A PI3-Kinase–Mediated Negative Feedback Regulates Neuronal Excitability

Abstract

Use-dependent downregulation of neuronal activity (negative feedback) can act as a homeostatic mechanism to maintain neuronal activity at a particular specified value. Disruption of this negative feedback might lead to neurological pathologies, such as epilepsy, but the precise mechanisms by which this feedback can occur remain incompletely understood. At one glutamatergic synapse, the Drosophila neuromuscular junction, a mutation in the group II metabotropic glutamate receptor gene (DmGluRA) increased motor neuron excitability by disrupting an autocrine, glutamate-mediated negative feedback. We show that DmGluRA mutations increase neuronal excitability by preventing PI3 kinase (PI3K) activation and consequently hyperactivating the transcription factor Foxo. Furthermore, glutamate application increases levels of phospho-Akt, a product of PI3K signaling, within motor nerve terminals in a DmGluRA-dependent manner. Finally, we show that PI3K increases both axon diameter and synapse number via the Tor/S6 kinase pathway, but not Foxo. In humans, PI3K and group II mGluRs are implicated in epilepsy, neurofibromatosis, autism, schizophrenia, and other neurological disorders; however, neither the link between group II mGluRs and PI3K, nor the role of PI3K-dependent regulation of Foxo in the control of neuronal excitability, had been previously reported. Our work suggests that some of the deficits in these neurological disorders might result from disruption of glutamate-mediated homeostasis of neuronal excitability.

The Metabotropic Glutamate Receptor Activates the Lipid Kinase PI3K in Drosophila Motor Neurons Through the Calcium/Calmodulin-Dependent Protein Kinase II and the Nonreceptor Tyrosine Protein Kinase DFak

“…Fixed larval tissues were incubated with a rabbit anti-Drosophila p-Akt (Ser505) polyclonal primary antibody (1:500 dilution; Cell Signaling Technologies). The antibody used was from a different lot from the antibody used in a previous report (Howlett et al 2008) and thus might not have identical properties. Larvae were then incubated with Rhodamine Red conjugated goat antirabbit IgG (1:500 dilution; Jackson ImmunoResearch), and Cy-2 conjugated antibodies against horseradish peroxidase (1:200 dilution;Jackson ImmunoResearch).…”

“…Transmission electron microscopy was performed as described previously (Lavery et al 2007;Howlett et al 2008). Briefly, larvae were grown, selected, and dissected as described above, fixed with glutaraldehyde and paraformaldehyde, stained with 0.5% OsO 4 and 2% uranyl acetate, and embedded in an eponate 12-araldite mixture.…”

“…In Drosophila, the DmGluRA 112b null mutation in the single mGluR increases motor neuron excitability by preventing activation of the lipid kinase PI3K (Bogdanik et al 2004;Howlett et al 2008). Given that glutamate is the major excitatory neurotransmitter at NMJs in insects such as Drosophila (Jan and Jan 1976), it was suggested that DmGluRA in motor neurons participate in a negative feedback: glutamate released from motor nerve terminals as a consequence of neuronal activity activates DmGluRA autoreceptors in motor nerve terminals.…”

“…In this view, DmGluRA 112b increases excitability by preventing PI3K activation and thus severing this negative feedback. This view is supported by the observation that transgene-induced PI3K inhibition increases neuronal excitability in a manner similar to DmGluRA 112b , whereas transgene-induced PI3K activation decreases neuronal excitability (Howlett et al 2008). However, the molecular mechanism by which glutamate/DmGluRA activates PI3K was not elucidated.…”

“…In Drosophila larval motor neurons, glutamate activation of the single mGluR, called DmGluRA, downregulates neuronal excitability (Bogdanik et al 2004); glutamate both activates PI3K and requires PI3K activity for this downregulation (Howlett et al 2008). Because glutamate is the excitatory neurotransmitter at the Drosophila neuromuscular junction (NMJ) (Jan and Jan 1976), it was hypothesized that this DmGluRA-mediated downregulation of neuronal excitability carried out a negative feedback on activity: glutamate released from motor nerve terminals would activate DmGluRA autoreceptors, which would then depress excitability.…”