Publications

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.

To identify novel risk genes and better understand the molecular pathway underlying Alzheimer's disease (AD), whole-exome sequencing was performed in 215 early-onset AD (EOAD) patients and 255 unrelated healthy controls of Han Chinese ethnicity. Subsequent validation, computational annotation and in vitro functional studies were performed to evaluate the role of candidate variants in EOAD. We identified two rare missense variants in the phosphodiesterase 11A (PDE11A) gene in individuals with EOAD. Both variants are located in evolutionarily highly conserved amino acids, are predicted to alter the protein conformation and are classified as pathogenic. Furthermore, we found significantly decreased protein levels of PDE11A in brain samples of AD patients. Expression of PDE11A variants and knockdown experiments with specific short hairpin RNA (shRNA) for PDE11A both resulted in an increase of AD-associated Tau hyperphosphorylation at multiple epitopes in vitro. PDE11A variants or PDE11A shRNA also caused increased cyclic adenosine monophosphate (cAMP) levels, protein kinase A (PKA) activation and cAMP response element-binding protein phosphorylation. In addition, pretreatment with a PKA inhibitor (H89) suppressed PDE11A variant-induced Tau phosphorylation formation. This study offers insight into the involvement of Tau phosphorylation via the cAMP/PKA pathway in EOAD pathogenesis and provides a potential new target for intervention.

Alpha-synuclein is the main protein component of Lewy bodies, the pathological hallmark of Parkinson's disease. However, genetic modifiers of cerebrospinal fluid (CSF) alpha-synuclein levels remain unknown. The use of CSF levels of amyloid beta1-42, total tau, and phosphorylated tau181 as quantitative traits in genetic studies have provided novel insights into Alzheimer's disease pathophysiology. A systematic study of the genomic architecture of CSF biomarkers in Parkinson's disease has not yet been conducted. Here, genome-wide association studies of CSF biomarker levels in a cohort of individuals with Parkinson's disease and controls (N = 1960) were performed. PD cases exhibited significantly lower CSF biomarker levels compared to controls. A SNP, proxy for APOE ε4, was associated with CSF amyloid beta1-42 levels (effect = - 0.5, p = 9.2 × 10-19). No genome-wide loci associated with CSF alpha-synuclein, total tau, or phosphorylated tau181 levels were identified in PD cohorts. Polygenic risk score constructed using the latest Parkinson's disease risk meta-analysis were associated with Parkinson's disease status (p = 0.035) and the genomic architecture of CSF amyloid beta1-42 (R2 = 2.29%; p = 2.5 × 10-11). Individuals with higher polygenic risk scores for PD risk presented with lower CSF amyloid beta1-42 levels (p = 7.3 × 10-04). Two-sample Mendelian Randomization revealed that CSF amyloid beta1-42 plays a role in Parkinson's disease (p = 1.4 × 10-05) and age at onset (p = 7.6 × 10-06), an effect mainly mediated by variants in the APOE locus. In a subset of PD samples, the APOE ε4 allele was associated with significantly lower levels of CSF amyloid beta1-42 (p = 3.8 × 10-06), higher mean cortical binding potentials (p = 5.8 × 10-08), and higher Braak amyloid beta score (p = 4.4 × 10-04). Together these results from high-throughput and hypothesis-free approaches converge on a genetic link between Parkinson's disease, CSF amyloid beta1-42, and APOE.

Apart from amyloid β deposition and tau neurofibrillary tangles, Alzheimer's disease (AD) is a neurodegenerative disorder characterized by neuronal loss and astrocytosis in the cerebral cortex. The goal of this study is to investigate genetic factors associated with the neuronal proportion in health and disease. To identify cell-autonomous genetic variants associated with neuronal proportion in cortical tissues, we inferred cellular population structure from bulk RNA-Seq derived from 1536 individuals. We identified the variant rs1990621 located in the TMEM106B gene region as significantly associated with neuronal proportion (p value = 6.40 × 10-07) and replicated this finding in an independent dataset (p value = 7.41 × 10-04) surpassing the genome-wide threshold in the meta-analysis (p value = 9.42 × 10-09). This variant is in high LD with the TMEM106B non-synonymous variant p.T185S (rs3173615; r2 = 0.98) which was previously identified as a protective variant for frontotemporal lobar degeneration (FTLD). We stratified the samples by disease status, and discovered that this variant modulates neuronal proportion not only in AD cases, but also several neurodegenerative diseases and in elderly cognitively healthy controls. Furthermore, we did not find a significant association in younger controls or schizophrenia patients, suggesting that this variant might increase neuronal survival or confer resilience to the neurodegenerative process. The single variant and gene-based analyses also identified an overall genetic association between neuronal proportion, AD and FTLD risk. These results suggest that common pathways are implicated in these neurodegenerative diseases, that implicate neuronal survival. In summary, we identified a protective variant in the TMEM106B gene that may have a neuronal protection effect against general aging, independent of disease status, which could help elucidate the relationship between aging and neuronal survival in the presence or absence of neurodegenerative disorders. Our findings suggest that TMEM106B could be a potential target for neuronal protection therapies to ameliorate cognitive and functional deficits.

Epidemiologic studies have reported inconsistent results regarding an association between Parkinson disease (PD) and cutaneous melanoma (melanoma). Identifying shared genetic architecture between these diseases can support epidemiologic findings and identify common risk genes and biological pathways. Here, we apply polygenic, linkage disequilibrium-informed methods to the largest available case-control, genome-wide association study summary statistic data for melanoma and PD. We identify positive and significant genetic correlation (correlation: 0.17, 95% CI 0.10-0.24; P = 4.09 × 10-06) between melanoma and PD. We further demonstrate melanoma and PD-inferred gene expression to overlap across tissues (correlation: 0.14, 95% CI 0.06 to 0.22; P = 7.87 × 10-04) and highlight seven genes including PIEZO1, TRAPPC2L, and SOX6 as potential mediators of the genetic correlation between melanoma and PD. These findings demonstrate specific, shared genetic architecture between PD and melanoma that manifests at the level of gene expression.

Apolipoprotein E (APOE) ε4 genotype is associated with increased risk of dementia in Parkinson's disease (PD), but the mechanism is not clear, because patients often have a mixture of α-synuclein (αSyn), amyloid-β (Aβ), and tau pathologies. APOE ε4 exacerbates brain Aβ pathology, as well as tau pathology, but it is not clear whether APOE genotype independently regulates αSyn pathology. In this study, we generated A53T αSyn transgenic mice (A53T) on Apoe knockout (A53T/EKO) or human APOE knockin backgrounds (A53T/E2, E3, and E4). At 12 months of age, A53T/E4 mice accumulated higher amounts of brainstem detergent-insoluble phosphorylated αSyn compared to A53T/EKO and A53T/E3; detergent-insoluble αSyn in A53T/E2 mice was undetectable. By immunohistochemistry, A53T/E4 mice displayed a higher burden of phosphorylated αSyn and reactive gliosis compared to A53T/E2 mice. A53T/E2 mice exhibited increased survival and improved motor performance compared to other APOE genotypes. In a complementary model of αSyn spreading, striatal injection of αSyn preformed fibrils induced greater accumulation of αSyn pathology in the substantia nigra of A53T/E4 mice compared to A53T/E2 and A53T/EKO mice. In two separate cohorts of human patients with PD, APOE ε4/ε4 individuals showed the fastest rate of cognitive decline over time. Our results demonstrate that APOE genotype directly regulates αSyn pathology independent of its established effects on Aβ and tau, corroborate the finding that APOE ε4 exacerbates pathology, and suggest that APOE ε2 may protect against αSyn aggregation and neurodegeneration in synucleinopathies.

BACKGROUND: Low frequency coding variants in TREM2 are associated with Alzheimer disease (AD) risk and cerebrospinal fluid (CSF) TREM2 protein levels are different between AD cases and controls. Similarly, TREM2 risk variant carriers also exhibit differential CSF TREM2 levels. TREM2 has three different alternative transcripts, but most of the functional studies only model the longest transcript. No studies have analyzed TREM2 expression levels or alternative splicing in brains from AD and cognitively normal individuals. We wanted to determine whether there was differential expression of TREM2 in sporadic-AD cases versus AD-TREM2 carriers vs sex- and aged-matched normal controls; and if this differential expression was due to a particular TREM2 transcript.

METHODS: We analyzed RNA-Seq data from parietal lobe brain tissue from AD cases with TREM2 variants (n = 33), AD cases (n = 195) and healthy controls (n = 118), from three independent datasets using Kallisto and the R package tximport to determine the read count for each transcript and quantified transcript abundance as transcripts per million.

RESULTS: The three TREM2 transcripts were expressed in brain cortex in the three datasets. We demonstrate for the first time that the transcript that lacks the transmembrane domain and encodes a soluble form of TREM2 (sTREM2) has an expression level around 60% of the canonical transcript, suggesting that around 25% of the sTREM2 protein levels could be explained by this transcript. We did not observe a difference in the overall TREM2 expression level between cases and controls. However, the isoform which lacks the 5' exon, but includes the transmembrane domain, was significantly lower in TREM2- p.R62H carriers than in AD cases (p = 0.007).

CONCLUSION: Using bulk RNA-Seq data from three different cohorts, we were able to quantify the expression level of the three TREM2 transcripts, demonstrating: (1) all three transcripts of them are highly expressed in the human cortex, (2) that up to 25% of the sTREM2 may be due to the expression of a specific isoform and not TREM2 cleavage; and (3) that TREM2 risk variants do not affect expression levels, suggesting that the effect of the TREM2 variants on CSF levels occurs at post-transcriptional level.

BACKGROUND: Alzheimer's disease (AD) is the most common form of dementia. This neurodegenerative disorder is associated with neuronal death and gliosis heavily impacting the cerebral cortex. AD has a substantial but heterogeneous genetic component, presenting both Mendelian and complex genetic architectures. Using bulk RNA-seq from the parietal lobes and deconvolution methods, we previously reported that brains exhibiting different AD genetic architecture exhibit different cellular proportions. Here, we sought to directly investigate AD brain changes in cell proportion and gene expression using single-cell resolution.

METHODS: We generated unsorted single-nuclei RNA sequencing data from brain tissue. We leveraged the tissue donated from a carrier of a Mendelian genetic mutation, PSEN1 p.A79V, and two family members who suffer from sporadic AD, but do not carry any autosomal mutations. We evaluated alternative alignment approaches to maximize the titer of reads, genes, and cells with high quality. In addition, we employed distinct clustering strategies to determine the best approach to identify cell clusters that reveal neuronal and glial cell types and avoid artifacts such as sample and batch effects. We propose an approach to cluster cells that reduces biases and enable further analyses.

RESULTS: We identified distinct types of neurons, both excitatory and inhibitory, and glial cells, including astrocytes, oligodendrocytes, and microglia, among others. In particular, we identified a reduced proportion of excitatory neurons in the Mendelian mutation carrier, but a similar distribution of inhibitory neurons. Furthermore, we investigated whether single-nuclei RNA-seq from the human brains recapitulate the expression profile of disease-associated microglia (DAM) discovered in mouse models. We also determined that when analyzing human single-nuclei data, it is critical to control for biases introduced by donor-specific expression profiles.

CONCLUSION: We propose a collection of best practices to generate a highly detailed molecular cell atlas of highly informative frozen tissue stored in brain banks. Importantly, we have developed a new web application to make this unique single-nuclei molecular atlas publicly available.