Impaired lysosomal acidity maintenance in acid lipase-deficient cells leads to defective autophagy.

The lysosome is an acid organelle that contains a variety of hydrolytic enzymes and plays a significant role in intracellular degradation to maintain cellular homeostasis. Genetic variants in lysosome-related genes can lead to severe congenital diseases, such as lysosomal storage diseases. In the present study, we investigated the impact of depleting lysosomal acid lipase A (LIPA), a lysosomal esterase that metabolizes esterified cholesterol or triglyceride, on lysosomal function. Under nutrient-rich conditions, LIPA gene KO (LIPAKO) cells exhibited impaired autophagy, whereas, under starved conditions, they showed normal autophagy. The cause underlying the differential autophagic activity was increased sensitivity of LIPAKO cells to ammonia, which was produced from l-glutamine in the medium. Further investigation revealed that ammonia did not affect upstream signals involved in autophagy induction, autophagosome-lysosome fusion, and hydrolytic enzyme activities in LIPAKO cells. On the other hand, LIPAKO cells showed defective lysosomal acidity upon ammonia loading. Microscopic analyses revealed that lysosomes of LIPAKO cells enlarged, whereas the amount of lysosomal proton pump V-ATPase did not proportionally increase. Since the enlargement of lysosomes in LIPAKO cells was not normalized under starved conditions, this is the primary change that occurred in the LIPAKO cells, and autophagy was affected by impaired lysosomal function under the specific conditions. These findings expand our comprehension of the pathogenesis of Wolman's disease, which is caused by a defect in the LIPA gene, and suggest that conditions, such as hyperlipidemia, may easily disrupt lysosomal functions.

A case series of Fabry diseases with CKD in Japan.

BACKGROUND: It is well known that kidney injury is vital organ damage in Fabry disease (FD). Renin-angiotensin system (RAS) inhibitors are known to reduce proteinuria in patients with chronic kidney disease (CKD) by dilating the glomerular export arteries and reducing intraglomerular pressure. This improvement in intraglomerular pressure, although lowering the glomerular filtration rate, is thought to prevent renal damage and be renoprotective in the long term. RAS inhibitors may be effective in FD patients with proteinuria to prevent the progression of kidney disease, however, the degree to which they are used in clinical practice is unknown. METHODS: The J-CKD-DB-Ex is a comprehensive multicenter database that automatically extracts medical data on CKD patients. J-CKD-DB-Ex contains data on 187,398 patients in five medical centers. FD patients were identified by ICD-10. Clinical data and prescriptions of FD patients between January 1 of 2014, and December 31 of 2020 were used for the analysis. RESULTS: We identified 39 patients with FD from the J-CKD-DB-Ex including those with suspected FD. We confirmed 22 patients as FD. Half of the patients received RAS inhibitors. RAS inhibitors tended to be used in CKD patients with more severe renal impairment. CONCLUSIONS: This case series revealed the actual clinical practice of FD patients with CKD. In particular, we found cases in which patients had proteinuria, but were not treated with RAS inhibitors. The database was shown to be useful in assessing the clinical patterns of patients with rare diseases.

A Case of Mucopolysaccharidosis II Caused by a Novel Variant with Skin Linear Hyperpigmented Streaks along Blaschko's Lines.

We report a case of an eight-year-old boy with mucopolysaccharidosis (MPS) II with atypical skin lesions of hyperpigmented streaks along Blaschko's lines. This case presented with mild symptoms of MPS such as hepatosplenomegaly, joint stiffness, and quite mild bone deformity, which was the reason for the delay in diagnosis until the age of seven years. However, he showed an intellectual disability that did not meet the diagnostic criteria for an attenuated form of MPS II. Iduronate 2-sulfatase activity was reduced. Clinical exome sequencing of DNA from peripheral blood revealed a novel pathogenic missense variant (NM_000202.8(IDS_v001):c.703C>A, p.(Pro235Thr)) in the IDS gene, which was confirmed in the mother with a heterozygous state. His brownish skin lesions differed from the Mongolian blue spots or "pebbling" of the skin that are observed in MPS II.

Hematopoietic Disorders, Renal Impairment and Growth in Mucopolysaccharidosis-Plus Syndrome.

Mucopolysaccharidoses (MPS) are rare lysosomal storage disorders (LSD) characterized by the excessive accumulation of glycosaminoglycans (GAG). Conventional MPS, caused by inborn deficiencies of lysosomal enzymes involved in GAG degradation, display various multisystemic symptoms-including progressive neurological complications, ophthalmological disorders, hearing loss, gastrointestinal and hepatobiliary issues, cardiorespiratory problems, bone and joint abnormalities, dwarfism, and coarse facial features. Mucopolysaccharidosis-Plus Syndrome (MPSPS), an autosomal recessive disease caused by a mutation in the endo-lysosomal tethering protein VPS33A, shows additional renal and hematopoietic abnormalities ("Plus symptoms") uncommon in conventional MPS. Here, we analyze data from biochemical, histological, and physical examinations-particularly of blood counts and kidney function-to further characterize the clinical phenotype of MPSPS. A series of blood tests indicate hematopoietic symptoms including progressive anemia and thrombocytopenia, which correlate with histological observations of hypoplastic bone marrow. High urinary excretion of protein (caused by impairments in renal filtration), hypoalbuminemia, and elevated levels of creatinine, cholesterol, and uric acid indicate renal dysfunction. Histological analyses of MPSPS kidneys similarly suggest the extensive destruction of glomerular structures by foamy podocytes. Height and weight did not significantly deviate from the average, but in some cases, growth began to decline at around six months or one year of age.

Biallelic VPS35L pathogenic variants cause 3C/Ritscher-Schinzel-like syndrome through dysfunction of retriever complex.

BACKGROUND: 3C/Ritscher-Schinzel syndrome is characterised by congenital cranio-cerebello-cardiac dysplasia, where CCDC22 and WASHC5 are accepted as the causative genes. In combination with the retromer or retriever complex, these genes play a role in endosomal membrane protein recycling. We aimed to identify the gene abnormality responsible for the pathogenicity in siblings with a 3C/Ritscher-Schinzel-like syndrome, displaying cranio-cerebello-cardiac dysplasia, coloboma, microphthalmia, chondrodysplasia punctata and complicated skeletal malformation. METHODS: Exome sequencing was performed to identify pathogenic variants. Cellular biological analyses and generation of knockout mice were carried out to elucidate the gene function and pathophysiological significance of the identified variants. RESULTS: We identified compound heterozygous pathogenic variants (c.1097dup; p.Cys366Trpfs*28 and c.2755G>A; p.Ala919Thr) in the VPS35L gene, which encodes a core protein of the retriever complex. The identified missense variant lacked the ability to form the retriever complex, and the frameshift variant induced non-sense-mediated mRNA decay, thereby confirming biallelic loss of function of VPS35L. In addition, VPS35L knockout cells showed decreased autophagic function in nutrient-rich and starvation conditions, as well as following treatment with Torin 1. We also generated Vps35l-/- mice and demonstrated that they were embryonic lethal at an early stage, between E7.5 and E10.5. CONCLUSIONS: Our results suggest that biallelic loss-of-function variants in VPS35L underlies 3C/Ritscher-Schinzel-like syndrome. Furthermore, VPS35L is necessary for autophagic function and essential for early embryonic development. The data presented here provide a new insight into the critical role of the retriever complex in fetal development.

Autophagosome-lysosome fusion in neurons requires INPP5E, a protein associated with Joubert syndrome.

Autophagy is a multistep membrane traffic pathway. In contrast to autophagosome formation, the mechanisms underlying autophagosome-lysosome fusion remain largely unknown. Here, we describe a novel autophagy regulator, inositol polyphosphate-5-phosphatase E (INPP5E), involved in autophagosome-lysosome fusion process. In neuronal cells, INPP5E knockdown strongly inhibited autophagy by impairing the fusion step. A fraction of INPP5E is localized to lysosomes, and its membrane anchoring and enzymatic activity are necessary for autophagy. INPP5E decreases lysosomal phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2), one of the substrates of the phosphatase, that counteracts cortactin-mediated actin filament stabilization on lysosomes. Lysosomes require actin filaments on their surface for fusing with autophagosomes. INPP5E is one of the genes responsible for Joubert syndrome, a rare brain abnormality, and mutations found in patients with this disease caused defects in autophagy. Taken together, our data reveal a novel role of phosphoinositide on lysosomes and an association between autophagy and neuronal disease.

Mucopolysaccharidosis-Plus Syndrome.

Previously, we reported a novel disease of impaired glycosaminoglycans (GAGs) metabolism without deficiency of known lysosomal enzymes-mucopolysaccharidosis-plus syndrome (MPSPS). MPSPS, whose pathophysiology is not elucidated, is an autosomal recessive multisystem disorder caused by a specific mutation p.R498W in the VPS33A gene. VPS33A functions in endocytic and autophagic pathways, but p.R498W mutation did not affect both of these pathways in the patient's skin fibroblast. Nineteen patients with MPSPS have been identified: seventeen patients were found among the Yakut population (Russia) and two patients from Turkey. Clinical features of MPSPS patients are similar to conventional mucopolysaccharidoses (MPS). In addition to typical symptoms for conventional MPS, MPSPS patients developed other features such as congenital heart defects, renal and hematopoietic disorders. Diagnosis generally requires evidence of clinical picture similar to MPS and molecular genetic testing. Disease is very severe, prognosis is unfavorable and most of patients died at age of 10-20 months. Currently there is no specific therapy for this disease and clinical management is limited to supportive and symptomatic treatment.

A brain-enriched Drp1 isoform associates with lysosomes, late endosomes, and the plasma membrane.

Dynamin-related protein 1 (Drp1) constricts mitochondria as a mechanochemical GTPase during mitochondrial division. The Drp1 gene contains several alternative exons and produces multiple isoforms through RNA splicing. Here we performed a systematic analysis of Drp1 transcripts in different mouse tissues and identified a previously uncharacterized isoform that is highly enriched in the brain. This Drp1 isoform is termed Drp1ABCD because it contains four alterative exons: A, B, C, and D. Remarkably, Drp1ABCD is located at lysosomes, late endosomes, and the plasma membrane in addition to mitochondria. Furthermore, Drp1ABCD is concentrated at the interorganelle interface between mitochondria and lysosomes/late endosomes. The localizations of Drp1ABCD at lysosomes, late endosomes, and the plasma membrane require two exons, A and B, that are present in the GTPase domain. Drp1ABCD assembles onto these membranes in a manner that is regulated by its oligomerization and GTP hydrolysis. Experiments using lysosomal inhibitors show that the association of Drp1ABCD with lysosomes/late endosomes depends on lysosomal pH but not their protease activities. Thus, Drp1 may connect mitochondria to endosomal-lysosomal pathways in addition to mitochondrial division.

Mutation in VPS33A affects metabolism of glycosaminoglycans: a new type of mucopolysaccharidosis with severe systemic symptoms.

Mucopolysaccharidoses (MPS) are a group of genetic deficiencies of lysosomal enzymes that catabolize glycosaminoglycans (GAG). Here we describe a novel MPS-like disease caused by a specific mutation in the VPS33A gene. We identified several Yakut patients showing typical manifestations of MPS: coarse facial features, skeletal abnormalities, hepatosplenomegaly, respiratory problems, mental retardation, and excess secretion of urinary GAG. However, these patients could not be diagnosed enzymatically as MPS. They showed extremely high levels of plasma heparan sulphate (HS, one of GAG); 60 times the normal reference range and 6 times that of MPS patients. Additionally, most patients developed heart, kidney, and hematopoietic disorders, which are not typical symptoms for conventional MPS, leading to a fatal outcome between 1 and 2-years old. Using whole exome and Sanger sequencing, we identified homozygous c.1492C > T (p.Arg498Trp) mutations in the VPS33A gene of 13 patients. VPS33A is involved in endocytic and autophagic pathways, but the identified mutation did not affect either of these pathways. Lysosomal over-acidification and HS accumulation were detected in patient-derived and VPS33A-depleted cells, suggesting a novel role of this gene in lysosomal functions. We hence propose a new type of MPS that is not caused by an enzymatic deficiency.

Unfolded protein response is activated in Krabbe disease in a manner dependent on the mutation type.

Krabbe disease, one of the autosomal-recessive lysosomal storage disorders (LSDs), is caused by a deficiency of galactocerebrosidase (GALC) activity, resulting in the intracellular accumulation of psychosine, which is cytotoxic for neuronal cells. Genetically pathogenic mutations result in conformational changes in GALC and disrupt the lysosmal trafficking of cargos, which subsequently accumulate in the endoplasmic reticulum (ER). Recently, ER stress together with the activation of the unfolded protein response (UPR) has been suggested to play a key role in the pathogenesis of LSDs. In this study, we hence investigated whether the UPR is activated in Krabbe disease using COS-7 cells expressing pathogenic GALC mutants and skin fibroblasts (SFs) from Krabbe disease patients with various phenotypes, using a combination of semiquantitative and quantitative real-time polymerase chain reactions. We found that UPR activation in Krabbe disease depends on the mutations and cell types, and there is the possibility that multiple pathways, involving ER chaperones, inositol-requiring kinase 1, and protein kinase regulated by RNA-like ER kinase are activated by mutations associated with the infantile form. These results indicate that in Krabbe disease, each misfolded/unfolded protein evokes different UPR activation depending on the mutation, and that the activated pathways affect the phenotypes.

Challenge of phenotype estimation for optimal treatment of Krabbe disease.

Krabbe disease is an autosomal recessive, inherited demyelinating disease caused by deficiency of the lysosomal enzyme galactocerebrosidase. It is recognized as one of the predominant genetic diseases showing leukodystrophy from infancy to adulthood. The clinical phenotype and genotype for this disease show considerable variation worldwide, which makes accurate diagnosis difficult. Effective therapy is limited, although hematopoietic stem cell transplantation at an early stage has been established to some extent. We report here the long-term clinical effect on juvenile Krabbe disease for two brothers who underwent hematopoietic stem cell transplantation at an early stage of their disease. We review research into genotype-phenotype correlation for the possibility of early diagnosis at a presymptomatic stage. Medical care for this intractable disease will improve in the near future as a result of the increasing awareness of its molecular pathology and improvements in medical treatment. © 2016 Wiley Periodicals, Inc.

HOPS, CORVET and newly-identified Hybrid tethering complexes contribute differentially towards multiple modes of endocytosis.

Vesicular transport driven by membrane trafficking systems conserved in eukaryotes is critical to cellular functionality and homeostasis. It is known that homotypic fusion and vacuole protein sorting (HOPS) and class C core endosomal vacuole tethering (CORVET) interact with Rab-GTPases and SNARE proteins to regulate vesicle transport, fusion, and maturation in autophagy and endocytosis pathways. In this study, we identified two novel "Hybrid" tethering complexes in mammalian cells in which one of the subunits of HOPS or CORVET is replaced with the subunit from the other. Substrates taken up by receptor-mediated endocytosis or pinocytosis were transported by distinctive pathways, and the newly identified hybrid complexes contributed to pinocytosis in the presence of HOPS, whereas receptor-mediated endocytosis was exclusively dependent on HOPS. Our study provides new insights into the molecular mechanisms of the endocytic pathway and the function of the vacuolar protein sorting-associated (VPS) protein family.

Prenatal Diagnosis of Mucopolysaccharidosis-Plus Syndrome (MPSPS).

Mucopolysaccharidosis-plus syndrome (MPSPS) is an autosomal-recessive disorder caused by c.1492C>T (p.R498W) in the VPS33A gene. MPSPS is a severe disorder that causes a short lifespan in patients. Currently, there is no specific treatment for patients. The Yakut population is more prone to this disease than others. Diagnosing MPSPS relies on clinical manifestations, and genetic testing (GT) is used to confirm the diagnosis. In this research, we examined two pregnancy cases, one of which involved a prenatal diagnosis for MPSPS. Notably, neither pregnant woman had a known family history of the disorder. During their pregnancies, both women underwent prenatal ultrasonography, which revealed increased prenasal thickness during the second trimester. In the first case, ultrasonography indicated increased prenasal thickness in the second trimester, but a definitive diagnosis was not made at that time. The patient was eventually diagnosed with MPSPS at 11 months of age. On the contrary, in the second case, GT uncovered that the parents were carriers of MPSPS. Consequently, a placental biopsy was performed, leading to an early diagnosis of MPSPS. This study emphasizes the importance of ultrasonography findings in prenatal MPSPS diagnosis. Combining ultrasonography with GT can be a valuable approach to confirming MPSPS at an early stage, allowing for the appropriate planning of delivery methods and medical care. Ultimately, this comprehensive approach can significantly enhance the quality of life of both affected patients and their parents.

A case of Marfanoid-progeroid-lipodystrophy syndrome: experimental proof of skipping exons and escaping nonsense-mediated decay.

We report a Japanese patient with tall stature, dolichocephaly, prominent forehead, narrow nasal ridge, mild retrognathia, subcutaneous fat reduction, bilateral entropion of both eyelids, high arched palate, long fingers, and mild hyperextensible finger joints as a case of Marfanoid-progeroid-lipodystrophy syndrome. Genetic investigation revealed a heterozygous variant NC_000015.10(NM_000138.5):c.8226+5G>A in the FBN1 gene. Skipping of exon 65 and escaping nonsense-mediated decay followed by frameshift were experimentally confirmed in the proband's mRNA.

Lysosomal storage causes cellular dysfunction in mucolipidosis II skin fibroblasts.

Mucolipidosis II (ML-II) is a fatal inherited metabolic disease caused by deficiency of GlcNAc-phosphotransferase, which plays a role in generating the mannose 6-phosphate recognition marker on lysosomal enzymes. In ML-II, many lysosomal acid hydrolases are mistargeted out of cells, and lysosomes become filled with undigested substrates, which explains inclusion cell disease as an alternative name for this disease. In this study, we revealed various cellular phenotypes in ML-II skin fibroblasts. We quantitated phospholipid and cholesterol within cells and showed ~2-fold accumulation in ML-II as compared with normal cells. Lysosomal pH of ML-II cells was higher than that of normal cells (5.29 ± 0.08 versus 4.79 ± 0.10, p < 0.001). The proliferated lysosomes in ML-II cells were accumulated ~3-fold in amount as compared with normal cells. Intracellular logistics including endocytosis and mannose 6-phosphate receptor recycling were impaired in ML-II cells. To confirm whether these ML-II cellular phenotypes derive from deficient lysosomal acid hydrolases within lysosomes, we performed supplementation of lysosomal enzymes using a partially purified total enzyme mixture, which was derived from the conditioned culture medium of normal skin fibroblasts after NH(4)Cl treatment. This supplementation corrected all of the previously described ML-II phenotypes. In addition, the autophagic and mitochondrial impairment that we have previously reported improved, and inclusion bodies disappeared on electron micrography following total lysosomal enzyme supplementation. Our results indicate that various cellular phenotypes in ML-II are caused by the deficiency of many lysosomal enzymes and massive accumulation of undigested substrates.

Genistein reduces heparan sulfate accumulation in human mucolipidosis II skin fibroblasts.

Genistein, a soy isoflavone, reduces glycosaminoglycan synthesis and its effect on mucopolysaccharidoses has been tested. In this report, we examined the effect of genistein in human mucolipidosis II skin fibroblasts in vitro. Heparan sulfate was accumulated within both cells and in extracellular spaces in mucolipidosis II. Genistein reduced the amount of heparan sulfate in cultured cells dose dependently and also inhibited cell growth dose dependently.

Long-term efficacy of hematopoietic stem cell transplantation on brain involvement in patients with mucopolysaccharidosis type II: a nationwide survey in Japan.

Hematopoietic stem cell transplantation (HSCT) has not been indicated for patients with mucopolysaccharidosis II (MPS II, Hunter syndrome), while it is indicated for mucopolysaccharidosis I (MPS I) patients <2 years of age and an intelligence quotient (IQ) of ≥ 70. Even after the approval of enzyme replacement therapy for both of MPS I and II, HSCT is still indicated for patients with MPS I severe form (Hurler syndrome). To evaluate the efficacy and benefit of HSCT in MPS II patients, we carried out a nationwide retrospective study in Japan. Activities of daily living (ADL), IQ, brain magnetic resonance image (MRI) lesions, cardiac valvular regurgitation, and urinary glycosaminoglycan (GAG) were analyzed at baseline and at the most recent visit. We also performed a questionnaire analysis about ADL for an HSCT-treated cohort and an untreated cohort (natural history). Records of 21 patients were collected from eight hospitals. The follow-up period in the retrospective study was 9.6 ± 3.5 years. ADL was maintained around baseline levels. Cribriform changes and ventricular dilatation on brain MRI were improved in 9/17 and 4/17 patients, respectively. Stabilization of brain atrophy was shown in 11/17 patients. Cardiac valvular regurgitation was diminished in 20/63 valves. Urinary GAG concentration was remarkably lower in HSCT-treated patients than age-matched untreated patients. In the questionnaire analysis, speech deterioration was observed in 12/19 patients in the untreated cohort and 1/7 patient in HSCT-treated cohort. HSCT showed effectiveness towards brain or heart involvement, when performed before signs of brain atrophy or valvular regurgitation appear. We consider HSCT is worthwhile in early stages of the disease for patients with MPS II.

Identification of CUL4A-DDB1-WDFY1 as an E3 ubiquitin ligase complex involved in initiation of lysophagy.

Macroautophagy is a bulk degradation system in which double membrane-bound structures called autophagosomes to deliver cytosolic materials to lysosomes. Autophagy promotes cellular homeostasis by selectively recognizing and sequestering specific targets, such as damaged organelles, protein aggregates, and invading bacteria, termed selective autophagy. We previously reported a type of selective autophagy, lysophagy, which helps clear damaged lysosomes. Damaged lysosomes become ubiquitinated and recruit autophagic machinery. Proteomic studies using transfection reagent-coated beads and further evaluations reveal that a CUL4A-DDB1-WDFY1 E3 ubiquitin ligase complex is essential to initiate lysophagy and clear damaged lysosomes. Moreover, we show that LAMP2 is ubiquitinated by the CUL4A E3 ligase complex as a substrate on damaged lysosomes. These results reveal how cells selectively tag damaged lysosomes to initiate autophagy for the clearance of lysosomes.

A novel nonsense variant in ARID1B causing simultaneous RNA decay and exon skipping is associated with Coffin-Siris syndrome.

Coffin-Siris syndrome (CSS) is a congenital disorder that is characterized by an absent/hypoplastic fifth distal phalanx, psychomotor developmental delay, and coarse facial features. One of the causative genes, ARID1B (AT-rich interactive domain-containing protein 1B), encodes components of the BAF chromatin remodeling complexes. Here, we report a case of a 3-year 8-month-old male with a novel nonsense variant (NM_001374820.1:c.4282C > T, p.(Gln1428*)) in the ARID1B gene, which was identified with whole-exome sequencing. He showed clinical symptoms of cleft soft palate, distinctive facial features (flat nasal bridge, thick eyebrows, and long eyelashes), right cryptorchidism, and hypertrichosis that partially overlapped with CSS. One of the most characteristic features of CSS is absent/hypoplastic fifth distal phalanx. He showed no obvious clinical finding in the lengths of his fingers or in the formation of his fingernails. However, radiographic analyses of the metacarpophalangeal bones revealed shortening of all the distal phalanges and fifth middle phalanges, suggesting brachydactyly. We performed mRNA analyses and revealed that both nonsense-mediated decay and nonsense-associated altered splicing were simultaneously caused by the c.4282C > T nonsense variant. The proband's clinical manifestations fit the previously reported criteria of disease for CSS or intellectual disability with ARID1B variant. Altogether, we suggest that c.4282C > T is a pathogenic variant that causes this clinical phenotype.

Pathology of the first autopsy case diagnosed as mucolipidosis type III α/ß suggesting autophagic dysfunction.

Mucolipidosis type III (MLIII) is a mild form of Mucolipidosis type II (MLII, I-cell disease) of late onset, of which almost no pathological study has been reported, as it is a very rare disease. We encountered the case of a 23-year-old man of Japanese and Caucasian mixed parentage diagnosed with MLIII by enzyme assay and genotyping. He died suddenly due to severe dilated cardiomyopathy. On the day after his death, autopsy was performed, and accumulation of Luxol Fast Blue (LFB) positive material was found to be most severe in the neuronal cells of dorsal root ganglions (DRG). Electromicroscopic DRG revealed the neuronal cytoplasm was filled with a zebra-body-like membranous matrix. We tried immunohistochemistry to investigate the mechanism of such accumulation in the DRG that resulted in double positive anti-ubiquitin antibody (FK-2) and anti-LC3 antibody (as specific marker for autophagy) staining, and speculated activating of autophagosome pathway, and 'zebra-body' should be suspected as dysfunctional autophagosome. We also detected foamy cell proliferation in the dura mater, Auerbach's plexus (peripheral nervous system), podocytes of almost all glomeruli, cartilage tissue in lumbar discs, and in cardiac muscle. We tried FK-2 and anti-LC3 antibody staining also for the podocytes, the area with the most marked proliferation of foamy cells, but the result was negative. This led us to speculate that these pathological findings, namely, accumulation of LFB-positive material and foamy fibroblast proliferation, might be the forms of dysfunctional autophagosome at various stages of development. This pathological study of MLIII supports the theory that MLIII is a mild type of MLII because of the close similarity of their pathological findings.