Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
2000-10-06
2003-08-05
Kunz, Gary (Department: 1647)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C514S903000, C530S350000
Reexamination Certificate
active
06602851
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the fields of neurobiology and developmental biology. More specifically, the present invention relates to the identification of novel neuregulin splice variant isoforms.
2. Description of the Related Art
Traumatic injury of adult mammalian peripheral nerve results in degeneration of axon segments and myelin distal to the injury site with concomitant Schwann cell dedifferentiation and proliferation. These changes in Schwann cell morphology are essential for subsequent axonal regeneration (Hall and Gregson, 1977; Pellegrino et al., 1986; Fawcett and Keynes, 1990; Nadim et al., 1990) and are accompanied by increased Schwann cell expression of molecules promoting neurite sprouting [e.g., neurotrophic factors and cell adhesion molecules; reviewed in Fawcett and Keynes, 1990; Fu and Gordon, 1997]. The signals responsible for repressing myelin protein synthesis, inducing expression of molecules supportive of axonal regeneration, and stimulating Schwann cell mitogenesis in injured nerve are poorly understood. It is likely, however, that these signaling molecules include several members of the neuregulin (NRG) family of growth and differentiation factors.
The neuregulin (NRG) family of growth and differentiation factors is thought to form a complex network of intercellular signaling molecules mediating multiple important developmental, maintenance and regenerative functions throughout the nervous system. For instance, neuregulins are highly expressed by sensory and motor neurons during development (Chen et al., 1994; Falls et al., 1993; Ho et al., 1995; Marchionni et al., 1993) and have been implicated as axon-derived signals influencing the differentiation, survival and proliferation of associated Schwann cells during this same period (reviewed in (Topilko et al., 1996; Lemke, 1996)). Neuregulins are also highly potent mitogens for neonatal Schwann cells in vitro (Brockes et al., 1980; Goodearl et al., 1993; Levi et al., 1995) and repress expression of myelin protein zero (P
0
) and myelin basic protein in these same cells (Cheng and Mudge, 1996). Furthermore, axon-associated NRGs are a component of the “axon-associated mitogen” found on the neurites of neonatal dorsal root ganglion (DRG) neurons (Morrissey et al., 1995). Based on these developmental and in vitro observations, it is hypothesized that neuregulins, potentially released from the injured axon, similarly induce the Schwann cell dedifferentiation and proliferation during the Wallerian degeneration which follows traumatic injury of peripheral nerve and which is essential for subsequent axonal regeneration (Hall and Gregson, 1977; Pellegrino et al., 1986; Fawcett and Keynes, 1990; Nadim et al., 1990).
These molecules are indeed expressed with the temporal and spatial distribution expected for postaxotomy mediators of Schwann cell proliferation and/or other effects in axotomized rat sciatic nerve (Carroll et al., 1997). However, Schwann cells themselves apparently produce neuregulin, a finding consistent with recent reports of neuregulin expression by cultured neonatal Schwann cells in vitro (Raabe et al., 1996; Rosenbaum et al., 1997). Furthermore, the dorsal root ganglia (DRG) sensory and spinal cord motor neurons projecting into the sciatic nerve express the erbB receptors necessary for neuregulin responsiveness during embryogenesis and adulthood. Also, recombinant neuregulin is a survival factor for embryonic day 15 spinal cord motor neurons in vitro. It is therefore likely that neuregulin signaling proceeds bidirectionally between these cell types or that Schwann cell- and neuron-derived neuregulins act in an autocrine fashion.
Since astrocytes, oligodendrocytes and many populations of central nervous system (CNS) neurons similarly express both neuregulins and neuregulin receptors, these same possibilities may need to be considered in the brain. Given the potential complexity of neuregulin signaling among glia and neurons, the question arises as to how neuregulin signaling might be compartmentalized or otherwise regulated. This control may be facilitated, in part, by the synthesis of distinct forms of neuregulin by each expressing cell type. Cloning of neuregulin family members (Wen et al., 1992; Marchionni et al., 1993; Carroll et al., 1997; Falls et al., 1993; Ho et al., 1995; Carroll et al., 1997; Yang et al., 1998) demonstrated these molecules to be structurally diverse proteins translated from alternatively spliced mRNAs transcribed from a single locus. Neuregulins may be divided into three subfamilies, each defined by their unique N terminus and known as the heregulin (HRG)
eu differentiation factor (NDF)/mesenchymal, glial growth factor (GGF) and sensory and motor neuron-derived factor [SMDF; also known as cysteine-rich domain (CRD)-neuregulin] subfamilies.
The structures of various members of the neu differentiation factor subfamily have been thoroughly studied. The seven known neu differentiation factor isoforms are synthesized as either directly secretable forms or as transmembrane precursors requiring proteolytic cleavage for release (Wen et al., 1994). These proteins possess distinct epidermal growth factor (EGF)-like domains (&agr; and &bgr; isoforms) resulting in differences in receptor affinity (Wen et al., 1994) and ability to induce biological effects (Marikovsky et al., 1995; Pinkas-Kramarski et al., 1996).
The EGF-like domain, which consists of a common region fused to either &agr;- or &bgr;-domains, is essential for biologic activity. Truncated &bgr;-neuregulin molecules containing only the EGF-like domain bind to the neuregulin receptor with an affinity similar to that of the full-length factor (Holmes et al., 1992; Peles et al., 1993) and are capable of inducing a variety of biologic responses (Holmes et al., 1992; Peles et al., 1993; Chu et al., 1995; Levi et al., 1995; Syroid et al., 1996).
In spite of their similar structures, neuregulin &agr; and &bgr; EGF-like domains are not functionally equivalent; &bgr;-neuregulins have an affinity for erbB receptors an order of magnitude greater than &agr;-neuregulins (Wen et al., 1994). Furthermore, &agr;-neuregulins are nonmitogenic for some, but not all, cell types which proliferate in response to &bgr;-neuregulins (Pinkas-Kramarski et al., 1996).
Further variability in other regions may alter glycosylation (Wen et al., 1994), protease-mediated release from the cell membrane (Wen et al., 1994) and direct signaling by transmembrane precursors (Wang et al., 1998). In addition to the unique amino termini (the functions of which are currently unknown), the mesenchymal and GGF (but not the SMDF) neuregulin subfamilies contain an immunoglobulin-like domain (Ben-Baruch and Yarden, 1994; Peles and Yarden, 1993; Ho et al., 1995) mediating neuregulin interactions with cell surface glycoproteins, with resultant concentration and specific localization of the factor (Sudhalter et al., 1996). Splice variants in the glial growth factor and mesenchymal neuregulin subfamilies also may contain serine and threonine-rich spacer domains which serve as the site of o- and n-linked glycosylation (Wen et al., 1994; Carroll et al., 1997); this glycosylation is non-essential for biologic activity and the precise function(s) of this region is as yet unknown.
Neuregulins may be synthesized as either transmembrane precursors or directly secretable forms. This distinction depends upon the juxtamembrane domain, which is immediately C terminal to the EGF-like domain. Four juxtamembrane domains, designated 1 to 4, have been identified in the rat. In this regard, the ‘3’ juxtamembrane domain is notable in that it, unlike other juxtamembrane domains, contains a termination codon, thus leading to truncation of the factor and synthesis in a directly secretable form. In all other neuregulin isoforms, the juxtamembrane domain is followed by a transmembrane domain which anchors the factor in the cell membrane and is itself coupled to one of three possible cytoplasmic domains (de
Adler Benjamin Aaron
Gucker Stephen
Kunz Gary
UAB Research Foundation
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