The task of restraining microglia’s neurotoxic tendencies may fall to a small GTPase called RhoA, according to a study published June 23 in Cell Reports. Scientists led by João Relvas, University of Porto, Portugal, found that deleting RhoA from microglia unleashed a pro-inflammatory cascade that addled neuronal synapses and sapped memory—all in mice. Mice missing RhoA in their microglia produced more Aβ; on the flip side, Aβ aggregates squelched RhoA, tripping off a vicious cycle that hobbled microglial function and exacerbated neuronal damage. The researchers pieced together the signaling pathways involved, and used pharmacological inhibitors to stem the damage. Their findings cast RhoA as a stopgap against inflammation and amyloidosis in the brain.
- Deleting RhoA in mouse microglia causes neuroinflammation, synaptic loss, memory deficits.
- Without RhoA, mouse microglia stoke amyloidosis.
- Aβ accumulation inhibits RhoA, dampens plaque clearance.
RhoA belongs to the family of Rho GTPases, which toggle between active, GTP-bound and inactive, GDP-bound states. When activated, Rho GTPases influence many cellular functions. They include the dynamic rearrangement of the cytoskeleton that happens during cell division and—importantly for the current study—during microglial activation. In particular, RhoA and its downstream effectors have been implicated in stroke, Alzheimer’s, Parkinson’s, and amyotrophic lateral sclerosis (Droppelmann et al., 2014). RhoA reportedly plays a role in the handling and toxicity of Aβ (Lee et al., 2019; Sycheva et al., 2019; Zhang et al., 2019; Lee et al., 2019). Alas, RhoA and other small GTPases are active in multiple cell types, making it difficult to pin down exactly how RhoA influences neurodegeneration.
RhoA Says “WhoA!” to Inflammation. In healthy conditions (left), RhoA restrains pro-inflammatory signaling in microglia. Without RhoA (right), Src trips off a neurotoxic, amyloidogenic cascade. [Courtesy of Socodato et al., Cell Reports, 2020.]
To home in on the role of RhoA in microglial function, co-first authors Renato Socodato and Camila Portugal and colleagues generated conditional knockout mice, in which deletion of RhoA can be induced in CX3CR1-expressing cells. In the brain, that means microglia, in addition to infiltrating myeloid cells. Forty days after the researchers ablated RhoA from microglia, they observed fewer microglia in the hippocampi, cortices, and striata of the conditional knockouts compared with control mice. The remaining microglia expressed markers of necrosis, projected fewer processes, and pumped out more of the pro-inflammatory cytokine TNF-α. This suggested that RhoA supports microglial survival and homeostasis.
The researchers report that cultured primary mouse microglia lacking RhoA secreted TNF-α, which spurred the autocrine release of glutamate. In neurons cultured with conditioned media from these RhoA-deficient microglia, neurites beaded up, a telltale sign of excitotoxicity. In the mice missing RhoA in their microglia, the hippocampus lost synapses, and long-term potentiation weakened. Hippocampal neurons took a hit, dropping in number by nearly a quarter compared with controls. Mice lacking microglial RhoA also had faltered in a novel object recognition test.
Sapping Synapses? Compared with untreated control mice (left), mice missing RhoA in their microglia (middle) had fewer functional synapses (yellow). Treatment with the Src blocker AZD (right) protected synapses. Quantitation of synaptic puncta (right) represents three images of each of four hippocampal sections per mouse. [Courtesy of Socodato et al., Cell Reports, 2020.]
Using pharmacological inhibitors, knockdown approaches, and dominant-negative mutants, the researchers pieced together the signaling pathways responsible for the neurotoxic consequences of RhoA deficiency in microglia. Under healthy conditions, RhoA bolsters expression of Csk, a repressor of Src family kinases. Absent RhoA, Csk’s repression of Src slips, which unleashes TNF-α secretion, glutamate release, and excitotoxicity. The researchers found that injecting the Src blocker AZD0530 into the abdomens of RhoA-deficient mice counteracted the effects of RhoA deficiency.
How about Aβ production or deposition? The researchers report elevated levels of Aβ40 and Aβ42 peptides, and their amyloidogenic precursor, β-CTF, in the brains of mice lacking microglial RhoA. They also detected an excess of fibrillar Aβ wound into plaque-like structures. The researchers hypothesized that the excess glutamate released from RhoA-deficient microglia may have incited activity-dependent Aβ production by nearby neurons (Lesné et al., 2005; Dec 2005 news).
If RhoA deficiency in microglia sparks Aβ production in neurons, might Aβ accumulation also dampen RhoA activity in microglia? In microglia isolated from 4-month-old APP/PS1 mice, the researchers observed less activated RhoA-GTP, lower expression of Csk, and more phosphorylated Src than in microglia from non-transgenic mice. Treating a microglial cell line with synthetic Aβ42 oligomers dampened RhoA activity and triggered TNF-α and glutamate release. The researchers believe Aβ oligomers dampen RhoA activity in microglia, fueling a loop in which TNF-α triggers glutamate release, which overexcites neurons and generates more Aβ. Alzforum was unable to obtain comment from the authors of this study.
Even before plaques formed, APP/PS1 mice lost synapses in the hippocampus, and their microglia became less ramified, a sign that the cells were rousing from homeostasis. Treating the mice with weekly doses of the Src inhibitor AZD0530 for one month substantially preserved synapses and microglial ramification, the researchers reported.
Oleg Butovsky of Brigham and Women’s Hospital in Boston called the work a technical tour de force exploring the role of RhoA signaling in microglial regulation. He found the induction of amyloidosis in mice lacking microglial RhoA particularly striking.
At the same time, Butovsky said, the role of RhoA in neurodegenerative processes remains unclear (for review, see Aguilar et al., 2017). For example, in gene-expression studies from his group, microglia encircling Aβ plaques expressed more RhoA than did microglia farther away from plaques (Krasemann et al., 2017). Some AD mouse models have elevated RhoA activity (Pozueta et al., 2013; Park et al., 2017).
How RhoA tracks with neurodegeneration in humans needs to be explored. Some studies have spotted the protein mingling with tau (Huesa et al., 2009). The GTPase is expressed throughout the body, and in multiple cell types, making RhoA or its downstream effectors tricky therapeutic targets, Butovsky added.
To Richard Ransohoff of Third Rock Ventures, Boston, the findings seem at odds with a well-documented example of RhoA inhibition in humans, i.e., statin use. “If RhoA were functioning in human brain microglia in vivo as the authors suggest for mouse cells, then it would be anticipated that statin treatment, by inhibiting Rho family member prenylation and signaling, should worsen neuroinflammation,” he wrote. “However, this result has not been observed and, in fact, results opposite in direction have been reported. It would be useful for the authors to address this disconnect.” Along those lines, a recent mouse study found that suppressing RhoA signaling does not affect the inflammatory status of peripheral immune cells (Akula et al., 2019).
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