Axonal swellings and degeneration in mice lacking the major proteolipid of myelin.

Glial cells produce myelin and contribute to axonal morphology in the nervous system. Two myelin membrane proteolipids, PLP and DM20, were shown to be essential for the integrity of myelinated axons. In the absence of PLP-DM20, mice assembled compact myelin sheaths but subsequently developed widespread axonal swellings and degeneration, associated predominantly with small-caliber nerve fibers. Similar swellings were absent in dysmyelinated shiverer mice, which lack myelin basic protein (MBP), but recurred in MBP*PLP double mutants. Thus, fiber degeneration, which was probably secondary to impaired axonal transport, could indicate that myelinated axons require local oligodendroglial support.

Insights into thymic purine metabolism and adenosine deaminase deficiency revealed by transgenic mice overexpressing ecto-5'-nucleotidase (CD73).

The adenosine producing enzyme ecto-5'-nucleotidase (5'-NT) is not normally expressed during thymocyte development until the medullary stage. To determine whether earlier expression would lead to adenosine accumulation and/or be deleterious for thymocyte maturation, thymic purine metabolism, and T cell differentiation were studied in lckNT transgenic mice overexpressing 5'-NT in cortical thymocytes under the control of the lck proximal promoter. In spite of a 100-fold elevation in thymic 5'-NT activity, transgenic adenosine levels were unchanged and T cell immunity was normal. Inosine, the product of adenosine deamination, was elevated more than twofold, however, indicating that adenosine deaminase (ADA) can prevent the accumulation of adenosine, even with a dramatic increase in 5'-NT activity, and demonstrating the availability of 5'-NT substrates in the thymus for the first time. Thymic adenosine concentrations of mice treated with the ADA inhibitor 2'-deoxycoformycin (dCF) were elevated over 30-fold, suggesting that high ADA activity, rather than an absence of 5'-NT, is mainly responsible for low thymic adenosine levels. The adenosine concentrations in dCF-treated mice are sufficient to cause adenosine receptor-mediated thymocyte apoptosis in vitro, suggesting that adenosine accumulation could play a role in ADA-deficient severe combined immunodeficiency.

The neurobiology of aging.

Basic principles of the neurobiology of aging were reviewed within selected topic areas chosen for their potential relevance to epileptogenesis in the aging brain. The availability of National Institute on Aging-supported aged mouse and rat strains and other biological resources for studies of aging and age-associated diseases was presented, and general principles of animal use in gerontological research were discussed. Neurobiological changes during normal brain aging were compared and contrasted with neuropathological events of Alzheimer's disease (AD) and age-associated memory impairment (AAMI). Major themes addressed were the loss of synaptic connections as vulnerable neurons die and circuits deteriorate in AD, the absence of significant neuron loss but potential synaptic alteration in the same circuits in AAMI, and the effects of decreased estrogen on normal aging. The "calcium hypothesis of brain aging" was examined by a review of calcium dyshomeostasis and synaptic communication in aged hippocampus, with particular emphasis on the role of L-type voltage-gated calcium channels during normal aging. Established and potential mechanisms of hippocampal plasticity during aging were discussed, including long-term potentiation, changes in functional connectivity, and increased gap junctions, the latter possibly being related to enhanced network excitability. Lastly, application of microarray gene chip technology to aging brain studies was presented and use of the hippocampal "zipper slice" preparation to study aged neurons was described.

Aberrant dendrite growth in central nervous system white matter induced by a mutant proteolipid protein transgene.

Transgenic mice were produced that carry a construct encoding a mutant form of the DM20 isoform of myelin proteolipid protein. The transgene is under the direction of the human Plp gene promoter, which has previously been shown to direct tissue-specific expression of transgenes. Two lines of mice were generated with this construct, both of which express the transgene at extremely low levels. Central nervous system myelination proceeds normally in the transgenic mice. However, in aged transgenic mice, areas of dendrite processes synapsed with axonal termini were observed within the white matter of the spinal cord. This phenotype was accompanied by focal areas of astrocytic hypertrophy and an increase in apoptotic cell death in white matter but not gray matter. One interpretation of these findings is that expression of the mutant DM20 alters signaling between oligodendrocytes and neurons, producing abnormal neurite outgrowth.

Embryonic development of central nervous system myelination in a reptilian species, Eumeces fasciatus.

The myelin proteolipid proteins are a vital component of the vertebrate central nervous system (CNS), contributing essential functions to the development of the myelinating cells of the CNS and to the structure of CNS myelin. Alternative splicing of the proteolipid protein (PLP) gene to produce two related isoforms occurs in Mammalia, Aves, and Reptilia, but not Amphibia. As part of a long-term investigation into the function of the different isoforms of PLP, embryonic development, myelination, and PLP gene expression in reptilian CNS were examined. PLP gene expression was already substantial by day 19 (stage 39) of the 27-day Eumeces fasciatus egg incubation period. By day 21 of incubation, also stage 39, PLP mRNA was at peak levels; there was a significant amount of CNS myelination as demonstrated by electron microscopy of the spinal cord; and the reflexive motor response was evident. Although most axons were myelinated by the time of hatching, myelin sheaths continued to increase in size and compactness after hatching. The correlation of physiological development, CNS myelination, and expression of the PLP gene in the lizard corresponded well with the developmental pattern seen in mammals.

Program for testing biological interventions to promote healthy aging.

The National Institute on Aging (NIA) sponsored a workshop on September, 1999 to discuss the feasibility of establishing a program to evaluate potential intervention strategies to decelerate the rate of aging in mammals. The ultimate goal is to identify promising interventions in animals that might lead to clinical trials in humans. The participants discussed various animal models, biological endpoints and possible structure of such a program. The ability to implement such a program will require a decision by NIA staff about whether the anticipated benefits to be derived from identification of effective interventions under well controlled conditions in an animal model, in this case the mouse, would justify the anticipated cost of the testing program.

Principles of animal use for gerontological research.

This essay presents some practical advice and suggestions for those who wish to use mice and rats in experiments on the biology of aging. Ten principles set forth guidance on choice of ages, choice of stocks, the importance of specific pathogen-free status, the uses of necropsy data, the dangers of pooling samples from different individuals, planning ahead for loss of aged mice to death and disease, the use of cost-adjusted power calculations, and the dangers of inferring causal associations from correlated age effects.

Myelin proteolipid DM20: evidence for function independent of myelination.

DM20 is a proteolipid protein that has been extensively studied for its role in central nervous system myelination. We demonstrate that DM20 expression is widespread and independent of myelination. In the Schwann cells and neurons of the peripheral nervous system, DM20 is not incorporated into the membrane as it is in the central nervous system (CNS), but remains cytoplasmic. Mutations that severely reduce the amount of DM20 mRNA in CNS myelinating cells have little effect on DM20 expression in nonmyelinating cells of the peripheral nervous system and embryonic CNS. Most importantly, the combination of wild-type DM20 from the endogenous X-linked gene and mutant DM20 expressed from an autosomal transgene results in embryonic lethality. We propose a function for DM20 to explain these diverse findings based on the ability of DM20 to form multimeric complexes, and hypothesize that the DM20 complex participates in intracellular molecular transport.

Design of aging intervention studies: the NIA interventions testing program.

The field of biogerontology has made great strides towards understanding the biological processes underlying aging, and the time is ripe to look towards applying this knowledge to the pursuit of aging interventions. Identification of safe, inexpensive, and non-invasive interventions that slow the aging process and promote healthy aging could have a significant impact on quality of life and health care expenditures for the aged. While there is a plethora of supplements and interventions on the market that purport to slow aging, the evidence to validate such claims is generally lacking. Here we describe the development of an aging interventions testing program funded by the National Institute on Aging (NIA) to test candidate interventions in a model system. The development of this program highlights the challenges of long-term intervention studies and provides approaches to cope with the stringent requirements of a multi-site testing program.

Gene expression and oligodendrocyte development in the myelin deficient rat.

The proteolipid proteins play a major role in the structure of the CNS myelin sheath, but they have also been implicated in the oligodendrocyte development leading to myelination. Mutations in the PLP gene result in severe dysmyelination and a paucity of mature oligodendrocytes. The myelin deficient (md) rat, carrying a Thr75-->Pro substitution present in both isoforms of proteolipid protein (PLP and DM20), is the most severely affected of the PLP mutants described to date. The expression of myelin associated genes was quantitated to determine the effect of the mutation on oligodendrocyte development in vivo. At 5 days postnatal, gene expression in the md rat approximated that in age-matched control rats, but as they matured, there was a progressive inhibition of gene expression in the md rats. The genes expressed late in the myelination program (PLP and MBP) were affected more dramatically than those expressed earlier in oligodendrocyte development (CNP and GPDH). The results indicate that the later stages of oligodendrocyte maturation and myelin elaboration are inhibited.

A-11: cell type-specific and single-active-X transcription controls of newly found gene in cultured human cells.

We describe the isolation and characterization of a human X-chromosomal gene that is subject to both single-active-X control and tissue-specific control. The A-11 gene was identified by a cDNA that hydridizes to a 3.2-kb EcoR1 fragment of genomic DNA on the long arm of the human X chromosome. A-11 transcripts are normally present in fibroblasts but not in B- or T-lymphoblasts. However, A-11 transcription was activated in four of 11 independent, gamma ray-induced B-lymphoblastoid HLA antigen-loss mutants. Cell hybrids with a human fibroblast-derived active X contained A-11 transcripts but hybrids carrying the human inactive X did not. Azacytidine, a potent inhibitor of DNA methylation, readily reactivated the A-11 locus on the inactive X in hybrid cells, indicating that differential methylation is likely to be involved in the single-active-X control of A-11 transcription in fibroblasts. Failure of cells to remethylate DNA synthesized to repair gamma ray-induced damage may also have resulted in the activation of A-11 transcription among the lymphoblastoid mutants. The A-11 locus provides an opportunity to study the relationship between two types of transcriptional regulation of a gene.

Genomic DNA analysis from mouse toe lysates.

The protocol described in this report provides a simple, accurate and efficient assay for detection of transgenes and mutations in large colonies of rodents, using crude lysates prepared from the digit cut from the animals for identification purposes. This can be done as early as 6 days of age, minimizing trauma to the mice and allowing assays to be completed long before weaning.

Molecular analysis of glial cell development in the canine 'shaking pup' mutant.

The shaking pup, a canine mutant, carries a point mutation in the myelin proteolipid protein (PLP) gene that causes dysmyelination of the central nervous system (CNS) with resultant tremor, seizures, and other persistent neurological deficits. The developmental potential of glial cells in the shaking pup CNS and peripheral nervous system (PNS) was evaluated by quantitative analysis of the expression of several glial-specific genes. All of the myelin-associated genes demonstrated developmental patterns of expression similar to those observed in the controls, but at significantly reduced levels. Expression of the genes for the major CNS myelin proteins, PLP and the myelin basic protein, are most dramatically affected in the shaking pup, although reduced expression levels are observed for other oligodendrocyte-specific genes such as 2',3'-cyclic nucleotide 3'phosphodiesterase and glucose phosphate dehydrogenase. The pattern of gene expression in the shaking pup indicates that the oligodendrocytes experience an inhibition in development after the myelination program has begun. There appears to be little evidence for an astrocytic response to the dysmyelinating condition at the RNA level, but we present evidence for ectopic expression of P0 mRNA in the CNS. Expression of the P0 and PLP genes in the sciatic nerve appears to be normal, reinforcing previous reports that PNS myelination is unaffected by the mutation in the PLP gene.

Myelin proteolipid protein: function in myelin structure is distinct from its role in oligodendrocyte development.

The myelin proteolipid proteins PLP and DM20 are essential for the compaction of central nervous system myelin and they play an important role in the maturation of the oligodendrocyte. The specific function of the less abundant DM20 isoform is still unknown, but rescue experiments previously indicated that both isoforms are necessary for oligodendrocyte maturation. In vitro experiments have suggested DM20 may assist in the translocation of PLP into the membrane. We tested this hypothesis in vivo, by investigating whether wild-type PLP derived from a transgene could be incorporated into the myelin membrane of Plp mutant rumpshaker mice. We previously demonstrated that expression of the PLP transgene alone in a more severe Plp mutant, jimpy mouse, did not result in PLP incorporation into the myelin. Here we report that there was significantly more PLP in white matter from rumpshaker expressing the PLP transgene than their nontransgenic rumpshaker littermates and that myelin structure was improved. The delay in oligodendrocyte development was not alleviated by expression of the PLP transgene however, supporting an essential role for DM20 in oligodendrocyte maturation.