Neurofilament mRNA Research Articles

Simultaneous up‐regulation of neurofilament proteins during the postnatal development of the rat nervous system

The expression of neurofilament (NF) proteins was examined during postnatal development of the rat nervous system in order to elucidate the nature of NF expression during the period of transition from the embryonic (immature) to the adult (mature) stages of NF expression. mRNAs to the light (NF-L), mid-sized (NF-M), and heavy (NF-H) NF proteins were compared by Northern blots and by in situ hybridizations, in NF-rich (i.e., DRG, spinal cord and brainstem) and in NF-poor (i.e., cerebellum and cerebral cortex) regions of the developing rat nervous system. NF proteins were analyzed by gel electrophoresis and by immunoblots. In each tissue, the expression of NF-H was delayed compared to that of NF-L and NF-M, as previously reported. The present study now shows that the delayed expression of NF-H is accompanied by parallel up-regulations in the expressions of NF-L and NF-M, both at the levels of mRNA and protein. Similar rates of increase of all three NF mRNAs occur between postnatal days 5 (P5) and 24 (P24) in rat spinal cord and DRG. Furthermore, the postnatal up-regulation of NF expression is characterized by a progressive accumulation of all three NF proteins in the tissues. The findings indicate that the adult pattern of NF expression (with high levels of expression of all three NF proteins) becomes established during the postnatal period of development, raising questions as to the nature of factors that coregulate the expression of NF subunits.

Expression and assembly of a human neurofilament protein in transgenic mice provide a novel neuronal marking system.

To investigate the regulation of neurofilament gene expression, we have generated several lines of transgenic mice carrying multiple copies of a cloned human neurofilament (NF-L) gene. We show that a 21.5-kb DNA fragment including the human NF-L gene contains essential information for correct expression in nervous tissue of transgenic mice. The integrated genes are arranged in multiple tandem arrays, but the extent of transgene expression does not correlate with copy number nor does it influence the expression of the endogenous neurofilament genes. However, the levels of human NF-L protein recovered in neurofilament preparations from brains of transgenic mice correlate directly with the relative abundance of human NF-L mRNA detected in each line. There is an apparent delay in the accumulation of human NF-L protein during development, as determined by immunoblotting with a human-specific monoclonal antibody. Finally, immunohistochemical localization of the human NF-L protein results in the specific staining of neurons and their processes in transgenic mice.

Identification of gene products expressed in the developing chick visual system: characterization of a middle-molecular-weight neurofilament cDNA.

A cDNA library enriched for transcripts that accumulate from embryonic day 7 until day 1 posthatching was constructed from poly(A)+ RNA of chick optic lobe tissue. From this library, three recombinants were isolated that correspond to neural mRNAs appearing during the major period of synaptogenesis in the retinotectal system. One of these recombinants (OZ 11) was identified as a middle-molecular weight neurofilament (NF-M) cDNA which, together with two overlapping clones, encodes part of the rod and the entire tail region of NF-M. In situ hybridization revealed NF-M mRNA to be highly expressed in regions of the chick central nervous system, which contain multipolar and/or long-projection neurons.

Cloning and developmental expression of the murine neurofilament gene family

DNA clones encoding the 3 mouse neurofilament (NF) genes have been isolated by cross-hybridization with a previously described NF-L cDNA probe from the rat. Screening of a λgt10 cDNA library prepared from mouse brain RNA led to the cloning of an NF-L cDNA of 2.0 kb that spans the entire coding region of 541 amino acids and of an NF-M cDNA that covers 219 amino acids from the internal α-helical region and the carboxy-terminal domains of the protein. These cDNA clones were used as probes to screen mouse genomic libraries, and cosmid clones containing both NF-L and NF-M sequences were isolated as well as overlapping cosmids containing the NF-H gene. This strongly suggests that the 3 neurofilament genes are organised in a cluster and derived by gene duplication of a common ancestral gene. RNA blot analyses using specific DNA probes for each of the genes indicate that NF mRNAs are differentially expressed during brain development. The NF-L and NF-M mRNAs are detected early in the embryonal brain, with a progressive increase in their levels during development, while the NF-H mRNA is barely detectable at embryonal stages and accumulates later in the postnatal brain.

Elevated Levels of Retinal Neurofilament mRNA Accompany Optic Nerve Regeneration

RNA isolated from goldfish retinas before and during optic nerve regeneration, when translated in vitro, directed the synthesis of neurofilament proteins that are normally found in high levels in the optic nerve. The major neurofilament proteins of the goldfish optic nerve comprise a group of four isoelectric variants of molecular weight 58,000 (58K) which we have identified previously as ON1-ON4. The levels of ON1 and ON2 within the optic nerve had been shown to decrease shortly after optic nerve crush and then increase to precrush levels during the regeneration process. Employing two-dimensional electrophoretic analysis of in vitro translation products and immunoprecipitations with antibodies specific for the ON proteins and an anti-intermediate filament monoclonal antibody, we show that ON1 and ON2 are encoded by mRNA synthesized in the retinas. The synthesis of ON3 and ON4 by retina RNA was undetected. This confirms data from previous ex vivo experiments that indicated that ON1 and ON2 are of neuronal origin whereas ON3 and ON4 are nonneuronal. ON1 and ON2 synthesis increases dramatically during optic nerve regeneration to levels 10- and 30-fold over precrush levels, respectively. In addition to ON1 and ON2, the synthesis of a previously unidentified 52K protein is observed at relatively high levels 20 and 32 days after optic nerve crush, but is unobserved before regeneration. Thus, optic nerve regeneration can be correlated with specific changes in intermediate filament gene expression within the retina.

Effects of nerve growth factor and dibutyryl cyclic AMP on neurofilament, γ-enolase and THY-1 mRNA levels in cultured pheochromocytoma and neuroblastoma cells

Effects of nerve growth factor and dibutyryl cyclic AMP on neurofilament, γ-enolase and THY-1 mRNA levels in cultured pheochromocytoma and neuroblastoma cells

OPTIC NERVE REGENERATION WITH RETURN OF VISION IN ANURANS

OPTIC NERVE REGENERATION WITH RETURN OF VISION IN ANURANS