Molecular and clinical characterization of the equine-like G3 rotavirus that caused the first outbreak in Japan, 2016.

Since 2013, equine-like G3 rotavirus (eG3) strains have been detected throughout the world, including in Japan, and the strains were found to be dominant in some countries. In 2016, the first eG3 outbreak in Japan occurred in Tomakomai, Hokkaido prefecture, and the strains became dominant in other Hokkaido areas the following year. There were no significant differences in the clinical characteristics of eG3 and non-eG3 rotavirus infections. The eG3 strains detected in Hokkaido across 2 years from 2016 to 2017 had DS-1-like constellations (i.e. G3-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2), and the genes were highly conserved (97.5-100 %). One strain, designated as To16-12 was selected as the representative strain for these strains, and all 11 genes of this strain (To16-12) exhibited the closest identity to one foreign eG3 strain (STM050) seen in Indonesia in 2015 and two eG3 strains (IS1090 and MI1125) in another Japanese prefecture in 2016, suggesting that this strain might be introduced into Japan from Indonesia. Sequence analyses of VP7 genes from animal and human G3 strains found worldwide did not identify any with close identity (>92 %) to eG3 strains, including equine RV Erv105. Analysis of another ten genes indicated that the eG3 strain had low similarity to G2P[4] strains, which are considered traditional DS-1-like strains, but high similarity to DS-1-like G1P[8] strains, which first appeared in Asia in 2012. These data suggest that eG3 strains were recently generated in Asia as mono-reassortant strain between DS-1-like G1P[8] strains and unspecified animal G3 strains. Our results indicate that rotavirus surveillance in the postvaccine era requires whole-genome analyses.

Rescue from Stx2-Producing E. coli-Associated Encephalopathy by Intravenous Injection of Muse Cells in NOD-SCID Mice.

Shiga toxin-producing Escherichia coli (STEC) causes hemorrhagic colitis, hemolytic uremic syndrome, and acute encephalopathies that may lead to sudden death or severe neurologic sequelae. Current treatments, including immunoglobulin G (IgG) immunoadsorption, plasma exchange, steroid pulse therapy, and the monoclonal antibody eculizumab, have limited effects against the severe neurologic sequelae. Multilineage-differentiating stress-enduring (Muse) cells are endogenous reparative non-tumorigenic stem cells that naturally reside in the body and are currently under clinical trials for regenerative medicine. When administered intravenously, Musecells accumulate to the damaged tissue, where they exert anti-inflammatory, anti-apoptotic, anti-fibrotic, and immunomodulatory effects, and replace damaged cells by differentiating into tissue-constituent cells. Here, severely immunocompromised non-obese diabetic/severe combined immunodeficiency (NOD-SCID) mice orally inoculated with 9 × 109 colony-forming units of STEC O111 and treated 48 h later with intravenous injection of 5 × 104 Muse cells exhibited 100% survival and no severe after-effects of infection. Suppression of granulocyte-colony-stimulating factor (G-CSF) by RNAi abolished the beneficial effects of Muse cells, leading to a 40% death and significant body weight loss, suggesting the involvement of G-CSF in the beneficial effects of Muse cells in STEC-infected mice. Thus, intravenous administration of Muse cells could be a candidate therapeutic approach for preventing fatal encephalopathy after STEC infection.

Ultrastructure of spinal anterior horn cells in human Niemann-Pick type C (NPC) patient and mouse model of NPC with retroposon insertion in NPC1 genes.

Niemann-Pick disease type C (NPC) is a neurovisceral lipid-storage disease. Although NPC patients show lipid storage in anterior horn cells of the spinal cord, little information is available regarding the electron microscopic analyses of the morphologies of intra-endosomal lipid like-materials in the anterior horn cells of NPC patients. In this study, we elucidated the intra-endosomal ultrastructures in spinal anterior horn cells in an NPC patient, as well as in mutant BALB/c NPC1-/- mice with a retroposon insertion in the NPC1 gene. These morphologies were classified into four types: vesicle, multiple concentric sphere (MCS), membrane, and rose flower. The percentages of the composition in the NPC patient and NPC1-/- mice were: vesicle (55.5% and 14.9%), MCS (15.7% and 3.4%), membrane (23.6% and 57.1%), and rose flower (5.2% and 24.6%), respectively. Formation of the intra-endosomal structures could proceed as follows: (i) a vesicle or MCS buds off the endosome into the lumen; (ii) when a vesicle breaks down, a membrane is formed; and (iii) after an MCS breaks down, a rose flower structure is formed. Our new finding in this study is that ultrastructural morphology is the same between the NPC patient and NPC1-/- mice, although there are differences in the composition.

Immunohistochemical and ultrastructural evidence for the pathogenesis of white matter degeneration in patients with panencephalopathic-type Creutzfeldt-Jakob disease: Inducible nitric oxide synthase overexpression in bizarre astrocytes.

Excessive production of nitric oxide (NO) due to the overinduction of inducible nitric oxide synthase (iNOS) has a severe cytotoxic effect, which may relate to the pathogenesis of neurodegenerative disorders. In this study, we report the novel finding that iNOS is overinduced in a large number of bizarre astrocytes in the white matter of patients with panencephalopathic (PE)-type Creutzfeldt-Jakob disease (CJD). This study was carried out on brain tissue from seven patients with PE-type CJD. As controls, 12 normal individuals and nine patients with cerebral infarction were examined. We identified a large number of bizarre astrocytes in the degenerative cerebral white matter in PE-type CJD. Using immunohistochemistry, only bizarre astrocytes in PE-type CJD showed strong immunoreactivity for both iNOS and superoxide dismutase 1 (SOD1). Ultrastructural examination demonstrated that these bizarre astrocytes contained many free polyribosome-like granules. No significant iNOS immunoreactivity was observed in either the astrocytes of patients with cerebral infarcts or in the normal controls. This study suggests that the iNOS-overexpressing astrocytes, especially iNOS-overexpressing bizarre astrocytes, could play an important role in the development of white matter lesions in PE-type CJD. Our data also suggest that the bizarre astrocytes could be protecting themselves from the cytotoxicity of NO by producing SOD1. These immunohistochemical findings are supported by the ultrastructural observation of numerous polyribosome granules restricted to the cytoplasm of these bizarre astrocytes.

Evidence that glial cells attenuate G47R transthyretin accumulation in the central nervous system.

Amyloidogenic protein forms amyloid aggregations at membranes leading to dysfunction of amyloid clearance and amyloidosis. Glial cells function in the clearance and degradation of amyloid ß (Aß) in the brain. This study aimed to clarify the reason why amyloid transthyretin (ATTR) rarely accumulates in the CNS. We pathologically analyzed the relationship between amyloid deposition with basement membranes or glial cells in a rare case of ATTR leptomeningeal amyloidosis. In addition, we compared the cytotoxicity of ATTR G47R, the amyloidosis-causing mutation in the case studied (n = 1), and Aß in brains from patients with cerebral amyloid angiopathy (n = 6). In the subarachnoid space of the ATTR G47R case, most amyloids accumulated at the components of basement membranes. On the CNS surface, ATTR accumulations were retained by astrocytic end feet. In areas where glial end feet enveloped ATTR, ubiquitination and micro-vacuolation of ATTR was evident. The colocalization of GFAP and ubiquitin was also evident. The accumulation of ATTR G47R in the CNS was negatively correlated with the prevalence of astrocytes. Quantitatively, amyloid deposits along the vessels were mostly partial in cerebral Aß angiopathy cases and nearly complete along the basement membrane in the ATTR G47R case. The vascular expressions of type IV collagen and smooth muscle actin were severely reduced in areas with ATTR G47R deposition, but not in areas with Aß deposition. The vascular protein level recovered in the ATTR G47R case when vessels entered into areas of parenchyma that were rich in astrocytes. In addition, the strong interactions between the transthyretin variant and basement membranes may have led to dysfunction of transthyretin clearance and leptomeningeal amyloidosis. The present study was the first to show that glial cells may attenuate G47R transthyretin accumulation in the CNS.

Neuroaxonal dystrophy in PLA2G6 knockout mice.

The PLA2G6 gene encodes group VIA calcium-independent phospholipase A2 (iPLA2 ß), which belongs to the PLA2 superfamily that hydrolyses the sn-2 ester bond in phospholipids. In the nervous system, iPLA2 ß is essential for remodeling membrane phospholipids in axons and synapses. Mutated PLA2G6 causes PLA2G6-associated neurodegeneration (PLAN) including infantile neuroaxonal dystrophy (INAD) and adult-onset dystonia-parkinsonism (PARK14), which have unique clinical phenotypes. In the PLA2G6 knockout (KO) mouse, which is an excellent PLAN model, specific membrane degeneration takes place in neurons and their axons, and this is followed by axonal spheroid formation. These pathological findings are similar to those in PLAN. This review details the evidence that membrane degeneration of mitochondria and axon terminals is a precursor to spheroid formation in this disease model. From a young age before the onset, many mitochondria with damaged inner membranes appear in PLA2G6 KO mouse neurons. These injured mitochondria move anterogradely within the axons, increasing in the distal axons. As membrane degeneration progresses, the collapse of the double membrane of mitochondria accompanies axonal injury near impaired mitochondria. At the axon terminals, the membranes of the presynapses expand irregularly from a young age. Over time, the presynaptic membrane ruptures, causing axon terminal degeneration. Although these processes occur in different degenerating membranes, both contain tubulovesicular structures, which are a specific ultrastructural marker of INAD. This indicates that two unique types of membrane degeneration underlie PLAN pathology. We have shown a new pathological mechanism whereby axons degenerate due to defective maintenance and rupture of both the inner mitochondrial and presynaptic membranes. This degeneration mechanism could possibly clarify the pathologies of PLAN, Parkinson disease and neurodegeneration with iron accumulation (NBIA), which are assumed to be due to the primary degeneration of axons.

Autopsy case of spinocerebellar ataxia type 31 with severe dementia at the terminal stage.

Spinocerebellar ataxia type 31 (SCA31) is an autosomal dominant cerebellar ataxia commonly observed in Japan. However, few neuropathological examinations have been conducted. Here we report the case of a 76-year-old Japanese male SCA31 patient. He noticed dysarthria and difficulty walking at 65 years old. His symptoms subsequently deteriorated, although he could still walk with assistance at 70 years. At 73 years, when he could no longer walk, he was admitted to our hospital. He showed severe limb and truncal ataxia. His father and older brother had shown the same symptoms. Brain magnetic resonance imaging showed cerebellar atrophy of the anterior lobe and white matter hyperintensities. He was diagnosed with SCA31 by genetic analysis. Gradually, his cognitive functions and ability to communicate declined. He died of respiratory failure at the age of 76. Neuropathological examination revealed severe Purkinje cell loss that was accentuated in the anterior lobe of the cerebellum. Furthermore, the remaining Purkinje cells showed abnormal processes (that is, halo-like amorphous materials), as has been reported previously. Severe deposition of hyperphosphorylated tau-positive neurites, many senile plaques and amyloid angiopathy were observed in the neocortex. Our findings suggest that in SCA31, accelerated tau and amyloid pathology in the neocortex might induce dementia at the terminal stage.

Molecular evolutionary analysis of novel NSP4 mono-reassortant G1P[8]-E2 rotavirus strains that caused a discontinuous epidemic in Japan in 2015 and 2018.

In the 2010s, several unusual rotavirus strains emerged, causing epidemics worldwide. This study reports a comprehensive molecular epidemiological study of rotaviruses in Japan based on full-genome analysis. From 2014 to 2019, a total of 489 rotavirus-positive stool specimens were identified, and the associated viral genomes were analyzed by next-generation sequencing. The genotype constellations of those strains were classified into nine patterns (G1P[8] (Wa), G1P[8]-E2, G1P[8] (DS-1), G2P[4] (DS-1), G3P[8] (Wa), G3P[8] (DS-1), G8P[8] (DS-1), G9P[8] (Wa), and G9P[8]-E2). The major prevalent genotype differed by year, comprising G8P[8] (DS-1) (37% of that year's isolates) in 2014, G1P[8] (DS-1) (65%) in 2015, G9P[8] (Wa) (72%) in 2016, G3P[8] (DS-1) (66%) in 2017, G1P[8]-E2 (53%) in 2018, and G9P[8] (Wa) (26%) in 2019. The G1P[8]-E2 strains (G1-P[8]-I1-R1-C1-M1-A1-N1-T1-E2-H1) isolated from a total of 42 specimens in discontinuous years (2015 and 2018), which were the newly-emerged NSP4 mono-reassortant strains. Based on the results of the Bayesian evolutionary analyses, G1P[8]-E2 and G9P[8]-E2 were hypothesized to have been generated from distinct independent inter-genogroup reassortment events. The G1 strains detected in this study were classified into multiple clusters, depending on the year of detection. A comparison of the predicted amino acid sequences of the VP7 epitopes revealed that the G1 strains detected in different years encoded VP7 epitopes harboring distinct mutations. These mutations may be responsible for immune escape and annual changes in the prevalent strains.

Correlation between pathology and neuromelanin MR imaging in Parkinson's disease and dementia with Lewy bodies.

INTRODUCTION: Direct correlation between neuropathological findings and postmortem neuromelanin MR imaging (NmMRI) was performed in the substantia nigra pars compacta (SNc) to clarify the pathological background of the signal changes in normal, Parkinson's disease (PD), and dementia with Lewy bodies (DLB) cases. METHODS: NmMRI of 10 % formalin-fixed autopsied midbrains was performed in three cases (normal control, DLB, and PD) with a 3T imaging system, using a 3D gradient echo T1-weighted sequence with a magnetization transfer contrast pulse. Neuropathological examinations of the midbrains were performed, and the density of neuromelanin-positive neurons (number per square millimeter) was determined. The extent of iron deposition in the midbrain was also evaluated using ferritin immunohistochemistry. Furthermore, we directly correlated the contrast signal ratio in the SNc and the density of neuromelanin-containing neurons. RESULTS: Diffuse hyperintense areas in the SNc reflected well-preserved neuromelanin-containing neurons in the normal control case, whereas an iso-intense area in the SNc showed severe loss of neuromelanin-containing neurons in the DLB and PD cases. Increased signal intensity in the SNc was apparently not influenced by iron deposition. Furthermore, a significant positive correlation between signal intensity and the density of neuromelanin-containing neurons was seen in the SNc. CONCLUSION: Based on the direct correlation between postportem NmMRI and neuropathological findings, signal intensity in the SNc is closely related to the quantity of neuromelanin-containing neurons but is not influenced by iron deposition.

An integrated genetic analysis of epileptogenic brain malformed lesions.

Focal cortical dysplasia is the most common malformation during cortical development, sometimes excised by epilepsy surgery and often caused by somatic variants of the mTOR pathway genes. In this study, we performed a genetic analysis of epileptogenic brain malformed lesions from 64 patients with focal cortical dysplasia, hemimegalencephy, brain tumors, or hippocampal sclerosis. Targeted sequencing, whole-exome sequencing, and single nucleotide polymorphism microarray detected four germline and 35 somatic variants, comprising three copy number variants and 36 single nucleotide variants and indels in 37 patients. One of the somatic variants in focal cortical dysplasia type IIB was an in-frame deletion in MTOR, in which only gain-of-function missense variants have been reported. In focal cortical dysplasia type I, somatic variants of MAP2K1 and PTPN11 involved in the RAS/MAPK pathway were detected. The in-frame deletions of MTOR and MAP2K1 in this study resulted in the activation of the mTOR pathway in transiently transfected cells. In addition, the PTPN11 missense variant tended to elongate activation of the mTOR or RAS/MAPK pathway, depending on culture conditions. We demonstrate that epileptogenic brain malformed lesions except for focal cortical dysplasia type II arose from somatic variants of diverse genes but were eventually linked to the mTOR pathway.

Neuroaxonal dystrophy in calcium-independent phospholipase A2ß deficiency results from insufficient remodeling and degeneration of mitochondrial and presynaptic membranes.

Infantile neuroaxonal dystrophy (INAD) is a fatal neurodegenerative disease characterized by the widespread presence of axonal swellings (spheroids) in the CNS and PNS and is caused by gene abnormality in PLA2G6 [calcium-independent phospholipase A(2)ß (iPLA(2)ß)], which is essential for remodeling of membrane phospholipids. To clarify the pathomechanism of INAD, we pathologically analyzed the spinal cords and sciatic nerves of iPLA(2)ß knock-out (KO) mice, a model of INAD. At 15 weeks (preclinical stage), periodic acid-Schiff (PAS)-positive granules were frequently observed in proximal axons and the perinuclear space of large neurons, and these were strongly positive for a marker of the mitochondrial outer membrane and negative for a marker of the inner membrane. By 100 weeks (late clinical stage), PAS-positive granules and spheroids had increased significantly in the distal parts of axons, and ultrastructural examination revealed that these granules were, in fact, mitochondria with degenerative inner membranes. Collapse of mitochondria in axons was accompanied by focal disappearance of the cytoskeleton. Partial membrane loss at axon terminals was also evident, accompanied by degenerative membranes in the same areas. Imaging mass spectrometry showed a prominent increase of docosahexaenoic acid-containing phosphatidylcholine in the gray matter, suggesting insufficient membrane remodeling in the presence of iPLA(2)ß deficiency. Prominent axonal degeneration in neuroaxonal dystrophy might be explained by the collapse of abnormal mitochondria after axonal transportation. Insufficient remodeling and degeneration of mitochondrial inner membranes and presynaptic membranes appear to be the cause of the neuroaxonal dystrophy in iPLA(2)ß-KO mice.

Mutant SOD1 impairs axonal transport of choline acetyltransferase and acetylcholine release by sequestering KAP3.

Mutations in the superoxide dismutase 1 (sod1) gene cause familial amyotrophic lateral sclerosis (FALS), likely due to the toxic properties of misfolded mutant SOD1 protein. Here we demonstrated that, starting from the pre-onset stage of FALS, misfolded SOD1 species associates specifically with kinesin-associated protein 3 (KAP3) in the ventral white matter of SOD1(G93A)-transgenic mouse spinal cord. KAP3 is a kinesin-2 subunit responsible for binding to cargos including choline acetyltransferase (ChAT). Motor axons in SOD1(G93A)-Tg mice also showed a reduction in ChAT transport from the pre-onset stage. By employing a novel FALS modeling system using NG108-15 cells, we showed that microtubule-dependent release of acetylcholine was significantly impaired by misfolded SOD1 species. Furthermore, such impairment was able to be normalized by KAP3 overexpression. KAP3 was incorporated into SOD1 aggregates in human FALS cases as well. These results suggest that KAP3 sequestration by misfolded SOD1 species and the resultant inhibition of ChAT transport play a role in the dysfunction of ALS.

A pediatric intramedullary spinal cord tumor with unusual solid-cystic and papillary features: a case report.

Spinal cord tumors are rare in children. We report a novel case of pediatric intramedullary spinal cord tumor with unusual solid-cystic and papillary features. Clinically, the patient presented at the age of 3 years with motor deficit and urinary incontinence, and MRI demonstrated multilocular cystic lesions in the thoracic spine. Histologically the tumor consisted of solid, sheet-like components and branching papillary structures, and immunohistochemistry demonstrated positive reactivity for epithelial membrane antigen, cytokeratins (7, AE1/3, CAM5.2), E-cadherin and transthyretin, and negativity for GFAP, S-100 protein, synaptophysin and neurofilament. These histological and immunohistochemical findings appeared to be unique, and were not compatible with the features of classical ependymoma or choroid plexus papilloma. The clinical behavior, characterized by relatively rapid tumor regrowth after surgical resection and a relatively high MIB-1 labeling index, suggest that this tumor might have had moderate malignant potential. This pediatric case appears to be particularly informative with regard to the tumor biology or tumorigenesis of intramedullary spinal cord tumor with unusual solid-cystic and papillary features.

Estimating the germicidal effect of upper-room UVGI system on exhaled air of patients based on ventilation efficiency.

Upper room (UR)-ultraviolet germicidal (UVGI) systems, one of several disinfection applications of UV, target airborne infectious diseases in rooms of buildings such as healthcare facilities. Previous studies have introduced many experiments showing the germicidal effect of UR-UVGI systems. In this study, a novel numerical method of estimating the germicidal effect of UR-UVGI systems for air exhaled by ward patients was introduced. The method adopts and modifies the concept of ventilation efficiency because the germicidal effect depends upon how the air containing airborne infectious particles flows and stays within UV-radiated area. A case study based on a four-patient ward showed that UV doses were correlated with the age of the air exhaled by a source patient, as expected. Moreover, the UV doses were considerably affected by the position of the UR-UVGI system. Inactivation rates of the influenza virus estimated using the UV doses, were in the range of 48-74%, and those of Mycobacterium tuberculosis were 68-90% in the breathing area of a neighboring patient. The results indicate not directly the decreased concentration of airborne infectious particles, but the possibility of inactivation caused by the UR-UVGI system, which is useful for system optimization.

Nuclear TAR DNA binding protein 43 expression in spinal cord neurons correlates with the clinical course in amyotrophic lateral sclerosis.

TAR DNA binding protein 43 (TDP-43) has been considered a signature protein in frontotemporal dementia and amyotrophic lateral sclerosis (ALS), but not in ALS associated with the superoxide dismutase 1 (SOD1) gene mutations (ALS1). To clarify how TDP may be involved in ALS pathogenesis, clinical and pathological features in cases of sporadic ALS ([SALS] n = 18) and ALS1 (n = 6) were analyzed. In SALS patients with rapid clinical courses, TDP mislocalization (i.e. cytoplasmic staining and TDP-positive cytoplasmic inclusions) in anterior horn cells was frequent. In SALS patients with slow clinical courses, TDP-43 mislocalization was rare. In an ALS1 patient with the SOD1 gene mutation C111Y, there were numerous TDP-positive inclusions and colocalization of SOD1 and TDP. In mutant SOD1 transgenic (G93A) mice at the end stage (median, 256 days), TDP-positive inclusions and TDP colocalization with SOD1 were also observed; nuclear TDP-43 immunoreactivity was highly correlated with life span in these mice. In both humans and mice, nuclei that stained strongly for TDP were large and circular; weakly stained nuclei were atrophic or deformed. In conclusion, low levels of TDP expression in the nucleus cor relate with a rapid clinical course in SALS and in ALS1 model mice, suggesting that nuclear TDP may play a protective role against motor neuron death resulting from different underlying etiologies.

Ossified Metaplastic Spinal Meningioma Without Psammomatous Calcification: A Case Report.

Meningiomas constitute approximately 25% of primary spinal cord tumors, and 1% to 5% are calcified. Ossification is a rare event and the etiology of ossification in meningiomas is not well known. We present the case of a 29-year-old female with a rare case of ossified thoracic spinal metaplastic meningioma. The tumor was successfully resected, and pathology confirmed ossified metaplastic meningioma. On histopathological examination, only mature bone tissue and tumor cells were present in the region containing no psammoma bodies, suggesting that the tumor cells had transitioned to mature osteocytes.

Functional analysis of a double-point mutation in the KCNJ2 gene identified in a family with Andersen-Tawil syndrome.

Andersen-Tawil syndrome (ATS) is a skeletal muscle channelopathy with autosomal dominant inheritance resulting in periodic paralysis, arrhythmia characterized by QT prolongation, and dysmorphic features. The KCNJ2 gene has been identified as the causative gene of ATS. Herein, we reported 2 cases of a 21-year-old man and his mother, with episodic paralytic attacks and/or arrhythmia, which are characteristic of ATS. Both G144A, a reported ATS mutation, and V296F, a novel mutation, were identified in the KCNJ2 gene on the same allele from the proband and his mother, but not from his father. In the present study, we investigated the functional effect of these variants on the potassium channel Kir2.1 and the significance of the double mutation. G144A, V296F, and G144A-V296F mutant channels expressed in cultured cells revealed a loss-of-function effect of these mutations on Kir2.1. The K+ currents of G144A and G144A-V296F channels were more suppressed than that of V296F channel alone, whereas was no difference between G144A and G144A-V296F. To our knowledge, a double mutation in the KCNJ2 gene has not been reported previously. While either of 2 mutations potentially causes ATS, the G144A mutation might cause the dominant effect on the patients' clinical presentation.

Endosomal accumulation of Toll-like receptor 4 causes constitutive secretion of cytokines and activation of signal transducers and activators of transcription in Niemann-Pick disease type C (NPC) fibroblasts: a potential basis for glial cell activation in the NPC brain.

Niemann-Pick disease type C (NPC) is an inherited lipid storage disorder caused by mutations in NPC1 or NPC2 genes. Loss of function of either protein results in the endosomal accumulation of cholesterol and other lipids, progressive neurodegeneration, and robust glial cell activation. Here, we report that cultured human NPC fibroblasts secrete interferon-beta, interleukin-6 (IL-6), and IL-8, and contain increased levels of signal transducers and activators of transcription (STATs). These cells also contained increased levels of Toll-like receptor 4 (TLR4) that accumulated in cholesterol-enriched endosomes/lysosomes, and small interfering RNA knockdown of this receptor reduced cytokine secretion. In the NPC1-/- mouse brain, glial cells expressed TLR4 and IL-6, whereas both glial and neuronal cells expressed STATs. Genetic deletion of TLR4 in NPC1-/- mice reduced IL-6 secretion by cultured fibroblasts but failed to alter STAT levels or glial cell activation in the brain. In contrast, genetic deletion of IL-6 normalized STAT levels and suppressed glial cell activation. These findings indicate that constitutive cytokine secretion leads to activation of STATs in NPC fibroblasts and that this secretion is partly caused by an endosomal accumulation of TLR4. These results also suggest that similar signaling events may underlie glial cell activation in the NPC1-/- mouse brain.