Sara Lewis Abstract
Sara Lewis
Ph.D.Candidate
Neuroscience GIDP
Genetics Society of America Drosophila Research Conference
Chicago, Illinois
March 4-8, 2015
ABSTRACT:
Pak (p21-activated kinase) mutations cause defects in brain structure and neurite-arbor morphogenesis through regulation of non-muscle myosin.
Authors:
Sara A Lewis,1,2,3, Linda L Restifo1,2,3
1University of Arizona Neuroscience Graduate Program, Tucson, AZ, 85721, 2UA Neuroscience, Tucson, AZ, 85721, 3UA Neurology, Tucson, AZ, 85724
Drosophila Pak is the ortholog of human p21-activated kinase 3 (PAK3). PAK3 mutations cause intellectual disability with microcephaly and craniofacial defects, but little is known about the cellular neuropathology. Pak mediates Cdc42 and Rac GTPase activity in response to extracellular cues, such as adhesion and growth factors. Pak inhibits myosin function and F-actin cleavage by cofilin, thereby promoting F-actin stabilization. We used nonsense mutations from independent mutageneses to reveal that Pak is required for mushroom body (MB) morphogenesis during metamorphosis. Pak-mutant brains showed missing and misdirected α and β lobes, and became worse over time. We used primary culture to identify mechanisms of Pak function at the single-neuron level. Cultured Pak-mutant larval CNS neurons have small neurite arbors with reduced length and branching. These neurites have increased curvature with regions of excessive width, reminiscent of the filagree defect caused by fascin deficiency. Initial neurite outgrowth and branch formation appeared normal in vitro, but were followed by reduction in branch number, suggesting excessive branch retraction. Cytoskeletal abnormalities were present in Pak-mutant growth cones and neurites. The MB-lobe and neurite-arbor phenotypes map to Pak based on deletions and transgenic rescue. Genetic interaction studies were used to elucidate mechanisms by which Pak regulates neurite-arbor morphology. Pak indirectly inhibits myosin regulatory light chain (MRLC) function, thereby reducing myosin-actin interactions. Transgenic addition of non-phosphorylatable MRLC (sqhA20, A21) suppressed the Pak-mutant neurite-arbor phenotype, restoring size and complexity. Genetic reductions in function of Pak and flw, encoding MRLC phosphatase, synergistically reduced neurite-arbor size. Distinct myosin isoforms can anchor polymerizing actin in growth cones, mediate retrograde actin flow, or retract branches. Together, our data support a working model whereby Pak stabilizes the nascent neurite arbor of cultured CNS neurons by inhibition of non-muscle myosin.
Intellectual disability (ID), formerly known as mental retardation, affects 1-3% of the population. ID is caused by abnormalities in brain development with mutations in many genes causing ID. I am studying a gene (PAK3) that causes a form of ID with small brain size. Additionally, this gene affects at least a dozen other of these ID-causing genes. Studying this gene improves understanding of mechanisms that regulate normal brain development and how they are defective in ID.
The PAK3 gene has been demonstrated in non-neuron cell types to interact with the actin cytoskeleton, or the network of proteins that provides the structure to determine the shape and size of a cell. I predicted that this gene is important for determining neuron size and shape; therefore small neurons could be causing the small brain size in children with these mutations. To test this, I am using a combination of neuroscience, cellular biology, molecular biology, and genetics.
I am examining brain structure and neurons in cell culture of fruit flies with mutations in the fly version of the PAK3 gene, Pak. A brain structure composed of bundled neurons which branch to form an “L” shape is normal early in development in Pak-mutant brains. However, some of these branches are missing later in development, with many adults having no branches. This suggests that branches that should have been maintained were lost. To test if Pak regulates size and shape of neurons, I examined neurons in cell culture. Neurons from the brains of Pak-mutant flies are small with few branches. The initial outgrowth of these Pak-mutant neurons is normal, but followed by a loss of branches. This suggests branches are being retracted, which agrees with what is observed in the brain.
I have investigated which proteins are the targets of Pak activity. Myosin is predicted to be inhibited by Pak; loss of Pak would lead to over-active myosin. Myosin binds onto the actin cytoskeleton and causes branch retraction. I demonstrated that Pak regulates myosin activity using interactions between genes in the predicted Pak-myosin pathway. I concluded that Pak regulates neuron size and shape by inhibiting branch retraction through myosin. This gives a target for future efforts of drug discovery to help people with this type of brain development defect.