Extracellular matrix alterations in the central nervous system of a mouse model of diastrophic dysplasia

Mutations in the diastrophic dysplasia sulfate transporter (DTDST or SLC26A2) gene cause a spectrum of recessively inherited chondrodysplasias of variable severity (1). The gene encodes for a sulfate/chloride membrane antiporter, crucial for the uptake of extracellular sulfate necessary for proteoglycan sulfation, that is expressed in cartilage but also in other tissues, including the nervous tissue. A recently generated Dtdst knock-in mouse (dtd mouse), with a non-lethal defect in the sulfation pathway causing cartilage undersulfation, reproduces the skeletal phenotype of human DTD and is a useful model to study DTDST disorders (2). Chondroitin sulfate proteoglycans (CSPGs) are widely expressed in the central nervous system, both dispersed in the neuropil and concentrated in perineuronal nets (PNNs) surrounding specific subsets of neurons (3), and play several important regulatory and structural functions in normal and pathological conditions (4, 5). In particular, PNNs are involved in the regulation of ionic homeostasis in the pericellular microenvironment of highly active neurons and may be implicated in the stabilization of synaptic contacts and in the limitation of neuroplasticity (6). Aim of our study was therefore to verify whether the sulfation defect present in dtd mice can cause abnormalities also in proteoglycans expressed in the central nervous system. Adult (P60) dtd mice (n = 5) and their wild-type littermates were perfused with paraformaldehyde. Macroscopic inspection of brains removed from the skulls showed reduction in size of dtd brains compared to wild-type ones. Coronal vibratome and paraffin sections from brains and spinal cords were processed for the morphological, cytochemical and immunocytochemical analysis. Thionin staining showed minor cytoarchitectural alterations in dtd mice brains, but allowed the detection of several darkly stained neurons, reminiscent of necrotic cells, in the cerebral cortex and hippocampus. The general distribution of CSPGs was visualized by lectin-cytochemical staining with biotinylated Wisteria floribunda agglutinin (WFA), which selectively binds to N-acetylgalactosamine residues of chondroitin sulfate glycosaminoglycans (CS-GAGs) and consistently labels PNNs (7); immunocytochemistry was used to detect the protein cores of selected CSPGs (aggrecan; brevican; phosphacan/RPTPζ/β) and their GAGs moieties. Double immunofluorescence was used to visualize simultaneously CSPGs and neuronal or glial markers. The observation of coronal brain and spinal cord sections showed a consistent and generalized reduction in the labeling intensity for all the tested CSPGs markers in samples from the dtd mice compared to the wild-type littermates. In particular, PNNs were less numerous in the cingulate and sensorimotor cerebral cortex, in the hippocampus, in the reticular thalamic nucleus and in the spinal cord. A moderate increase in astrogliosis, revealed by labeling for glial fibrillary acid protein, was detected in dtd mice brains. Since the majority of PNNs in the rodent brain envelop a subset of inhibitory neurons containing the calcium-binding protein parvalbumin (PV) (7), we performed double-labeling experiments to verify whether PV-neurons were also affected by the mutation. In the brains of dtd mice we found that PV-positive neurons showed alterations consisiting in: irregular laminar position in the cerebral cortex; increased somatic labeling; decreased labeling in synaptic terminals. Conversely, a higher density of synaptic terminals was detected in dtd mice brains using synaptophysin as a marker of both inhibitory and excitatory synapses. Collectively our results strongly suggest that the undersulfation caused by the Dtdst mutation impairs the formation of normal CSPGs in the central nervous system. Supported by FIRST (University of Milan, to SDB) and Telethon-Italy (grant n. GGP06076, to AR). 1) Rossi, A. and Superti-Furga, A. (2001). Mutations in the diastrophic dysplasia sulfate transporter (DTDST) gene: 22 novel mutations, mutation review, associated skeletal phenotypes, and diagnostic relevance. Human Mutation, 17, 159-161. 2) Forlino, A., Piazza, R., Tiveron, C., Della Torre, S., Tatangelo, L., Bonafè, L., Gualeni, B., Romano, A., Pecora, F., Superti-Furga, A., Cetta, G., Rossi, A. (2005) A diastrophic dysplasia sulfate transporter (SLC26A2) mutant mouse: morphological and biochemical characterization of the resulting chondrodysplasia phenotype. Hum. Mol. Genet., 14, 859-871. 3) Celio, M.R., Spreafico, R., De Biasi, S., Vitellaro-Zuccarello, L. (1998) Perineuronal nets: past and present. Trends Neurosci, 21, 510-515. 4) Brückner, G., Härtig, W., Kacza, J., Seeger, G., Welt., K, Brauer, K. (1996 b) Extracellular matrix organization in various regions of rat brain grey matter. J. Neurocytol., 25, 333-346. 5) Brückner, G., Hausen, D., Härtig, W., Drlicek, M., Arendt, T., Brauer, K. (1999) Cortical areas abundant in extracellular matrix chondroitin sulphate proteoglycans are less affected by cytoskeletal changes in Alzheimer’s disease. Neuroscience, 92, 791–805. 6) Pizzorusso, T., Medini, P., Berardi, N., Chierzi, S., Fawcett, J.W., Maffei, L. (2002) Reactivation of ocular dominance plasticity in the adult visual cortex. Science, 298, 1248-1251. 7) Härtig, W., Brauer, K., Brückner, G. (1992) Wisteria floribunda agglutinin-labelled nets surround parvalbumin-containing neurons. Neuroreport, 3, 869-872.