Preprint Club
A cross-institutional Journal Club Initiative

Keywords

• Alzheimer’s disease

• Disease-Associated-Microglia (DAM)

• Sexual dimorphism

Main Findings

1. Cst7, a DAM signature gene, is upregulated in male and female microglia in close proximity to plaques in models of AD

2. This upregulation is more pronounced in female microglia

3. Cst7 exhibits sex-specific microglial functions as well as gene expression in models of AD

4. Cst7 deficient female microglia have increased phagocytosis of Aβ, but lysosomal degradation is not affected

The authors deleted the DAM-associated gene Cst7 in a murine model of amyloid-driven AD, the AppNL-G-F, to examine the role of Cst7 in microglia-driven AD pathology. Firstly, they compared the transcriptome of male and female microglia from disease animals (AppNL-G-FCst7+/+) to Cst7-deficient animals (AppNL-G-FCst7-/-). RNA-seq analysis revealed the deletion of Cst7 in diseased males led to a striking downregulation of classical proinflammatory mediators related to the NF-kB signalling. Whereas, in disease females Cst7 deletion led to an opposite effect, where most of proinflammatory and endolysosomal pathway-related genes were upregulated.  These findings suggested, for the first time, that the DAM gene Cst7 drives sex-dependent transcriptomic changes in microglia in murine model of AD. This sex-dependent microglia state is particularly important to consider in the context of AD as it has been well described to be more prevalent in females compared to males.

To confirm that transcriptome changes correlated with protein levels and to assess if Cst7 was really important for changes in microglial lysosomal activation in different areas of the brain, immunostainings of the cortex, hippocampus and dorsal subiculum were performed using IBA1 (microglia), Aβ clone 6E10 (Aβ plagues) and LAMP2 (lysosomes), on sagittal sections  from 12-month male and female AppWt/WtCst7+/+, AppNL-G-FCst7+/+ and AppNL-G-FCst7-/- mice. Strikingly, Cst7 deletion led to significantly decreased microglial and lysosomal burden in the dorsal subiculum in males but increased in females, followed by the same trend that was observed in cortex and hippocampus similar to RNA sequencing data. Here, stronger sex-dependent effects of Cst7deletion were observed in subiculum rather than other brain areas possibly because it is thought to be the initiating site of AD and contains not only more plaques but also plagues that have denser cores. This fact could provide a plausible explanation for why a greater effect of Cst7 knockdown is observed in this area.

Finally, the authors examined the functional role carried out by Cst7 by assessing the uptake/phagocytosis and degradation capacity of Aβ by microglia. Increased lysosomal capacity in Cst7 knockout diseased females led to a greater Aβ burden. However, despite reduced expression of lysosomal makers LAMP2 in males, Cst7 deletion did not affect microglial amyloid burden that was related to decreased expression of inflammatory genes such as Nlrp3and Il1b. Moreover, no significant differences were observed in terms of phagocytic capacity or degradation of the amyloids between AppNL-G-FCst7+/+ or AppNL-G-FCst7-/- mice.

To further investigate the functional role of Cst7, the authors used an in vitro model whereby primary microglia obtained from AppNL-G-FCst7+/+ or AppNL-G-FCst7-/- murine brains were cultured for up to three days maintaining their DAM signature over this period. Whereas Cst7 knockout led to a significant increase in microglial phagocytosis of myelin debris and the uptake of Aβ fragments, suggesting Cst7/CF negatively regulates phagocytosis in female microglia but not lysosomal proteolysis.

Together, these findings highlight new functional roles for DAM-associated, Cst7, suggesting it plays an integral part in the regulation of “two crucial processes of phagocytosis and inflammatory signalling, which are sexually dimorphic”.

Limitations

• microglial gene Cst7 in AD, the function of Cst7 was investigated solely in a murine model. Thus, further mechanistic studies of CF/CST7 as a key regulator of DAM microglia would be extremely valuable and possibly could solve the discrepancy for the validation in human microglia differentiated from iPSCs.

• This study only tackles the role of CF/Cst7 functioning in the AD context. More complex disease and non-disease models, especially involving tracking of time-dependent changes of Cst7/CF might be able to give an insight into the early onset of AD that appears around two decades prior to the first appearance of symptoms.

• While this preprint reveals a key mechanistic role of one of the most robustly upregulated microglial gene CF/Cst7 in amyloid-driven AD, the molecular mechanisms underpinning the effects of Cst7 deletion only in females and the accelerated microglial shift towards a DAM reactive state remain unknown. Further investigations involving altered hormone background in females (by ovariectomy/hormone replacement therapy) could bring valuable insight into other, possibly sex-specific mediators involved in CF/Cst7 regulation that leads to this sexually dimorphic microglial phenotype in AD.

Significance/Novelty

1. Describing a function of a disease associated microglia gene, particularly in the context of a neurodegenerative disease. To date there have been many transcriptomic studies that have identified the DAM microglia signature, but little work has been done on the functional output of these DAM microglia. The authors examine the role of one DAM-associated gene, consistently upregulated in several AD models providing insight into its functional role in the disease.

2. Sexual dimorphic microglia effect. There have been papers that have started to show that microglia from males and females react differentially and have different baseline levels of NF-KB and inflammatory mediators but this study highlights sex-specific effects of microglial-Cst7 in Alzheimer’s especially in females. This potentially could partly-explain the increased prevalence of AD in females compared to males and again points to microglia are key players in AD pathology.

Despite the limited human data on microglial CF, some studies reported discrepant undetectable expression of microglial Cst7 around plaques that may be partially due to the sex. Thus, sexual dimorphism of Cst7-dependent DAM/MGnD/ARM microglia phenotype could explain increased incidence and dramatic phenotype of AD mainly in females, encouraging the use of gender-specific treatment strategies for the patients.

Credit

Reviewed by Austeja Baleviciute as part of the cross-institutional journal club of the Immunology Institute of the Icahn School of Medicine, Mount Sinai and the Kennedy Institute of Rheumatology, University of Oxford.