Alan Peterson

α-Internexin Is Structurally and Functionally Associated with the Neurofilament Triplet Proteins in the Mature CNS

α-Internexin, a neuronal intermediate filament protein implicated in neurodegenerative disease, coexists with the neurofilament (NF) triplet proteins (NF-L, NF-M, and NF-H) but has an unknown function. The earlier peak expression of α-internexin than the triplet during brain development and its ability to form homopolymers, unlike the triplet, which are obligate heteropolymers, have supported a widely held view that α-internexin and neurofilament triplet form separate filament systems. Here, we demonstrate, however, that despite a postnatal decline in expression, α-internexin is as abundant as the triplet in the adult CNS and exists in a relatively fixed stoichiometry with these subunits. α-Internexin exhibits transport and turnover rates identical to those of triplet proteins in optic axons and colocalizes with NF-M on single neurofilaments by immunogold electron microscopy. α-Internexin also coassembles with all three neurofilament proteins into a single network of filaments in quadruple-transfected SW13vim(−) cells. Genetically deleting NF-M alone or together with NF-H in mice dramatically reduces α-internexin transport and content in axons throughout the CNS. Moreover, deleting α-internexin potentiates the effects of NF-M deletion on NF-H and NF-L transport. Finally, overexpressing a NF-H–LacZ fusion protein in mice induces α-internexin and neurofilament triplet to aggregate in neuronal perikarya and greatly reduces their transport and content selectively in axons. Our data show that α-internexin and the neurofilament proteins are functionally interdependent. The results strongly support the view that α-internexin is a fourth subunit of neurofilaments in the adult CNS, providing a basis for its close relationship with neurofilaments in CNS diseases associated with neurofilament accumulation.

Neurofilaments Bind Tubulin and Modulate Its Polymerization

Neurofilaments assemble from three intermediate-filament proteins, contribute to the radial growth of axons, and are exceptionally stable. Microtubules are dynamic structures that assemble from tubulin dimers to support intracellular transport of molecules and organelles. We show here that neurofilaments, and other intermediate-filament proteins, contain motifs in their N-terminal domains that bind unassembled tubulin. Peptides containing such motifs inhibit the in vitro polymerization of microtubules and can be taken up by cultured cells in which they disrupt microtubules leading to altered cell shapes and an arrest of division. In transgenic mice in which neurofilaments are withheld from the axonal compartment, axonal tubulin accumulation is normal but microtubules assemble in excessive numbers. These observations suggest a model in which axonal neurofilaments modulate local microtubule assembly. This capacity also suggests novel mechanisms through which inherited or acquired disruptions in intermediate filaments might contribute to pathogenesis in multiple conditions.

Axonal Neurofilaments Control Multiple Fiber Properties But Do Not Influence Structure or Spacing of Nodes of Ranvier

In the vertebrate nervous system, axon calibers correlate positively with myelin sheath dimensions and electrophysiological parameters including action potential amplitude and conduction velocity. Neurofilaments, a prominent component of the neuronal cytoskeleton, are required by axons to support their normal radial growth. To distinguish between fiber features that arise in response to absolute axon caliber and those that are under autonomous control, we investigated transgenic mice in which neurofilaments are sequestered in neuronal cell bodies. The neurofilament deficient axons in such mice achieve mature calibers only 50% of normal and have altered conduction properties. We show here that this primary axonal defect also induces multiple changes in myelin sheath composition and radial dimensions. Remarkably, other fundamental fiber features, including internodal spacing and the architecture and composition of nodes of Ranvier, remain unaltered. Thus, many fiber characteristics are controlled through mechanisms operating independently of absolute axon caliber and the neurofilament cytoskeleton.

Separate Proteolipid Protein/DM20 Enhancers Serve Different Lineages and Stages of Development

The gene encoding DM20 emerged in cartilaginous fish, descending from a bilaterian ancestor of the M6 proteolipid gene family. Its proteolipid protein (PLP) isoform appeared in amphibians, contains an additional 35 amino acids, and, in the mammalian CNS, is the dominant myelin protein in which it confers an essential neuroprotective function. During development, the DM20 isoform is prominent in a number of tissues, and plp/DM20 transcripts are detected in multiple progenitor populations, including those that continue to express plp/DM20 as they differentiate into myelinating oligodendrocytes. The locus also encodes isoforms with extended leader sequences that accumulate in the cell bodies of several types of neurons. Here, to locate and characterize regulatory sequences controlling the complex plp/DM20 transcription program, putative regulatory sequences, suggested by interspecies conservation, were ligated individually to a minimally promoted eGFPlacZ reporter gene. These constructs were inserted in single copy at a common site adjacent to the hypoxanthine-guanine phosphoribosyltransferase locus in embryonic stem cells and their in vivo expression programs were compared in transgenic mice. Most expressed developmental and cell-specific subprograms accommodated within the known expression phenotype of the endogenous plp/DM20 locus, thus defining multiple components of the combinatorial mechanism controlling its normal temporal and cell-specific program. Along with previously characterized nervous system enhancers, those described here should help expose the content and configuration of elements that are operational in multiple glial and neuronal lineages. The transgenic lines derived here also provide effective markers for multiple stages of glial and neuronal lineage progression.

A Methylation Mosaic Model for Mammalian Genome Imprinting

Publisher Summary This chapter presents a methylation mosaic model for mammalian genome imprinting. This model predicts that the paternally transmitted allele at some loci will be unavailable for appropriate expression, while at other loci the expression of the maternally transmitted allele will be affected. The introduction of new genetic material into the mouse genome by microinjection of DNA has provided a powerful tool with which to investigate gamete-of-origin-dependent DNA methylation. Matings between transgenic and nontransgenic animals yield offspring in which the transgene is inherited from only one parent. Therefore, potential gamete-of-origin-dependent differences in DNA methylation at these loci can be directly assayed in such offspring. If the gamete-of-origin- dependent methylation changes demonstrated for a number of hemizygous transgene loci are directly related to the developmental failure of gynogenotes and androgenotes, several additional observations made in these experiments are unexpected. In the simplest model, the hypomethylation of an allele would result in the expression of that allele, while the hypermethylation of that allele would result in no expression.

Expression of Human Neurofilament‐light Transgene in Mouse Neurons Transplanted into the Brain of Adult Rats

To investigate the expression of nerve cell‐specific transgene products in neural transplants, we implanted into the hippocampus of immunosuppressed adult Sprague – Dawley rats cell suspensions obtained from the septal region of the fetal brain of mice that carry the human neurofilament‐light (hNF‐L) gene. In grafts examined between 3 weeks and 7 months after transplantation, axons and nerve cell somata immunoreacted to antibodies specific to the human NF‐L subunit. Thus, the hNF‐L protein appears to be a suitable marker of these grafted neurons. Transgenic mice bearing the hNF‐L gene may be a convenient source of donor tissue or be used as hosts for neural transplantation studies. Furthermore, the hNF‐L promoter/enhancer elements in this transgene may help direct neuronal expression of heterologous genes that could influence nerve cell responses in either the transplant or host tissues.