Multicellular living organisms and unmodified parts thereof and – Method of using a transgenic nonhuman animal in an in vivo...
Reexamination Certificate
1999-06-25
2004-04-06
Shukla, Ram R. (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Method of using a transgenic nonhuman animal in an in vivo...
C435S004000, C435S007100, C435S007200, C435S007210, C435S325000
Reexamination Certificate
active
06717030
ABSTRACT:
INTRODUCTION
FIELD OF THE INVENTION
The present invention relates to: cells and non-human animals deficient for the protein kinase C isozyme &egr; (PKC&egr;); the use of PKC&egr; as a target for drugs; the use of modulators of PKC&egr; in methods of reducing anxiety, modulating alcohol consumption and self-administration of other drugs of abuse, altering the effects of alcohol, and treating conditions associated with insufficient activity of the GABA
A
receptor; and the identification of individuals with enhanced susceptibility to alcoholism or other forms of addiction.
BACKGROUND OF THE INVENTION
Anxiety is very common sensation that, if severe or persistent, can be quite disabling. Anxiety-related disorders are so prevalent that benzodiazepines, the most frequently prescribed anxiolytic agents, regularly appear in lists of the top 20 or 25 most frequently prescribed drugs. Given the undesirable side effects of benzodiazepines and other anxiety-reducing drugs, there is a need for new treatments for anxiety.
Alcoholism is the most common form of drug abuse and a major public health problem worldwide. Nevertheless, few drugs exist that modify alcohol intake and the genetic factors that influence alcohol's effects on brain and behavioral processes remain largely uncharacterized. Thus, there is a need for diagnostic tests that can identify individuals with a predisposition to becoming alcoholics and a need for treatments that can alter alcohol consumption.
The Lewin Group estimated the economic cost to U.S. society in 1992 due to alcohol and drug abuse to be $246 billion, $148 billion of which was attributed to alcohol abuse and alcoholism and $98 billion of which stemmed from drug abuse and dependence (H. Harwood et al., The Economic Costs of Alcohol and Drug Abuse in the United States, 1992, NIH Publication Number 98-4327 (September 1998)). When adjusted for inflation and population growth, the alcohol estimates for 1992 are very similar to cost estimates produced over the past 20 years, and the drug estimates demonstrate a steady and strong pattern of increase. The current estimates are significantly greater than the most recent detailed estimates developed for 1985 for alcohol and for drugs (Rice et al. 1990)—42 percent higher for alcohol and 50 percent greater for drugs over and above increases due to population growth and inflation
Protein kinase C (PKC) is a multigene family of phospholipid-dependent, serine-threonine kinases central to many signal transduction pathways. So far, ten members, i.e., isozymes, of the PKC family have been described, which are encoded by nine different genes. The ten isozymes are designated as the &agr;-, &bgr;I, &bgr;II, &ggr;-, &dgr;-, &egr;-, &xgr;-, &eegr;-, &igr;-, and &thgr;-isozymes. Nishizuka, 1992
, Science
258:607-614; Selbie et al., 1993
, J. Bio. Chem.
268:24296-24302. Based on sequence homology and biochemical properties, the PKC gene family has been divided in three groups. A first group, i.e., the &agr;, &bgr;1, &bgr;2, and &ggr; isozyme, designated as “conventional” PKCs, are regulated by calcium, diacylglycerol and phorbol esters. A second group, i.e., the &dgr;, &egr;, &thgr; and &eegr; isozymes, designated as “novel ” PKCs, are calcium-independent, but diacylglycerol and phorbol ester-sensitive. Finally, a third group, i.e., the &xgr;, and &igr; isozymes, designated as “atypical ” PKCs, are insensitive to calcium, diacylglycerol, and PMA. In addition, two related phospholipid-dependent kinases, PKC&mgr; and protein kinase D, share sequence homology in their regulatory domains to novel PKCs and may constitute a new subgroup. Johannes et al., 1994
, J. Biol. Chem.
269:6140-6148; Valverde et al., 1994
, Proc. Natl. Acad. Sci. USA
91:8572-8576.
A number of studies with tumor promoting phorbol esters suggest that PKC modulates neural differentiation. For example, phorbol esters induce neural tissue from ectoderm in Xenopus embryos (Otte et al., 1988
, Nature
334:618-620) and elicit neurite outgrowth from chick sensory ganglia (Mehta et al., 1993
, J. Neurochem.
60:972-98 1, Hsu et al., 1984
, Cancer Res.
44:4607-4614), chick ciliary ganglion neurons (Bixby, 1989
, Neuron
3:287-297), several human neuroblastoma cell lines (Pahlman et al., 1983
, Cell Diff:
12: 165-170; Spinelli et al., 1982
, Cancer Res.
42:5067-5073), and rat PC12 cells (Roivainen et al., 1993
, Brain Res.
624:85-93; Hall et al. 1988
, J. Biol. Chem.
263:4460-4466). Studies using purified isozymes, kinase-defective mutants, and transgenic or mutant cell lines have implicated PKC&agr;, -&bgr;, -&dgr;, -&egr;, and -&xgr; in the differentiation of nonneural cells (Berra et al., 1993
, Cell
74:555-563; Goodnight et al., 1994
, Adv. Cancer Res.
64:159-209; Gruber et al., 1992
, J. Biol Chem.
267:13356-13360; Macfarlane and Manzel, 1994
, J. Biol. Chem.
269:4327-4331; Powell et al., 1992
Proc. Natl. Acad. USA
89:146-151). Overexpression of PKC&agr; or -&bgr; in Xenopus embryos enhances neural induction (Otte and Moon, 1992
, Cell
68:1021-1029), but little else is known about the identity of specific PKC isozymes that regulate neural differentiation.
Recent evidence suggests that PKC&egr; plays a role in neural differentiation and plasticity. PKC&egr; is expressed predominantly in the nervous system and is particularly abundant in the hippocampus, olfactory tubercle, and layers I and II of cerebral cortex (Saito et al., 1993
, Brain Res.
607:241-248). Within immunoreactive neurons, it is localized to the Golgi apparatus and to axons and presynaptic nerve terminals (Saito et al., supra). PKC&egr; is activated by growth factors that stimulate neural differentiation such as insulin (Heidereich et al., 1990
, J. Biol. Chem.
265:15076-15082) and NGF (Ohmichi et al., 1993
, Biochem. J.
295:767-772). In addition, in developing chick brain, it is the major isozyme found in nondividing, differentiating neurons (Mangoura et al., 1993
, J. Neurosci. Res.
35:488-498).
Further evidence for involvement of PKC&egr; in neural differentiation has come from studies with PC12 cells. PC12 cells are derived from neural crest and, when treated with NGF or fibroblast growth factors, undergo dramatic biochemical and morphological differentiation, developing several characteristics of mature sympathetic neurons. Greene et al., 1991, in:
Culturing Nerve Cells
(Banker, G. and Goslin, K. eds) pp. 207-226, MIT Press, Cambridge, Mass. PKC-activating phorbol esters enhance NGF-induced activation of ERK1 and ERK2 mitogen-activated protein (MAP) kinases and neurite outgrowth in PC12 cells, suggesting that PKC modulates responses to NGF (Rolvainen et al., 1993, supra; Hall et al., 1988, supra; Rolvainen et al., 1995
, Proc. Natl. Acad. Sci. USA
92:1891-1895). Studies with ethanol-treated PC12 cells suggested that PKC&egr; is responsible for this effect. Like phorbol esters, ethanol increases NGF-induced MAP kinase activation and neurite outgrowth through a PKC-dependent mechanism (Roivainen et al., 1993, supra; Roivainen et al., 1995, supra). Ethanol promotes PKC-mediated phosphorylation in PC12 cells by increasing levels of messenger RNA and protein for two PKC isozymes, PKC&dgr; and PKC&egr; (Messing et al., 1991
, J. Biol. Chem.,
266:23428-23432; Roivainen et al., 1994
, Toward a Molecular Basis of Alcohol Use and Abuse
, pp.29-38). Recent data demonstrate that overexpression of PKC&egr;, but not of PKC&dgr;, enhances NGF-induced MAP kinase activation and neurite outgrowth (Hundle et al., 1995
, J. Biol. Chem.
270:30134-30140). These findings establish PKC&egr; as a positive modulator of neurite growth. They also suggest that PKC&egr; mediates the neurite-promoting effect of ethanol and phorbol esters in PC 12 cells.
A recent study suggests that PKC&egr; specifically mediates enhancement of MAP kinase activation and neurite growth by phorbol esters and ethanol in PC12 cells. PKC activation is generally associated with enzyme translocation to lipid containing structures in particulate fractions of cells. Specifically, studies with PC12 cell lines that stably express th
Hodge Clyde W.
Messing Robert O.
Haliday Emily H.
Quine Intellectual Property Law Group P.C.
Shukla Ram R.
The Regents of the University of California
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