Transgenic Models of Neurofibrillary Pathology

             Nerve cells within the brain have distinctive shapes and a high degree of organization. Cellular shape and organization are believed to be maintained by a group of cellular proteins collectively called the cytoskeleton. Among the components of the cytoskeleton are three proteins which based on their size are termed the light, mid-sized and heavy neurofilament proteins.              In several neurodegenerative diseases including Alzheimer’s disease, Parkinson’s disease and amyotrophic lateral sclerosis, neurofilaments accumulate abnormally within diseased neurons. Specifically in Alzheimer’s disease neurofilaments are found within structures called ryeurofibrillary tangles. Tangle formation is associated with, if not directly responsible for the neuronal cell death in Alzheimer’s disease. The causes of neurofibrillary tangle formation are not known and to date no animal model exists to study such changes.              Recently we found that certain features of human neurodegenerative diseases can be reproduced in transgenic mice. Transgenic mice are created by microinjecting a foreign gene into a fertilized mouse egg which is then re-implanted into a foster mother. The foreign gene becomes incorporated into all cells of the developing mouse embryo and depending on the gene’s particularly characteristics will be expressed in many tissues of the adult mouse. We found that when a human mid-sized neurofilament gene was inserted into a transgenic mouse a neurofibrillary pathology including neurofibrillary tangle formation developed in aging adult mice.                Here we propose to examine how well these animals model Alzheimer’s disease neurofibrillary changes by characterizing the composition of the mouse neurofibrillary tangles. We will also study the effects on the brain of the neurofilament accumulation in these lesions. This model offers an opportunity to study a selective over expression of one component of neurofibrillary tangles. If this animal model is found to mimic the changes seen in Alzheimer’s disease, it might be useful in testing drugs or other forms of therapy aimed at slowing or stopping the pathological process in Alzheimer’s disease.               Although much is now known about neurofilament composition, many questions remain about neurofilament structure and function. If the normal functions of neurofilaments were known the consequences of their disruption in chronic neurodegenerative diseases might be better understood. Neurofilaments are thought to protect against mechanical stress in large neurons and possibly to control nerve diameter. However the evidence for this is largely circumstantial. Much of our ignorance about neurofilament function stems from our inability to selectively perturb one or more neurofilament subunits in living cells. Neurofilaments might be disrupted by introducing compounds or antibodies which interfere with the their assembly or transport. However these approaches are primarily useful in cultured cells and have limited value in the intact animal. No naturally occurring mutants of neurofilaments are available to study the consequences the loss of one or more of the neurofilament subunits.                 A relatively new approach to inactivating mammalian genes in the mouse has been developed within the past few years utilizing cells termed embryonic stem cells (ES cells). These cells are derived from early stage mouse embryos. Target genes of interest are inactivated within the ES cell by replacing the normal gene with a mutated form which is functionally inactive. Following inactivation of the target gene in ES cells, the cells are introduced into mice by microinjecting the ES cells into a developing mouse embryo and then implanting the embryo into a foster mother. ES cells mix with the normal host cells and can colonize all tissues, including the reproductive organs. Subsequent interbreeding of these mice allows the generation of mice in which both copies of the target gene are mutated and no functional protein is produced. With this approach it is now feasible to inactivate probably any mouse gene. Gene targeting technology offers the opportunity to create animal models for human genetic diseases as has recently been accomplished by creating a mouse model of cystic fibrosis.             This approach appears ideal for genes such as the neurofilaments, whose functions will likely only be fully appreciated at the tissue level. Here we propose to create mice lacking the mouse mid-sized and heavy neurofilament genes. The consequences of neurofilament disruption on neural development and function will be assessed by examining fetal and adult mice for developmental or structural defects. Neurofilament loss may lead to altered neurofilament structure, changes in neuron size or effect other cytoskeletal components. Effects may occur either during development or as with the neurofilament over expression model described above only with aging. These studies will complement an already ongoing program designed to study neurofilament processing by prcxiucing mutated versions of the mid-sized human neurofilament in transgenic mice and should contribute to a better understanding of the mechanisms of neurofibrillary tangle formation and the normal functions of neurofilaments.