Neurofilament Assembly and Function

             Human neurodegenerative disease s in general, and Alzheimer’s Disease, Amyotrophic Lateral Sclerosis and Parkinson’s Diseases in particular, have a number of common and unifying characteristics. All affect the aging nervous system; they exhibit overlapping constellations of neuropathological changes; all are degenerative diseases showing neuronal changes and loss without indications of immunologic, infectious or vascular disorders. These diseases almost always include some abnormal intraneuronal inclusion and collections of neurofibrillar material . The composition of these material differs among the diseases but, where examined, they always contain a component derived from neurofilaments ..               The neurofilaments, the intermediate filaments of the neuron, are a polymer a ssembled from three protein subunits, NF(L), NF(M) and NF(H) that are evolutionarily related to each other and to the other intermediate filament (IF) proteins. Studies of the similarities in amino acid sequence, gene organization and immunocytoche mical crossreactivity provide convincing evidence that neurofilament genes evolved from a primordial IF gene prior to the diverge nce of the vertebrate and invertebrate species, some 800 million years ago. Neurofilaments and their mRNAs are present in a wide range of organisms and in each is found only in neurons. Thus both the structure of the neurofilament and its neuronal specificity have been preserved over a great span of evolutionary history. This conservation and specificity speaks of an important role for the neurofilaments, but no function has as yet been assigned to these or any IF protein , although speculations have been advanced.                     Mu ch of our ignorance about IF function stems from our present inability to selectively perturb their networks in living cells and observe the consequences, as can be done with actin fibers and microtubules through agents such as cytochalasin B and colchicine. In the case of the neurofilaments, the search for the function must be carried to the living cell (the neuron) because they are dynamic structures in which the sub-unit composition and topology of phosphate groups differ in the different compartments of the cell. In this application, we propose to produce altered forms of the neurofilament sub-units in cultured cells or in the neurons of transgenic mice and observe their interactions with the endogenous components, using immunomicroscopy and biochemical techniques. The system we propo se will allow the systematic exploration of the neurofilament subunits since NF subunits bearing small deletions, insertions or substitutions can be prepared by these methods and ultimately expressed in neurons of transgenic mice. In the present study we will produce only large deletions which remove whole domains of the NF(M), hoping to reveal classes of phenotypes. Subsequent explorations can employ more subtle alterations. ·               Using recombinant DNA techniques, we will alter the human NF(M) gene so that the encoded protein contains a he terologous 11 amino acid “tag” that can b e used to immunocytochemically identify the product irrespective of the regions deleted . Antipeptide antisera of exceptional titers have already been produced against this “tag” sequence. Transgenic mice and fibroblasts carrying this “tagged” gene will be produced and the patterns of expression of the “tagged ” NF(M) gene will be compared to that of the unaltered NF(M) gene in the transfected fibroblasts and transgenic mice we have already produced. The patterns of expressions of both transgenes (localization, intensity of fluorescent signal, cytoplasmic associations) will be assessed using immunornicroscopy. In the case of the transgenic mice, the expression of the transgenes will also be compared to that of the endogenous mouse NF (M) gene.                In a second part of this undertaking, sets of genes will be prepared which encode tagged NF(M) subunits deleted for portions of either the amino terminus, carboxy terminus or KSP repeat domains . These genes will be introduced into fibroblasts and their patterns of expression studied using immunornicroscopy. Constructs yielding informative results will be introduced into transgenic mice so that the altered NF subunit can be studied in neurons of transgenic mice.               An advantage of the proposed approach is the ability to first screen and investigate trial alterations of the neurofilament sub-units in transfected fibroblasts before investing heavily in the transgenic mouse paradigm. These fibroblast experiments should yield useful information about possible cloning artifacts, the appropriateness of the transcription of the transferred gene, the localization and stability of the encoded protein in vivo and the ability of the protein to interact with endogenous cytoskeletal elements. Selected constructs will be expressed in transgenic mice for detailed analysis of the cell biological or biochemical attributes of the encoded proteins. This experimental design will allow us to examine many alterations in the neurofilament sub-units in a cost-effective way and thereby to expose many cell biological aspects of neurofilaments. It also may be possible to mimic or even reproduce in mice certain phenomena associated with neurodegenerative diseases in transgenic mice.