Characterization of the mouse gene encoding the neuronal intermediate filament protein α-internexin

doi:10.1016/0378-1119(94)90163-5

Gene

Volume 149, Issue 2, 18 November 1994, Pages 289-292

https://doi.org/10.1016/0378-1119(94)90163-5 Get rights and content

Abstract

We have determined the complete nucleotide (nt) sequence of the coding region of the mouse gene encoding the neuronal intermediate filament protein, a-internexin (αINX). The mouse αINX gene (mαINX) contains three exons and two introns, and may be classified as a member of the type-IV intermediate filament multigene family. The nt sequence of the transcribed region of mαINX shows high homology to that of the rat gene. Comparison of the deduced amino-acid sequences between the mouse and rat gene products reveals that the head and rod domains are highly conserved. However, the tail domains show significant differences which may make it possible to raise specific antibodies that can distinguish the αINX of mouse from that of rat.

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We previously identified the E693Δ mutation in amyloid precursor protein (APP) in patients with Alzheimer's disease (AD) and then generated APP-transgenic mice expressing this mutation. As these mice possessed abundant Aβ oligomers from 8 months of age but no amyloid plaques even at 24 months of age, they are a good model to study pathological effects of amyloid β (Aβ) oligomers. The two-dimensional fluorescence difference in gel electrophoresis (2D-DIGE) technology, using a mixed-sample internal standard, is now recognized as an accurate method to determine and quantify proteins. In this study, we examined the proteins for which levels were altered in the hippocampus of 12-month-old APP_E693Δ-transgenic mice using 2D-DIGE and liquid chromatography–tandem mass spectrometry (LC–MS/MS). Fourteen proteins were significantly changed in the hippocampus of APP_E693Δ-transgenic mice. Actin cytoplasmic 1 (β-actin), heat shock cognate 71 kDa, γ-enolase, ATP synthase subunit β, tubulin β-2A chain, clathrin light chain B (clathrin) and dynamin-1 were increased. Heat shock-related 70 kDa protein 2, neurofilament light polypeptide (NFL), stress-induced-phosphoprotein 2, 60 kDa heat shock protein (HSP60), α-internexin, protein kinase C and casein kinase substrate in neurons protein 1 (Pacsin 1), α-enolase and β-actin were decreased. Western blotting also validated the changed levels of HSP60, NFL, clathrin and Pacsin 1 in APP_E693Δ-transgenic mice. The identified proteins could be classified as cytoskeleton, chaperons, neurotransmission, energy supply and signal transduction. Thus, proteomics by 2D-DIGE and LC–MS/MS has provided knowledge of the levels of proteins in the early stages of AD brain.
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Citation Excerpt :
The brain specific type IV intermediate filament protein α-internexin is mainly expressed by immature neurons preceding the expression of neuronal intermediate filament proteins (Kaplan et al., 1990), and is widely expressed during development and through adulthood. It is a major component of the intermediate filament network in small interneurons and cerebellar granule cells (Chien and Liem, 1994; Pachter and Liem, 1985). The neuron-specific cytoskeletal protein βΙΙΙ-tubulin is expressed at the earliest phase of neuronal differentiation and strongest during periods of axon guidance and maturation (Liu et al., 2007; Tischfield et al., 2010).
The embryonic stem cell test (EST) is a validated method to assess the developmental toxicity potency of chemicals. It was developed to reduce animal use and allow faster testing for hazard assessment. The cells used in this method are maintained and differentiated in media containing foetal calf serum. This animal product is of considerable variation in quality, and individual batches require extensive testing for their applicability in the EST. Moreover, its production involves a large number of foetuses and possible animal suffering. We demonstrate the serum-free medium and feeder cell-free maintenance of the mouse embryonic stem cell line D3 and investigate the use of specific growth factors for induction of cardiac differentiation. Using a combination of bone morphogenetic protein-2, bone morphogenetic protein-4, activin A and ascorbic acid, embryoid bodies efficiently differentiated into contracting myocardium. Additionally, examining levels of intracellular marker proteins by flow cytometry not only confirmed differentiation into cardiomyocytes, but demonstrated significant differentiation into neuronal cells in the same time frame. Thus, this approach might allow for simultaneous detection of developmental effects on both early mesodermal and neuroectodermal differentiation. The serum-free conditions for maintenance and differentiation of D3 cells described here enhance the transferability and standardisation and hence the performance of the EST.
Chapter 12 Cytoskeletal Abnormalities in Amyotrophic Lateral Sclerosis/Motor Neuron Disease
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This chapter discusses the role of neurofilaments (NFs) and other cytoskeletal proteins in motor neuron disease (MND) with particular emphasis on recent transgenic mouse studies. Evidence indicates that cytoskeletal abnormalities may contribute through different molecular mechanisms to the pathogenesis of MND. Experiments with transgenic mice and cultured neuronal cells suggest that certain types of neurofilamentous accumulations similar to those found in amyotrophic lateral sclerosis (ALS) could provoke neurodegeneration. Transgenic studies reveal that deleterious intermediate ﬁlament (IF) structures could result from a variety of primary causes, including NF gene mutations or overexpression of genes encoding peripherin or of proteins associated with microtubule-based transport. Severe motor neuron loss occurred frequently in transgenic mouse models exhibiting an axonal localization of IF swellings. This supports the axonal strangulation model by which axonal IF accumulations may impede the transport machinery.
Pathways to motor neuron degeneration in transgenic mouse models
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Amyotrophic lateral sclerosis (ALS) is an adult-onset neurological disorder characterized by the selective loss of motor neurons. A pathological hallmark of both sporadic and familial ALS is the presence of abnormal accumulations of neurofilament and peripherin proteins in motor neurons. In the past decade, transgenic mouse approaches have been used to address the role of such cytoskeletal abnormalities in motor neuron disease and also to unravel the pathogenesis caused by mutations in the gene coding for superoxide dismutase 1 (SOD1) that account for ~20% of familial ALS cases. In mouse models, disparate effects could result from different types of intermediate filament (IF) aggregates. Perikaryal IF accumulations induced by the overexpression of any of the three wild-type neurofilament proteins were quite well tolerated by motor neurons. Indeed, perikaryal swellings provoked by NF-H overexpression can even confer protection against toxicity of mutant SOD1. Other types of IF aggregates seem neurotoxic, such as those found in transgenic mice overexpressing either peripherin or an assembly-disrupting NF-L mutant. Moreover, understanding the toxicity of SOD1 mutations has been surprisingly difficult. The analysis of transgenic mice expressing mutant SOD1 has yielded complex results, suggesting that multiple pathways may contribute to disease that include the involvement of non-neuronal cells.
No requirement of α-internexin for nervous system development and for radial growth of axons
1999, Molecular Brain Research
α-Internexin is a type IV intermediate filament protein that is expressed abundantly in neurons during development of the peripheral and central nervous systems as well as in few neurons of the adult central nervous system. It has been suggested that α-internexin may act as a scaffold for the formation of neuronal intermediate filaments during early development. In addition, recent reports suggest that α-internexin could play a major role in two degenerative neurological disorders. We report here an analysis of mice with a targeted disruption of α-internexin gene. Unexpectedly, α-internexin −/− mice developed normally and did not exhibit overt phenotypes. Moreover, the absence of α-internexin did not interfere with neurite extension of cultured DRG neurons. The number and caliber of L4 ventral root axons remained unchanged in α-internexin −/− mice. In the retina, α-internexin begins to be expressed in retinal ganglion cells when their first axons reach the optic chiasma. Using HRP tracer, we show that the projection pattern of the RGC axons is not modified by the absence of α-internexin. Electron microscopy did not reveal significant differences in axonal calibers, in myelination of axons and in neurofilament structures between α-internexin −/− and control mice during development and at adult stage. These data indicate that α-internexin is not required for the polymerization of neurofilament in vivo. Mice deficient for both α-internexin and neurofilament light chain (NF-L) exhibited no over phenotypes as well. No intermediate filament structures were detectable in optic nerve of α-internexin −/−; NF-L −/− mice. Ours results do not support the hypothesis of a role for type IV intermediate filaments in axonal outgrowth during development of nervous system.
Gefiltin in zebrafish embryos: Sequential gene expression of two neurofilament proteins in retinal ganglion cells
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Neurogenesis is correlated with the progressive synthesis of diverse neuronal intermediate filaments (IF) proteins. This apparent developmental regulation of IF protein gene expression suggests that specific neurofilament proteins impart unique structural attributes that support the staged growth of the neuron. In the teleost visual pathway, the sequential expression of two IF genes, plasticin and gefiltin, is linked to the age of retinal ganglion cells (RGCs) and to the regeneration of optic axons after nerve injury. Given this pattern of plasticin and gefiltin expression, we hypothesized that the two proteins would be sequentially expressed in zebrafish retina during development. We analyzed the pattern of gefiltin expression during zebrafish development and compared it to our previous determination of plasticin expression (Canger et al. 1998). Gefiltin is expressed after plasticin, during the later stages of retinal development when axons grow past the optic chiasm and innervate their targets. Thus, during RGC development, expression of plasticin and gefiltin resembles that with optic nerve regeneration. Outside of the visual pathway, gefiltin is predominantly expressed in the central nervous system whereas plasticin is primarily expressed in the peripheral nervous system. These results suggest that the expression of these genes is regulated in a neuron-specific manner. In addition, since plasticin and gefiltin are co-expressed during RGC development, these findings suggest a more complex mechanism of transcriptional regulation which orchestrates the sequential expression of these genes.

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Characterization of the mouse gene encoding the neuronal intermediate filament protein α-internexin

Abstract

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

The predicted amino acid sequence of α-internexin is that of a novel neuronal intermediate filament protein

EMBO J.