Bassoon controls synaptic vesicle release via regulation of presynaptic phosphorylation and cAMP.

Neuronal presynaptic terminals contain hundreds of neurotransmitter-filled synaptic vesicles (SVs). The morphologically uniform SVs differ in their release competence segregating into functional pools that differentially contribute to neurotransmission. The presynaptic scaffold bassoon is required for neurotransmission, but the underlying molecular mechanisms are unknown. We report that glutamatergic synapses lacking bassoon feature decreased SV release competence and increased resting pool of SVs as assessed by imaging of SV release in cultured neurons. CDK5/calcineurin and cAMP/PKA presynaptic signalling are dysregulated, resulting in an aberrant phosphorylation of their downstream effectors synapsin1 and SNAP25, well-known regulators of SV release competence. An acute pharmacological restoration of physiological CDK5 and cAMP/PKA activity fully normalises the SV pools in neurons lacking bassoon. Finally, we demonstrate that CDK5-dependent regulation of PDE4 activity interacts with cAMP/PKA signalling and thereby controls SV release competence. These data reveal that bassoon organises SV pools in glutamatergic synapses via regulation of presynaptic phosphorylation and cAMP homeostasis and indicate a role of CDK5/PDE4/cAMP axis in the control of neurotransmitter release.

Bassoon inhibits proteasome activity via interaction with PSMB4.

Proteasomes are protein complexes that mediate controlled degradation of damaged or unneeded cellular proteins. In neurons, proteasome regulates synaptic function and its dysfunction has been linked to neurodegeneration and neuronal cell death. However, endogenous mechanisms controlling proteasomal activity are insufficiently understood. Here, we describe a novel interaction between presynaptic scaffolding protein bassoon and PSMB4, a ß subunit of the 20S core proteasome. Expression of bassoon fragments that interact with PSMB4 in cell lines or in primary neurons attenuates all endopeptidase activities of cellular proteasome and induces accumulation of several classes of ubiquitinated and non-ubiquitinated substrates of the proteasome. Importantly, these effects are distinct from the previously reported impact of bassoon on ubiquitination and autophagy and might rely on a steric interference with the assembly of the 20S proteasome core. In line with a negative regulatory role of bassoon on endogenous proteasome we found increased proteasomal activity in the synaptic fractions prepared from brains of bassoon knock-out mice. Finally, increased activity of proteasome and lower expression levels of synaptic substrates of proteasome could be largely normalized upon expression of PSMB4-interacting fragments of bassoon in neurons derived from bassoon deficient mice. Collectively, we propose that bassoon interacts directly with proteasome to control its activity at presynapse and thereby it contributes to a compartment-specific regulation of neuronal protein homeostasis. These findings provide a mechanistic explanation for the recently described link of bassoon to human diseases associated with pathological protein aggregation. Presynaptic cytomatrix protein bassoon (Bsn) interacts with PSMB4, the ß7 subunit of 20S core proteasome, via three independent interaction interfaces. Bsn inhibits proteasomal proteolytic activity and degradation of different classes of proteasomal substrates presumably due to steric interference with the assembly of 20S core of proteasome. Upon Bsn deletion in neurons, presynaptic substrates of the proteasome are depleted, which can be reversed upon expression of PSMB4-interacting interfaces of Bsn. Taken together, bsn controls the degree of proteasome degradation within the presynaptic compartment and thus, contributes to the regulation of synaptic proteome.

Aberrant neuronal activity-induced signaling and gene expression in a mouse model of RASopathy.

Noonan syndrome (NS) is characterized by reduced growth, craniofacial abnormalities, congenital heart defects, and variable cognitive deficits. NS belongs to the RASopathies, genetic conditions linked to mutations in components and regulators of the Ras signaling pathway. Approximately 50% of NS cases are caused by mutations in PTPN11. However, the molecular mechanisms underlying cognitive impairments in NS patients are still poorly understood. Here, we report the generation and characterization of a new conditional mouse strain that expresses the overactive Ptpn11D61Y allele only in the forebrain. Unlike mice with a global expression of this mutation, this strain is viable and without severe systemic phenotype, but shows lower exploratory activity and reduced memory specificity, which is in line with a causal role of disturbed neuronal Ptpn11 signaling in the development of NS-linked cognitive deficits. To explore the underlying mechanisms we investigated the neuronal activity-regulated Ras signaling in brains and neuronal cultures derived from this model. We observed an altered surface expression and trafficking of synaptic glutamate receptors, which are crucial for hippocampal neuronal plasticity. Furthermore, we show that the neuronal activity-induced ERK signaling, as well as the consecutive regulation of gene expression are strongly perturbed. Microarray-based hippocampal gene expression profiling revealed profound differences in the basal state and upon stimulation of neuronal activity. The neuronal activity-dependent gene regulation was strongly attenuated in Ptpn11D61Y neurons. In silico analysis of functional networks revealed changes in the cellular signaling beyond the dysregulation of Ras/MAPK signaling that is nearly exclusively discussed in the context of NS at present. Importantly, changes in PI3K/AKT/mTOR and JAK/STAT signaling were experimentally confirmed. In summary, this study uncovers aberrant neuronal activity-induced signaling and regulation of gene expression in Ptpn11D61Y mice and suggests that these deficits contribute to the pathophysiology of cognitive impairments in NS.

Correction: Aberrant neuronal activity-induced signaling and gene expression in a mouse model of RASopathy.

[This corrects the article DOI: 10.1371/journal.pgen.1006684.].

Lesion of the hippocampus selectively enhances LEC's activity during recognition memory based on familiarity.

The sense of familiarity for events is crucial for successful recognition memory. However, the neural substrate and mechanisms supporting familiarity remain unclear. A major controversy in memory research is whether the parahippocampal areas, especially the lateral entorhinal (LEC) and the perirhinal (PER) cortices, support familiarity or whether the hippocampus (HIP) does. In addition, it is unclear if LEC, PER and HIP interact within this frame. Here, we especially investigate if LEC and PER's contribution to familiarity depends on hippocampal integrity. To do so, we compare LEC and PER neural activity between rats with intact hippocampus performing on a human to rat translational task relying on both recollection and familiarity and rats with hippocampal lesions that have been shown to then rely on familiarity to perform the same task. Using high resolution Immediate Early Gene imaging, we report that hippocampal lesions enhance activity in LEC during familiarity judgments but not PER's. These findings suggest that different mechanisms support familiarity in LEC and PER and led to the hypothesis that HIP might exert a tonic inhibition on LEC during recognition memory that is released when HIP is compromised, possibly constituting a compensatory mechanism in aging and amnesic patients.