Transgenic mice expressing human p25

Details Transgenic mice expressing human p25 Transgenic mice expressing human p25

2000-02-02

2004-02-17

Crouch, Deborah (Department: 1632)

Multicellular living organisms and unmodified parts thereof and

Nonhuman animal

Transgenic nonhuman animal

C800S003000, C800S012000

Reexamination Certificate

active

06693226

ABSTRACT:

TECHNICAL FIELD
The invention provides transgenic, non-human animals and transgenic non-human mammalian cells harboring a transgene encoding a p25 polypeptide, an activator of the protein kinase cdk5. The invention also provides non-human animals and cells comprising a transgene encoding a p25 polypeptide and further comprising functional overexpression of p25, the p25 transgene and targeting constructs used to produce such transgenic cells and animals, transgenes encoding human p25 polypeptide sequences and methods for using the transgenic animals in pharmaceutical screening and as commercial research animals for modeling neurodegenerative disease such as Alzheimer's disease and p25/cdk5 biochemistry in vivo.
BACKGROUND OF THE INVENTION
Throughout the specification, a number of publications are cited. These publications are incorporated by reference in their entirety. A complete listing of the publications appears later in the specification.
Alzheimer's disease (AD) is a progressive, neurodegenerative disorder characterized by loss of cognitive function. The primary neuropathological lesions in AD are amyloid plaques and neurofibrillary tangles (NFTs). Amyloid plaques are composed primarily of amyloid beta (Ab) peptides, varying in length from 39-42 amino acids, which are derived from amyloid precursor protein (APP) (reviewed in 1). NFTs are composed of the microtubule binding protein tau that is hyperphosphorylated at epitopes which exist in a predominantly unphosphorylated state in disease-free brain (2-4). The respective roles that these lesions play in the neuronal loss and dementia observed in patients with AD remain controversial.
The precise mechanism of NFT formation is not clear but work from many laboratories suggests that hyperphosphorylation of tau may be an important event. The paired helical filament (PHF) is the fundamental unit of the NFT. Hyperphosporylation of tau at serine or threonine residues followed by proline (SP or TP), epitopes which are concentrated at the amino and carboxy termini of tau, results in loss of affinity for microtubules, and a presumed concomittant increase in the concentration of cytoplasmic tau (5-8). However, phosphorylation of tau at serine 262, which is not followed by proline, can also reduce the affinity of tau for microtubules (9). In vitro experiments with purified tau show that in the presence of endogenous cations (e.g., mRNA, heparin sulfate proteoglycan), tau polymerizes to form structures indistinguishable from the PHF seen in AD brain (10,11). The cation-dependent formation of PHF in vitro is independent of the phosphorylation state of tau, suggesting that the key permissive event in initiating PHF formation may be an increase in the cytoplasmic concentration of tau. In support of this hypothesis, recent evidence (12-14) indicates that the mutations in the tau gene associated with susceptibility to a form of inherited dementia called frontotemporal dementia and Parkinsonism linked to chromosome 17 (FTDP-17), reduce the affinity of tau for microtubules (15). Therefore these mutations, like hyperphosphorylation, may result in an increase in cytoplasmic tau concentrations. In the presence of endogenous cations, this increase is presumed to permit PHF formation followed by NFT assembly and, ultimately, neuronal death.
As with other phosphoproteins, the phosphorylation state of tau is the sum of protein kinase and protein phosphatase activity. Thus hyperphosphorylation of tau in AD may be due to an increase in kinase activity or a decrease in phosphatase activity. While many protein kinases phosphorylate tau at AD-relevant epitopes in vitro (reviewed in 16,17), only two have been co-purified with microtubules from mammalian brain, GSK3b and cdk5 (18). To our knowledge, only these two kinases will phosphorylate tau when transfected heterologously into mammalian cells (19,24). We chose to focus on cdk5 vs. GSK3b as the latter plays a role in energy metabolism and is expressed in an active form in all cells, while cdk5 is only active in neurons (vide infra).
The kinase cdk5 is a member of the cyclin-dependent protein kinase family and is expressed in nearly all cells (reviewed in 25,26). Unlike other members of the cdk family, there is no known cyclin which activates cdk5. Rather, the positive allosteric regulators of cdk5 are p35 (27), amino terminal proteolytic fragments of p35, e.g, p25, p23 or p21 (28,29) and p39 (30). These proteins share minimal amino acid sequence homology to cyclins (27-29) but computer modeling and biochemical experiments suggests that the mechanism of activation of cdk5 by p25/35 may be similar to that of cyclin A activation of cdk2 (31-33). The protein p25/35 is expressed predominantly in neurons implying that most cdk5 activity is concentrated in neuronal structures (27,28). The protein p35 has a relatively short half life within cells and is rapidly ubiquitinated, suggesting tight regulation of cdk5 activity in neurons (34). The kinase cdk5 plays a pivotal role in neuronal development as evidenced by the abnormal corticogenesis and perinatal lethality of cdk5 knockout mice (35) and the disturbances in neuronal migration and early death in p35 knock-out mice (36). In developmental studies in rodents, the peak catalytic activity of cdk5 occurs at E11 or 12, lending further support for the role of cdk5 in neurogenesis (37,38). Furthermore, in primary cultured neurons, cdk5/p35 is localized to growth cones suggesting a role in neurite outgrowth (39). Recently, evidence of signaling pathways which may modulate cdk5 activity have emerged. For example, it has been demonstrated that cdk5/p35 interacts with Rac and modulates PAK activity (40), and that laminin-enhanced outgrowth of cerebellar neurons is disrupted by suppression of p35 expression (41). A few substrates for cdk5 have been identified and are consistent with the presumed role in neurite outgrowth and plasma membrane dynamics. These include cytoskeletal proteins such as tau (42-45) and neurofilament (46-48), synaptic vesicle proteins such as synapsin and Munc-18 (49,50) and the retinoblastoma protein (51). Nevertheless, neither a clear picture of a signal transduction pathway(s) which regulates cdk5/p35 activity nor the role of both cdk5/p35 in mature brain have been elucidated.
A clear picture of the protein kinases responsible for the hyperphosphorylation of tau in AD is also lacking. However, evidence that cdk5 may a play a pathological role is accumulating. For example, in in vitro studies, cdk5 will phosphorylate up to eight different epitopes of tau, including those associated with AD and known to decrease the affinity of tau for microtubules (42-45). Additionally, heterologous co-transfection of cdk5/p25 with tau into mammalian cells also results in phosphorylation of several of these epitopes (24). Finally, immunohistochemical evidence suggests that cdk5 is proximal to NFTs in AD brain (52,53).
The development of experimental models of Alzheimer's disease that can be used to define further the underlying biochemical events involved in AD pathogenesis would be highly desirable. Such models could be employed, in one application, to screen for agents that alter the degenerative course of AD. For example, a model system of AD could be used to screen for environmental factors that induce or accelerate the pathogenesis of AD. Alternatively, an experimental model could be used to screen for agents that inhibit, prevent, or reverse the progression of AD. Such models could be employed to develop pharmaceuticals that are effective in preventing, arresting or reversing AD. Only humans and aged non-human primates develop any of the pathological features of AD. The expense and difficulty of using primates and the length of time required for developing the AD pathology makes extensive research on such animals prohibitive. Rodents do not develop AD, even at an extreme age. Despite various reports that certain treatments result in hyperphosphorylation of tau and/or neuronal death associated with the phosphorylation of tau, there is a need

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