The long term objectives of this program are to model aspects of human neurodegenerative diseases in mice. Murine models of Alzheimer's pathology would facilitate studies of lesion pathogenesis and might also be used in testing drugs designed to reverse or delay pathology. Recently several mutations in the amyloid precursor protein (APP) gene have been linked to familial Alzheimer's disease (FAD) and a form of congophilic angiopathy associated with hereditary intracerebral hemorrhage (ICH). Utilizing homologous recombination in ES cells we will introduce the FAD 717 mutation, the Swedish FAD double mutation and the codon 693 ICH mutation into the highly homologous mouse APP gene. Animals will be assessed for pathological and biochemical evidence of abnormal amyloid accumulation. Additionally since the functions of the various APP isoforms are unknown we will create mice dither lacking a functional murine APP gene or lacking the protease inhibitor (KPI) containing forms of mouse APP. Both mutations should be informative concerning the normal functions of APP. The neurons that are vulnerable in AD receive prominent glutamatergic imput and many of the cytoskeletal alterations seen in AD neurofibrillary tangles can be elicited by excitatory amino acids in cultured neurons. In all forms of excitotoxicity accumulating evidence supports a central role for altered regulation of intracellular calcium in neuronal damage. The ability to target selected glutamate receptor subunits (GluRs) to neurons makes it possible to test whether altering glutamate mediated calcium influx will alter sensitivity to excitotoxic injury. Neuronal GluR composition will be altered by targeting three rat glutamate receptors (GluR2, GluR6 and NMDAR1) """"""""tagged"""""""" with a heterologous amino acid sequence to allow differentiation of transgenic from endogenous mouse GluRs. These subunits were chosen because they are expected to either decrease (GluR2) or increase (GluR6 and NMDAR1) calcium influx in response to GluR activation. The human mid-sized neurofilament gene will be used as a targeting vector since it can direct expression to a wide variety of neurons including the cortical pyramidal cells which are heavily effected in AD. Finally we will extend our previous studies of neurofilament gene regulation by mapping tissue specific DNAse I hypersensitive sites in the human NF(M) gene. Hypersensitive regions will be assayed for their ability to direct neuron specific expression in transgenic mice. Ultimately we will seek to identify the transcription factors which interact with these regions. In addition to better delineating the tissue specific control regions of human NF(M) these studies may allow improved design of neuron specific targeting vectors for use in transgenic mice.
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