Neuroplastin deletion in glutamatergic neurons impairs selective brain functions and calcium regulation: implication for cognitive deterioration.

The cell adhesion molecule neuroplastin (Np) is a novel candidate to influence human intelligence. Np-deficient mice display complex cognitive deficits and reduced levels of Plasma Membrane Ca2+ ATPases (PMCAs), an essential regulator of the intracellular Ca2+ concentration ([iCa2+]) and neuronal activity. We show abundant expression and conserved cellular and molecular features of Np in glutamatergic neurons in human hippocampal-cortical pathways as characterized for the rodent brain. In Nptn lox/loxEmx1Cre mice, glutamatergic neuron-selective Np ablation resulted in behavioral deficits indicating hippocampal, striatal, and sensorimotor dysfunction paralleled by highly altered activities in hippocampal CA1 area, sensorimotor cortex layers I-III/IV, and the striatal sensorimotor domain detected by single-photon emission computed tomography. Altered hippocampal and cortical activities correlated with reduction of distinct PMCA paralogs in Nptn lox/loxEmx1Cre mice and increased [iCa2+] in cultured mutant neurons. Human and rodent Np enhanced the post-transcriptional expression of and co-localized with PMCA paralogs in the plasma membrane of transfected cells. Our results indicate Np as essential for PMCA expression in glutamatergic neurons allowing proper [iCa2+] regulation and normal circuit activity. Neuron-type-specific Np ablation empowers the investigation of circuit-coded learning and memory and identification of causal mechanisms leading to cognitive deterioration.

Structural analysis of brain ganglioside acetylation patterns in mice with altered ganglioside biosynthesis.

Gangliosides are sialylated membrane glycosphingolipids especially abundant in mammalian brain tissue. Sialic acid O-acetylation is one of the most common structural modifications of gangliosides which considerably influences their chemical properties. In this study, gangliosides extracted from brain tissue of mice with altered ganglioside biosynthesis (St8sia1 null and B4galnt1 null mice) were structurally characterized and their acetylation pattern was analyzed. Extracted native and alkali-treated gangliosides were resolved by high performance thin layer chromatography. Ganglioside mixtures as well as separated individual ganglioside fractions were further analyzed by tandem mass spectrometry. Several O-acetylated brain ganglioside species were found in knockout mice, not present in the wild-type mice. To the best of our knowledge this is the first report on the presence of O-acetylated GD1a in St8sia1 null mice and O-acetylated GM3 species in B4galnt1 null mice. In addition, much higher diversity of abnormally accumulated brain ganglioside species regarding the structure of ceramide portion was observed in knockout versus wild-type mice. Obtained findings indicate that the diversity of brain ganglioside structures as well as acetylation patterns in mice with altered ganglioside biosynthesis, is even higher than previously reported. Further investigation is needed in order to explore the effects of acetylation on ganglioside interactions with other molecules and consequently the physiological role of acetylated ganglioside species.

Cholesterogenic genes expression in brain and liver of ganglioside-deficient mice.

The aim of this study was to determine the effect of changed ganglioside profile on transcription of selected genes involved in cholesterol homeostasis. For that purpose, the expression of 11 genes related to cholesterol synthesis, regulation, and cholesterol transport was investigated in selected brain regions (frontal cortex, hippocampus, brain stem, cerebellum) and liver of St8sia1 knockout (KO) mice characterized by deficient synthesis of b- and c-series gangliosides and accumulation of a-series gangliosides. The expression of majority of the analyzed genes, as determined using quantitative real time PCR, was slightly higher in St8sia1 KO compared to wild-type (wt) controls. More prominent changes were observed in Hmgr, Cyp51, and Cyp46 expression in brain (hippocampus and brain stem) and Srebp1a, Insig2a, and Ldlr in liver. In addition, the expression of master transcriptional regulators, Srebp1a, Srebp1c, and Insig2a, as well as transporters Ldlr and Vldlr differed between liver and brain, and within brain regions in wt animals. Cyp46 expression was expectedly brain-specific, with brain region difference in both wt and St8sia1 KO. The established change in transcriptome of cholesterogenic genes is associated to specific alteration of ganglioside composition which indicates relationship between gangliosides and regulation of cholesterol metabolism.

Arylsulfatase a gene polymorphisms in relapsing remitting multiple sclerosis: genotype-phenotype correlation and estimation of disease progression.

Arylsulfatase A (ASA) is a lysosomal enzyme involved in catabolism of cerebroside-sulfate, major lipid constituent of oligodendrocyte membranes. Various polymorphisms in ASA gene have been described, leading to different levels of enzyme deficiency. Progressive demyelination occurs in metachromatic leukodystrophy (MLD), while the condition of ASA-pseudodeficiency (ASA-PD) is suggested to contribute to complex pathogenesis of multiple sclerosis (MS). This work presents usefulness of genotype-phenotype correlation in estimation of disease severity and progression. The presence of two most common mutations associated with ASA-PD was analyzed in 56 patients with diagnosis of relapsing-remitting multiple sclerosis, by polymerase chain reaction restriction fragment length polymorphism method. In MS patients confirmed as ASA-PD mutations carriers, arylsulfatase activity was determined in leukocyte homogenates by spectrophotometry. To determine whether there is a difference between disability level and/or disease progression in patients with or without mutations we have estimated disability level using Expanded disability status scale (EDSS) and disease progression using Multiple sclerosis severity score (MSSS). Correlation of genotypes and disease progression was statistically analyzed by Kruskal-Wallis test. Patients showing higher MSSS score and found to be carriers of both analyzed ASA-PD mutations were additionally examined using conventional magnetic resonance (MR) techniques. The presence of either one or both mutations was determined in 13 patients. Lower ASA activities were observed in all MS patients carrying the mutations. Nine of the mutations carriers had mild disability (EDSS=0-4.0), 1 had moderate disability (EDSS=4.5-5.5), and 3 had severe disability (EDSS > or = 6.0). On the other hand, only 3 MS patients who were mutation carriers showed MSSS values lower than 5.000 while in other MS patients-mutation carriers the MSSS values ranged from 5.267 to 9.453. Comparison of MR findings between MS patients, mutations carrier vs. non-carrier, matched for sex, age and disease duration, showed that the total number of lesions and the number of hypointense lesions on T1-weighted images was greater in MS patient carrying the ASA-PD mutations. Our results on genotype-phenotype correlation analysis indicate a possible contribution of detected arylsulfatase A gene polymorphisms to the clinical severity of multiple sclerosis, estimated by EDSS, MSSS and MR findings. The MSSS proved to be more appropriate indicator of disease progression and should be more frequently used in clinical practice especially for comparison of disease progression in different groups of patients and identification of factors that may influence disease progression such as the presence of gene polymorphisms.

Serum gangliosides in patients with brain tumors.

In order to determine possible differences in serum gangliosides content and composition before and after surgical removal of tumor, gangliosides isolated from preoperative and postoperative sera of patients with brain tumors were analyzed. Serum samples were collected from patients with glioblastoma, meningioma, acoustic neurinoma, haemangioma, oligodendroglioma and astrocytoma, one week before and one week after surgical removal of the tumor. Serum gangliosides were qualitatively and quantitatively analyzed by high performance thin layer chromatography and laser densitometry. Results showed changes of total gangliosides concentrations in analyzed postoperative sera comparing to preoperative sera. There was not a significant difference in ganglioside pattern of preoperative vs. preoperative sera. However, a postoperative decreased proportion of ganglioside GD3 was observed in sera derived from patients with complete tumor removal. The results of this study indicate that comparative quantitative and compositional analysis of both preoperative and postoperative serum gangliosides may provide useful information concerning tumor progression, surgical success and prognosis.

Kinetics and activity of arylsulfatase A in leukocytes derived from patients with cerebral palsy.

Activity and kinetics of arylsulfatase A (ASA, EC 3.1.6.8) were analyzed in leukocyte homogenates derived from patients suffering from cerebral palsy. Lower ASA activity was found in the patients' leukocytes than in controls, as determined by spectrophotometry using chromogenic substrate p-nitrocatechol sulfate (p-NCS). Kinetic parameters, K(m) and v(max), for leukocyte ASA were determined from the dependence of initial reaction velocities on the p-NCS concentrations. A slight difference in K(m) values was found for leukocyte enzyme in cerebral palsy (0.26 mmol L(-1)) compared to the control (0.21 mmol L(-1)), whereas v(max) value for leukocyte ASA in disease reached only 58% of the control value. In addition, the presence of the most common mutations associated with ASA pseudo-deficiency (N350S, 1524+95 A>G) and metachromatic leukodystrophy (P426L) was detected in all investigated patients. Changes in activity and kinetic parameters of leukocyte ASA in cerebral palsy are most probably related to the decrease of enzyme concentration; the detected mutations might at least partially contribute to the observed changes.

Croatian population data for arylsulfatase a pseudodeficiency-associated mutations in healthy subjects, and in patients with Alzheimer-type dementia and Down syndrome.

BACKGROUND: Arylsulfatase A (ASA) is a lysosomal enzyme involved in catabolism of cerebroside sulfate, whose deficiency causes metachromatic leukodystrophy, a rare autosomal recessive disorder characterized by storage of cerebroside sulfate, mainly in the nervous system. Low ASA activities have also been reported in healthy individuals and several neuropsychiatric disorders due to the condition termed ASA pseudodeficiency. The aim of this study was to establish frequency of two mutations associated with ASA pseudodeficiency in healthy individuals in the Croatian population as well as in persons with Alzheimer-type dementia and Down syndrome. METHODS: Presence of N350S and 1524+95 A-->G pseudodeficiency mutations was detected in genomic DNA extracted from leukocytes of healthy subjects (n = 125) and of patients with Alzheimer-type dementia (n = 18) and Down syndrome (n = 21). Arylsulfatase A activity was measured in leukocyte homogenates by spectrophotometry (lambda = 515 nm) using p-nitrocatechol sulfate as chromogenic substrate. RESULTS: Frequency of N350S mutation and mutation 1524+95 A-->G was estimated at 6.8 and 2.8% for healthy controls, 11 and 5.5% for Alzheimer-type dementia, and 12 and 9.5% for Down syndrome, respectively. Arylsulfatase A activity was slightly but not significantly decreased in leukocytes derived from subjects with dementia and Down syndrome in comparison with age-matched control samples. CONCLUSIONS: Frequency of two mutations associated with ASA pseudodeficiency in the Croatian population is slightly below the range reported for other populations. Additionally, despite the proposed role of arylsulfatase A pseudodeficiency as one of the predisposing factors for neuropsychiatric disorders, our preliminary results did not show significantly higher frequencies of either mutation in Alzheimer-type dementia or Down syndrome.