Injury type-dependent differentiation of NG2 glia into heterogeneous astrocytes.

The glial scar is comprised of a heterogeneous population of reactive astrocytes. NG2 glial cells (also known as oligodendrocyte progenitor cells or polydendrocytes) may contribute to this heterogeneity by differentiating into astrocytes in the injured CNS, but there have been conflicting reports about whether astrocytes comprise a significant portion of the NG2 cell lineage. By using genetic fate mapping after spinal cord injury (SCI) and experimental autoimmune encephalomyelitis (EAE) in mice, the goal of this study was to confirm and extend upon previous findings, which have shown that NG2 cell plasticity varies across CNS injuries. We generated mice that express tdTomato in NG2 lineage cells and express GFP under the Aldh1l1 or Glt1 promoter so that NG2 glia-derived astrocytes can be detected by their expression of GFAP and/or GFP. We found that astrocytes comprise approximately 25% of the total NG2 cell lineage in the glial scar by 4 weeks after mid-thoracic contusive SCI, but only 9% by the peak of functional deficit after EAE. Interestingly, a subpopulation of astrocytes expressed only GFP without co-expression of GFAP, uncovering their heterogeneity and the possibility of an underestimation of NG2 glia-derived astrocytes in previous studies. Additionally, we used high performance liquid chromatography to measure the level of tamoxifen and its metabolites in the spinal cord and show that genetic labeling of NG2 glia-derived astrocytes is not an artifact of residual tamoxifen. Overall, our data demonstrate that a heterogeneous population of astrocytes are derived from NG2 glia in an injury type-dependent manner.

Diagnosing lysosomal storage disorders: mucopolysaccharidosis type I.

Mucopolysaccharidosis type I (MPS I) is a lysosomal storage disorder due to deficiency of alpha iduronidase (IDUA). Progressive storage of dermatan and heparan sulfate throughout the body lead to a multiorgan presentation including short stature, dysostosis multiplex, corneal clouding, hearing loss, coarse facies, hepatosplenomegaly, and intellectual disability. Diagnosis of MPS I is based on IDUA enzyme analysis in leukocytes or dried blood spots (DBS) followed by molecular confirmation of the IDUA gene mutations in individuals with low enzyme activity. The advent of mass spectrometry methods for enzyme analysis in DBS has enabled high-throughput screening for MPS I in symptomatic individuals and newborn infants. The following unit provides the detailed analytical protocol for measurement of IDUA activity in DBS using tandem mass spectrometry.

Diagnosis of lysosomal storage disorders: Gaucher disease.

Gaucher Disease (GD) is a progressive lysosomal storage disorder caused by deficiency of glucocerebrosidase (GBA). The clinical phenotype follows a spectrum ranging from severe early-onset to milder late-onset disease. The absence of neurological involvement defines GD type I, whereas neuronopathic features define GD type II and III. Early diagnosis may be important for timely initiation of enzyme replacement therapy to prevent disease complications, although the enzyme does not cross the blood brain barrier. Diagnosis of GD can be readily achieved by analysis of GBA in leukocytes, fibroblasts, and/or dried blood spots using fluorometric, microfluidic or mass spectrometry-based assays. Low GBA activities are typically confirmed through molecular analysis of the GBA gene. GBA analysis in dried blood spots may be attractive for high-throughput screening of at-risk individuals and/or newborn infants. The method detailed in this unit is based on GBA analysis by tandem mass spectrometry following incubation of dried blood spots with the GBA-specific substrate D-glucosyl-ß1-1'-N-dodecanoyl-D-erythro-sphingosine [C12-glucocerebroside (C36H69NO8)] and internal standard N-myristoyl-D-erythro-sphingosine [C14-ceramide (C32H63NO3)]. GBA activities in more than 2,000 newborn infants showed a mean of 22.0 ± 13.8 µmol/hr/liter (median: 19.9 µmol/hr/liter; 95% CI: 21.41-22.59 µmol/hr/liter). GBA activities in an adult population (n >1,200) showed generally lower enzyme activities than newborns, with a mean of 9.87 ± 9.35 µmol/hr/liter (median: 8.06 µmol/hr/liter). GBA activities in ten adult patients with confirmed GD were less than 4.2 µmol/hr/liter and in seven infants and children with GD less than 1.24 µmol/hr/liter. This method is robust, sensitive, and suitable for high-throughput analysis of hundreds of samples.

Analysis of lyso-globotriaosylsphingosine in dried blood spots.

Recently, lyso-globotriaosylsphingosine (lyso-Gb3) was found to be elevated in plasma of treatment naive male patients and some female patients with Fabry Disease (FD). This study tested whether lyso-Gb3 could be analyzed in dried blood spots (DBS) from filter cards and whether concentrations are elevated in newborn infants with FD. Lyso-Gb3 concentrations were analyzed in DBS following extraction using a novel HPLC-mass spectrometry (MS)/MS method. Lyso-Gb3 levels in DBS were above the lower limit of quantitation (0.28 ng/mL) in 5/17 newborn FD infants (16 males; range: 1.02-8.81 ng/mL), but in none of the newborn controls, in all 13 patients (4 males) with classic FD (range: 2.06-54.1 ng/mL), in 125/159 Taiwanese individuals with symptomatic or asymptomatic FD who carry the late onset α-galactosidase A (GLA) mutation c.936+919G>A (IVS4+919G>A) (3.75±0.69 ng/mL; range: 0.418-3.97 ng/mL) and in 20/29 healthy controls (0.77±0.24 ng/mL; range: 0.507-1.4 ng/mL). The HPLC-MS/MS method for analysis of lyso-Gb3 is robust and yields reproducible results in DBS in patients with FD. However, concentrations of lyso-Gb3 were below the limit of quantitation in most newborn infants with FD rendering this approach not suitable for newborn screening. In addition, most females with the late onset mutation have undetectable lyso-Gb3 concentrations.

Diagnosing lysosomal storage disorders: Fabry disease.

Fabry disease (FD) is an X-linked lysosomal storage disorder due to deficiency of alpha galactosidase A (GLA). Progressive, intralysosomal accumulation of neutral glycosphingolipids in endothelial cells and podocytes leads to multi-organ involvement in affected males and to a lesser extent in affected females. Diagnosis of FD is based on GLA analysis in leukocytes or dried blood spots (DBS) in FD males while GLA activities may be within the normal range in FD females. The advent of fluorometric and mass spectrometry methods for enzyme analysis in DBS has simplified the diagnostic approach for FD males, facilitating high-throughput screening of at risk populations and newborn infants. However, the diagnostic mainstay for FD females remains molecular analysis of the GLA gene. The following unit will provide the detailed analytical protocol for measurement of GLA activity in DBS using tandem mass spectrometry.

Diagnosing lysosomal storage disorders: mucopolysaccharidosis type II.

Mucopolysaccharidosis type II (MPS II) is an X-linked lysosomal storage disorder caused by a deficiency of iduronate 2-sulfatase (IDS). Progressive, intralysosomal accumulation of the glycosaminoglycans (GAGs) dermatan and heparan sulfate in almost all tissues leads to multi-organ involvement in affected males but to virtual absence of symptoms in heterozygote female carriers due to preferential inactivation of the mutant allele. Diagnosis of MPS II in males is based on IDS analysis in leukocytes, fibroblasts, plasma, or dried blood spots (DBS), whereas IDS activities may be within the normal range in heterozygote females. The advent of fluorometric and mass spectrometry methods for enzyme analysis in DBS has simplified the diagnostic approach for MPS II males. Molecular analysis of the IDS gene confirms the diagnosis of MPS II in males and is the only diagnostic test to confirm carrier status in females. This unit provides detailed analytical protocols for measurement of IDS activity in DBS and plasma using a fluorometric assay.

Diagnosing lysosomal storage disorders: Pompe disease.

Pompe disease is a lysosomal storage disorder caused by a deficiency of acid alpha glucosidase (GAA). Diagnosis of Pompe disease is typically based on an enzyme analysis of blood or tissues, such as fibroblasts, followed by confirmation through molecular testing. The advent of fluorometric and mass spectrometry methods for enzyme analysis in dried blood spots (DBS) has simplified the diagnostic approach for Pompe disease, facilitating high-throughput screening of at-risk populations and newborn infants. The following unit will provide the detailed analytical protocol for measurement of GAA activity in DBS using tandem mass spectrometry.

Analysis of acid sphingomyelinase activity in dried blood spots using tandem mass spectrometry.

BACKGROUND: Niemann Pick disease (NP) is a rare, lysosomal storage disorder due to deficiency of the intra-lysosomal enzyme acid sphingomyelinase (ASM) resulting in intracellular accumulation of sphingomyelin. We evaluated a tandem mass spectrometry (MS/MS) method to analyze ASM activity in dried blood spots (DBS) that may be suitable for laboratory diagnosis of NP including high throughput screening of at-risk populations and potentially for newborn screening. METHODS: ASM activity was measured in 3.2 mm punches from DBS. The eluate was incubated with the ASM substrate (N-Hexanoyl-D-erythro-sphingosylphosphorylcholine [C6-sphingomyelin (C(29)H(59)N(2)O(6)P)]) and an internal standard (N-butyroyl-D-erythro-sphingosine [C4-ceramide (C(22)H(43)NO(3))]). ASM product and IS were analyzed using MS/MS in multiple reaction monitoring mode for transitions m/z 370.6>264.3 (ASM internal standard) and m/z 398.6>264.3 (ASM product). RESULTS: ASM activities were stable for up to 2 months at or below 4℃. Position of the punch in the DBS and/or hematocrit of the DBS had a limited effect on ASM activities. Both intra- and inter-assay variability were below 10%. There was no carry-over. The median ASM activity in 2,085 newborn infants was 9.5 µmol/h/L (mean 10.6) with a SD of 5.06 µmol/h/L. Six of 2,085 (0.3%) infants were found to have ASM activities below the cut-off of 2.5 µmol/h/L. ASM activities were below the cut-off level in all 10 previously diagnosed cases with NP (range: 0.16 to 2.08 µmol/h/L). CONCLUSIONS: This MS/MS method for the measurement of ASM activity in DBS is robust and suitable for laboratory diagnosis of NP.

Endocannabinoids modulate human epidermal keratinocyte proliferation and survival via the sequential engagement of cannabinoid receptor-1 and transient receptor potential vanilloid-1.

We have recently shown that lipid mediators of the emerging endocannabinoid system (ECS) are key players of growth control of the human pilosebaceous unit. In this study, we asked whether the prototypic endocannabinoid anandamide (N-arachidonoylethanolamine, AEA) has a role in growth and survival of epidermal keratinocytes (KCs). Using human cultured KCs and skin organ-culture models, and by employing combined pharmacological and molecular approaches, we provide early evidence that AEA markedly suppresses KC proliferation and induces cell death, both in vitro and in situ. Moreover, we present that these cellular actions are mediated by a most probably constitutively active signaling mechanism that involves the activation of the metabotropic cannabinoid receptor CB(1) and a sequential engagement of the "ionotropic cannabinoid receptor" transient receptor potential vanilloid-1 (TRPV1). Finally, we demonstrate that the cellular effects of AEA are most probably due to a Ca(2+) influx via the non-selective, highly Ca(2+)-permeable ion channel TRPV1, and the concomitant elevation of intracellular Ca(2+) concentration. The data reported here may encourage one to explore whether the targeted manipulation of the above signaling pathway of the cutaneous ECS could become a useful adjunct treatment strategy for hyperproliferative human dermatoses such as psoriasis or KC-derived skin tumors.

[Management of Fabry disease]. / Fabry-betegség - terápiás útmutató

Fabry disease is a rare, X-linked lysosomal storage disorder that leads to accumulation of globotriaosylceramide in different tissues of the body. The disease is progressive and the first symptoms usually present in childhood. Consequences of the disease are disability and premature death. The disease in females could be as severe as in males although women may be asymptomatic. The possibility of enzyme replacement therapy has made it necessary to elaborate a comprehensive guideline for the diagnosis and treatment follow-up. The guideline has been summarized by a Hungarian multi-disciplinary working group consisting of physicians who are involved in diagnosis and care of Fabry patients. Previous clinical studies, published articles, and recently established international treatment guidelines were reviewed by the group.

Newborn screening for Fabry disease by measuring GLA activity using tandem mass spectrometry.

BACKGROUND: Fabry disease (FD) is an X-linked lysosomal storage disorder caused by the deficiency of alpha-galactosidase A (GLA). We evaluated a tandem mass spectrometry method to measure GLA activity. METHODS: One 3.2mm punch from a dried blood spot sample (DBS) was incubated with substrate and internal standard in the reaction buffer for 22 h. The resulting product was quantified against internal standard using MS/MS. RESULTS: The median GLA activity of male newborn DBS (N=5025) was 9.85 + or - 6.4 micromol/h/l (CI 95% is 9.67-10.02 micromol/h/l); The median GLA activity of female newborns (N=4677) was 10.2 + or - 6.3 micromol/h/l (CI 95% is 10.02-10.38 micromol/h/l). The difference between the two subgroups is within assay analytical variation. The GLA activities in the DBS samples from 9 juvenile and adult males with previously identified FD were below 1.64 micromol/h/l. The GLA activities from 32 juvenile and adult females with confirmed FD were below 4.73 micromol/h/l. In 5 (16%) females GLA activities were above the 0.5th percentile of lower limit of CI 95% at 3.18 micromol/h/l. CONCLUSIONS: The MS/MS method for Fabry disease newborn screening is robust and can be readily multiplexed with other lysosomal disorders such as Pompe, Gaucher, Niemann-Pick, and Krabbe diseases.

[Fabry disease--diagnostic guideline]. / Fabry-betegség. Diagnosztikai útmutató.

Fabry disease is a rare, X-linked lysosomal storage disorder that leads to accumulation of globotriaosylceramide in different tissues of the body. The disease is progressive, first symptoms usually present in childhood. Consequencies of the diseases are disability and premature death. The disease in females could be as severe as in males although women may also be asymptomatic. The possibility of enzyme replacement therapy has made it necessary to elaborate a comprehensive guideline for the diagnosis and treatment follow-up. The guideline was established by a Hungarian multi-disciplinary working group, consisting of physicians who are involved in health care of Fabry patients. Previous clinical studies, published materials, and recently established international treatment guidelines were reviewed by the group.

Novel sequence variants of the alpha-galactosidase A gene in patients with Fabry disease.

We carried out molecular studies of 15 unrelated Hungarian families diagnosed with Fabry disease (FD). Genetic analysis of the alpha-galactosidase A gene was performed in 22 hemizygous males and 34 females. One of the female patients with severe disease phenotype showed homozygosity for the recurrent c.644A>G mutation due to parental consanguinity. The c.644A>G mutation that has previously been found mostly in patients with the cardiac variant of FD, was associated with renal but not cardiac involvement in this female and in two other family members. In nine families, eight novel sequence variants such as small deletions (c.363delT, c.477delT, c.746delAC) and single nucleotide changes (c.107T>C, c.493G>C, c.796G>T, c.866T>G, c.871G>A) were found in addition to six previously described private mutations. This report contributes to the identification of novel disease-causing mutations in FD, and increases our knowledge on demographics and molecular characteristics of this rare lysosomal storage disorder. This is the first comprehensive overview of molecular genetic features of Hungarian patients with FD.

Newborn screening for Pompe disease by measuring acid alpha-glucosidase activity using tandem mass spectrometry.

BACKGROUND: Pompe disease, caused by the deficiency of acid alpha-glucosidase (GAA), is a lysosomal storage disorder that manifests itself in its most severe form within the first months of life. Early detection by newborn screening is warranted, since prompt initiation of enzyme replacement therapy may improve morbidity and mortality. We evaluated a tandem mass spectrometry (MS/MS) method to measure GAA activity for newborn screening for Pompe disease. METHODS: We incubated 3.2-mm punches from dried blood spots (DBS) for 22 h with the substrate [7-benzoylamino-heptyl)-{2-[4-(3,4,5-trihydroxy-6-hydroxymethyl-tetrahydro-pyran-2-yloxy)-phenylcarbamoyl]- ethyl}-carbamic acid tert-butyl ester] and internal standard [7-d(5)-benzoylamino-heptyl)-[2-(4-hydroxy-phenylcarbamoyl)-ethyl]-carbamic acid tertbutyl ester]. We quantified the resulting product and internal standard using MS/MS. We assessed inter- and intrarun imprecision, carryover, stability, and correlation between enzyme activities and hematocrit and punch location and generated a Pompe disease-specific cutoff value using routine newborn screening samples. RESULTS: GAA activities in DBS from 29 known Pompe patients were <2 micromol/h/L. GAA activities in routine newborn screening samples were [mean (SD)] 14.7 (7.2) micromol/h/L (n = 10,279, median 13.3, 95% CI 14.46-14.74 micromol/h/L) and in normal adult samples 9.3 (3.3) micromol/h/L (n = 229, median 9, 95% CI 8.88-9.72 micromol/h/L). GAA activity was stable for 28 days between 37 degrees C and -80 degrees C. Carryover could not be observed, whereas intrarun and interrun imprecision were <10%. The limit of detection was 0.26 micromol/h/L and limit of quantification 0.35 micromol/h/L. CONCLUSIONS: The measurement of GAA activities in dry blood spots using MS/MS is suitable for high-throughput analysis and newborn screening for Pompe disease.