The myelin proteolipid protein gene modulates apoptosis in neural and non-neural tissues.

Mutations of the myelin proteolipid protein gene (Plp) are associated with excessive programmed cell death (PCD) of oligodendrocytes. We show for the first time that PLP is a molecule ubiquitously expressed in non-neural tissues during normal development, and that the level of native PLP modulates the level of PCD. We analyze three non-neural tissues, and show that native PLP is expressed in trophoblasts, spermatogonia, and cells of interdigital webbing. The non-neural cells that express high levels of native PLP also undergo PCD. The level of PLP expression modulates the level of PCD because mice that overexpress native PLP have increased PCD and mice deficient in PLP have decreased PCD. We show that overexpression of native PLP causes a dramatic acidification of extracellular fluid that, in turn, causes increased PCD. These studies show that the level of native PLP modulates the amount of PCD during normal development via a pH-dependent mechanism.

Analyses of proteolipid protein mutants show levels of proteolipid protein regulate oligodendrocyte number and cell death in vitro and in vivo.

Previous tissue culture studies indicate that the level of native proteolipid protein (PLP) or mutated PLP regulates the number of oligodendrocytes (Olgs). The regulation of Olg number is most likely due to toxicity of over-expression of native PLP or mis-sense mutations of PLP. We tested, in vivo and in vitro, the hypothesis that the absence of native PLP or reduced amounts of mutated PLP leads to an increase in numbers of Olgs and a corresponding decrease in the number of apoptotic Olgs. In cultures derived from PLP deficient mice, the number of Olgs is twofold greater than in wild-type mice. In primary glial cultures or in enriched OLG cultures, in which the synthesis of native PLP is blocked using antisense technology, the number of apoptotic cells is several-fold reduced. Injection of PLP antisense oligodeoxynucleotides into jimpy (jp) mice reduces the number of dying glia in spinal cord 3x compared to controls, and increased the number of myelinated fibers. These studies demonstrate that inhibition of native or mutant PLP synthesis directly reduces apoptosis. The regulation of apoptosis by PLP gene expression occurs independently of myelination, indicating that the PLP gene has multiple primary functions.

Hyperpolarization-activated, cyclic AMP-gated, HCN1-like cation channel: the primary, full-length HCN isoform expressed in a saccular hair-cell layer.

The expression of transcript for hyperpolarization-activated, cyclic nucleotide-sensitive cation channel (HCN) isoforms underlying hyperpolarization-activated, inward current (I(h)) has been determined for a model hair-cell preparation from the saccule of the rainbow trout, Oncorhynchus mykiss. Based upon identification from homology to known vertebrate HCN cDNA sequence, cloning of PCR products amplified with degenerate primers indicated an expression frequency of 7:2:1 (HCN1:HCN2:HCN4) for the hair-cell sheet compared with 1:1:7 for brain. Full-length sequence has been obtained for the HCN1-like isoform representing the primary HCN transcript expressed in the hair-cell preparation. The channel protein is 938 amino acids in length with 93% amino acid identity for the region extending from the S1-S6 membrane spanning domains through the voltage-pore and cyclic nucleotide-binding domains, compared with HCN1 for rabbit, rat, mouse and human. The N- and C-terminal regions are less homologous, with 39-51% and 43-44% amino acid identities, respectively. Compared with other vertebrate HCN1, the hair-cell HCN1 contains additional consensus phosphorylation sites associated with unique repeats in the carboxy terminus. The HCN1-like transcript has been localized to hair cells of the saccular sensory epithelia by in situ hybridization. Previous electrophysiological studies have identified I(h) as the sole inwardly rectifying ion channel in a specific population of hair cells of the saccule of frogs [J Neurophysiol (1995) 73:1484] and fish [J Physiol (1996) 495:665]. I(h) is an important determinant of the resting membrane potential, and for this population of hair cells, is predicted to maintain the membrane potential within a voltage range allowing the voltage-gated calcium channels to open, permitting "spontaneous" release of transmitter. The molecular properties of the HCN1-like isoform underlying I(h) expressed in the saccular hair cells of the teleost, trout, may consequently impact spontaneous release of transmitter from hair cells of the saccule.

Hypoxic-ischemic injury results in acute disruption of myelin gene expression and death of oligodendroglial precursors in neonatal mice.

Studies of ischemic brain injury in neonatal rodents have focused upon the pathophysiology of neuronal damage. Much less consideration has been given to white matter injury, even though it is a major contributor to chronic neurological dysfunction in children. In the human neonate, particularly in those born prematurely, periventricular white matter is highly susceptible to hypoxic--ischemic (H--I) injury. To understand the basis for this selective vulnerability, we examined myelin gene expression and cell death in the subventricular layer and the surrounding white matter of neonatal mice following H--I insult. Using an in situ hybridization technique that gives high resolution and is very sensitive, we examined myelin basic protein and proteolipid protein gene expression three and twenty-four hours after a H-I insult. To elicit unilateral forebrain hypoxic and ischemic injury, 9--10-day-old mice underwent right carotid artery ligation followed by timed (40--70 min) exposure to 10% oxygen. Twenty-four hours following H--I, myelin basic protein and proteolipid protein transcripts were markedly reduced in striatum, external capsule, fornix, and corpus callosum in the injured side. Three hours after lesioning (ligation+70 min hypoxic exposure) myelin basic protein gene transcripts were visibly reduced in the ipsilateral white matter tracts. Interestingly, some cells in the subventricular layer expressed proteolipid protein transcripts, and 3 h after a H--I insult they were degenerating in the injured but not contralateral side. TUNEL staining showed an increase in the number of positive cells in the injured subventricular layer and corpus callosum but the adjacent striatum did not show a corresponding change in the number of TUNEL labeled cells. Ultrastructural studies of the subventricular zone and corpus callosum 3 h after H--I revealed that many subventricular cells, glial cells in the corpus callosum, and callosal axons in the injured side had already degenerated. However, the subventricular cells, glia and axons in the contralateral corpus callosum were spared. Many cells in the injured corpus callosum exhibited a apoptotic morphology; yet more mature oligodendrocytes in this region appeared normal. Our results show that a H--I insult causes a surprisingly swift and dramatic degenerative response in the subventricular layer and adjacent white matter. Within 3 h after H--I, the programmed cell death cascade was initiated; internucleosomal DNA degradation took place in subventricular and glial cells; oligodendrocyte progenitors died and axonal degeneration in the ipsilateral corpus callosum was extensive. The swiftness of the subventricular and glial cell degeneration suggests the H--I insult directly targets glia, as well as neurons, and raises the provocative question of whether glia exert damaging effects upon neurons and axons. Since the severity of the H--I insult can be modulated by varying the duration of hypoxia, the model is ideal to study whether oligodendrocyte progenitors are more susceptible to death than mature oligodendrocytes, whether mature oligodendrocytes de-differentiate and then are induced to remyelinate surviving axons, and/or whether oligodendrocyte progenitors in the subventricular layer can be stimulated to proliferate, migrate, and remyelinate the surviving axons.

PPAR delta agonists stimulate oligodendrocyte differentiation in tissue culture.

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors of the nuclear hormone receptor superfamily that have been described as master genes that switch cells from an undifferentiated phenotype to a differentiated phenotype. In the present investigation, we examined the possibility that ligands for PPARs are potent activators of oligodendrocyte (OL) differentiation and/or proliferation. Primary glial cultures and enriched OL cultures of neonatal mouse cerebra were treated with three different PPAR agonists: a PPAR gamma-selective agonist, a PPAR delta-selective agonist, and a pan agonist selective for both PPAR gamma and delta. Treatment with PPAR gamma agonist does not have an effect on the differentiation of OLs; however, PPAR delta agonist and the pan agonist treatment accelerates the differentiation of OLs within 24 h of application in mixed glial cultures. The number of OLs with processes and huge membrane sheets increases two- to threefold in both groups. The increase in the size of the sheets is also mirrored by changes in the intensity and distribution of myelin basic protein (MBP) and proteolipid protein (PLP) mRNAs. As compared to controls, the PPAR delta agonist-treated groups contain more OLs that have MBP and PLP mRNA extending into distal processes. These results indicate that PPAR delta plays a significant role in the maturation of OLs and regulates the size of OL sheets. BrdU immunostaining reveals that these agonists do not significantly stimulate proliferation of OLs expressing glycolipids. The studies in enriched OL cultures reproduce the effects of the PPAR agonists seen in the mixed glial cultures, indicating that the effect of the PPAR agonists is directly on the OLs and not via astrocytes. In the enriched cultures, the total number of OLs increases significantly in the PPAR delta agonist-treated groups, but BrdU immunostaining does not show an increased proliferation of cells. These findings suggest that PPAR delta increases the survival of cells and/or prevents cell death in enriched cultures. Although PPAR delta is expressed in various cell types, its role as a factor in the transcriptional regulation of OL differentiation has not been explored. We show for the first time that a ligand that serves as an agonist for PPAR delta activates the program of OL differentiation in primary and enriched OL cultures.

The pathobiology of myelin mutants reveal novel biological functions of the MBP and PLP genes.

Substantial biological data indicate that the myelin basic protein (MBP) and myelin proteolipid protein (PLP/DM20) genes produce products with functions beyond that of serving as myelin structural proteins. Much of this evidence comes from studies on naturally-occurring and man-made mutations of these genes in mice and other species. This review focuses upon recent evidence showing the existence of other products of these genes that may account for some of these other functions, and recent studies providing evidence for alternative biological functions of PLP/DM20. The MBP and PLP/DM20 genes each encode the classic MBP and PLP isoforms, as well as a second family of proteins that are not involved in myelin structure. The biological roles of these other products of the genes are becoming clarified. The non-classic MBP gene products appear to be components of transcriptional complexes in the nucleus, and they also may be involved in signaling pathways in T-cells and in neural cells. The non-classic PLP/DM20 gene products appear to be components of intracellular transport vesicles in oligodendrocytes. There is evidence for other functions of the classic PLP/DM20 proteins, including a role in neural cell death mechanisms, autocrine and paracrine regulation of oligodendrocytes and neurons, intracellular transport and oligodendrocyte migration.

Differential expression of apoptotic markers in jimpy and in Plp overexpressors: evidence for different apoptotic pathways.

Point mutations and duplications of proteolipid protein (PLP) gene in mammals cause dysmyelination and oligodendrocyte cell death. The jimpy mouse, which has a lethal Plp point mutation, is the best characterized of the mutants; transgenic mice, which have additional copies of Plp gene, are less characterized. While oligodendrocyte death is a prominent feature in jimpy, the pathways leading to death have not been investigated in jimpy and Plp overexpressors. Using immunohistochemistry and immunobloting, we examined expression of cleaved caspase-3, Poly (ADP-ribose) polymerase (PARP), caspase-12, and mitochondrial apoptotic markers in spinal cord in jimpy males and Plp overexpressors. Compared to controls, cleaved caspase-3 is increased 10x in jimpy white matter spinal cord, and 3x in Plp overexpressor. In jimpy, the number of cleaved caspase-3 cells far exceeds the number of TUNEL(+) cells. The majority of cleaved caspase-3(+) cells were not TUNEL(+) and these cells exhibited staining in perikarya and in processes. Only 30% of the cleaved caspase-3(+) cells were TUNEL(+) and exhibited both nuclear and perinuclear staining. This observation suggests that activation of caspase-3 begins earlier and overlaps for a period of time with DNA fragmentation. In both Plp mutants, quantitative immunobloting of PARP showed a 45% increase in total as well as cleaved form, indicating that oligodendrocytes die via apoptosis. Most interestingly, cleavage of caspase-12, a caspase associated with unfolded protein response, is dramatically increased in jimpy but not at all in Plp overexpressors. Mitochondrial markers cytochrome c and Bcl-X(L) are upregulated in both Plp mutants but levels of expression are different between mutants, suggesting that apoptosis in these two Plp mutants follows different pathways. In jimpy, mitochondrial apoptotic markers may play a role in amplifying the apoptotic signal. Our data shows for the first time, in vivo, that mutations in Plp gene increase oligodendrocyte death by activating the caspase cascade but the trigger to upregulate this cascade follows different pathways.

High-resolution in situ hybridization and TUNEL staining with free-floating brain sections.

We applied in situ hybridization and the TUNEL technique to free-floating (vibratomed) sections of embryonic and postnatal mouse CNS. Full-length cDNAs specific for oligodendrocyte- or astrocyte-specific genes were labeled with digoxigenin using the random primer method. With paraformaldehyde-fixed sections, the nonradioactive in situ hybridization method provides detection of individual, very small glial progenitor cells in embryonic development. Small, isolated cells expressing oligodendrocyte specific messages can be detected in the neuroepithelium at embryonic and postnatal stages. The technique can be completed within 3 days and is as sensitive as the radioactive method. Likewise, the TUNEL method using DAB as the chromogen on free-floating sections provides excellent resolution. These DAB-stained sections can be embedded in plastic and thin-sectioned to visualize the ultrastructure of apoptotic cells. Both in situ hybridization and TUNEL methods can be applied to the same section, the tissue embedded in plastic, and semithin sections cut. The high resolution obtained with this combined procedure makes it possible to determine whether brain cells expressing glia-specific messages are undergoing apoptosis.

Programmed cell death without DNA fragmentation in the jimpy mouse: secreted factors can enhance survival.

Jimpy is one of many related mutations affecting the myelin proteolipid protein gene that causes severe hypomyelination in the central nervous system (CNS). Underlying the hypomyelination is a failure of oligodendrocytes (OLs) to differentiate, and the premature death of large numbers of OLs during the developmental period. Previous light and electron microscopic evidence suggested that jimpy OLs die in a manner consistent with programmed cell death. We have used TUNEL staining as a biochemical marker for apoptosis in conjunction with immunostaining for OL and myelin markers. At 13 - 14 days postnatal, a time when the number of dying OLs in jimpy CNS is increased more than five times normal, there are only modest increases (70% in spinal cord; 20% in cerebral cortex) in TUNEL labeled cells in mutant CNS tissues. The results in vitro are similar, and only a small per cent of TUNEL labeled cells have the antigenic phenotype of OLs. The discrepancy between numbers of dying and TUNEL labeled cells suggests either that most jimpy OLs do not undergo programmed cell death or that the biochemical pathways leading to their death do not involve DNA fragmentation which is detected by the TUNEL method. We also present evidence that jimpy OLs show increased survival and enhanced differentiation when they are grown in vitro in medium conditioned by cells lines which express products of the proteolipid protein gene. Cell lines expressing proteolipid protein and the alternatively spliced DM20 protein have differential effects on cell numbers and production of myelin-like membranes.

Proteolipid protein regulates the survival and differentiation of oligodendrocytes.

Proteolipid protein (PLP) has been postulated to play a critical role in the early differentiation of oligodendrocytes (OLs) in addition to its known role as a structural component of myelin. To identify this early function, we blocked the synthesis of PLP in glial cultures with antisense oligodeoxynucleotides that targeted the PLP initiation codon. Primary glial cultures were incubated with phosphorothioate-protected oligodeoxynucleotides (S-ODNs) for up to 11 d. PLP in OLs was reduced >90%. OLs treated with antisense S-ODNs appeared strikingly healthy as judged by (1) immunocytochemical staining for myelin glycolipids and myelin basic protein, (2) their prolonged survival compared with untreated cultures, and (3) their ability to re-establish membrane sheets after removal of the S-ODNs. Our studies show that PLP is required for elaboration and stability of the myelin membrane sheets made by most OLs, but it is not necessary for the network of processes established by OLs. More importantly, the number of OLs in the antisense-treated cultures was nearly sevenfold greater after a 10-11 d incubation with S-ODNs than in control cultures. The number of proliferating OL progenitors was not increased in the antisense-treated cultures, indicating that the increase in the number of OLs was attributable to prolonged OL survival. The tissue culture studies reveal that the absence of PLP/DM20 has the positive effect of promoting OL survival but the negative effect of preventing their full differentiation. This finding clarifies many of the paradoxical findings seen in the PLP mutants, the PLP overexpressers, and the PLP- animals.

Epigenetic factors up-regulate expression of myelin proteins in the dysmyelinating jimpy mutant mouse.

Proteolipid protein (PLP) is a major structural component of central nervous system (CNS) myelin. Evidence exists that PLP or the related splice variant DM-20 protein may also play a role in early development of oligodendrocytes (OLs), the cells that form CNS myelin. There are several naturally occurring mutations of the PLP gene that have been used to study the roles of PLP both in myelination and in OL differentiation. The PLP mutation in the jimpy (jp) mouse has been extensively characterized. These mutants produce no detectable PLP and exhibit an almost total lack of CNS myelin. Additionally, most OLs in affected animals die prematurely, before producing myelin sheaths. We have studied cultures of jp CNS in order to understand whether OL survival and myelin formation require production of normal PLP. When grown in primary cultures, jp OLs mimic the relatively undifferentiated phenotype of jp OLs in vivo. They produce little myelin basic protein (MBP), never immunostain for PLP, and rarely elaborate myelin-like membranes. We report here that jp OLs grown in medium conditioned by normal astrocytes synthesize MBP and incorporate it into membrane expansions. Some jp OLs grown in this way stain with PLP antibodies, including an antibody to a peptide sequence specific for the mutant jp PLP. This study shows that: (1) an absence of PLP does not necessarily lead to dysmyelination or OL death; (2) OLs are capable of translating at least a portion of the predicted jp PLP; (3) the abnormal PLP made in the cultured jp cells is not toxic to OLs. These results also highlight the importance of environmental factors in controlling OL phenotype.

Programmed cell death in the dysmyelinating mutants.

A large number of genetic mutants that are missing a particular myelin protein or that have an aberrant myelin protein composition have been described. These mutations usually cause dysmyelination in the PNS or CNS. Similarly, the nervous system of animals experimentally altered to block synthesis of myelin proteins have recently been generated that show aberrations in the myelin sheath. For both groups of animals, the numbers of myelinating cells remain relatively stable and glial cell death is minimal. The exception is animals with mutations in the proteolipid protein (PLP) gene which exhibit extensive death of oligodendrocytes (OLs). The degree of OL death in the PLP mutants generally correlates with the amount of dysmyelination. Dying OLs in the PLP mutants exhibit the classical features of apoptotic cells. Programmed cell death (PCD) is often, but not necessarily, manifested by cleavage of DNA into abundant oligonucleosomal fragments. Detection of these abundant DNA fragments was examined in normal and jimpy (jp) mice using the TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) method. In normal spinal cord and brain, at least twice as many cells exhibited DNA fragmentation when compared to numbers of pyknotic glia observed microscopically. In jp spinal cord and brain, roughly one-half of cells exhibited DNA fragmentation when compared to numbers of pyknotic glia observed microscopically. PCD of cells in normal development involving DNA fragmentation has been previously described and our results support that conclusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Oligodendrocytes in female carriers of the jimpy gene make more myelin than normal oligodendrocytes.

The female carrier of the jimpy (jp) gene is a model system to study the plasticity of neuroglial cells and the mechanisms they use to compensate for a temporary deficit in myelin. Myelin in the female carriers is reduced 30-40% during the first postnatal month but is normal in adults. We hypothesized that the number of oligodendrocytes (OLs) in the female carriers is increased, based upon previous data showing OL proliferation is increased but the number of dying OLs is only slightly elevated in development. To test this hypothesis, antibodies to carbonic anhydrase (CA)II, an OL-specific marker, were used to quantify the number of OLs in the spinal cords of 1-month-old and adult female carriers. Contrary to expectations, the number of OLs is significantly reduced in the dorsal funiculus and grey matter by 21% in adult female carriers compared to controls. A reduction of lesser magnitude is present in the 1-month-old animals. Electron microscopic montages prepared from normal and carrier dorsal funiculus were used to count total numbers of glia. Ultrastructural quantification shows a similar reduction in the number of OLs and confirms the validity of the CAII immunostaining as a means to quantify OLs. These data show that there are 21% fewer OLs in the central nervous system (CNS) of adult female carriers but normal amounts of myelin. Presumably, some OLs in the carrier CNS are maintaining more myelin than their counterparts in normal CNS would. These findings demonstrate that (1) a reduction in number of OLs does not necessarily involve a reduction in the amount of myelin, and (2) OLs have considerable flexibility in the amount of myelin they can make.

Postmitotic oligodendrocytes generated during postnatal cerebral development are derived from proliferation of immature oligodendrocytes.

The phenotype of proliferating glia is examined during postnatal rodent development by combining immunocytochemistry (ICC) with 3H-thymidine autoradiography (ARG) to identify cells in the S phase of the cell cycle. Antibodies (ABs) which are specific for cells in the oligodendrocyte (OL) lineage were utilized, with emphasis placed upon the proliferation of OLs as it remains unclear whether this cell type divides in situ. The results show that proliferating cells stain with ABs which are specific for OLs and myelin glycolipids. The proliferating OLs (oligodendroblasts), although they do not appear to have formed myelin sheaths, have quite elaborate and distinctive morphologies. These oligodendroblasts give rise to very long, thin processes which in turn have additional branches. Their cytoarchitecture corresponds closely to cells described as oligodendroblasts with electron microscopy and whose processes often appear to be in the initial phase of myelination (Skoff et al: J. Comp. Neurol. 169:291-312, 1976a). These proliferating OLs are still quite immature because the expression of myelin specific proteins is only occasionally observed in 3H-thymidine labeled cells. The phenotype of the oligodendroblasts is quite different from that of proliferating astrocytes (astroblasts). As shown in previous studies (Skoff; Dev. Biol. 139:149-163, 1990), the astroblasts, which are identified by the presence of glial fibrillary acidic protein (GFAP), usually have thick, stubby processes, and both their nucleus and cytoplasm are larger and of lighter density than those found in oligodendroblasts. In early myelinating regions of the cerebrum, glycolipid positive cells account for the majority of the 3H-thymidine labeled cells. This data, when combined with the quantification of proliferating astrocytes (ASs) from previous immunocytochemical and electron microscopic studies, indicate that oligodendroblasts and astroblasts constitute the vast majority of the proliferating glia in the brain and in optic nerve at times when ASs and OLs are being generated. In normal postnatal cerebral development, the immature ASs and OLs which proliferate are the direct, immediate precursors for most postmitotic ASs and OLs.

Analysis of myelin proteolipid protein and F0 ATPase subunit 9 in normal and jimpy CNS.

Membrane fractions and chloroform-methanol (C-M) extracts of jimpy (jp) and normal CNS at 17-20 days were examined by immunoblot and sequence analysis to determine whether myelin proteolipid protein (FLP) or DM-20 could be detected in jp CNS. No reactivity was detected in jp samples with several PLP antibodies (Abs) except with one Ab to amino acids 109-128 of normal PLP. Proteins in the immunoreactive bands approximately 26 M(r) comigrating with PLP were sequenced for the first 10-12 residues. A sequence corresponding to PLP was found in normal CNS, as expected, but not in the band from jp CNS. Our results provide no evidence for an aberrant form of PLP in jp CNS at 17-20 days. This and other studies suggest that the abnormalities in jp brain are not due to toxicity of the mutant jp PLP/DM-20 proteins. Interestingly, a sequence identical to the amino terminus of the mature proton channel subunit 9 of mitochondrial F0 ATPase was detected in the immunoreactive bands approximately 26 M(r) in both normal and jp samples. This identification was supported by reactivity with an Ab to the F0 subunit and by labeling with dicyclohexylcarbodiimide (DCCD). In contrast to PLP isolated from whole CNS, PLP isolated from myelin was devoid of F0 subunit 9 based on sequence analysis and lack of reactivity with an Ab to the F0 subunit, yet still reacted with DCCD. This finding rules out the possibility that contaminating F0 ATPase gives rise to the DCCD binding exhibited by PLP and confirms the possibility that PLP has proton channel activity, as suggested by Lin and Lees (1,2).

The pH of jimpy glia is increased: intracellular measurements using fluorescent laser cytometry.

The jimpy mutation lies in the gene which codes for myelin proteolipid protein, and the brains and spinal cords of jimpy mice contain little myelin and no measurable proteolipid protein. It has been thought that the mutation affected only the myelin forming oligodendroglial cells, but there is now considerable evidence that astroglia are also a target of the mutation since jimpy astrocytes exhibit a prominent gliosis along with defects in metabolism and proliferation. Because cell proliferation is associated with an increase in intracellular pH, we investigated whether the pH of jimpy glia was abnormal. Using a pH sensitive fluorescent dye and a laser cytometry system we measured the intracellular pH of individual cells in cultures derived from both jimpy and normal brains. The relative pH of flat astrocytes in jimpy cultures was higher than in normal cultures by an average of 0.24 pH units, and these increased values were evident 2-3 days after plating. At this in vitro age the cultures contain only a few oligodendrocytes, none of which express detectable proteolipid protein. The pH of the process-bearing cell population, which contains the oligodendrocytes as well as some astrocytes and presumptive glial precursors, was also increased but not until 7 days in culture. The finding that a mutation in the myelin proteolipid protein gene can alter the normal pH of astrocytes is quite unexpected since, as far as is known, astrocytes do not make proteolipid protein. These results and others discussed in this paper support the hypothesis that either proteolipid protein itself, or some other product of the gene, may have an important role in central nervous system development.

Jimpy mutation affects astrocytes: lengthening of the cell cycle in vitro.

The jimpy mutation has been identified as a point mutation in the gene coding for the major myelin proteolipid protein. The most prominent effect of the mutation is an extreme reduction in central nervous system myelin in affected mice. However, both oligodendrocytes and astrocytes, the two types of central nervous system macroglia, have been shown to exhibit more subtle developmental and metabolic changes as a result of the mutation. These include early death and proliferation abnormalities in jimpy oligodendrocytes, and hypertrophy, increased pH and abnormal responses to high K+ in jimpy astrocytes. In the present study, we examine the effect of the mutation of the cell cycle of astrocytes. Using an immunocytochemical method to chart the percent of labeled mitoses, we find the total cell cycle to be lengthened in jimpy astrocytes by 5-6 h, with increases in several different phases. Since there is no evidence that astrocytes make myelin proteolipid protein, the results support previous studies which suggest that this gene may code for other proteins playing an important role in the development of many cell types.

Double-labeling in situ hybridization analysis of mRNAs for carbonic anhydrase II and myelin basic protein: expression in developing cultured glial cells.

We applied in situ hybridization to analyze the location and the developmental changes in the distribution of the transcripts for carbonic anhydrase II (CAII) and myelin basic protein (MBP) in mouse primary cultured glial cells. Both mRNAs were localized to the oligodendrocyte using double-labeling in situ hybridization. No evidence for CAII transcripts in astrocytes was obtained, indicating that CAII is expressed only by oligodendrocytes in normal rodent glia. As early as 48 h after plating, CAII and MBP mRNAs are present in a few, small round cells. Message is present 2-4 days before levels of these proteins can be detected in similar primary glial cultures. The intensity of labeling for MBP and CAII mRNA positive cells increases significantly during the second week but then decreases after the end of the third week. Only the oligodendrocyte perikaryon and a few processes are positive during the first week. In contrast, at 14 days, a large number of cell processes in addition to the cell bodies are heavily stained for both mRNAs. Both mRNAs could be detected far away from the cell body, up to 250 microns in some cell processes. Some segments on a cell process accumulate higher levels of mRNA than other areas. These areas may correspond to the accumulation of free ribosomes and to starting points for the membrane sheets elaborated by cultured oligodendrocytes. The developmental profile for timing and distribution of these two messages mimics closely their in situ pattern.