In Fruit Flies, MicroRNA Protects Synapses Against Excitotoxicity

With their ability to quickly fine-tune gene expression, microRNAs are a natural fit to regulate synaptic activity in response to the changing needs of the nervous system. Few of these micro-managers are known to work at the presynaptic side, but now researchers have identified a new one, miR-1000. This microRNA has a similar sequence to the mammalian miR-137. It tamps down expression of the glutamate transporter that loads presynaptic vesicles with the neurotransmitter. Glutamate excitotoxicity crippled neurons in flies missing miR-1000, and neurodegeneration ensued, reported senior author Stephen Cohen and colleagues at the Institute of Molecular and Cell Biology in Singapore in the February 2 Nature Neuroscience online.

Extra excitation.  The neuromuscular junctions of flies lacking miR-1000 (right) contain more and larger synaptic boutons (labeled for VGluT in red) than flies with only one copy of the microRNA gene (left). White indicates the muscle and blue, nuclei. [Image courtesy of Verma et al., Nature Neuroscience.]

MiR-1000 joins several other microRNAs known to participate in synaptic regulation, mostly from studies of rodent hippocampal neurons. Several microRNAs act at the postsynaptic end. For example, three different microRNAs act on synapse strength and glutamate response in rats or mice (Schratt et al., 2006; Saba et al., 2012; Harraz et al., 2012). Less is known about microRNA activity at the presynaptic terminal, though, for example, one microRNA appears to mediate synapse density and glutamate receptor expression in rats (Cohen et al., 2011, reviewed inKaplan et al., 2013).

Stephen Cohen, who recently moved to the University of Copenhagen, Denmark, was not specifically looking for synaptic microRNAs. He has been screening microRNA knockouts in Drosophila for any kind of neurodegenerative defects (see Oct 2009 Interview). First author Pushpa Verma observed two telltale signs of neurodegeneration in flies lacking miR-1000: They were lackluster climbing the walls of their vials, and they died young. The mutants lived for three weeks after emerging from their pupae as adults, whereas normal flies make it twice as long. To determine if the flies’ neurons were degenerating, Verma examined their brains with an antibody to activated caspase-3, a marker for apoptosis. Sure enough, the brains of 2-day-old adults already contained activated caspase, and 10-day-olds had 10 times as much. Vacuoles also dotted the brains of 10-day-old flies, a sign of age-related neurodegeneration. Verma concluded that the flies underwent early onset, progressive neurodegeneration.

What caused the phenotype? MicroRNAs typically have hundreds of targets, and a computer program predicted that miR-1000 might regulate 374 different genes. Verma focused on just seven known to function in the nervous system. Most microRNAs turn off their targets, and expression of only one, the vesicular glutamate transfer (VGluT) ramped up in miR-1000 knockouts. If excess VGluT was the problem in these mutants, Verma reasoned, then she should be able to fix it by reducing their VGLuT. Sure enough, when she crossed miR-1000 knockouts with flies with diminished VGluT production, she saw a rescue of the phenotype. The double mutants climbed better and lived longer.

One obvious explanation for the neurodegeneration Verma observed was too much glutamate in presynaptic vesicles, leading to excitotoxicity. Verma and Cohen reasoned that if the presynaptic termini were releasing excess glutamate, then reducing glutamate receptor activity on the postsynaptic end should alleviate the problem. First, Verma treated the miR-1000 mutant flies with the receptor blocker memantine. This improved their climbing skills. She also reduced expression of glutamate receptors by knocking out one copy of their genes. Again, this fixed the climbing problems, and extended the flies’ lifespans.

Glutamate signaling should lead to enhanced neural activity. Because in flies glutamate is the neurotransmitter of neuromuscular junctions, and they are relatively easy to dissect, Verma examined them in the miR-1000 mutant larvae. The junctions had more synaptic boutons than normal larvae, and those boutons were bigger (see image above). When she recorded spontaneous activity from the neurons at the junctions, she saw unusually large and frequent action potentials.

Cohen suggested that miR-1000’s normal role is to fine-tune synaptic activity. That does not mean VGluT regulation is miR-1000’s only task. Indeed, the related mammalian miR-137 may be a tumor suppressor that is silenced in some cancers (reviewed in Chen et al., 2013). The particular job of a microRNA often varies by cell type and developmental stage, explained Guoping Feng of the Massachusetts Institute of Technology in Cambridge, who was not involved in the study.

To explore this neurodegeneration mechanism in mammals, Verma depleted miR-137 in mouse primary cortical neuron cultures. The cells made caspase-3, indicating apoptosis. When she depleted miR-137 in the brains of adult mice, they produced more of the mammalian VGluT2.

Does this fly finding explain anything about human neurodegenerative disease? “It doesn’t—yet,” Cohen said. Researchers are still struggling to understand how microRNAs contribute to healthy physiology and disease (see Oct 2009 news). However, there are hints that the neuroprotective mechanism Verma described could, perhaps, be involved in human disorders. MicroRNAs, including miR-137, have been implicated in psychiatric disorders such as schizophrenia, noted Feng (Geaghan and Cairns, 2014; Ripke et al., 2011; Kwon et al., 2013). In addition, one study found unusually low levels of miR-137 in seven brains from people who had Alzheimer’s (see Oct 2011 news).

Glutamate excitoxicity has already been fingered as a possible mechanism in AD as well as Parkinson’s and amyotrophic lateral sclerosis (Ong et al., 2013; Blandini, 2010; Apr 2010 news). Cohen speculated that variants in miR-137, or its target sequence in the glutamate transporter gene, might make a small difference to glutamate loading and cause excitotoxicity that could, over decades, increase risk for neurodegeneration.

That possibility would be worth investigating, commented Fen-Biao Gao of the University of Massachusetts Medical School in Worcester, who did not participate in the study. However, he added that it might be difficult to find the evidence for a miR-137 defect in human postmortem tissues because only certain vulnerable neurons in the brain or spinal cord might have the problem. Looking at the organs as a whole, scientists might miss something, he said.

Reference:
Verma P, Augustine GJ, Ammar MR, Tashiro A, Cohen SM. A neuroprotective role for microRNA miR-1000 mediated by limiting glutamate excitotoxicity. Nat Neurosci. 2015 Mar;18(3):379-85. Epub 2015 Feb 2. [PubMed].


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