LTP in hippocampal neurons is associated with a CaMKII-mediated increase in GluA1 surface expression.

The use of hippocampal dissociated neuronal cultures has enabled the study of molecular changes in endogenous native proteins associated with long-term potentiation. Using immunofluorescence labelling of the active (Thr286-phosphorylated) alpha-Ca(2+) /calmodulin-dependent protein kinase II (CaMKII) we found that CaMKII activity was increased by transient (3 × 1 s) depolarisation in 18- to 21-day-old cultures but not in 9- to 11-day-old cultures. The increase in Thr286 phosphorylation of CaMKII required the activation of NMDA receptors and was greatly attenuated by the CaMKII inhibitor KN-62. We compared the effects of transient depolarisation on the surface expression of GluA1 and GluA2 subunits of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor and found a preferential recruitment of the GluA1 subunit. CaMKII inhibition prevented this NMDA receptor-dependent delivery of GluA1 to the cell surface. CaMKII activation is therefore an important factor in the activity-dependent recruitment of native GluA1 subunit-containing alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors to the cell surface of hippocampal neurons.

Disruption of the interaction between myosin VI and SAP97 is associated with a reduction in the number of AMPARs at hippocampal synapses.

Myosin VI is an actin-based motor protein that is enriched at the postsynaptic density and appears to interact with alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate-type glutamate receptors (AMPARs) via synapse associated protein 97 (SAP97). Here, we find that a Flag epitope-tagged dominant negative construct that inhibits the interaction between SAP97 and myosin VI (Flag-myoVI-DN) causes a dramatic reduction in the number of synapses and the surface expression of AMPARs in cultured hippocampal neurons. Furthermore, we find that Flag-myoVI-DN also prevents the rapid delivery of AMPARs to synapses that can be induced by the transient activation of N-methyl-d-aspartate receptors. The Flag-myoVI-DN induced decrease in surface AMPARs is not because of reduced AMPAR subunit protein synthesis. Using whole-cell recording, we show that Flag-myoVI-DN also prevents the activity-induced increase in miniature excitatory postsynaptic current frequency that is normally associated with recruitment of AMPARs to the cell surface at synaptic sites that lack these receptors (i.e. 'silent' synapses). Together, these results indicate that myosin VI/SAP97 plays an important role in trafficking and activity-dependent recruitment of AMPARs to synapses.

CIC, a member of a novel subfamily of the HMG-box superfamily, is transiently expressed in developing granule neurons.

We describe here the identification and characterization of a new gene, Cic, in both human and mouse genomes. These are orthologs of the Drosophila gene capicua, and represent a new subfamily of the HMG-box superfamily. Expression of the Cic gene is predominantly restricted to immature granule cells in the cerebellum, hippocampus and olfactory bulb in the CNS. This gene is therefore implicated in CNS development, in particular in granule cell development.

Novel strategy to study gene expression and function in developing cerebellar granule cells.

The advent of techniques for global analyses of cell biology, such as genomics and proteomics, opens the way to rapid progress in understanding the molecular control of developing tissues. However, such studies in the CNS are hindered by the complexity of this tissue. In particular, few approaches allow cells to be isolated that are enriched for specific stages of their maturation. We describe a new strategy to study gene expression and function in cerebellar granule cells. In these experiments, we have used square pulse electroporation to introduce fluorescent dye or DNA constructs into immature granule cell precursors in situ. This method only labels granule cell precursors in the superficial part of the external granule layer. Combining this labelling with fluorescent activated cell sorting (FACS) allows the transfected cells to be isolated at any time during their subsequent development, thus providing a means of analysing granule cells as they undergo maturation. This transfection method can be used to study events in the normal maturation of granule cells or the effects of introduced transgenes. Such studies can be carried out on cells purified from primary cultures or cells in situ using cerebellar slice cultures. Our strategy provides a new route to detailed analysis of the role of genes in controlling many aspects of granule cell biology. These approaches will allow recent global analyses to be more readily applied to subpopulations of cells in complex tissues.

Differential expression of SOX4 and SOX11 in medulloblastoma.

Primitive neuroectodermal tumors (PNETs) are composed of immature neuronal precursor cells and sometimes more mature neuronal cell types. Medulloblastomas, occuring in the cerebellum, represent the most common PNET and are broadly classified into two subgroups: classical and desmoplastic. Desmoplastic medulloblastomas exhibit a slightly better prognosis than classical medulloblastomas. However, there are currently no good molecular markers available to distinguish clinical outcome and similar treatment is used for most patients with associated complications. It has been shown that neoplastic cells in these tumors recapitulate stages in maturation of normal human neuroblasts; therefore, embryological studies of the earliest events in the development of the cerebellum may provide useful information about the molecular behavior of the tumor. Transcription factors such as Sox proteins involved in neural development may also play a role in the etiology of brain tumors. Sox4 in particular has been implicated in the biology of several other types of cancer. We have studied the expression of Sox4, and the closely related Sox11 gene, in medulloblastomas. Sox4 and Sox11 were strongly expressed in most classical medulloblastomas but only weakly in desmoplastic medulloblastomas. The expression profile of these two genes in developing cerebellum was also analyzed. Our results suggest that strong Sox4 and Sox11 expression in classical medulloblastomas reflects their maturation-dependent expression during normal cerebellum development, and that they may therefore provide markers to divide tumors into clinically relevant subgroups.