The complexity of intermediate filaments in the nervous system.
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Animals↗
Biomedical subjects
R K Liem
Publications and source records attributed to R K Liem.
Assembly properties of amino- and carboxyl-terminally truncated neurofilament NF-H proteins with NF-L and NF-M in the presence and absence of vimentin.
To understand the assembly characteristics of the high-molecular-weight neurofilament protein (NF-H), carboxyl- and amino-terminally deleted NF-H proteins were examined by transiently cotransfecting mutant NF-H constructs with the other neurofilament triplet proteins, low- and middle-molecular-weight neurofilament protein (NF-L and NF-M, respectively), in the presence or absence of cytoplasmic vimentin. The results confirm that NF-H can coassemble with vimentin and NF-L but not with NF-M into filamentous networks. Deletions from the amino-terminus show that the N-terminal head is necessary for the coassembly of NF-H with vimentin, NF-L, or NF-M/vimentin. However, headless NF-H or NF-H from which the head and a part of the rod is removed can still incorporate into an NF-L/vimentin network. Deletion of the carboxyl-terminal tail of NF-H shows that this region is not essential for coassembly with vimentin but is important for coassembly with NF-L into an extensive filamentous network. Carboxyl-terminal deletion into the alpha-helical rod results in a dominant-negative mutant, which disrupts all the intermediate filament networks. These results indicate that NF-L is the preferred partner of NF-H over vimentin and NF-M, the head region of NF-H is important for the formation of NF-L/NF-H filaments, and the tail region of NF-H is important to form an extensive network of NF-L/NF-H filaments.
Base Sequence↗
Phosphorylation of the high molecular weight neurofilament protein (NF-H) by Cdk5 and p35.
The high molecular weight neurofilament protein (NF-H) is highly phosphorylated in the axon. The phosphorylation sites have been identified as KSP (Lys-Ser-Pro) repeats in the tail domain of NF-H. These KSP sequences are present more than 50 times in the NF-H tail, and most of these sites are normally phosphorylated in vivo. These KSP sites can be further divided into two separate consensus sequences, KSPXK and KSPXY (where Y is not K). The extensive phosphorylation of NF-H has been proposed to play a critical role in the determination of axonal diameter. Recent studies have shown that Cdk5, a kinase related to the cell cycle-dependent kinase Cdc2, is expressed in the brain and associates with the cytoskeleton. In vitro phosphorylation studies have shown that Cdk5 in conjunction with its activator, p35, is able to phosphorylate histone H1, dephosphorylated NF-H, as well as a synthetic peptide with the repetitive KSP motif. We have cloned the cDNAs for rat Cdk5 and p35 by reverse transcription-polymerase chain reaction and cDNA library screening and studied the phosphorylation of NF-H both in vivo and in vitro. By transient transfection assays, we have shown that NF-H can only be extensively phosphorylated in the presence of both Cdk5 and p35. This phosphorylation can be inhibited by a Cdk5-dominant negative mutant, an observation which further supports that Cdk5 is a kinase that is able to phosphorylate NF-H. By immunoprecipitating Cdk5 and p35 from the transfected cells, we have been able to show that the KSPXK repeats are the preferred phosphorylation sites for Cdk5, while the KSPXY repeats are not directly phosphorylated by Cdk5 and p35.
Amino Acid Sequence↗
Characterization of interactions between the neurofilament triplet proteins by the yeast two-hybrid system.
In the adult axon, the neurofilaments (NFs) are heteropolymers formed from the low (NFL), middle (NFM), and high (NFH) molecular weight neurofilament triplet proteins (NFTPs). All three proteins have the basic intermediate filament protein tripartite structure, which consists of a short amino-terminal head region, an alpha-helical rod region of approximately310 amino acids, and a carboxyl-terminal tail region of variable length. In vitro polymerization studies have shown that only NFL can assemble into homopolymeric 10-nm filaments. The assembly of intermediate filaments, including the NFs, begins with the formation of a coiled-coil dimer involving the alpha-helical rod domains of two molecules. In order to determine whether homodimers or heterodimers of NFTPs are the preferred intermediates in the assembly of NFs, we have used the yeast two-hybrid system to study the interactions between the different NFTPs. By monitoring the activity of the lacZ reporter gene product, we are able to show that the interactions of NFL with NFL, NFM, or NFH are stronger than the interactions of NFM with NFM or NFH and the interaction of NFH with NFH. These results imply that NFM and NFH are more likely to form heterodimers with NFL than homodimers and are consistent with the inability of NFM and NFH to self-polymerize in vitro and in vivo.
Animals↗
alpha-Internexin is the only neuronal intermediate filament expressed in developing cerebellar granule neurons.
We have used immunocytochemistry and in situ hybridization to examine the distribution of neuronal intermediate filament proteins and their mRNAs in the developing mouse cerebellum. First, we demonstrate that alpha-internexin is abundantly expressed in the developing cerebellum and is the only neuronal intermediate filament protein expressed in developing, including migrating, granule neurons. Second, in granule neuron reaggregates in vitro, alpha-internexin is the only neuronal intermediate filament protein highly expressed in the processes of the cultured granule neurons. This in vitro observation is consistent with results from immunocytochemistry and in situ hybridization studies of developing granule neurons in vivo, which suggest that alpha-internexin is the major neuronal intermediate filament protein in developing granule neurons. Finally, the neurofilament triplet proteins are expressed later, and coexist with alpha-internexin in other cells, including Purkinje cells and interneurons in the mature mouse cerebellum. These changes in neuronal intermediate filament composition may regulate neuronal maturation and axonal stability in cerebellar development. Furthermore, alpha-internexin may play a key role in neurite outgrowth and the establishment of neuronal cytoarchitecture.
Age Factors↗
Intermediate filaments in the nervous system: implications in cancer.
In this review, we describe the different intermediate filament (IF) proteins, their assembly into IFs, the functions of IFs and their relation to disease with a particular emphasis on the intermediate filaments expressed in the nervous system. In the mammalian nervous system, seven intermediate filament proteins are known to be expressed in neurons or neuroblasts. These include the three neurofilament triplet proteins, which are present in both central and peripheral neurons; alpha-internexin, which is the first neuronal intermediate filament protein expressed in the developing mammalian nervous system and present primarily in CNS neurons in the adult nervous system; peripherin, which is most abundant in the PNS; vimentin, which is expressed in neuronal progenitor cells along with nestin, as well as in a few adult neurons. In contrast to these neuron-specific IF proteins, the glial fibrillary acidic protein (GFAP) is glial specific and expressed in mature astrocytes. Vimentin and nestin are also expressed in glial progenitor cells and vimentin is expressed along with GFAP in some mature astrocytes. As a whole, the expression of IF proteins is tissue specific and developmentally regulated. As a result, IF proteins are good markers for determining the cell origin and differentiation status of tumor cells. For example, peripherin is expressed in neuroblastomas, GFAP in astrocytomas and neurofilaments in tumors of neuronal origin. However, tumor cells may express IF patterns which are irrelevant to their cell origin. Therefore, one has to be very careful in using IF patterns as sole indicators of cell origin and differentiation status of tumors.
Animals↗
Activation of the alpha-internexin promoter by the Brn-3a transcription factor is dependent on the N-terminal region of the protein.
The Brn-3a, Brn-3b, and Brn-3c proteins are closely related POU (Pit-Oct-Unc) family transcription factors which are expressed predominantly in neuronal cells. We have identified the alpha-internexin gene as the first reported, neuronally expressed, target gene whose promoter activity is modulated by these factors. Both the Brn-3a and Brn-3c factors can activate the alpha-internexin promoter while Brn-3b represses it and can prevent activation by Brn-3a. Using chimeric constructs containing different regions of Brn-3a or Brn-3b, we show that activation of the alpha-internexin promoter requires the N-terminal region of Brn-3a. In contrast the activation by Brn-3a but not Brn-3b of an artificial promoter containing a synthetic Brn-3 binding site can be shown using the same constructs to be dependent on the POU domain of Brn-3a. Moreover, the isolated POU domain of Brn-3a can activate this artificial promoter but not the alpha-internexin promoter. Hence Brn-3a contains two distinct transactivation domains, at the N terminus and within the POU domain, whose effect is dependent upon the target promoter. The relationship of gene transactivation by Brn-3a to its ability to transform primary cells which is also dependent on the N-terminal region of the protein is discussed.
Base Sequence↗
The neuronal intermediate filament, alpha-internexin is transiently expressed in amacrine cells in the developing mouse retina.
We have investigated the expression of intermediate filament proteins in the developing mouse retina by immunohistochemistry. Antibodies against alpha-internexin, the three neurofilament subunits (NF-L, NF-M, NF-H), vimentin, and glial fibrillary acidic protein (GFAP) were used to determine the relative expression of these proteins at different post-natal stages of mouse retinal development. alpha-Internexin is widely distributed in the process of amacrine cells, horizontal cells and retinal ganglion cells before post-natal day 5 (P5). At this age, NF-L and NF-M are detected primarily in the processes of horizontal cells and retinal ganglion cells, but are rarely found in amacrine cell processes. After P5, alpha-internexin is found to colocalize with other neuronal intermediate filaments in the cell processes of horizontal and ganglion cells, but its expression is barely detectable in amacrine cells processes. NF-H is not encountered in either the horizontal cell processes or the ganglion nerve fibers until P5. Vimentin is present in all glial cells (astrocytes and Müller cells) and some horizontal cell processes during development, while GFAP is found only in astrocyte processes of the mature retina. The transient presence of alpha-internexin in amacrine cells only in early development suggests that the protein may play a role in the plasticity of neuronal connections in the retina.
Animals↗
Translation initiation and assembly of peripherin in cultured cells.
The peripherin gene has three potential ATG translation initiation sites at positions 38, 56, and 290. The second ATG has been proposed to be the initiation codon used for translation of the protein, but there is no experimental evidence for this conjecture. We have isolated a full-length peripherin cDNA (designated as p61-11) from a rat brain cDNA library. Upon sequencing, we found that this cDNA contains a point mutation at the second potential translation initiation codon, which changes this ATG to ACG. When expressed in SW13 cl.2 vim- cells, a cell line without any detectable cytoplasmic intermediate filaments, the protein product of p61-11 cannot form a filamentous network and the major product is 45 kDa in size, which is most likely initiated from the third ATG. The protein product from the first ATG (57 kDa in size) of p61-11 is also detected albeit in smaller amounts. We introduced a frame-shift mutation upstream of the third ATG in p61-11 to create p61-11FS and showed that the third ATG is able to initiate translation efficiently even in the presence of the first ATG, and the 45 kDa protein leads to a diffuse nonfilamentous staining pattern in vim- cells confirming that the first ATG may not be the preferred translation initiation codon, since it cannot suppress a downstream ATG. We increased the translation efficiency from the first ATG of p61-11 by mutating the three nucleotides preceding this first ATG and thereby placing it in a better Kozak consensus sequence for translation initiation. The resulting 57 kDa protein is able to form a filamentous network in vim- cells. We corrected the mutation in the original p61-11 by polymerase chain reaction and generated two peripherin constructs: perM1M2 (which contains all three translation initiation codons) and per delta 1M2 (the first ATG is deleted, but the other two are present). When transfected, their protein products, about 57 kDa in size, form filamentous networks in the absence of other cytoplasmic intermediate filaments. Since there is no 45 kDa protein detected for these latter two constructs, it is reasonable to conclude that in the presence of the second ATG, little or no translation is initiated from the third ATG. Taken together, these results strongly suggest that the second ATG is the preferred translation initiation codon for the peripherin gene.
Adenocarcinoma↗
Characterization of the mouse gene encoding the neuronal intermediate filament protein alpha-internexin.
We have determined the complete nucleotide (nt) sequence of the coding region of the mouse gene encoding the neuronal intermediate filament protein, alpha-internexin (alpha INX). The mouse alpha INX gene (m alpha INX) 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 m alpha INX 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 alpha INX of mouse from that of rat.
Amino Acid Sequence↗
Expression of the gene for the neuronal intermediate filament protein alpha-internexin coincides with the onset of neuronal differentiation in the developing rat nervous system.
While neurofilaments have long been considered early markers of neuronal differentiation, they cannot be detected in most newly postmitotic neurons of the developing central nervous system (CNS). Here we show that these neurons already express the neuronal intermediate filament protein alpha-internexin at high levels. alpha-internexin is expressed by most, if not all, neurons as they begin differentiation and shows no overlap with vimentin, whose expression in the CNS is restricted to mitotic neuronal precursors. In the adult, alpha-internexin is the only intermediate filament gene expressed by the cerebellar granule cells, the source of the thin-caliber parallel fibers; conversely, neurofilament proteins are highly expressed in large neurons, which express alpha-internexin at low levels. These data suggest that neuronal intermediate filaments may regulate axonal stability and/or diameter through changes not only in their number, but also in their subunit composition.
Animals↗
Reexpression of glial fibrillary acidic protein rescues the ability of astrocytoma cells to form processes in response to neurons.
Astroglial cells play an important role in orchestrating the migration and positioning of neurons during central nervous system development. Primary astroglia, as well as astrocytoma cells will extend long stable processes when co-cultured with granule neurons. In order to determine the function of the glial fibrillary acidic protein (GFAP), the major intermediate filament protein in astroglia and astrocytoma cells, we suppressed the expression of GFAP by stable transfection of an anti-sense GFAP construct in human astrocytoma U251MG cells. The resulting AS2-U251 cells can no longer extend stable processes in the presence of granule neurons. To show that this effect is due specifically to the absence of GFAP, we reintroduced a fully encoding rat brain GFAP cDNA into these AS2-U251 cells. The resulting rat GFAP appeared as a filamentous network and the reexpression of GFAP rescued the ability of these astrocytoma cells to form stable processes when co-cultured with neurons. From these results, it is clear that the glial specific intermediate filament protein, GFAP, is required for process extension of these astrocytoma cells in response to granule neurons.
Animals↗
The endless story of the glial fibrillary acidic protein.
All intermediate filament proteins consist of an alpha-helical rod domain flanked by non-helical N-terminal head and C-terminal tail domains. The roles of the non-helical domains of various intermediate filament proteins in the assembly and co-assembly of higher-order filamentous structures have been studied by many groups but with quite contradictory results. Type III intermediate filaments are unique in that they can form homopolymers both in vitro and in vivo. The expression and assembly characteristics of carboxy- and amino-terminal deletion mutants of glial fibrillary acidic protein (GFAP), an astrocyte-specific type III intermediate filament protein, were examined by transient transfections of either vimentin-positive or vimentin-negative variants of human adrenocarcinoma-derived SW13 cell lines. Whereas complete deletion of the C-terminal tail domain of GFAP results in the formation of polymorphic aggregates, both intranuclear and cytoplasmic in self-assembly experiments, efficient co-assembly of these tail-less GFAP mutants with vimentin can be achieved as long as the KLLEGEE sequence at the C-terminal end of the rod domain is preserved. Up to one-fifth of the C-terminal end of the tail domain can be deleted without affecting the capability of GFAP to self-assemble. The highly conserved RDG-containing motif in the tail domain may be important for self-assembly but is not sufficient. The entire head domain seems to be required for self-assembly. All N-terminal deletion mutants of GFAP share the same phenotype of diffuse cytoplasmic staining when expressed in vimentin-negative SW13 cells. Although co-assembly with vimentin can still be achieved with completely head-less GFAP, preservation of some of the head domain greatly enhanced the efficiency. Our results form the basis for further, more detailed mapping of the essential regions in filament assembly of GFAP and other type III IFs.
Amino Acid Sequence↗
Molecular biology of neuronal intermediate filaments.
In the past few years, several neuronal intermediate filament proteins have been characterized. While ongoing investigations have continued to shed light on their developmental expression, the importance of different domains of the proteins for assembly, the elements in their genes necessary for tissue-specific expression, and the role of phosphorylation of neurofilaments, the function(s) of these structures remain a matter of speculation.
Animals↗
Assembly of type IV neuronal intermediate filaments in nonneuronal cells in the absence of preexisting cytoplasmic intermediate filaments.
We report here on the in vivo assembly of alpha-internexin, a type IV neuronal intermediate filament protein, in transfected cultured cells, comparing its assembly properties with those of the neurofilament triplet proteins (NF-L, NF-M, and NF-H). Like the neurofilament triplet proteins, alpha-internexin coassembles with vimentin into filaments. To study the assembly characteristics of these proteins in the absence of a preexisting filament network, transient transfection experiments were performed with a non-neuronal cell line lacking cytoplasmic intermediate filaments. The results showed that only alpha-internexin was able to self-assemble into extensive filamentous networks. In contrast, the neurofilament triplet proteins were incapable of homopolymeric assembly into filamentous arrays in vivo. NF-L coassembled with either NF-M or NF-H into filamentous structures in the transfected cells, but NF-M could not form filaments with NF-H. alpha-internexin could coassemble with each of the neurofilament triplet proteins in the transfected cells to form filaments. When all but 2 and 10 amino acid residues were removed from the tail domains of NF-L and NF-M, respectively, the resulting NF-L and NF-M deletion mutants retained the ability to coassemble with alpha-internexin into filamentous networks. These mutants were also capable of forming filaments with other wild-type neurofilament triplet protein subunits. These results suggest that the tail domains of NF-L and NF-M are dispensable for normal coassembly of each of these proteins with other type IV intermediate filament proteins to form filaments.
Amino Acid Sequence↗
Expression of high molecular weight tau in the central and peripheral nervous systems.
Using a novel PCR approach, we have cloned a cDNA encoding the entire high molecular weight tau molecule from rat dorsal root ganglia. The resulting 2080 bp cDNA differs from low molecular weight rat brain tau by the insertion of a novel 762 bp region (exon 4a) between exons 4 and 5. This cDNA clone is identical in sequence with a high molecular weight tau (HMW) cDNA from rat PC12 tumor cells and is closely related to a HMW tau cDNA from mouse N115 tumor cells. In vitro transcription/translation produces a protein that migrates on SDS-PAGE with the same apparent molecular weight as HMW tau purified from rat sciatic nerve. The HMW tau protein is generated from an 8 kb mRNA, which can be detected by northern blots in peripheral ganglia, but not in brain. A more sensitive assay using PCR and Southern blot analysis demonstrates the presence of exon 4a in spinal cord and in retina. In combination with immunohistochemical studies of spinal cord, these data suggest that HMW tau, though primarily in the peripheral nervous system, is also expressed in limited areas of the central nervous system, although its presence cannot be detected in the cerebral cortices.
Amino Acid Sequence↗
Primary structure of high molecular weight tau present in the peripheral nervous system.
The tau proteins are a family of brain microtubule binding proteins that are required during axonal outgrowth and are found in neurofibrillary tangles in Alzheimer disease. A protein of higher molecular weight, immunologically related to tau, is expressed in the adult peripheral system and in cultured neuronal cell lines of neural crest origin. The predicted amino acid sequence of the high molecular weight tau from N115 cells has been determined from the sequence of its 2340-base-pair cDNA. High molecular weight tau contains an open reading frame encoding 733 amino acid residues. It contains sequences homologous to those present in the N-, middle, and C-terminal domains of adult brain tau proteins, including four homologous repeats, which are the tubulin binding sites, and an amino acid stretch, which is present only in the N-terminal domain of the mature brain variants. The middle region contains a previously unidentified nonhomologous stretch of 237 amino acid residues as well as a domain of 66 residues homologous to exon 6 of the bovine gene that is absent in all bovine, rat, and mouse tau cDNAs sequenced so far. A cDNA probe specific to the nonhomologous tau insert hybridizes to the 8- to 9-kilobase tau mRNA in N115 cells but not to the 6-kilobase tau mRNA in brain. Probes for the domains common to brain tau isoforms hybridize to both messages. The sequence of high molecular weight tau protein also suggests that it, like low molecular weight tau, is an elongated hydrophilic molecule. This cDNA should allow us to study the role of the domains specific to these tau forms in the specialization of the peripheral nervous system and for study of their expression in normal and pathological states.
Amino Acid Sequence↗
Structure of the gene for the neuronal intermediate filament protein alpha-internexin and functional analysis of its promoter.
We have isolated the gene encoding the neuronal intermediate filament protein alpha-internexin using low stringency hybridization conditions and an NF-M (neurofilament middle molecular weight subunit) cDNA probe. This gene consists of three exons and two introns. The sequence data and the exon-intron organization of the gene establish its classification as a type IV intermediate filament gene. Transient transfection experiments showed that up to 5 kilobases of the alpha-internexin promoter region transcribed equally well in both the alpha-internexin-expressing and -nonexpressing cell lines. The results also demonstrated that the region from -77 to +73 relative to the transcription start site was sufficient for accurate basal transcription, but inclusion of the -254 to -78 region was required for efficient transcription. Sequence analysis shows that the -254 to -78 region contains several potential positive regulatory elements.
Amino Acid Sequence↗