Since the advent of knockout technologies using mouse embryonic stem cells in the late 1980s, there has been an explosion of murine models to profile human diseases. The understanding of the genetic contribution to these diseases has been further enhanced with the incorporation of tissue-specific gene deletion strategies through the use of the Cre-lox and FLP-FRT site-specific recombination systems. Autophagy, a crucial regulator of cell energy homeostasis, is also a companion process to the ubiquitin-proteasome system to assist in the turnover of proteins. Two distinct types of mouse models have been engineered to characterize autophagy. The first type is based on the reporter model system to both detect and quantitate the
2. Transgenic models for autophagy detection
GFP-LC3. The best characterized and most widely used detection model is the GFP-LC3 transgenic mouse generated by Mizushima and colleagues . This robustly expressing transgenic mouse, in which LC3 is driven by a constitutive CAG promoter, displays punctate GFP fluorescence that corresponds to autophagosomes. With this transgenic model, quantitation of autophagosomes is feasible using a high resolution fluorescence microscopy. This reporter has been crossed into many of the knockout and floxed autophagy models generated in the field. For example,
GFP-GABARAP. GFP-GABARAP transgenic mice were originally generated to address the question of the role of GABARAP in podocytes. Since GABARAP was reported to be highly expressed in podocytes, a pCAG-GFP-GABARAP transgenic mouse was produced in order to examine subcellular localization in this specialized cell type. The expression level of GFP-GABARAP is low, yet visible, ameliorating many of the potential effects of highly expressing fluorescent proteins. In podocytes, GFP-GABARAP co-localized with p62 aggregates but not LC3II. Although it was shown that GABARAP was not the preferred Atg8 ortholog for conjugation in podocytes, this may prove to be a valuable reporter model in other tissues .
As we will see as a common thread in many of the disease models featured in this chapter, there is no consensus in the literature as to whether autophagy is protective or maladaptive. The contribution of autophagy to cancer development is the most demonstrable example of the Janus-like role of autophagy in disease pathogenesis, and perhaps the most thoroughly investigated. Autophagy is hypothecated to contribute to tumor progression through two distinct mechanisms. Tumor growth is initially restricted when the centrally-located cells undergo nutrient deprivation, hypoxia, and if prolonged, subsequently undergo necrosis. This metabolic stress induces autophagy to maintain energy homeostasis and prevent necrosis until neo-vascularization occurs. Angiogenesis is potentially able to restore a nutrient supply throughout the tumor, promoting tumor survival and facilitating growth and metastasis. Additionally, transformed cells that harbor defects in apoptosis and autophagy pathways tumor cells undergo chronic necrosis, which foments vascularization of the necrotic area, thus promoting tumorigenesis. Conversely, intact autophagy can be cytoprotective by eliminating damaged or aged organelles and degrading aggregated or misfolded proteins, thus preventing accumulation of tumorigenic and/or cytotoxic cellular inclusions.
These two scenarios are manifested in mouse models of autophagy disruption. When
2.2. Heart disease
Coronary heart disease (CHD) is the leading cause of death of men and women in the United States. CHD is caused by narrowing of the arteries that supply the heart. In mouse models a standard treatment is to induce an ischemic event by surgically occluding one or more coronary artery. This occlusion may be relieved at a later time (ischemia/reperfusion) or remain (permanent occlusion). Interestingly, autophagy is induced in both ischemia/reperfusion (I/R) events and permanent occlusion events but the outcomes are differential. During I/R events autophagy is activated, however in
In addition to CAD, congenital mutations and other pathological conditions (e.g. type 2 diabetes) my result in cardiomyopathies. In a recent study, Choi et al showed that in a cardiomyopathy model resulting from a mutation in A-type lamins (A/C) resulted in active mTOR in cardiomyocytes, which inhibited autophagy activation . This lack of autophagy activation was proposed to lead to an energy deficit and was detrimental to the survival of the cardiomyocytes and resulted in disease progression. Further evidence of the protective role of autophagy was obtained when autophagy was reactivated by the mTOR-inhibiting drug rapamycin. In mice mutant for A-type lamins, rapamycin treatment attenuated the cardiomyopathy phenotype. In another approach, a mouse model of diabetic cardiomyopathy, generated by diet alteration to include a surplus of saturated fatty acids, has revealed that autophagy was activated in response to pressure increase resultant from cardiac hypertrophy. Autophagy induction was measured by increased Becn1 and LC3B mRNA, as well as increased GFP-LC3 puncta in the diabetic hypertrophy model. In this system, isolated cardiomyocytes that were autophagy impaired did not develop hypertrophy, indicating that increased autophagy was required for hypertrophy. Furthermore, isolated cardiomyocytes treated with myristate led to an increase in Becn1 and Atg7 expression through a ceramide-dependent mechanism . The role of autophagy is complex and seems to be highly context specific with regard to heart disease. It is apparent that the method by which autophagy is inhibited or induced and in which type of model differentially dictates whether autophagy will result in a positive or negative outcome.
2.3. Neurodegenerative diseases (e.g. Alzheimer’s and Parkinson’s disease)
Post-mitotic neurons rely heavily on basal autophagy to clear old, damaged organelles and potentially harmful protein aggregates. Several neurodegenerative disorders result from expansions of poly-glutamine or poly-alanine stretches, including Huntington’s disease, Parkinson’s disease, spinocerebral ataxias, and fronto-temporal dementia. Mutant proteins involved in these diseases have an increased propensity to aggregate and poison the cell; accordingly, the disease progression is directly related to the amount of protein aggregates formed in these patients. In normal physiology, neurons rely on both the proteasomal degradation system and autophagy to maintain protein and energy homeostasis. It has been shown that autophagy is used preferentially to remove large aggregated proteins or multi- protein plaques. In a rat model of Machado-Joseph disease (spinocerebral ataxia type 3) overexpression of Becn1 reduced both mutant ataxin-3 accumulation and ubiquitin-positive inclusions . It seems that autophagy is used as a compensatory mechanism to relieve the toxic effects of these mutant protein aggregates in the cell, serving a protective role. Neuronal-specific conditional deletion of
Autophagy and its association with aging have been explored in two distinct contexts: 1) the impact of autophagy on increasing lifespan or longevity, and 2) the role of autophagy in age-related disease states. Longevity‑promoting regimens, including caloric restriction (CR) and inhibition of TOR with rapamycin, resveratrol or the natural polyamine spermidine, have been associated with autophagy induction . CR can improve heart function through autophagy, as long-term CR preserved cardiac contractile function with improved cardiomyocyte function and lessened cardiac remodeling . Rapamycin prolonged median and maximal lifespan of both male and female mice when fed beginning at 600 days of age; this supplementation led to a life span increase of 14% for females and 9% for males. In addition, rapamycin-treated mice beginning at 270 days of age also increased survival in both males and females . Lifelong administration of rapamycin extended the lifespan of female 129/Sv mice, as 22.9% of rapamycin-treated mice survived the age of death of the last control mouse. Rapamycin also inhibited age-related weight gain, decreased aging rate, and delayed spontaneous cancer formation . Although rapamycin and caloric restriction both increase the life span of mice, they probably do not occur through similar mechanisms. Dietary restricted mice (40% food restriction) and rapamycin-treated mice both exhibited increased levels of autophagy . The fat mass was similar between control and rapamycin-treated mice, but lower for the caloric restricted mice. There were also striking differences in insulin sensitivity and expression of cell cycle and sirtuin genes in mice fed rapamycin compared with dietary restriction. Spermidine, a natural polyamine whose intracellular concentration declines during human aging, extended the lifespan of yeast, flies and worms, and human immune cells. In addition, spermidine administration potently inhibited oxidative stress in aging mice .
Basal autophagy helps to reduce the deleterious effects from oxidative stress, heat stress and cytoplasmic protein aggregates. During the aging process, basal autophagy levels gradually decline so that the cell is not equipped to deal with these stressors. Since many age-related diseases correlate with a decline in basal autophagy, a targeted therapeutic strategy would be to increase the levels of productive autophagy to reduce the severity of the disease. However, it is important to keep in mind that a tight regulation of basal autophagy levels is important, as too much autophagy could have a negative outcome. For example, in the
2.5. Lysosomal storage disorders
The group of degenerative disorders included in umbrella term lysosomal storage disorders (LSDs) is a heterogeneous and emerging list of diseases that commonly present with an inability to metabolize a normal cellular substrate. The metabolic defect may reside with the ability of the lysosome to degrade the substrate, or a blockade of autophagic flux, most often inhibiting fusion of the autophagosome and lysosome. A reduction in autophagic flux may result in an increase in autophagosome like structures in the cytoplasm as well as uncharacteristically large autophagosome like structures being formed. Consequently, failure to eliminate/recycle the autophagosomal contents induces cellular stress and may result in death.
Pompe disease, the first LSD to be characterized, is caused by an inability to synthesize acid α-glucosidase (GAA), a lysosomal enzyme needed to breakdown glycogen. Pompe mice (GAA KO) phenocopied the human condition, and abnormal autophagosomal and autolysosomal structures were seen intracellularly. When Pompe mice were crossed with
Multiple sulfatase deficiency (MSD) is a disease where affected individuals have a reduction in the activity of all sulfatases due to mutations in Sulfatase Modifying Factor 1 (SUMF1), an enzyme responsible for post-translational modification of all sulfatases. A s
Mucopolysaccharidosis type VI (MPS VI) is caused by a specific sulfatase deficiency (N-acetylgalactosamine-4-sulfate) and patients may be short in stature and suffer from joint stiffness and destruction, cardiac valve abnormalities and corneal clouding. In a rat model of MPS VI, an increased number of autophagic structures were identified by electron microscopy .
Niemann Pick type C disease is a metabolic disorder characterized by the accumulation of lipids in late endosomes/lysosomes. The vast majority of cases are due to mutations in the
Mucolipidosis type IV disease (MLIV) is caused by mutations in the MCOLN1 gene which encodes a lysosomal cation channel. Affected patients suffer from psychomotor delays and multiple ophthalmic pathologies. The
2.6. Infectious disease and immunity (e.g. Crohn’s disease)
The innate immune system is the first line of defense against pathogens; it is evolutionarily more ancient than the adaptive immune system and is deployed quickly and effectively despite its lack of pathogen specificity or memory. Viruses, bacteria, and parasites can be eliminated in an autophagic process involved in innate immunity defense termed ‘xenophagy.’ Invading bacteria can generally be classified as vacuolar (e.g.
The more complicated adaptive immune system also relies on autophagy in many capacities. Studies inducing ablation of autophagy proteins have revealed an essential role for autophagy in maintaining normal numbers of B cells, T cells and hematopoietic stem cell survival and function.
Genome wide association studies of Crohn’s Disease identified two autophagy associated genes, Atg16L and IRGM. A naturally occurring insertion/deletion mutation was identified in the 5’UTR (untranslated region) of IRGM (immunity-related GTPase family, M) which disrupted a transcription factor binding site . In another study, a SNP in the coding region of IRGM was identified that affected a microRNA binding site . These identified mutations suggested that IRGM expression level changes were associated with Crohn’s disease in humans. A mouse knockout model of
The relationship between Crohn’s disease and the autophagic process is more developed in terms of investigating the
2.7. Muscle atrophy
Muscle atrophy is a symptom of a multitude of pathological states including but not limited to fasting conditions, denervation, inactivity, cancer, cardiac failure, and diabetes. Autophagy has been shown to be active in muscular atrophy and other myopathies however due to the nature of the methods used it cannot be said with certainty whether autophagy is promoting atrophy or is activated as a cytoprotective mechanism and coincides with pathology.
In a mouse tissue-specific mouse model generated to investigate the effect of superoxide dismutase 1 (SOD1) ablation on skeletal muscle, it was found that mutants developed muscle atrophy, reduction in contractile force and abnormal mitochondria. Significant upregulation of the mitophagy (specific and selective form of autophagy wherein mitochondria are preferentially enveloped and degraded) gene
As discussed previously, a muscle-specific
Cerebral ischemia is achieved in mice most often by surgical intervention and results in global, restricted, or cerebral directed ischemia depending on the method selected. Furthermore in some models ischemia is reversed, allowing reperfusion of the cerebral tissue. Autophagy is induced by hypoxia/ischemia events; however it is unclear whether autophagy is protective or maladaptive in stroke models. Responsibility of much of the dissenting opinions may be attributed to the variety of techniques used to induce ischemia events, these have been reviewed at length by Hossmann in . Neonatal mice subjected to hypoxic/ischemic (H/I) brain injury responded with a robust autophagic response in neurons and hippocampal neuron death.
2.9. Type 2 diabetes
Type 2 diabetes (T2D) is a complex disease that manifests in tissues, especially adipose, muscles, and liver, becoming resistant to insulin signaling and causing hyperglycemia. Pancreatic β–cells initially respond by increasing their production of insulin, but prolonged insulin resistance results in atresia of β–cells and a marked reduction in insulin production. Due to the high metabolic demands placed on β–cells it follows that autophagy would be play an important role in the pathophysiology of this chronic disease. In wild type C57BL/6 mice β–cells, unlike most organs, autophagosomes are sparingly observed after a period of starvation. However, when mice were fed high fat diets for 12 weeks, autophagosomes were readily observed. These results were confirmed
2.10. Reproductive infertility
The role of autophagy during folliculogenesis is a comparatively new topic of study. In rats, LC3II expression was characterized in follicles of varying developmental stages. Primordial follicles exhibited only weak expression, but antral follicles exhibited robust expression restricted to granulosa cells. Staining was not observed in the oocyte proper or theca cells in any stage follicle . In mice, expression studies also indicate a role for autophagy during folliculogenesis. An expression profile of
In conclusion, a variety of mouse models have been established and interrogated to understand the implications of autophagy in human disease. These genetic-based models are primarily either reliant upon: 1) the generation of an autophagy-defective mouse to characterize a given disease state, or 2) the characterization of autophagy within a pre-established murine model. As this review has shown, conditional knockout models have been extremely useful in disease profiling. The next wave of studies will invariably utilize inducible-based systems for conditional knockouts, genetic-based rescue experiments of disease models, or pharmaceutical-based modification of autophagy. A prevailing theme in the field is that autophagy can either be beneficial or deleterious depending on the disease and its progression state, a theme which must be addressed in designing and implementing appropriate treatment regimes.
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