Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

Pathogenic VCP/TER94 Alleles Are Dominant Actives and Contribute to Neurodegeneration by Altering Cellular ATP Level in a Drosophila IBMPFD Model

Inclusion body myopathy with Paget's disease of bone and frontotemporal dementia (IBMPFD) is caused by mutations in Valosin-containing protein (VCP), a hexameric AAA ATPase that participates in a variety of cellular processes such as protein degradation, organelle biogenesis, and cell-cycle regulation. To understand how VCP mutations cause IBMPFD, we have established a Drosophila model by overexpressing TER94 (the sole Drosophila VCP ortholog) carrying mutations analogous to those implicated in IBMPFD. Expression of these TER94 mutants in muscle and nervous systems causes tissue degeneration, recapitulating the pathogenic phenotypes in IBMPFD patients. TER94-induced neurodegenerative defects are enhanced by elevated expression of wild-type TER94, suggesting that the pathogenic alleles are dominant active mutations. This conclusion is further supported by the observation that TER94-induced neurodegenerative defects require the formation of hexamer complex, a prerequisite for a functional AAA ATPase. Surprisingly, while disruptions of the ubiquitin-proteasome system (UPS) and the ER–associated degradation (ERAD) have been implicated as causes for VCP–induced tissue degeneration, these processes are not significantly affected in our fly model. Instead, the neurodegenerative defect of TER94 mutants seems sensitive to the level of cellular ATP. We show that increasing cellular ATP by independent mechanisms could suppress the phenotypes of TER94 mutants. Conversely, decreasing cellular ATP would enhance the TER94 mutant phenotypes. Taken together, our analyses have defined the nature of IBMPFD–causing VCP mutations and made an unexpected link between cellular ATP level and IBMPFD pathogenesis

TDP-43-Based Animal Models of Neurodegeneration: New Insights into ALS Pathology and Pathophysiology

The clinical and pathological overlap between amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) suggests these diseases share common underlying mechanisms, a suggestion underscored by the discovery that TDP-43 inclusions are a key pathologic feature in both ALS and FTLD. This finding, combined with the identification of TDP-43 mutations in ALS, directly implicates this DNA/RNA binding protein in disease pathogenesis in ALS and FTLD. However, many key questions remain, including what is the normal function of TDP-43, and whether disease-associated mutations produce toxicity in the nucleus, cytoplasm or both. Furthermore, although pathologic TDP-43 inclusions are clearly associated with many forms of neurodegeneration, whether TDP-43 aggregation is a key step in the pathogenesis in ALS, FTLD and other disorders remains to be proven. This review will compare the features of numerous recently developed animal models of TDP-43-related neurodegeneration, and discuss how they contribute to our understanding of the pathogenesis of human ALS and FTLD