The pathogenesis and clinical heterogeneity of Parkinson's disease (PD) have been evaluated from molecular, pathophysiological, and clinical perspectives. High-throughput proteomic analysis of cerebrospinal fluid (CSF) opened new opportunities for scrutinizing this heterogeneity. To date, this is the most comprehensive CSF-based proteomics profiling study in PD with 569 patients (350 idiopathic patients, 65 GBA + mutation carriers and 154 LRRK2 + mutation carriers), 534 controls, and 4135 proteins analyzed. Combining CSF aptamer-based proteomics with genetics we determined protein quantitative trait loci (pQTLs). Analyses of pQTLs together with summary statistics from the largest PD genome wide association study (GWAS) identified 68 potential causal proteins by Mendelian randomization. The top causal protein, GPNMB, was previously reported to be upregulated in the substantia nigra of PD patients. We also compared the CSF proteomes of patients and controls. Proteome differences between GBA + patients and unaffected GBA + controls suggest degeneration of dopaminergic neurons, altered dopamine metabolism and increased brain inflammation. In the LRRK2 + subcohort we found dysregulated lysosomal degradation, altered alpha-synuclein processing, and neurotransmission. Proteome differences between idiopathic patients and controls suggest increased neuroinflammation, mitochondrial dysfunction/oxidative stress, altered iron metabolism and potential neuroprotection mediated by vasoactive substances. Finally, we used proteomic data to stratify idiopathic patients into "endotypes". The identified endotypes show differences in cognitive and motor disease progression based on previously reported protein-based risk scores.Our findings not only contribute to the identification of new therapeutic targets but also to shape personalized medicine in CNS neurodegeneration.

Background Alzheimer disease (AD) has substantial genetic, molecular, and cellular heterogeneity associated with its etiology. Much of our current understanding of the main AD molecular events associated with the amyloid hypothesis ( APP, PSEN1 and PSEN2 ) and neuroimmune modulation ( TREM2 and MS4A ) is based on genetic studies including GWAS. However, the functional genes, downstream transcriptional ramifications, and the cell-type-specific effects of many GWAS loci remain poorly understood. Understanding these effects can point us to the cellular processes involved in AD and uncover potential therapeutic targets. Methods We applied a genetic-based approach to our sample selection; our cohort included carriers of AD pathogenic mutations ( APP, PSEN1) , risk variants in TREM2 , and the resilience variant (rs1582763) in the MS4A cluster associated with cerebrospinal fluid (CSF) soluble TREM2 levels. We performed single-nucleus RNA-sequencing (snRNA-seq) of 1,102,459 nuclei from the human parietal cortex of these carriers. Following initial unbiased clustering and cell-type annotation, we performed deep subclustering analysis per cell type to identify unique cellular transcriptional states associated with these genetic variants. We identified differentially expressed genes between cell states and genetic variant carriers/controls, and performed differential cell proportion analyses to determine key differences among these carriers. We analyzed sequencing data from human dorsolateral prefrontal cortex and mouse models to replicate the enrichment of unique cell states in genetic variant carriers. Finally, we leveraged these cell-state differential expression results to link genes in AD GWAS loci to their functional cell types. Findings We identified cell-specific expression states influenced by AD genetic factors for neurons and glia. Autosomal dominant AD (ADAD) brains exhibited unique transcriptional states in all cell types. TREM2 variant carrier brains were also enriched for specific microglia and oligodendrocyte subpopulations. Carriers of the resilience MS4A variant were enriched for an altered activated-microglia expression state. We mapped AD GWAS genes to their potential functional cell types, and some, including PLCG2 and SORL1 , were expressed in a broader range of brain cell types than previously reported. Interpretation AD pathogenic, risk, or resilience variants are sufficient to alter the transcriptional and cellular landscape of human brains. Overall, our results suggest that the genetic architecture contributes to the cortical cellular heterogeneity associated with disease status, which is a critical factor to consider when designing drug trials and selecting the treatment program for AD patients. Our findings suggest that integrating genetic and single-cell molecular data facilitates our understanding of the heterogeneity of pathways, biological processes and cell types modulated by genetic risk factors for AD. Funding US National Institutes of Health, Hope Center, Archer foundation, Alzheimer Association, CZI.

Regulation of glial activation and neuroinflammation are critical factors in the pathogenesis of Alzheimer’s disease (AD). YKL-40, a primarily astrocytic protein encoded by the gene Chi3l1 , is a widely studied cerebrospinal fluid biomarker that increases with aging and early in AD. However, the function of Chi3l1 /YKL-40 in AD is unknown. In a cohort of patients with AD, we observed that a variant in the human CHI3L1 gene, which results in decreased CSF YKL-40 expression, was associated with slower AD progression. At baseline, Chi3l1 deletion in mice had no effect on astrocyte activation while modestly promoting microglial activation. In a mouse APP/PS1 model of AD, Chi3l1 deletion decreased amyloid plaque burden and increased periplaque expression of the microglial lysosomal marker CD68, suggesting that Chi3l1 may suppress glial phagocytic activation and promote amyloid accumulation. Accordingly, Chi3l1 knockdown increased phagocytosis of zymosan particles and of β-amyloid peptide in both astrocytes and microglia in vitro. We further observed that expression of Chi3l1 is regulated by the circadian clock, as deletion of the core clock proteins BMAL1 or CLOCK/NPAS2 strongly suppresses basal Chi3l1 expression, whereas deletion of the negative clock regulators PER1/PER2 increased Chi3l1 expression. Basal Chi3l1 mRNA was nonrhythmic because of a long mRNA half-life in astrocytes. However, inflammatory induction of Chi3l1 was gated by the clock. Our findings reveal Chi3l1 /YKL-40 as a modulator of glial phagocytic activation and AD pathogenesis in both mice and humans and suggest that the astrocyte circadian clock regulates inflammatory Chi3l1 induction.

Parietal cortex RNA-sequencing (RNA-seq) data were generated from individuals with and without Alzheimer disease (AD; n(control) = 13; n(AD) = 83) from the Knight Alzheimer Disease Research Center (Knight ADRC). Using this and an independent (Mount Sinai Brain Bank (MSBB)) AD RNA-seq dataset, cortical circular RNA (circRNA) expression was quantified in the context of AD. Significant associations were identified between circRNA expression and AD diagnosis, clinical dementia severity and neuropathological severity. It was demonstrated that most circRNA-AD associations are independent of changes in cognate linear messenger RNA expression or estimated brain cell-type proportions. Evidence was provided for circRNA expression changes occurring early in presymptomatic AD and in autosomal dominant AD. It was also observed that AD-associated circRNAs coexpressed with known AD genes. Finally, potential microRNA-binding sites were identified in AD-associated circRNAs for miRNAs predicted to target AD genes. Together, these results highlight the importance of analyzing non-linear RNAs and support future studies exploring the potential roles of circRNAs in AD pathogenesis.

Alzheimer disease (AD) is a complex disorder with diversity in phenotype, onset and progression of clinical symptoms. Ultimately, AD is characterized by the deposition of amyloid beta (Aβ) and phosphorylated tau protein aggregates in the brain. We hypothesize that different clinical AD manifestations may result from common altered pathways relevant to the development of pathology. In our effort to identify novel players to the missing heritability of AD, we have performed genomic and transcriptomic integrative analysis. We leveraged genomic data (https://doi.org/10.3389/fnins.2018.00209) and transcriptomic data (doi:10.1186/s13073-018-0551-4) from autosomal dominant AD (ADAD), early onset AD (EOAD), late onset AD (LOAD) patients and healthy controls (HC), which was available in our laboratory. Transcriptomic data was generated using parietal tissue from brain donors from the Knight-ADRC at WUSM. After QC, 103 samples remained for analysis: 16 HC, 19 ADAD, 13 EOAD and 55 LOAD. Statistical analyses were performed with DESeq2. Through transcriptome-wide PCA, we can infer that AD and HC present largely different transcriptomic profiles (Figure 1a). Differential gene expression analysis identified AGFG2 as a significantly differentiated gene across AD etiologies and vs HC (Figure 1b). It was nominally significant in gene-based analysis in the familial cohort (CMC P=0.012) and several variants within this gene segregated according to disease status. AGFG2 is a member of the HIV-1 Rev binding protein family and plays a role in the Rev export pathway (https://doi.org/10.1038/s41593-018-0216-z). In the brain, it is primarily expressed in astrocytes. Recent studies using siRNA in HEK293 cells stably over-expressing a mCherry-APP695wt-YFP found AGFG2 among the top eight genes with genome-wide significance, suggesting that these genes could be involved in the AD process via the regulation of APP metabolism (https://doi.org/10.1007/s00401-016-1652-z). The ADSP consortia recently published new rare variants in STAG3 associated with AD; interestingly, the associated variant is an eQTL for AGFG2 (https://doi.org/10.1038/s41380-018-0112-7).

Umber Dube, Jorge L Del-Aguila, Zeran Li, John P Budde, Shan Jiang, Simon Hsu Laura Ibanez, 4 Maria Victoria Fernandez, Fabiana Farias, Joanne Norton, Jen Gentsch, Fengxian Wang, the 5 Dominantly Inherited Alzheimer Network (DIAN), Stephen Salloway, Colin L Masters, Jae-Hong Lee, 6 Neill R Graff-Radford, Jasmeer P Chhatwal, Randall J Bateman, John C Morris, Celeste M Karch, 7 Oscar Harari, and Carlos Cruchaga 8

Background Alzheimer’s disease (AD) is characterized by neuronal loss and astrocytosis in the cerebral cortex. However, the specific effects that pathological mutations and coding variants associated with AD have on the cellular composition of the brain are often ignored. Methods We developed and optimized a cell-type-specific expression reference panel and employed digital deconvolution methods to determine brain cellular distribution in three independent transcriptomic studies. Results We found that neuronal and astrocyte relative proportions differ between healthy and diseased brains and also among AD cases that carry specific genetic risk variants. Brain carriers of pathogenic mutations in APP , PSEN1 , or PSEN2 presented lower neuron and higher astrocyte relative proportions compared to sporadic AD. Similarly, the APOE ε 4 allele also showed decreased neuronal and increased astrocyte relative proportions compared to AD non-carriers. In contrast, carriers of variants in TREM2 risk showed a lower degree of neuronal loss compared to matched AD cases in multiple independent studies. Conclusions These findings suggest that genetic risk factors associated with AD etiology have a specific imprinting in the cellular composition of AD brains. Our digital deconvolution reference panel provides an enhanced understanding of the fundamental molecular mechanisms underlying neurodegeneration, enabling the analysis of large bulk RNA-sequencing studies for cell composition and suggests that correcting for the cellular structure when performing transcriptomic analysis will lead to novel insights of AD.

BACKGROUND:A relationship quantitative trait locus exists when the correlation between multiple traits varies by genotype for that locus. Relationship quantitative trait loci (rQTL) are often involved in gene-by-gene (G×G) interactions or gene-by-environmental interactions, making them a powerful tool for detecting G×G.METHODS:We performed genome-wide association studies to identify rQTL between tau and Aβ42 and ptau and Aβ42 with over 3000 individuals using age, gender, series, APOE ε2, APOE ε4, and two principal components for population structure as covariates. Each significant rQTL was separately screened for interactions with other loci for each trait in the rQTL model. Parametric bootstrapping was used to assess significance.RESULTS:We found four significant tau/Aβ42 rQTL from three unique locations and six ptau/Aβ42 rQTL from five unique locations. G×G screens with these rQTL produced four significant G×G interactions (one Aβ42, two ptau, and one tau) with four rQTL where each second locus was from a unique location. On follow-up, rs1036819 and rs74025622 were associated with Alzheimer's disease (AD) case/control status; rs15205 and rs79099429 were associated with rate of decline.CONCLUSIONS:The two most significant rQTL (rs8027714 and rs1036819) for ptau/Aβ42 are on different chromosomes and both are strong hits for pelvic organ prolapse. While diseases of the nervous system can cause pelvic organ prolapse, it is unlikely related to the ptau/Aβ42 relationship but may suggest that these two loci share a pathway. In addition to a ptau/Aβ42 rQTL and association with AD case/control status, rs1036819 is a strong rQTL for case/control status/Aβ42 and for tau/Aβ42. It resides in the ZFAT gene, which is related to autoimmune thyroid disease. For tau, rs9817620 interacts with the tau/Aβ42 rQTL rs74025622. It is in the CHL1 gene, which is a neural cell adhesion molecule and may be involved in signal transduction pathways. CHL1 is related to BACE1, which is a β-secretase enzyme that initiates production of the β-amyloid peptide involved in AD and is a primary drug target. Overall, there are numerous loci that affect the relationship between these important AD endophenotypes and some are due to interactions with other loci. Some affect the risk of AD and/or rate of progression.

A genome-wide survival analysis of 14,406 Alzheimer's disease (AD) cases and 25,849 controls identified eight previously reported AD risk loci and 14 novel loci associated with age at onset. Linkage disequilibrium score regression of 220 cell types implicated the regulation of myeloid gene expression in AD risk. The minor allele of rs1057233 (G), within the previously reported CELF1 AD risk locus, showed association with delayed AD onset and lower expression of SPI1 in monocytes and macrophages. SPI1 encodes PU.1, a transcription factor critical for myeloid cell development and function. AD heritability was enriched within the PU.1 cistrome, implicating a myeloid PU.1 target gene network in AD. Finally, experimentally altered PU.1 levels affected the expression of mouse orthologs of many AD risk genes and the phagocytic activity of mouse microglial cells. Our results suggest that lower SPI1 expression reduces AD risk by regulating myeloid gene expression and cell function.

Alzheimer disease (AD), Frontotemporal lobar degeneration (FTD), Amyotrophic lateral sclerosis (ALS) and Parkinson disease (PD) have a certain degree of clinical, pathological and molecular overlap. Previous studies indicate that causative mutations in AD and FTD/ALS genes can be found in clinical familial AD. We examined the presence of causative and low frequency coding variants in the AD, FTD, ALS and PD Mendelian genes, in over 450 families with clinical history of AD and over 11,710 sporadic cases and cognitive normal participants from North America. Known pathogenic mutations were found in 1.05% of the sporadic cases, in 0.69% of the cognitively normal participants and in 4.22% of the families. A trend towards enrichment, albeit non-significant, was observed for most AD, FTD and PD genes. Only PSEN1 and PINK1 showed consistent association with AD cases when we used ExAC as the control population. These results suggest that current study designs may contain heterogeneity and contamination of the control population, and that current statistical methods for the discovery of novel genes with real pathogenic variants in complex late onset diseases may be inadequate or underpowered to identify genes carrying pathogenic mutations.

APOE4 is the strongest genetic risk factor for late-onset Alzheimer disease. ApoE4 increases brain amyloid-β pathology relative to other ApoE isoforms. However, whether APOE independently influences tau pathology, the other major proteinopathy of Alzheimer disease and other tauopathies, or tau-mediated neurodegeneration, is not clear. By generating P301S tau transgenic mice on either a human ApoE knock-in (KI) or ApoE knockout (KO) background, here we show that P301S/E4 mice have significantly higher tau levels in the brain and a greater extent of somatodendritic tau redistribution by three months of age compared with P301S/E2, P301S/E3, and P301S/EKO mice. By nine months of age, P301S mice with different ApoE genotypes display distinct phosphorylated tau protein (p-tau) staining patterns. P301S/E4 mice develop markedly more brain atrophy and neuroinflammation than P301S/E2 and P301S/E3 mice, whereas P301S/EKO mice are largely protected from these changes. In vitro, E4-expressing microglia exhibit higher innate immune reactivity after lipopolysaccharide treatment. Co-culturing P301S tau-expressing neurons with E4-expressing mixed glia results in a significantly higher level of tumour-necrosis factor-α (TNF-α) secretion and markedly reduced neuronal viability compared with neuron/E2 and neuron/E3 co-cultures. Neurons co-cultured with EKO glia showed the greatest viability with the lowest level of secreted TNF-α. Treatment of P301S neurons with recombinant ApoE (E2, E3, E4) also leads to some neuronal damage and death compared with the absence of ApoE, with ApoE4 exacerbating the effect. In individuals with a sporadic primary tauopathy, the presence of an ε4 allele is associated with more severe regional neurodegeneration. In individuals who are positive for amyloid-β pathology with symptomatic Alzheimer disease who usually have tau pathology, ε4-carriers demonstrate greater rates of disease progression. Our results demonstrate that ApoE affects tau pathogenesis, neuroinflammation, and tau-mediated neurodegeneration independently of amyloid-β pathology. ApoE4 exerts a 'toxic' gain of function whereas the absence of ApoE is protective.

Alzheimer’s disease (AD) pathology appears several years before clinical symptoms, so identifying ways to detect individuals in the preclinical stage is imperative. The cerebrospinal fluid (CSF) Tau/Aβ42 ratio is currently the best known predictor of AD status and cognitive decline, and the ratio of CSF levels of chitinase-3-like 1 protein (CHI3L1, YKL-40) and amyloid beta (Aβ42) were reported as predictive, but individual variability and group overlap inhibits their utility for individual diagnosis making it necessary to find ways to improve sensitivity of these biomarkers.

Low frequency coding variants in TREM2 are associated with increased Alzheimer disease (AD) risk, while loss of functions mutations in the gene lead to an autosomal recessive early-onset dementia, named Nasu-Hakola disease (NHD). TREM2 can be detected as a soluble protein in cerebrospinal fluid (CSF) and plasma, and its CSF levels are elevated in inflammatory CNS diseases. We measured soluble TREM2 (sTREM2) in the CSF of a large AD case–control dataset ( n = 180) and 40 TREM2 risk variant carriers to determine whether CSF sTREM2 levels are associated with AD status or mutation status. We also performed genetic studies to identify genetic variants associated with CSF sTREM2 levels. CSF, but not plasma, sTREM2 was highly correlated with CSF total tau and phosphorylated-tau levels ( r = 0.35, P < 1×10 −4 ; r = 0.40, P < 1×10 −4 , respectively), but not with CSF Aβ42. AD cases presented higher CSF sTREM2 levels than controls ( P = 0.01). Carriers of NHD-associated TREM2 variants presented significantly lower CSF sTREM2 levels, supporting the hypothesis that these mutations lead to reduced protein production/function (R136Q, D87N, Q33X or T66M; P = 1×10 −3 ). In contrast, CSF sTREM2 levels were significantly higher in R47H carriers compared to non-carriers ( P = 6×10 −3 ), suggesting that this variant does not impact protein expression and increases AD risk through a different pathogenic mechanism than NHD variants. In GWAS analyses for CSF sTREM2 levels the most significant signal was located on the MS4A gene locus ( P = 5.45 × 10 −07 ) corresponding to one of the SNPs reported to be associated with AD risk in this locus. Furthermore, SNPs involved in pathways related to virus cellular entry and vesicular trafficking were overrepresented, suggesting that CSF sTREM2 levels could be an informative phenotype for AD.

Much of the heritability of late-onset Alzheimer's disease (LOAD) remains unaccounted for, despite progress from genome-wide association studies. The overall contribution of rare variants to the risk of LOAD remains to be determined and, to date, few rare variants in LOAD have been identified and functionally characterized. We hypothesized that families with multiple affected individuals are enriched for genetic risk factors. In previous studies, we performed whole-exome sequencing in 14 families with LOAD and found an enrichment of coding variants in the phospholipase D3 (PLD3) gene and Unc-5 Homolog C (UNC5C). We have generated whole exome sequencing for additional 103 families (380 individuals). Enrichment of coding exons and flanking intronic regions was performed using VCRome. This step was performed by the Genome Institute at Washington University. Captured DNA was sequenced by paired-end reads on the HiSeq 2000 sequencer (Illumina). Sequencing data and variant calling was performed with the BWA/GATK pipeline. To identify novel variants and genes associated with AD risk, we performed analyses within families (Mendelscan: http://gmt.genome.wustl.edu/packages/mendelscan/) and across families (GSKAT). We found more than 700,000 variants. Around 20% of those variants were located on coding regions. Of these 53.8% were missense and 40.8% were synonymous. We also found between 12 to 324 low frequency variants (MAF<1%) segregating with disease status within the sequencing individuals. The number of segregating variants depended on the number of sequenced samples per family. First, we tested whether the genes known to be involved on AD, showed a significant association with AD. PSEN1, 2, APOE, PLD3, SORL1, AKAP9 showed a nominal association, but none of the GWAS genes. In the G-SKAT analyses we did not found any gene that passed multiple test correction, but we found three genes with a suggestive association (p=9x10−5): PROM2, BEST1 and HIST1H1C. We have identified several new candidate genes for risk for AD, however additional studies are needed to validate their association with AD. We plan to perform additional segregation analyses in the rest of the family members, as well as deep resequencing in large datasets to validate the role of those genes in AD risk.