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Diet supplement dampens brain seizures
Seizure disorders, like epilepsy, are associated with a pathological hyper-excitability in brain neurons. Unfortunately, there are limited treatments that can prevent this. However, University of Alabama at Birmingham, researchers find they can alter biochemical reactions in brain proteins with a dietary supplement - glucosamine.
Glucosamine rapidly dampened hyper-excitability in both rat and mouse brains. As one of the most common supplements used, glucosamine is neither a vitamin nor a mineral, but an amino sugar which helps synthesize some proteins and lipids. Found in the exoskeletons of crustaceans and other arthropods aa well as the cell walls of fungi and many other higher organisms. it is one of the most abundant commercially produced monosaccharides. Results from the research may lead to a novel therapeutic target for treating seizure disorders.
Proteins are the workhorses of living cells, and their activities are tightly regulated in response to changing neural conditions. Adding or removing a phosphoryl group is a well-known mechanism to regulate proteins. It is estimated that human proteins may have as many as 230,000 sites for phosphorylation. Phosphorylation is important as it is the mechanism regulating equilibrium between insulin and water in a cell.
A lesser-known regulator is N-acetylglucosamine to proteins, usually controlled by glucose, the primary fuel of neurons. Several years ago, neuroscientist Lori McMahon PhD, professor of cell, developmental and integrative biology at UAB, found out from her colleague John Chatham DPhil, a UAB professor of pathology and a cardiac physiologist, that brain cells have the second-highest amounts of proteins with N-acetylglucosamine, or O-GlcNAcylation, in the body. At the time, very little was known about how O-GlcNAcylation might affect brain function, so McMahon and Chatham started working together.
In 2014, McMahon and Chatham reported that acute increases in protein O-GlcNAcylation caused long-term synaptic depression, reducing neuronal synaptic strength in the hippocampus of the brain. This was the first time acute changes in O-GlcNAcylation of neuronal proteins were shown to directly change synaptic function.
As neural excitability in the hippocampus is a key feature of seizures and epilepsy, McMahon and Chatham hypothesized that increasing protein O-GlcNAcylation might dampen the pathological hyper-excitability associated with these brain disorders. GlcNAcylation of neuronal proteins were shown to directly change synaptic function.
That turned out to be the case, as reported in the Journal of Neuroscience study, "Acute increases in protein O-GlcNAcylation dampen epileptiform activity in hippocampus." The study was led by corresponding author McMahon and first author Luke Stewart, a doctoral student in the Neuroscience Theme of the Graduate Biomedical Sciences Program. Stewart is co-mentored by McMahon and Chatham.
"Our findings support the conclusion that protein O-GlcNAcylation is a regulator of neuronal excitability, and it represents a promising target for further research on seizure disorder therapeutics." Researchers caution, however, that the dampening mechanism is likely to be complex.
Glucose, the major fuel for neurons, also controls levels of protein O-GlcNAcylation on proteins. However, high levels of the dietary supplement glucosamine, or an inhibitor of the enzyme that removes O-GlcNAcylation, leads to rapid increases in O-GlcNAc levels.
In experiments with hippocampal brain slices treated to induce a stable and ongoing hyper-excitability, UAB researchers found that an acute increase in protein O-GlcNAcylation significantly decreased the sudden bursts of electrical activity known as epileptiform activity in area CA1 of the hippocampus. An increased protein O-GlcNAcylation in normal cells also protected against a later induction of drug-induced hyper-excitability.
The effects were seen in brain slices treated with both glucosamine and an inhibitor of the enzyme that removes O-GlcNAc groups. They also found that treatment with glucosamine alone for as short a time as 10 minutes was able to dampen ongoing drug-induced hyper-excitability.
In common with the long-term synaptic depression provoked by increased O-GlcNAcylation, the dampening of hyper-excitability required the GluA2 subunit of the AMPA receptor, which is a glutamate-gated ion channel responsible for fast synaptic transmission in the brain. This finding suggested a conserved mechanism for the two changes provoked by increased O-GlcNAcylation - synaptic depression and dampening of hyper-excitability.
Researchers also found the spontaneous firing of pyramidal neurons in another region of the hippocampus, area CA3, was reduced by increased O-GlcNAcylation in normal brain slices and in drug-induced slices. This reduction in spontaneous firing of CA3 pyramidal neurons likely contributes to decreased hyper-excitability in area CA1 as CA3 neurons directly excite those in CA1.
Similar to the findings for brain slices, mice that were treated to increase O-GlcNAcylation before getting drug-induced hyper-excitability had fewer brain activity spikes associated with epilepsy — called interictal spikes. Several drug-induced hyperexcitable mice had convulsive seizures during the experiments - this occurred in both increased O-GlcNAcylation mice as well as control mice. Brain activity during the seizures differed between these two groups: The peak power of the brain activity for the mice with increased O-GlcNAcylation occurred at a lower frequency, as compared with the control mice.
O-GlcNAcylation is a ubiquitous and dynamic post-translational modification involving the O-linkage of ?-N-acetylglucosamine to serine/threonine residues of membrane, cytosolic, and nuclear proteins. This modification is similar to phosphorylation and regarded as a key regulator of cell survival and homeostasis. Previous studies have shown that phosphorylation of serine residues on synaptic proteins is a major regulator of synaptic strength and long-term plasticity, suggesting that O-GlcNAcylation of synaptic proteins is likely as important as phosphorylation; however, few studies have investigated its role in synaptic efficacy. We recently demonstrated that acutely increasing O-GlcNAcylation induces a novel form of LTD at CA3-CA1 synapses, O-GlcNAc LTD. Here, using hippocampal slices from young adult male rats and mice, we report that epileptiform activity at CA3-CA1 synapses, generated by GABAAR inhibition, is significantly attenuated when protein O-GlcNAcylation is pharmacologically increased. This dampening effect is lost in slices from GluA2 KO mice, indicating a requirement of GluA2-containing AMPARs, similar to expression of O-GlcNAc LTD. Furthermore, we find that increasing O-GlcNAcylation decreases spontaneous CA3 pyramidal cell activity under basal and hyperexcitable conditions. This dampening effect was also observed on cortical hyper-excitability during in vivo EEG recordings in awake mice where the effects of the proconvulsant pentylenetetrazole are attenuated by acutely increasing O-GlcNAcylation. Collectively, these data demonstrate that the post-translational modification, O-GlcNAcylation, is a novel mechanism by which neuronal and synaptic excitability can be regulated, and suggest the possibility that increasing O-GlcNAcylation could be a novel therapeutic target to treat seizure disorders and epilepsy.
SIGNIFICANCE STATEMENT We recently reported that an acute pharmacological increase in protein O-GlcNAcylation induces a novel form of long-term synaptic depression at hippocampal CA3-CA1 synapses (O-GlcNAc LTD). This synaptic dampening effect on glutamatergic networks suggests that increasing O-GlcNAcylation will depress pathological hyper-excitability. Using in vitro and in vivo models of epileptiform activity, we show that acutely increasing O-GlcNAc levels can significantly attenuate ongoing epileptiform activity and prophylactically dampen subsequent seizure activity. Together, our findings support the conclusion that protein O-GlcNAcylation is a regulator of neuronal excitability, and it represents a promising target for further research on seizure disorder therapeutics.
Authors: Luke T. Stewart, Anas U. Khan, Kai Wang, Diana Pizarro, Sandipan Pati, Susan C. Buckingham, Michelle L. Olsen, John C. Chatham and Lori L. McMahon
Co-authors: Anas Khan, Kai Wang and Michelle Olsen, UAB Department of Cell, Developmental and Integrative Biology; Diana Pizarro and Sandip Pati, UAB Department of Neurology; and Sue Buckingham, UAB Department of Neurobiology.
At UAB, McMahon holds the Jarman F. Lowder Endowed Professorship in Neuroscience.
Search Terms: glutamate transmission, neuronal excitation, O-GlcNAc, post-translational modification, synaptic circuits. Funding for the research came from the National Institutes of Health grant NS076312 and NIH pre-doctoral fellowship NS095568.
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Some of the many Glucosamine products on the market. Image Credit: public domain