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The molecular machinery responsible for cytosolic accumulation of misfolded TDP-43 in amyotrophic lateral sclerosis (ALS) remains elusive. Here we identified a cullin-2 (CUL2) RING complex as a novel ubiquitin ligase for fragmented forms of TDP-43. The von Hippel Lindau protein (VHL), a substrate binding component of the complex, preferentially recognized misfolded TDP-43 at Glu246 in RNA-recognition motif 2. Recombinant full-length TDP-43 was structurally fragile and readily cleaved, suggesting that misfolded TDP-43 is cleared by VHL/CUL2 in a step-wise manner via fragmentation. Surprisingly, excess VHL stabilized and led to inclusion formation of TDP-43, as well as mutant SOD1, at the juxtanuclear protein quality control center. Moreover, TDP-43 knockdown elevated VHL expression in cultured cells, implying an aberrant interaction between VHL and mislocalized TDP-43 in ALS. Finally, cytoplasmic inclusions especially in oligodendrocytes in ALS spinal cords were immunoreactive to both phosphorylated TDP-43 and VHL. Thus, our results suggest that an imbalance in VHL and CUL2 may underlie oligodendrocyte dysfunction in ALS, and highlight CUL2 E3 ligase emerges as a novel therapeutic potential for ALS.

The single amino acid mutation G26R in human apolipoprotein A-I (apoA-IIowa) is the first mutation that was associated with familial AApoA1 amyloidosis. The N-terminal fragments (amino acid residues 1–83) of apoA-I containing this mutation deposit as amyloid fibrils in patients’ tissues and organs, but the mechanisms of cellular degradation and cytotoxicity have not yet been clarified. In this study, we demonstrated degradation of apoA-IIowa fibrils via the autophagy-lysosomal pathway in human embryonic kidney 293 cells. ApoA-IIowa fibrils induced an increase in lysosomal pH and the cytosolic release of the toxic lysosomal protease cathepsin B. The mitochondrial dysfunction caused by apoA-IIowa fibrils depended on cathepsin B and was ameliorated by increasing the degradation of apoA-IIowa fibrils. Thus, although apoA-IIowa fibril transport to lysosomes and fibril degradation in lysosomes may have occurred, the presence of an excess number of apoA-IIowa fibrils, more than the lysosomes could degrade, may be detrimental to cells. Our results thus provide evidence that the target of apoA-IIowa fibrils is lysosomes, and we thereby gained a novel insight into the mechanism of AApoA1 amyloidosis.

Macrophages in hyperlipidemic conditions accumulate cholesterol esters and develop into foamy transformed macrophages. During this transformation, macrophages demonstrate endoplasmic reticulum fragmentation and consequently produce acyl coenzyme A : cholesterol acyltransferase 1 (ACAT1-positive late endosomes (ACAT1-LE). ACAT1-LE-positive macrophages effectively esterify modified or native low-density lipoprotein-derived free cholesterol, which results in efficient cholesterol esterification as well as atherosclerotic plaque formation. These macrophages show significant cholesterol ester formation even when free cholesterol egress from late endosomes is impaired, which indicates that free cholesterol is esterified at ACAT1-LE. Genetic blockade of cholesterol egress from late endosomes causes Niemann-Pick disease type C (NPC), an inherited lysosomal storage disease with progressive neurodegeneration. Induction of ACAT1-LE in macrophages with the NPC phenotype led to significant recovery of cholesterol esterification. In addition, in vivo ACAT1-LE induction significantly extended the lifespan of mice with the NPC phenotype. Thus, ACAT1-LE not only regulates intracellular cholesterol metabolism but also ameliorates NPC pathophysiology.

Desmoplastic (scirrhous) invasion and lymph node metastasis are critical for the treatment and prognosis of invasive ductal carcinoma of the breast. Despite being an anti-angiogenic therapeutic candidate, Thrombospondin-1 (TSP-1) promotes invasion and metastasis of some carcinomas. To clarify the effect of TSP-1 on invasion and metastasis, we obtained 101 invasive ductal carcinomas of the breast with axillary lymph node resection. All tumors were histologically divided into two categories, carcinomas with, and those with non- /minimal desmoplastic component. Immunohistochemistry for TSP-1 was performed on all primary tumors and axillary lymph nodes with tumor metastasis. Fifty-four (53.5%) of 101 tumors were recognized as positive for TSP-1 in the cytoplasm of tumor cells. Histological study showed that significantly more cancers with desmoplastic components (46/69, 66.7%) manifested TSP-1 expression than did cancers with no- or minimal (less than 20%) desmoplasia (8/32, 25.0% ; p< 0.001). Axillary lymph node metastasis was significantly higher in TSP-1-positive- (28/54, 51.9%) than TSP-1-negative cancers (11/47, 23.4% ; p<0.005). The present study indicates that tumor cells in the desmoplastic component strongly expressed TSP-1 in invasive ductal carcinoma of the breast and TSP-1 participates in invasion of these tumors. Our findings also suggest that TSP-1 promotes lymph node metastasis and TSP-1 potentially could be a predictive marker for metastasis.

Large cell neuroendocrine carcinoma (LCNEC) is a rare poorly differentiated carcinoma with neuroendocrine differentiation showing aggressive clinical behavior. We herein report a case of gallbladder LCNEC, which was difficult to differentiate from poorly differentiated adenocarcinoma. An imprint cytology was very useful for the final diagnosis in this case. A 56-year-old male with left exophthalmos was admitted to the hospital. Radiological examinations revealed the presence of a left gallbladder tumor with orbital metastasis. The histological diagnosis was poorly differentiated adenocarcinoma, and intensive chemoradiotherapy was administered. Unfortunately, the patient died of extensive metastases 36 months after the initial onset of symptoms. An autopsy revealed a tumor mass in the gallbladder associated with multiple liver and peritoneal metastases. Imprint cytology of the main tumor revealed cytological features of LCNEC, and additional histological examinations confirmed this diagnosis. Although performing a histological examination is important for making a final diagnosis, imprint cytology is powerful tool for differential diagnosis of LCNEC, especially in patients with carcinoma with poor differentiation.