Brain protein synthesis rates and energy sensing in sulfur-amino acid restricted mice
Date
2022
Authors
Martinez, Wenceslao, author
Hamilton, Karyn, advisor
LaRocca, Thomas, committee member
Moreno, Julie, committee member
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Abstract
Protein homeostasis (proteostasis) is the maintenance of the cellular proteome through protein synthesis, folding, trafficking, and degradation. Loss of proteostasis is considered one of the hallmarks of aging and is a driver for age-related neurodegenerative diseases. Dietary sulfur amino acid restriction (SAAR), a life-/healthspan extending treatment, activates mechanisms that maintain proteostasis in the liver. However, it is unknown if dietary SAAR activates mechanisms promoting proteostatic maintenance in the brain. To address this knowledge gap, wild-type male C57Bl/B6 mice were fed one of two levels of SAAR (expressed as % kcal); 0.18% methionine and 0% cysteine (Low SAAR) or 0.12% methionine and 0% cysteine (High SAAR), or a matched control diet sufficient (0.85-0.88%) in methionine. Deuterium enriched water was used to measure rates of newly synthesized proteins and DNA (as a marker of cellular proliferation). Brains were collected at days 1, 3, 7, 14, 21, and 35 of treatment. Mitochondrial, cytosolic, and mixed fractions of frontal cortex were analyzed for rates of protein synthesis and cell proliferation using GC/MS. A one phase association was used to determine the rate of the rise of newly synthesized protein and DNA to capture the kinetic parameter k (1/d). Phosphorylated and total protein content for AMP-protein kinase (AMPK), ribosomal Protein S6 (RPS6) and eukaryotic Initiation Factor 2 (eIF2) were measured at day 1 (acute) and day 35 (long-term) via western blot. Mitochondrial protein synthesis rates were significantly greater in the Low SAAR diet compared to the matched control diet, but did not differ in the cytosolic and mixed fractions. Protein synthesis rates in all fractions of the High SAAR diet were not different from control. There was no significant difference in cell proliferation rates between the SAAR diets and their control matched diets. However, in the High SAAR diet, as reflected by greater protein synthesis to DNA synthesis ratios, more newly synthesized proteins were allocated toward mitochondrial proteome maintenance rather than cell proliferation compared to control. At day 1, eIF2 activation tended to greater (p=0.0922) in the Low SAAR diet compared to control, but was not different at day 35 in the Low SAAR or High SAAR diets. AMPK activation did not differ in the Low SAAR or High SAAR diet compared to their controls at day 1 and day 35. RPS6 activation was not significantly different at day 1 or day 35 in either SAAR diet compared to their controls. This is the first study to simultaneously assess rates of protein synthesis and cell proliferation in the frontal cortex during dietary SAAR, as well as assess activation of key energy sensing proteins. The results from this study show that despite restriction of the sulfur amino acids, rates of protein synthesis were maintained in the cytosolic and mixed fractions with Low SAAR, while the rate of mitochondrial protein synthesis was greater than the control group with Low SAAR but not High SAAR. Dietary SAAR also promoted allocation of more newly synthesized proteins towards maintenance of the existing proteome, with less for new cell proliferation.
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Embargo Expires: 05/24/2024