CASK gene-related neurodevelopmental disorders confer features like small head size (microcephaly), short stature, and intellectual disability, and a breakthrough fly neuron model may aid in identifying pharmaceutical treatments.
- Despite approximately 700 million years of evolutionary history since flies and humans separated as species, the CASK gene shows striking DNA sequence preservation.
- A CASK mutation recapitulates reduced brain volume in flies, and varying doses of dysfunctional CASK gene copies prompt progressively severe degrees of abnormally shaped neurons, depending on the mutant gene copy dose.
- Using a newly developed device to extract these CASK mutant-related abnormal neurons may provide a means to test pharmaceutical compounds that potentially improve their shape and possibly alleviate CASK-related disorders.
CASK-related disorders are rare and debilitating, manifesting with features that can include microcephaly and intellectual disability. Their prevalence is unknown, and 2.19 females are affected with CASK-related disorders for every affected male. Limited treatment options like anti-seizure medications, hearing and vision aids, along with physical therapy are used to alleviate symptoms. However, research geared toward finding ways to counteract the neurological underpinnings of CASK-related disorders to impede disease progression has remained in a primitive state.
Now, published in Neural Development, Restifo and colleagues from the University of Arizona have uncovered that the fly CASK gene involved in these disorders codes for 52% to 87% of the same protein building blocks (amino acids) seen in humans, showing high similarity. As such, a mutation in the fly CASK gene recapitulates smaller brain size in flies, as seen in CASK disorder patients with microcephaly. Moreover, at the microscopic level, the CASK mutation induces “bushy” neurons having shorter and denser projections (neurites). What’s more, more severe “bushy” neuronal disfigurations occur in a gene copy additive manner, where one mutant copy has a milder effect than two mutant copies. Furthermore, genetically manipulating mutant flies to restore normal CASK gene activity partially reverses the abnormal “bushy” neuronal shape, confirming that the mutant CASK gene drives these neuronal disfigurations. These findings provide researchers with a new fly neuron model with which to test pharmaceutical compounds. Future research may reveal newly identified compounds to correct the “bushy” CASK mutant neuron shape, providing new potential therapeutics to counteract these disorders.
CASK Mutations Induce Fly Microcephaly Features and Short, Dense Neuron Projections
To confirm that the CASK gene mutation recapitulates features of microcephaly in flies, similar to its effects in humans, Restifo and colleagues measured fly brain sizes. Since, like humans, the fly has two copies of each gene, Restifo and colleagues utilized flies with two mutant copies of the CASK gene, rendering the gene non-functional. As such, the flies with two mutant copies of CASK displayed substantially reduced brain volumes. These data show that CASK mutations impeding the gene’s function confer flies with smaller brains, similar to effects seen in humans with CASK mutations.
In search of neurological markers for CASK-related disorders in flies, Restifo and colleagues examined fly neurons under a microscope. Intriguingly, flies with CASK mutations exhibited “bushy” neurons that were shorter in length and showed more dense neurite formations. Interestingly, two copies of the mutant gene conferred a more severe “bushy” neuron shape than one mutant copy. These results suggest that, genetically, the dose of mutant copies has an additive effect on the “bushy” neuron trait, where one mutant copy has an intermediate influence between two copies and none, a phenomenon called semi-dominance.
Since restoring normal gene function to reverse the abnormally shaped “bushy” neurons would support the CASK mutation’s essential role in inducing “bushy” neurons, the UArizona researchers tested whether genetically driving normal CASK activity reverses the abnormal neuron shape. As expected, driving normal CASK gene activity in CASK mutant flies partially reversed the “bushy” neuronal shape as shown with increased neurite length and area, along with reduced branch density. These findings provide further evidence for the CASK mutation’s key role in inducing abnormally shaped neurons.
To more efficiently and reliably analyze fly neurons, Restifo and colleagues engineered a device called a microfluidic system that dissociates neurons from the nervous system. This new microfluidic system speeds up the fly neuron extraction process for analyses. Previously, researchers have undergone extensive training to learn the neuron extraction process, but with a device that reliably dissociates neurons from the nervous system, research efficiency increases. To confirm the reliability of the microfluidic system, Restifo and colleagues verified that although this technique dissociated neurons with statistically longer neurites, they found a significant “bushy” neuronal presentation from CASK mutant flies compared to non-mutant flies.
“Our discoveries of the CASK-[loss of function] bushy neurite-arbor phenotype and an automated microfluidic system for CNS tissue dissociation provide a novel cell-based assay for first-line drug discovery,” say Restifo and colleagues.
Identifying Compounds to Alleviate CASK-Related Disorders
With this new fly neuron model for CASK-related disorders that display a “bushy” neuron shape and with the newly engineered microfluidic system for neuron extraction, researchers have a more efficient means to test pharmaceuticals’ effects on these neuron disfigurations. Along those lines, the improved efficiency of this process will allow for the screening of a multitude of candidate drugs for CASK-related disorders. Furthermore, this new model doesn’t rely on experimentation involving vertebrates like rodents, which researchers try to minimize.
If future research utilizing this CASK-related disorder fly neuron model identifies drugs that improve the “bushy” neuron shape, researchers can then test the drugs in rodents, non-human primates, and ultimately, perform human trials in individuals with CASK mutations. These studies could help to pinpoint pharmaceuticals that ameliorate the progression of CASK-related disorders in infants with debilitating CASK mutations.
Tello JA, Jiang L, Zohar Y, Restifo LL. Drosophila CASK regulates brain size and neuronal morphogenesis, providing a genetic model of postnatal microcephaly suitable for drug discovery. Neural Dev. 2023 Oct 7;18(1):6. doi: 10.1186/s13064-023-00174-y. PMID: 37805506; PMCID: PMC10559581.
Moog, U. & Kutsche, K. CASK Disorders. in GeneReviews® (eds. Adam, M. P. et al.) (University of Washington, Seattle, 1993).