Differential cyclin D1 requirements of proliferating Schwann cells during development and after injury.

Neurons regulate Schwann cell proliferation, but little is known about the molecular basis of this interaction. We have examined the possibility that cyclin D1 is a key regulator of the cell cycle in Schwann cells. Myelinating Schwann cells express cyclin D1 in the perinuclear region, but after axons are severed, cyclin D1 is strongly upregulated in parallel with Schwann cell proliferation and translocates into Schwann cell nuclei. During development, cyclin D1 expression is confined to the perinuclear region of proliferating Schwann cells and the analysis of cyclin D1-null mice indicates that cyclin D1 is not required for this type of Schwann cell proliferation. As in the adult, injury to immature peripheral nerves leads to translocation of cyclin D1 to Schwann cell nuclei and injury-induced proliferation is impaired in both immature and mature cyclin D1-deficient Schwann cells. Thus, our data indicate that the molecular mechanisms regulating proliferation of Schwann cells during development or activated by axonal damage are fundamentally different.

Modification of representational difference analysis applied to the isolation of forskolin-regulated genes from Schwann cells.

Many aspects of the response of Schwann cells to axonal cues can be induced in vitro by the adenylyl cyclase activator forskolin, yet the role of cAMP signaling in regulating Schwann cell differentiation remains unclear. To define better the relationship between cAMP signaling and Schwann cell differentiation, we used a modification of cDNA representational difference analysis (RDA) that permits the analysis of small amounts of mRNA and identified additional genes that are differentially expressed by forskolin-treated and untreated Schwann cells. The genes that we have identified, including MKP3, a regulator of ERK signaling, and the sphingosine-1-phosphate receptor edg3/lp(B3), may play important roles in mediating Schwann cell differentiation.

Proteolipid protein mRNA stability is regulated by axonal contact in the rodent peripheral nervous system.

Proteolipid protein (PLP) and its alternatively spliced isoform, DM20, are the main intrinsic membrane proteins of compact myelin in the CNS. PLP and DM20 are also expressed by Schwann cells, the myelin-forming cells in the PNS, and are necessary for normal PNS function in humans. We have investigated the expression of PLP in the PNS by examining transgenic mice expressing a LacZ transgene under the control of the PLP promoter. In these animals, myelinating Schwann cells expressed beta-galactosidase more prominently than nonmyelinating Schwann cells. PLP/DM20 mRNA levels, but not those of LacZ mRNA, increased during sciatic nerve development and decreased after axotomy, with resultant Wallerian degeneration. PLP/DM20 transcription rates, in nuclear run off experiments, however, did not increase in developing rat sciatic nerve despite robust increases in PLP/DM20 mRNA levels during the same period. In RNAse protection studies, PLP mRNA levels fell to undetectable levels following nerve transection whereas levels of DM20 were essentially unchanged despite both being transcribed from the same promoter. Finally, cotransfection studies demonstrated that PLP-GFP, but not DM20-GFP mRNA is down-regulated in Schwann cells cultured in the absence of forskolin. Taken together these data demonstrate that steady state levels of PLP mRNA are regulated at a posttranscriptional level in Schwann cells, and that this regulation is mediated by Schwann cell-axonal contact. Since the difference between these two mRNAs is a 105-bp sequence in PLP and not in DM20, this sequence is likely to play a role in the regulation of PLP mRNA.

Analysis of the human MBP promoter in primary cultures of oligodendrocytes: positive and negative cis-acting elements in the proximal MBP promoter mediate oligodendrocyte-specific expression of MBP.

Since the regulation of myelin basic protein expression depends primarily on the initiation of transcription, we analyzed the 5' flanking region of the human myelin basic protein gene in transient transfection studies in primary cultures of developing oligodendrocytes. We demonstrated that 149 base pairs 5' of the initiation of transcription was sufficient to direct oligodendrocyte-specific expression of myelin basic protein. The capsite of the fusion transcript was identical with that of the endogenous myelin basic protein transcript, and chloramphenicol acetyl transferase reporter gene expression was restricted to oligodendrocytes in these cultures. Within this 149 base pair region, one distal, negative cis-acting segment, containing a consensus nuclear factor I site, and one proximal, positive cis-acting segment were identified. The distal segment behaved more negatively in Cos-7 cells than in oligodendrocytes, reducing expression to background levels. Furthermore, these functionally important cis-acting segments bound oligodendrocyte nuclear proteins in a pattern differing from other cells, including Cos-7 cells. Interestingly, the distal segment increased heterologous SV40 promoter activity in oligodendrocytes but had no effect on the SV40 promoter in Cos-7 cells. We conclude that the functionally negative distal segment may mediate oligodendrocyte-specific expression of MBP by restricting its expression in other cells. These experiments strongly support using primary cultures of oligodendrocytes for analyzing the myelin-specific promoters.

Genetic separation of hypoxanthine and guanine-xanthine phosphoribosyltransferase activities by deletion mutations in Salmonella typhimurium.

Certain proAB deletion mutants of Salmonella typhimurium were found to be simultaneously deleted in a gene required for the utilization of guanine and xanthine (designated gxu). These mutants were resistant to 8-azaguanine and when carrying an additional pur mutation were unable to use guanine or xanthine as a purine source. The defect was correlated with deficiencies in the uptake and phosphoribosyltransferase activities for guanine and xanthine. Hypoxanthine and adenine activities were unaltered. The deficiency was restored to normal by transduction to pro(+) and in F' merodiploids.

Genetic eparation of the inosinic acid cyclohydrolase-transformylase complex of Salmonella typhimurium.

Genetic and enzymatic analyses were made with the purH mutants of Salmonella typhimurium. These mutants are purine auxotrophs which are deficient in the conversion of phosphoribosyl-aminoimidazolecarboxamide (AIC) to inosine-5'-monophosphate (IMP). Two steps are required for this process: phosphoribosyl-AIC transformylase (EC 2.1.2.3) and IMP cyclohydrolase (EC 3.5.4.10). Genetic analysis identified two complementation groups, I and II, and a third group of noncomplementing mutants (I-II). Mutations in gene I lead to complete loss of transformylase activity and no loss of cyclohydrolase activity if the mutation is of the missense type, but partial loss if it is of the chain-terminating type (nonsense or frameshift). Gene II mutants are all of the missense type and show normal transformylase activity but no cyclohydrolase activity. The noncomplementing mutants (I-II) are all of the chain-terminating type and are completely deficient in both activities. The results are explained and discussed in terms of subunit interactions of a stable enzyme complex.