Morphological and functional aspects of the epidermis of the sea lamprey Petromyzon marinus throughout development.

The development of the epidermis of sea lamprey Petromyzon marinus along the whole life cycle was studied using conventional staining techniques and lectin histochemistry. The epidermis undergoes variations in morphology and thickness throughout development. The simple cuboidal epithelium found in the epidermis of prolarvae becomes stratified cubic in the adult by increasing the number of cell layers. The cuticle thickness undergoes a steady increase during the larval period. There are changes in the glycoconjugate composition of the three main cell types of the P. marinus epidermis, mucous, granular and skein cells, which are more pronounced after metamorphosis. The Alcian blue-periodic acid Schiff (AB-PAS) histochemical method shows the presence of both acidic and neutral glycoconjugates in the mucous cells, indicating their secretory function. Moreover, lectin analysis reveals a mucous secretion containing glycoconjugates such as sulphated glycosaminoglycans (N-acetylglucosamine and N-acetylgalactosamine) and N-glycoproteins rich in mannose. Although granular cells are AB-PAS negative, they exhibit a similar glycoconjugate composition to the mucous cells. Moreover, granular cells show sialic acid positivity in larvae but this monosaccharide residue is not detected after metamorphosis. The skein cells, a unique cell of lampreys, are negative to AB-PAS staining but they mostly contain l-fucose and sialic acid residues, which also disappear after metamorphosis. The function of the granular and skein cells is still unknown but the role of their glycoconjugate composition is discussed. In addition, a different cellular origin is suggested for these two types of cells.

Spontaneous unraveling of hagfish slime thread skeins is mediated by a seawater-soluble protein adhesive.

Hagfishes are known for their ability to rapidly produce vast quantities of slime when provoked. The slime is formed via the interaction between seawater and two components released by the slime glands: mucin vesicles from gland mucous cells, which swell and rupture in seawater to form a network of mucus strands, and intermediate filament-rich threads, which are produced within gland thread cells as tightly coiled bundles called skeins. A previous study showed that the unraveling of skeins from Atlantic hagfish (Myxine glutinosa) requires both the presence of mucins and hydrodynamic mixing. In contrast, skeins from Pacific hagfish (Eptatretus stoutii) unravel in the absence of both mucins and mixing. We tested the hypothesis that spontaneous unraveling of E. stoutii skeins is triggered by the dissolution of a seawater-soluble protein adhesive and the release of stored strain energy within the coiled thread. Here we show that, as predicted by this hypothesis, unraveling can be initiated by a protease under conditions in which unraveling does not normally occur. We also demonstrate, using high resolution scanning electron microscopy, that the treatment of skeins with solutions that cause unraveling also leads to the disappearance of surface and inter-thread features that remain when skeins are washed with stabilizing solutions. Our study provides a mechanism for the deployment of thread skeins in Pacific hagfish slime, and raises the possibility of producing novel biomimetic protein adhesives that are salt, temperature and kosmotrope sensitive.

Co-localization of Bunina bodies and TDP-43 inclusions in lower motor neurons in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is characterized by motor neuron involvement with Bunina bodies (BBs) and transactivation response DNA protein 43 (TDP-43) inclusions. We examined the spinal cord (n = 20), hypoglossal nucleus (n = 6) and facial nucleus (n = 5) from ALS patients to elucidate the relationship between BBs and TDP-43 inclusions. BBs were found in the anterior horn in 16 of 20 cases, in the hypoglossal nucleus in all six cases and in the facial nucleus in four out of five cases. TDP-43 inclusions were found in each region of all the cases. Co-localization of BBs and TDP-43 inclusions was found in 15.2% of total neurons in the anterior horn, 29.2% in the hypoglossal nucleus and 17.3% in the facial nucleus. The frequency of TDP-43 inclusions was significantly higher in neurons with BBs than in those without in each region. Ultrastructurally, TDP-43-positive filamentous structures were intermingled with BBs. These findings suggest that there is a close relationship in the occurrence between BBs and TDP-43 inclusions.

From reductionism to synthesis: The case of hagfish slime.

Reductionist strategies aim to understand the mechanisms of complex systems by studying individual parts and their interactions. In this review, we discuss how reductionist approaches have shed light on the structure, function, and production of a complex biomaterial - hagfish defensive slime. Hagfish slime is an extremely dilute hydrogel-like material composed of seawater, mucus, and silk-like proteins that can deploy rapidly. Despite being composed almost entirely of water, hagfish slime has remarkable physical properties, including high strength and toughness. While hagfish slime has a promising future in biomimetics, including the development of eco-friendly high-performance fibers, recreating hagfish slime in the lab has been a difficult challenge. Over the past two decades, reductionist experiments have provided a wealth of information about the individual components of hagfish slime. However, a reductionist approach provides a limited understanding because hagfish defensive slime, like most biological phenomena, is more than just the sum of its parts. We end by providing some thoughts about how the knowledge generated in the last few decades might be synthesized into a working model that can explain hagfish slime structure and function.

Novel versatile 3D bio-scaffold made of natural biocompatible hagfish exudate for tissue growth and organoid modeling.

Hagfish exudate is a natural biological macromolecule made of keratin intermediate filament protein skeins and mucin vesicles. Here, we successfully examined this remarkable biomaterial as a substrate for three-dimensional (3D) cell culturing purposes. After the sterilization with chloroform vapor, Dulbecco's modified eagle medium was mixed with the exudate to rupture the vesicles and skeins; a highly soft, adherent, fibrous and biocompatible hydrogel was formed. A variety of cells, including Hela-FUCCI, NMuMG-FUCCI, 10T1/2 and C2C12, was cultured on the hagfish exudate. A remarkable 3D growth by ~2.5 folds after day 3, ~5 folds after day 5, ~10 folds after day 7 and ~15 folds after day 14 were seen compared to day one of culturing in the hagfish exudate scaffold. In addition, the phase contrast, fluorescent and confocal microscopy observations confirmed the organoid shape formation within the three-week culture. The viability of cells was almost 100% indicating the great in vitro and in vivo potential of this exceptional biomaterial with no cytotoxic effect.

Pathogenic LRRK2 requires secondary factors to induce cellular toxicity.

Pathogenic mutations in the leucine-rich repeat kinase 2 (LRRK2) gene belong to the most common genetic causes of inherited Parkinson's disease (PD) and variations in its locus increase the risk to develop sporadic PD. Extensive research efforts aimed at understanding how changes in the LRRK2 function result in molecular alterations that ultimately lead to PD. Cellular LRRK2-based models revealed several potential pathophysiological mechanisms including apoptotic cell death, LRRK2 protein accumulation and deficits in neurite outgrowth. However, highly variable outcomes between different cellular models have been reported. Here, we have investigated the effect of different experimental conditions, such as the use of different tags and gene transfer methods, in various cellular LRRK2 models. Readouts included cell death, sensitivity to oxidative stress, LRRK2 relocalization, α-synuclein aggregation and neurite outgrowth in cell culture, as well as neurite maintenance in vivo. We show that overexpression levels and/or the tag fused to LRRK2 affect the relocalization of LRRK2 to filamentous and skein-like structures. We found that overexpression of LRRK2 per se is not sufficient to induce cellular toxicity or to affect α-synuclein-induced toxicity and aggregate formation. Finally, neurite outgrowth/retraction experiments in cell lines and in vivo revealed that secondary, yet unknown, factors are required for the pathogenic LRRK2 effects on neurite length. Our findings stress the importance of technical and biological factors in LRRK2-induced cellular phenotypes and hence imply that conclusions based on these types of LRRK2-based assays should be interpreted with caution.

Autophagy Is a Common Degradation Pathway for Bunina Bodies and TDP-43 Inclusions in Amyotrophic Lateral Sclerosis.

Bunina bodies (BBs) coexisting with TDP-43-immunoreactive (TDP-43-IR) skein-like inclusions (SIs) and round inclusions (RIs) in lower motor neurons are a frequent feature of sporadic amyotrophic lateral sclerosis (sALS). Since previous studies have shown that BBs and TDP-43-IR inclusions are often detected in association with autophagy-related structures (autophagosomes and autolysosomes), we examined the anterior horn cells (AHCs) of the spinal cord from 15 patients with sALS and 6 control subjects, using antibodies against autophagy-related proteins (LC3, cathepsin B, and cathepsin D). Among AHCs with SIs, 43.9% contained BBs, whereas 51.7% of AHCs with RIs did so. The cytoplasm of AHCs showed diffuse immunoreactivity for LC3, cathepsin B and cathepsin D in both sALS and controls. Ultrastructurally, SIs and mature BBs contained autophagosomes and autolysosomes. Mature BBs were localized in the vicinity of SIs. RIs also contained autophagosomes, autolysosomes, and early-stage BBs. These findings suggest that autophagy is a common degradation pathway for BBs and TDP-43-IR inclusions, which may explain their frequent coexistence.

Amyotrophic lateral sclerosis with appearance of many skein-like inclusions in anterior horn cells.

We report an autopsy case of amyotrophic lateral sclerosis (ALS), in which an abnormally large number of skein-like inclusions (SLIs) was found in anterior horn cells. The patient was a 73-year-old man, who presented with dysarthria. His motor neuron symptoms were predominantly of the upper-neuron type, and cognitive impairment was also noted. He died of septic shock 13 months after onset of the first neurological symptoms. Autopsy revealed marked loss of upper motor neurons, severe degeneration of the pyramidal tract, mild to moderate loss of anterior horn cells, and the appearance of many SLIs, which were immunoreactive for both pTDP-43 (phosphorylated transactivation responsive DNA-binding protein of 43 kDa) and ubiquitin, in anterior horn cells. Intra-axonal pTDP-43-positive granules arranged in a bead-like fashion were also found. The appearance of pTDP-43-positive intracytoplasmic inclusions in the brain was mostly restricted to the motor cortex. An Alzheimer type tau-pathology was found mainly in the hippocampus (Braak stage III), and many argyrophilic grains were distributed in the limbic area. Atypical ALS showing a rapid clinical course associated with cognitive impairment and predominant involvement of the upper motor neurons has recently been reported. The present case shares some clinical and pathologic findings with this type of atypical ALS. The appearance of a large number of SLIs is an unusual finding. Although its pathologic significance remains unknown, it cannot simply be ascribed to the relative preservation of anterior horn cells.

Anterior Cingulate Cortex TDP-43 Pathology in Sporadic Amyotrophic Lateral Sclerosis.

Neuronal constituents of the human anterior cingulate cortex displayed morphological changes related to the 43-kDa transactive response DNA-binding protein (TDP-43) in advanced pathological stages of sporadic amyotrophic lateral sclerosis (sALS). By using nonphosphorylation-dependent TDP-43 immunocytochemistry, it was seen that the changes in susceptible pyramidal cells of the superficial cellular layers II-IIIab differed from those in the deep layers IIIc-Vb: A complete loss of nuclear TDP-43 expression (i.e. nuclear clearing) in the small projection neurons of layers II-IIIab was consistently accompanied by the development of somatic skein-like TDP-43-immunopositive inclusions. In contrast, in the large pyramidal cells of layers IIIc-Vb and von Economo neurons of layer Vb, skein-like inclusions were lacking or, when aggregated TDP-43 was present, the aggregates presented as dash-like TDP-43-immunopositive particles in the vicinity of the cell nucleus. The cytoskeleton of projection neurons in layers II-IIIab is neurofilament-sparse in contrast to that of the large neurons in layers IIIc-Vb, which are rich in neurofilaments and also heat shock proteins that function as their molecular chaperones. The disparities between the two neuronal populations may contribute to the two differing immunocytochemical profiles reported here. Some implications of the findings for the pathogenesis and progression of TDP-43 pathology in sALS are discussed.

Larval epidermis of the red eye tree frog Agalychnis callidryas (Anura, Hylidae): ultrastructural investigation on the Kugelzellen, specialized forms of the constitutive skein cell line.

An ultrastructural study was carried out on the epidermis of Agalychnis callidryas tadpoles during limb development. Larval epidermis consisted of four cell layers: basal, lower intermediate, upper intermediate, and surface or apical layers. Basal cells represented the stem compartment of intermediate cells: both belong to the skein cell (SC) lineage, described in several anuran species, on account of the conspicuous intracytoplasmic tonofilament bundles. Apical cells were secretory in nature and released mucus on the body surface. Intermediate SCs exhibited a hydrated central cytoplasm and peripheral tonofilament bundles. They closely resembled the epidermal ball-like cells, Kugelzellen (KZn) of Xenopus laevis tadpoles, and possibly shared their turgor-stiffness properties. In A. callidryas, the stratification of intermediated SCs on their stem cell layer provided the chance to study their cytodifferentiation in a suitable sequence, until basal cell differentiation shifted toward the keratinocyte lineage in premetamorphic stages. Present data assign A. callidryas to the anuran species with a constitutive SC population in larval epidermis, and demonstrate that KZn express the ultimate specialization of such cell line. SCs were arranged in the fashion of a random-rubble stone groundwork, and possessed long processes. These cytoplasmic outgrowths contained a tonofilament axial rod and held together contiguous cells. Ultrastructural findings suggest that this complex structure may impart compressive as well as sliding strengths to the larval epidermis, representing a possible adaption to the fresh water environment.