Ueli Suter

Two conserved domains in the NGF propeptide are necessary and sufficient for the biosynthesis of correctly processed and biologically active NGF.

The three members of the neurotrophin family (NGF, BDNF and NT‐3) are synthesized as large precursor proteins which undergo proteolytic processing to yield biologically active, mature neurotrophic factors. We have used in vitro mutagenesis to examine the pro‐region in the NGF precursor protein as a first step towards a general understanding of the role of propeptides in the biosynthesis of neurotrophins. Our results demonstrate that only two small domains within the NGF propeptide are required for the expression and secretion of properly processed and biologically active, recombinant mouse NGF in COS‐7 cells. Domain I plays an important role in the expression of active NGF while domain II is involved in proteolytic processing. Both domains are partially conserved between the propeptides of NGF proteins isolated from different species as well as BDNF and NT‐3.

Progress in the molecular understanding of hereditary peripheral neuropathies reveals new insights into the biology of the peripheral nervous system.

Since the first description of the autosomal dominant inherited peripheral neuropathy Charcot-Marie-Tooth (CMT) disease over a century ago, there has been considerable disagreement, based on morphological abnormalities of both the axons of peripheral nerves and their surrounding Schwann cells, as to whether this disorder is due primarily to an autonomous Schwann cell defect or an autonomous neuronal defect. Recently, the Schwann cell protein peripheral myelin protein 22 (PMP-22) has been implicated in the molecular pathogenesis of hereditary peripheral neuropathies in mice and humans. Reinterpretations of morphological studies of the diseased nerves in light of these findings strongly suggest that Schwann cells have a much more pronounced influence on their ensheathed axons than previously anticipated.

The Regulated Secretion and Vectorial Targeting of Neurotrophins in Neuroendocrine and Epithelial Cells

The varied roles that neurotrophins play in the development and activity-dependent plasticity of the nervous system presumably require that the sites and quantity of neurotrophin release be precisely regulated. As a step toward understanding how different neurotrophins are sorted and secreted by neurons, we expressed nerve growth factor (NGF), brain-derived neurotrophic factor, and neurotrophin-3 in cell lines used as models for neuronal protein sorting. All three neurotrophins were secreted by a regulated pathway in transfected AtT-20 and PC12 neuroendocrine cells, with a 3-6-fold increase in neurotrophin release in response to 8-bromo-cAMP or depolarization, respectively. To determine if the propeptide directs the intracellular sorting of mature NGF, we examined mutants in which regions spanning the propeptide were deleted. These mutants underwent regulated release in every case in which expression could be detected. Similarly, NGF sorting was not significantly altered by mutations which specifically abolished N-glycosylation or proteolytic processing sites within the NGF precursor. Finally, we found that all three neurotrophins were secreted 65-75% basolaterally by polarized Madin-Darby canine kidney epithelial cells. These findings suggest that the determinants of regulated neurotrophin secretion lie within the mature neurotrophin moiety and that NGF, brain-derived neurotrophic factor, and neurotrophin-3 are likely to be sorted similarly and released in a regulated manner by neurons.

The genetics of myelin

Myelin formation and maintenance requires complex interactions between neurons and glia, and between the integral protein and lipid components of the myelin sheath. Many of the underlying mechanisms may be examined by studying the perturbations caused by spontaneous and targeted mutations in myelin protein genes. This review summarizes the progress in our understanding of these mutations with an emphasis on integrating the recent advances in the genetics of myelin into a more generalized view of myelin organization and function.

The in vivo expression of type B CD23 mRNA in B-chronic lymphocytic leukemic cells is associated with an abnormally low CD23 upregulation by IL-4 : comparison with their normal cellular counterparts

We recently reported that the sera of chronic lymphocytic leukemia (CLL) patients contained 3-500 times more soluble CD23 (or IgE-BF) than the sera of patients with other lymphoproliferative diseases or normal individuals and that their B cells (B-CLLs) overexpressed CD23 Ag. In the present report, we extended these studies and showed that CD5+ B cells from all CLL patients (n = 15) co-express CD23 Ag. We next identified two additional major differences between B-CLLs and normal adult B cells. First, in contrast to normal adult B cells which exclusively express type A CD23 mRNA, freshly isolated B-CLLs expressed both type B and type A CD23 mRNA. Second, although IL-4 is a potent inducer of type B CD23 mRNA on normal B cells, an optimal concentration of IL-4 infranormally upregulated CD23 on highly purified B-CLLs both at the protein and at the molecular levels. However, co-stimulation of CLL PBMC with phytohemagglutinin (PHA) and IL-4 strongly upregulated CD23 on B-CLLs, reconstituting the high level of CD23 expression observed in vivo. We next attempted to relate B-CLLs to the CD5+ B cell subpopulations present in peripheral blood mononuclear cells (PBMC, n = 3), cord blood mononuclear cells (CBMC, n = 6) and tonsillar lymphocytes (TONS, n = 3) by analysing their co-expression of CD20, CD5 and CD23 Ag and their phenotypic regulation by IL-4. Our results indicated that B-CLLs presented some features in common with the CD23+ umbilical cord blood B cells in as much as, like in B-CLLs; (i) all CD23+ cord blood cells co-expressed CD5 Ag, (ii) freshly isolated CBMC expressed both type A and type B CD23 mRNA, and finally (iii) these cells weakly re-expressed CD23 Ag upon IL-4 stimulation as compared to adult PBMC.

Mutation of tryptophan-21 in mouse nerve growth factor (NGF) affects binding to the fast NGF receptor but not induction of neurites on PC12 cells

By using in vitro DNA mutagenesis, we replaced the tryptophan residue at position 21 in mouse nerve growth factor (NGF) with either phenylalanine, leucine or serine. Yield, biological activity, immunological reactivity and receptor binding of the recombinant proteins were determined. All three mutants were produced at considerably lower yields than wild-type NGF, with the serine mutant being undetectable. The results of competitive binding assays show that tryptophan-21 is involved in recognition of the fast NGF receptor of PC12 cells. However, specific biological activity of NGF is not altered by the replacement of tryptophan-21. Our results therefore suggest that biological activity of NGF is not directly coupled to binding to the fast NGF receptor.

Chapter 14: Isolation of transcriptionally regulated sequences associated with neuronal and non-neuronal cell interactions

Publisher Summary This chapter describes the use of differential hybridization to identify DNA sequences encoding proteins associated with the regeneration of injured rat sciatic nerve. In addition, it describes the characterization of one of the isolated proteins, which is involved in neuronal/non-neuronal cell interactions, to illustrate the power of this approach. There are two general approaches toward identifying the proteins involved in nerve regeneration. The first of these is to study the expression and localization, during nerve regeneration of proteins that are identified in other systems. Another approach is to identify proteins, which change their rates of protein synthesis during nerve regeneration. This leads to the identification of several proteins whose role in nerve regeneration is not yet identified. The chapter describes an extension of the second approach, and it takes advantage of techniques that use nucleic acids instead of proteins to identify sequences whose expression is regulated during nerve regeneration. This procedure successfully identified a number of known and unknown cDNA sequences, the expression of which were either induced or repressed after nerve injury. The association of this protein with myelin, as well as the growth-arrested state of cells suggests an important role for this protein in neuron-glia interactions.

Chapter 32 The molecular basis of the neuropathies of mouse and human

Publisher Summary This chapter discusses the discovery of a gene for peripheral myelin protein 22 (PMP22) and its involvement in trembler neurological mutants. The cDNA for PMP22 was found in three independent library screens, two comparing the cDNAs from libraries made from normal rat sciatic nerve and from sciatic nerve after injury and the other comparing the cDNAs of quiescent and proliferating fibroblasts. In the latter instance, the gene was referred to as the growth arrest specific gene-3 because of its potential involvement in controlling growth in these cells. The cDNA encodes a small, extremely hydrophobic protein of 160 amino acid residues, with four putative membrane spanning domains and one asparagine-linked glycosylation site. Expression of PMP22 correlates with myelin formation and myelin-specific gene expression during sciatic nerve development and its down-regulation after nerve injury follows that of the myelin protein Po. In situ hybridization confirmed that PMP22 mRNA is synthesized by Schwann cells. PMP22 mRNA expression is clearly regulated by axon contact and is found mainly in the peripheral nervous system (PNS). The chapter discusses PMP22 and the inherited peripheral neuropathies in human, along with human peripheral neuropathies associated with mutations in other PNS myelin genes.