Biomedical subjects

James I Morgan

Publications and source records attributed to James I Morgan.

11 recordsLinked to original sources

PubMed2006

Cbln1 is essential for interaction-dependent secretion of Cbln3.

Cbln1 and the orphan glutamate receptor GluRdelta2 are pre- and postsynaptic components, respectively, of a novel transneuronal signaling pathway regulating synapse structure and function. We show here that Cbln1 is secreted from cerebellar granule cells in complex with a related protein, Cbln3. However, cbln1- and cbln3-null mice have different phenotypes and cbln1 cbln3 double-null mice have deficits identical to those of cbln1 knockout mice. The basis for these discordant phenotypes is that Cbln1 and Cbln3 reciprocally regulate each other's degradation and secretion such that cbln1-null mice lack both Cbln1 and Cbln3, whereas cbln3-null mice lack Cbln3 but have an approximately sixfold increase in Cbln1. Unlike Cbln1, Cbln3 cannot form homomeric complexes and is secreted only when bound to Cbln1. Structural modeling and mutation analysis reveal that, by constituting a steric clash that is masked upon binding Cbln1 in a "hide-and-run" mechanism of endoplasmic reticulum retention, a single arginine confers the unique properties of Cbln3.

Amino Acid Sequence↗

PubMed2006

The carboxypeptidase-like substrate-binding site in Nna1 is essential for the rescue of the Purkinje cell degeneration (pcd) phenotype.

The Purkinje cell degeneration (pcd) phenotype is characterized by adult onset neurodegeneration resulting from mutations in Nna1, a gene encoding an intracellular protein with a putative metallocarboxypeptidase domain. As Nna1 is also induced in axotomized motor neurons, the elucidation of its function can shed light on previously unsuspected mechanisms common to degenerative and regenerative responses. Structural modeling revealed that Nna1 and three related gene products constitute a new subfamily of metallocarboxypeptidases with a distinctive substrate-binding site. To test whether the metallocarboxypeptidase domain is functionally essential, transgenic mice were generated that expressed Nna1 or a substrate-binding site mutant of Nna1 selectively in Purkinje cells using the L7/pcp2 promoter. When bred onto a homozygous pcd(3J) background, wild type but not mutant Nna1 rescued ataxic behavior and Purkinje cell loss. Therefore, loss of Nna1 in Purkinje cells leads directly to their degeneration and Nna1's carboxypeptidase domain is essential for survival of these neurons.

Amino Acid Sequence↗

PubMed2006

The Purkinje cell degeneration (pcd) mouse: an unexpected molecular link between neuronal degeneration and regeneration.

The spontaneous autosomal recessive mouse mutation, Purkinje cell degeneration (pcd), was first identified through its ataxic behavior. Since its discovery in the 1970s, the strain has undergone extensive investigation, although another quarter century elapsed until the mutant gene (agtpbp1 a.k.a. Nna1) underlying the pcd phenotype was identified. As Nna1 was initially discovered as a gene induced in motor neurons following axotomy the finding that its loss leads to selective neuronal degeneration points to a novel and unexpected common molecular mechanism contributing to the apparently opposing processes of degeneration and regeneration. The elucidation of this mechanism may of course have significant implications for an array of neurological disorders. Here we will first review the principle features of the pcd phenotype and then discuss the functional implications of more recent findings emanating from the characterization of Nna1, the protein that is lost in pcd. We also provide new data on the genetic dissection of the cell death pathways operative in pcd(3J) mice, proving that granule cell death and Purkinje cell death in these mice have distinct molecular bases. We also provide new information on the structure of mouse Nna1 as well as Nna1 protein levels in pcd(3J) mice.

Animals↗

PubMed2006

The influence of phosphorylation on the activity and structure of the neuronal IQ motif protein, PEP-19.

PEP-19 is a 7.6 kDa neuronally expressed polypeptide that contains a single calmodulin-binding IQ motif. The calmodulin-binding activity of several neuronal IQ motif proteins is regulated by phosphorylation of a conserved serine. We propose that the serine residue within the IQ motif of PEP-19 is phosphorylated, and that phosphorylation modifies the activity of PEP-19. Camstatin, a functionally active 25-residue fragment of PEP-19's IQ motif, binds calmodulin and inhibits neuronal nitric oxide synthase. A truncated camstatin-in which the IQ motif serine is the only phosphorylatable residue-was screened against 42 different kinases. Truncated camstatin is selectively phosphorylated by four isoforms of protein kinase C. Furthermore, treatment of full-length PEP-19 with PKCgamma catalyzes phosphorylation of the same serine residue. Fluorescent anisotropy shows that phosphorylation of camstatin inhibits its binding to calmodulin. NMR solution structures indicate that both camstatin and phospho-camstatin exist in similar dynamic turn-like conformations. This suggests that camstatin's greater affinity for calmodulin is due not to a change in the conformation of the phospho-peptide, but rather, to a disruption of hydrophobic interactions between phospho-camstatin and calmodulin caused by the presence of the hydrophilic phosphate group. The H(alpha) chemical shifts and the circular dichroism spectra of the camstatins are consistent with those of "nascent helices". We submit that PEP-19 is a PKC substrate, and that the phosphorylation state of PEP-19 may play a role in the modulation of calmodulin-dependent signaling.

Amino Acid Motifs↗

PubMed2005

Cbln1 is essential for synaptic integrity and plasticity in the cerebellum.

Cbln1 is a cerebellum-specific protein of previously unknown function that is structurally related to the C1q and tumor necrosis factor families of proteins. We show that Cbln1 is a glycoprotein secreted from cerebellar granule cells that is essential for three processes in cerebellar Purkinje cells: the matching and maintenance of pre- and postsynaptic elements at parallel fiber-Purkinje cell synapses, the establishment of the proper pattern of climbing fiber-Purkinje cell innervation, and induction of long-term depression at parallel fiber-Purkinje cell synapses. Notably, the phenotype of cbln1-null mice mimics loss-of-function mutations in the orphan glutamate receptor, GluR delta2, a gene selectively expressed in Purkinje neurons. Therefore, Cbln1 secreted from presynaptic granule cells may be a component of a transneuronal signaling pathway that controls synaptic structure and plasticity.

Age Factors↗

PubMed2005

The structure and proteolytic processing of Cbln1 complexes.

The hexadecapeptide cerebellin is present in the brains of many vertebrate species and is derived from a larger protein, Cbln1 (cerebellin 1 precursor protein). Although cerebellin has features of a neuropeptide, Cbln1 belongs to the C1q/tumor necrosis factor superfamily of secreted proteins, suggesting that it is the biologically active molecule and the proteolytic events that generate cerebellin serve another function. Therefore, we assessed whether Cbln1 undergoes proteolytic processing and determined what consequences the cleavage events necessary to produce cerebellin have on the structure of Cbln1. Substantial degradation of Cbln1 was evident in the synaptic compartment of cerebellum and lysates of cultured cerebellar neurons and cells transfected with Cbln1 expression vectors. However, only uncleaved Cbln1 containing the cerebellin motif was released and assembled into hexameric complexes. Using yeast two hybrid and mammalian expression systems we show that the cleavages required to produce cerebellin influence the subunit stoichiometry of Cbln1 complexes. Cleavage at the N-terminus of the cerebellin sequence in Cbln1 yields trimeric complexes by separating the trimer-mediating C-terminal C1q domain from conserved N-terminal cysteine residues that mediate higher order oligomerization. Cleavage at the C-terminus of the cerebellin motif disrupts the C1q domain and abolishes subunit interactions. Functional implications of these data are discussed.

Amino Acid Sequence↗

PubMed2004

Expression of Bcl-2 extends the survival of olfactory receptor neurons in the absence of an olfactory bulb.

In the olfactory neuroepithelium, the number of olfactory receptor neurons (ORNs) is maintained at a relatively constant level by a precise balance between the elimination of mature receptors and proliferation of their precursors. However, little is known of the mechanisms that couple alterations in receptor death rates to changes in precursor proliferation. To investigate this relationship, we generated a line of mice expressing Bcl-2, a protein with anti-apoptotic properties, in mature olfactory receptor neurons using the Olfactory Marker Protein (OMP) promoter. OMP-bcl-2 transgenic mice showed selective expression of Bcl-2 in mature sensory neurons of the olfactory neuroepithelium (ONE) and vomeronasal organ. Olfactory bulbectomy (OBX) resulted in the death of mature receptor neurons followed by the sustained proliferation of their precursors in wild-type and OMP-bcl-2 transgenic mice. The persistently enhanced proliferation of olfactory neuroblasts that followed bulbectomy was indistinguishable between transgenic and non-transgenic mice. However, receptor neurons that were subsequently born in the absence of the bulb had longer life spans in OMP-bcl-2 mice. The increased proliferation of neuroblasts and extended life spans combined to restore near normal numbers of olfactory receptors in bulbectomized OMP-bcl-2 mice. A model is proposed to explain the dissociation of death and proliferation in OMP-bcl-2 transgenic mice.

Animals↗

PubMed2004

Identification of candidate Purkinje cell-specific markers by gene expression profiling in wild-type and pcd(3J) mice.

The identification of mRNAs that have restricted expression patterns in the brain represents powerful tools with which to characterize and manipulate the nervous system. Here, we describe a strategy using microarray technology (Affymetrix Mouse Genome 430 2.0 Arrays) to identify mRNA transcripts that are candidate markers of cerebellar Purkinje neurons. Initially, gene expression profiles were compared between cerebella of 4-month-old Purkinje cell degeneration (pcd(3J)) mice, in which most Purkinje cells had already degenerated and wild-type littermates with a normal complement of Purkinje neurons. Of 14,563 probe sets expressed in wild-type cerebellum, 797 showed a significant (p<0.0001) reduction in pcd(3J) mice. These probes could represent transcripts with varying levels of specificity for Purkinje cells as well as transcripts in other cell types that decline as a secondary consequence of Purkinje cell loss. Ranking of the probe signals revealed that well-known Purkinje cell-specific transcripts such as calbindin and L7/pcp2 clustered in a group that was <33% of wild-type levels. Therefore, to identify potentially new Purkinje cell-specific transcripts that cluster with the known markers, more stringent selection criteria were applied (<33% of wild-type signal and p<0.0001). With these criteria, 55 independent transcripts were identified of which 33 were annotated genes and 22 were ESTs and RIKEN cDNAs. A literature search revealed that 25 of the 33 annotated genes were expressed in Purkinje cells, with no data being available on the other 8. Thus, the additional 8 annotated and 22 un-annotated genes are clustered with many genes expressed in Purkinje cells making them candidate markers. To confirm the microarray data, eight representative annotated genes were selected including five reported to be in Purkinje neurons and three for which no data was available. Semi-quantitative RT-PCR demonstrated reduced expression of all eight transcripts in cerebella from pcd(3J) mice. The promoters of genes expressed selectively in subsets of neurons can be used to direct heterologous gene expression in transgenic mice and the more restricted the expression pattern the greater their utility. Therefore, microarray analysis was used to assess expression levels of all 55 transcripts in cerebral cortex, striatum, substantia nigra and ventral tegmental area. This permitted the identification of a set of genes whose promoters might have utility for selectively targeting gene expression to cerebellar Purkinje cells.

Animals↗

PubMed2003

Mouse embryos cloned from brain tumors.

Cancer cells escape from growth control by accumulating genetic and epigenetic alterations. In rare instances, epigenetic changes alone are oncogenic. Furthermore, agents that modify DNA methylation or chromatin structure can restore a normal phenotype to cells harboring oncogenic mutations. However, it is unclear to what extent epigenetic reprogramming can reverse oncogenesis. Using somatic nuclear transfer, we show that medulloblastomas arising in Ptc1+/- mice can direct preimplantation development. Additionally, blastocysts derived from medulloblastoma nuclei form postimplantation embryos with typical cell layers. Thus, tumor cells can be epigenetically reprogrammed into normal cell types. This approach could lead to a general strategy for assessing genetic and epigenetic contributions to tumorigenesis.

Animals↗

PubMed2002

Purkinje cell degeneration (pcd) phenotypes caused by mutations in the axotomy-induced gene, Nna1.

The classical recessive mouse mutant, Purkinje cell degeneration (pcd), exhibits adult-onset degeneration of cerebellar Purkinje neurons, retinal photoreceptors, olfactory bulb mitral neurons, and selected thalamic neurons, and has defective spermatogenesis. Here we identify Nna1 as the gene mutated in the original pcd and two additional pcd alleles (pcd2J and pcd3J). Nna1 encodes a putative nuclear protein containing a zinc carboxypeptidase domain initially identified by its induction in spinal motor neurons during axonal regeneration. The present study suggests an unexpected molecular link between neuronal degeneration and regeneration, and its results have potential implications for neurodegenerative diseases and male infertility.

Alleles↗

PubMed2002

Differential post-transcriptional regulation of p21WAF1/Cip1 levels in the developing nervous system following gamma-irradiation.

Radiation-induced death in the developing brain is p53-dependent. However, genetic studies indicate that the signalling pathways that couple irradiation to p53 expression can vary between different developing neural populations [Herzog et al. (1998) Science, 280, 1089-1091]. Here we establish that signalling downstream of p53 also exhibits brain region-specific differences that are associated with the relative vulnerability of some cell populations to radiation-induced killing in the mouse. Following gamma-irradiation, p53 and p21WAF1/cip1, but not Bax, protein levels increased in the developing cerebellum. In contrast, neither p21WAF1/cip1 nor Bax protein levels were elevated in the retina following irradiation, despite increased p53 expression. In the retina, p53 expression was associated with cells destined to die, whereas in the cerebellum, p53 was expressed in both radiation-sensitive and radiation-resistant neuroblasts of the external granule cell layer. Although p21WAF1/cip1 mRNA was expressed in all p53-positive neuroblasts after irradiation, p21WAF1/cip1 protein was only detected in radiation-resistant neuroblasts of the cerebellum. Thus, p21WAF1/cip1 was subject to post-transcriptional regulation with p21WAF1/cip1 protein only accumulating in cells destined to survive irradiation. Nevertheless, p21WAF1/cip1 function was not essential for radiation resistance, as postmitotic neuroblasts in the external granule cell layer were spared in p21WAF1/cip1 knockout mice.

Animals↗