Multicellular living organisms and unmodified parts thereof and – Nonhuman animal – Transgenic nonhuman animal
Reexamination Certificate
2000-01-21
2001-11-27
Crouch, Deborah (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Nonhuman animal
Transgenic nonhuman animal
C800S003000
Reexamination Certificate
active
06323391
ABSTRACT:
BACKGROUND OF THE INVENTION
Stabilization of neurofilament (NF) mRNAs is a critical phenomena in determining levels of NF expression, axonal size and rate of axonal conduction. By regulating NF mRNA stability, the neuron is able to establish fundamental functional parameters of its own phenotype. The stabilization of NF mRNA and increased levels of NF expression are strongly influenced by the nature of target cell innervation and, presumably, by feedback signals from the target cells to the parent neuron. The feedback signals regulating NF expression arise during a developmental timeframe in which feedback signals from the target cells are also promoting the survival and further development of the parent neuron. The latter phenomena are believed to involve growth factors produced by the target cell which interact with receptors on the parent neuron and prevent the parent neuron from undergoing apoptosis. Neurons that reach and innervate target cells acquire growth factors and survive, while neurons that fail to innervate target cells do not acquire growth factors and undergo apoptosis. Growth factors thereby enable the developing neurons to override an intrinsic program of apoptosis, as exemplified by the inability of the developing neuron to survive when separated from the target cell by nerve transection or when grown in vitro in the absence of growth factors. During the next phase of development (between 0 and 4 weeks of postnatal development), neurons lose their dependence on growth factors for survival, as exemplified by their ability to survive in vitro in the absence of growth factors as well as by their ability to survive a nerve transection (Schwartz et al.,
J. Neurosci. Res.,
1990, 27:193-201). The weaning of neurons of their dependence on growth factors for survival reflects a change in the expression of genes regulating apoptosis, possibly due to the recruitment of a new set of genes which serve to override apoptosis in the absence of growth factors. While the identity of these anti-apoptosis genes are unknown, it is significant that they impart a vital and unique property to the neuron during the same developmental timeframe in which there is dramatic upregulation in the expression of the three NF genes (Schlaepfer, W. W. and Bruce, J.,
J. Neurosci. Res.,
1990, 25:39-49). The dramatic increase of NF expression is due to the stabilization of NF mRNAs (Schwartz et al.,
J. Biol. Chem.
1992, 267:24596-24600 and Schwartz et al.,
Mol. Brain Res.,
1994, 27:215-220) and is mediated by factors that bind to the NF mRNAs (Ca
ete-Soler et al.,
J. Biol. Chem.,
1998, 273:12650-12654; and Ca
ete-Soler et al.,
J. Biol. Chem.,
1998, 273:12655-12661). If these same factors regulate the expression of anti-apoptotic genes that maintain neuronal homeostasis in the absence of growth factors, the factors themselves would be an important vehicle by which to identify the anti-apoptotic gene products that maintain neuronal homeostasis.
The possibility that the same regulatory factors alter post-transcriptional expression of NF genes and gene products maintaining neuronal homeostasis derives from studies of motor neuron degeneration in transgenic mice bearing neurofilament transgenes. Although the pathogenesis of motor neuron degeneration due to expression of a mutant NF-L transgene (Lee et al.,
Neuron,
1994, 13:975-988) or overexpression of a wild-type NF-L (Xu et al.,
Cell,
1993, 73:23-33) or NF-H (Cote et al.,
Cell,
1993, 35-45) transgene in transgenic mice is presently unknown, it has been generally assumed that the neuropathic effects result from expression of NF protein by the transgene. It has been further assumed that the additional expression of NF protein by the transgene causes motor neuron degeneration by disrupting NF assembly or transport in NF-rich motor neurons (Collard et al.,
Nature,
1995,375:61-64; Bruijn, L. I. and Cleveland, D. W.,
Neuropathol. Appl. Neurobiol.,
1996,22:373-387).
The interpretation that the pathogenesis of experimental motor neuron degeneration is due to alterations in protein function, however, is problematic on several grounds. While NF accumulations are prima facie evidence of disrupted NF transport, they do not indicate whether disrupted transport is a cause, rather than a result, of neuronal degeneration. Accumulation of NFs is a frequent and readily detectable pathological change that does not necessarily lead to a progressive loss of neuronal viability, even with massive accumulations of NFs in motor neurons (Eyer, J. and Peterson, A. C.,
Neuron,
1994, 12:389-405). Nor is there appreciable loss of neuronal viability from a marked depletion of NFs due to ablation of NF-L (Zhu et al.,
Exp. Neurol.,
1997, 148:299-316) or medium neurofilament subunit (NF-M) (Elder et al.,
J. Cell Biol.,
1998, 141:727-739) or a spontaneous nonsense mutation of NF-L (Yamashaki et al.,
Lab. Invest.,
1992, 66:734-743). Finally, there is the issue of specificity, as to why a widely and abundantly expressed neuronal protein should lead to the selective degeneration of a very small subset of neurons. It is also unclear why experimental motor neuron degeneration occurs from overexpression of a mouse NF-L (Xu et al.,
Cell,
1993, 73:23-33) or a human heavy neurofilament subunit (NF-H; Cote et al.,
Cell,
1993, 73:35-46) transgene, but not from overexpression of an NF-M (Wong et al.,
J. Cell Biol.,
1995, 130:1413-1422) or a chimeric NF-H/lacZ (Eyer, J. and Peterson, A. C.,
Neuron,
1994, 12:389-405) transgene.
More recently, the effects of NF expression on other models of motor neuron degeneration have been examined by cross breeding transgenic lines of mice. These studies have shown that neither the time-course nor neuropathological effects of primary sensory neuronal degeneration in (wst/wst) wasted mice or primary motor neuron degeneration in SOD-1G37R mutant mice are altered by the presence of a mutant NF-H transgene (NF-H/lacZ) causing massive maldistribution of NFs within the afflicted neurons (Eyer et al.,
Nature,
1998, 391:584-587). On the other hand, the additional expression from a wild-type, full-length human NF-H transgene was found to prolong the lifespan and reduce the neuropathologic effects on motor neurons of the same SOD-1G37R transgenic mice (Couillard-Despres et al.,
Proc. Nat'l Acad. Sci. USA,
1998, 95:9629-9630). Motor neuron degeneration by an SOD-1 transgene was also slowed by ablation of the endogenous NF-L gene, thereby markedly reducing NF expression (Williamson et al.,
Proc. Nat'l Acad. Sci USA,
1998, 95:9631-9636). Paradoxically, the ablation of the NF-L gene enhanced the pathological effects of the mutant SOD-1 transgene on primary sensory neurons.
The severe neuropathic effects that result from low level expression of a mutant NF-L transgene (Lee et al.
Neuron,
1994, 13:975-988) contrast with the mild neuropathic effects that result from overexpression of the wild-type NF-L (Xu et al.,
Cell,
1993, 73:23-33) transgene, thus indicating that a mutation in the NF-L transgene markedly enhances the neuropathic effects of the transgene in transgenic mice. This mutant NF-L transgene, however, contained two separate mutations, namely, a leucine-to-proline point mutation in the rod domain of the protein and a 36 bp c-myc tag that was appended to the carboxyl terminus of the protein. The c-myc tag was added in order to mark the NF-L protein from the transgene and distinguish it from the wild-type NF-L protein encoded by the endogenous NF-L gene of the mouse. The leucine-to-proline point mutation in the rod domain was intended to create a dominant disassembling subunit that leads to the disassembly of all NFs in the cell (Gill et al.
J. Cell Biol.,
1990, 111:2005-2019). Although the neuropathic effects of the transgene were attributed to the point mutation in the rod domain, this interpretation was not supported by a close examination of degenerating motor neurons. Close examination showed that expression of the mutant NF-L subunit did not lead to a granular disintegration of NFs, as characteristic of
Ca{overscore (n)}ete-Soler Rafaela
Schlaepfer William W.
Baker Anne-Marie
Crouch Deborah
Licata & Tyrrell P.C.
Trustees of the University of Pennsylvania
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