(S)-(-)-blebbistatin O-benzoate has the potential to improve atopic dermatitis symptoms in NC/Nga mice by upregulating epidermal barrier function and inhibiting type 2 alarmin cytokine induction.

Atopic dermatitis is a multi-pathogenic disease characterized by chronic skin inflammation and barrier dysfunction. Therefore, improving the skin's ability to form an epidermal barrier and suppressing the production of cytokines that induce type 2 inflammatory responses are important for controlling atopic dermatitis symptoms. (-)-Blebbistatin, a non-muscle myosin II inhibitor, has been suggested to improve pulmonary endothelial barrier function and control inflammation by suppressing immune cell migration; however, its efficacy in atopic dermatitis is unknown. In this study, we investigated whether (S)-(-)-blebbistatin O-benzoate, a derivative of (-)-blebbistatin, improves dermatitis symptoms in a mite antigen-induced atopic dermatitis model using NC/Nga mice. The efficacy of the compound was confirmed using dermatitis scores, ear thickness measurements, serum IgE levels, histological analysis of lesions, and filaggrin expression analysis, which is important for barrier function. (S)-(-)-Blebbistatin O-benzoate treatment significantly reduced the dermatitis score and serum IgE levels compared to those in the vehicle group (p < 0.05). Furthermore, the histological analysis revealed enhanced filaggrin production and a decreased number of mast cells (p < 0.05), indicating that (S)-(-)-blebbistatin O-benzoate improved atopic dermatitis symptoms in a pathological model. In vitro analysis using cultured keratinocytes revealed increased expression of filaggrin, loricrin, involucrin, and ceramide production pathway-related genes, suggesting that (S)-(-)-blebbistatin O-benzoate promotes epidermal barrier formation. Furthermore, the effect of (S)-(-)-blebbistatin O-benzoate on type 2 alarmin cytokines, which are secreted from epidermal cells upon scratching or allergen stimulation and are involved in the pathogenesis of atopic dermatitis, was evaluated using antigens derived from mite feces. The results showed that (S)-(-)-blebbistatin O-benzoate inhibited the upregulation of these cytokines. Based on the above, (S)-(-)-blebbistatin O-benzoate has the potential to be developed as an atopic dermatitis treatment option that controls dermatitis symptoms by suppressing inflammation and improving barrier function by acting on multiple aspects of the pathogenesis of atopic dermatitis.

Ultraviolet Protection From a Patented Amino Acid Complex Technology.

OBJECTIVE:   This study aimed to investigate the ultraviolet (UV) protection/repair benefits of a patented Amino Acid Complex (AAComplex). METHODS: I) AAComplex was incubated with dermal fibroblasts, with/without UVA, and collagen I was measured with a GlasBoxPlus device. II) A lotion, with/without AAComplex (1%) was applied topically to skin explants, following UVA irradiation, and quantified for health-related biomarkers (TNFalpha, histamine, and MMP-1). III) A broad spectrum sunscreen with SPF 46 and a skincare serum containing AAComplex (2%) were assessed using epidermal equivalents, in the presence of UV irradiation, for effects on IL-1alpha, thymine dimers, Ki-67, filaggrin and Nrf2. RESULTS: I) Collagen I synthesis in dermal fibroblasts was significantly decreased after UVA compared to without UV. The presence of AAComplex prevented this decrease. II) UVA irradiation of skin explants increased histamine, TNFα, and MMP-1. Hydrocortisone aceponate cream significantly decreases all 3 biomarkers. AAComplex contained lotion also significantly decreased all 3 biomarkers, the no AAComplex control lotion only reduced histamine. III) With the regimen of sunscreen + AAComplex contained skincare serum, the significant reduction in IL-1alpha was observed along with a complete recovery of Ki-67 and stimulation of filaggrin and Nrf2T. No thymine dimer positive cell was observed indicating the most positive skin impact from the regiment.  Conclusion: This research using different human skin models demonstrated that AAComplex can provide protection and damage repair caused by UV, at the ingredient level also when formulated in a serum or lotion formula. Skin may be best protected from UV damage when the regimen is used.   J Drugs Dermatol. 2024;23(5):366-375. doi:10.36849/JDD.7916.

Lagerstroemia macrocarpa extract inhibits Th2-mediated STAT6 signaling pathway in human keratinocytes.

In this study, we examined physiological functions as a key material to develop cosmeceuticals using extracts of Lagerstroemia macrocarpa Wall. Ex Kurz (L. macrocarpa). Initially, the L. macrocarpa extract was treated by different concentration and antioxidant assay (DPPH and ABTS) were performed to measure free radical scavenging ability. In the cytotoxicity experiment, the extract was treated into human epidermal keratinocytes with different concentrations to measure cytotoxicity. We found that the extract induces differentiation markers such as keratin (KRT)1, KRT2, KRT9, KRT10 in keratinocytes. Furthermore, the extract significantly induces involucrin (IVL), loricrin (LOR), claudin1 (CLDN1), and filaggrin (FLG) expression, suggesting that it may enhance skin barrier functions. Especially, the extract restored FLG expression inhibited by interleukin (IL)-4/IL-13 in in vitro atopic dermatitis-like model. Therefore, we expect L. macrocarpa extract will be an effective material to develop the therapeutic and cosmeceutical of atopic dermatitis.

Decreased TET2/5-hmC reduces the integrity of the epidermal barrier via epigenetic dysregulation of filaggrin in psoriatic lesions.

BACKGROUND: TET2 participates in tumor progression and intrinsic immune homeostasis via epigenetic regulation. TET2 has been reported to be involved in maintaining epithelial barrier homeostasis and inflammation. Abnormal epidermal barrier function and TET2 expression have been detected in psoriatic lesions. However, the mechanisms underlying the role of TET2 in psoriasis have not yet been elucidated. OBJECTIVE: To define the role of TET2 in maintaining epithelial barrier homeostasis and the exact epigenetic mechanism in the dysfunction of the epidermal barrier in psoriasis. METHODS: We analyzed human psoriatic skin lesions and datasets from the GEO database, and detected the expression of TET2/5-hmC together with barrier molecules by immunohistochemistry. We constructed epidermal-specific TET2 knockout mice to observe the effect of TET2 deficiency on epidermal barrier function via toluidine blue penetration assay. Further, we analyzed changes in the expression of epidermal barrier molecules by immunofluorescence in TET2-specific knockout mice and psoriatic model mice. RESULTS: We found that decreased expression of TET2/5-hmC correlated with dysregulated barrier molecules in human psoriatic lesions. Epidermal-specific TET2 knockout mice showed elevated transdermal water loss associated with abnormal epidermal barrier molecules. Furthermore, we observed that TET2 knockdown in keratinocytes reduced filaggrin expression via filaggrin promoter methylation. CONCLUSION: Aberrant epidermal TET2 affects the integrity of the epidermal barrier through the epigenetic dysregulation of epidermal barrier molecules, particularly filaggrin. Reduced TET2 expression is a critical factor contributing to an abnormal epidermal barrier in psoriasis.

Filament structure and subcellular organization of the bacterial intermediate filament-like protein crescentin.

The protein crescentin is required for the crescent shape of the freshwater bacterium Caulobacter crescentus (vibrioides). Crescentin forms a filamentous structure on the inner, concave side of the curved cells. It shares features with eukaryotic intermediate filament (IF) proteins, including the formation of static filaments based on long and parallel coiled coils, the protein's length, structural roles in cell and organelle shape determination and the presence of a coiled coil discontinuity called the "stutter." Here, we have used electron cryomicroscopy (cryo-EM) to determine the structure of the full-length protein and its filament, exploiting a crescentin-specific nanobody. The filament is formed by two strands, related by twofold symmetry, that each consist of two dimers, resulting in an octameric assembly. Crescentin subunits form longitudinal contacts head-to-head and tail-to-tail, making the entire filament non-polar. Using in vivo site-directed cysteine cross-linking, we demonstrated that contacts observed in the in vitro filament structure exist in cells. Electron cryotomography (cryo-ET) of cells expressing crescentin showed filaments on the concave side of the curved cells, close to the inner membrane, where they form a band. When comparing with current models of IF proteins and their filaments, which are also built from parallel coiled coil dimers and lack overall polarity, it emerges that IF proteins form head-to-tail longitudinal contacts in contrast to crescentin and hence several inter-dimer contacts in IFs have no equivalents in crescentin filaments. Our work supports the idea that intermediate filament-like proteins achieve their shared polymerization and mechanical properties through a variety of filament architectures.

Differential distribution of intermediate filament proteins in the bovine and ovine tongues.

Intermediate filaments constitute the most heterogeneous class among the major classes of cytoskeletal proteins of mammalian cells. The 40 or more intermediate filament proteins have been classified into five types which show very specific rules of expression in specialized cell types. This study aimed to investigate the immunohistochemical distribution of cytokeratins (CKs) 8, 18, and 19 as well as the intermediate filaments vimentin, laminin, and desmin in bovine and ovine tongues. Immunohistochemical staining was performed for CKs 8, 18, 19, vimentin, laminin, and desmin. Our results revealed similar immunostaining intensity and distribution among various CKs, contrasting with distinct patterns for vimentin, laminin, and desmin. Immunoreactions were primarily localized in serous acini and ductal epithelium for cytokeratins, while vimentin and laminin were evident in connective tissue, endothelium, serous acini, and desmin in striated and smooth muscles. This study highlighted the absence of CKs 8, 18, 19, vimentin, and desmin in the lingual epithelium of bovine and ovine tongues. These findings enabled the classification of epithelial cells based on their specific cytokeratin patterns. Furthermore, vimentin was identified in mesodermal tissues and organs, desmin in muscle tissue, and laminin played crucial roles in basement membrane formation, nerve tissue regeneration, innervation of epithelial taste buds, and tissue separation and connection. Our findings provide essential insights into intermediate filament dynamics at the cellular and tissue levels. They serve as a foundation for future studies using systematic molecular biological techniques in this field.

Human filaggrin monomer does not seem to be a proteasome target.

Absence of a functional proteasome in the suprabasal layers of the epidermis is responsible for keratosis linearis with ichthyosis congenital and sclerosing keratoderma syndrome. Patient epidermis shows hypergranulosis associated with abnormally shaped keratohyalin granules and abnormal distribution of filaggrin in the Stratum granulosum and Stratum corneum. This suggests that the proteasome is involved in the degradation of filaggrin. To test this hypothesis, the proteasome proteolytic activity was inhibited in 3D reconstructed human epidermis (RHE) with the specific clasto-lactacystin ß-lactone inhibitor. Confirming the efficacy of inhibition, ubiquitinated proteins accumulated in treated RHEs as compared to controls. Levels of urocanic acid (UCA) and pyrrolidone carboxylic acid (PCA), the end products of filaggrin degradation, were reduced. However, neither filaggrin accumulation nor appearance of filaggrin-derived peptides were observed. On the contrary, the amount of filaggrin was shown to decrease, and a similar tendency was observed for profilaggrin, its precursor. Accumulation of small cytoplasmic vesicles associated with a significant increase in autophagy markers indicated activation of the autophagy process upon proteasome inhibition. Taken together, these results suggest that the perturbation of UCA and PCA production after proteasome inhibition was probably due to down-regulation of filaggrin expression rather than to blocking of filaggrin proteolysis.

Plectin Deficiency in Fibroblasts Deranges Intermediate Filament and Organelle Morphology, Migration, and Adhesion.

Plectin, a highly versatile and multifunctional cytolinker, has been implicated in several multisystemic disorders. Most sequence variations in the human plectin gene (PLEC) cause epidermolysis bullosa simplex with muscular dystrophy (EBS-MD), an autosomal recessive skin-blistering disorder associated with progressive muscle weakness. In this study, we performed a comprehensive cell biological analysis of dermal fibroblasts from three different patients with EBS-MD, where PLEC expression analyses revealed preserved mRNA levels in all cases, whereas full-length plectin protein content was significantly reduced or completely absent. Downstream effects of pathogenic PLEC sequence alterations included massive bundling of vimentin intermediate filament networks, including the occurrence of ring-like nuclei-encasing filament bundles, elongated mitochondrial networks, and abnormal nuclear morphologies. We found that essential fibroblast functions such as wound healing, migration, or orientation upon cyclic stretch were significantly impaired in the cells of patients with EBS-MD. Finally, EBS-MD fibroblasts displayed reduced adhesion capacities, which could be attributed to smaller focal adhesion contacts. Our study not only emphasizes plectin's functional role in human skin fibroblasts, it also provides further insights into the understanding of EBS-MD-associated disease mechanisms.

Nuclear roles for non-lamin intermediate filament proteins.

The nuclear-localized lamins have long been thought to be the only intermediate filaments (IFs) with an impact on the architecture, properties, and functions of the nucleus. Recent studies, however, uncovered significant roles for IFs other than lamins (here referred to as "non-lamin IFs") in regulating key properties of the nucleus in various cell types and biological settings. In the cytoplasm, IFs often occur in the perinuclear space where they contribute to local stiffness and impact the shape and/or the integrity of the nucleus, particularly in cells under stress. In addition, selective non-lamin IF proteins can occur inside the nucleus where they partake in fundamental processes including nuclear architecture and chromatin organization, regulation of gene expression, cell cycle progression, and the repair of DNA damage. This text reviews the evidence supporting a role for non-lamin IF proteins in regulating various properties of the nucleus and highlights opportunities for further study.

Inflammation-related proteins in blood after dermal exposure to some common chemicals depend on the skin barrier gene filaggrin - a human experimental study.

Filaggrin (FLG), a skin barrier protein, is associated with higher dermal uptake of some chemicals in carriers of loss-of-function (null) mutations. This study investigates FLG mutations and systemic effects following dermal exposure to chemicals. Individuals (n = 23 FLG null, n = 31 FLG wt) were simultaneously exposed to pyrimethanil, pyrene, oxybenzone, and nickel ions for 4 h. Pre- and post-exposure, 25-hydroxyvitamin D3 (25(OH)D3, LC-MS/MS) and 92 inflammation-related proteins (proximity-extension assay) were measured. FLG null carriers exhibited significantly higher 25(OH)D3 concentrations than wt carriers, both pre- and post-exposure. Eleven proteins differed in abundance post- vs pre-exposure among FLG null carriers, and 22 proteins among wt carriers (three proteins overlapped). Twelve proteins showed median differences (post- vs pre-exposure) between FLG null and wt carriers. Overall, FLG null carriers showed an increase, while FLG wt carriers showed a decrease in inflammation-related proteins. These findings suggest FLG-dependent differences in susceptibility to systemic effects following simultaneous dermal chemical exposure.

Expression of hornerin in skin lesions of atopic dermatitis and skin diseases.

We have previously identified the filaggrin (FLG)-like protein, hornerin (HRNR). Recently, there have been several reports regarding the relationship between HRNR and atopic dermatitis (AD). In the present study, we examined HRNR expression in the skin lesions of seven unrelated patients with AD to clarify the role of HRNR in the pathogenesis of AD. HRNR was detected in chronic AD lesions (n = 4), whereas no HRNR signals were observed in acute AD lesions (n = 3). HRNR was detected in the cytokeratin 6-expressing epidermis, and Ki67-positive keratinocytes were more abundant in the HRNR-positive epidermis. These findings suggest that HRNR may be associated with epidermal hyperproliferation in AD lesions. Next, we examined HRNR expression in skin diseases associated with hyperkeratosis. HRNR signals were irregularly observed in different cells from those expressing FLG in epidermolytic ichthyosis and actinic keratosis. Therefore, HRNR may play a unique role in the molecular process of cornification.

Plasticity of cytoplasmic intermediate filament architecture determines cellular functions.

Cytoplasmic intermediate filaments endow cells with mechanical stability. They are subject to changes in morphology and composition if needed. This remodeling encompasses entire cells but can also be restricted to specific intracellular regions. Intermediate filaments thereby support spatially and temporally defined cell type-specific functions. This review focuses on recent advances in our understanding of how intermediate filament dynamics affect the underlying regulatory pathways. We will elaborate on the role of intermediate filaments for the formation and maintenance of surface specializations, cell migration, contractility, organelle positioning, nucleus protection, stress responses and axonal conduction velocity. Together, the selected examples highlight the modulatory role of intermediate filament plasticity for multiple cellular functions.

Lamins as structural nuclear elements through evolution.

Lamins are nuclear intermediate filament proteins with important, well-established roles in humans and other vertebrates. Lamins interact with DNA and numerous proteins at the nuclear envelope to determine the mechanical properties of the nucleus, coordinate chromatin organization, and modulate gene expression. Many of these functions are conserved in the lamin homologs found in basal metazoan organisms, including Drosophila and Caenorhabditis elegans. Lamin homologs have also been recently identified in non-metazoans, like the amoeba Dictyostelium discoideum, yet how these proteins compare functionally to the metazoan isoforms is only beginning to emerge. A better understanding of these distantly related lamins is not only valuable for a more complete picture of eukaryotic evolution, but may also provide new insights into the function of vertebrate lamins.

How do disordered head domains assist in the assembly of intermediate filaments?

The dominant structural feature of intermediate filament (IF) proteins is a centrally located α-helix. These long α-helical segments become paired in a parallel orientation to form coiled-coil dimers. Pairs of dimers further coalesce in an anti-parallel orientation to form tetramers. These early stages of intermediate filament assembly can be accomplished solely by the central α-helices. By contrast, the assembly of tetramers into mature intermediate filaments is reliant upon an N-terminal head domain. IF head domains measure roughly 100 amino acids in length and have long been understood to exist in a state of structural disorder. Here, we describe experiments favoring the unexpected idea that head domains self-associate to form transient structural order in the form of labile cross-ß interactions. We propose that this weak form of protein structure allows for dynamic regulation of IF assembly and disassembly. We further offer that what we have learned from studies of IF head domains may represent a simple, unifying template for understanding how thousands of other intrinsically disordered proteins help to establish dynamic morphological order within eukaryotic cells.

Engineering a Biomimetic In Vitro Model of Bruch's Membrane Using Hagfish Slime Intermediate Filament Proteins.

Bruch's membrane resides in the subretinal tissue and regulates the flow of nutrients and waste between the retinal pigment epithelial (RPE) and vascular layers of the eye. With age, Bruch's membrane becomes thicker, stiffer, and less permeable, which impedes its function as a boundary layer in the subretina. These changes contribute to pathologies such as age-related macular degeneration (AMD). To better understand how aging in Bruch's membrane affects surrounding tissues and to determine the relationship between aging and disease, an in vitro model of Bruch's membrane is needed. An accurate model of Bruch's membrane must be a proteinaceous, semipermeable, and nonporous biomaterial with similar mechanical properties to in vivo conditions. Additionally, this model must support RPE cell growth. While models of subretinal tissue exist, they typically differ from in vivo Bruch's membrane in one or more of these properties. This study evaluates the capability of membranes created from recombinant hagfish intermediate filament (rHIF) proteins to accurately replicate Bruch's membrane in an in vitro model of the subretinal tissue. The physical characteristics of these rHIF membranes were evaluated using mechanical testing, permeability assays, brightfield microscopy, and scanning electron microscopy. The capacity of the membranes to support RPE cell culture was determined using brightfield and fluorescent microscopy, as well as immunocytochemical staining. This study demonstrates that rHIF protein membranes are an appropriate biomaterial to accurately mimic both healthy and aged Bruch's membrane for in vitro modeling of the subretinal tissue.

Axonal Transport Defect in Gigaxonin Deficiency Rescued by Tubastatin A.

Giant axonal neuropathy (GAN) is a disease caused by a deficiency of gigaxonin, a mediator of the degradation of intermediate filament (IF) proteins. A lack of gigaxonin alters the turnover of IF proteins, provoking accumulation and disorganization of neurofilaments (NFs) in neurons, a hallmark of the disease. However, the effects of IF disorganization on neuronal function remain unknown. Here, we report that cultured embryonic dorsal root ganglia (DRG) neurons derived from Gan-/- mice exhibit accumulations of IF proteins and defects in fast axonal transport of organelles. Kymographs generated by time-lapse microscopy revealed substantial reduction of anterograde movements of mitochondria and lysosomes in axons of Gan-/- DRG neurons. Treatment of Gan-/- DRG neurons with Tubastatin A (TubA) increased the levels of acetylated tubulin and it restored the normal axonal transport of these organelles. Furthermore, we tested the effects of TubA in a new mouse model of GAN consisting of Gan-/- mice with overexpression of peripherin (Prph) transgene. Treatment of 12-month-old Gan-/-;TgPer mice with TubA led to a slight amelioration of motor function, especially a significant improvement of gait performance as measured by footprint analyses. Moreover, TubA treatment reduced the abnormal accumulations of Prph and NF proteins in spinal neurons and it boosted the levels of Prph transported into peripheral nerve axons. These results suggest that drug inhibitors of histone deacetylase aiming to enhance axonal transport should be considered as a potential treatment for GAN disease.

Independent Membrane Binding Properties of the Caspase Generated Fragments of the Beaded Filament Structural Protein 1 (BFSP1) Involves an Amphipathic Helix.

BACKGROUND: BFSP1 (beaded filament structural protein 1) is a plasma membrane, Aquaporin 0 (AQP0/MIP)-associated intermediate filament protein expressed in the eye lens. BFSP1 is myristoylated, a post-translation modification that requires caspase cleavage at D433. Bioinformatic analyses suggested that the sequences 434-452 were α-helical and amphipathic. METHODS AND RESULTS: By CD spectroscopy, we show that the addition of trifluoroethanol induced a switch from an intrinsically disordered to a more α-helical conformation for the residues 434-467. Recombinantly produced BFSP1 fragments containing this amphipathic helix bind to lens lipid bilayers as determined by surface plasmon resonance (SPR). Lastly, we demonstrate by transient transfection of non-lens MCF7 cells that these same BFSP1 C-terminal sequences localise to plasma membranes and to cytoplasmic vesicles. These can be co-labelled with the vital dye, lysotracker, but other cell compartments, such as the nuclear and mitochondrial membranes, were negative. The N-terminal myristoylation of the amphipathic helix appeared not to change either the lipid affinity or membrane localisation of the BFSP1 polypeptides or fragments we assessed by SPR and transient transfection, but it did appear to enhance its helical content. CONCLUSIONS: These data support the conclusion that C-terminal sequences of human BFSP1 distal to the caspase site at G433 have independent membrane binding properties via an adjacent amphipathic helix.

A New Mouse Model of Giant Axonal Neuropathy with Overt Phenotypes and Neurodegeneration Driven by Neurofilament Disorganization.

Research on pathogenic mechanisms underlying giant axonal neuropathy (GAN), a disease caused by a deficiency of gigaxonin, has been hindered by the lack of appropriate animal models exhibiting substantial symptoms and large neurofilament (NF) swellings, a hallmark of the human disease. It is well established that intermediate filament (IF) proteins are substrates for gigaxonin-mediated degradation. However, it has remained unknown to what extent NF accumulations contribute to GAN pathogenesis. Here, we report the generation of a new mouse model of GAN that is based on crossing transgenic mice overexpressing peripherin (Prph) with mice knockout for Gan The Gan-/-;TgPer mice developed early onset sensory-motor deficits along with IF accumulations made up of NF proteins and of Prph, causing swelling of spinal neurons at a young age. Abundant inclusion bodies composed of disorganized IFs were also detected in the brain of Gan-/-;TgPer mice. At 12 months of age, the Gan-/-;TgPer mice exhibited cognitive deficits as well as severe sensory and motor defects. The disease was associated with neuroinflammation and substantial loss of cortical neurons and spinal neurons. Giant axons (≥160 µm2) enlarged by disorganized IFs, a hallmark of GAN disease, were also detected in dorsal and ventral roots of the Gan-/-;TgPer mice. These results, obtained with both sexes, support the view that the disorganization of IFs can drive some neurodegenerative changes caused by gigaxonin deficiency. This new mouse model should be useful to investigate the pathogenic changes associated with GAN disease and for drug testing.SIGNIFICANCE STATEMENT Research on pathogenic mechanism and treatment of GAN has been hampered by the lack of animal models exhibiting overt phenotypes and substantial neurofilament disorganization, a hallmark of the disease. Moreover, it remains unknown whether neurologic defects associated with gigaxonin deficiency in GAN are because of neurofilament disorganization as gigaxonin may also act on other protein substrates to mediate their degradation. This study reports the generation of a new mouse model of GAN based on overexpression of Prph in the context of targeted disruption of gigaxonin gene. The results support the view that neurofilament disorganization may contribute to neurodegenerative changes in GAN disease. The Gan-/-;TgPer mice provide a unique animal model of GAN for drug testing.