Kousaku Ohno

The Niemann-Pick C1 Protein Resides in a Vesicular Compartment Linked to Retrograde Transport of Multiple Lysosomal Cargo

Niemann-Pick C disease (NP-C) is a neurovisceral lysosomal storage disorder. A variety of studies have highlighted defective sterol trafficking from lysosomes in NP-C cells. However, the heterogeneous nature of additional accumulating metabolites suggests that the cellular lesion may involve a more generalized block in retrograde lysosomal trafficking. Immunocytochemical studies in fibroblasts reveal that theNPC1 gene product resides in a novel set of lysosome-associated membrane protein-2 (LAMP2)(+)/mannose 6-phosphate receptor(−) vesicles that can be distinguished from cholesterol-enriched LAMP2(+) lysosomes. Drugs that block sterol transport out of lysosomes also redistribute NPC1 to cholesterol-laden lysosomes. Sterol relocation from lysosomes in cultured human fibroblasts can be blocked at 21 °C, consistent with vesicle-mediated transfer. These findings suggest that NPC1(+) vesicles may transiently interact with lysosomes to facilitate sterol relocation. Independent of defective sterol trafficking, NP-C fibroblasts are also deficient in vesicle-mediated clearance of endocytosed [14C]sucrose. Compartmental modeling of the observed [14C]sucrose clearance data targets the trafficking defect caused by mutations in NPC1 to an endocytic compartment proximal to lysosomes. Low density lipoprotein uptake by normal cells retards retrograde transport of [14C]sucrose through this same kinetic compartment, further suggesting that it may contain the sterol-sensing NPC1 protein. We conclude that a distinctive organelle containing NPC1 mediates retrograde lysosomal transport of endocytosed cargo that is not restricted to sterol.

Chemical chaperone therapy for brain pathology in GM1-gangliosidosis

We synthesized a galactose derivative, N-octyl-4-epi-β-valienamine (NOEV), for a molecular therapy (chemical chaperone therapy) of a human neurogenetic disease, β-galactosidosis (GM1-gangliosidosis and Morquio B disease). It is a potent inhibitor of lysosomal β-galactosidase in vitro. Addition of NOEV in the culture medium restored mutant enzyme activity in cultured human or murine fibroblasts at low intracellular concentrations, resulting in a marked decrease of intracellular substrate storage. Short-term oral administration of NOEV to a model mouse of juvenile GM1-gangliosidosis, expressing a mutant enzyme protein R201C, resulted in significant enhancement of the enzyme activity in the brain and other tissues. Immunohistochemical stain revealed a decrease in the amount of GM1 and GA1 in neuronal cells in the fronto-temporal cerebral cortex and brainstem. However, mass biochemical analysis did not show the substrate reduction observed histochemically in these limited areas in the brain probably because of the brief duration of this investigation. Chemical chaperone therapy may be useful for certain patients with β-galactosidosis and potentially other lysosomal storage diseases with central nervous system involvement.

Niemann-Pick C1 Disease: The I1061T Substitution Is a Frequent Mutant Allele in Patients of Western European Descent and Correlates with a Classic Juvenile Phenotype

Niemann-Pick type C (NPC) disease is an autosomal recessive lipid-storage disorder usually characterized by hepatosplenomegaly and severe progressive neurological dysfunction, resulting from mutations affecting either the NPC1 gene (in 95% of the patients) or the yet-to-be-identified NPC2 gene. Our initial study of 25 patients with NPC1 identified a T3182-->C transition that leads to an I1061T substitution in three patients. The mutation, located in exon 21, affects a putative transmembrane domain of the protein. PCR-based tests with genomic DNA were used to survey 115 unrelated patients from around the world with all known clinical and biochemical phenotypes of the disease. The I1061T allele constituted 33 (14.3%) of the 230 disease-causing alleles and was never found in controls (>200 alleles). The mutation was particularly frequent in patients with NPC from Western Europe, especially France (11/62 alleles) and the United Kingdom (9/32 alleles), and in Hispanic patients whose roots were in the Upper Rio Grande valley of the United States. The I1061T mutation originated in Europe and the high frequency in northern Rio Grande Hispanics results from a founder effect. All seven unrelated patients who were homozygous for the mutation and their seven affected siblings had a juvenile-onset neurological disease and severe alterations of intracellular LDL-cholesterol processing. The mutation was not found (0/40 alleles) in patients with the severe infantile neurological form of the disease. Testing for this mutation therefore has important implications for genetic counseling of families affected by NPC.

Enhanced autophagy and mitochondrial aberrations in murine GM1-gangliosidosis

G(M1)-gangliosidosis is an autosomal recessive lysosomal lipid storage disorder, caused by mutations of the lysosomal beta-galactosidase (beta-gal) and results in the accumulation of G(M1). The underlying mechanisms of neurodegeneration are poorly understood. Here we demonstrate increased autophagy in beta-gal-deficient (beta-gal(-/-)) mouse brains as evidenced by elevation of LC3-II and beclin-1 levels. Activation of autophagy in the beta-gal(-/-) brain was found to be accompanied with enhanced Akt-mTOR and Erk signaling. In addition, the mitochondrial cytochrome c oxidase activity was significantly decreased in brains and cultured astrocytes from beta-gal(-/-) mouse. Mitochondria isolated from beta-gal(-/-) astrocytes were morphologically abnormal and had a decreased membrane potential. These cells were more sensitive to oxidative stress than wild type cells and this sensitivity was suppressed by ATP, an autophagy inhibitor 3-methyladenine and a pan-caspase inhibitor z-VAD-fmk. These results suggest activation of autophagy leading to mitochondrial dysfunction in the brain of G(M1)-gangliosidosis.

Accumulation of cholera toxin and GM1 ganglioside in the early endosome of Niemann–Pick C1-deficient cells

We investigated intracellular trafficking of GM1 ganglioside in Niemann–Pick C1 (NPC1)-deficient Chinese hamster ovary cells [NPC1(−) cells] by using cholera toxin (CT) as a probe. Both the holotoxin and the B subunit (CTB) accumulated in GM1-enriched intracellular vesicles of NPC1(−) cells. CTB-labeled vesicles contained the early endosome marker Rab5 but not lysosome-associated membrane protein 2 and were not labeled with either Texas red–transferrin or Lysotracker, indicating that they represent early endosomes. Similarly, CT accumulated in intracellular vesicles of human NPC fibroblasts that contained both Rab5 and early endosomal antigen 1. CTB accumulation in NPC1(−) cells was abolished by expression of wild-type NPC1 but not by mutant proteins with a mutation either in the NPC domain or the sterol-sensing domain. A part of these mutant NPC1 proteins expressed in NPC1(−) cells was localized on CTB-labeled vesicles. U18666A treatment of “knock in” cells [NPC1(−) cells that stably expressed wild-type NPC1] caused CTB accumulation similar to that in NPC1(−) cells, and a part of wild-type NPC1was localized on CTB-labeled vesicles in drug-treated cells. Finally, CT tracer experiments in NPC1(−) cells revealed retarded excretion of internalized toxin into the culture medium and an increase in the intracellular release of A subunits. In accordance with the latter result, CT was more effective in stimulating cAMP formation in NPC1(−) than in wild-type cells. These results suggest that transport of CT/GM1 complexes from the early endosome to the plasma membrane depends on the function of NPC1, whereas transport to the Golgi apparatus/endoplasmic reticulum does not.

Niemann-Pick type C disease: Accelerated neurofibrillary tangle formation and amyloid β deposition associated with apolipoprotein E ε4 homozygosity

Niemann–Pick type C disease is a neurovisceral storage disorder. Neurofibrillary tangles similar to those in Alzheimers disease have been reported in most juvenile/adult patients without amyloid β protein (Aβ) deposits. Recently, we found deposits of Aβ in the form of diffuse plaques in three (31‐ and 32‐year‐old sisters and a 37‐year‐old man) of nine Niemann–Pick type C disease patients, who presented with most severe tauopathy and with numerous neurofibrillary tangles. Aβ deposits were not detected in any of the control brains of patients younger than age 42 years. These three patients with Aβ deposit all were homozygotes of apolipoprotein E ε4. Our study suggested that NPC1 gene mutations combined with homozygosity of apolipoprotein E ε4 alleles could manifest neuropathology similar to that of Alzheimers disease. Investigation of these patients may provide an important clue for understanding the pathogenesis of Alzheimers disease.

Effects of cyclodextrin in two patients with Niemann-Pick Type C disease

Niemann-Pick Type C disease (NPC) is an autosomal recessive lysosomal storage disorder characterized by progressive neurological deterioration. Currently, there are no effective treatments for NPC, although miglustat has shown some effectiveness in stabilizing neurological status in juvenile-onset NPC patients. Recent studies have demonstrated the efficacy of hydroxypropyl-β-cyclodextrin (HPB-CD) in NPC mice. Herein, we describe the effects of HPB-CD in two patients with NPC. The two patients received HPB-CD infusions twice (Patient 2) or thrice (Patient 1) weekly, starting with a dose of 80 mg/kg per dose that was increased gradually to 2g/kg per dose (Patient 2) or 2.5 g/kg per dose (Patient 1). Although HPB-CD did not improve the neurological deficits in either patient, it was partially effective in improving hepatosplenomegaly and central nervous system dysfunction, especially during the first 6 months of treatment. No adverse effects were observed over the course of treatment, although Patient 1 exhibited transient cloudiness of the lungs with fever after 2 years. For more effective treatment of NPC patients with HPB-CD, it is necessary to improve drug delivery into the central nervous system.

Chaperone activity of bicyclic nojirimycin analogues for Gaucher mutations in comparison with N-(n-nonyl)deoxynojirimycin.

Gaucher disease (GD), the most prevalent lysosomal storage disorder, is caused by mutations of lysosomal β‐glucosidase (acid β‐Glu, β‐glucocerebrosidase); these mutations result in protein misfolding. Some inhibitors of this enzyme, such as the iminosugar glucomimetic N‐(n‐nonyl)‐1‐deoxynojirimycin (NN‐DNJ), are known to bind to the active site and stabilize the proper folding for the catalytic form, acting as “chemical chaperones” that facilitate transport and maturation of acid β‐Glu. Recently, bicyclic nojirimycin (NJ) analogues with structure of sp2 iminosugars were found to behave as very selective, competitive inhibitors of the lysosomal β‐Glu. We have now evaluated the glycosidase inhibitory profile of a series of six compounds within this family, namely 5‐N,6‐O‐(N′‐octyliminomethylidene‐NJ (NOI‐NJ), the 6‐thio and 6‐amino‐6‐deoxy derivatives (6S‐NOI‐NJ and 6N‐NOI‐NJ) and the corresponding galactonojirimycin (GNJ) counterparts (NOI‐GNJ, 6S‐NOI‐GNJ and 6N‐NOI‐GNJ), against commercial as well as lysosomal glycosidases. The chaperone effects of four selected candidates (NOI‐NJ, 6S‐NOI‐NJ, 6N‐NOI‐NJ, and 6S‐NOI‐GNJ) were further evaluated in GD fibroblasts with various acid β‐Glu mutations. The compounds showed enzyme enhancement on human fibroblasts with N188S, G202R, F213I or N370S mutations. The chaperone effects of the sp2 iminosugar were generally stronger than those observed for NN‐DNJ; this suggests that these compounds are promising candidates for clinical treatment of GD patients with a broad range of β‐Glu mutations, especially for neuronopathic forms of Gaucher disease.

Genotype-phenotype relationship of Niemann-Pick disease type C: a possible correlation between clinical onset and levels of NPC1 protein in isolated skin fibroblasts

Editor—Niemann-Pick disease type C (NP-C, MIM 257220) is a fatal autosomal recessive disorder characterised by progressive neurological deterioration and hepatosplenomegaly. NP-C patients can be classified into four major groups according to the onset of neurological symptoms, that is, early infantile, late infantile, juvenile, and adult forms, and the earlier the clinical onset the more quickly progressive are the symptoms and the shorter is the life span.1-4Complementation analysis using cultured skin fibroblasts indicated the presence of at least two subgroups of NP-C, NPC1 (the major subgroup that comprises >90% of NP-C patients) and NPC2 (the minor subgroup).2-4 In 1997, the NPC1 gene ( NPC1 ) (accession No AF002020) that is responsible for the NPC1 subgroup was identified by positional cloning.5 6 The number of NPC1 mutations known to date is not far off 100,7-11 taking into account the accumulated data from seven groups presented in a recent international workshop (International Workshop, The Niemann-Pick C Lesion and the Role of Intracellular Lipid Sorting in Human Disease, Bethesda, USA, October 1999). Because the genomic structure of NPC1 was unknown, initial mutation screening was performed on RT-PCR products or partial genomic amplicons. In our previous study using RT-PCR products, we identified 14 different mutations in 19 alleles from 11 patients, and failed to detect mutations in the remaining three alleles.8 Mutation screening using RT-PCR products has several drawbacks compared with screening using genomic amplicons. For example, mutations that reduce the mRNA stability may escape the screening.12 13 To refine the screening method, we screened a CITB human BAC library (Research Genetics, Huntsville, AL) and isolated a clone 386K10 that contained all the 25 exons of NPC1 and a 2 kb fragment of 5′UTR. Our analysis using 386K10 confirmed the exon/intron boundary sequences reported by Morris et al …

Neural substrates of sarcasm: A functional magnetic-resonance imaging study

The understanding of sarcasm reflects a complex process, which involves recognizing the beliefs of the speaker. There is a clear association between deficits in mentalizing, which is the ability to understand other peoples behavior in terms of their mental state, and the understanding of sarcasm in individuals with autistic spectrum disorders. This suggests that mentalizing is important in pragmatic non-literal language comprehension. To highlight the neural substrates of sarcasm, 20 normal adult volunteers underwent functional magnetic-resonance imaging. We used scenario-reading tasks, in which sentences describing a certain situation were presented, followed by the protagonists comments regarding that situation. Depending on the situation, the semantic content of the comments was classified as sarcastic, non-sarcastic, or contextually unconnected. As the combination of the first and second sentences represented discourse-level information that was not encoded in the individual sentences, sarcasm detection was represented as the differential activation induced by the second sentences. Sarcasm detection activated the left temporal pole, the superior temporal sulcus, the medial prefrontal cortex, and the inferior frontal gyrus (Brodmanns area [BA] 47). The left BA 47 was activated more prominently by sarcasm detection than by the first sentence. These findings indicate that the detection of sarcasm recruits the medial prefrontal cortex, which is part of the mentalizing system, as well as the neural substrates involved in reading sentences. The left BA 47 might therefore be where mentalizing and language processes interact during sarcasm detection.