11 May 2011. Spinocerebellar ataxias, a collection of movement diseases in which cerebellar neurons die, are closely linked with polyglutamine repeats and protein aggregation (Paulson, 2009). But that is not the whole story: Some ataxias, for example, spinocerebellar ataxia 13 (SCA13), are due to mutations in ion channels. Even in disease caused by polyglutamine expansion, there is some evidence that ion channels are not working properly, said Henry Paulson of the University of Michigan in Ann Arbor.
Now, researchers from the University of California, Los Angeles, show in a new zebrafish model for SCA13 how malfunction of the ion channel might contribute to disease. The paper appears in the May 4 Journal of Neuroscience. In humans, SCA13 is caused by dominant-negative mutations in one of the four identical subunits of the Kv3.3 potassium channel. Mutations in even one subunit render channels non-functional (see ARF related news story on Waters et al., 2006). The new study, led by first author Fadi Issa and senior author Diane Papazian, determined that the channel is expressed in primary motor neurons, among the earliest to develop in the embryonic spinal cord. The cerebellum develops later.
To mimic the disease, the scientists injected zebrafish embryos with RNA encoding the dysfunctional subunit containing an R420H mutation. This particular mutation causes late-onset ataxia in people with SCA13. Issa examined the effects of the mutation in embryos two or three days after fertilization.
The Kv3.3 channel normally drives persistent, high-frequency firing in motor neurons. With the mutant subunit interfering with the formation of the tetrameric channels, the firing rate dropped, and many of the caudal primary neurons the scientists examined were spent before the end of a 60-millisecond stimulation in patch-clamp experiments. “The fundamental issue is that each and every neuron seems to be firing less frequently, and that reduces their output,” said Pierre Drapeau of the University of Montréal, Canada, whose lab Issa visited for electrophysiology training. “Though we've known for several years that causative mutations occurred in the KCNC3 voltage-gated potassium channel, this is the first report demonstrating altered neuronal excitability in an animal model of disease,” wrote Michael Waters, University of Florida, Gainesville. (See full comment below.)
This faulty firing could lead to neurodegeneration in a variety of ways, Papazian said. For example, calcium enters the cells via voltage-dependent influx. A lifetime of gradually altered calcium levels could eventually damage a cell, she suggested. Calcium concentrations have also been fingered as potential mechanisms for neurodegeneration in Alzheimer’s disease (Supnet and Bezprozvanny, 2010).
Issa also looked for behavioral effects from the mutant channel. Morphologically speaking, the zebrafish embryos developed normally, but their early movement was altered. Issa used the well-known startle response (Korn and Faber, 2005), prodding the embryo’s tails with a pin so they will dart away. He kept the mutant RNA levels low so the response would still occur. Fish with unimpaired Kv3.3 function flip so that they face 180 degrees away from the stimulus and can swim in the opposite direction. But the fish with the mutant Kv3.3 turned at much more variable angles, averaging only 143 degrees away from the prod. “The startle response was no longer reliable in moving the animal directly away from the stimulus,” Papazian said. The response reminded her of people with SCA13, who tend to take small steps of variable length rather than walking with a regular stride (Palliyath et al., 1998).
Because the RNA treatment only lasted a few days, the researchers were unable to look for long-term effects in adult fish. Another caveat is that Issa’s fish are an embryonic, spinal cord model for an adult-onset, cerebellar disease. Papazian and colleagues are currently working to achieve longer-term expression, as well as on a rat model that may provide answers in adult animals. Most ataxias have been linked to potentially toxic protein aggregates, but the researchers did not examine for those in this fish model. Either way, the research confirms, to Paulson’s mind, that “we need to be thinking about channel dysfunction in human ataxias,” he told ARF. He further suggested that modulators of channel function might be effective treatments.—Amber Dance.
Issa FA, Mazzochi C, Mock AF, Papazian DM. Spinocerebellar ataxia type 13 mutant potassium channel alters neuronal excitability and causes locomotor deficits in zebrafish. J Neurosci. 2011 May 4:31(18):6831-41. Abstract