Svjetlana Kalanj Bognar

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.

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.

Neuroplastin Expression in the Hippocampus of Mice Lacking Complex Gangliosides

We report changes in neuroplastin gene and protein expression in the hippocampus of B4galnt1 null mice, which lacks complex ganglioside structures, compared with that of wild-type mice. Neuroplastin mRNA expression was significantly higher in the hippocampi of B4galnt1 null mice than in wild-type mice. Moreover, Western blot analysis shows increased neuroplastin protein levels of neuroplastin-55 isoform in B4galnt1 null hippocampal homogenates. Immunohistochemistry revealed a substantially different distribution of neuroplastin immunoreactivity in sagittal sections of the hippocampi derived from B4galnt1 null in comparison with those from wild-type mice. Most strikingly, B4galnt1 null mice had relatively little neuroplastin immunoreactivity in the pyramidal layer of CA1 and CA3, whereas wild-type mice had strong neuroplastin staining of pyramidal cells. Results of this study support the hypothesis that alterations of brain ganglioside expression influence the expression of neuroplastin. As both neuroplastin and gangliosides have important roles in synaptic transmission, synaptic plasticity, and neurite outgrowth, it will be of particular interest to unravel the molecular mechanisms underlying the relationship between ganglioside composition and neuroplastin transcript and protein expression in the mammalian nervous system.

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 Nptnlox/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 Nptnlox/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.

Role of gangliosides in brain aging and neurodegeneration

Gangliosides are membrane glycosphingolipids bearing sialic acid residues. Within membranes, gangliosides are specifically enriched in highly organized domains, lipid rafts, and are attributed with diverse functions such as intercellular interactions, cell recognition, neurotransmission, and signal transduction. The highest concentration and variability of ganglioside structures are found in the human brain. Specific temporal and regional distribution of brain gangliosides has been reported; moreover, gangliosides may serve as markers of neurodevelopmental stages, aging and neurodegeneration. Brain ganglioside content and composition as well as ganglioside metabolism are altered in Alzheimer’s disease. It appears that the alterations of ganglioside metabolism leading to changes in membrane physico-chemical properties are not merely a consequence of primary pathology, but may also be involved in the early pathogenesis of Alzheimer’s disease through documented effects on APP proteolytic processing and amyloid aggregation. Investigations of glycolipid metabolic alterations which accompany neurodegenerative disorders provide insight into pathogenetic mechanisms and enable recognition of diagnostic markers as well as molecular structures acting as therapeutic tools interfering with cascade of pathological events.

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.