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.
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
, is a widely ...studied cerebrospinal fluid biomarker that increases with aging and early in AD. However, the function of
/YKL-40 in AD is unknown. In a cohort of patients with AD, we observed that a variant in the human
gene, which results in decreased CSF YKL-40 expression, was associated with slower AD progression. At baseline,
deletion in mice had no effect on astrocyte activation while modestly promoting microglial activation. In a mouse APP/PS1 model of AD,
deletion decreased amyloid plaque burden and increased periplaque expression of the microglial lysosomal marker CD68, suggesting that
may suppress glial phagocytic activation and promote amyloid accumulation. Accordingly,
knockdown increased phagocytosis of zymosan particles and of β-amyloid peptide in both astrocytes and microglia in vitro. We further observed that expression of
is regulated by the circadian clock, as deletion of the core clock proteins BMAL1 or CLOCK/NPAS2 strongly suppresses basal
expression, whereas deletion of the negative clock regulators PER1/PER2 increased
expression. Basal
mRNA was nonrhythmic because of a long mRNA half-life in astrocytes. However, inflammatory induction of
was gated by the clock. Our findings reveal
/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
induction.
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.
To determine whether the extent of overlap of the genetic architecture among the sporadic late-onset Alzheimer's Disease (sLOAD), familial late-onset AD (fLOAD), sporadic early-onset AD (sEOAD), and ...autosomal dominant early-onset AD (eADAD).
Polygenic risk scores (PRSs) were constructed using previously identified 21 genome-wide significant loci for LOAD risk.
We found that there is an overlap in the genetic architecture among sEOAD, fLOAD, and sLOAD. The highest association of the PRS and risk (odds ratio OR = 2.27; P = 1.29 × 10−7) was observed in sEOAD, followed by fLOAD (OR = 1.75; P = 1.12 × 10−7) and sLOAD (OR = 1.40; P = 1.21 × 10−3). The PRS was associated with cerebrospinal fluid ptau181-Aβ42 on eADAD (P = 4.36 × 10−2).
Our analysis confirms that the genetic factors identified for LOAD modulate risk in sLOAD and fLOAD and also sEOAD cohorts. Specifically, our results suggest that the burden of these risk variants is associated with familial clustering and earlier onset of AD. Although these variants are not associated with risk in the eADAD, they may be modulating age at onset.
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.
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.
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.
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.
Soluble triggering receptor expressed on myeloid cells 2 (sTREM2) in cerebrospinal fluid (CSF) has been associated with Alzheimer's disease (AD). TREM2 plays a critical role in microglial activation, ...survival, and phagocytosis; however, the pathophysiological role of sTREM2 in AD is not well understood. Understanding the role of sTREM2 in AD may reveal new pathological mechanisms and lead to the identification of therapeutic targets. We performed a genome-wide association study (GWAS) to identify genetic modifiers of CSF sTREM2 obtained from the Alzheimer's Disease Neuroimaging Initiative. Common variants in the membrane-spanning 4-domains subfamily A (
) gene region were associated with CSF sTREM2 concentrations (rs1582763;
= 1.15 × 10
); this was replicated in independent datasets. The variants associated with increased CSF sTREM2 concentrations were associated with reduced AD risk and delayed age at onset of disease. The single-nucleotide polymorphism rs1582763 modified expression of the
and
genes in multiple tissues, suggesting that one or both of these genes are important for modulating sTREM2 production. Using human macrophages as a proxy for microglia, we found that MS4A4A and TREM2 colocalized on lipid rafts at the plasma membrane, that sTREM2 increased with
overexpression, and that silencing of
reduced sTREM2 production. These genetic, molecular, and cellular findings suggest that MS4A4A modulates sTREM2. These findings also provide a mechanistic explanation for the original GWAS signal in the
locus for AD risk and indicate that TREM2 may be involved in AD pathogenesis not only in
risk-variant carriers but also in those with sporadic disease.
Genetic studies of Alzheimer disease (AD) have prioritized variants in genes related to the amyloid cascade, lipid metabolism, and neuroimmune modulation. However, the cell-specific effect of ...variants in these genes is not fully understood. Here, we perform single-nucleus RNA-sequencing (snRNA-seq) on nearly 300,000 nuclei from the parietal cortex of AD autosomal dominant (APP and PSEN1) and risk-modifying variant (APOE, TREM2 and MS4A) carriers. Within individual cell types, we capture genes commonly dysregulated across variant groups. However, specific transcriptional states are more prevalent within variant carriers. TREM2 oligodendrocytes show a dysregulated autophagy-lysosomal pathway, MS4A microglia have dysregulated complement cascade genes, and APOEε4 inhibitory neurons display signs of ferroptosis. All cell types have enriched states in autosomal dominant carriers. We leverage differential expression and single-nucleus ATAC-seq to map GWAS signals to effector cell types including the NCK2 signal to neurons in addition to the initially proposed microglia. Overall, our results provide insights into the transcriptional diversity resulting from AD genetic architecture and cellular heterogeneity. The data can be explored on the online browser ( http://web.hararilab.org/SNARE/ ).
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.
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.
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).
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.
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;
r
2
= 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.
More than 20 genetic loci have been associated with risk for Alzheimer’s disease (AD), but reported genome-wide significant loci do not account for all the estimated heritability and provide little ...information about underlying biological mechanisms. Genetic studies using intermediate quantitative traits such as biomarkers, or endophenotypes, benefit from increased statistical power to identify variants that may not pass the stringent multiple test correction in case–control studies. Endophenotypes also contain additional information helpful for identifying variants and genes associated with other aspects of disease, such as rate of progression or onset, and provide context to interpret the results from genome-wide association studies (GWAS). We conducted GWAS of amyloid beta (Aβ
42
), tau, and phosphorylated tau (ptau
181
) levels in cerebrospinal fluid (CSF) from 3146 participants across nine studies to identify novel variants associated with AD. Five genome-wide significant loci (two novel) were associated with ptau
181
, including loci that have also been associated with AD risk or brain-related phenotypes. Two novel loci associated with Aβ
42
near
GLIS1
on 1p32.3 (
β
= −0.059,
P
= 2.08 × 10
−8
) and within
SERPINB1
on 6p25 (
β
= −0.025,
P
= 1.72 × 10
−8
) were also associated with AD risk (
GLIS1
: OR = 1.105,
P
= 3.43 × 10
−2
), disease progression (
GLIS1
:
β
= 0.277,
P
= 1.92 × 10
−2
), and age at onset (
SERPINB1
:
β
= 0.043,
P
= 4.62 × 10
−3
). Bioinformatics indicate that the intronic
SERPINB1
variant (rs316341) affects expression of
SERPINB1
in various tissues, including the hippocampus, suggesting that
SERPINB1
influences AD through an Aβ-associated mechanism. Analyses of known AD risk loci suggest
CLU
and
FERMT2
may influence CSF Aβ
42
(
P
= 0.001 and
P
= 0.009, respectively) and the
INPP5D
locus may affect ptau
181
levels
(P
= 0.009); larger studies are necessary to verify these results. Together the findings from this study can be used to inform future AD studies.
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