Hexanucleotide expansions within intron 1 of the C9ORF72 gene not only lead to the production of toxic products; they also douse the normal expression of the gene. The consequences of this loss play out dramatically within microglia, according to a study published June 15 in Neuron. Researchers led by Rita Sattler of Barrow Neurological Institute in Phoenix and Robert Baloh of Cedars-Sinai Medical Center in Los Angeles reported that in C9ORF72 knockout mice, microglia rev up expression of interferon genes, accumulate distended lysosomes, and kick synapse destruction into high gear as the mice age. In a mouse model of amyloidosis, C9ORF72 knockouts had fewer, smaller plaques, but also fewer synapses due to overzealous pruning by microglia. In all, the findings pinpoint the inflammatory and degenerative consequences of C9ORF72 loss of function within microglia, and highlight the destructive nature of the interferon response in the brain.
- Microglia lacking C9ORF72 made more interferon.
- They accumulated lysosomes and destroyed synapses.
- 5xFAD mice sans C9 accumulated fewer plaques but lost more synapses, memory.
Hexanucleotide expansions in the C9ORF72 gene lead to amyotrophic lateral sclerosis and/or frontotemporal dementia. Repeat-laden transcripts from the mutated gene weave into RNA foci, while polydipeptide repeats translated from multiple reading frames form protein aggregates. The disruptive expansions also dampen expression of the lysosomal protein, which is involved in autophagy. All told, these toxic gain- and loss of-function mechanisms synergize—churning out a mess of toxic aggregates while also hobbling the cell’s ability to clean them up (Nov 2018 conference news; Zhu et al., 2020).
Without C9, Microglia Dine. Healthy microglia that express C9ORF72 leave synapses intact. Without C9, microglia rev up interferon genes with age or during amyloidosis, and overprune synapses. [Courtesy of Lall et al., Neuron, 2021.]
Baloh and colleagues recently reported that in myeloid cells from C9ORF72 knockout mice and from C9 carriers who had ALS/FTD, flagging lysosomal function leads to an accumulation of, among others, the STING protein, a driver of the type I interferon response (McCauley et al., 2020). Interferon escalation and heightened inflammation were known consequences of C9ORF72 knockout (Mar 2016 news).
Could C9 deficiency also mess with microglia? First author Deepti Lall and colleagues addressed this by comparing the transcriptomes of wild-type mouse microglia to those missing one or both copies of C9ORF72. Based on single cell RNA-seq transcriptional profiling, the researchers identified 16 cell clusters, most of which expressed canonical microglial markers P2ry12, TMEM119, and TREM2. Cells in two of these clusters expressed signatures resembling those of activated response microglia (ARM) and interferon response microglia (IRM) that were previously described (Sala Frigerio et al., 2019). ARMs have some similarities with disease associated microglia (DAM) and neurodegenerative microglia (MGnD) described in mouse models of amyloidosis (Apr 2019 news; Jun 2017 news; Sep 2017 news). Lall and colleagues dubbed this cluster activated response microglia (ARM), which was marked by elevated expression of Clec7a, Itgax, ApoE.
IRMs are defined by elevated interferon-stimulated genes, which are akin to those blamed for synapse loss in amyloidosis models and detected in postmortem samples from AD patients (Roy et al., 2020). The proportion of microglia in the ARM and IRM clusters was similar among genotypes. However, the intensity with which cells in each cluster expressed the full ARM or IRM signature differed. In C9 knockouts and in heterozygotes, the IRM signature intensified among IRM cells, while the ARM signature dampened among ARM cells. Notably, these findings meshed with bulk RNA sequencing of microglia, which detected a rise in IRM genes in aged, but not young, C9-deficient mice relative to wild-type.
In keeping with Baloh’s earlier work, the researchers found more STING in C9-deficient microglia, as well as higher expression of downstream, interferon-stimulated genes. C9-less microglia also amassed lysosomes, suggesting a poorly functioning disposal system.
How would these changes influence the way microglia behave? In the motor cortices of year-old C9 knockouts, the researchers found slightly more microglia than in wild-type. They also found more of the complement protein C1q, which is produced by microglia and known to instigate synaptic pruning. Sure enough—more microglia in C9 knockout mice were loaded up with synaptic material than were microglia in wild-type mice, while neurons in C9 knockouts had fewer synapses, fewer dendritic branches, and smaller neurites. Notably, this synaptic dearth was not detected in mice expressing a single copy of C9. In keeping with their sparse synapses, C9 double knockouts had trouble remembering the location of the escape hole in the Barnes maze test of spatial memory.
Are microglial solely to blame for synaptic and behavioral deficits? Recent studies have found that C9-deficiency in neurons can also cause synaptic defects (Xiao et al., 2019; Ho et al., 2019). To eliminate that from the equation, the researchers generated mice lacking C9ORF72 expression only in myeloid cells, including microglia. In these mice, microglia accumulated lysosomes, and synapse numbers were down. This suggested that without C9, microglia transform from neuron protectors to synapse slayers, even when neurons make C9.
Appetite for Amyloid
Microglia are known to overzealously prune synapses in multiple neurodegenerative diseases, including AD. Although C9ORF72 expansions are primarily associated with ALS/FTD, rare cases of C9 expansion carriers with AD or other neurodegenerative diseases have been reported (see Harms et al., 2013; Apr 2014 news). To investigate whether C9 deficiency affects how microglia deal with plaques and synapses in a mouse model of amyloidosis, the researchers generated 5xFAD mice on a C9-deficient background. In 3-month-olds, which are starting to develop amyloid plaques, the scientists spotted no obvious effects of C9 deficiency on plaque deposition. However, by 6 months, 5xFAD mice lacking C9ORF72 had fewer plaques, and those that remained were smaller and more compact than the aggregates in C9-replete 5xFAD mice. In the C9 knockouts, about twice as many microglia crowded around each plaque as in control mice.
What baited more microglia to Aβ plaques in C9 knockout 5xFAD mice? While changes in the microglia themselves could explain it, the researchers also spotted IgG antibodies glommed onto plaques in the C9 knockouts. These antibodies were not present in 5xFAD controls. B cells producing anti-Aβ antibodies were also detected in the spleens and lymph nodes of C9 knockouts, in agreement with the known tendency of C9 knockout mice to churn out autoantibodies. To what extent these anti-Aβ antibodies contributed to microglial plaque clearance remains unclear.
Alas, the stepped-up plaque removal in C9-deficient 5xFAD mice was all for naught, as once again C9 deficiency stoked microglial appetite for synapses. In 4-month-old C9-knockout 5xFAD mice, the researchers found microglia loaded with lysosomes and synaptic proteins, and they detected extensive synapse loss compared to 5xFAD controls. Notably, in C9 knockouts without amyloidosis, these degenerative phenotypes do not emerge until animals are a year old, suggesting that extracellular Aβ accelerated the effects of C9 deficiency. The 5xFAD mice lacking C9 also had more severe spatial memory deficits. In all, the findings suggest that while removing C9ORF72 helps microglia clear plaques, it also triggers synaptic damage that culminates in memory loss.
“These findings corroborate our previous work showing that IFN-activated microglia are intimately involved in synapse removal, both in wild-type animals and models of Aβ plaque pathology,” commented Wei Cao of Baylor College of Medicine in Houston. “It is interesting to see the increased number of C9ORF72-/- microglia clustered around amyloid plaques; however, it remains to be illuminated if and how the balance between IRMs and ARMs is skewed in 5XFAD by the lack of C9ORF72,” he added. “Better understanding the cross-regulation of different microglial activation states will offer more insights on their functional involvements in pathophysiological processes.”
Sattler told Alzforum that the investigators are using human induced pluripotent stem cell (iPSC)-derived microglia and neurons to understand more about how C9ORF72 loss alters cell functions and interactions. For example, they are investigating whether C9-deficient microglia amass lysosomes because they consume too much debris, and/or because of a slowdown in lysosomal digestion and autophagy. Another lingering question from the study is why only C9ORF72 knockout mice, not heterozygotes, were ravaged by synaptic loss, and what that means for people with ALS or FTD who still carry one functional copy? Sattler noted that it is common for heterozygous mouse models, as in the case of progranulin deficiency, to fall short of replicating the full spectrum of disease in people, who live longer and are different genetically. She believes that studies in cells derived from ALS/FTD IPSCs will more closely model the human disease.—Jessica Shugart
- It’s ‘And,’ Not ‘Either-Or’: C9ORF72 Mechanisms of Action are Linked
- C9ORF72 Knockout Causes Inflammation, not Neurodegeneration
- Parsing How Alzheimer’s Genetic Risk Works Through Microglia
- Hot DAM: Specific Microglia Engulf Plaques
- ApoE and Trem2 Flip a Microglial Switch in Neurodegenerative Disease
- C9ORF72 Repeats Expand into New Disorders—Cause, or Coincidence?
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