Multicellular living organisms and unmodified parts thereof and – Method of using a transgenic nonhuman animal in an in vivo...
Reexamination Certificate
2000-07-20
2004-05-11
Nguyen, Dave T. (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Method of using a transgenic nonhuman animal in an in vivo...
C800S012000, C800S018000, C435S320100
Reexamination Certificate
active
06734336
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to gene-targeted, non-human mammals comprising a human mutation in the non-human mammalian presenilin 1 (PS-1) FAD gene, methods of identifying compounds for treating Alzheimer's disease, and to methods of treating Alzheimer's disease.
BACKGROUND OF THE INVENTION
Alzheimer's disease (AD) is an age-dependent neurodegenerative disorder that leads to profound behavioral changes and dementia. Hallmark pathologies include the atrophy of brain gray matter as a result of the massive loss of neurons and synapses, and protein deposition in the form of both intraneuronal neurofibrillary tangles and extracellular amyloid plaques within the brain parenchyma. In addition, affected areas of the AD brain exhibit a reactive gliosis that appears to be a response to brain injury. Surviving neurons from vulnerable populations in AD show signs of metabolic compromise as indicated by alterations in the cytoskeleton (Wang et al.,
Nature Med
., 1996, 2, 871-875), Golgi complex (Salehi et al.,
J. Neuropath. Exp. Neurol
., 1995, 54, 704-709) and the endosomal-lysosomal system (Cataldo et al.,
Neuron
, 1995, 14, 671-680).
Approximately 10 to 30% of AD cases are inherited in an autosomal dominant fashion and are referred to as “familial Alzheimer's disease” or “FAD.” Genetic linkage studies have revealed that FAD is heterogeneous and a majority of the cases have been linked to gene mutations on chromosomes 1, 14, 19, or 21 (reviewed in Siman and Scott,
Curr. Opin. Biotech
., 1996, 7, 601-607). Importantly, these individuals have been shown to develop the classical symptomatic and pathological profiles of the disease confirming that the mutations are associated with the development of the disease rather than a related syndrome. The locus on chromosome 14 is associated with a significant fraction of FAD, and mutations at the locus have been mapped to a single-copy gene, termed “S182” or “presenilin 1” (PS-1), that encodes a 467 amino acid protein (Sherrington et al.,
Nature
, 1995, 375, 754-760; Clark et al.,
Nature Genet
., 1995, 11, 219-222). A closely related gene, “STM2” or “presenilin 2” (PS-2), located on chromosome 1, has been linked to two additional FAD kindreds including the descendants of German families from the Volga valley of Russia (Levy-Lahad et al.,
Science
, 1995, 269, 973-977; Rogaev et al.,
Nature
, 1995, 376, 775-778). PS-1 and PS-2 share an overall 67% amino acid sequence homology, and primary structure analysis indicates they are integral membrane proteins with 6 to 8 trans-membrane domains (Slunt et al.,
Amyloid—Int. J Exp. Clin. Invest
., 1995, 2, 188-190; Doan et al.,
Neuron
, 1996, 17, 1023-1030). Much of the information on function of the presenilins stems from the identification of a presenilin homolog in
C. elegans
termed “SEL-12,” a 6 to 8 trans-membrane protein that appears to participate in an intracellular signaling pathway mediated by the lin-12/glp-1/Notch family (Levitan and Greenwald,
Nature
, 1995, 377, 351-354). PS-1 and SEL-12 proteins share a 49% sequence homology and have similar membrane orientations. Importantly, both human PS-1 and PS-2 can rescue the mutant sel-12 phenotype in
C. elegans
, indicating a role for the presenilins in Notch signaling (Levitan et al.,
Proc. Natl. Acad. Sci. USA
, 1996, 93, 14940-14944).
FAD linked to the presenilins is highly penetrant and the aggressive nature of the disease suggests that the mutant protein participates in a seminal pathway of AD pathology. To date, over seventy FAD mutations have been identified in PS-1, and three FAD mutations have been found in PS-2. Most of the FAD mutations occur in conserved positions between the two presenilin proteins, suggesting that they are affecting functionally or structurally important amino acid residues. Interestingly, many of the mutated amino acids are also conserved in SEL-12. All but two of the presenilin mutations are missense mutations. One exception results in an aberrant RNA splicing event that climninates exon 9, creating an internally-deleted mutant protein (Perez-Tur et al.,
NeuroReport
, 1995, 7, 297-301; Sato et al.,
Hum. Mutat. Suppl
., 1998, 1, S91-94; and Prihar et al.,
Nature Med
., 1999, 5, 1090). The other results in two deletion transcripts (&Dgr;4 and &Dgr;4cryptic) and one full-length transcript with the amino acid Thr inserted between 113 and 114 (DeJonghe et al.,
Hum. Molec. Genet
., 1999, 8, 1529-1540). The latter transcript leads to the AD pathophysiology. These latter points, along with the genetic dominance of the disease, argue that disease pathogenesis in the presenilin kindreds requires the production of a mutant presenilin protein having a novel and detrimental function, rather than the simple loss or reduction of normal presenilin levels. The mutations do appear to disrupt normal presenilin function however, because mutant presenilins are not able to rescue or fully rescue the sel-12 phenotype (Levitan et al.,
Proc. Natl. Acad. Sci. USA
, 1996, 93, 14940-14944).
Expression profiles of the presenilins have been examined at a gross level but, so far, these analyses have yielded little information on the mechanism of disease pathogenesis. Both presenilin 1 and 2 are widely expressed in the CNS and peripheral tissues. In brain, expression is enriched in neurons but is apparent in both AD-vulnerable and resistant areas. Cellular localization studies indicate that the proteins accumulate primarily in the Golgi complex and endoplasmic reticulum but no significant alterations in expression levels or subcellular distribution have been attributed to the FAD mutations (Kovacs et al.,
Nature Med
., 1996, 2, 224-229).
The presenilin proteins are processed proteolytically through two intracellular pathways. Under normal conditions, accumulation of 30 kD N-terminal and 20 kD C-terminal proteolytic fragments occurs in the absence of the full-length protein. This processing pathway is highly regulated and appears to be relatively slow, accounting for turnover of only a minor fraction of the full-length protein. The remaining fraction appears to be rapidly degraded in a second pathway by the proteasome (Thinakaran et al.,
Neuron
, 1996, 17, 181-190; Kim et al.,
J. Biol. Chem
., 1997, 272, 11006-11010). Proteolytic metabolism of PS-1 variants linked to FAD appears to be different, but the relevance of the change to pathogenesis is not known (Lee, et al.,
Nature Med
., 1997, 3, 756-760).
One pathogenic role for the mutant presenilins in FAD appears to be related to effects on processing of the amnyloid precursor protein (APP) and production of the A&bgr; peptide, the primary proteinaceous component of the extracellular neuritic plaque in the AD brain. Elevated serum levels of the longer form of A&bgr; (A&bgr;42), considered to be the more pathogenic species of the A&bgr; peptides, have been measured in patients bearing PS-1 and PS-2 mutations (Scheuner et al.,
Nature Med
., 1996, 2, 864-870). Additionally, FAD brains with PS-1 mutations have large amounts of A&bgr; deposition (Lemere et al.,
Nature Med
., 1996, 2, 1146-1150; Mann et al.,
Ann. Neurol
., 1996, 40, 149-156; Gómez-Isla et al.,
Ann. Neurol
., 1997, 41, 809-813). Elevated levels of A&bgr;1-42 were also found in cells transfected with mutant PS-1 or PS-2 and in mice expressing mutant PS-1 (Borchelt et al.,
Neuron
, 1996, 17, 1005-1013; Duff et al.,
Nature
, 1996, 383, 710-713; Citron et al.,
Nature Med
., 1997, 3, 67-72; Murayama et al.,
Prog. Neuro
-
Psychopharmacol. Biol. Psychiatr
., 1999, 23, 905-913; Murayama et al.,
Neurosci. Lett
., 1999, 265, 61-63; Nakano et al.,
Eur. J. Neurosci
., 1999, 11, 2577-2581). The mechanism by which the mutant presenilins affect APP processing is not known, but these results do support a causative role of increased A&bgr;42 production in the development of FAD. Importantly, it is possible that mutant presenilins influence other AD pathogenic processes as well, such as presumptive intracellular signaling and cell death pathways involved directly or indirectly in neuro
Dorfman Karen
Reaume Andrew G.
Scott Richard W.
Cephalon Inc.
Nguyen Dave T.
Woodcock & Washburn LLP
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