Mariana Pavel

Autophagy and Neurodegeneration: Pathogenic Mechanisms and Therapeutic Opportunities

Autophagy is a conserved pathway that delivers cytoplasmic contents to the lysosome for degradation. Here we consider its roles in neuronal health and disease. We review evidence from mouse knockout studies demonstrating the normal functions of autophagy as a protective factor against neurodegeneration associated with intracytoplasmic aggregate-prone protein accumulation as well as other roles, including in neuronal stem cell differentiation. We then describe how autophagy may be affected in a range of neurodegenerative diseases. Finally, we describe how autophagy upregulation may be a therapeutic strategy in a wide range of neurodegenerative conditions and consider possible pathways and druggable targets that may be suitable for this objective.

Polyglutamine tracts regulate beclin 1-dependent autophagy

Nine neurodegenerative diseases are caused by expanded polyglutamine (polyQ) tracts in different proteins, such as huntingtin in Huntington’s disease and ataxin 3 in spinocerebellar ataxia type 3 (SCA3). Age at onset of disease decreases with increasing polyglutamine length in these proteins and the normal length also varies. PolyQ expansions drive pathogenesis in these diseases, as isolated polyQ tracts are toxic, and an N-terminal huntingtin fragment comprising exon 1, which occurs in vivo as a result of alternative splicing, causes toxicity. Although such mutant proteins are prone to aggregation, toxicity is also associated with soluble forms of the proteins. The function of the polyQ tracts in many normal cytoplasmic proteins is unclear. One such protein is the deubiquitinating enzyme ataxin 3 (refs 7, 8), which is widely expressed in the brain. Here we show that the polyQ domain enables wild-type ataxin 3 to interact with beclin 1, a key initiator of autophagy. This interaction allows the deubiquitinase activity of ataxin 3 to protect beclin 1 from proteasome-mediated degradation and thereby enables autophagy. Starvation-induced autophagy, which is regulated by beclin 1, was particularly inhibited in ataxin-3-depleted human cell lines and mouse primary neurons, and in vivo in mice. This activity of ataxin 3 and its polyQ-mediated interaction with beclin 1 was competed for by other soluble proteins with polyQ tracts in a length-dependent fashion. This competition resulted in impairment of starvation-induced autophagy in cells expressing mutant huntingtin exon 1, and this impairment was recapitulated in the brains of a mouse model of Huntington’s disease and in cells from patients. A similar phenomenon was also seen with other polyQ disease proteins, including mutant ataxin 3 itself. Our data thus describe a specific function for a wild-type polyQ tract that is abrogated by a competing longer polyQ mutation in a disease protein, and identify a deleterious function of such mutations distinct from their propensity to aggregate.

Autophagy regulates Notch degradation and modulates stem cell development and neurogenesis.

Autophagy is a conserved, intracellular, lysosomal degradation pathway. While mechanistic aspects of this pathway are increasingly well defined, it remains unclear how autophagy modulation impacts normal physiology. It is, however, becoming clear that autophagy may play a key role in regulating developmental pathways. Here we describe for the first time how autophagy impacts stem cell differentiation by degrading Notch1. We define a novel route whereby this plasma membrane-resident receptor is degraded by autophagy, via uptake into ATG16L1-positive autophagosome-precursor vesicles. We extend our findings using a physiologically relevant mouse model with a hypomorphic mutation in Atg16L1, a crucial autophagy gene, which shows developmental retention of early-stage cells in various tissues where the differentiation of stem cells is retarded and thus reveal how modest changes in autophagy can impact stem cell fate. This may have relevance for diverse disease conditions, like Alzheimers Disease or Crohns Disease, associated with altered autophagy.

Study of the Optimum Dose of Ferromagnetic Nanoparticles Suitable for Cancer Therapy Using MFH

At present, a successful realization of the magnetic fluid hyperthermia (MFH) therapy is conditioned by some unsolved problems. One of these problems is the choice of the correct particle concentration in order to achieve a defined temperature increase in the tumor tissue. A computer-based model was created using COMSOL: Multiphysics in order to simulate the heat dissipation within the tissue for typical configurations of the tumor position in relation to neighboring blood vessels as well as particle distribution within the tumor. The temperature achieved on the tumor border was investigated taking into account physiological parameters of different types of tissues. Using the correct nanoparticle dosage and considering their specific loss power, it is possible to estimate the efficiency of this therapeutic method. If the tumor shape and position are known by suitable medical imaging techniques (e.g., MRI, CT), simulations like this one could provide data in order to achieve the optimum dose and particle distribution in the tumor.

Study of the Optimum Injection Sites for a Multiple Metastases Region in Cancer Therapy by Using MFH

Metastases represent the final stage in cancer progression. Their early diagnosis and appropriate treatment are very important in order to maintain a high survival prognostic. The interest in MFH (magnetic fluid hyperthermia) and cancer therapy has noticeably increased in the last years. There are still numerous problems that need to be solved before a clinical model may be tested. The goal of this paper is to both quantify the optimum dose of magnetic material and optimize injection sites in order to achieve a therapeutic temperature of 42degC that may induce apoptosis in tumor cells. A successful realization of this therapy implies a heating zone of at least 2 mm around the tumor. finite element method (FEM) simulations of spherical metastases in liver and breast tissues near a blood vessel were performed using COMSOL multiphysics (heat transfer module) in order to simulate the temperature field produced by ferromagnetic nanoparticles within the tumor and healthy tissues. A systematical variation of tumor diameter and particle dosage was performed for every physical parameter for the tumor tissues mentioned above (e.g., tissue density, tumor/tissue perfusion rate) in order to understand the interdependence of these parameters and their effects on hyperthermia therapy.

Ferromagnetic Nanoparticles Dose Based on Tumor Size in Magnetic Fluid Hyperthermia Cancer Therapy

The interest in magnetic fluid hyperthermia (MFH) and cancer therapy has noticeably increased in the last years. At present, a successful realization of this interdisciplinary research is hampered by some unsolved problems. One of these problems this paper intended to clarify is how to find an estimate of the appropriate dosage of magnetic nanoparticles that injected into the tumor would help achieve an optimum temperature of 42degC, thus resulting in an increase of the susceptibility for apoptosis in tumor cells. We created a computational model in COMSOL: Multiphysics in order to analyze the heat dissipation within the tumor tissue. By considering various types of tissues with their respective physical and physiological properties (breast, liver, and skin tissues) and also by taking into account the amount of heat generated through the Brownian rotation and the Neel relaxation, it has been studied the tumor border temperature achieved for various concentrations of magnetic nanoparticles in their superparamagnetic behavior. Distinct simulations of a spherical tumor located in a cubical region of a volume of 1.2-3.5 cm3 within the tissue were designed. We performed a systematical variation of tumor diameter and particle dosage for every physical parameter of above mentioned tumor tissues (e.g., tissue density, tumor/tissue perfusion rate). By this systematization we intended to understand the interdependence of these parameters and their effects on hyperthermia therapy.

Mammalian autophagy and the plasma membrane

Autophagy (literally ‘self‐eating’) is an evolutionarily conserved degradation process where cytoplasmic components are engulfed by vesicles called autophagosomes, which are then delivered to lysosomes, where their contents are degraded. Under stress conditions, such as starvation or oxidative stress, autophagy is upregulated in order to degrade macromolecules and restore the nutrient balance. The source of membranes that participate in the initial formation of phagophores is still incompletely understood and many intracellular structures have been shown to act as lipid donors, including the endoplasmic reticulum, Golgi, nucleus, mitochondria and the plasma membrane. Here, we focus on the contributions of the plasma membrane to autophagosome biogenesis governed by ATG16L1 and ATG9A trafficking, and summarize the physiological and pathological implications of this macroautophagy route, from development and stem cell fate to neurodegeneration and cancer.

CCT complex restricts neuropathogenic protein aggregation via autophagy

Aberrant protein aggregation is controlled by various chaperones, including CCT (chaperonin containing TCP-1)/TCP-1/TRiC. Mutated CCT4/5 subunits cause sensory neuropathy and CCT5 expression is decreased in Alzheimers disease. Here, we show that CCT integrity is essential for autophagosome degradation in cells or Drosophila and this phenomenon is orchestrated by the actin cytoskeleton. When autophagic flux is reduced by compromise of individual CCT subunits, various disease-relevant autophagy substrates accumulate and aggregate. The aggregation of proteins like mutant huntingtin, ATXN3 or p62 after CCT2/5/7 depletion is predominantly autophagy dependent, and does not further increase with CCT knockdown in autophagy-defective cells/organisms, implying surprisingly that the effect of loss-of-CCT activity on mutant ATXN3 or huntingtin oligomerization/aggregation is primarily a consequence of autophagy inhibition rather than loss of physiological anti-aggregation activity for these proteins. Thus, our findings reveal an essential partnership between two key components of the proteostasis network and implicate autophagy defects in diseases with compromised CCT complex activity.

Polyglutamine tracts regulate autophagy

ABSTRACT Expansions of polyglutamine (polyQ) tracts in different proteins cause 9 neurodegenerative conditions, such as Huntington disease and various ataxias. However, many normal mammalian proteins contain shorter polyQ tracts. As these are frequently conserved in multiple species, it is likely that some of these polyQ tracts have important but unknown biological functions. Here we review our recent study showing that the polyQ domain of the deubiquitinase ATXN3/ataxin-3 enables its interaction with BECN1/beclin 1, a key macroautophagy/autophagy initiator. ATXN3 regulates autophagy by deubiquitinating BECN1 and protecting it from proteasomal degradation. Interestingly, expanded polyQ tracts in other polyglutamine disease proteins compete with the shorter ATXN3 polyQ stretch and interfere with the ATXN3-BECN1 interaction. This competition results in decreased BECN1 levels and impaired starvation-induced autophagy, which phenocopies the loss of autophagic function mediated by ATXN3. Our findings describe a new autophagy-protective mechanism that may be altered in multiple neurodegenerative diseases.

Contact inhibition controls cell survival and proliferation via YAP/TAZ-autophagy axis

Contact inhibition enables noncancerous cells to cease proliferation and growth when they contact each other. This characteristic is lost when cells undergo malignant transformation, leading to uncontrolled proliferation and solid tumor formation. Here we report that autophagy is compromised in contact-inhibited cells in 2D or 3D-soft extracellular matrix cultures. In such cells, YAP/TAZ fail to co-transcriptionally regulate the expression of myosin-II genes, resulting in the loss of F-actin stress fibers, which impairs autophagosome formation. The decreased proliferation resulting from contact inhibition is partly autophagy-dependent, as is their increased sensitivity to hypoxia and glucose starvation. These findings define how mechanically repressed YAP/TAZ activity impacts autophagy to contribute to core phenotypes resulting from high cell confluence that are lost in various cancers.At high cell density or when plated on soft matrix, YAP/TAZ are redistributed from the nucleus to the cytosol, becoming transcriptionally inactive. Here the authors show that at high cell density, autophagosome formation is impaired due to reduced YAP/TAZ-dependent transcription of actomyosin genes