Research
Our laboratory has been using spontaneous mutations in rodents as genetic tools to understand functions of genes in health and disease.
We have described a naturally occurring mutation (Nuc1) in the Sprague-Dawley rat with a novel eye phenotype. Nuc1 is inherited as a single Mendelian locus with viable, but severely affected homozygotes and an intermediate phenotype in heterozygotes. We recently reported that the mutation causing Nuc1 is a 27 base pair insertion in exon 6 of the βA3/A1-crystallin gene on rat chromosome 10.
Our data suggest, for the first time, that βA3/A1-crystallin is essential for the normal functioning of retinal astrocytes and that it has a critical role in the maturation of the neurons and vessels in the developing eye of the rat. In the Nuc1 homozygous rat, the development of the retinal neurons and the vasculature is abnormal. An association between retinal neurogenesis and vasculogenesis is supported by numerous reports in the literature. There are also numerous reports describing an expanding role for glia in neuronal and vascular development. Each of these areas remains the focus of active investigation with progress that promises to produce an integrated understanding of vasculogenesis, neurogenesis and glial function in retinal development. The Nuc1 mutation serves as a useful genetic tool with which to explore these relationships, while serving as a model in which to develop mechanistic insights into normal retinal development as well as various retinal disease processes.
Our current studies focus on (1) various biochemical parameters of the normal and mutant βA3/A1-crystallin protein in astrocytes, including sub-cellular localization, quaternary structure, and protein-protein interactions. (2) apparent abnormal migration of Nuc1 astrocytes and how the βA3/A1-crystallin mutation may mediate this effect through disruption of normal regulation of intermediate filaments, (3) interactions between retinal ganglion cells and astrocytes, (4) possible compromise of the structural and functional integrity and cellular organization of retinal vessels, and (5) the potential roles of the Notch, VEGF, MAPK/ERK, and PI-3 kinase/Akt pathways in astrocytes that may mediate the remodeling of neuronal and vascular cells during development.
In addition, we are also using the Cre-loxP system to delete βA3/A1-crystallin selectively from astrocytes early in development, and are using transgenic technology to target expression of wildtype and mutant βA3/A1-crystallin specifically to astrocytes to generate both gain-of-function and loss-of-function mouse lines. Using these genetically engineered mouse models, we will be able to examine the effects of gain or loss of function mutations in βA3/A1-crystallin on astrocyte development, as well as on neuronal and vascular remodeling in the retina and the brain.
Our second genetic tool is a novel spontaneous rat mutant that displays neuro-developmental abnormalities. The phenotype first appeared spontaneously in a litter of inbred Sprague-Dawley rats. We have established that the condition is inherited in a Mendelian fashion as a single autosomal recessive trait, and have used genetic linkage analysis to map the gene to rat chromosome 1. Hind limbs of the affected animals are extended and abducted so severely that they do not effectively support the animal’s weight. Because of this unusual appearance and gait anomaly, we have named the mutant strain frogleg.
Neonatal frogleg rats typically are smaller than normal with a thin rough coat, but reach normal weight by 2-3 months of age. They exhibit impaired standing and exploring behavior and splayed hind limbs. X-ray and CT analysis indicated no abnormalities in the skeletal structure of the hind limbs, yet the frogleg hind limbs have abnormal internal rotation, abduction and hyperextension. Brains from the mutant animals are dramatically lower in weight than those of age-matched control animals and have markedly dilated ventricles. Preliminary studies also showed abnormalities in the peripheral nervous system (PNS). Schwann cells in the sciatic nerves of frogleg rats are morphologically abnormal and the sciatic nerve compound muscle action potential (CMAP) is also significantly reduced. This may indicate a possible abnormality in the axon-glia interaction.
Hereditary motor and sensory neuropathies can be caused by mutations in genes that are expressed in Schwann cells or the axons they ensheath. New genetic causes for inherited neuropathies such as the frogleg mutation may help investigators to better understand the complex interactions between Schwann cells and axons as they relate to such processes as myelination and axon function in the PNS. Once the gene responsible for the frogleg mutation is identified, it will provide insights as to how the encoded protein behaves in the cell and how the mutated protein causes inherited neuropathy, particularly in the context of the Schwann cell and axon. Autosomal recessive neuropathies are relatively rare but are clinically more severe than autosomal dominant forms, and the frogleg mutant model may provide a new perspective on the mechanisms leading to these devastating human diseases.
