Intermediate Filament Proteins and Their Associated Diseases
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《新英格兰医药杂志》
The cytoskeleton consists of three abundant families of fibrillary proteins: microfilaments, microtubules, and intermediate filaments.1,2 Intermediate filament proteins derive their name from their diameter, which is intermediate between the diameters of microfilaments and microtubules.1,2 They differ from actin microfilaments and tubulin microtubules in their large number, their distribution in the cytoplasm and nucleus, their diverse primary structure (Table 1), their nonpolar architecture, their relative insolubility, and their nucleotide-independent dynamics.1,2,3 The human genome contains at least 65 functional genes encoding intermediate filament proteins, placing them among the 100 largest gene families in humans.4 More than 30 diseases are related to mutations in these genes (Table 2). The majority of them are rare (affecting fewer than 200,000 patients in the United States) or difficult to treat,7 but collectively they affect most tissues, and not all of them are rare, as exemplified by the association of keratin mutations with end-stage liver disease. Intermediate filament proteins have long been considered unique to multicellular eukaryotic organisms,8 in contrast to microfilaments and microtubules, which have prokaryotic ancestors. However, crescentin, which is found in several curved bacteria, including Caulobacter crescentus and Helicobacter pylori, was recently identified as an intermediate filament–like ancestor protein that accounts for the morphologic features of caulobacter.9
Table 1. Intermediate Filament Proteins.
Table 2. Disorders Associated with Mutations in Genes Encoding Intermediate Filaments.
General Features of Intermediate Filaments
Structure
All intermediate filament proteins have a prototypical structure consisting of a coiled-coil, -helix rod domain (two polypeptide -helixes wound around each other) that is interrupted by linkers and flanked by N-terminal head and C-terminal tail domains (Figure 1). 2,3 The simplest soluble unit of intermediate filament proteins is a tetramer consisting of two antiparallel dimers; each dimer, in the case of keratins, consists of one type I keratin molecule and one type II keratin molecule. A high-resolution architectural model of intermediate filaments is lacking, although the mature, 10-to-12-nm fiber is believed to contain 32 monomers in diameter, with important structural polymorphisms among members of the protein family.2,3 Cytoplasmic intermediate filaments can assume various network configurations depending on the cell type and manifest variable differentiation-related patterns, such as those in apical poles (e.g., pancreatic keratins), Z lines (desmin), and axonal processes (neurofilaments).2,3,10
Figure 1. Structural Organization of Intermediate Filament Proteins.
The schematic diagram shows an intermediate filament protein dimer, which self-associates to form higher-order noncovalent oligomeric structures. Intermediate filament tetramers consist of two dimers aligned in antiparallel fashion. The dimers may be heteropolymers (e.g., one type I and one type II keratin) or homopolymers, as in the case of many other intermediate filament proteins. Each intermediate filament molecule consists of a central, -helical coiled-coil rod domain (green), 310 to 352 amino acids in size, that is interrupted by linker regions (purple), each consisting of 8 to 17 amino acids. The rod domain begins and ends with highly conserved sequence motifs (consisting of 8 to 12 amino acids ) that, when mutated, result in the most severe disease phenotypes.2,3,10,11,12,13,14,15,16,17 The rod is flanked by head and tail domains (blue) that provide most of the structural heterogeneity of intermediate filament proteins and contain all the known post-translational modifications, including phosphorylation (PO4), O-linked N-acetylglucosamine (GlcNAc) glycosylation, transglutamination, and farnesylation (only in lamins, which also contain a nuclear localization signal).10,18,19,20,21,22,23 Lamins, keratins (type I but not type II), vimentin, and desmin are caspase substrates during apoptosis and are cleaved at a highly conserved aspartate residue within the motif X1X2X3D (where X1 denotes an aliphatic amino acid, X2 an acidic amino acid, X3 an aliphatic amino acid or methionine, and D aspartate).10,24,25 Cleavage at other aspartate residues can also be found. Intermediate filament proteins interact with several binding partners that can be categorized as linkers, bundlers, chaperones, kinases, apoptosis-related proteins, and nuclear proteins.10,26,27,28,29
Regulation
Intermediate filament proteins are regulated by several post-translational modifications, including farnesylation, phosphorylation, glycosylation, and transglutamination, and by an accumulating number of associated proteins (Figure 1). These modifications and protein associations contribute in key ways to the function and dynamics of intermediate filaments.10,23,29 For example, phosphorylation regulates the filaments' organization and solubility, association with interacting proteins, and susceptibility to degradation during apoptosis.10,19,20,29 Alterations in intermediate filament gene expression and protein phosphorylation also serve as markers of tissue injury.10,14,30,31,32,33 In addition, transglutamination of intermediate filaments and their associated proteins under physiologic conditions is essential for the formation of the protective, cornified cell envelope that contributes to the structure of the skin barrier.21 In pathologic conditions, intermediate filament transglutamination and other alterations cause the formation of a variety of intermediate filament–containing inclusion bodies (Figure 2A).13,34,35 Intermediate filament proteins are also cleaved by caspases during apoptosis at a conserved motif (Figure 1 and Figure 2B),10,24,25 and mutations that alter the degradation of keratins during apoptosis have been identified within this motif.10
Figure 2. Alterations in Intermediate Filament Proteins in Disease States.
Panel A shows intermediate filament inclusion bodies. The most common intermediate filament inclusions are Mallory bodies, which are seen in a variety of liver diseases, particularly those that are alcohol-related.34 Shown at upper left (subpanel a) is a liver specimen (stained with chromotrope aniline blue) from a patient with alcoholic hepatitis. Several hepatocytes contain Mallory bodies (arrows), which were confirmed to contain keratins 8 and 18 with the use of specific immunohistochemical staining (inset). At upper right (subpanel b) is an immunoelectron micrograph of a Mallory body from the same liver, double-labeled with antibodies to keratins 8 and 18 (18-nm gold particles; single-headed arrows) and to MM120-1, a nonkeratin Mallory body component (10-nm gold particles; double-headed arrows). At bottom left (subpanel c) is a micrograph (hematoxylin and eosin) of a specimen of the brain stem from a child with Alexander disease caused by an arginine-to-histidine mutation at position 239 in glial fibrillary acidic protein. Arrows indicate the extensive and characteristic Rosenthal-fiber inclusions (R) surrounding the blood vessel (V); the inset shows an electron micrograph of the fibrous deposits.13 At bottom right (subpanel d) is a micrograph (modified trichrome stain) of two muscle fibers, showing desmin deposits (arrows) from a patient with a desmin mutation; the inset shows an electron micrograph of the granulofilamentous deposits.35 Panel B shows examples of the cell specificity of intermediate filament proteins and their alterations in human disease. At left (subpanel a), on double staining of specimens of cirrhotic liver and normal liver (inset) with antibodies against vimentin (green) and keratins 8 and 18 (red), there is almost no staining of mesenchymal elements by vimentin in the normal liver but extensive staining in the fibrotic liver.36 At right (subpanel b), on staining with antibodies against keratins 8 and 18 (red) and keratin 18 apoptotic fragment (green, which appears yellow because of overlap with keratins 8 and 18),37 apoptosis is nearly absent in the normal liver (inset), but there is substantial caspase activation and keratin degradation in the cirrhotic liver. Panel C shows a transmission electron micrograph of a basal epidermal keratinocyte from a patient with an arginine-to-cysteine mutation at position 125 in keratin 14, which caused Dowling–Meara epidermolysis bullosa simplex, the most severe form of this disease. Aggregates of keratin proteins that are not seen in normal keratinocytes are present (arrows). The cell appears otherwise normal in this unstressed situation and contains normal-appearing keratin filaments (kf). N denotes nucleus, and m normal melanin deposits.29,38
Functions
Shared and Tissue-Specific Functions
Difficulties in unraveling the functions of intermediate filament proteins are related to their poor solubility, the lack of specific pharmacologic agents to inhibit them, their apparent absence in genetically tractable organisms such as yeast,8 and the lack of an essential biologic function when they are tested in culture systems.1,2 The best understood function of the intermediate filaments is that of a scaffold for maintaining cell and tissue integrity.1,2,3,10,11,29 The diseases that are related to intermediate filaments and that manifest as tissue, cell, or nuclear fragility in the skin, cornea, or muscle and some of the laminopathies (Table 2) support this view.2,11,15 In addition to scaffolding, intermediate filament proteins protect against nonmechanical stress and decrease susceptibility to apoptosis.24,25,37,39 Other roles of intermediate filaments include tissue-specific functions such as those of neurofilaments in dendritic arborization and in the radial growth of myelinated axons.1,2,3,14,40
Disease-Related Aspects
An important aspect of the genes encoding intermediate filaments is their selective expression in certain cell types and during differentiation (Table 1).1,2,3 This feature is relevant to the association of mutations in these genes with a broad range of tissue-specific diseases (Table 2). The clinical manifestations of abnormal intermediate filament proteins can be due to mutations with high penetrance, such as those that cause epidermolysis bullosa simplex2,11,12,38,41; mutations predisposing to disease, such as end-stage liver disease, which may be associated with mutations in the genes encoding keratins 8 and 1810,36; neurofilament mutations, which may predispose to amyotrophic lateral sclerosis14,16; and aberrant accumulation of intermediate filament–rich inclusions or aggregates, such as Mallory bodies in liver disease and Rosenthal fibers in Alexander disease (Figure 2A and Figure 2C). 13,34 Furthermore, intermediate filament proteins can serve as markers of the tissue origin of poorly differentiated tumors (keratins define epithelial tissues, whereas vimentin defines mesenchymal origin ),42 as tumor markers in serum,43 and as a means of detecting micrometastases.44
Cytoplasmic Intermediate Filament–Related Diseases
Diseases of the Skin and Epithelium
Ultrastructural studies performed in the 1980s showed that epidermal basal cells from patients with epidermolysis bullosa simplex contain dense cytoplasmic aggregates (Figure 2C).45 In the late 1980s and early 1990s, various experiments showed that keratin mutations cause keratin aggregation in cultured cells and mechanical fragility of epithelial cells in vivo.46,47 Soon thereafter, mutations in the genes encoding keratins 5 and 14 were described in patients with epidermolysis bullosa simplex,38,41,48 which is associated with blistering in the epidermal basal-cell compartment (where keratins 5 and 14 are the major keratins) after mild frictional trauma, such as rubbing of the skin (Figure 3A).2,11 Next, dominant mutations in the genes encoding keratin 1 and its partner, keratin 10, which are expressed in the suprabasal epidermis, were associated with epidermolytic hyperkeratosis.49,50 These studies established that keratin diseases are characterized largely by fragility of the affected tissue and that in some cases they are accompanied by hyperplasia of the tissues expressing the mutant keratins.2,10,11
Figure 3. Intermediate Filament Diseases as a Reflection of Their Tissue-Specific Expression.
Shown are photographs of patients with intermediate filament–related disorders; the intermediate filament proteins that, when mutated, cause the disorder are indicated.11 Panel A shows hereditary blistering in a baby with the Dowling–Meara form of epidermolysis bullosa simplex, caused by a heterozygous arginine-to-cysteine mutation at position 125 in keratin 14. Blisters occur because of structural weakening of the epidermal basal cells, where keratins 5 and 14 are predominantly expressed. As shown in Panel B, mutations in keratins 1 and 10 in epidermolytic hyperkeratosis generally cause widespread hyperkeratosis (evident in this photograph) and skin fragility. The slit-lamp image in Panel C reveals microcysts (arrows) in a severely affected patient with a keratin 12 mutation. The intermediate filament cytoskeleton of the anterior corneal epithelium consists largely of keratins 3 and 12. Mutations in these keratins cause Meesman corneal dystrophy, which is characterized by corneal microcysts due to cell fragility. It can be asymptomatic in some cases, but in others it leads to irritation, photophobia, the sensation of a foreign body, and in a few cases, corneal scarring and loss of visual acuity.
A broad range of skin and other epithelial diseases have been associated with keratin mutations (Table 2 and Figure 3).2,10,11 In general, these disorders are caused by dominant-negative mutations, most of which are missense or small in-frame insertion or deletion mutations, primarily affecting the keratin rod domain (as listed in the Human Intermediate Filament Mutation Database).51 Mutations affecting the helix boundary motifs of the rod domain (Figure 1) generally cause severe disease and have high penetrance.2,10,11,12 Mutations occurring elsewhere in the gene usually cause less severe disease or lead to disease susceptibility, as seen in mutations in the genes encoding neurofilaments and keratins 8 and 18. Less disruptive mutations can also lead to a clinically distinct skin disease affecting only pressure-exposed palm and sole epidermis, as in epidermolytic hyperkeratosis.52 Mutations in the keratins 8 and 18 genes that do not disrupt filament formation have been identified as a risk factor for hepatic cirrhosis. In addition, keratin 8 variants may be associated with a predisposition to disease in some cases of inflammatory bowel disease and chronic pancreatitis (Table 2), but confirmatory studies are needed.53,54
Diseases of the Hair
Keratins are also subdivided into "soft" (epithelial and organ) keratins and "hard" (trichocyte) keratins of specialized epithelial cells that terminally differentiate to form hair and nail.55 Monilethrix is a keratin-related hair disorder characterized by beading and fragility of the hair, which are caused by mutations in Hb1 and Hb6, the genes encoding human-hair keratins Hb1 and Hb6.11,56 Several other epithelial keratins are expressed in specific hair follicle regions. Of these, sequence variants in keratin 6hf are implicated as risk factors in the loose-anagen syndrome57 and pseudofolliculitis barbae58 (Table 2). The overlapping distribution of keratins in hair follicles probably accounts for the relative paucity of known intermediate filament disorders primarily affecting the hair. However, it is likely that keratin genes, because of their multitude and their compartmentalization within hair substructures, play a part in other hair disorders and in normal variations in hair characteristics.
Myopathies and Lens Disorders
In desmin myopathy, identified in 1998 as the first nonkeratin intermediate filament disease,59 the muscle disease is similar to that in epidermolysis bullosa simplex with muscular dystrophy60 (discussed below). Gain-of-function mutations in B-crystallin also cause desmin-related myopathy, whereas loss-of-function mutations cause congenital cataract.61 B-crystallin is a chaperone that regulates the organization of several cytoplasmic intermediate filaments, including desmin in muscle and phakinin and filensin in the eye. Similarly, mutations in phakinin underlie autosomal-dominant cataract,62,63 and it is likely that mutations in filensin, its partner protein, cause an analogous disease.
Neurodegenerative Disorders
Mutations in neuronal intermediate filament proteins are related to several neurologic diseases: mutations in glial fibrillary acidic protein in Alexander disease, a neurodegenerative disorder13,64; mutations in neurofilament light chains in axonal Charcot–Marie–Tooth disease65; and mutations in neurofilament heavy chains14,16,40 and peripherin66 as potential causes of sporadic amyotrophic lateral sclerosis (Table 2).
Interacting-Protein–Related Diseases
Proteins that interact with intermediate filaments in desmosome and hemidesmosome cell-junction complexes are associated with several diseases.2,26,67 The loss of plectin, a cytoskeletal linker protein with critical functions in keratin organization in skin and desmin organization in muscle sarcomeres, causes the recessive disorder epidermolysis bullosa simplex with muscular dystrophy.68 Disturbances of other desmosomal proteins (e.g., plakophilin, plakoglobin, desmoplakin, desmogleins, and corneodesmosin) have been linked to ectodermal dysplasia, hair abnormalities, and cardiomyopathy.
Pathogenesis of Cytoplasmic Intermediate Filament–Related Diseases
Organization and Redundancy
Depending on their nature and location within the protein backbone, intermediate filament mutations exert a wide range of effects on the formation or organization of intermediate filament polymers.2,11,12,69 Defective intermediate filaments that are associated with severe disease are usually incorrectly polymerized or substantially reorganized, whereas those associated with mild disease may not have obvious changes in their network configuration or cause obvious changes in cellular architecture.2,10,11,12 The ability of a mutant intermediate filament protein to "poison" the function of the intermediate filament network also depends not only on its intrinsic properties, but also on the number and abundance of potentially redundant intermediate filament proteins in a cell and on the ability of a cell to overexpress alternative intermediate filament protein.3,10,11,70,71,72
Cell Fragility
Loss of cellular or nuclear integrity after short- or long-term exposure to physical trauma (e.g., pressure, stretching, or heat) is a hallmark in several intermediate filament disorders, pointing to fragility as a major contributor to pathogenesis (Figure 4).2,10,11,12 Perinuclear filaments collapse in cells expressing disease-causing mutant intermediate filament proteins,2,38 and a similar collapse in keratin filaments correlates with a marked reduction in cytoplasmic resistance to deformation in cultured cells.73 Cell fragility can be engendered by intrinsic defects in the polymer backbone or an inability of the assembled intermediate filaments to achieve the normal network architecture. Accordingly, analysis of the micromechanical properties of keratins reconstituted in vitro showed that an arginine-to-cysteine mutation at position 125 in keratin 14, which causes severe epidermolysis bullosa simplex, substantially decreases the strength of keratin polymers, particularly under filament-deforming conditions.74 Mutations that alter post-translational modifications or interactions with other cellular proteins can also promote cell fragility by affecting the properties and function of intermediate filaments. For example, mutations in keratins 8 and 18 alter keratin solubility, phosphorylation, and glycosylation,10,53 and a mutation in keratin 14 alters cytoskeletal dynamics and solubility when the mutated gene is transfected in epithelial cells.75
Figure 4. Physiologic Effects of Intermediate Filament Diseases.
The diagram highlights the physiologic effects that are shared by affected cells and tissues of patients with cytoplasmic and nuclear intermediate filament mutations and effects that are somewhat specific to each type of mutation. The genetic background of a mutation carrier and epigenetic (e.g., DNA methylation) and environmental (e.g., toxins) factors may influence the presence and type of disease presentation.
Disruption of Organelle and Protein Targeting
Mutations may perturb or enhance interactions between intermediate filaments and organelles or granules or cause subcellular mistargeting of proteins, with consequent alterations in cell function or an enhanced susceptibility to apoptosis. For example, the proline-to-leucine mutation at position 24 in keratin 5 causes epidermolysis bullosa simplex with mottled pigmentation in association with aberrant distribution of mitochondria and melanin granules2,76; mice lacking keratin 8 have subcellular mistargeting of their liver39 and intestinal proteins77,78; and myocytes from desmin-null mice show an aberrant distribution of mitochondria.79 Cell malfunctioning and death may also occur by gain-of-function mechanisms unrelated to the normal functions of cytoplasmic and nuclear intermediate filaments. The presence of cytoplasmic aggregates or inclusions, as is seen in disorders associated with changes in desmin35 and glial fibrillary acidic protein13 and several keratin-based disorders,2,11,34 probably reflects the inability of the host cell to handle mutant intermediate filament proteins properly and is a potential source of stress that can lead to cell death. For example, cell lines established from patients with epidermolysis bullosa simplex, who have mutations in the genes encoding keratins 5 and 14, not all of which lead to keratin aggregation, have increased sensitivity to osmotic stress and enhanced activation of stress kinases.80
Genetics, Epigenetics, and Environment
The contributions of patients' genetic background and of environmental and epigenetic factors that remain to be defined are also important (Figure 4). Examples include the identification of neurofilament and keratin 8 and 18 mutations that may predispose to amyotrophic lateral sclerosis14,16,40 and liver disease,10,36 respectively; the finding that an arginine-to-cysteine mutation at position 94 in keratin 17 causes either steatocystoma multiplex or pachyonychia congenita type 211; and the strain dependence of phenotypes in keratin-null mice.70,81
Laminopathies
Diseases
Mutations in the LMNA gene, which encodes the developmentally regulated lamin A and C isoforms, have been implicated in 10 disorders (Table 2).5,15,17,82,83,84,85,86,87,88,89 These conditions are generally late-onset diseases that affect differentiated tissues in a highly selective fashion, as in dilated cardiomyopathies with conduction defects, or in a more systemic fashion, as in premature aging syndromes. Several of these disorders were reproduced in mice with null or point-mutated LMNA alleles.90,91,92
Like mutations associated with cytoplasmic intermediate filament–related disorders, mutations in nonlamin proteins can cause diseases that mimic laminopathies (Table 2). For instance, mutations in peroxisome-proliferator–activated receptor cause familial partial lipodystrophy.93 Multiple lines of experimentation have established that the nuclear lamina participates in determining the shape, size, and integrity of the nucleus, the number and positioning of nuclear pores, and the organization of transcriptionally silent chromatin.15,17,28,85,94 Owing in part to the timing of their appearance and pathologic features, the muscular lesions elicited by lamin A and C mutations are consistent with defects translating into loss of nuclear integrity after repeated trauma, analogous to the findings in cytoplasmic intermediate filament–related disorders. Yet, most of the tissues affected in laminopathies are not exposed to substantial trauma and, unlike cytoplasmic intermediate filament disorders, a large fraction of the disease-causing mutations are located within the nonhelical, tail domain of lamins A and C.15,17 Hence, a full understanding of those disorders will require further experimentation.
Pathogenesis
The pathogenesis of laminopathies is probably related to other lamin functions that are just beginning to be understood, including the control of gene expression and effects on the endoplasmic reticulum, which is contiguous with the nuclear envelope.15,17,28,94 The first function is supported by the binding of lamin to chromatin,95 specific transcription factors (including sterol-response element-binding protein,96 a factor involved in adipocyte-specific gene expression), and proteins associated with nuclear processes, such as DNA replication and RNA processing.28,94 The second possible function stems from the demonstration that key nuclear-envelope components (e.g., emerin) are aberrantly distributed to endoplasmic reticulum cisternae in the fibroblasts of patients with null or mutant lamins A and C.15,97 How the function of the endoplasmic reticulum may be disrupted by these mutations is not understood. Such disruption can either elicit a general cellular stress response or alter the cellular response to specific stresses, such as heat shock.15,17,98
Additional studies are needed to ascertain the mechanisms underlying the variable effect of lamin mutations on a large number of tissues. In this regard, the atomic structure of the lamin A tail domain reveals that mutations in lamins A and C that cause familial partial lipodystrophy and Emery–Dreifuss muscular dystrophy (Table 2) affect this domain differentially and may act by disrupting interactions with key associated proteins.15,17,99,100
Therapeutic Approaches
Identification of the molecular basis of disorders related to intermediate filament proteins has provided definitive diagnoses and prognoses in affected families and has facilitated genetic counseling and first-trimester prenatal testing for severe disorders.101 Despite the usefulness of genotype–phenotype correlations, there are some cases in which the same keratin mutation leads to different phenotypes in different kindreds.11 This points to the existence of genes that can modulate the effects of mutations in intermediate filament genes (Figure 4), as has been formally established in studies of null-mutant mouse models.70,81 It is hoped that the identification of these genes will help to define compensatory mechanisms that can be exploited to therapeutic advantage.
Most, if not all, current therapies for intermediate filament–related diseases are directed toward ameliorating tissue-specific damage or, in the case of laminopathies, toward ameliorating associated metabolic derangements, such as diabetes and hyperlipidemia. The dominant negative effect exerted by polymeric intermediate filament proteins2,10,11,12 suggests that conventional gene replacement may not be effective. However, data from a mouse model of epidermolysis bullosa simplex indicates that a 50 percent reduction in mutant-allele expression can prevent blistering.102 Although the therapeutic threshold may be higher in humans, this result is encouraging since it shows that overexpression of a wild-type intermediate filament allele might be beneficial. Likewise, compensatory overexpression of wild-type keratin 15 in a family with recessive epidermolysis bullosa simplex due to the absence of keratin 14 probably accounts for the milder phenotype observed in that kindred.71 Methods based on the use of therapeutic RNA molecules (either ribozymes or short inhibitory RNAs) are being developed for selectively silencing mutant alleles. The latter method was effective in down-regulating mutant keratin 14 allele expression in cultured cells.75
Conversely, down-regulation of wild-type intermediate filament genes may also offer therapeutic advantages. For example, the absence of glial fibrillary acidic protein and vimentin in double-null mouse retina allowed successful neuron engraftment that would otherwise have been blunted by intermediate filament–related reactive gliosis.103 Further testing of these methods in animal and other models, as research on delivery systems for humans is undertaken, is warranted.
Another potential method for treating keratin-related diseases is ectopic expression of other homopolymeric intermediate filament proteins, such as desmin, to supplement the abnormal keratin cytoskeleton.104,105 Type III intermediate filaments (Table 1) cannot polymerize with keratins and are therefore unaffected by mutant keratin subunits. Ectopic desmin expression in the epidermis of transgenic mice allows the formation of a parallel desmin network without detrimental effects.105 However, the desmin transgene does not rescue the lethal keratin 5–null phenotype, possibly because of suboptimal expression levels.105 Similarly, the treatment of keratin disorders may exploit the likely functional redundancy of this large protein family, as demonstrated by the overexpression of normal keratins in tissues affected by a keratin mutation.72 Candidates for such manipulation include the wound-inducible keratins (keratins 6, 16, and 17)32 and other injury-inducible keratins (keratins 8, 18, 19, and 20).30,33 Alternatively, small molecules that can activate compensating keratins in the absence of injury, so that they may supplement defective keratins, may be identified. Conversely, drugs that can down-regulate the expression of mutant keratin (or other intermediate filament proteins) may be equally beneficial.
A common feature of many intermediate filament–related disorders, including skin disorders, liver disease, desmin myopathy, and Alexander disease, is the occurrence of cytoplasmic inclusion bodies. The presence of these abnormal deposits, rather than the loss of cytoskeletal integrity per se, may contribute to the disease process, although this idea remains to be conclusively demonstrated.2,13,14,34,35 Thus, pharmacologic or other therapies aimed at accelerating the turnover or formation of these aggregates may prove beneficial in treating intermediate filament–related diseases.106 Alternatively, genetic or drug-related strategies aimed at reversing the enhanced propensity toward apoptosis when intermediate filament proteins are altered may be beneficial. This idea is supported by the reversal of the mitochondrial defects and cardiac dysfunction in desmin-null mice by means of cardiac overexpression of the anti-apoptotic protein Bcl-2.107
Future Perspectives
Several intermediate filament proteins remain unassociated with human disease (e.g., lamins B1 and B2; vimentin; keratins 7, 19, and 20; -internexin; syncoilin; and synemin), but it remains highly possible that mutations in the genes encoding some or all of these proteins will be found to be related to certain human diseases. The phenotype of a given intermediate filament–associated disease may be genetically heterogeneous, with several mutations remaining to be defined in non–intermediate filament genes. Clinically relevant areas of growth in research related to intermediate filament proteins will probably include improvements in prenatal and postnatal diagnostic capabilities; refinement of the use of intermediate filament proteins as markers of disease progression; improved understanding of environmental, genetic, and epigenetic modifiers; and therapeutic manipulation of the expression of these proteins. As the structure, function, and regulatory mechanisms of intermediate filament proteins unfold, aided in part by relevant animal models, well-tailored therapeutic strategies can be developed to improve on current treatment limitations. An essential goal of therapy is to alter the natural history of end-organ damage in intermediate filament–related disorders, which in many cases (such as that of the laminopathies) can be progressive or of late onset.
Supported by a Department of Veterans Affairs Merit Award , National Institutes of Health (NIH) grants DK47918 and DK52951, and the Broad Medical Research Program (to Dr. Omary); by NIH grants AR44232 and AR42047 (to Dr. Coulombe); and by the Dystrophic Epidermolysis Bullosa Research Association UK, the Pachyonychia Congenita Project, and the Wellcome Trust (to Dr. McLean).
We are indebted to our laboratory members for their work over the years; Drs. Gisele Bonne, Bruno Eymard, Hans Goebel, Nam-On Ku, Albee Messing, Robert Oshima, Howard Worman, and Kurt Zatloukal for providing important figures and comments; to Kris Morrow for assistance in the preparation of the figures; and to Carolyn Taylor for assistance in the preparation of the reference list; and all the patients and families who have participated in clinical and genetic studies that made it possible to identify the molecular basis of intermediate filament–related diseases.
Source Information
From the Department of Medicine, Palo Alto Veterans Affairs Medical Center and Stanford University, Palo Alto, Calif. (M.B.O.); the Departments of Biological Chemistry and Dermatology, Johns Hopkins University School of Medicine, Baltimore (P.A.C.); and the Human Genetics Unit, University of Dundee, Ninewells Hospital and Medical School, Dundee, United Kingdom (W.H.I.M.).
Address reprint requests to Dr. Omary at the Palo Alto VA Medical Center, 3801 Miranda Ave., Mail Code 154J, Palo Alto, CA 94304.
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Table 1. Intermediate Filament Proteins.
Table 2. Disorders Associated with Mutations in Genes Encoding Intermediate Filaments.
General Features of Intermediate Filaments
Structure
All intermediate filament proteins have a prototypical structure consisting of a coiled-coil, -helix rod domain (two polypeptide -helixes wound around each other) that is interrupted by linkers and flanked by N-terminal head and C-terminal tail domains (Figure 1). 2,3 The simplest soluble unit of intermediate filament proteins is a tetramer consisting of two antiparallel dimers; each dimer, in the case of keratins, consists of one type I keratin molecule and one type II keratin molecule. A high-resolution architectural model of intermediate filaments is lacking, although the mature, 10-to-12-nm fiber is believed to contain 32 monomers in diameter, with important structural polymorphisms among members of the protein family.2,3 Cytoplasmic intermediate filaments can assume various network configurations depending on the cell type and manifest variable differentiation-related patterns, such as those in apical poles (e.g., pancreatic keratins), Z lines (desmin), and axonal processes (neurofilaments).2,3,10
Figure 1. Structural Organization of Intermediate Filament Proteins.
The schematic diagram shows an intermediate filament protein dimer, which self-associates to form higher-order noncovalent oligomeric structures. Intermediate filament tetramers consist of two dimers aligned in antiparallel fashion. The dimers may be heteropolymers (e.g., one type I and one type II keratin) or homopolymers, as in the case of many other intermediate filament proteins. Each intermediate filament molecule consists of a central, -helical coiled-coil rod domain (green), 310 to 352 amino acids in size, that is interrupted by linker regions (purple), each consisting of 8 to 17 amino acids. The rod domain begins and ends with highly conserved sequence motifs (consisting of 8 to 12 amino acids ) that, when mutated, result in the most severe disease phenotypes.2,3,10,11,12,13,14,15,16,17 The rod is flanked by head and tail domains (blue) that provide most of the structural heterogeneity of intermediate filament proteins and contain all the known post-translational modifications, including phosphorylation (PO4), O-linked N-acetylglucosamine (GlcNAc) glycosylation, transglutamination, and farnesylation (only in lamins, which also contain a nuclear localization signal).10,18,19,20,21,22,23 Lamins, keratins (type I but not type II), vimentin, and desmin are caspase substrates during apoptosis and are cleaved at a highly conserved aspartate residue within the motif X1X2X3D (where X1 denotes an aliphatic amino acid, X2 an acidic amino acid, X3 an aliphatic amino acid or methionine, and D aspartate).10,24,25 Cleavage at other aspartate residues can also be found. Intermediate filament proteins interact with several binding partners that can be categorized as linkers, bundlers, chaperones, kinases, apoptosis-related proteins, and nuclear proteins.10,26,27,28,29
Regulation
Intermediate filament proteins are regulated by several post-translational modifications, including farnesylation, phosphorylation, glycosylation, and transglutamination, and by an accumulating number of associated proteins (Figure 1). These modifications and protein associations contribute in key ways to the function and dynamics of intermediate filaments.10,23,29 For example, phosphorylation regulates the filaments' organization and solubility, association with interacting proteins, and susceptibility to degradation during apoptosis.10,19,20,29 Alterations in intermediate filament gene expression and protein phosphorylation also serve as markers of tissue injury.10,14,30,31,32,33 In addition, transglutamination of intermediate filaments and their associated proteins under physiologic conditions is essential for the formation of the protective, cornified cell envelope that contributes to the structure of the skin barrier.21 In pathologic conditions, intermediate filament transglutamination and other alterations cause the formation of a variety of intermediate filament–containing inclusion bodies (Figure 2A).13,34,35 Intermediate filament proteins are also cleaved by caspases during apoptosis at a conserved motif (Figure 1 and Figure 2B),10,24,25 and mutations that alter the degradation of keratins during apoptosis have been identified within this motif.10
Figure 2. Alterations in Intermediate Filament Proteins in Disease States.
Panel A shows intermediate filament inclusion bodies. The most common intermediate filament inclusions are Mallory bodies, which are seen in a variety of liver diseases, particularly those that are alcohol-related.34 Shown at upper left (subpanel a) is a liver specimen (stained with chromotrope aniline blue) from a patient with alcoholic hepatitis. Several hepatocytes contain Mallory bodies (arrows), which were confirmed to contain keratins 8 and 18 with the use of specific immunohistochemical staining (inset). At upper right (subpanel b) is an immunoelectron micrograph of a Mallory body from the same liver, double-labeled with antibodies to keratins 8 and 18 (18-nm gold particles; single-headed arrows) and to MM120-1, a nonkeratin Mallory body component (10-nm gold particles; double-headed arrows). At bottom left (subpanel c) is a micrograph (hematoxylin and eosin) of a specimen of the brain stem from a child with Alexander disease caused by an arginine-to-histidine mutation at position 239 in glial fibrillary acidic protein. Arrows indicate the extensive and characteristic Rosenthal-fiber inclusions (R) surrounding the blood vessel (V); the inset shows an electron micrograph of the fibrous deposits.13 At bottom right (subpanel d) is a micrograph (modified trichrome stain) of two muscle fibers, showing desmin deposits (arrows) from a patient with a desmin mutation; the inset shows an electron micrograph of the granulofilamentous deposits.35 Panel B shows examples of the cell specificity of intermediate filament proteins and their alterations in human disease. At left (subpanel a), on double staining of specimens of cirrhotic liver and normal liver (inset) with antibodies against vimentin (green) and keratins 8 and 18 (red), there is almost no staining of mesenchymal elements by vimentin in the normal liver but extensive staining in the fibrotic liver.36 At right (subpanel b), on staining with antibodies against keratins 8 and 18 (red) and keratin 18 apoptotic fragment (green, which appears yellow because of overlap with keratins 8 and 18),37 apoptosis is nearly absent in the normal liver (inset), but there is substantial caspase activation and keratin degradation in the cirrhotic liver. Panel C shows a transmission electron micrograph of a basal epidermal keratinocyte from a patient with an arginine-to-cysteine mutation at position 125 in keratin 14, which caused Dowling–Meara epidermolysis bullosa simplex, the most severe form of this disease. Aggregates of keratin proteins that are not seen in normal keratinocytes are present (arrows). The cell appears otherwise normal in this unstressed situation and contains normal-appearing keratin filaments (kf). N denotes nucleus, and m normal melanin deposits.29,38
Functions
Shared and Tissue-Specific Functions
Difficulties in unraveling the functions of intermediate filament proteins are related to their poor solubility, the lack of specific pharmacologic agents to inhibit them, their apparent absence in genetically tractable organisms such as yeast,8 and the lack of an essential biologic function when they are tested in culture systems.1,2 The best understood function of the intermediate filaments is that of a scaffold for maintaining cell and tissue integrity.1,2,3,10,11,29 The diseases that are related to intermediate filaments and that manifest as tissue, cell, or nuclear fragility in the skin, cornea, or muscle and some of the laminopathies (Table 2) support this view.2,11,15 In addition to scaffolding, intermediate filament proteins protect against nonmechanical stress and decrease susceptibility to apoptosis.24,25,37,39 Other roles of intermediate filaments include tissue-specific functions such as those of neurofilaments in dendritic arborization and in the radial growth of myelinated axons.1,2,3,14,40
Disease-Related Aspects
An important aspect of the genes encoding intermediate filaments is their selective expression in certain cell types and during differentiation (Table 1).1,2,3 This feature is relevant to the association of mutations in these genes with a broad range of tissue-specific diseases (Table 2). The clinical manifestations of abnormal intermediate filament proteins can be due to mutations with high penetrance, such as those that cause epidermolysis bullosa simplex2,11,12,38,41; mutations predisposing to disease, such as end-stage liver disease, which may be associated with mutations in the genes encoding keratins 8 and 1810,36; neurofilament mutations, which may predispose to amyotrophic lateral sclerosis14,16; and aberrant accumulation of intermediate filament–rich inclusions or aggregates, such as Mallory bodies in liver disease and Rosenthal fibers in Alexander disease (Figure 2A and Figure 2C). 13,34 Furthermore, intermediate filament proteins can serve as markers of the tissue origin of poorly differentiated tumors (keratins define epithelial tissues, whereas vimentin defines mesenchymal origin ),42 as tumor markers in serum,43 and as a means of detecting micrometastases.44
Cytoplasmic Intermediate Filament–Related Diseases
Diseases of the Skin and Epithelium
Ultrastructural studies performed in the 1980s showed that epidermal basal cells from patients with epidermolysis bullosa simplex contain dense cytoplasmic aggregates (Figure 2C).45 In the late 1980s and early 1990s, various experiments showed that keratin mutations cause keratin aggregation in cultured cells and mechanical fragility of epithelial cells in vivo.46,47 Soon thereafter, mutations in the genes encoding keratins 5 and 14 were described in patients with epidermolysis bullosa simplex,38,41,48 which is associated with blistering in the epidermal basal-cell compartment (where keratins 5 and 14 are the major keratins) after mild frictional trauma, such as rubbing of the skin (Figure 3A).2,11 Next, dominant mutations in the genes encoding keratin 1 and its partner, keratin 10, which are expressed in the suprabasal epidermis, were associated with epidermolytic hyperkeratosis.49,50 These studies established that keratin diseases are characterized largely by fragility of the affected tissue and that in some cases they are accompanied by hyperplasia of the tissues expressing the mutant keratins.2,10,11
Figure 3. Intermediate Filament Diseases as a Reflection of Their Tissue-Specific Expression.
Shown are photographs of patients with intermediate filament–related disorders; the intermediate filament proteins that, when mutated, cause the disorder are indicated.11 Panel A shows hereditary blistering in a baby with the Dowling–Meara form of epidermolysis bullosa simplex, caused by a heterozygous arginine-to-cysteine mutation at position 125 in keratin 14. Blisters occur because of structural weakening of the epidermal basal cells, where keratins 5 and 14 are predominantly expressed. As shown in Panel B, mutations in keratins 1 and 10 in epidermolytic hyperkeratosis generally cause widespread hyperkeratosis (evident in this photograph) and skin fragility. The slit-lamp image in Panel C reveals microcysts (arrows) in a severely affected patient with a keratin 12 mutation. The intermediate filament cytoskeleton of the anterior corneal epithelium consists largely of keratins 3 and 12. Mutations in these keratins cause Meesman corneal dystrophy, which is characterized by corneal microcysts due to cell fragility. It can be asymptomatic in some cases, but in others it leads to irritation, photophobia, the sensation of a foreign body, and in a few cases, corneal scarring and loss of visual acuity.
A broad range of skin and other epithelial diseases have been associated with keratin mutations (Table 2 and Figure 3).2,10,11 In general, these disorders are caused by dominant-negative mutations, most of which are missense or small in-frame insertion or deletion mutations, primarily affecting the keratin rod domain (as listed in the Human Intermediate Filament Mutation Database).51 Mutations affecting the helix boundary motifs of the rod domain (Figure 1) generally cause severe disease and have high penetrance.2,10,11,12 Mutations occurring elsewhere in the gene usually cause less severe disease or lead to disease susceptibility, as seen in mutations in the genes encoding neurofilaments and keratins 8 and 18. Less disruptive mutations can also lead to a clinically distinct skin disease affecting only pressure-exposed palm and sole epidermis, as in epidermolytic hyperkeratosis.52 Mutations in the keratins 8 and 18 genes that do not disrupt filament formation have been identified as a risk factor for hepatic cirrhosis. In addition, keratin 8 variants may be associated with a predisposition to disease in some cases of inflammatory bowel disease and chronic pancreatitis (Table 2), but confirmatory studies are needed.53,54
Diseases of the Hair
Keratins are also subdivided into "soft" (epithelial and organ) keratins and "hard" (trichocyte) keratins of specialized epithelial cells that terminally differentiate to form hair and nail.55 Monilethrix is a keratin-related hair disorder characterized by beading and fragility of the hair, which are caused by mutations in Hb1 and Hb6, the genes encoding human-hair keratins Hb1 and Hb6.11,56 Several other epithelial keratins are expressed in specific hair follicle regions. Of these, sequence variants in keratin 6hf are implicated as risk factors in the loose-anagen syndrome57 and pseudofolliculitis barbae58 (Table 2). The overlapping distribution of keratins in hair follicles probably accounts for the relative paucity of known intermediate filament disorders primarily affecting the hair. However, it is likely that keratin genes, because of their multitude and their compartmentalization within hair substructures, play a part in other hair disorders and in normal variations in hair characteristics.
Myopathies and Lens Disorders
In desmin myopathy, identified in 1998 as the first nonkeratin intermediate filament disease,59 the muscle disease is similar to that in epidermolysis bullosa simplex with muscular dystrophy60 (discussed below). Gain-of-function mutations in B-crystallin also cause desmin-related myopathy, whereas loss-of-function mutations cause congenital cataract.61 B-crystallin is a chaperone that regulates the organization of several cytoplasmic intermediate filaments, including desmin in muscle and phakinin and filensin in the eye. Similarly, mutations in phakinin underlie autosomal-dominant cataract,62,63 and it is likely that mutations in filensin, its partner protein, cause an analogous disease.
Neurodegenerative Disorders
Mutations in neuronal intermediate filament proteins are related to several neurologic diseases: mutations in glial fibrillary acidic protein in Alexander disease, a neurodegenerative disorder13,64; mutations in neurofilament light chains in axonal Charcot–Marie–Tooth disease65; and mutations in neurofilament heavy chains14,16,40 and peripherin66 as potential causes of sporadic amyotrophic lateral sclerosis (Table 2).
Interacting-Protein–Related Diseases
Proteins that interact with intermediate filaments in desmosome and hemidesmosome cell-junction complexes are associated with several diseases.2,26,67 The loss of plectin, a cytoskeletal linker protein with critical functions in keratin organization in skin and desmin organization in muscle sarcomeres, causes the recessive disorder epidermolysis bullosa simplex with muscular dystrophy.68 Disturbances of other desmosomal proteins (e.g., plakophilin, plakoglobin, desmoplakin, desmogleins, and corneodesmosin) have been linked to ectodermal dysplasia, hair abnormalities, and cardiomyopathy.
Pathogenesis of Cytoplasmic Intermediate Filament–Related Diseases
Organization and Redundancy
Depending on their nature and location within the protein backbone, intermediate filament mutations exert a wide range of effects on the formation or organization of intermediate filament polymers.2,11,12,69 Defective intermediate filaments that are associated with severe disease are usually incorrectly polymerized or substantially reorganized, whereas those associated with mild disease may not have obvious changes in their network configuration or cause obvious changes in cellular architecture.2,10,11,12 The ability of a mutant intermediate filament protein to "poison" the function of the intermediate filament network also depends not only on its intrinsic properties, but also on the number and abundance of potentially redundant intermediate filament proteins in a cell and on the ability of a cell to overexpress alternative intermediate filament protein.3,10,11,70,71,72
Cell Fragility
Loss of cellular or nuclear integrity after short- or long-term exposure to physical trauma (e.g., pressure, stretching, or heat) is a hallmark in several intermediate filament disorders, pointing to fragility as a major contributor to pathogenesis (Figure 4).2,10,11,12 Perinuclear filaments collapse in cells expressing disease-causing mutant intermediate filament proteins,2,38 and a similar collapse in keratin filaments correlates with a marked reduction in cytoplasmic resistance to deformation in cultured cells.73 Cell fragility can be engendered by intrinsic defects in the polymer backbone or an inability of the assembled intermediate filaments to achieve the normal network architecture. Accordingly, analysis of the micromechanical properties of keratins reconstituted in vitro showed that an arginine-to-cysteine mutation at position 125 in keratin 14, which causes severe epidermolysis bullosa simplex, substantially decreases the strength of keratin polymers, particularly under filament-deforming conditions.74 Mutations that alter post-translational modifications or interactions with other cellular proteins can also promote cell fragility by affecting the properties and function of intermediate filaments. For example, mutations in keratins 8 and 18 alter keratin solubility, phosphorylation, and glycosylation,10,53 and a mutation in keratin 14 alters cytoskeletal dynamics and solubility when the mutated gene is transfected in epithelial cells.75
Figure 4. Physiologic Effects of Intermediate Filament Diseases.
The diagram highlights the physiologic effects that are shared by affected cells and tissues of patients with cytoplasmic and nuclear intermediate filament mutations and effects that are somewhat specific to each type of mutation. The genetic background of a mutation carrier and epigenetic (e.g., DNA methylation) and environmental (e.g., toxins) factors may influence the presence and type of disease presentation.
Disruption of Organelle and Protein Targeting
Mutations may perturb or enhance interactions between intermediate filaments and organelles or granules or cause subcellular mistargeting of proteins, with consequent alterations in cell function or an enhanced susceptibility to apoptosis. For example, the proline-to-leucine mutation at position 24 in keratin 5 causes epidermolysis bullosa simplex with mottled pigmentation in association with aberrant distribution of mitochondria and melanin granules2,76; mice lacking keratin 8 have subcellular mistargeting of their liver39 and intestinal proteins77,78; and myocytes from desmin-null mice show an aberrant distribution of mitochondria.79 Cell malfunctioning and death may also occur by gain-of-function mechanisms unrelated to the normal functions of cytoplasmic and nuclear intermediate filaments. The presence of cytoplasmic aggregates or inclusions, as is seen in disorders associated with changes in desmin35 and glial fibrillary acidic protein13 and several keratin-based disorders,2,11,34 probably reflects the inability of the host cell to handle mutant intermediate filament proteins properly and is a potential source of stress that can lead to cell death. For example, cell lines established from patients with epidermolysis bullosa simplex, who have mutations in the genes encoding keratins 5 and 14, not all of which lead to keratin aggregation, have increased sensitivity to osmotic stress and enhanced activation of stress kinases.80
Genetics, Epigenetics, and Environment
The contributions of patients' genetic background and of environmental and epigenetic factors that remain to be defined are also important (Figure 4). Examples include the identification of neurofilament and keratin 8 and 18 mutations that may predispose to amyotrophic lateral sclerosis14,16,40 and liver disease,10,36 respectively; the finding that an arginine-to-cysteine mutation at position 94 in keratin 17 causes either steatocystoma multiplex or pachyonychia congenita type 211; and the strain dependence of phenotypes in keratin-null mice.70,81
Laminopathies
Diseases
Mutations in the LMNA gene, which encodes the developmentally regulated lamin A and C isoforms, have been implicated in 10 disorders (Table 2).5,15,17,82,83,84,85,86,87,88,89 These conditions are generally late-onset diseases that affect differentiated tissues in a highly selective fashion, as in dilated cardiomyopathies with conduction defects, or in a more systemic fashion, as in premature aging syndromes. Several of these disorders were reproduced in mice with null or point-mutated LMNA alleles.90,91,92
Like mutations associated with cytoplasmic intermediate filament–related disorders, mutations in nonlamin proteins can cause diseases that mimic laminopathies (Table 2). For instance, mutations in peroxisome-proliferator–activated receptor cause familial partial lipodystrophy.93 Multiple lines of experimentation have established that the nuclear lamina participates in determining the shape, size, and integrity of the nucleus, the number and positioning of nuclear pores, and the organization of transcriptionally silent chromatin.15,17,28,85,94 Owing in part to the timing of their appearance and pathologic features, the muscular lesions elicited by lamin A and C mutations are consistent with defects translating into loss of nuclear integrity after repeated trauma, analogous to the findings in cytoplasmic intermediate filament–related disorders. Yet, most of the tissues affected in laminopathies are not exposed to substantial trauma and, unlike cytoplasmic intermediate filament disorders, a large fraction of the disease-causing mutations are located within the nonhelical, tail domain of lamins A and C.15,17 Hence, a full understanding of those disorders will require further experimentation.
Pathogenesis
The pathogenesis of laminopathies is probably related to other lamin functions that are just beginning to be understood, including the control of gene expression and effects on the endoplasmic reticulum, which is contiguous with the nuclear envelope.15,17,28,94 The first function is supported by the binding of lamin to chromatin,95 specific transcription factors (including sterol-response element-binding protein,96 a factor involved in adipocyte-specific gene expression), and proteins associated with nuclear processes, such as DNA replication and RNA processing.28,94 The second possible function stems from the demonstration that key nuclear-envelope components (e.g., emerin) are aberrantly distributed to endoplasmic reticulum cisternae in the fibroblasts of patients with null or mutant lamins A and C.15,97 How the function of the endoplasmic reticulum may be disrupted by these mutations is not understood. Such disruption can either elicit a general cellular stress response or alter the cellular response to specific stresses, such as heat shock.15,17,98
Additional studies are needed to ascertain the mechanisms underlying the variable effect of lamin mutations on a large number of tissues. In this regard, the atomic structure of the lamin A tail domain reveals that mutations in lamins A and C that cause familial partial lipodystrophy and Emery–Dreifuss muscular dystrophy (Table 2) affect this domain differentially and may act by disrupting interactions with key associated proteins.15,17,99,100
Therapeutic Approaches
Identification of the molecular basis of disorders related to intermediate filament proteins has provided definitive diagnoses and prognoses in affected families and has facilitated genetic counseling and first-trimester prenatal testing for severe disorders.101 Despite the usefulness of genotype–phenotype correlations, there are some cases in which the same keratin mutation leads to different phenotypes in different kindreds.11 This points to the existence of genes that can modulate the effects of mutations in intermediate filament genes (Figure 4), as has been formally established in studies of null-mutant mouse models.70,81 It is hoped that the identification of these genes will help to define compensatory mechanisms that can be exploited to therapeutic advantage.
Most, if not all, current therapies for intermediate filament–related diseases are directed toward ameliorating tissue-specific damage or, in the case of laminopathies, toward ameliorating associated metabolic derangements, such as diabetes and hyperlipidemia. The dominant negative effect exerted by polymeric intermediate filament proteins2,10,11,12 suggests that conventional gene replacement may not be effective. However, data from a mouse model of epidermolysis bullosa simplex indicates that a 50 percent reduction in mutant-allele expression can prevent blistering.102 Although the therapeutic threshold may be higher in humans, this result is encouraging since it shows that overexpression of a wild-type intermediate filament allele might be beneficial. Likewise, compensatory overexpression of wild-type keratin 15 in a family with recessive epidermolysis bullosa simplex due to the absence of keratin 14 probably accounts for the milder phenotype observed in that kindred.71 Methods based on the use of therapeutic RNA molecules (either ribozymes or short inhibitory RNAs) are being developed for selectively silencing mutant alleles. The latter method was effective in down-regulating mutant keratin 14 allele expression in cultured cells.75
Conversely, down-regulation of wild-type intermediate filament genes may also offer therapeutic advantages. For example, the absence of glial fibrillary acidic protein and vimentin in double-null mouse retina allowed successful neuron engraftment that would otherwise have been blunted by intermediate filament–related reactive gliosis.103 Further testing of these methods in animal and other models, as research on delivery systems for humans is undertaken, is warranted.
Another potential method for treating keratin-related diseases is ectopic expression of other homopolymeric intermediate filament proteins, such as desmin, to supplement the abnormal keratin cytoskeleton.104,105 Type III intermediate filaments (Table 1) cannot polymerize with keratins and are therefore unaffected by mutant keratin subunits. Ectopic desmin expression in the epidermis of transgenic mice allows the formation of a parallel desmin network without detrimental effects.105 However, the desmin transgene does not rescue the lethal keratin 5–null phenotype, possibly because of suboptimal expression levels.105 Similarly, the treatment of keratin disorders may exploit the likely functional redundancy of this large protein family, as demonstrated by the overexpression of normal keratins in tissues affected by a keratin mutation.72 Candidates for such manipulation include the wound-inducible keratins (keratins 6, 16, and 17)32 and other injury-inducible keratins (keratins 8, 18, 19, and 20).30,33 Alternatively, small molecules that can activate compensating keratins in the absence of injury, so that they may supplement defective keratins, may be identified. Conversely, drugs that can down-regulate the expression of mutant keratin (or other intermediate filament proteins) may be equally beneficial.
A common feature of many intermediate filament–related disorders, including skin disorders, liver disease, desmin myopathy, and Alexander disease, is the occurrence of cytoplasmic inclusion bodies. The presence of these abnormal deposits, rather than the loss of cytoskeletal integrity per se, may contribute to the disease process, although this idea remains to be conclusively demonstrated.2,13,14,34,35 Thus, pharmacologic or other therapies aimed at accelerating the turnover or formation of these aggregates may prove beneficial in treating intermediate filament–related diseases.106 Alternatively, genetic or drug-related strategies aimed at reversing the enhanced propensity toward apoptosis when intermediate filament proteins are altered may be beneficial. This idea is supported by the reversal of the mitochondrial defects and cardiac dysfunction in desmin-null mice by means of cardiac overexpression of the anti-apoptotic protein Bcl-2.107
Future Perspectives
Several intermediate filament proteins remain unassociated with human disease (e.g., lamins B1 and B2; vimentin; keratins 7, 19, and 20; -internexin; syncoilin; and synemin), but it remains highly possible that mutations in the genes encoding some or all of these proteins will be found to be related to certain human diseases. The phenotype of a given intermediate filament–associated disease may be genetically heterogeneous, with several mutations remaining to be defined in non–intermediate filament genes. Clinically relevant areas of growth in research related to intermediate filament proteins will probably include improvements in prenatal and postnatal diagnostic capabilities; refinement of the use of intermediate filament proteins as markers of disease progression; improved understanding of environmental, genetic, and epigenetic modifiers; and therapeutic manipulation of the expression of these proteins. As the structure, function, and regulatory mechanisms of intermediate filament proteins unfold, aided in part by relevant animal models, well-tailored therapeutic strategies can be developed to improve on current treatment limitations. An essential goal of therapy is to alter the natural history of end-organ damage in intermediate filament–related disorders, which in many cases (such as that of the laminopathies) can be progressive or of late onset.
Supported by a Department of Veterans Affairs Merit Award , National Institutes of Health (NIH) grants DK47918 and DK52951, and the Broad Medical Research Program (to Dr. Omary); by NIH grants AR44232 and AR42047 (to Dr. Coulombe); and by the Dystrophic Epidermolysis Bullosa Research Association UK, the Pachyonychia Congenita Project, and the Wellcome Trust (to Dr. McLean).
We are indebted to our laboratory members for their work over the years; Drs. Gisele Bonne, Bruno Eymard, Hans Goebel, Nam-On Ku, Albee Messing, Robert Oshima, Howard Worman, and Kurt Zatloukal for providing important figures and comments; to Kris Morrow for assistance in the preparation of the figures; and to Carolyn Taylor for assistance in the preparation of the reference list; and all the patients and families who have participated in clinical and genetic studies that made it possible to identify the molecular basis of intermediate filament–related diseases.
Source Information
From the Department of Medicine, Palo Alto Veterans Affairs Medical Center and Stanford University, Palo Alto, Calif. (M.B.O.); the Departments of Biological Chemistry and Dermatology, Johns Hopkins University School of Medicine, Baltimore (P.A.C.); and the Human Genetics Unit, University of Dundee, Ninewells Hospital and Medical School, Dundee, United Kingdom (W.H.I.M.).
Address reprint requests to Dr. Omary at the Palo Alto VA Medical Center, 3801 Miranda Ave., Mail Code 154J, Palo Alto, CA 94304.
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