Endoplasmic reticulum stress-induced caspase-4 activation mediates apoptosis and neurodegeneration in INCL

Infantile neuronal ceroid lipofuscinosis (INCL), a neurodegenerative storage disorder of childhood, is caused by mutations in the palmitoyl-protein thioesterase-1 (PPT1) gene. PPT1 cleaves thioester linkages in S-acylated (palmitoylated) proteins and its mutation causes abnormal intracellular accumulation of fatty-acylated proteins and peptides leading to INCL pathogenesis. Although apoptosis is the suggested cause of neurodegeneration in INCL, the molecular mechanism(s) of apoptosis remains unclear. Using the PPT1-knockout (PPT1-KO) mice that mimic INCL, we previously reported that one mechanism of apoptosis involves endoplasmic reticulum (ER) stress-induced caspase-12 activation. However, the human caspase-12 gene contains several mutations, which make it functionally inactive. Thus, it has been suggested that human caspase-4 is the counterpart of murine caspase-12. Here we report that in the human INCL brain ER stress-induced activation of unfolded protein response (UPR) mediates caspase-4 and caspase-3 activation and apoptosis. Moreover, we show that the INCL brain contains high level of growth-associated protein-43 (GAP-43), which is known to undergo palmitoylation. We also demonstrate that transfection of cultured INCL cells with a green fluorescent protein-GAP-43 cDNA construct shows abnormal localization of this protein in the ER. Further, INCL cells manifest evidence of ER stress and UPR (elevated levels of Grp-78/Bip and GADD153), caspase-4 as well as caspase-3 activation and cleavage of poly(ADP)-ribose polymerase, a compelling sign of apoptosis. Most importantly, we show that inhibition of caspase-4 activity protects INCL cells from undergoing apoptosis. Our results provide insight into at least one of the molecular mechanisms of apoptosis in INCL and may allow the identification of potential targets for therapeutic intervention.     

Mutagenic activities and physicochemical properties of selected nitrobenzanthrones

Mutagenic activity of nine nitro derivatives of benzanthrone, namely 1-nitro-, 2-nitro-, 3-nitro-, 9-nitro-, 11-nitro-, 1,9-dinitro-, 3,9-dinitro-, 3,11-dinitro- and 3,9,11-trinitrobenzanthrone were tested with Salmonella strains TA98, TA100, YG1021 and YG1024 in both the presence and absence of an S9 mix. Each compound exhibited mutagenic activity with all the strains. Among these nine isomers, 3-nitrobenzantrone exhibited the most mutagenic activity with all the strains without the S9 mix. The mutagenic activities of the dinitro and trinitro derivatives of benzanthrone were lower than that of the 3-nitro derivative; this is evident from the mutagenic activity of nitrated polyaromatic hydrocarbons (PAH), which is generally enhanced with an increase in nitration. The physicochemical properties of nitrated benzanthrone (reduction potential, hydrophobicity and orientation of nitro groups to the aromatic ring) demonstrated that mononitrated benzanthrone exhibits a lower reduction potential than mononitroPAHs such as 1-nitropyrene and 3-nitrofluoranthene, but was almost equivalent to that of dinitroPAH. Moreover, the mutagenic activity of mononitrobenzanthrones clearly depend on the reduction potential of each compound; however, this tendency was not observed in polynitrobenzanthrones, probably because the reduction of the nitro groups to amino groups of polynitrated benzanthrone might be predominant without a sufficient formation of corresponding hydroxyamines. These results suggest that aromatic compounds that contain keto groups, when nitrated, may act as potentially powerful direct-acting mutagens.     

Lactoferrin and surfactant protein A exhibit distinct binding specificity to F protein and differently modulate respiratory syncytial virus infection

Surfactant protein A (SP-A) and lactoferrin (LF) play important roles in innate immune systems in the respiratory mucous membranes. We investigated how SP-A and LF act against respiratory syncytial virus (RSV) infection. The present study indicated that RSV-induced IL-8 secretion from HEp-2 cells was up-regulated by SP-A (170% of control) but down-regulated by LF (23% of control). RSV infectivity determined by viral titers and the uptake of FITC-labeled RSV were also increased by SP-A, but decreased by LF. To clarify the mechanism of these opposite effects, we examined the interactions of SP-A and LF with RSV F protein, the most important surface glycoprotein for viral penetration. RSV F protein was found to be the ligand for both SP-A and LF, but the manners of binding were different. LF directly interacted with the F(1) subunit, which involved antigenic sites of F protein. Contrarily, SP-A associated with the F(2) subunit, which was highly glycosylated. SP-A but not LF failed to interact with deglycosylated F protein. Moreover, SP-A initiated the hemolyzing fusion activity of F protein. These results suggest that SP-A and LF modulate RSV infection by different binding specificity to F protein.     

Prevention of amyloid beta-induced memory impairment by fluvastatin, associated with the decrease in amyloid beta accumulation and oxidative stress in amyloid beta injection mouse model

Alzheimer's disease (AD), the most common cause of dementia in the elderly, is characterized by amyloid beta (Abeta)-containing plaques and neurofibrillary tangles, and synaptic and neuronal loss, along with progressive cognitive impairment. Although growing evidence suggests the beneficial effects of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) on AD, this notion is still controversial. To evaluate the efficacy of statins for Abeta-induced cognitive impairment, we employed an Abeta injection model. Using this model, the present study demonstrated that pretreatment with fluvastatin, but not post-treatment just after Abeta exposure, prevented Abeta-induced memory impairment. We also observed that fluvastatin significantly decreased Abeta accumulation and oxidative stress after Abeta injection. Mice treated with simvastatin, but not fluvastatin, did not demonstrate the prevention of Abeta-induced memory impairment, and showed no significant decrease in oxidative stress. More importantly, fluvastatin significantly prevented the loss of neurons in the basal forebrain induced by Abeta. Overall, the present study demonstrated that fluvastatin significantly prevented memory impairment induced by Abeta. The beneficial effects of fluvastatin might be explained by the preservation of neurons through a significant decrease in Abeta accumulation and oxidative stress. In clinical practice, the timing of the start of fluvastatin treatment might be critical in achieving a beneficial effect on cognitive function.     

Preoperative diagnosis of clear cell sarcoma of the kidney by detection of BCOR internal tandem duplication in circulating tumor DNA

Clear cell sarcoma of the kidney (CCSK) is the second most common renal malignancy in children. The prognosis is poorer in CCSK than in Wilms’ tumor, and multimodal treatment including surgery, intensive chemotherapy, and radiation is required to improve the outcome for children with CCSK. Histological evaluation is required for the diagnosis. However, biopsies of tumors to obtain diagnostic specimens are not routinely performed because of the risk of spreading tumor cells during the procedure. Recently, internal tandem duplication (ITD) of BCOR has been recognized as a genetic hallmark of CCSK. We herein established a novel BCOR‐ITD‐specific polymerase chain reaction method with well‐designed primers, and then performed a liquid biopsy for cell‐free DNA (cfDNA) obtained from plasma of three children with nonmetastatic renal tumors (stage II) and from one control. BCOR‐ITD was positively detected in the cfDNA of two cases, both of which were later diagnosed as CCSK based on histological feature of the resected tumor specimen, while it was not detected for a normal control and a patient diagnosed with Wilms’ tumor. Our study is the first one of preoperative circulating tumor DNA assay in pediatric renal tumors. The liquid biopsy method enables less invasive, preoperative diagnosis of CCSK with no risk of tumor spillage, which can avoid iatrogenic upstaging.     

Fine‐scale geographic clustering pattern of human T‐cell leukemia virus type 1 infection among blood donors in Kyushu‐Okinawa,Japan

Human T‐cell leukemia virus type I (HTLV‐1) infection is endemic in Japan, particularly clustered in the southwestern district, Kyushu‐Okinawa, which consists of eight prefectures that further consist of 274 municipalities. However, no information is available about the fine‐scale distribution of HTLV‐1 infection within Kyushu‐Okinawa. To assess the municipal‐level distribution of people with HTLV‐1 infection in Kyushu‐Okinawa, we performed a cross‐sectional study using a fine‐scale geographic information system map based on HTLV‐1 screening test results from the Japanese Red Cross database from September 2012 to February 2014. Of the 881 871 (646 914 male, 234 957 female) screened blood donors, 981 were seropositive for HTLV‐1 by confirmatory test. The seroprevalence was 0.11% (95% confidence interval [CI] 0.10%‐0.12%) for all, 0.094% (95% CI, 0.09%‐0.10%) for male, and 0.16% (95% CI, 0.14%‐0.18%) for female individuals. The sex‐ and age‐specific HTLV‐1 seroprevalence varied significantly across municipalities; particularly, the seroprevalence among women aged 50 years was significantly higher than that of men in both the mainland of Kyushu‐Okinawa and the satellite island, in all of which the seroprevalence of HTLV‐1 was more than 1.2%. These results show that, even in the Kyushu‐Okinawa district, there are endemic clusters of HTLV‐1 in small areas. This suggests that public health education programs are needed to eliminate new HTLV‐1 infection in these areas.     

Antigen‐specific airway IL‐33 production depends on FcγR‐mediated incorporation of the antigen by alveolar macrophages in sensitized mice

Although interleukin (IL)‐33 is a candidate for the aggravation of asthma, the mechanisms underlying antigen‐specific IL‐33 production in the lung are unclear. Therefore, we analysed the mechanisms in mice. Intra‐tracheal administration of ovalbumin (OVA) evoked increases in IL‐33 and IL‐33 mRNA in the lungs of both non‐sensitized and OVA‐sensitized mice, and the increases in the sensitized mice were significantly higher than in the non‐sensitized mice. However, intra‐tracheal administration of bovine serum albumin did not increase the IL‐33 level in the OVA‐sensitized mice. Depletion of neither mast cells/basophils nor CD4⁺ cells abolished the OVA‐induced IL‐33 production in sensitized mice, suggesting that the antigen recognition leading to the IL‐33 production was not related with either antigen‐specific IgE‐bearing mast cells/basophils or memory CD4⁺ Th2 cells. When a fluorogenic substrate‐labelled OVA (DQ‐OVA) was intra‐tracheally administered, the lung cells of sensitized mice incorporated more DQ‐OVA than those of non‐sensitized mice. The lung cells incorporating DQ‐OVA included B‐cells and alveolar macrophages. The allergic IL‐33 production was significantly reduced by treatment with anti‐FcγRII/III mAb. Depletion of alveolar macrophages by clodronate liposomes significantly suppressed the allergic IL‐33 production, whereas depletion of B‐cells by anti‐CD20 mAb did not. These results suggest that the administered OVA in the lung bound antigen‐specific IgG Ab, and then alveolar macrophages incorporated the immune complex through FcγRII/III on the cell surface, resulting in IL‐33 production in sensitized mice. The mechanisms underlying the antigen‐specific IL‐33 production may aid in development of new pharmacotherapies.     

Vestibular and cochlear nerve enhancement on MRI and its correlation with vestibulocochlear functional deficits in patients with Ramsay Hunt syndrome

ObjectiveThe correlation between enhancement of the vestibulocochlear nerves on gadolinium-enhanced magnetic resonance imaging (MRI) and vestibulocochlear functional deficits was examined in patients with Ramsay Hunt syndrome (RHS).MethodsNineteen patients with RHS who showed herpes zoster oticus, peripheral facial palsy, and vertigo were enrolled. Canal paresis (CP) in the caloric test, abnormal response to ocular and cervical vestibular myogenic potentials (oVEMP and cVEMP), and refractory sensorineural hearing loss were evaluated. MRI images perpendicular to the internal auditory canal were reconstructed to identify the superior (SVN) and inferior vestibular nerves (IVN) and the cochlear nerve (CV). The signal intensity increase (SIinc) of the four-nerve enhancement was calculated as an index.ResultsAmong RHS patients, 79%, 53%, 17% and 26% showed CP in the caloric test, abnormal responses to oVEMP and cVEMP, and refractory sensorineural hearing loss, respectively. SIinc rates of the SVN were significantly increased in RHS patients with CP in the caloric test, and with abnormal responses to oVEMP and cVEMP. SIinc rates of the SVN tended to increase in RHS patients with refractory sensorineural hearing loss (p = 0.052). SIinc rates of the IVN were significantly increased in RHS patients with abnormal responses to oVEMP and cVEMP, and refractory sensorineural hearing loss, but not in those with CP in the caloric test. SIinc rates of the CN were significantly increased in RHS patients with CP in the caloric test, abnormal response to oVEMP and refractory sensorineural hearing loss, but not in those with abnormal response to cVEMP.ConclusionIn patients with RHS, the origin of vertigo may be superior vestibular neuritis, which is affected by reactive varicella-zoster virus from the geniculate ganglion of the facial nerve through the faciovestibular anastomosis. The results also suggested that in some RHS patients, inferior vestibular neuritis contributes to the development of vertigo and that the origin of refractory sensorineural hearing loss is cochlear neuritis.