Effects of aging, diet and potential genetic interventions on the levels of Smurf2 and its interacting partners in Zebrafish (Danio Rerio) brain
Author(s)
Advisor
Adams, Michelle MarieDate
2020-09Publisher
Bilkent University
Language
English
Type
ThesisItem Usage Stats
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Abstract
Aging is a natural process that is ultimate combination of numerous intrinsic
and extrinsic changes in an organism. Contrary the common belief, brain aging is not
a loss of neurons while it has been shown that subtle cellular and synaptic alterations
have contribution to brain aging. Therefore, the molecular and cellular alterations
may give more insight into the brain aging process. There are some hallmarks of
aging that are common features in different organisms including genomic instability,
telomere attrition, cellular senescence. There are some common factors with the
ability to regulate more than one of the hallmarks of aging such as Smurf2. HECTdomain E3 ubiquitin ligase Smurf2 has several roles in the cellular processes for example, telomere attrition and cellular senescence. Moreover, its gene expression is
higher in the aged brain. Although there are several publications about Smurf2, most
of them focused on its role in cancer. We believed that Smurf2 levels should be examined in terms of brain aging. The first aim of the study was to examine the levels
of Smurf2 and its interacting partners across lifespan. Although the Smurf2 protein
level was not increased significantly in the whole zebrafish brain, its protein level
was upregulated significantly in telencephalon and cerebellum. Also, subcellular
protein fractionation demonstrated an enriched Smurf2 level in the cytosolic part. In
the case of gene expression levels, smurf2 level was significantly higher in aged
whole brain although its expression was downregulated during aging in telencephalon
and cerebellum. In addition, the levels of mdm2, ep300a and sirt1 were lower in the
aged telencephalon. According to multivariate analysis there is a potential balance
between Smurf2-mediated ubiquitination, ep300a-mediated acetylation and Sirt1-
mediated deacetylation but with advancing age, this balance may disrupt and other
regulatory genes should also take a role to sustain cellular stability. The second aim
was to investigate the roles of Smurf2 on brain aging with the help of genetic
interventions including inducible knockin, stable knockout or transient knockdown.
Since stable knockin and knockout models should be genotyped before further
investigations, the genotyping and phenotyping methods were employed to find an
efficient and reliable way. Also, transient knockdown via Vivo-morpholino was
applied to adult brain and efficient post injection times of two different morpholinos
were identified in order to examine the effects of Smurf2 knockdown in both young
and old zebrafish. Lastly, it was aimed to examine the effects of non-genetic
interventions including dietary regimens and pharmacological compounds on the
gene expression of smurf2 and its interacting partners and the levels of the neuronal
proteins and proliferation/senescence proteins. The opposing short-term dietary
regimens, overfeeding and caloric restriction, were altered the levels of neuronal
proteins, HuC and DCAMKL1, and their relation with proliferation and senescence
proteins during aging. Also, the gene expression levels of smurf2 and interacting
partners except tp53 was not influenced by dietary regimens and aging in terms of
whole brain. Also, multivariate analysis indicated that the correlations among smurf2,
mdm2, ep300a and sirt1 were conserved in both young and old ages independent to
dietary regimen which may imply that the balance between ubiquitination, acetylation
and deacetylation is maintained in order to provide cellular stability during aging.
Heclin, an inhibitor of HECT E3 ligases, were employed to inhibit Smurf2 activity.
Before using in adult zebrafish, heclin was applied to embryos to see its effects. The
higher dose of heclin decreased the survival ratio and altered the gene expression
levels of downstream gene drastically. So, moderate dose of heclin should be applied
to the adult brain and neuronal markers should be examined to observe target effects
rather than off-target, unspecific impacts. Taken together, Smurf2 has potential roles
during aging and it could be a promising target to delay the brain aging process and
probably the onset of age-related cognitive decline.
Keywords
ZebrafishAging
Brain
Smurf2
Caloric restriction
Overfeeding
Genetic interventions
Heclin
Neuronal proteins