Previous studies have suggested that there are genes whose expression levels are associated with chronological age. However, which genes show consistent age association across studies, and which are specific to a given organism or tissue remains unresolved. Here, we re-assessed this question using two independently ascertained series of human brain samples from two anatomical regions, the frontal lobe of the cerebral cortex and cerebellum. Using microarrays to estimate gene expression, we found sixty associations between expression and chronological age that were statistically significant and were replicated in both series in at least one tissue. There were a greater number of significant associations in the frontal cortex compared to the cerebellum. We then repeated the analysis in a subset of samples using laser capture microdissection to isolate purkinje neurons from the cerebellum. We were able to replicate five gene associations from either frontal cortex or cerebellum in the Purkinje cell dataset, suggesting that there is a subset of genes have robust changes withs aging. Of these, the most consistent and strongest association was with expression of RHBDL3, a rhomboid protease family member. We confirmed several hits using an independent technique (qRT-PCR) and in an independent published sample series that used a different array platform. We also interrogated larger patterns of age related gene expression using weighted gene correlation network analysis (WGCNA). We found several modules that showed significant associations with chronological age and, of these, several that showed negative associations were enriched for genes encoding components of mitochondria. Overall, our results show that there is a distinct and reproducible gene signature for aging in the human brain.
Autosomal recessive parkinsonism genes contribute to maintenance of mitochondrial function. Two of these, PINK1 and parkin, act in a pathway promoting autophagic removal of depolarized mitochondria. Although recruitment of parkin to mitochondria is PINK1-dependent, additional components necessary for signaling are unclear. We performed a screen for endogenous modifiers of parkin recruitment to depolarized mitochondria and identified hexokinase 2 (HK2) as a novel modifier of depolarization-induced parkin recruitment. Hexose kinase activity was required for parkin relocalization, suggesting the effects are shared among hexokinases including the brain-expressed hexokinase 1 (HK1). Knockdown of both HK1 and HK2 led to a stronger block in parkin relocalization than either isoform alone, and expression of HK2 in primary neurons promoted YFP-parkin recruitment to depolarized mitochondria. Mitochondrial parkin recruitment was attenuated with AKT inhibition, which is known to modulate HK2 activity and mitochondrial localization. We, therefore, propose that Akt-dependent recruitment of hexokinases is a required step in the recruitment of parkin prior to mitophagy.
The complexity of the adult brain is a result of both developmental processes and experience-dependent circuit formation. One way to look at the differences between embryonic and adult brain is to examine gene expression. Previous studies have used microarrays to address this in a global manner. However, the transcriptome is more complex than gene expression levels alone, as alternative splicing and RNA editing generate a diverse set of mature transcripts. Here, we developed a high-resolution transcriptome dataset of mouse cerebral cortex at embryonic and adult stages using RNA sequencing (RNA-Seq). We found many differences in gene expression, splicing and RNA editing between embryonic and adult cerebral cortex. Each dataset was validated technically and biologically, and in each case we found our RNA-Seq observations to have predictive validity. We propose this dataset and analysis to be a helpful resource for understanding gene expression in the embryonic and adult cerebral cortex.
Obsessive-compulsive disorder (OCD) is a common, debilitating neuropsychiatric
illness with complex genetic etiology. The International OCD Foundation Genetics
Collaborative (IOCDF-GC) is a multi-national collaboration established to discover the
genetic variation predisposing to OCD. A set of individuals affected with DSM-IV OCD, a
subset of their parents, and unselected controls, were genotyped with several different
Illumina SNP microarrays. After extensive data cleaning, 1,465 cases, 5,557
ancestry-matched controls and 400 complete trios remained, with a common set of 469,410
autosomal and 9,657 X-chromosome SNPs. Ancestry-stratified case-control association
analyses were conducted for three genetically-defined subpopulations and combined in two
meta-analyses, with and without the trio-based analysis. In the case-control analysis, the
lowest two p-values were located within DLGAP1
(p=2.49×10-6 and p=3.44×10-6), a
member of the neuronal postsynaptic density complex. In the trio analysis, rs6131295, near
BTBD3, exceeded the genome-wide significance threshold with a
p-value=3.84 × 10-8. However, when trios were meta-analyzed
with the combined case-control samples, the p-value for this variant was
3.62×10-5, losing genome-wide significance. Although no SNPs were
identified to be associated with OCD at a genome-wide significant level in the combined
trio-case-control sample, a significant enrichment of methylation-QTLs (p<0.001)
and frontal lobe eQTLs (p=0.001) was observed within the top-ranked SNPs
(p<0.01) from the trio-case-control analysis, suggesting these top signals may
have a broad role in gene expression in the brain, and possibly in the etiology of
Obsessive-compulsive disorder; GWAS; Genetic; Genomic; Neurodevelopmental disorder; DLGAP
MATR3 is an RNA/DNA binding protein that interacts with TDP-43, a major disease protein linked to amyotrophic lateral sclerosis (ALS) and fronto-temporal dementia. Using exome sequencing, we identified mutations in MATR3 in ALS kindreds. We also observed MATR3 pathology in the spinal cords of ALS cases with and without MATR3 mutations. Our data provide additional evidence supporting the role of aberrant RNA processing in motor neuron degeneration.
Parkinson’s disease (PD) has a number of known genetic risk factors. Clinical and epidemiological studies have suggested the existence of intermediate factors that may be associated with additional risk of PD. We construct genetic risk profiles for additional epidemiological and clinical factors using known genome-wide association studies (GWAS) loci related to these specific phenotypes to estimate genetic comorbidity in a systematic review. We identify genetic risk profiles based on GWAS variants associated with schizophrenia and Crohn’s disease as significantly associated with risk of PD. Conditional analyses adjusting for SNPs near loci associated with PD and schizophrenia or PD and Crohn’s disease suggest that spatially overlapping loci associated with schizophrenia and PD account for most of the shared comorbidity, while variation outside of known proximal loci shared by PD and Crohn’s disease accounts for their shared genetic comorbidity. We examine brain methylation and expression signatures proximal to schizophrenia and Crohn’s disease loci to infer functional changes in the brain associated with the variants contributing to genetic comorbidity. We compare our results with a systematic review of epidemiological literature, while the findings are dissimilar to a degree; marginal genetic associations corroborate the directionality of associations across genetic and epidemiological data. We show a strong genetically defined level of comorbidity between PD and Crohn’s disease as well as between PD and schizophrenia, with likely functional consequences of associated variants occurring in brain.
Germ-line genetic control of gene expression occurs via expression quantitative trait loci (eQTLs). We present a large, exon-specific eQTL data set covering ten human brain regions. We found that cis-eQTL signals (within 1 Mb of their target gene) were numerous, and many acted heterogeneously among regions and exons. Co-regulation analysis of shared eQTL signals produced well-defined modules of region-specific co-regulated genes, in contrast to standard coexpression analysis of the same samples. We report cis-eQTL signals for 23.1% of catalogued genome-wide association study hits for adult-onset neurological disorders. The data set is publicly available via public data repositories and via http://www.braineac.org/. Our study increases our understanding of the regulation of gene expression in the human brain and will be of value to others pursuing functional follow-up of disease-associated variants.
Genome wide association studies have nominated many genetic variants for common human traits, including diseases, but in many cases the underlying biological reason for a trait association is unknown. Subsets of genetic polymorphisms show a statistical association with transcript expression levels, and have therefore been nominated as expression quantitative trait loci (eQTL). However, many tissue and cell types have specific gene expression patterns and so it is not clear how frequently eQTLs found in one tissue type will be replicated in others. In the present study we used two appropriately powered sample series to examine the genetic control of gene expression in blood and brain. We find that while many eQTLs associated with human traits are shared between these two tissues, there are also examples where blood and brain differ, either by restricted gene expression patterns in one tissue or because of differences in how genetic variants are associated with transcript levels. These observations suggest that design of eQTL mapping experiments should consider tissue of interest for the disease or other trait studied.
Significance: Studies of sporadic cases, toxin models, and genetic causes of Parkinson's disease suggest that mitochondrial dysfunction may be an early feature of pathogenesis. Recent Advances: Compelling evidence of a causal relationship between mitochondrial function and disease was found with the identification of several genes for recessive parkinsonism, PINK1, DJ-1, and parkin. There is evidence that each of these regulates responses to cellular stresses, including oxidative stress and depolarization of the mitochondrial membrane. Specifically, PINK1 and parkin modulate mitochondrial dynamics by promoting autophagic removal of depolarized mitochondria. Mutations in all genes linked to Parkinson's disease lead to enhanced sensitivity to mitochondrial toxins and oxidative stress. Critical Issues: Both increased mitochondrial damage due to complex 1 inhibition, mishandling of calcium, oxidant stress, or impaired clearance of dysfunctional mitochondria would lead to the accumulation of nonfunctional organelles and could contribute to neuronal dysfunction. However, several unanswered questions remain about the underlying mechanism(s) involved. Future Directions: PINK1 and parkin have been demonstrated to regulate mitochondrial dynamics, but the pathways linking PINK1 activity to parkin function are still unclear and warrant further investigation. Antioxid. Redox Signal. 16, 869–882.
Three genes have been identified that cause, in humans, autosomally inherited parkinsonism. These are PARK2, encoding the E3 ubiquitin ligase parkin; PINK1, a mitochondrial kinase; and PARK7, which codes for the protein DJ-1. In several experimental systems, it has been shown that all three proteins impact mitochondrial function and/or oxidative stress responses. These are probably related because mitochondria produce oxidative stress in neurons. Moreover, it is clear that there are relationships between these genes, with a single pathway linking PINK1 and parkin and a parallel relationship with DJ-1. Work in progress in the field is aimed at understanding these relationships in more depth.
Mutations in three genes that cause autosomal recessive parkinsonism have functions related to mitochondrial maintenance during cellular stress. A single pathway links PINK1 and parkin; DJ-1 may be in a parallel pathway.
By combining experimental neuron models and mathematical tools, we developed a “systems” approach to deconvolve cellular mechanisms of neurodegeneration underlying the most common known cause of Parkinson's disease (PD), mutations in leucine-rich repeat kinase 2 (LRRK2). Neurons ectopically expressing mutant LRRK2 formed inclusion bodies (IBs), retracted neurites, accumulated synuclein, and died prematurely, recapitulating key features of PD. Degeneration was predicted from the levels of diffuse mutant LRRK2 that each neuron contained, but IB formation was neither necessary nor sufficient for death. Genetic or pharmacological blockade of its kinase activity destabilized LRRK2 and lowered its levels enough to account for the moderate reduction in LRRK2 toxicity that ensued. By contrast, targeting synuclein, including neurons made from PD patient-derived induced pluripotent cells, dramatically reduced LRRK2-dependent neurodegeneration and LRRK2 levels. These findings suggest that LRRK2 levels are more important than kinase activity per se in predicting toxicity and implicate synuclein as a major mediator of LRRK2-induced neurodegeneration.
LRRK2; mechanisms; Parkinson's disease; single cell; synuclein
Progressive supranuclear palsy (PSP) is the most common atypical parkinsonian disorder. Abnormal tau inclusions, in selected regions of the brain, are a hallmark of the disease and the H1 haplotype of MAPT, the gene encoding tau, is the major risk factor in PSP. A 3-repeat and 4-repeat tau isoform ratio imbalance has been strongly implicated as a cause of disease. Thus, understanding tau isoform regional expression in disease and pathology-free states is crucial to elucidating mechanisms involved in PSP and other tauopathies. We used a tau-isoform specific fluorescent assay to investigate relative 4R-tau expression in 6 different brain regions in PSP cases and healthy controls. We identified marked difference in 4R-tau relative expression, both across brain regions and between MAPT haplotypes. Highest 4R-tau expression levels were identified in the globus pallidus as compared to pons, cerebellum and frontal cortex. 4R-tau expression levels were related to both the MAPT H1 and H1c haplotypes. Similar regional variation was seen in both PSP cases and controls.
Background: LRRK2 mutations are causative for Parkinson disease, but regulation of LRRK2 remains elusive.
Results: Arsenite induces loss of LRRK2 Ser910/Ser935 phosphorylation and 14-3-3 binding, increased self-association, attenuated kinase activity and GTP binding, and translocation to centrosomes.
Conclusion: LRRK2 is regulated by arsenite-induced signaling and oxidative stress.
Significance: Understanding LRRK2 regulation will provide novel approaches toward developing therapeutic tools targeting LRRK2 activity.
Mutations in the gene encoding leucine-rich repeat kinase 2 (LRRK2) are a common genetic cause of Parkinson disease, but the mechanisms whereby LRRK2 is regulated are unknown. Phosphorylation of LRRK2 at Ser910/Ser935 mediates interaction with 14-3-3. Pharmacological inhibition of its kinase activity abolishes Ser910/Ser935 phosphorylation and 14-3-3 binding, and this effect is also mimicked by pathogenic mutations. However, physiological situations where dephosphorylation occurs have not been defined. Here, we show that arsenite or H2O2-induced stresses promote loss of Ser910/Ser935 phosphorylation, which is reversed by phosphatase inhibition. Arsenite-induced dephosphorylation is accompanied by loss of 14-3-3 binding and is observed in wild type, G2019S, and kinase-dead D2017A LRRK2. Arsenite stress stimulates LRRK2 self-association and association with protein phosphatase 1α, decreases kinase activity and GTP binding in vitro, and induces translocation of LRRK2 to centrosomes. Our data indicate that signaling events induced by arsenite and oxidative stress may regulate LRRK2 function.
Leucine-rich Repeat Kinase 2 (LRRK2); Neurodegenerative Disease; Oxidative Stress; Parkinson Disease; Phosphorylation; Arsenite
Brain-derived neurotrophic factor (BDNF) has been implicated in the pathophysiology of psychiatric and neurological disorders and in the mechanisms of antidepressant pharmacotherapy. Psychiatric and neurological conditions have also been associated with reduced brain levels of N-acetyl-aspartate (NAA), which has been used as a putative marker of neural integrity. However, few studies have explored the relationship between BDNF polymorphisms and NAA levels directly. Here, we present data from a single-voxel proton magnetic resonance spectroscopy study of 64 individuals and explore the relationship between BDNF polymorphisms and prefrontal NAA level. Our results indicate an association between a single nucleotide polymorphism (SNP) within BDNF, known as rs1519480, and reduced NAA level (p=0.023). NAA levels were further predicted by age and Asian ancestry. There was a significant interaction between rs1519480 and age on NAA level (p=0.031) Specifically, the effect of rs1519480 on NAA level became significant at age ≥ 34.17. NAA level decreased with advancing age for genotype TT (p=0.001) but not for genotype CT (p=0.82) or CC (p=0.34). Additional in silico analysis of 142 postmortem brain samples revealed an association between the same SNP and reduced BDNF mRNA expression in the prefrontal cortex. The rs1519480 SNP influences BDNF mRNA expression and has an impact on prefrontal NAA level over time. This genetic mechanism may contribute to interindividual variation in cognitive performance seen during normal aging, as well as contributing to the risk for developing psychiatric and neurological conditions.
BDNF; N-acetyl-aspartate; proton magnetic resonance spectroscopy; SNP; genetic variation
Mitochondrial DNA damage is thought to be a causal contributor to aging as mice with inactivating mutations in polymerase gamma (Polg) develop a progeroid phenotype. To further understand the molecular mechanisms underlying this phenotype, we used iTRAQ and RNA-Seq to determine differences in protein and mRNA abundance respectively in the brains of one year old Polg mutator mice compared to control animals. We found that mitochondrial respiratory chain proteins are specifically decreased in abundance in the brains of the mutator mice, including several nuclear encoded mitochondrial components. However, we found no evidence that the changes we observed in protein levels were the result of decreases in mRNA expression. These results show that there are post-translational effects associated with mutations in Polg.
The dysregulation of mitochondrial function has been implicated in the pathogenesis of Parkinson disease. Mutations in the parkin, PINK1 and DJ-1 genes all result in recessive parkinsonism. Although the protein products of these genes have not been fully characterized, it has been established that all three contribute to the maintenance of mitochondrial function. PINK1 and parkin act in a common pathway to regulate the selective autophagic removal of depolarized mitochondria, but the relationship between DJ-1 and PINK1- and/or parkin-mediated effects on mitochondria and autophagy is less clear. We have shown that loss of DJ-1 leads to mitochondrial phenotypes including reduced membrane potential, increased fragmentation and accumulation of autophagic markers. Supplementing DJ-1-deficient cells with glutathione reverses both mitochondrial and autophagic changes suggesting that DJ-1 may act to maintain mitochondrial function during oxidative stress and thereby alter mitochondrial dynamics and autophagy indirectly.
mitochondria; oxidative stress; Parkinson disease; PINK1; parkin; DJ-1
Parkinson's disease (PD) is the second most common neurodegenerative disease affecting 1–2% in people >60 and 3–4% in people >80. Genome-wide association (GWA) studies have now implicated significant evidence for association in at least 18 genomic regions. We have studied a large PD-meta analysis and identified a significant excess of SNPs (P < 1 × 10−16) that are associated with PD but fall short of the genome-wide significance threshold. This result was independent of variants at the 18 previously implicated regions and implies the presence of additional polygenic risk alleles. To understand how these loci increase risk of PD, we applied a pathway-based analysis, testing for biological functions that were significantly enriched for genes containing variants associated with PD. Analysing two independent GWA studies, we identified that both had a significant excess in the number of functional categories enriched for PD-associated genes (minimum P = 0.014 and P = 0.006, respectively). Moreover, 58 categories were significantly enriched for associated genes in both GWA studies (P < 0.001), implicating genes involved in the ‘regulation of leucocyte/lymphocyte activity’ and also ‘cytokine-mediated signalling’ as conferring an increased susceptibility to PD. These results were unaltered by the exclusion of all 178 genes that were present at the 18 genomic regions previously reported to be strongly associated with PD (including the HLA locus). Our findings, therefore, provide independent support to the strong association signal at the HLA locus and imply that the immune-related genetic susceptibility to PD is likely to be more widespread in the genome than previously appreciated.
Recent genome wide association studies have identified CLU, CR1, ABCA7
BIN1, PICALM and MS4A6A/MS4A6E in addition to the long established APOE, as loci for Alzheimer’s disease. We have systematically examined each of these loci to assess whether common coding variability contributes to the risk of disease. We have also assessed the regional expression of all the genes in the brain and whether there is evidence of an eQTL explaining the risk. In agreement with other studies we find that coding variability may explain the ABCA7 association, but common coding variability does not explain any of the other loci. We were not able to show that any of the loci had eQTLs within the power of this study. Furthermore the regional expression of each of the loci did not match the pattern of brain regional distribution in Alzheimer pathology.
Although these results are mainly negative, they allow us to start defining more realistic alternative approaches to determine the role of all the genetic loci involved in Alzheimer’s disease.
Alzheimer’s disease; genetic risk; GWAS
Several methods now exist for identifying and quantifying many biological events in parallel and in a relatively unbiased fashion. For gene expression experiments, cloning approaches have been supplemented with microarray platforms over the past few years. The focus of this review is on deep sequencing, a new set of techniques that can be used to both identify RNA species and quantify them in a massively parallel fashion. Deep sequencing has some advantages over other methods, driven largely by the high depth of coverage for any library of nucleic acids. This allows, for example, estimates of alternative splicing and untranslated region utilization. We will discuss how deep sequencing methods are being applied to characterization of gene expression in the brain and how these technologies might develop over the next few years.
Alternative splicing; Deep sequencing; Gene expression; massively parallel signature sequencing; Next generation sequencing; non-coding RNA; small RNA
Background: Mutations in the gene encoding leucine-rich repeat kinase 2 (LRRK2) cause Parkinson disease.
Results: LRRK2 binds directly to three β-tubulin isoforms at the luminal face of microtubules and suppresses α-tubulin acetylation. Interaction is weakened by the R1441G LRRK2 GTPase domain mutant.
Conclusion: LRRK2 modulates microtubule stability.
Significance: Deregulation of microtubule-dependent processes likely contribute to neurodegeneration in Parkinson disease.
Mutations in LRRK2, encoding the multifunctional protein leucine-rich repeat kinase 2 (LRRK2), are a common cause of Parkinson disease. LRRK2 has been suggested to influence the cytoskeleton as LRRK2 mutants reduce neurite outgrowth and cause an accumulation of hyperphosphorylated Tau. This might cause alterations in the dynamic instability of microtubules suggested to contribute to the pathogenesis of Parkinson disease. Here, we describe a direct interaction between LRRK2 and β-tubulin. This interaction is conferred by the LRRK2 Roc domain and is disrupted by the familial R1441G mutation and artificial Roc domain mutations that mimic autophosphorylation. LRRK2 selectively interacts with three β-tubulin isoforms: TUBB, TUBB4, and TUBB6, one of which (TUBB4) is mutated in the movement disorder dystonia type 4 (DYT4). Binding specificity is determined by lysine 362 and alanine 364 of β-tubulin. Molecular modeling was used to map the interaction surface to the luminal face of microtubule protofibrils in close proximity to the lysine 40 acetylation site in α-tubulin. This location is predicted to be poorly accessible within mature stabilized microtubules, but exposed in dynamic microtubule populations. Consistent with this finding, endogenous LRRK2 displays a preferential localization to dynamic microtubules within growth cones, rather than adjacent axonal microtubule bundles. This interaction is functionally relevant to microtubule dynamics, as mouse embryonic fibroblasts derived from LRRK2 knock-out mice display increased microtubule acetylation. Taken together, our data shed light on the nature of the LRRK2-tubulin interaction, and indicate that alterations in microtubule stability caused by changes in LRRK2 might contribute to the pathogenesis of Parkinson disease.
Lrrk2; Microtubules; Molecular Genetics; Parkinson Disease; Tubulin; GTPase Mutation; RocCOR; Cytoskeletal Dynamics; Growth Cone; Tubulin Acetylation
Mutations in Leucine-rich repeat kinase 2 (LRRK2) are a common cause of inherited Parkinson’s disease (PD). The protein is large and complex, but pathogenic mutations cluster in a region containing GTPase and kinase domains. LRRK2 can autophosphorylate in vitro within a dimer pair, although the significance of this reaction is unclear. Here, we mapped the sites of autophosphorylation within LRRK2 and found several potential phosphorylation sites within the GTPase domain. Using mass spectrometry, we found that Thr1343 is phosphorylated and, using kinase dead versions of LRRK2, show that this is an autophosphorylation site. However, we also find evidence for additional sites in the GTPase domain and in other regions of the protein suggesting that there may be multiple autophosphorylation sites within LRRK2. These data suggest that the kinase and GTPase activities of LRRK2 may exhibit complex autoregulatory interdependence.
Parkinson’s disease; kinase; GTPase; autophosphorylation
The levels of microRNAs (miRNAs) are altered under different conditions such as cancer, senescence, and aging. Here, we have identified differentially expressed miRNAs in skeletal muscle from young and old rhesus monkeys using RNA sequencing. In old muscle, several miRNAs were upregulated, including miR-451, miR-144, miR-18a and miR-15a, while a few miRNAs were downregulated, including miR-181a and miR-181b. A number of novel miRNAs were also identified, particularly in old muscle. We also examined the impact of caloric restriction (CR) on miRNA abundance by reverse transcription (RT) followed by real-time, quantitative (q)PCR analysis and found that CR rescued the levels of miR-181b and chr1:205580546, and also dampened the age-induced increase in miR-451 and miR-144 levels. Our results reveal that there are changes in expression of known and novel miRNAs with skeletal muscle aging and that CR may reverse some of these changes to a younger phenotype.
gene expression; posttranscriptional gene regulation; muscle aging; muscle diseases
Mutations in parkin, PTEN-induced kinase 1 (PINK1) and DJ-1 can all cause autosomal recessive forms of Parkinson disease. Recent data suggest that these recessive parkinsonism-associated genes converge within a single pathogenic pathway whose dysfunction leads to the loss of substantia nigra pars compacta neurons. The major common functional effects of all three genes relate to mitochondrial and oxidative damage, with a possible additional involvement of the ubiquitin proteasome system. This review highlights the role of the mitochondrial kinase, PINK1, in protection against mitochondrial dysfunction and how this might relate to loss of substantia nigra neurons in recessive parkinsonism.
parkinsonism; parkin; Drp1; fission; fusion; oxidative stress
In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.
LC3; autolysosome; autophagosome; flux; lysosome; phagophore; stress; vacuole