matched controls, age 4 to 30 years, revealed significantly lower levels of methionine synthase mRNA in autistic subjects. The greatest decrease occurred in the youngest subjects, when levels in control subjects were normally at their highest, implying an adaptive response to elevated levels of oxidative stress. Thus, autism likely results from a disruption of both normal redox regulation and methylation status.

Exon-specific studies probing folate-binding and cap domain exons in SH-SY5Y human neuroblastoma cells highlighted a combinatorial approach to MS composition. It is likely that different isoforms are generated, depending on the cellular redox status, as an adaptive response to oxidative stress. Non-neuronal cell lines HEK, HepG2 and LN-18 do not show similar splicing modifications, suggesting that redox-dependent splicing is not a feature of all cell types and is more prominent in neuronal cells.

Tumor necrosis factor-alpha, a pro-inflammatory cytokine, is able to induce neuroinflammation, which is a common underlying problem in autism. Tumor necrosis factoralpha treatment significantly reduced both cobalamin-binding and cap domain mRNA in SHSY5Y human neuroblastoma cells, while increasing homocysteine levels. Additionally, cysteine and glutathione levels were increased, despite lower cysteine uptake, indicating an increase in transsulfuration. Methionine synthase activity was inhibited by tumor necrosis factor-alpha, which, when coupled with the mRNA data, indicates transcriptional changes in methionine synthase that can affect cellular redox and methylation activity.

Alternative splicing of the cap domain may reflect previously unrecognized alterations in methionine synthase in response to oxidative stress and aging. The resultant link between redox status and methylation activity is likely to be an important factor in neurodevelopmental, neuropsychiatric and neurodegenerative disorders. These findings represent an important adaptive response to counter progressive oxidation with aging and further indicate that autism may be a neurometabolic redox disorder in which lower levels of methionine synthase activity play an important role.



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