In vitro and in vivo models of Huntington's disease show alterations in the endocannabinoid system

In this study, we analyzed the components of the endocannabinoid system (ECS) in R6/2 mice, a widely used model of Huntington's disease (HD). We measured the endogenous content of N-arachidonoylethanolamine and 2-arachidonoylglycerol and the activity of their biosynthetic enzymes (N-acyl-phosphatidylethanolamine-hydrolyzing phospholipase D and diacylglycerol lipase, respectively) and hydrolytic enzymes [fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase, respectively] and of their target receptors (type 1 cannabinoid receptor, type 2 cannabinoid receptor, and transient receptor potential vanilloid-1) in the brains of wild-type and R6/2 mice of different ages, as well as in the striatum and cortex of 12-week-old animals. In addition, we measured FAAH activity in lymphocytes of R6/2 mice. In the whole brains of 12-week-old R6/2 mice, we found reductions in N-acyl-phosphatidylethanolamine-hydrolyzing phospholipase D activity, diacylglycerol lipase activity and cannabinoid receptor binding, mostly associated with changes in the striatum but not in the cortex, as well as an increase in 2-arachidonoylglycerol content as compared with wild-type littermates, without any other change in ECS elements. Then, our analysis was extended to HD43 cells, an inducible cellular model of HD derived from rat ST14A cells. In both induced and noninduced conditions, we demonstrated a fully functional ECS. Overall, our data suggest that the ECS is differently affected in mouse and human HD, and that HD43 cells are suitable for high-throughput screening of FAAH-oriented drugs affecting HD progression

In vitro and in vivo models of Huntington's disease show alterations in the endocannabinoid system

In this study, we analyzed the components of the endocannabinoid system (ECS) in R6/2 mice, a widely used model of Huntington's disease (HD). We measured the endogenous content of N-arachidonoylethanolamine and 2-arachidonoylglycerol and the activity of their biosynthetic enzymes (N-acyl-phosphatidylethanolamine-hydrolyzing phospholipase D and diacylglycerol lipase, respectively) and hydrolytic enzymes [fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase, respectively] and of their target receptors (type 1 cannabinoid receptor, type 2 cannabinoid receptor, and transient receptor potential vanilloid-1) in the brains of wild-type and R6/2 mice of different ages, as well as in the striatum and cortex of 12-week-old animals. In addition, we measured FAAH activity in lymphocytes of R6/2 mice. In the whole brains of 12-week-old R6/2 mice, we found reductions in N-acyl-phosphatidylethanolamine-hydrolyzing phospholipase D activity, diacylglycerol lipase activity and cannabinoid receptor binding, mostly associated with changes in the striatum but not in the cortex, as well as an increase in 2-arachidonoylglycerol content as compared with wild-type littermates, without any other change in ECS elements. Then, our analysis was extended to HD43 cells, an inducible cellular model of HD derived from rat ST14A cells. In both induced and noninduced conditions, we demonstrated a fully functional ECS. Overall, our data suggest that the ECS is differently affected in mouse and human HD, and that HD43 cells are suitable for high-throughput screening of FAAH-oriented drugs affecting HD progression

Divergent phenotype of two siblings human leukocyte antigen identical, affected by nonclassical and classical congenital adrenal hyperplasia caused by 21-hydroxylase deficiency

Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive disorders most often caused by enzyme 21-hydroxylase deficiency. Most mutations causing enzymatic deficiency are generated by recombinations between the active gene CYP21 and the pseudogene CYP21P. Only 1-2% of affected alleles result from spontaneous mutations. The phenotype of CAH varies greatly, usually classified as classical or nonclassical, depending on variable degree in 21-hydroxylase activity. Here we report a divergent phenotype of two human leukocyte antigen identical siblings, affected by nonclassical and classical CAH caused by 21-hydroxylase deficiency due to different genotype

Functional and structural analysis of four novel mutations of CYP21A2 gene in italian patients with 21-hydroxylase deficiency

Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder mainly caused by defects in the 21-hydroxylase gene (CYP21A2), coding for the enzyme 21-hydroxylase (21-OH). About 95% of the mutations arise from gene conversion between CYP21A2 and the inactive pseudogene CYP21A1P: only 5% are novel CYP21A2 mutations, in which functional analysis of mutant enzymes has been helpful to correlate genotype-phenotype. In the present study, we describe 3 novel point mutations (p.L122P, p.Q481X, and p.E161X) in 3 Italian patients with CAH: the fourth mutation (p.M150R) was found in the carrier state. Molecular modeling suggests a major impact on 21-hydroxylase activity, and functional analysis after expression in COS-7 cells confirms reduced enzymatic activity of the mutant enzymes. Only the p.M150R mutation affected the activity to a minor extent, associated with NC CAH. CYP21A2 genotyping and functional characterization of each disease-causing mutation has relevance both for treatment and genetic counseling to the patients

The endocannabinoid system shows different alterations in in vitro and in vivo models of Huntington’s disease

Introduction: In this investigation we analyzed the main components of the so-called “endocannabinoid system” (ECS) in R6/2 mice, a widely used model of Huntington’s disease (HD). Methods: We measured the endogenous content of anandamide (AEA) and 2-arachidonoylglycerol (2-AG), of their biosynthetic (NAPE-PLD and DAGL, respectively) and hydrolytic enzymes (FAAH and MAGL, respectively), and of their target receptors (CB1, CB2 and TRPV1) in the brain of wild-type and R6/2 mice of different ages. In addition, we measured FAAH activity in lymphocytes of R6/2 mice, in order to evaluate whether central ECS alterations were mirrored by peripheral cells. Results: In 12-week-old R6/2 mice we found a reduction of NAPE-PLD and DAGL activity, and of CB binding, as well as an increase in 2-AG content when compared to wild-type littermates, without any other change in ECS elements. Our analysis was extended to HD43 cells, an inducible cellular model of HD derived from rat ST14A cells. In both induced and non-induced conditions we demonstrated a fully functional ECS, and we showed that HD43 cells replicate the decrease in FAAH activity (half of that measured in ST14A cells) already observed in human brain and lymphocytes of HD patients. Conclusions: Overall, our data suggest that ECS is differently affected in mouse and human HD, and that HD43 cells are suitable for high throughput screening of FAAH-oriented drugs affecting HD progression. [...

Cerebrospinal fluid levels of the endocannabinoid anandamide are reduced in patients with untreated newly diagnosed temporal lobe epilepsy.

PURPOSE: The endocannabinoid system is involved in excitatory/inhibitory balance mechanisms within the central nervous system (CNS). Growing evidence shows that its perturbation leads to development of epileptic seizures in experimental models, thus indicating that endocannabinoids play an intrinsic protective role in suppressing pathologic neuronal excitability. Experimental data also demonstrate that the endocannabinoid anandamide (AEA) can antagonize epileptic discharges in hippocampal tissue. The objective of our study was to measure endocannabinoids levels in the cerebrospinal fluid (CSF) of drug-naive patients affected by temporal lobe epilepsy (TLE). METHODS: We measured the levels of both AEA and the other endocannabinoid, 2-arachidonoylglycerol (2-AG), in the CSF of drug-naive patients with TLE. RESULTS: A significant reduction of AEA was found in the CSF of patients with compared with healthy controls (epileptic patients = 2.55 +/- 1.78 pmol/ml; healthy controls = 11.65 +/- 7.53 pmol/ml; n = 9 for both groups, p < 0.01). 2-AG levels, however, were not affected (epileptic patients = 209.5 +/- 146.56; healthy controls = 159.6 +/- 110.2) (n = 6 for both groups, p = 0.48).DISCUSSION: Our findings seem to be consistent with experimental evidence demonstrating a significant prevention of epileptic seizures induced by endocannabinoids in models of epilepsy. Furthermore, they support the hypothesis that AEA may be involved in its pathogenesis, suggesting a hypothetical primary impairment of the endocannabinoid system in untreated TLE. The actual role of this in vivo dysregulation still remains unclear

Missense mutations in the TGM2 gene encoding transglutaminase 2 are found in patients with early-onset type 2 diabetes

Transglutaminase 2 (TG2 or TGM2) is a multi-functional enzyme which catalyzes transamidation reactions or acts as a G-protein in intracellular signalling. Tgm2-/- Mice lacking TG2 activity are glucose intolerant and show impairment of insulin secretion, suggesting an important physiological role for TG2 in the pancreatic beta cell. We have previously described a TGM2 heterozygous missense mutation ((c.998A>G, p.N333S) in a 14 year-old patient with insulin-treated diabetes and in his diabetic father. The aim of this study was to further investigate the role of TG2 in early-onset type 2 diabetes. We analysed the TGM2 gene in 205 patients with clinically defined Maturity Onset Diabetes of the Young (MODY) or early-onset type 2 diabetes. We found two novel heterozygous mutations (c.989T>G, p.M330R; c.992T>A, p.I331N), which were not detected in 300 normoglycemic controls. All mutations were in residues which are located close to the catalytic site and impaired transamidating activity in vitro. Gene expression of TGM family genes and localization of TG2 in normal human pancreas indicated that TG2 is the only transglutaminase significantly expressed in human pancreatic islet cells. We conclude that reduced TG2 activity can contribute to disorders of glucose metabolism possibly via an impairment of insulin secretion

Missense mutations in the TGM2 gene encoding transglutaminase 2 are found in patients with early-onset type 2 diabetes. Mutation in brief no. 982. Online

Transglutaminase 2 (TG2 or TGM2) is a multi-functional enzyme which catalyzes transamidation reactions or acts as a G-protein in intracellular signalling. Tgm2-/- Mice lacking TG2 activity are glucose intolerant and show impairment of insulin secretion, suggesting an important physiological role for TG2 in the pancreatic beta cell. We have previously described a TGM2 heterozygous missense mutation ((c.998A>G, p.N333S) in a 14 year-old patient with insulin-treated diabetes and in his diabetic father. The aim of this study was to further investigate the role of TG2 in early-onset type 2 diabetes. We analysed the TGM2 gene in 205 patients with clinically defined Maturity Onset Diabetes of the Young (MODY) or early-onset type 2 diabetes. We found two novel heterozygous mutations (c.989T>G, p.M330R; c.992T>A, p.I331N), which were not detected in 300 normoglycemic controls. All mutations were in residues which are located close to the catalytic site and impaired transamidating activity in vitro. Gene expression of TGM family genes and localization of TG2 in normal human pancreas indicated that TG2 is the only transglutaminase significantly expressed in human pancreatic islet cells. We conclude that reduced TG2 activity can contribute to disorders of glucose metabolism possibly via an impairment of insulin secretion