The PI3K-dependent activation of AKT results in the inhibition of GSK3β in most signaling pathways. These kinases regulate multiple neuronal processes including the control of synapse number as shown for Drosophila and rodents. Alzheimer disease’s patients exhibit high levels of circulating GSK3β and, consequently, pharmacological strategies based on GSK3β antagonists have been designed. The approach, however, has yielded inconclusive results so far. Here, we carried out a comparative study in Drosophila and rats addressing the role of GSK3β in synaptogenesis. In flies, the genetic inhibition of the shaggy-encoded GSK3β increases the number of synapses, while its upregulation leads to synapse loss. Likewise, in three weeks cultured rat hippocampal neurons, the pharmacological inhibition of GSK3β increases synapse density and Synapsin expression. However, experiments on younger cultures (12 days) yielded an opposite effect, a reduction of synapse density. This unexpected finding seems to unveil an age- and dosage-dependent differential response of mammalian neurons to the stimulation/inhibition of GSK3β, a feature that must be considered in the context of human adult neurogenesis and pharmacological treatments for Alzheimer’s disease based on GSK3β antagonists.

The number of synapses is a major determinant of behavior and many neural diseases exhibit deviations in that number. However, how signaling pathways control this number is still poorly understood. Using the Drosophila larval neuromuscular junction, we show here a PI3K-dependent pathway for synaptogenesis which is functionally connected with other previously known elements including the Wit receptor, its ligand Gbb, and the MAPkinases cascade. Based on epistasis assays, we determined the functional hierarchy within the pathway. Wit seems to trigger signaling through PI3K, and Ras85D also contributes to the initiation of synaptogenesis. However, contrary to other signaling pathways, PI3K does not require Ras85D binding in the context of synaptogenesis. In addition to the MAPK cascade, Bsk/JNK undergoes regulation by Puc and Ras85D which results in a narrow range of activity of this kinase to determine normalcy of synapse number. The transcriptional readout of the synaptogenesis pathway involves the Fos/Jun complex and the repressor Cic. In addition, we identified an antagonistic pathway that uses the transcription factors Mad and Medea and the microRNA bantam to down-regulate key elements of the pro-synaptogenesis pathway. Like its counterpart, the anti-synaptogenesis signaling uses small GTPases and MAPKs including Ras64B, Ras-like-a, p38a and Licorne. Bantam downregulates the pro-synaptogenesis factors PI3K, Hiw, Ras85D and Bsk, but not AKT. AKT, however, can suppress Mad which, in conjunction with the reported suppression of Mad by Hiw, closes the mutual regulation between both pathways. Thus, the number of synapses seems to result from the balanced output from these two pathways.

SummarySynapse transmission depends on the precise structural and functional assembly between pre-and postsynaptic elements. This tightly regulated interaction has been thoroughly characterised in vivo in the Drosophila glutamatergic larval neuromuscular junction (NMJ) synapse, a suitable model to explore synapse formation, dynamics and plasticity. Previous findings have demonstrated that presynaptic upregulation of phosphoinositide 3-kinase (PI3K) increases synapse number, generating new functional contacts and eliciting changes in behaviour. Here, we show that genetically driven overexpression of PI3K in the presynaptic element also leads to a correlated increase in the levels of glutamate receptor (GluRII) subunits and the number of postsynaptic densities (PSDs), without altering GluRII formation and assembly dynamics. In addition to GluRIIs, presynaptic PI3K activity also modifies the expression of the postsynaptic protein Discs large (Dlg). Remarkably, PI3K specifically overexpressed in the final larval stages is sufficient for the formation of NMJ synapses. No differences in the number of synapses and PSDs were detected when PI3K was selectively expressed in the postsynaptic compartment. Taken together, these results demonstrate that PI3K-dependent synaptogenesis plays an instructive role in PSD formation and growth from the presynaptic side.

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The MCM2-7 complex is a highly conserved hetero-hexameric protein complex, critical for DNA unwinding at the replicative fork during DNA replication. Overexpression or mutation in MCM2-7 genes is linked to and may drive several cancer types in humans. In mice, mutations in MCM2-7 genes result in growth retardation and mortality. All six MCM2-7 genes are also expressed in the developing mouse CNS, but their role in the CNS is not clear. Here, we use the central nervous system (CNS) of Drosophila melanogaster to begin addressing the role of the MCM complex during development, focusing on the specification of a well-studied neuropeptide expressing neuron: the Tv4/FMRFa neuron. In a search for genes involved in the specification of the Tv4/FMRFa neuron we identified Mcm5 and find that it plays a highly specific role in the specification of the Tv4/FMRFa neuron. We find that other components of the MCM2-7 complex phenocopies Mcm5, indicating that the role of Mcm5 in neuronal subtype specification involves the MCM2-7 complex. Surprisingly, we find no evidence of reduced progenitor proliferation, and instead find that Mcm5 is required for the expression of the type I BMP receptor Tkv, which is critical for the FMRFa expression. These results suggest that the MCM2-7 complex may play roles during CNS development outside of its well-established role during DNA replication.

During Drosophila epithelial development, signalling specialized filopodial or cytonemes establish direct contacts between distant cells to facilitate the formation of the Hedgehog (Hh) signalling gradient. However, the regulatory mechanisms for Hh cytoneme initiation and their dynamic behaviour are still unknown. Here we show that Hh cytoneme dynamics in Drosophila epithelia depends on the Epidermal Growth Factor (EGF) signalling pathway through its ligand Spitz. We describe that the EGF pathway is required to maintain basal plasma membrane levels of the adhesion protein and Hh co-receptor Ihog, able to modify cyoneme behaviour. Furthermore, we describe that EGF signalling promotes Ihog interaction with actin cytoskeleton regulators, such as the actin cross-linker Filamin A, Cheerio (Che) in Drosophila. Cheerio is recruited by Ihog towards focalized small regions of the plasma membrane to contribute to the cytoneme stabilization function of Ihog. Finally, we describe that EGF regulation and PI3K signalling act synergically on Hh cytoneme behaviour, revealing a new form of cooperation between major tissue organizers.