The molecular architecture of the cytomatrix of presynaptic nerve terminals is poorly understood. Here we show that Bassoon, a novel protein of >400,000 M r, is a new component of the presynaptic cytoskeleton. The murine bassoon gene maps to chromosome 9F. A comparison with the corresponding rat cDNA identified 10 exons within its protein-coding region. The Bassoon protein is predicted to contain two double-zinc fingers, several coiled-coil domains, and a stretch of polyglutamines (24 and 11 residues in rat and mouse, respectively). In some human proteins, e.g., Huntingtin, abnormal amplification of such poly-glutamine regions causes late-onset neurodegeneration. Bassoon is highly enriched in synaptic protein preparations. In cultured hippocampal neurons, Bassoon colocalizes with the synaptic vesicle protein synaptophysin and Piccolo, a presynaptic cytomatrix component. At the ultrastructural level, Bassoon is detected in axon terminals of hippocampal neurons where it is highly concentrated in the vicinity of the active zone. Immunogold labeling of synaptosomes revealed that Bassoon is associated with material interspersed between clear synaptic vesicles, and biochemical studies suggest a tight association with cytoskeletal structures. These data indicate that Bassoon is a strong candidate to be involved in cytomatrix organization at the site of neurotransmitter release.

Mutant mice lacking the central region of the presynaptic active zone protein Bassoon were generated to establish the role of this protein in the assembly and function of active zones as sites of synaptic vesicle docking and fusion. Our data show that the loss of Bassoon causes a reduction in normal synaptic transmission, which can be attributed to the inactivation of a significant fraction of glutamatergic synapses. At these synapses, vesicles are clustered and docked in normal numbers but are unable to fuse. Phenotypically, the loss of Bassoon causes spontaneous epileptic seizures. These data show that Bassoon is not essential for synapse formation but plays an essential role in the regulated neurotransmitter release from a subset of glutamatergic synapses.

NMDA (N-methyl-D-aspartate) receptors and calcium can exert multiple and very divergent effects within neuronal cells, thereby impacting opposing occurrences such as synaptic plasticity and neuronal degeneration. The neuronal Ca2+ sensor Caldendrin is a postsynaptic density component with high similarity to calmodulin. Jacob, a recently identified Caldendrin binding partner, is a novel protein abundantly expressed in limbic brain and cerebral cortex. Strictly depending upon activation of NMDA-type glutamate receptors, Jacob is recruited to neuronal nuclei, resulting in a rapid stripping of synaptic contacts and in a drastically altered morphology of the dendritic tree. Jacob's nuclear trafficking from distal dendrites crucially requires the classical Importin pathway. Caldendrin binds to Jacob's nuclear localization signal in a Ca2+-dependent manner, thereby controlling Jacob's extranuclear localization by competing with the binding of Importin-α to Jacob's nuclear localization signal. This competition requires sustained synapto-dendritic Ca2+ levels, which presumably cannot be achieved by activation of extrasynaptic NMDA receptors, but are confined to Ca2+ microdomains such as postsynaptic spines. Extrasynaptic NMDA receptors, as opposed to their synaptic counterparts, trigger the cAMP response element-binding protein (CREB) shut-off pathway, and cell death. We found that nuclear knockdown of Jacob prevents CREB shut-off after extrasynaptic NMDA receptor activation, whereas its nuclear overexpression induces CREB shut-off without NMDA receptor stimulation. Importantly, nuclear knockdown of Jacob attenuates NMDA-induced loss of synaptic contacts, and neuronal degeneration. This defines a novel mechanism of synapse-to-nucleus communication via a synaptic Ca2+-sensor protein, which links the activity of NMDA receptors to nuclear signalling events involved in modelling synapto-dendritic input and NMDA receptor–induced cellular degeneration.

cDNA clones encoding proteins related to the aggrecan/versican family of proteoglycan core proteins have been isolated with antisera against rat brain synaptic junctions. Two sets of overlapping cDNAs have been characterized that differ in their 3-terminal regions. Northern analyses with probes derived from unique regions of each set were found to hybridize with two brain-specific transcripts of 3.3 and 3.6 kilobases (kb). The 3.6-kb transcript encodes a polypeptide that exhibits 82% sequence identity with bovine brevican and is thought to be the rat ortholog of brevican. Interestingly, the polypeptide deduced from the open reading frame of the 3.3-kb transcript is truncated just carboxyl-terminal of the central domain of brevican and instead contains a putative glypiation signal. Antibodies raised against a bacterially expressed glutathione S-transferase-brevican fusion protein have been used to show that both soluble and membrane-bound brevican isoforms exist. Treatment of the crude membrane fraction and purified synaptic plasma membranes with phosphatidylinositolspecific phospholipase C revealed that isoforms of brevican are indeed glycosylphosphatidylinositol-anchored to the plasma membrane. Moreover, digestions with chondroitinase ABC have indicated that rat brevican, like its bovine ortholog, is a conditional chondroitin sulfate proteoglycan. Immunohistochemical studies have shown that brevican is widely distributed in the brain and is localized extracellularly. During postnatal development, amounts of both soluble and phosphatidylinositol-specific phospholipase C-sensitive isoforms increase, suggesting a role for brevican in the terminally differentiating and the adult nervous system.

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Neuroplastins are essential for learning and memory. Retrograde amnesia after an associative learning task can be induced by ablation of the neuroplastin gene. The inducible neuroplastin-deficient mouse model provides a new and unique means to analyze the molecular and cellular mechanisms underlying retrograde amnesia and memory.

Olfactory recognition memory was tested in adult male mice using a social discrimination task. The testing was conducted to begin to characterize the role of protein synthesis and the specific brain regions associated with activity in this task. Long-term olfactory recognition memory was blocked when the protein synthesis inhibitor anisomycin was injected 20 min before, immediately after, or 6 h after sampling. No effect was observed when anisomycin was administered 3 h or 18 h after sampling. Immunohistochemical analysis of Fos expression revealed that sampling-like exposure to a juvenile increased the activity of a subset of cells in the accessory olfactory bulb and the brain areas that are associated with it. Additionally, increased Fos expression was measured in the main olfactory bulb and the piriform cortex, whereas no signs of activation were seen in the cortical nucleus of the amygdala, all components of the main olfactory system. No increases in Fos immunoreactivity were observed after 4 h. Our data suggest that long-lasting olfactory recognition memory requires two stages of protein synthesis. The first stage takes place within 1–2 h and the second stage between 6–7 h after sampling. The first but not the second stage is paralleled by an increase in the number of Fos-immunoreactive cells in brain areas associated with both the main and accessory olfactory systems. It therefore appears that the role of the second stage of protein synthesis in recognition memory depends on the integrity of the first stage of protein synthesis

Mutations in the ARHGEF6 gene, encoding the guanine nucleotide exchange factor αPIX/Cool-2 for the Rho GTPases Rac1 and Cdc42, cause X-linked intellectual disability (ID) in humans. We show here that αPix/Arhgef6 is primarily expressed in neuropil regions of the hippocampus. To study the role of αPix/Arhgef6 in neuronal development and plasticity and gain insight into the pathogenic mechanisms underlying ID, we generated αPix/Arhgef6-deficient mice. Gross brain structure in these mice appeared to be normal; however, analysis of Golgi-Cox-stained pyramidal neurons revealed an increase in both dendritic length and spine density in the hippocampus, accompanied by an overall loss in spine synapses. Early-phase long-term potentiation was reduced and long-term depression was increased in the CA1 hippocampal area of αPix/Arhgef6-deficient animals. Knockout animals exhibited impaired spatial and complex learning and less behavioral control in mildly stressful situations, suggesting that this model mimics the human ID phenotype. The structural and electrophysiological alterations in the hippocampus were accompanied by a significant reduction in active Rac1 and Cdc42, but not RhoA. In conclusion, we suggest that imbalance in activity of different Rho GTPases may underlie altered neuronal connectivity and impaired synaptic function and cognition in αPix/Arhgef6 knockout mice.