Unique molecular signature in mucolipidosis type IV microglia.

BACKGROUND: Lysosomal storage diseases (LSD) are a large family of inherited disorders characterized by abnormal endolysosomal accumulation of cellular material due to catabolic enzyme and transporter deficiencies. Depending on the affected metabolic pathway, LSD manifest with somatic or central nervous system (CNS) signs and symptoms. Neuroinflammation is a hallmark feature of LSD with CNS involvement such as mucolipidosis type IV, but not of others like Fabry disease. METHODS: We investigated the properties of microglia from LSD with and without major CNS involvement in 2-month-old mucolipidosis type IV (Mcoln1-/-) and Fabry disease (Glay/-) mice, respectively, by using a combination of flow cytometric, RNA sequencing, biochemical, in vitro and immunofluorescence analyses. RESULTS: We characterized microglia activation and transcriptome from mucolipidosis type IV and Fabry disease mice to determine if impaired lysosomal function is sufficient to prime these brain-resident immune cells. Consistent with the neurological pathology observed in mucolipidosis type IV, Mcoln1-/- microglia demonstrated an activation profile with a mixed neuroprotective/neurotoxic expression pattern similar to the one we previously observed in Niemann-Pick disease, type C1, another LSD with significant CNS involvement. In contrast, the Fabry disease microglia transcriptome revealed minimal alterations, consistent with the relative lack of CNS symptoms in this disease. The changes observed in Mcoln1-/- microglia showed significant overlap with alterations previously reported for other common neuroinflammatory disorders including Alzheimer's, Parkinson's, and Huntington's diseases. Indeed, our comparison of microglia transcriptomes from Alzheimer's disease, amyotrophic lateral sclerosis, Niemann-Pick disease, type C1 and mucolipidosis type IV mouse models showed an enrichment in "disease-associated microglia" pattern among these diseases. CONCLUSIONS: The similarities in microglial transcriptomes and features of neuroinflammation and microglial activation in rare monogenic disorders where the primary metabolic disturbance is known may provide novel insights into the immunopathogenesis of other more common neuroinflammatory disorders. TRIAL REGISTRATION: ClinicalTrials.gov, NCT01067742, registered on February 12, 2010.

Modeling Niemann-Pick disease type C1 in zebrafish: a robust platform for in vivo screening of candidate therapeutic compounds.

Niemann-Pick disease type C1 (NPC1) is a rare autosomal recessive lysosomal storage disease primarily caused by mutations in NPC1 NPC1 is characterized by abnormal accumulation of unesterified cholesterol and glycolipids in late endosomes and lysosomes. Common signs include neonatal jaundice, hepatosplenomegaly, cerebellar ataxia, seizures and cognitive decline. Both mouse and feline models of NPC1 mimic the disease progression in humans and have been used in preclinical studies of 2-hydroxypropyl-ß-cyclodextrin (2HPßCD; VTS-270), a drug that appeared to slow neurological progression in a Phase 1/2 clinical trial. However, there remains a need to identify additional therapeutic agents. High-throughput drug screens have been useful in identifying potential therapeutic compounds; however, current preclinical testing is time and labor intensive. Thus, development of a high-capacity in vivo platform suitable for screening candidate drugs/compounds would be valuable for compound optimization and prioritizing subsequent in vivo testing. Here, we generated and characterize two zebrafish npc1-null mutants using CRISPR/Cas9-mediated gene targeting. The npc1 mutants model both the early liver and later neurological disease phenotypes of NPC1. LysoTracker staining of npc1 mutant larvae was notable for intense staining of lateral line neuromasts, thus providing a robust in vivo screen for lysosomal storage. As a proof of principle, we were able to show that treatment of the npc1 mutant larvae with 2HPßCD significantly reduced neuromast LysoTracker staining. These data demonstrate the potential value of using this zebrafish NPC1 model for efficient and rapid in vivo optimization and screening of potential therapeutic compounds.This article has an associated First Person interview with the first author of the paper.

Gastrointestinal Tract Pathology in a BALB/c Niemann-Pick Disease Type C1 Null Mouse Model.

BACKGROUND: Niemann-Pick disease, type C (NPC) is a rare lysosomal storage disorder characterized by progressive neurodegeneration, splenomegaly, hepatomegaly, and early death. NPC is caused by mutations in either the NPC1 or NPC2 gene. Impaired NPC function leads to defective intracellular transport of unesterified cholesterol and its accumulation in late endosomes and lysosomes. A high frequency of Crohn disease has been reported in NPC1 patients, suggesting that gastrointestinal tract pathology may become a more prominent clinical issue if effective therapies are developed to slow the neurodegeneration. The Npc1 nih mouse model on a BALB/c background replicates the hepatic and neurological disease observed in NPC1 patients. Thus, we sought to characterize the gastrointestinal tract pathology in this model to determine whether it can serve as a model of Crohn disease in NPC1. METHODS: We analyzed the gastrointestinal tract and isolated macrophages of BALB/cJ cNctr-Npc1m1N/J (Npc1-/-) mouse model to determine whether there was any Crohn-like pathology or inflammatory cell activation. We also evaluated temporal changes in the microbiota by 16S rRNA sequencing of fecal samples to determine whether there were changes consistent with Crohn disease. RESULTS: Relative to controls, Npc1 mutant mice demonstrate increased inflammation and crypt abscesses in the gastrointestinal tract; however, the observed pathological changes are significantly less than those observed in other Crohn disease mouse models. Analysis of Npc1 mutant macrophages demonstrated an increased response to lipopolysaccharides and delayed bactericidal activity; both of which are pathological features of Crohn disease. Analysis of the bacterial microbiota does not mimic what is reported in Crohn disease in either human or mouse models. We did observe significant increases in cyanobacteria and epsilon-proteobacteria. The increase in epsilon-proteobacteria may be related to altered cholesterol homeostasis since cholesterol is known to promote growth of this bacterial subgroup. CONCLUSIONS: Macrophage dysfunction in the BALB/c Npc1-/- mouse is similar to that observed in other Crohn disease models. However, neither the degree of pathology nor the microbiota changes are typical of Crohn disease. Thus, this mouse model is not a good model system for Crohn disease pathology reported in NPC1 patients.

An Efficient Approach to Evaluate Reporter Ion Behavior from MALDI-MS/MS Data for Quantification Studies Using Isobaric Tags.

Protein quantification, identification, and abundance determination are important aspects of proteome characterization and are crucial in understanding biological mechanisms and human diseases. Different strategies are available to quantify proteins using mass spectrometric detection, and most are performed at the peptide level and include both targeted and untargeted methodologies. Discovery-based or untargeted approaches oftentimes use covalent tagging strategies (i.e., iTRAQ, TMT), where reporter ion signals collected in the tandem MS experiment are used for quantification. Herein we investigate the behavior of the iTRAQ 8-plex chemistry using MALDI-TOF/TOF instrumentation. The experimental design and data analysis approach described is simple and straightforward, which allows researchers to optimize data collection and proper analysis within a laboratory. iTRAQ reporter ion signals were normalized within each spectrum to remove peptide biases. An advantage of this approach is that missing reporter ion values can be accepted for purposes of protein identification and quantification without the need for ANOVA analysis. We investigate the distribution of reporter ion peak areas in an equimolar system and a mock biological system and provide recommendations for establishing fold-change cutoff values at the peptide level for iTRAQ data sets. These data provide a unique data set available to the community for informatics training and analysis.

Cholesterol Biosynthesis and Trafficking in Cortisol-Producing Lesions of the Adrenal Cortex.

CONTEXT: Cortisol-producing adenomas (CPAs), primary pigmented nodular adrenocortical disease (PPNAD), and primary macronodular adrenocortical hyperplasia (PMAH) cause ACTH-independent Cushing syndrome (CS). Investigation of their pathogenesis has demonstrated their integral link to the cAMP-dependent protein kinase signaling pathway. OBJECTIVE: The aim of this study was to identify differences in cholesterol biosynthesis among different CS-causing adrenocortical tumors. Because of the concomitant associations of cAMP levels with cholesterol and with steroid biosynthesis, we hypothesized that benign cortisol-producing tumors would display aberration of these pathways. DESIGN AND SETTING: Twenty-three patients with CPA, PPNAD, or PMAH who underwent adrenalectomy for CS were included in the study. Preoperative biochemical analyses were performed, and excised adrenal tissues were studied. MAIN OUTCOME MEASURES: Serum, urinary hormone levels, serum lipid profiles, and anthropometric data were obtained preoperatively. Adrenal tissues were analyzed for total protein, cholesterol, and neutral sterol content by mass spectrometry and expression of HMGCR, LDLR, ABCA1, DHCR24, and STAR genes. RESULTS: There were differences in cholesterol content and markers of cholesterol biosynthesis and metabolism that distinguished CPAs from PMAH and PPNAD; cholesterol, lathosterol, and lathosterol/cholesterol ratio were significantly higher in CPAs. ABCA1 mRNA was lower among CPAs compared to tissues from bilateral adrenocortical hyperplasia (PMAH and PPNAD), and mRNA expression of LDL-R, DCHR24, and HMGCR tended to be higher in CPA tumor tissues. CONCLUSION: CPAs displayed characteristics of "cholesterol-starved" tissues when compared to PPNAD and PMAH and appeared to have increased intrinsic cholesterol production and uptake from the periphery, as well as decreased cholesterol efflux. This has implications for a potential new way of treating these tumors.

Pituitary adenoma with paraganglioma/pheochromocytoma (3PAs) and succinate dehydrogenase defects in humans and mice.

CONTEXT: Germline mutations in genes coding succinate dehydrogenase (SDH) subunits A, B, C, and D have been identified in familial paragangliomas (PGLs)/pheochromocytomas (PHEOs) and other tumors. We described a GH-secreting pituitary adenoma (PA) caused by SDHD mutation in a patient with familial PGLs. Additional patients with PAs and SDHx defects have since been reported. DESIGN: We studied 168 patients with unselected sporadic PA and with the association of PAs, PGLs, and/or pheochromocytomas, a condition we named the 3P association (3PAs) for SDHx germline mutations. We also studied the pituitary gland and hormonal profile of Sdhb(+/-) mice and their wild-type littermates at different ages. RESULTS: No SDHx mutations were detected among sporadic PA, whereas three of four familial cases were positive for a mutation (75%). Most of the SDHx-deficient PAs were either prolactinomas or somatotropinomas. Pituitaries of Sdhb(+/-) mice older than 12 months had an increased number mainly of prolactin-secreting cells and several ultrastructural abnormalities such as intranuclear inclusions, altered chromatin nuclear pattern, and abnormal mitochondria. Igf-1 levels of mutant mice tended to be higher across age groups, whereas Prl and Gh levels varied according to age and sex. CONCLUSION: The present study confirms the existence of a new association that we termed 3PAs. It is due mostly to germline SDHx defects, although sporadic cases of 3PAs without SDHx defects also exist. Using Sdhb(+/-) mice, we provide evidence that pituitary hyperplasia in SDHx-deficient cells may be the initial abnormality in the cascade of events leading to PA formation.

Heterogeneous genetic background of the association of pheochromocytoma/paraganglioma and pituitary adenoma: results from a large patient cohort.

CONTEXT: Pituitary adenomas and pheochromocytomas/paragangliomas (pheo/PGL) can occur in the same patient or in the same family. Coexistence of the two diseases could be due to either a common pathogenic mechanism or a coincidence. OBJECTIVE: The objective of the investigation was to study the possible coexistence of pituitary adenoma and pheo/PGL. DESIGN: Thirty-nine cases of sporadic or familial pheo/PGL and pituitary adenomas were investigated. Known pheo/PGL genes (SDHA-D, SDHAF2, RET, VHL, TMEM127, MAX, FH) and pituitary adenoma genes (MEN1, AIP, CDKN1B) were sequenced using next generation or Sanger sequencing. Loss of heterozygosity study and pathological studies were performed on the available tumor samples. SETTING: The study was conducted at university hospitals. PATIENTS: Thirty-nine patients with sporadic of familial pituitary adenoma and pheo/PGL participated in the study. OUTCOME: Outcomes included genetic screening and clinical characteristics. RESULTS: Eleven germline mutations (five SDHB, one SDHC, one SDHD, two VHL, and two MEN1) and four variants of unknown significance (two SDHA, one SDHB, and one SDHAF2) were identified in the studied genes in our patient cohort. Tumor tissue analysis identified LOH at the SDHB locus in three pituitary adenomas and loss of heterozygosity at the MEN1 locus in two pheochromocytomas. All the pituitary adenomas of patients affected by SDHX alterations have a unique histological feature not previously described in this context. CONCLUSIONS: Mutations in the genes known to cause pheo/PGL can rarely be associated with pituitary adenomas, whereas mutation in a gene predisposing to pituitary adenomas (MEN1) can be associated with pheo/PGL. Our findings suggest that genetic testing should be considered in all patients or families with the constellation of pheo/PGL and a pituitary adenoma.

Disorders of cholesterol metabolism and their unanticipated convergent mechanisms of disease.

Cholesterol plays a key role in many cellular processes, and is generated by cells through de novo biosynthesis or acquired from exogenous sources through the uptake of low-density lipoproteins. Cholesterol biosynthesis is a complex, multienzyme-catalyzed pathway involving a series of sequentially acting enzymes. Inherited defects in genes encoding cholesterol biosynthetic enzymes or other regulators of cholesterol homeostasis result in severe metabolic diseases, many of which are rare in the general population and currently without effective therapy. Historically, these diseases have been viewed as discrete disorders, each with its own genetic cause and distinct pathogenic cascades that lead to its specific clinical features. However, studies have recently shown that three of these diseases have an unanticipated mechanistic convergence. This surprising finding is not only shedding light on details of cellular cholesterol homeostasis but also suggesting novel approaches to therapy.

Relative acidic compartment volume as a lysosomal storage disorder-associated biomarker.

Lysosomal storage disorders (LSDs) occur at a frequency of 1 in every 5,000 live births and are a common cause of pediatric neurodegenerative disease. The relatively small number of patients with LSDs and lack of validated biomarkers are substantial challenges for clinical trial design. Here, we evaluated the use of a commercially available fluorescent probe, Lysotracker, that can be used to measure the relative acidic compartment volume of circulating B cells as a potentially universal biomarker for LSDs. We validated this metric in a mouse model of the LSD Niemann-Pick type C1 disease (NPC1) and in a prospective 5-year international study of NPC patients. Pediatric NPC subjects had elevated acidic compartment volume that correlated with age-adjusted clinical severity and was reduced in response to therapy with miglustat, a European Medicines Agency­approved drug that has been shown to reduce NPC1-associated neuropathology. Measurement of relative acidic compartment volume was also useful for monitoring therapeutic responses of an NPC2 patient after bone marrow transplantation. Furthermore, this metric identified a potential adverse event in NPC1 patients receiving i.v. cyclodextrin therapy. Our data indicate that relative acidic compartment volume may be a useful biomarker to aid diagnosis, clinical monitoring, and evaluation of therapeutic responses in patients with lysosomal disorders.

Efficacy of N-acetylcysteine in phenotypic suppression of mouse models of Niemann-Pick disease, type C1.

Niemann-Pick disease, type C1 (NPC1), which arises from a mutation in the NPC1 gene, is characterized by abnormal cellular storage and transport of cholesterol and other lipids that leads to hepatic disease and progressive neurological impairment. Oxidative stress has been hypothesized to contribute to the NPC1 disease pathological cascade. To determine whether treatments reducing oxidative stress could alleviate NPC1 disease phenotypes, the in vivo effects of the antioxidant N-acetylcysteine (NAC) on two mouse models for NPC1 disease were studied. NAC was able to partially suppress phenotypes in both antisense-induced (NPC1ASO) and germline (Npc1-/-) knockout genetic mouse models, confirming the presence of an oxidative stress-related mechanism in progression of NPC1 phenotypes and suggesting NAC as a potential molecule for treatment. Gene expression analyses of NAC-treated NPC1ASO mice suggested NAC affects pathways distinct from those initially altered by Npc1 knockdown, data consistent with NAC achieving partial disease phenotype suppression. In a therapeutic trial of short-term NAC administration to NPC1 patients, no significant effects on oxidative stress in these patients were identified other than moderate improvement of the fraction of reduced CoQ10, suggesting limited efficacy of NAC monotherapy. However, the mouse model data suggest that the distinct antioxidant effects of NAC could provide potential treatment of NPC1 disease, possibly in concert with other therapeutic molecules at earlier stages of disease progression. These data also validated the NPC1ASO mouse as an efficient model for candidate NPC1 drug screening, and demonstrated similarities in hepatic phenotypes and genome-wide transcript expression patterns between the NPC1ASO and Npc1-/- models.

Succinate dehydrogenase (SDH) D subunit (SDHD) inactivation in a growth-hormone-producing pituitary tumor: a new association for SDH?

BACKGROUND: Mutations in the subunits B, C, and D of succinate dehydrogenase (SDH) mitochondrial complex II have been associated with the development of paragangliomas (PGL), gastrointestinal stromal tumors, papillary thyroid and renal carcinoma (SDHB), and testicular seminoma (SDHD). AIM: Our aim was to examine the possible causative link between SDHD inactivation and somatotropinoma. PATIENTS AND METHODS: A 37-yr-old male presented with acromegaly and hypertension. Other family members were found with PGL. Elevated plasma and urinary levels of catecholamines led to the identification of multiple PGL in the proband in the neck, thorax, and abdomen. Adrenalectomy was performed for bilateral pheochromocytomas (PHEO). A GH-secreting macroadenoma was also found and partially removed via transsphenoidal surgery (TTS). Genetic analysis revealed a novel SDHD mutation (c.298_301delACTC), leading to a frame shift and a premature stop codon at position 133 of the protein. Loss of heterozygosity for the SDHD genetic locus was shown in the GH-secreting adenoma. Down-regulation of SDHD protein in the GH-secreting adenoma by immunoblotting and immunohistochemistry was found. A literature search identified other cases of multiple PGL and/or PHEO in association with pituitary tumors. CONCLUSION: We describe the first kindred with a germline SDHD pathogenic mutation, inherited PGL, and acromegaly due to a GH-producing pituitary adenoma. SDHD loss of heterozygosity, down-regulation of protein in the GH-secreting adenoma, and decreased SDH enzymatic activity supports SDHD's involvement in the pituitary tumor formation in this patient. Older cases of multiple PGL and PHEO and pituitary tumors in the literature support a possible association between SDH defects and pituitary tumorigenesis.

Increasing cholesterol synthesis in 7-dehydrosterol reductase (DHCR7) deficient mouse models through gene transfer.

Smith-Lemli-Opitz syndrome (SLOS) is caused by deficiency in the terminal step of cholesterol biosynthesis: the conversion of 7-dehydrocholesterol (7DHC) to cholesterol (C), catalyzed by 7-dehydrocholesterol reductase (DHCR7). This disorder exhibits several phenotypic traits including dysmorphia and mental retardation with a broad range of severity. There are few proven treatment options. That most commonly used is a high cholesterol diet that seems to enhance the quality of life and improve behavioral characteristics of patients, although these positive effects are controversial. The goal of our study was to investigate the possibility of restoring DHCR7 activity by gene transfer. We constructed an adeno-associated virus (AAV) vector containing the DHCR7 gene. After we infused this vector into affected mice, the introduced DHCR7 gene could be identified in liver, mRNA was expressed and a functional enzyme was produced. Evidence of functionality came from the ability to partially normalize the serum ratio of 7DHC/C in treated animals, apparently by increasing cholesterol production with concomitant decrease in 7DHC precursor. By 5 weeks after treatment the mean ratio (for 7 animals) had fallen to 0.05 while the ratio for untreated littermate controls had risen to 0.14. This provides proof of principle that gene transfer can ameliorate the genetic defect causing SLOS and provides a new experimental tool for studying the pathogenesis of this disease. If effective in humans, it might also offer a possible alternative to exogenous cholesterol therapy. However, it would not offer a complete cure for the disorder as many of the negative implications of defective synthesis are already established during prenatal development.

HEM dysplasia and ichthyosis are likely laminopathies and not due to 3beta-hydroxysterol Delta14-reductase deficiency.

Mutations of the lamin B receptor (LBR) have been shown to cause HEM dysplasia in humans and ichthyosis in mice. LBR is a bifunctional protein with both a lamin B binding and a sterol Delta(14)-reductase domain. It previously has been proposed that LBR is the primary sterol Delta(14)-reductase and that HEM dysplasia and ichthyosis are inborn errors of cholesterol synthesis. However, DHCR14 also encodes a sterol Delta(14)-reductase and could provide enzymatic redundancy with respect to cholesterol synthesis. To test the hypothesis that LBR and DHCR14 both function as sterol Delta(14)-reductases, we obtained ichthyosis mice (Lbr(-/-)) and disrupted Dhcr14. Heterozygous Lbr and Dhcr14 mice were intercrossed to test for a digenic phenotype. Lbr(-/-), Dhcr14(Delta4-7/Delta4-7) and Lbr(+/-):Dhcr14(Delta4-7/Delta4-7) mutant mice have distinct physical and biochemical phenotypes. Dhcr14(Delta4-7/Delta4-7) mice are essentially normal, whereas Lbr(+/-):Dhcr14(Delta4-7/Delta4-7) mice are growth retarded and neurologically abnormal. Neither of these mutants resembles the ichthyosis mouse and biochemically, no sterol abnormalities were detected in either liver or kidney tissue. In contrast, relatively small transient elevations of Delta(14)-sterols were observed in Lbr(-/-) and Dhcr14(Delta4-7/Delta4-7) brain tissue, and marked elevations were seen in Lbr(+/-):Dhcr14(Delta4-7/Delta4-7) brain. Pathological evaluation demonstrated vacuolation and swelling of the myelin sheaths in the spinal cord of Lbr(+/-):Dhcr14(Delta4-7/Delta4-7) mice consistent with a demyelinating process. This was not observed in either Lbr(-/-) or Dhcr14 (Delta4-7/Delta4-7) mice. Our data support the conclusions that LBR and DHCR14 provide substantial enzymatic redundancy with respect to cholesterol synthesis and that HEM dysplasia and ichthyosis are laminopathies rather than inborn errors of cholesterol synthesis.

Cholesterol deficiency in a mouse model of Smith-Lemli-Opitz syndrome reveals increased mast cell responsiveness.

Mutation of the 3beta-hydroxysterol delta7-reductase gene (Dhcr7-/-) results in Smith-Lemli-Opitz syndrome (SLOS). Patients, and genetically altered mice, are unable to produce cholesterol and accumulate 7-dehydrocholesterol (DHC) in serum and tissue. This causes multiple growth and developmental abnormalities as well as immune system anomalies including allergy. Because cholesterol is a key component of liquid-ordered membranes (lipid rafts) and these domains have been implicated in regulating mast cell activation, we examined whether mast cell responsiveness is altered in this model. Mast cells derived from Dhcr7-/- mice (DHCR KO) showed constitutive cytokine production and hyper-degranulation after stimulation of the high affinity IgE receptor (Fc epsilonRI). DHCR KO mast cells, but not wild-type mast cells, accumulated DHC in lipid rafts. DHC partially disrupted lipid raft stability and displaced Lyn kinase protein and activity from lipid rafts. This led to down-regulation of some Lyn-dependent signaling events but increased Fyn kinase activity and Akt phosphorylation. The Lyn-dependent phosphorylation of Csk-binding protein, which negatively regulates Fyn activity, was decreased. This phenotype reproduces some of the characteristics of Lyn-null mast cells, which also demonstrate hyper-degranulation. These findings provide the first evidence of lipid raft dysfunction in SLOS and may explain the observed association of allergy with SLOS.