Binninger, David

Methionine sulfoxide reductase (MSR) is an important antioxidant to help mitigate oxidative stress that contributes to age-associated neurodegenerative diseases, such as Alzheimer’s Disease and Parkinson’s Disease. In MSR deficient Drosophila melanogaster (fruit flies), larvae show a developmental delay like that seen when wild-type larvae are reared on nutrient deficit culture medium. These investigators further showed that serotonin levels were depressed in these nutrient deficient larvae. The overarching aim of this study was to better understand the role of serotonin in MSR regulated physiology.
Supplementing food with serotonin partially rescued the slower mouth hook movements (MHM) observed in the MSR-deficient flies. However, supplementation with serotonin altering drugs that cross the blood brain barrier (5-hydroxytryptophan, fluoxetine, or paravi chlorophenylalanine) did not rescue MHM and caused impairments to the growth of larvae during development. This study indicates that serotonin regulates feeding behavior partially through the regulation of MSR production but acts independently to regulate development.

The oxidation of methionine (Met) into methionine sulfoxide (met-(o)) leads to deleterious modifications to a variety of cellular constituents. These deleterious alterations can be reversed by enzymes known as methionine sulfoxide reductases (Msr). The Msr (MsrA and MsrB) family of enzymes have been studied extensively for their biological roles in reducing oxidized Met residues back into functional Met. A wide range of studies have focused on Msr both in vivo and in vitro using a variety of model organisms. More specifically, studies have noted numerous processes affected by the overexpression, under expression, and silencing of MsrA and MsrB. Collectively, the results of these studies have shown that Msr is involved in lifespan and the management of oxidative stress. More recent evidence is emerging that supports existing biological functions of Msr and theorizes the involvement of Msr in numerous biological pathways.

Drosophila melanogaster tolerates several hours of anoxia (the absence of
oxygen) by entering a protective coma. A burst of reactive oxygen species (ROS) is
produced when oxygen is reintroduced to the cells. ROS causes oxidative damage to
critical cellular molecules, which contribute to aging and development of certain agerelated
conditions. The amino acid, methionine, is susceptible to oxidation, although this
damage can be reversed by methionine sulfoxide reductases (Msr). This project
investigates the effect of Msr-deficiency on anoxia tolerance in Drosophila throughout
the lifespan of the animal. The data show that the time for recovery from the
protective comma as well as the survival of the animals lacking any Msr activity
depends on how quickly the coma is induced by the anoxic conditions. Insight into
the roles(s) of Msr genes under anoxic stress can lead us to a path of designing
therapeutic drugs around these genes in relation to stroke.

Biological homeostasis relies on protective mechanisms that respond to cellular oxidation
caused primarily by free radical reactions. Methionine sulfoxide reductases Msr are a class of
enzymes that reverse oxidative damage to methionine. The focus of this study is on the
relationship between Msr and dopamine in Drosophila. Dopaminergic neurons in drosophila
have comparable roles to those found in humans. A deficit in dopamine leads to the onset of
many neurological disorders including the loss of fine motor control—a neurodegenerative
condition characteristic of Parkinson’s disease PD. We have found that dopamine levels in the
heads of MsrAΔ/ΔBΔ/Δ mutants are significantly reduced in comparison
to the wild type. In addition, we have found that TH protein and expression levels are markedly
reduced in an Msr-deficient system. Our findings suggest that it is possible the Msr system plays
an important role in maintaining dopaminergic neurons alive, and thus, is protectant of the CNS.

Oxidative damage is an inevitable consequence of aerobic respiration. Methionine
sulfoxide reductases (Msr) are a group of enzymes that function to repair oxidized
methionine residues in both free methionine and methionine in proteins. MsrA was the
first of these enzymes to be discovered and is the most thoroughly studied. It is thought to
play a role in both the aging process and probably several neurodegenerative diseases. I
recently obtained a strain of Drosophila that was reported to have a P-element transposon
located within Exon 2 (part of the open reading frame) of the eip71cd gene, which is the
Drosophila homolog of MsrA. Thus, the transposon insertion should disrupt expression
of the msrA gene. I did a series of experiments to "jump out" the P-element in an effort
to recover two types of isogenic strains. The first would be a null mutation of the MsrA
gene created by deletion of flanking genomic DNA when the P-element excised from the
chromosome. The second would be a precise excision of the P-element, which would
restore the genetic locus to its original structure. This study looks at the effect of a null
mutant of the MsrA gene on aging and resistance to oxidative stress.

The inevitable aging process can be partially attributed to the accumulation of
oxidative damage that results from the action of free radicals. Methionine sulfoxide
reductases (Msr) are a class of enzymes that repair oxidized methionine residues. The
two known forms of Msr are MsrA and MsrB which reduce the R- and S- enantiomers of
methionine sulfoxide, respectively. Our lab has created the first genetic animal model
that is fully deficient for any Msr activity. Previously our lab showed that these animals
exhibit a 20 hour delay in development of the third instar larvae (unpublished data). My
studies have further shown that the prolonged third-instar stage is due to a reduced
growth rate associated with slower food intake and a markedly slower motility. These
Msr-deficient animals also exhibit decreased egg-laying that can be attributed to a lack of
female receptivity to mating.

Oxidative stress is considered a major factor in the etiology of age related diseases and the aging process itself. Organisms have developed mechanisms to protect against oxidative damage resulting from increased production of reactive oxygen species during aging. One of the major antioxidant systems is the methionine sulfoxide reductase (Msr) enzyme family. The two major Msr enzymes, MsrA and MsrB, can stereospecifically reduce the S and R epimers, respectively, of methionine sulfoxide in proteins back to methionine. This study, using Drosophila melanogaster, decribes the first animal system lacking both MsrA and MsrB. The loss of either MsrA or MsrB had no effect on lifespan in Drosophila, but loss of MsrB results in a slight decrease in locomotor activity from middle age onward. Double mutants lacking both forms of Msr have a significantly decreased lifespan and decreased locomotor activity at all ages examined. The double Msr mutants had no detectable increase in protein oxidation or decrease in mitochondrial function and were not more sensitive to oxidative stress. These results suggested that other cellular antioxidant systems were protecting the flies against oxidative damage and the decreased life span observed in the double knockouts was not due to widespread oxidative damage. However, one cannot exclude limited oxidative damage to a specific locus or cell type. In this regard, it was observed that older animals, lacking both MsrA and MsrB, have significantly reduced levels of dopamine, suggesting there might be oxidative damage to the dopaminergic neurons. Preliminary results also suggest that the ratio of F to G actin is skewed towards G actin in all mutants. The present results could have relevance to the loss of dopaminergic neurons in Parkinson’s disease.

Aging is a biological process that has many detrimental effects due to the
accumulation of oxidative damage to key biomolecules due to the action of free
radicals. Methionine sulfoxide reductase (Msr) functions to repair oxidative
damage to methionine residues. Msr comes in two forms, MsrA and MsrB, each
form has been shown to reduce a specific enantiomer of bound and free oxidized
methionine. Effects of Msr have yet to be studied in the major developmental
stages of Drosophila melanogaster despite the enzymes elevated expression
during these stages. A developmental timeline was determined for MsrA mutant,
MsrB mutant, and double null mutants against a wild type control. Results show
that the Msr double mutant is delayed approximately 20 hours in the early/mid
third instar stage while each of the single mutants showed no significant difference to the wild type. Data suggests that the reasoning of this phenomenon
is due to an issue gaining mass.

Biological homeostasis relies on protective mechanisms that respond to cellular oxidation caused primarily by free radical reactions. Methionine sulfoxide reductases (Msr) are a class of enzymes that reverse oxidative damage to methionine in proteins. The focus of this study is on the relationship between Msr and dopamine levels in Drosophila. Dopaminergic neurons in Drosophila have comparable roles to those found in humans. A deficit in dopamine leads to the onset of many neurological disorders including the loss of fine motor control—a neurodegenerative condition characteristic of Parkinson’s disease (PD). We found that dopamine levels in the heads of MsrAΔ/ΔBΔ/Δ mutants are significantly reduced in comparison to MsrA ⁺/⁺ B⁺/⁺ heads. In addition, wefound protein and expression levels are markedly reduced in an Msr-deficient system. Our findings suggest an important role for the Msr system in the CNS.

Drosophila melanogaster can withstand hours of oxygen deprivation (anoxia) by entering a protective coma called spreading depression. When oxygen is reintroduced to the cells, a burst of reactive oxygen species (ROS) causes oxidative damage. Methionine is susceptible to oxidation to form methionine sulfoxide. This oxidation is reversible where methionine sulfoxide reductase (Msr) A and B reduce the S and R enantiomers, respectively. In this study, MsrA and MsrB single deletion lines were exposed to one hour of anoxia and the Drosophila Activity Monitor (DAM) recorded their recovery times. RNA interference (RNAi) lines were used to mimic the effect of these deletion lines by ubiquitously knocking down their expression. My current data indicates that MsrA loss-of-function strains recover significantly faster than the MsrB loss-of-function lines with increasing age. Insight into the roles of Msr genes under anoxic stress could lead to a better understanding of how these genes contribute to aging.