Drosophila melanogaster Mutated in its GBA1b Ortholog Recapitulates Neuronopathic Gaucher Disease

Gaucher disease (GD) results from mutations in the GBA1 gene, which encodes lysosomal glucocerebrosidase (GCase). The large number of mutations known to date in the gene lead to a heterogeneous disorder, which is divided into a non-neuronopathic, type 1 GD, and two neurological, type 2 and type 3, forms. We studied the two fly GBA1 orthologs, GBA1a and GBA1b. Each contains a Minos element insertion, which truncates its coding sequence. In the GBA1a(m/m) flies, which express a mutant protein, missing 33 C-terminal amino acids, there was no decrease in GCase activity or substrate accumulation. However, GBA1b(m/m) mutant flies presented a significant decrease in GCase activity with concomitant substrate accumulation, which included C14:1 glucosylceramide and C14:0 glucosylsphingosine. GBA1b(m/m) mutant flies showed activation of the Unfolded Protein Response (UPR) and presented inflammation and neuroinflammation that culminated in development of a neuronopathic disease. Treatment with ambroxol did not rescue GCase activity or reduce substrate accumulation; however, it ameliorated UPR, inflammation and neuroinflammation, and increased life span. Our results highlight the resemblance between the phenotype of the GBA1b(m/m) mutant fly and neuronopathic GD and underlie its relevance in further GD studies as well as a model to test possible therapeutic modalities

Drosophila melanogaster Mutated in its GBA1b Ortholog Recapitulates Neuronopathic Gaucher Disease

Gaucher disease (GD) results from mutations in the GBA1 gene, which encodes lysosomal glucocerebrosidase (GCase). The large number of mutations known to date in the gene lead to a heterogeneous disorder, which is divided into a non-neuronopathic, type 1 GD, and two neurological, type 2 and type 3, forms. We studied the two fly GBA1 orthologs, GBA1a and GBA1b. Each contains a Minos element insertion, which truncates its coding sequence. In the GBA1a(m/m) flies, which express a mutant protein, missing 33 C-terminal amino acids, there was no decrease in GCase activity or substrate accumulation. However, GBA1b(m/m) mutant flies presented a significant decrease in GCase activity with concomitant substrate accumulation, which included C14:1 glucosylceramide and C14:0 glucosylsphingosine. GBA1b(m/m) mutant flies showed activation of the Unfolded Protein Response (UPR) and presented inflammation and neuroinflammation that culminated in development of a neuronopathic disease. Treatment with ambroxol did not rescue GCase activity or reduce substrate accumulation; however, it ameliorated UPR, inflammation and neuroinflammation, and increased life span. Our results highlight the resemblance between the phenotype of the GBA1b(m/m) mutant fly and neuronopathic GD and underlie its relevance in further GD studies as well as a model to test possible therapeutic modalities

Immune cell C/EBPβ deficiency is associated with hepatic mononuclear defects and spontaneous hepatitis but not steatohepatitis induced liver fibrosis

BACKGROUND: CCAAT/enhancer-binding protein ß (C/EBPß) is a transcription factor known to be involved in macrophage differentiation and function, steatohepatitis and liver fibrosis. METHODS: Immune restricted C/EBPß deficient and control mice were investigated in steady-state and in the CDA-HFD steatohepatitis model. Mice were assessed for weight change, liver biochemical profile, histology and hepatic phagocytes composition. RESULTS: Flow cytometry analysis of hepatic nonparenchymal cells revealed reduced numbers of hepatic monocytes and Kupffer cells and an increase in hepatic MHC class II positive myeloid cells in immune cells restricted C/EBPß deficient mice. Immune-restricted C/EBPß deficiency resulted in decreased weight gain and appearance of mild spontaneous liver inflammation. Nevertheless, In the CDA-HFD steatohepatitis model, immune restricted C/EBPß deficient and proficient mice exhibit similar grade of hepatic steatosis, liver enzymes levels and fibrosis stage. CONCLUSIONS: Immune-restricted C/EBPß deficiency leads to significant alteration in hepatic mononuclear phagocytes composition associated with spontaneous mild hepatitis. Steatohepatitis associated fibrosis is not dependent on C/EBPß expression by immune cells

RGS2 expression predicts amyloid-β sensitivity, MCI and Alzheimer's disease: genome-wide transcriptomic profiling and bioinformatics data mining

Alzheimer's disease (AD) is the most frequent cause of dementia. Misfolded protein pathological hallmarks of AD are brain deposits of amyloid-β (Aβ) plaques and phosphorylated tau neurofibrillary tangles. However, doubts about the role of Aβ in AD pathology have been raised as Aβ is a common component of extracellular brain deposits found, also by in vivo imaging, in non-demented aged individuals. It has been suggested that some individuals are more prone to Aβ neurotoxicity and hence more likely to develop AD when aging brains start accumulating Aβ plaques. Here, we applied genome-wide transcriptomic profiling of lymphoblastoid cells lines (LCLs) from healthy individuals and AD patients for identifying genes that predict sensitivity to Aβ. Real-time PCR validation identified 3.78-fold lower expression of RGS2 (regulator of G-protein signaling 2; P=0.0085) in LCLs from healthy individuals exhibiting high vs low Aβ sensitivity. Furthermore, RGS2 showed 3.3-fold lower expression (P=0.0008) in AD LCLs compared with controls. Notably, RGS2 expression in AD LCLs correlated with the patients' cognitive function. Lower RGS2 expression levels were also discovered in published expression data sets from postmortem AD brain tissues as well as in mild cognitive impairment and AD blood samples compared with controls. In conclusion, Aβ sensitivity phenotyping followed by transcriptomic profiling and published patient data mining identified reduced peripheral and brain expression levels of RGS2, a key regulator of G-protein-coupled receptor signaling and neuronal plasticity. RGS2 is suggested as a novel AD biomarker (alongside other genes) toward early AD detection and future disease modifying therapeutics

ANGPTL4 promotes the progression of cutaneous melanoma to brain metastasis.

In an ongoing effort to identify molecular determinants regulating melanoma brain metastasis, we previously identified Angiopoietin-like 4 (ANGPTL4) as a component of the molecular signature of such metastases. The aim of this study was to determine the functional significance of ANGPTL4 in the shaping of melanoma malignancy phenotype, especially in the establishment of brain metastasis. We confirmed that ANGPTL4 expression is significantly higher in cells metastasizing to the brain than in cells from the cutaneous (local) tumor from the same melanoma in a nude mouse xenograft model, and also in paired clinical specimens of melanoma metastases than in primary melanomas from the same patients. In vitro experiments indicated that brain-derived soluble factors and transforming growth factor β1 (TGFβ1) up-regulated ANGPTL4 expression by melanoma cells. Forced over-expression of ANGPTL4 in cutaneous melanoma cells promoted their ability to adhere and transmigrate brain endothelial cells. Over-expressing ANGPTL4 in cells derived from brain metastases resulted in the opposite effects. In vivo data indicated that forced overexpression of ANGPTL4 promoted the tumorigenicity of cutaneous melanoma cells but did not increase their ability to form brain metastasis. This finding can be explained by inhibitory activities of brain-derived soluble factors. Taken together these findings indicate that ANGPTL4 promotes the malignancy phenotype of primary melanomas of risk to metastasize to the brain

In Vivo and Ex Vivo Evaluation of L-Type Calcium Channel Blockers on Acid β-Glucosidase in Gaucher Disease Mouse Models

Gaucher disease is a lysosomal storage disease caused by mutations in acid β-glucosidase (GCase) leading to defective hydrolysis and accumulation of its substrates. Two L-type calcium channel (LTCC) blockers—verapamil and diltiazem—have been reported to modulate endoplasmic reticulum (ER) folding, trafficking, and activity of GCase in human Gaucher disease fibroblasts. Similarly, these LTCC blockers were tested with cultured skin fibroblasts from homozygous point-mutated GCase mice (V394L, D409H, D409V, and N370S) with the effect of enhancing of GCase activity. Correspondingly, diltiazem increased GCase protein and facilitated GCase trafficking to the lysosomes of these cells. The in vivo effects of diltiazem on GCase were evaluated in mice homozygous wild-type (WT), V394L and D409H. In D409H homozygotes diltiazem (10 mg/kg/d via drinking water or 50–200 mg/kg/d intraperitoneally) had minor effects on increasing GCase activity in brain and liver (1.2-fold). Diltiazem treatment (10 mg/kg/d) had essentially no effect on WT and V394L GCase protein or activity levels (<1.2-fold) in liver. These results show that LTCC blockers had the ex vivo effects of increasing GCase activity and protein in the mouse fibroblasts, but these effects did not translate into similar changes in vivo even at very high drug doses

Effects of IKAP/hELP1 Deficiency on Gene Expression in Differentiating Neuroblastoma Cells: Implications for Familial Dysautonomia

Familial dysautonomia (FD) is a developmental neuropathy of the sensory and autonomous nervous systems. The IKBKAP gene, encoding the IKAP/hELP1 subunit of the RNA polymerase II Elongator complex is mutated in FD patients, leading to a tissue-specific mis-splicing of the gene and to the absence of the protein in neuronal tissues. To elucidate the function of IKAP/hELP1 in the development of neuronal cells, we have downregulated IKBKAP expression in SHSY5Y cells, a neuroblastoma cell line of a neural crest origin. We have previously shown that these cells exhibit abnormal cell adhesion when allowed to differentiate under defined culture conditions on laminin substratum. Here, we report results of a microarray expression analysis of IKAP/hELP1 downregulated cells that were grown on laminin under differentiation or non-differentiation growth conditions. It is shown that under non-differentiation growth conditions, IKAP/hELP1 downregulation affects genes important for early developmental stages of the nervous system, including cell signaling, cell adhesion and neural crest migration. IKAP/hELP1 downregulation during differentiation affects the expression of genes that play a role in late neuronal development, in axonal projection and synapse formation and function. We also show that IKAP/hELP1 deficiency affects the expression of genes involved in calcium metabolism before and after differentiation of the neuroblastoma cells. Hence, our data support IKAP/hELP1 importance in the development and function of neuronal cells and contribute to the understanding of the FD phenotype

CDH3-Related Syndromes: Report on a New Mutation and Overview of the Genotype-Phenotype Correlations

Hypotrichosis with juvenile macular dystrophy (HJMD) and ectodermal dysplasia, ectrodactyly and macular dystrophy (EEM) are both caused by mutations in the CDH3 gene. In this report, we describe a family with EEM syndrome caused by a novel CDH3 gene mutation and review the mutation spectrum and limb abnormalities in both EEM and HJMD. A protein structure model showing the localization of different mutations causing both syndromes is presented. The CDH3 gene was sequenced and investigation of the mutations performed using a protein structure model. The conservation score was calculated by ConSurf. We identified a novel CDH3 gene mutation, p.G277V, which resides in a conserved residue located on a β-strand in the second cadherin domain. Review of the data on previously published mutations showed intra-familial and inter-familial variations in the severity of the limb abnormalities. Syndactyly was the most consistent clinical finding present in all the patients regardless of mutation type. The results of our study point to a phenotypic continuum between HJMD and EEM. It is important for genetic counseling to keep in mind the possible clinical/phenotypic overlap between these 2 syndromes and to be aware of the possible risk of limb abnormalities in future pregnancies in families with HJMD syndrome. CDH3 gene mutation screening is recommended in patients with both these syndromes as part of the work-up in order to offer appropriate genetic counseling

Overexpression of human glucocerebrosidase containing different-sized leaders.

Gaucher disease results from impaired activity of the lysosomal enzyme glucocerebrosidase. Aiming at overexpressing the human glucocerebrosidase and testing the efficiency of the two in-frame ATGs of its gene in directing synthesis of an active enzyme, it was coupled to the T7 RNA polymerase promoter in a vaccinia virus-derived expression vector (pTM-1). cDNAs containing either one or both ATGs of the glucocerebrosidase mRNA were linked to the T7 polymerase promoter. Recombinant viruses were produced and used for infecting human cells in tissue culture. The results demonstrated that both ATGs directed translation of active glucocerebrosidase, resulting in a 10-fold increase in enzymic activity. Most of the protein remained sensitive to endoglycosidase H. The active enzyme represented a small fraction of the expressed glucocerebrosidase. The recombinant enzyme had the same Km and optimal pH towards the artificial substrate 4-methylumbelliferyl glucopyranoside as the authentic endogenous human enzyme. Measurements of intracellular enzymic activity directed by the cDNAs with either one or both ATGs in cells loaded with a fluorescent glucosylceramide demonstrated a 30% increase in activity directed by the cDNAs containing the first ATG over that containing the second ATG. This indicates that the protein synthesized from the first ATG, with a 38 amino acid leader, is translocated through the endoplasmic reticulum more readily than its counterpart directed by the second ATG, with a 19 amino acid leader. The elevation in glucocerebrosidase activity and the reproducibility of the data leads us to propose the use of the vaccinia virus-derived expression system as a tool for studying glucocerebrosidase mutants in Gaucher disease