Structure of the Mouse Myelin P 2 Protein Gene

Myelin P 2 protein is a small (14,800 Da) protein found in peripheral and central nervous system myelin. To investigate the regulation of expression of this myelin protein, a mouse genomic library was screened with a rabbit P 2 cDNA (pSN2.2–2), and a single positive phage clone containing a 20-kb insert was obtained. This insert contained a single internal Sail restriction site and several Eco RI sites. The Eco RI fragments from this insert were subcloned into Bluescript. The rabbit P 2 cDNA (pSN2.2–2) hybridized with a 4-kb Eco RI fragment, and this 4-kb fragment was then sequenced after the creation of nested deletions. The mouse gene contained four exons: exon 1 coded for amino acids 1–24, exon 2 for amino acids 24–81, exon 3 for amino acids 82–115, and exon 4 for amino acids 116–131. The three introns were 1.2 kb, 150 bp, and 1.3 kb in length. Primer extension analysis revealed two transcription start sites at +1 and +47, consistent with the presence of two P 2 mRNAs, with the larger transcript appearing more abundant. The amino acid sequences predicted from the mouse DNA indicate that the mouse protein differs from the rabbit protein at 16 different positions, with most of the differences located in exon 3. When the gene structure of fatty acid binding protein (FABP) genes were compared, the P 2 gene had the same overall structure as others in the FABP family, suggesting a common ancestral gene for members of this family. The 5′-flanking region contains candidate TATA and CAAT sequences, as well as two AP-l binding sites that may be important in modulation of the expression of this gene.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65134/1/j.1471-4159.1991.tb02101.x.pd

Partial Structure and Mapping of the Human Myelin P 2 Protein Gene

The myelin P 2 protein, a 14,800-Da cytosolic protein found primarily in peripheral nerves, belongs to a family of fatty acid binding proteins. Although it is similar in amino acid sequence and tertiary structure to fatty acid binding proteins found in the liver, adipocytes, and intestine, its expression is limited to the nervous system. It is detected only in myelin-producing cells of the central and peripheral nervous systems, i.e., the oligodendrocytes and Schwann cells, respectively. As part of a program to understand the regulation of expression of this gene, to determine its function in myelin-producing cells, and to study its role in peripheral nerve disease, we have isolated and characterized overlapping human genomic clones encoding the P 2 protein. We report here on the partial structure of this gene, and on its localization within the genome. By using a panel of human-hamster somatic cell hybrids and by in situ hybridization, we have mapped the human P 2 gene to segment q21 on the long arm of chromosome 8. This result identifies the myelin P 2 gene as a candidate gene for autosomal recessive Charcot-Marie-Tooth disease type 4A.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65484/1/j.1471-4159.1994.63062010.x.pd

Suitability of external controls for drug evaluation in Duchenne muscular dystrophy

OBJECTIVE: To evaluate the suitability of real-world data (RWD) and natural history data (NHD) for use as external controls in drug evaluations for ambulatory Duchenne muscular dystrophy (DMD). METHODS: The consistency of changes in the 6-minute walk distance (Δ6MWD) was assessed across multiple clinical trial placebo arms and sources of NHD/RWD. Six placebo arms reporting 48-week Δ6MWD were identified via literature review and represented 4 sets of inclusion/exclusion criteria (n = 383 patients in total). Five sources of RWD/NHD were contributed by Universitaire Ziekenhuizen Leuven, DMD Italian Group, The Cooperative International Neuromuscular Research Group, ImagingDMD, and the PRO-DMD-01 study (n = 430 patients, in total). Mean Δ6MWD was compared between each placebo arm and RWD/NHD source after subjecting the latter to the inclusion/exclusion criteria of the trial for baseline age, ambulatory function, and steroid use. Baseline covariate adjustment was investigated in a subset of patients with available data. RESULTS: Analyses included ∼1,200 patient-years of follow-up. Differences in mean Δ6MWD between trial placebo arms and RWD/NHD cohorts ranged from -19.4 m (i.e., better outcomes in RWD/NHD) to 19.5 m (i.e., worse outcomes in RWD/NHD) and were not statistically significant before or after covariate adjustment. CONCLUSIONS: We found that Δ6MWD was consistent between placebo arms and RWD/NHD subjected to equivalent inclusion/exclusion criteria. No evidence for systematic bias was detected. These findings are encouraging for the use of RWD/NHD to augment, or possibly replace, placebo controls in DMD trials. Multi-institution collaboration through the Collaborative Trajectory Analysis Project rendered this study feasible

The Simian Virus 40 Large T Antigen Does Not Inhibit Translation of the 14-kDa Myelin Basic Protein mRNA in Reticulocyte Lysates or in Transfected Cells

Viral T antigens are transcription factors that have been suspected of inhibiting expression of the myelin basic protein (MBP) mRNA at the translational level in vitro and in vivo. The effect of simian virus 40 (SV40) large T antigen (T-ag) was examined on the translation of the 14-kDa MBP mRNA in reticulocyte lysates and on MBP expression after transfection into cells that express SV40 T-ag. SV40 T-ag did not inhibit translation of 14-kDa MBP cRNAs in cell-free translations even at 30 µ M (∼600 µg/ml) T-ag. Permanent transfection of COS-1 cells (which endogenously express SV40 T-ag) with the 14-kDa MBP cDNA resulted in the expression of the 14-kDa MBP as determined by western blot analysis. Permanent transfection of N20.1 cells, an oligodendrocyte line immortalized with a temperature-sensitive SV40 T-ag, with the 14-kDa MBP cDNA construct also resulted in the expression of the 14-kDa MBP under conditions in which the cells expressed functional SV40 T-ag. These results indicate that SV40 T-ag does not prevent expression of the MBP gene at the translational level and that in those immortalized oligodendrocyte lines that express MBP mRNA, but not MBP protein, some factor other than the SV40 large T-ag is responsible for the posttranscriptional regulation.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65286/1/j.1471-4159.1995.64020928.x.pd

Neuregulin Signaling through a PI3K/Akt/Bad Pathway in Schwann Cell Survival

β-Neuregulin (βNRG) is a potent Schwann cell survival factor that binds to and activates a heterodimeric ErbB2/ErbB3 receptor complex. We found that NRG receptor signaling rapidly activated phosphoinositide 3-kinase (PI3K) in serum-starved Schwann cells, while PI3K inhibitors markedly exacerbated apoptosis and completely blocked NRG-mediated rescue. NRG also rapidly signaled the phosphorylation of mitogen-activated protein kinase (MAPK) and the serine/threonine kinase Akt. The activation of Akt and MAPK in parallel pathways downstream from PI3K resulted in the phosphorylation of Bad at different serine residues. PI3K inhibitors that blocked NRG-mediated rescue also blocked the phosphorylation of Akt, MAPK, and Bad. However, selective inhibition of MEK-dependent Bad phosphorylation downstream from PI3K had no effect on NRG-mediated survival. Conversely, ectopic expression of wild-type Akt not only enhanced Bad phosphorylation but also enhanced autocrine- and NRG-mediated Schwann cell survival. Taken together, these results demonstrate that NRG receptor signaling through a PI3K/Akt/Bad pathway functions in Schwann cell survival

Characterization of Insulin-Like Growth Factor-I and Its Receptor and Binding Proteins in Transected Nerves and Cultured Schwann Cells

The insulin-like growth factors (IGFs) are trophic factors whose growth-promoting actions are mediated via the IGF-I receptor and modulated by six IGF binding proteins (IGFBPs). In this study, we observed increased transcripts of both IGF-I and IGF-I receptor after rat sciatic nerve transection. Schwann cells (SCs) were the main source of IGF-I and IGFBP-5 immunoreactivity until 7 days after nerve transection, when invading macrophages in the distal nerve stumps were strongly IGF-I positive. In vitro, IGF-I promoted SC mitogenesis. Northern analysis revealed that SCs expressed IGF-I receptor and IGFBP-5. IGF-I treatment increased the intensity of IGFBP-5 without affecting gene expression. Des(1–3)IGF-I, an IGF-I analogue with low affinity for IGFBP, had no such effect. Incubation of recombinant human IGFBP-5 with SC conditioned media revealed IGF-I protection of IGFBP-5 from proteolysis, implying the presence of an IGFBP-5 protease in SC conditioned media. Collectively, these data support the concept that, in response to nerve injury, invading macrophages produce IGF-I and SC express the IGF-I receptor, to facilitate regeneration. This regenerative process may be augmented further by the ability of SC to secrete IGFBPs, which in turn may increase local IGF-I bioavailability.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65391/1/j.1471-4159.1996.66020525.x.pd