Long-term use of benzodiazepines, commonly prescribed for anxiety and insomnia, may impair cognition and increase the risk of developing dementia. How? By activating microglia to prune synapses, according to researchers led by Jochen Herms and Mario Dorostkar at Ludwig-Maximilians University, Munich. In the February 28 Nature Neuroscience, they reported that diazepam, aka Valium, upregulated the mitochondrial receptor TSPO in microglia, triggering overzealous trimming of dendritic spines. Mice fed high doses of diazepam lost spines more quickly and had poorer memories than animals given low doses. Overall, this could explain the link between benzodiazepines and dementia.
- Diazepam binds the mitochondrial translocator protein in microglia.
- The glia ramped up synaptic pruning.
- Given diazepam, mice did worse on learning tasks.
“This thorough study adds to a growing body of evidence that microglia are critical enactors of synaptic form and function,” Kim Green, University of California, Irvine, wrote to Alzforum.
Though largely replaced by selective serotonin reuptake inhibitors, benzodiazepines are still commonly prescribed for anxiety and insomnia, despite evidence tying them to cognitive impairment and higher risk of developing dementia (Sep 2014 news; Ferreira et al., 2021; Penninkilampi et al. 2018).
To find out why these drugs weaken cognition, first author Yuan Shi fed 4-month-old mice 5 mg/kg diazepam once per day for one week or 1 mg/kg daily for eight weeks to mimic sedative and anxiolytic effects, respectively. The animals expressed green fluorescent protein in hippocampal and cortical neurons, allowing the researchers to detect synapse changes. Shi captured two-photon microscopy images from live mice through a cranial window, or confocal microscopy images from brain slices taken postmortem. Compared to controls, mice given the high dose had lost about 13 percent of dendritic spines in the somatosensory cortex one week later, which grew back over the next eight weeks. The animals that received the lower dose lost a similar fraction of their spines more slowly over the eight weeks of treatment and grew them back within three weeks.
This synaptic disruption manifested as a learning impairment. One day after their last sedative dose, mice showed no more interest in new objects or places than in familiar ones. Likewise, mice given the lower dose had trouble remembering familiar objects, though they did remember which arm of a Y-maze they had traversed.
How did diazepam cause synaptic pruning? Benzodiazepines work by binding GABA-A receptors; however, high-dose diazepam shriveled spines and impaired memory even when its binding to the GABA-A receptor was blocked by the competitive inhibitor flumazenil, or when diazepam was given to mice with mutated GABA-A receptors that don’t bind the drug.
The mitochondrial translocator protein, TSPO, also binds benzodiazepines. In fact, TSPO was identified first as a benzodiazepine receptor outside of the CNS. The protein also happens to be upregulated in the brains of people with Alzheimer’s (Kreisl et al., 2013). Could TSPO be involved in the synaptic pruning?
In TSPO knockout mice, diazepam did not alter synapses or cognition, hinting that the drug may work through TSPO. In support of this, synapses also withered in wild-type mice given the TSPO ligand XBD173. Indeed, diazepam-treated mice bound more 18FGE-180, a TSPO PET ligand, than did control animals, suggesting the drug upregulates the mitochondrial protein. All told, the researchers believe that benzodiazepines impair cognition through TSPO.
How could TSPO be involved in thinning synapses? The scientists suspected microglia were to blame because they upregulate TSPO upon activation and are known to engulf dendritic spines (Dec 2014 conference news; Apr 2016 news). Indeed, TSPO strongly co-localized with microglia in brain slices from diazepam-treated mice, and microglia isolated from these animals contained more TSPO than did microglia from controls. Though diazepam did not change the number of microglia in the mice, the cells were bigger and had longer processes with more branches (see image below). They accumulated 50 percent more puncta containing the synaptic marker PSD95, indicating they had swallowed chunks of synapses, and they contained more C1q, the complement protein that serves as a synaptic “eat me” signal for microglia.
Further, after one week of diazepam treatment, the researchers saw GFP-labeled microglia flocking to dendritic spines. Supporting the idea that microglia mediate the cognitive effects, mice depleted of microglia remembered familiar objects and places just as well as control animals even after diazepam treatment.
What does all this mean for AD risk? “Alzheimer's symptoms appear after many synapses are gone, so chronic diazepam use may increase disease risk simply by reducing the number of synapses,” Herms told Alzforum. He is currently testing the effects of diazepam and TSPO ligands on dendritic spines in AD transgenic mouse models.
Makoto Higuchi, National Institute of Radiological Sciences, Chiba, Japan, noted contrasting results from the TSPO-specific diazepam derivative Ro5-4864. In mouse models of tauopathy, it was neuroprotective by lowering TSPO expression, mitigating gliosis, and alleviating cognitive deficits. “It is conceivable that diazepam, its analogs, and other TSPO ligands exert distinct effects on homeostatic and disease-associated microglial species,” he wrote (full comment below).—Chelsea Weidman Burke
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- Ferreira P, Ferreira AR, Barreto B, Fernandes L. Is there a link between the use of benzodiazepines and related drugs and dementia? A systematic review of reviews. Eur Geriatr Med. 2021 Aug 17; PubMed.
- Penninkilampi R, Eslick GD. A Systematic Review and Meta-Analysis of the Risk of Dementia Associated with Benzodiazepine Use, After Controlling for Protopathic Bias. CNS Drugs. 2018 Jun;32(6):485-497. PubMed.
- Kreisl WC, Lyoo CH, McGwier M, Snow J, Jenko KJ, Kimura N, Corona W, Morse CL, Zoghbi SS, Pike VW, McMahon FJ, Turner RS, Innis RB, . In vivo radioligand binding to translocator protein correlates with severity of Alzheimer's disease. Brain. 2013 Jul;136(Pt 7):2228-38. PubMed.
- Shi Y, Cui M, Ochs K, Brendel M, Strübing FL, Briel N, Eckenweber F, Zou C, Banati RB, Liu GJ, Middleton RJ, Rupprecht R, Rudolph U, Zeilhofer HU, Rammes G, Herms J, Dorostkar MM. Long-term diazepam treatment enhances microglial spine engulfment and impairs cognitive performance via the mitochondrial 18 kDa translocator protein (TSPO). Nat Neurosci. 2022 Mar;25(3):317-329. Epub 2022 Feb 28 PubMed. Nat Neurosci.