Modelling Autophagy in Cellular Processes

Monica Gupta, PhD

Tuesday, December 18th, 2018

Autophagy in Cellular Processes

Autophagy is derived from Greek and means self(auto)-eating(phagy). This process occurs in all eukaryotic cell types that contain a lysosomal compartment. In fact, basal autophagy contributes to long-lived protein degradation, organelle turnover in the cytoplasm, and the recycling of macromolecules to maintain bioenergetics¹.

The purpose of autophagy is not solely the simple elimination of materials, but rather the dynamic recycling of cellular components that produces new building blocks and energy for cellular renovation and homeostasis¹^,². The process of autophagy is conserved among yeast to mammals.

Macroautophagy begins with the formation of a cup-shaped double membrane structure that engulfs a portion of cytoplasm. This structure then encloses to form a mature vesicle called an autophagosome, which subsequently fuses with a lysosome, leading to the degradation of intra-autophagosomal components by lysosomal enzymes (Figure 1). Schematic overview of autophagy

Figure 1

Physiological and Pathological Roles of Autophagy

In mammals there are various diseases which result from impaired autophagy. What follows is a list of diseases associated with dysregulated autophagy²:

Organ-Tissue/Cell type	Role of Autophagy	Disease associated with impaired autophagy
All cells-general function	Constitutive autophagy induced by starvation resulting in degradation of cytoplasmic contents into simpler production	Aging, cell death², cancer³
Brain-neurons & microglia	Prevention of protein aggregate formation, mitochondria degradation in microglial cells and neurons	Parkinson's disease⁴ Alzheimer's disease⁴
Liver-hepatocytes	Prevention of hepatocytes degradation; suppression of tumors	Hepatocellular carcinoma²
Bone marrow-macrophages, dendritic cells, monocytes	Maintenance of hematopoietic stem cells, degradation of intracellular pathogens	Microbial Infection and impaired immunity⁵
Lymphoid system	Regulation of cytokine production	Microbial infection⁵
Pancreas- islets of langerhans	B-cell adaptation to high fat diet	Diabetes²
Adipose tissues	Adipogenesis	Obesity²
Intestine-paneth cells	Aberrant expression of inflammatory cytokines	Crohn's disease²

Murine Models to Study Autophagy

One of the contributing factors for the increased knowledge of physiological roles of autophagy is availability of mouse models to study autophagy. Essentially, two types of gene-modified mouse models have played critical roles: "autophagy-monitoring mice"⁶ and "autophagy-deficient mice"⁷. Monitoring the autophagic process and measuring autophagic flux in different tissues and organelles is critical to investigate the function of autophagy under specific stimuli. Analysis of autophagy-deficient mice has led to conceptual findings in this field.

Taconic Biosciences offers various murine models to study diverse aspects of autophagy in different cell types.

Mouse Model	Taconic Model #	Roles in Autophagy
Atg4b KO	TF2950	Atg4b is a member of ATG4-family cysteine proteases. These proteases have an important function as an essential factor in the Atg8 conjugation and deconjugation system, one of the unique mechanisms in autophagy. Inhibition of Atg4b results in inhibition of autophagy⁸.
Atg4c KO	TF2255	Atg4c is a member of ATG4-family cysteine proteases⁸.
Atg4d KO	TF1385	Atg4d is a member of ATG4-family cysteine proteases⁸.
TNFα Tg	1006	Tnfα induced autophagy play a critical role in induction of apoptosis/cell death in placental cells during pregnancy. Induction of autophagy has been implicated in placental dysfunction during pregnancies⁹. TNFα induce autophagy in vitro and in vivo in osteoclasts of human rheumatoid arthritis (RA) thus regulating osteoclast differentiation and bone resorption and play a central role in RA¹⁰.
SumF1 KO	TF3462	Deficiency of SumF1 is associated with lysosomal storage disorders (LSDs) which results in severe neurodegeneration due to inability of degradation of intracellular proteins by the process of autophagy¹¹.
Dram 2 KO	TF3276	Dram-2 interacts with central protein of autophagy LC3 to enhance autophagosome formation in DPN-mediated estrogen receptorβ activated autophagy in Hodgkin lymphoma (HL) cells. The induction of autophagy reduces 60% growth of lymphoma cells by inhibiting cell proliferation³.
Nlrp3 KO	12935	Autophagy keeps NLRP3 inflammasome activation in check by degrading oxidized mitochondria in macrophages thus limiting the NLRP3 inflammasome driven auto-inflammatory chronic inflammation¹².
Pik3c3 KO	TF1967	Pik3c3 play a crucial role in mTOR driven autophagic lysosome reformation (ALR). Defects in the ALR process results in increase neuronal death¹³.
2045	2045	Stat1 inhibits autophagy in mouse embryonic fibroblasts by transcriptional inhibition of the expression of a key autophagy protein ULK1¹⁴.
CIEA NOG mouse ®	NOG	NOG mice were used to demonstrate the role of QSOX1 protein (Quiescin Sulfhydryl oxidase) in inhibiting tumor growth and progression in breast cancer cells by downregulating autophagy¹⁵.

Our understanding of autophagy and its role in different cellular processes has improved substantially over the past decade. Researchers have discovered connections between autophagy and cancer, neurodegeneration, and even lifespan extension. Availability and access to improved animal models are major contributing factors in the discovery and rapid progress in the field of autophagy.

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References:

1. Ravikumar, B. et al. Regulation of Mammalian Autophagy in Physiology and Pathophysiology. Physiological Reviews 90, 1383-1435 (2010).

2. Mizushima, N. & Komatsu, M. Autophagy: Renovation of Cells and Tissues. Cell 147, 728-741 (2011).

3. Pierdominici, M. et al. Estrogen receptor β ligation inhibits Hodgkin lymphoma growth by inducing autophagy. Oncotarget 8, (2017).

4. Lee, J.-A. Autophagy in neurodegeneration: two sides of the same coin. BMB reports 42, 324-330 (2009).

5. Levine, B., Mizushima, N. & Virgin, H. W. Autophagy in immunity and inflammation. Nature 469, 323-335 (2011).

6. Mizushima, N., Yamamoto, A., Matsui, M., Yoshimori, T. & Ohsumi, Y. In vivo analysis of autophagy in response to nutrient starvation using transgenic mice expressing a fluorescent autophagosome marker. Molecular biology of the cell 15, 1101-1111 (2004).

7. Komatsu, M. Impairment of starvation-induced and constitutive autophagy in Atg7-deficient mice. The Journal of Cell Biology 169, 425-434 (2005).

8. Maruyama, T. & Noda, N. N. Autophagy-regulating protease Atg4: structure, function, regulation and inhibition. The Journal of Antibiotics 71, 72-78 (2018).

9. Cha, H.-H. et al. Autophagy Induced by Tumor Necrosis Factor α Mediates Intrinsic Apoptosis in Trophoblastic Cells. Reproductive Sciences 21, 612-622 (2014).

10. Zheng, L. et al. Role of autophagy in tumor necrosis factor-α-induced apoptosis of osteoblast cells. Journal of Investigative Medicine 65, 1014-1020 (2017).

11. Settembre, C. et al. A block of autophagy in lysosomal storage disorders. Human Molecular Genetics 17, 119-129 (2008).

12. Zhong, Z., Sanchez-Lopez, E. & Karin, M. Autophagy, NLRP3 inflammasome and auto-inflammatory/immune diseases. Clin Exp Rheumatol 34, 12-6 (2016).

13. Munson, M. J. & Ganley, I. G. MTOR, PIK3C3, and autophagy: Signaling the beginning from the end. Autophagy 11, 2375-2376 (2015).

14. Goldberg, A. A. et al. Regulation of ULK1 Expression and Autophagy by STAT1. Journal of Biological Chemistry 292, 1899-1909 (2017).

15. Poillet, L. et al. QSOX1 Inhibits Autophagic Flux in Breast Cancer Cells. PLoS ONE 9, e86641 (2014).

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