Novel mTORC1 Mechanism Suggests Therapeutic Targets for COMPopathies.

Cartilage oligomeric matrix protein (COMP) is a large, multifunctional extracellular protein that, when mutated, is retained in the rough endoplasmic reticulum (ER). This retention elicits ER stress, inflammation, and oxidative stress, resulting in dysfunction and death of growth plate chondrocytes. While identifying the cellular pathologic mechanisms underlying the murine mutant (MT)-COMP model of pseudoachondroplasia, increased midline-1 (MID1) expression and mammalian target of rapamycin complex 1 (mTORC1) signaling was found. This novel role for MID1/mTORC1 signaling was investigated since treatments shown to repress the pathology also reduced Mid1/mTORC1. Although ER stress-inducing drugs or tumor necrosis factor α (TNFα) in rat chondrosarcoma cells increased Mid1, oxidative stress did not, establishing that ER stress- or TNFα-driven inflammation alone is sufficient to elevate MID1 expression. Since MID1 ubiquitinates protein phosphatase 2A (PP2A), a negative regulator of mTORC1, PP2A was evaluated in MT-COMP growth plate chondrocytes. PP2A was decreased, indicating de-repression of mTORC1 signaling. Rapamycin treatment in MT-COMP mice reduced mTORC1 signaling and intracellular retention of COMP, and increased proliferation, but did not change inflammatory markers IL-16 and eosinophil peroxidase. Lastly, mRNA from tuberous sclerosis-1/2-null mice brain tissue exhibiting ER stress had increased Mid1 expression, confirming the relationship between ER stress and MID1/mTORC1 signaling. These findings suggest a mechanistic link between ER stress and MID1/mTORC1 signaling that has implications extending to other conditions involving ER stress.

Extended ultrastructural characterization of chordoma cells: the link to new therapeutic options.

Chordomas are rare bone tumors, developed from the notochord and largely resistant to chemotherapy. A special feature of this tumor is the heterogeneity of its cells. By combining high pressure freezing (HPF) with electron tomography we were able to illustrate the connections within the cells, the cell-cell interface, and the mitochondria-associated endoplasmic reticulum membrane complex that appears to play a special role among the characteristics of chordoma. These lipid raft-like regions are responsible for lipid syntheses and for calcium signaling. Compared to other tumor cells, chordoma cells show a close connection of rough endoplasmic reticulum and mitochondria, which may influence the sphingolipid metabolism and calcium release. We quantified levels of ceramide and glycosylceramide species by the methyl tert-butyl ether extraction method and we assessed the intracellular calcium concentration with the ratiometric fluorescent dye Fura-2AM. Measurements of the changes in the intracellular calcium concentration revealed an increase in calcium due to the application of acetylcholine. With regard to lipid synthesis, glucosylceramide levels in the chordoma cell line were significantly higher than those in normal healthy cells. The accumulation of glycosylceramide in drug resistant cancer cells has been confirmed in many types of cancer and may also account for drug resistance in chordoma. This study aimed to provide a deep morphological description of chordoma cells, it demonstrated that HPF analysis is useful in elucidating detailed structural information. Furthermore we demonstrate how an accumulation of glycosylceramide in chordoma provides links to drug resistance and opens up the field for new research options.

Infection and injury of human astrocytes by tick-borne encephalitis virus.

Tick-borne encephalitis (TBE), a disease caused by tick-borne encephalitis virus (TBEV), represents the most important flaviviral neural infection in Europe and north-eastern Asia. In the central nervous system (CNS), neurons are the primary target for TBEV infection; however, infection of non-neuronal CNS cells, such as astrocytes, is not well understood. In this study, we investigated the interaction between TBEV and primary human astrocytes. We report for the first time, to the best of our knowledge, that primary human astrocytes are sensitive to TBEV infection, although the infection did not affect their viability. The infection induced a marked increase in the expression of glial fibrillary acidic protein, a marker of astrocyte activation. In addition, expression of matrix metalloproteinase 9 and several key pro-inflammatory cytokines/chemokines (e.g. tumour necrosis factor α, interferon α, interleukin (IL)-1ß, IL-6, IL-8, interferon γ-induced protein 10, macrophage inflammatory protein, but not monocyte chemotactic protein 1) was upregulated. Moreover, we present a detailed description of morphological changes in TBEV-infected cells, as investigated using three-dimensional electron tomography. Several novel ultrastructural changes were observed, including the formation of unique tubule-like structures of 17.9 ±0.15 nm diameter with associated viral particles and/or virus-induced vesicles and located in the rough endoplasmic reticulum of the TBEV-infected cells. This is the first demonstration that TBEV infection activates primary human astrocytes. The infected astrocytes might be a potential source of pro-inflammatory cytokines in the TBEV-infected brain, and might contribute to the TBEV-induced neurotoxicity and blood-brain barrier breakdown that occurs during TBE. The neuropathological significance of our observations is also discussed.

Predictive factors of late venous aortocoronary graft failure: ultrastructural studies.

BACKGROUND: Venous aortocoronary graft arterialization may precede a preterm occlusion in some coronary artery bypass grafting (CABG) patients. The aim of the present study was to identify ultrastructural variations in the saphenous vein wall that may have an impact on the development of venous graft disease in CABG patients. METHODS: The study involved 365 consecutive patients with a mean age of 62.9 ± 9.4 years who underwent isolated CABG. The thickness and area of the whole venous wall, the tunica intima, the tunica media and the adventitia and the number and shape (length, thickness and length/thickness ratio) of the nuclei in the medial smooth muscle cells nuclei in the distal saphenous vein segments were evaluated by ultrastructural studies. Patients were followed up for 41 to 50 months (mean 45.1 ± 5.1). Saphenous vein graft patency was assessed by follow-up coronary angiography. Logistic regression models were used to identify independent risk factors for late graft failure. RESULTS: In 71 patients significant lesions in the saphenous vein grafts were observed. The whole venous wall thickness (437.5 µm vs. 405.5 µm), tunica media thickness (257.2 µm vs. 211.5 µm), whole venous wall area (2.23 mm(2) vs. 2.02 mm(2)) and tunica media area (1.09 mm(2) vs. 0.93 mm(2)) were significantly larger for this group of patients than for those without graft disease. In the latter group more elongated smooth muscle cell nuclei (higher length/thickness ratio) were found in the tunica media of the saphenous vein segments. Thickening of the saphenous vein tunica media and chunky smooth muscle cell nuclei were identified as independent risk factors for graft disease development. CONCLUSIONS: Saphenous vein tunica media hypertrophy (resulting in wall thickening) and chunky smooth muscle cell nuclei might predict the development of venous graft disease.

Juvenile ALS with basophilic inclusions is a FUS proteinopathy with FUS mutations.

BACKGROUND: Juvenile amyotrophic lateral sclerosis (ALS) with basophilic inclusions is a form of ALS characterized by protein deposits in motor neurons that are morphologically and tinctorially distinct from those of classic sporadic ALS. The nosologic position of this type of ALS in the molecular pathologic and genetic classification of ALS is unknown. METHODS: We identified neuropathologically 4 patients with juvenile ALS with basophilic inclusions and tested the hypothesis that specific RNA binding protein pathology may define this type of ALS. Immunohistochemical findings prompted us to sequence the fused in sarcoma (FUS) gene. RESULTS: Motor symptoms began between ages 17 and 22. Disease progression was rapid without dementia. No family history was identified. Basophilic inclusions were strongly positive for FUS protein but negative for TAR DNA binding protein 43 (TDP-43). Granular and compact FUS deposits were identified in glia and neuronal cytoplasm and nuclei. Ultrastructure of aggregates was in keeping with origin from fragmented rough endoplasmic reticulum. Sequencing of all 15 exons of the FUS gene in 3 patients revealed a novel deletion mutation (c.1554_1557delACAG) in 1 individual and the c.1574C>T (P525L) mutation in 2 others. CONCLUSION: Juvenile ALS with basophilic inclusions is a FUS proteinopathy and should be classified as ALS-FUS. The FUS c.1574C>T (P525L) and c.1554_1557delACAG mutations are associated with this distinct phenotype. The molecular genetic relationship with frontotemporal lobar degeneration with FUS pathology remains to be clarified.

Chondroid chordoma of the skull base: immunohistochemical and ultrastructural study of two cases with special reference to microtubules within rough-surfaced endoplasmic reticulum.

Two cases of skull base chordoma (case 1, a 57-year-old woman; case 2, a 69-year-old woman) were investigated immunohistochemically and ultrastructurally. The tumors showed histopathological features typical of chondroid chordoma and contained both classical chordomatous and hyaline cartilaginous components. Tumor cells were immunoreactive for cytokeratin, vimentin, and S-100 protein, but negative for microtubule-associated protein 2 and class III beta-tubulin (tub-B3). Tumor cells of case 2 were immunoreactive for tau-protein and class II beta-tubulin (tub-B2), whereas those of case 1 were negative. Ultrastructurally, tumor cells in both cases showed the presence of abundant glycogen granules, well-developed intracellular organelles, and desmosome-like junctions. In case 2, several microtubules were closely packed and ran parallel or in random directions within the dilated cisterns of rough-surfaced endoplasmic reticulum (rough ER). "Microtubules within rough ER" has been described in several neoplasms, including classical and chondroid chordomas. Although previous reports documented the tub-B3 immunoreactivity in chordomas, our results suggested that, in our case 2, the predominant isoform of beta-tubulin in microtubules within rough ER was not tub-B3 but tub-B2.

Pathological characteristics of gastric leiomyoblastoma.

AIM: To determine the pathological characteristics of gastric leiomyoblastoma. METHODS: All tissues were obtained during surgery or gastroscopy. Tissue specimens for examination by light microscope were 1 cm x 1 cm x 1 cm in size, fixed in 40 g/L neutral buffered formaldehyde, embedded in paraffin, and stained with hematoxylin and eosin. The fresh tissues obtained for electron microscopy were 1 mm x 1 mm x 1 mm in size, and fixed in phosphate buffered 30 g/L glutaraldehyde, postfixed in 10 g/L osmium tetroxide and dehydrated in graded alcohol, embebbed in Epon 812. Ultrathin sections of 50 nm were stained with uranyl acetate and lead citrate and examined under a JEM-2000 EX transmission electron microscope. RESULTS: The most important histopathological feature of leiomyoblastoma was the predominance of large, rounded or polygonal cells with characteristic perinuclear clear zone in cytoplasms. The tumor cells arranged in patch, cell junction or junctional complex could be found occasionally between cells under electron microscope. Most of the neoplastic cytoplasms were filled with myofilaments, dense bodies, and dense patches. Rough endoplasmic reticulum dilatated as lakes, and large quantities of protein secretions of intermediate electron density were found in the dilated cisternae. Intracisternal segregation could also be found. The nuclei were round or oval, and anomalous nuclei were found in part of cells. CONCLUSION: The diagnosis of gastric leiomyoblastoma can be confirmed by electron microscopy. The clear appearance of tumor cells is due to the dilation of rough endoplasmic reticulum, not fat droplets, glycogens or mucus in cytoplasm.

Ultrastructural study of synovitis induced by trauma to the rat temporomandibular joint (TMJ).

BACKGROUND: Electron microscopy was used to examine the histologic effect of trauma on the rat temporomandibular joint synovial membrane. METHODS: Trauma to the TMJ in male Wister rats (100-200 g) was introduced through repeated forced condylar hypermobility. Ultrastructural observations were made 5 days and 6 weeks after the trauma. RESULTS: The early response of the synovial membrane was synovial hyperplasia, type A synovial cell loss, dilation of the r-ER in the type B synovial cells and fibrin deposition on the synovial surfaces. The late response included degeneration of synovial cells with swollen mitochondria and cell projections, and cell fragmentation. Large amount of fibrin deposition on opposing surface layers was also noticed. CONCLUSION: The type A cell loss and fibrin deposition followed by the occurrence of fibrinous materials at opposing surface layers of the synovial membrane suggest that traumatic synovitis causes synovial adhesions.

Extremely low frequency (ELF) pulsed-gradient magnetic fields inhibit malignant tumour growth at different biological levels.

Extremely low frequency (ELF) pulsed-gradient magnetic field (with the maximum intensity of 0.6-2.0 T, gradient of 10-100 T.M(-1), pulse width of 20-200 ms and frequency of 0.16-1.34 Hz treatment of mice can inhibit murine malignant tumour growth, as seen from analyses at different hierarchical levels, from organism, organ, to tissue, and down to cell and macromolecules. Such magnetic fields induce apoptosis of cancer cells, and arrest neoangiogenesis, preventing a supply developing to the tumour. The growth of sarcomas might be amenable to such new method of treatment.

The PERK eukaryotic initiation factor 2 alpha kinase is required for the development of the skeletal system, postnatal growth, and the function and viability of the pancreas.

Phosphorylation of eukaryotic initiation factor 2 alpha (eIF-2 alpha) is typically associated with stress responses and causes a reduction in protein synthesis. However, we found high phosphorylated eIF-2 alpha (eIF-2 alpha[P]) levels in nonstressed pancreata of mice. Administration of glucose stimulated a rapid dephosphorylation of eIF-2 alpha. Among the four eIF-2 alpha kinases present in mammals, PERK is most highly expressed in the pancreas, suggesting that it may be responsible for the high eIF-2 alpha[P] levels found therein. We describe a Perk knockout mutation in mice. Pancreata of Perk(-/-) mice are morphologically and functionally normal at birth, but the islets of Langerhans progressively degenerate, resulting in loss of insulin-secreting beta cells and development of diabetes mellitus, followed later by loss of glucagon-secreting alpha cells. The exocrine pancreas exhibits a reduction in the synthesis of several major digestive enzymes and succumbs to massive apoptosis after the fourth postnatal week. Perk(-/-) mice also exhibit skeletal dysplasias at birth and postnatal growth retardation. Skeletal defects include deficient mineralization, osteoporosis, and abnormal compact bone development. The skeletal and pancreatic defects are associated with defects in the rough endoplasmic reticulum of the major secretory cells that comprise the skeletal system and pancreas. The skeletal, pancreatic, and growth defects are similar to those seen in human Wolcott-Rallison syndrome.

A recurrent RNA-splicing mutation in the SEDL gene causes X-linked spondyloepiphyseal dysplasia tarda.

Spondyloepiphyseal dysplasia tarda (SEDL) is a genetically heterogeneous disorder characterized by mild-to-moderate short stature and early-onset osteoarthritis. Both autosomal and X-linked forms have been described. Elsewhere, we have reported the identification of the gene for the X-linked recessive form, which maps to Xp22.2. We now report characterization of an exon-skipping mutation (IVS3+5G-->A at the intron 3 splice-donor site) in two unrelated families with SEDL. Using reverse transcriptase (RT)-PCR, we demonstrated that the mutation resulted in elimination of the first 31 codons of the open reading frame. The mutation was not detected in 120 control X chromosomes. Articular cartilage from an adult who had SEDL and carried this mutation contained chondrocytes with abundant Golgi complexes and dilated rough endoplasmic reticulum (ER). RT-PCR experiments using mouse/human cell hybrids revealed that the SEDL gene escapes X inactivation. Homologues of the SEDL gene include a transcribed retropseudogene on chromosome 19, as well as expressed genes in mouse, rat, Drosophila melanogaster Caenorhabditis elegans, and Saccharomyces cerevisiae. The latter homologue, p20, has a putative role in vesicular transport from ER to Golgi complex. These data suggest that SEDL mutations may perturb an intracellular pathway that is important for cartilage homeostasis.

Pale bodies in hepatocellular carcinoma.

Histochemical, immunohistochemical and ultrastructural studies were performed on cases of hepatocellular carcinoma (HCC) with pale bodies (PB). HCC containing PBs was observed in 3 (5.5%) of 55 consecutively resected HCC cases. Histologically, a large number of hepatocytes displayed pale or eosinophilic staining of the cytoplasm, resulting in ground-glass appearance. They were aggregated in nodular pattern, or diffusely intermixed with other malignant hepatocytes. PBs were negative for periodic-acid Schiff and Masson's trichrome staining. The inclusions showed a strong positive reaction for fibrinogen and some of them were weakly positive for albumin but negative for hepatitis B surface antigen, hepatitis B core antigen, alpha-fetoprotein and alpha-1-antitrypsin. Ultrastructurally, PBs were membrane-bound and contained granular materials of moderate electron density, and were closely related to dilated rough endoplasmic reticulum. These findings support that PBs are secretory fibrinogen accumulated in cystic ER and that such intracellular accumulation possibly reflects a defective transport of fibrinogen.

Dilated rough endoplasmic reticulum corresponding to septate cytoplasmic vacuoles in papillary thyroid carcinoma.

Septate cytoplasmic vacuoles, which are one of the cytologic findings characteristic of papillary thyroid carcinoma, are small, uniform, and well-defined vacuoles, with defined strands of cytoplasm separating them. We report on a case with histologic and ultrastructural findings corresponding to septate cytoplasmic vacuoles, which have not been previously described. The patient was a 51-yr-old man with a mass in the left anterior of the neck, measuring 4.0 x 3.5 cm in diameter. Aspiration cytology smears revealed findings typical of papillary thyroid carcinoma, including papillary tissue fragments, intranuclear inclusions, nuclear grooves, powdery chromatin, and psammoma bodies. The tumor cells which were located at the periphery of small three-dimensional clusters, and others which showed a sheet-like arrangement, demonstrated uniform, small vacuoles in relatively abundant, dense cytoplasm. Histologically, a small number of papillary thyroid carcinoma cells with multiple vacuoles were observed, which were limited to the hobnail-shaped cells located at papillary configurations or floating tumor cells within the papillary lumen. Ultrastructurally, the hobnail-shaped tumor cells demonstrated various-sized dilated rough endoplasmic reticulum and rich heterochromatin. We emphasize that septate cytoplasmic vacuoles correspond to dilated rough endoplasmic vacuoles and are probably related to degenerative changes.

Myofibroblastoma of the neck in a heifer.

A 1.5-year-old Holstein heifer had a subcutaneous tumor mass (20 cm diameter) on the ventral portion of the neck, and the tumor was diagnosed as a locally invasive myofibroblastoma. It consisted of moderately cellular fibrous tissue, and the interlobular septum of the thymus was invaded by tumor cells. The neoplastic cells were positive for alpha smooth muscle actin and vimentin, but not for desmin. Electron microscopy disclosed the presence of moderately developed rough endoplasmic reticulum and microfilaments with focal densities.

Intercalated duct cells in the chicken pancreatic islet with special reference to the alloxan administration.

The intercalated duct cells were observed in the A and B islets of the chicken pancreas. These cells adhered with each other by intercellular junctional complexes at the apical side. They had many microvilli projecting into the lumen. Abluminally, they displayed extended slender cytoplasmic processes between islet endocrine cells. Administration of alloxan resulted to denser cytoplasm and a more prominent thickening of cytoplasmic processes of the intercalated duct cells, although the blood glucose levels did not show appreciable changes by the treatment. The intercalated duct epithelial cells appeared clearly as stellate cells. The lysosomes increased in size and number with passage of time after alloxan administration. The present findings may suggest that intercalated ducts are not only anatomically important as a structure passing through the islet but also play physiologically by protecting the islet endocrine cells.

Activation of the autophagy, c-FOS and ubiquitin expression, and nucleolar alterations in Schwann cells precede demyelination in tellurium-induced neuropathy.

We have used an experimental model of tellurium (Te)-induced demyelinating neuropathy in the rat to study cellular mechanisms involved in the early response of myelinating Schwann cells (SCs) to injury, prior to demyelination. Starting at postnatal day 21, weaned rats were fed a diet containing 1.1% elemental Te. The animals were killed daily within the 1st week of Te diet and the sciatic nerves were processed for the ultrastructural and immunocytochemical studies. Immunohistochemistry revealed that Te induces an increased nuclear expression of c-Fos in SCs. By electron microscopy analysis, the early cytoplasmic alteration was a dramatic disorganization of the rough endoplasmic reticulum (ER) with cisternal dilations and redistribution and loss of membrane-bound ribosomes. This was followed by a prominent activation of the macroautophagy in SCs. This process involved the formation of autophagosomes containing well-preserved cell organelles, autolysosomes with cellular remnants in various phases of degeneration and lysosomes. Te treatment also induced the expression of free ubiquitin in the perikaryal region of the SC cytoplasm. Immunogold electron microscopy showed the subcellular distribution of ubiquitin in the cytosol, around of dilated ER cisterns and in the matrix of autolysosomes and residual bodies. At the nucleolar level, fibrillarin immunofluorescence revealed nucleolar segregation in SCs exposed to Te. The ultrastructural study confirmed the segregation of the nucleolar components with a peripheral distribution of the dense fibrillar component. These results support the hypothesis that the depletion of cholesterol induced by Te treatment triggers a stress response in myelinating SCs mediated by immediate early genes of the fos family. The cellular response includes a severe disruption of the protein synthesis machinery, namely the rough ER and nucleolus, with the subsequent activation of both ubiquitin and autophagic pathways of proteins and cell organelle degradation. This cytoplasmic remodeling may represent a cytoprotective mechanism in the response of SCs to a neurotoxic stress. Furthermore, it must be a prerequisite for the induction of phenotypic changes and cell repair mechanisms in SCs.

Ultrastructure of hepatocyte abnormalities in perimetastatic areas.

The ultrastructural pathology of non-invaded hepatocytes located in close proximity to metastases from diverse gastrointestinal carcinomas was studied. Observed abnormalities included swelling of rough and smooth endoplasmic reticulum, proliferation of lysosomes, and mitochondrial alterations as presence of granules and paracrystalline inclusions, marked pleomorphism, and lack of cristae. These results show that, contrary to the classical conception, the non-invaded cells surrounding primary tumours or their metastases could be abnormal.