A microglia clonal inflammatory disorder in Alzheimer’s Disease

Reviewer #1 (Public Review):

In the manuscript "A microglia clonal inflammatory disorder in Alzheimer's Disease", Vicario et al. provide a compelling study elucidating a potential contribution of somatic mutations within the microglia population of the CNS that accelerates microglia activation and disease-associated gene signatures in Alzheimer's disease. Here they especially identified an "enrichment" of pathological SNVs in microglia, but not the peripheral blood, that are associated with clonal proliferative disorders and neurological diseases in a subset of patients with AD. Convincingly, they identified P-SNVs in microglia of AD patients located within the ring domain of CBL, a negative regulator of MAPK signaling. They further provide mechanistic insights into how these variants result in MAPK over-activation and subsequently in a pro-inflammatory phenotype in human microglia-like cells in vitro.

Overall, this study provides clear and detailed evidence from an AD patient cohort pointing to a potential contribution of microglia-specific somatic mutations to disease onset and/or progression in a subset of patients with Alzheimer's disease.

Strengths:
As outlined above, the study identified P-SNVs in microglia of AD patients associated with clonal proliferative disorders, but also gave an in-depth analysis of re-occurring P-SNVs located within the ring domain of CBL, a negative regulator of MAPK signaling. They further provide mechanistic insights into how these variants result in MAPK over-activation and subsequently in a pro-inflammatory phenotype in HEK cells, BV2 cells, MAC cells, and human microglia-like cells in vitro.

Great care was taken here to validate their hypotheses at each step, as well as to identify the limitations of the possible conclusions. For example, they highlight that the pathway proposed to be affected may be an explanation for a subset of AD patients, and emphasize that it is yet unclear whether this accumulation of pathological SNVs is a cause or consequence of disease progression

The study clearly supports an enrichment of P-SNVs in several genes associated with clonal proliferative disorders in microglia and nicely separates this from SNVs associated with clonal hematopoiesis in the peripheral blood found in AD patients and controls.

The authors further acknowledged that several age-matched control patients were diagnosed with cancer or tumor-associated diseases and carefully dissected the occurring SNVs in these patients are not associated with the P-SNVs identified in the microglial compartment of the AD cohort.

Weaknesses:

Even though the study is overall very convincing, several points could help to connect the seen somatic variants in microglia more with a potential role in disease progression. The connection of P-SNVs in the genes chosen from neurological disorders was not further highlighted by the authors.

The authors show in snRNA-seq data that a disease-associated microglia state seems to be enriched in patients with somatic variants in the CBL ring domain, however, this analysis could be deepened. For example, how this knowledge may translate to patient benefits when the relevant cell populations appear concentrated in a single patient sample (Figure 5; AD52) is unclear; increasing the analyzed patient pool for Figure 5 and showcasing the presence of this microglia state of interest in a few more patients with driving mutations for CBL or other MAPK pathway associated mutations would lend their hypotheses further credibility.

A potential connection between P-SNVs in microglia and disease pathology and symptoms was not further explored by the authors.

A recent preprint (Huang et al., 2024) connected the occurrence of somatic variants in genes associated with clonal hematopoiesis in microglia in a large cohort of AD patients, this study is not further discussed or compared to the data in this manuscript.

Reviewer #2 (Public Review):

Summary:

In this study, Vicaro et al. aimed to quantify and characterize mosaic mutations in human sporadic Alzheimer's disease (AD) brain samples. They focused on three broad classes of brain cells, neurons that express the marker NeuN, microglia that express the marker PU.1, and double-negative cells that presumably comprise all other brain cell types, including astrocytes, oligodendrocytes, oligodendrocyte progenitor cells, and endothelial cells. The authors find an enrichment of potentially pathogenic somatic mutations in AD microglia compared to controls, with MAPK pathway genes being particularly enriched for somatic mutations in those cells. The authors report a striking enrichment for mutations in the gene CBL and use in vitro functional assays to show that these mutations indeed induce MAPK pathway activation.

The current state of the AD and somatic mutation fields puts this work into context. First, AD is a devastating disease whose prevalence is only increasing as the population of the U.S. is aging, necessitating the investigation of novel features of AD to identify new therapeutic opportunities. Second, microglia have recently come into focus as important players in AD pathogenesis. Many AD risk genes are selectively expressed in microglia, and microglia from AD brain samples show a distinct transcriptional profile indicating an inflammatory phenotype. The authors' previous work shows that a genetic mouse model of mosaic BRAF activation in macrophages (including microglia) displays a neurodegenerative phenotype similar to AD (Mass et al., 2017, doi:10.1038/nature23672). Third, new technological developments have allowed for identifying mosaic mutations present in only a small fraction of or even single cells. Together, these data form a rationale for studying mosaic mutations in microglia in AD. In light of the authors' findings regarding MAPK pathway gene somatic mutations, it is also important to note that MAPK has previously been implicated in AD neuroinflammation in the literature.

Strengths:

The study demonstrated several strengths.

Firstly, the authors used two methods to identify mosaic mutations:
(1) deep (~1,100x) DNA sequencing of a targeted panel of 716 genes they hypothesized might, if mutated somatically, play a role in AD, and
(2) deep (400x) whole-exome sequencing (WES) to identify clonal mosaics outside of those 716 genes.

A second strength is the agreement between these experiments, where WES found many variants identified in the panel experiment, and both experiments revealed somatic mutations in MAPK pathway genes.

Third, the authors demonstrated in several in vitro systems that many mutations they identified in MAPK genes activate MAPK signaling. Finally, the authors showed that in some human brain samples, single-cell gene expression analysis revealed that cells bearing a mosaic MAPK pathway mutation displayed dysregulated inflammatory signaling and dysregulation in other pathways. This single-cell analysis was in agreement with their in vitro analyses.

Weaknesses:

The study also showed some weaknesses. The sample size (45 AD donors and 44 controls) is small, reflected in the relatively modest effect sizes and p-values observed. This weakness is partially ameliorated by the authors' extensive molecular and functional validation of mutation candidates. Another weakness is the lack of discussion of whether the genes found to be mutated somatically in AD show any AD-risk alleles in the population. If they did, it would further support the authors' conclusions that they are playing a role in AD. Finally, as the authors point out, this study cannot conclude whether microglial mosaic mutations cause AD or are an effect of AD. Future studies may shed more light on this important question.

Conclusions and Impact:

Considering the study's aims, strengths, and weaknesses, I conclude that the authors achieved their goal of characterizing the role of mosaic mutations in human AD. Their data strongly suggest that mosaic MAPK mutations in microglia are associated with AD. The impacts of this study remain to be seen, but they could include attempts to target CBL or other mutated genes in the treatment of AD. This work also suggests a similar approach to identifying potentially causative somatic mutations in other neurodegenerative diseases.