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.

[1]  J. Hardy,et al.  Mutation analysis of patients with neuronal intermediate filament inclusion disease (NIFID) , 2006, Neurobiology of Aging.

[2]  C. Gilbert,et al.  Axons and Synaptic Boutons Are Highly Dynamic in Adult Visual Cortex , 2006, Neuron.

[3]  T. Gotow,et al.  Aggregate formation and phosphorylation of neurofilament-L Pro22 Charcot-Marie-Tooth disease mutants. , 2006, Human molecular genetics.

[4]  R. Nixon,et al.  Deleting the phosphorylated tail domain of the neurofilament heavy subunit does not alter neurofilament transport rate in vivo , 2006, Neuroscience Letters.

[5]  M. Docquier,et al.  No widespread induction of cell death genes occurs in pure motoneurons in an amyotrophic lateral sclerosis mouse model. , 2005, Human molecular genetics.

[6]  D. Cleveland,et al.  Altered axonal architecture by removal of the heavily phosphorylated neurofilament tail domains strongly slows superoxide dismutase 1 mutant-mediated ALS. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[7]  N. Cairns,et al.  Neuronal intranuclear inclusions are ultrastructurally and immunologically distinct from cytoplasmic inclusions of neuronal intermediate filament inclusion disease , 2005, Acta Neuropathologica.

[8]  A. Petzold Neurofilament phosphoforms: Surrogate markers for axonal injury, degeneration and loss , 2005, Journal of the Neurological Sciences.

[9]  S. Endo,et al.  p55 protein is a member of PSD scaffold proteins in the rat brain and interacts with various PSD proteins. , 2005, Brain research. Molecular brain research.

[10]  J. Ratnam,et al.  Neurofilament‐light increases the cell surface expression of the N‐methyl‐d‐aspartate receptor and prevents its ubiquitination , 2005, Journal of neurochemistry.

[11]  C. Theiss,et al.  Impairment of anterograde and retrograde neurofilament transport after anti-kinesin and anti-dynein antibody microinjection in chicken dorsal root ganglia. , 2005, European journal of cell biology.

[12]  S. Endo,et al.  A novel scaffold protein, TANC, possibly a rat homolog of Drosophila rolling pebbles (rols), forms a multiprotein complex with various postsynaptic density proteins , 2005, The European journal of neuroscience.

[13]  M. Smith,et al.  Neurofilament proteins in neurodegenerative diseases , 2004, Cellular and Molecular Life Sciences CMLS.

[14]  V. Meininger,et al.  A Frameshift Deletion in Peripherin Gene Associated with Amyotrophic Lateral Sclerosis* , 2004, Journal of Biological Chemistry.

[15]  J. Trojanowski,et al.  Clinical and neuropathologic variation in neuronal intermediate filament inclusion disease , 2004, Neurology.

[16]  S. Chin,et al.  A Pathogenic Peripherin Gene Mutation in a Patient with Amyotrophic Lateral Sclerosis , 2004, Brain pathology.

[17]  J. Trojanowski,et al.  alpha-internexin is present in the pathological inclusions of neuronal intermediate filament inclusion disease. , 2004, The American journal of pathology.

[18]  J. Trojanowski,et al.  α-Internexin aggregates are abundant in neuronal intermediate filament inclusion disease (NIFID) but rare in other neurodegenerative diseases , 2004, Acta Neuropathologica.

[19]  S. Züchner,et al.  The novel neurofilament light (NEFL) mutation Glu397Lys is associated with a clinically and morphologically heterogeneous type of Charcot-Marie-Tooth neuropathy , 2004, Neuromuscular Disorders.

[20]  Sameer B. Shah,et al.  NF-M is an essential target for the myelin-directed “outside-in” signaling cascade that mediates radial axonal growth , 2003, The Journal of cell biology.

[21]  G. Elder,et al.  Human midsized neurofilament subunit induces motor neuron disease in transgenic mice , 2003, Experimental Neurology.

[22]  J. Julien,et al.  Neurofilament Transport In Vivo Minimally Requires Hetero-Oligomer Formation , 2003, The Journal of Neuroscience.

[23]  P. Janmey,et al.  Mechanisms of Mitochondria-Neurofilament Interactions , 2003, The Journal of Neuroscience.

[24]  Nick C Fox,et al.  Neurofilament inclusion body disease: a new proteinopathy? , 2003, Brain : a journal of neurology.

[25]  Ian G. McKeith,et al.  Patients with a novel neurofilamentopathy: dementia with neurofilament inclusions , 2003, Neuroscience Letters.

[26]  Xinran Liu,et al.  Abnormal neurofilament transport caused by targeted disruption of neuronal kinesin heavy chain KIF5A , 2003, The Journal of cell biology.

[27]  Ammar Al-Chalabi,et al.  Neurofilaments and neurological disease. , 2003, BioEssays : news and reviews in molecular, cellular and developmental biology.

[28]  J. Lupski,et al.  Mutations in the neurofilament light chain gene (NEFL) cause early onset severe Charcot-Marie-Tooth disease. , 2003, Brain : a journal of neurology.

[29]  G. Sobue,et al.  Identification of novel sequence variants in the neurofilament‐light gene in a Japanese population: analysis of Charcot‐Marie‐Tooth disease patients and normal individuals , 2002, Journal of the peripheral nervous system : JPNS.

[30]  J. Julien,et al.  Myosin Va binding to neurofilaments is essential for correct myosin Va distribution and transport and neurofilament density , 2002, The Journal of cell biology.

[31]  J. Zidar,et al.  A novel NF-L mutation Pro22Ser is associated with CMT2 in a large Slovenian family , 2002, Neurogenetics.

[32]  N. Calcutt,et al.  Gene replacement in mice reveals that the heavily phosphorylated tail of neurofilament heavy subunit does not affect axonal caliber or the transit of cargoes in slow axonal transport , 2002, The Journal of cell biology.

[33]  D. Sibley,et al.  Neurofilament-M Interacts with the D1 Dopamine Receptor to Regulate Cell Surface Expression and Desensitization , 2002, The Journal of Neuroscience.

[34]  W. Streit,et al.  Axonally Transported Peripheral Signals Regulate α-Internexin Expression in Regenerating Motoneurons , 2002, The Journal of Neuroscience.

[35]  J. Undamatla,et al.  Differential expression and localization of neuronal intermediate filament proteins within newly developing neurites in dissociated cultures of Xenopus laevis embryonic spinal cord. , 2001, Cell motility and the cytoskeleton.

[36]  C. van Broeckhoven,et al.  Further evidence that neurofilament light chain gene mutations can cause Charcot‐Marie‐Tooth disease type 2E , 2001, Annals of neurology.

[37]  U. di Porzio,et al.  Neurofilament homeostasis and motoneurone degeneration , 2000, BioEssays : news and reviews in molecular, cellular and developmental biology.

[38]  R. Mills,et al.  Tubulin and Neurofilament Proteins Are Transported Differently in Axons of Chicken Motoneurons , 2000, Cellular and Molecular Neurobiology.

[39]  O. Evgrafov,et al.  A new variant of Charcot-Marie-Tooth disease type 2 is probably the result of a mutation in the neurofilament-light gene. , 2000, American journal of human genetics.

[40]  Q. Zhu,et al.  Disruption of Type IV Intermediate Filament Network in Mice Lacking the Neurofilament Medium and Heavy Subunits , 1999, Journal of neurochemistry.

[41]  J. Julien,et al.  No requirement of α-internexin for nervous system development and for radial growth of axons , 1999 .

[42]  F. Araya,et al.  Ultrastructural pathology of a Chilean case of tropical spastic paraparesis/human T-cell lymphotropic type I-associated myelopathy (TSP/HAM). , 1999, Ultrastructural pathology.

[43]  M. Faussone‐Pellegrini,et al.  The cytoskeleton of the myenteric neurons during murine embryonic life , 1999, Anatomy and Embryology.

[44]  R. Liem,et al.  Overexpression of α-Internexin Causes Abnormal Neurofilamentous Accumulations and Motor Coordination Deficits in Transgenic Mice , 1999, The Journal of Neuroscience.

[45]  T. Crawford,et al.  Neurofilament-dependent Radial Growth of Motor Axons and Axonal Organization of Neurofilaments Does Not Require the Neurofilament Heavy Subunit (NF-H) or Its Phosphorylation , 1998, The Journal of cell biology.

[46]  V. Tung,et al.  Antagonistic Roles of Neurofilament Subunits NF-H and NF-M Against NF-L in Shaping Dendritic Arborization in Spinal Motor Neurons , 1998, The Journal of cell biology.

[47]  Mark Ellisman,et al.  Specific interaction of HTLV tax protein and a human type IV neuronal intermediate filament protein. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[48]  R. Huganir,et al.  Splice Variant-Specific Interaction of the NMDA Receptor Subunit NR1 with Neuronal Intermediate Filaments , 1998, The Journal of Neuroscience.

[49]  J. Julien,et al.  Delayed Maturation of Regenerating Myelinated Axons in Mice Lacking Neurofilaments , 1997, Experimental Neurology.

[50]  J. Bamburg,et al.  Axonal transport and distribution of cyclophilin A in chicken neurones , 1997, Brain Research.

[51]  T. Fujii,et al.  Excitable membranes and synaptic transmission: postsynaptic mechanisms. Localization of α-internexin in the postsynaptic density of the rat brain , 1997, Brain Research.

[52]  J. Gallo,et al.  Incorporation of NF‐L into keratin filaments in transfected epithelial cells , 1997, Neuroreport.

[53]  B. Giasson,et al.  Developmentally regulated stabilization of neuronal intermediate filaments in rat cerebral cortex , 1997, Neuroscience Letters.

[54]  T. Kumanishi,et al.  Isolation of cDNA clones of the rat mRNAs expressed preferentially in the prenatal stages of brain development. , 1996, Brain research. Developmental brain research.

[55]  C. Mason,et al.  alpha-Internexin is the only neuronal intermediate filament expressed in developing cerebellar granule neurons. , 1996, Journal of neurobiology.

[56]  R. Porchet,et al.  Differential expression and modification of neurofilament triplet proteins during cat cerebellar development , 1996, The Journal of comparative neurology.

[57]  U. Lendahl,et al.  Nestin mRNA expression correlates with the central nervous system progenitor cell state in many, but not all, regions of developing central nervous system. , 1995, Brain research. Developmental brain research.

[58]  D. Gajdusek,et al.  Ultrastructural pathology of human T-cell lymphotropic virus type I encephalomyelopathy in a white patient with adult T-cell leukemia/lymphoma. , 1994, Ultrastructural pathology.

[59]  R. Liem,et al.  Expression of the gene for the neuronal intermediate filament protein α‐internexin coincides with the onset of neuronal differentiation in the developing rat nervous system , 1994, The Journal of comparative neurology.

[60]  J. Griffin,et al.  Phosphorylation‐Dependent Immunoreactivity of Neurofilaments and the Rate of Slow Axonal Transport in the Central and Peripheral Axons of the Rat Dorsal Root Ganglion , 1994, Journal of neurochemistry.

[61]  A. Peterson,et al.  Neurofilament-deficient axons and perikaryal aggregates in viable transgenic mice expressing a neurofilament-β-galactosidase fusion protein , 1994, Neuron.

[62]  R. Liem,et al.  Assembly of type IV neuronal intermediate filaments in nonneuronal cells in the absence of preexisting cytoplasmic intermediate filaments , 1993, The Journal of cell biology.

[63]  L. Cork,et al.  Increased expression of neurofilament subunit NF-L produces morphological alterations that resemble the pathology of human motor neuron disease , 1993, Cell.

[64]  M. Costa,et al.  Selective distribution of the 66-kDa neuronal intermediate filament protein in the sensory and autonomic nervous system of the guinea-pig , 1992, Brain Research.

[65]  P. Burton,et al.  Neurofilaments are prominent in bullfrog olfactory axons but are rarely seen in those of the tiger salamander, Ambystoma tigrinum , 1992, The Journal of comparative neurology.

[66]  R. Goldman,et al.  Some neural intermediate filaments contain both peripherin and the neurofilament proteins , 1991, Journal of neuroscience research.

[67]  M. Kaplan,et al.  Alpha-internexin, a novel neuronal intermediate filament protein, precedes the low molecular weight neurofilament protein (NF-L) in the developing rat brain , 1990, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[68]  R. McKay,et al.  CNS stem cells express a new class of intermediate filament protein , 1990, Cell.

[69]  M. Vitadello,et al.  Expression of neurofilament proteins in granule cells of the cerebellum , 1990, Brain Research.

[70]  F. Chiu,et al.  Characterization of a novel 66 kd subunit of mammalian neurofilaments , 1989, Neuron.

[71]  D. Cleveland,et al.  Expression of NF-L and NF-M in fibroblasts reveals coassembly of neurofilament and vimentin subunits , 1989, The Journal of cell biology.

[72]  D. Dahl,et al.  Expression of neurofilament immunoreactivity in developing rat cerebellum in vitro and in vivo , 1989, Neuroscience Letters.

[73]  K. Tokuyasu Application of cryoultramicrotomy to immunocytochemistry , 1986, Journal of microscopy.

[74]  F. Grosveld,et al.  Specific detection of neuronal cell bodies: in situ hybridization with a biotin-labeled neurofilament cDNA probe. , 1986, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[75]  R. Nixon,et al.  Multiple fates of newly synthesized neurofilament proteins: evidence for a stationary neurofilament network distributed nonuniformly along axons of retinal ganglion cell neurons , 1986, The Journal of cell biology.

[76]  R. Liem,et al.  Microtubule-associated proteins bind specifically to the 70-kDa neurofilament protein. , 1985, The Journal of biological chemistry.

[77]  J. Pachter,et al.  alpha-Internexin, a 66-kD intermediate filament-binding protein from mammalian central nervous tissues , 1985, The Journal of cell biology.

[78]  K. Angelides,et al.  Characterization of mammalian neurofilament triplet proteins. Subunit stoichiometry and morphology of native and reconstituted filaments. , 1985, The Journal of biological chemistry.

[79]  J. Pachter,et al.  The differential appearance of neurofilament triplet polypeptides in the developing rat optic nerve. , 1984, Developmental biology.

[80]  N. Hirokawa,et al.  Organization of mammalian neurofilament polypeptides within the neuronal cytoskeleton , 1984, The Journal of cell biology.

[81]  R. Nixon Increased axonal proteolysis in myelin-deficient mutant mice. , 1982, Science.

[82]  Jean-Pierre Julien,et al.  Functions of intermediate filaments in neuronal development and disease. , 2004, Journal of neurobiology.

[83]  D. Dickson,et al.  Neuroaxonal dystrophy in HTLV-1-associated myelopathy/tropical spastic paraparesis: neuropathologic and neuroimmunologic correlations , 2004, Acta Neuropathologica.

[84]  Q. Zhu,et al.  No requirement of alpha-internexin for nervous system development and for radial growth of axons. , 1999, Brain research. Molecular brain research.

[85]  J. Julien,et al.  Interactions between peripherin and neurofilaments in cultured cells: disruption of peripherin assembly by the NF-M and NF-H subunits. , 1999, Biochemistry and cell biology = Biochimie et biologie cellulaire.

[86]  T. Shea,et al.  Neuronal intermediate filament protein alpha-internexin facilitates axonal neurite elongation in neuroblastoma cells. , 1999, Cell motility and the cytoskeleton.

[87]  A. Brown Visualization of single neurofilaments by immunofluorescence microscopy of splayed axonal cytoskeletons. , 1997, Cell motility and the cytoskeleton.

[88]  J. Mandell,et al.  Compartmentation of alpha-internexin and neurofilament triplet proteins in cultured hippocampal neurons , 1996, Journal of neurocytology.

[89]  R. Nixon,et al.  Dynamics of neuronal intermediate filaments: a developmental perspective. , 1992, Cell motility and the cytoskeleton.

[90]  F. Gros,et al.  Peripherin, a new member of the intermediate filament protein family. , 1983, Developmental neuroscience.

[91]  G. Warren,et al.  Immunoelectron microscopy using thin, frozen sections: application to studies of the intracellular transport of Semliki Forest virus spike glycoproteins. , 1983, Methods in enzymology.