Scientists have zeroed in on a certain set of cortical neurons as instigators of a toxic circuit in a mouse model of TDP-43 proteinopathy. Somatostatin interneurons amp up their activity when the mice are just a few weeks old, resulting in hyperexcitability and excitotoxicity in downstream pyramidal neurons in layer 5 (L5PNs) of the cortex, report the authors of a study in the February 23 Nature Neuroscience online. The researchers fixed the problem by ablating the somatostatin interneurons. This suggests that if the same circuit were to go awry in people with TDP-43-based diseases, such as amyotrophic lateral sclerosis and frontotemporal dementia, then those neurons could be a possible target for therapeutics.
Excitotoxicity is a well-known culprit in the demise of motor neurons in ALS. In fact, the only FDA-approved treatment for the disease, riluzole, works by preventing the release of excess glutamate, which becomes excitotoxic. Glutamatergic layer 5 pyramidal neurons, which include upper motor neurons, transmit signals to the spinal cord and other subcortical regions. In the same region of the brain are two populations of interneurons, categorized by their expression of either somatostatin or parvalbumin. Joint senior authors Yun Li and Da-Ting Lin propose that somatostatin interneurons inhibit parvalbumin interneurons, which in turn temper the activity of pyramidal neurons. In toto, this system gives the brain a way to fine-tune motor activity.
Li and Lin, a married couple, began their studies at the Jackson Laboratory in Bar Harbor, Maine, and continued them after moving to the National Institute on Drug Abuse in Baltimore. The scientists first got interested in the activity of cortical layer 5 pyramidal neurons (L5PNs) in TDP-43 mice when they used two-photon microscopy to image live neurons in the brain through a window in the skull (see Apr 2012 news series). They crossed mice expressing yellow fluorescent protein (YFP) in L5PNs with mice expressing the ALS-linked A315T variant of human TDP-43 in all neurons (see Oct 2009 news). By a few months of age, this well-known model of TDP-43 pathology starts to have trouble moving; death comes soon afterward. Compared with normal mice, these transgenic mice have fewer motor neurons at death, and the remaining ones contain ubiquitinated protein aggregates.
When study first author Lifeng Zhang peered into the brains of YFP/A315T double transgenics, he saw numerous blebs along the layer 5 dendrites in mice as young as six weeks—well before motor symptoms or ubiquitin inclusions arise. Li recognized this blebbing as a sign of excitotoxicity. Zhang and co-first author Wen Zhang (no relation) then looked to see what might cause this. First, they measured action potentials in brain slices from three-week-old mice. Already, pyramidal neurons were hyperactive, firing more often than the same neurons in brains from wild-type littermates. Notably, weaker inhibitory currents modulated the pyramidal neurons, while afferent excitatory currents were as strong as in slices from normal tissue. In essence, the neurons were getting the right amount of stimulation, but too little inhibition.
To pinpoint why, the authors investigated the somatostatin and parvalbumin interneurons as possible troublemakers. In brain slices from TDP-43 mice, somatostatin interneurons were hyperactive, while parvalbumin interneurons were hypoactive. If somatostatin interneurons inhibit parvalbumin interneurons, which in turn inhibit layer 5 cortical motor neurons, then hyperactive somatostatin interneurons would elicit more action from the L5PNs, the authors concluded (see image above). These motor neurons would then release excess glutamate, Lin said, which could come back to haunt them.
Zhang and Zhang tested this theory in two ways. First, they used optogenetics to stimulate or inhibit somatostatin interneurons in brain slices from wild-type mice, measuring the effects on pyramidal neurons. Sure enough, activating the somatostatin interneurons reduced the parvalbumin inhibitory currents coming into pyramidal cells, and this led to hyperexcitability and suppression of L5PNs. Shutting off the somatostatin interneurons had the opposite effect, tightening inhibition of the pyramidal neurons.
Second, the scientists tested whether ablating somatostatin interneurons in TDP-43 mice would revive pyramidal neurons. They expressed the receptor for diphtheria toxin in the mouse somatostatin interneurons, then injected the toxin into the cortex once the animals reached six weeks old. Two weeks later, they sacrificed the mice and tested brain slices. The inhibitory currents coming into the layer 5 pyramidal neurons were stronger than those in slices from untreated animals, if not completely back to wild-type levels. In another set of mice sacrificed six weeks after the diphtheria treatment, the authors found fewer ubiquitin aggregates, and more motor neurons, than in untreated TDP-43 mice.
The authors do not know if correcting the activity of their microcircuit could alleviate symptoms in people with ALS, or even in mouse models. The mice they studied were younger than when symptoms began in the TDP-43-A315T mice, and Li notice no major changes in their mobility. The authors were unable to assess effects on lifespan because these animals die of bowel blockage before the motor neuron disease becomes as severe as in human ALS (see Sep 2012 news).
For Li, the next question will be what makes the somatostatin interneurons at the top of the circuit hyperexcitable. She plans to sequence RNAs from those neurons to identify altered expression patterns caused by the mutant TDP-43.
Does the same microcircuit go awry in people with ALS or FTD? “It is too early to make conclusions about whether similar mechanisms are occurring in ALS,” commented Anna King of the University of Tasmania in Australia, who was not involved in the study (see full comment below). However, King noted that studies in people hint that dysfunction of interneuron populations could contribute to upper motor neuron hyperexcitability in ALS.
For example, transcranial magnetic stimulation (TMS) detects unusually high activity in the motor cortices of people with ALS, often well before the physical symptoms arise (see Sep 2015 news). Steve Vucic of the University of Sydney, who uses TMS to study and help diagnose ALS, said interneuron and layer 5 defects could neatly explain the early cortical hyperexcitability he observes in people. He added that the new findings highlight the importance of upper motor neurons, not just the spinal cord, in ALS (see Jan 2015 news; news).
Vucic and King both think the new paper suggests that modulating the activity of the somatostatin-parvalbumin-L5PN microcircuit could eventually become a treatment for ALS. Lin has some ideas as to how one might do that. If scientists could identify a receptor or ion channel specific to somatostatin interneurons, he suggested, they could seek out small molecules that tune them down. Alternatively, he speculated that physicians could use TMS to focally tune the microcircuit, for example, by activating parvalbumin interneurons to turn down the L5PNs. While current TMS technology cannot activate neurons quite so specifically, Lin suspects that future improvements might make this feasible.—Amber Dance
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