Phyllis-Jean Linton

Impaired mitophagy at the heart of injury

Recent publications link mitophagy mediated by PINK1 and Parkin with cardioprotection and attenuation of inflammation and cell death. The field is in need of methods to monitor mitochondrial turnover in vivo to support the development of new therapies targeting mitochondrial turnover.

Measuring Cardiac Autophagic Flux In Vitro and In Vivo

Autophagy is a lysosomal-dependent catabolic pathway that recycles various cytoplasmic-borne components, such as organelles and proteins, through the lysosomes. This process creates energy and biomolecules that are used to maintain homeostasis and to serve as an energy source under conditions of acute stress. Autophagic flux is a measure of efficiency or throughput of the pathway. Here, we describe a method for determining autophagic flux in vitro and in vivo using the autophagosomal/lysosomal fusion inhibitors chloroquine or bafilomycin A1 and then probing for the autophagosomal marker LC3-II via Western Blot.

Assessing Basal and Acute Autophagic Responses in the Adult Drosophila Nervous System: The Impact of Gender, Genetics and Diet on Endogenous Pathway Profiles.

The autophagy pathway is critical for the long-term homeostasis of cells and adult organisms and is often activated during periods of stress. Reduced pathway efficacy plays a central role in several progressive neurological disorders that are associated with the accumulation of cytotoxic peptides and protein aggregates. Previous studies have shown that genetic and transgenic alterations to the autophagy pathway impacts longevity and neural aggregate profiles of adult Drosophila. In this study, we have identified methods to measure the acute in vivo induction of the autophagy pathway in the adult fly CNS. Our findings indicate that the genotype, age, and gender of adult flies can influence pathway responses. Further, we demonstrate that middle-aged male flies exposed to intermittent fasting (IF) had improved neuronal autophagic profiles. IF-treated flies also had lower neural aggregate profiles, maintained more youthful behaviors and longer lifespans, when compared to ad libitum controls. In summary, we present methodology to detect dynamic in vivo changes that occur to the autophagic profiles in the adult Drosophila CNS and that a novel IF-treatment protocol improves pathway response in the aging nervous system.

Autophagy in the Immune System

Autophagy is a degradative process that delivers cytoplasmic constituents into lysosomes, enabling the reuse of intracellular constituents and supplying an amino acid pool during periods of starvation. Mice deficient in the autophagy-related proteins (Atgs) Atg3, Atg5, or Atg7 die within one day of birth, indicating that autophagy is essential for survival during neonatal starvation. Autophagy is also involved in the clearance of old/damaged organelles, long-lived proteins, insoluble protein aggregates and lipid droplets, thereby regulating cellular homeostasis, cell death/survival, and lipid metabolism. As such, one can envision autophagy having an integral role in many physiological systems. This chapter will focus on the role of autophagy in the immune system.

Indigestible mitochondria cause heartburn

The link between impaired autophagic flux (autophagus interruptus), damaged mitochondria, and myocardial inflammation has been further tightened with the recent paper by Oka and colleagues, in which failure to degrade mitochondrial DNA exacerbated myocardial inflammation in the context of pressure overload. Using mice with cardiac-specific deletion of the lysosomal DNase II that were subjected to aortic banding, Otsus group showed that mitochondrial DNA accumulated in lysosomes and resulted in TLR9-dependent production of inflammatory cytokines.