ABSTRACT
Synapse development and neuronal activity represent fundamental processes for the establishment of cognitive function. Structural organization as well as signalling pathways from receptor stimulation to gene expression regulation are mediated by synaptic activity and misregulated in neurodevelopmental disorders such as autism spectrum disorder (ASD) and intellectual disability (ID). Deleterious mutations in the PTCHD1 (Patched domain containing 1) gene have been described in male patients with X-linked ID and/or ASD. The structure of PTCHD1 protein is similar to the Patched (PTCH1) receptor; however, the cellular mechanisms and pathways associated with PTCHD1 in the developing brain are poorly determined. Here we show that PTCHD1 displays a C-terminal PDZ-binding motif that binds to the postsynaptic proteins PSD95 and SAP102. We also report that PTCHD1 is unable to rescue the canonical sonic hedgehog (SHH) pathway in cells depleted of PTCH1, suggesting that both proteins are involved in distinct cellular signalling pathways. We find that Ptchd1 deficiency in male mice (Ptchd1-/y) induces global changes in synaptic gene expression, affects the expression of the immediate-early expression genes Egr1 and Npas4 and finally impairs excitatory synaptic structure and neuronal excitatory activity in the hippocampus, leading to cognitive dysfunction, motor disabilities and hyperactivity. Thus our results support that PTCHD1 deficiency induces a neurodevelopmental disorder causing excitatory synaptic dysfunction.
Subject(s)
Cognitive Dysfunction/metabolism , Membrane Proteins/deficiency , Synapses/metabolism , Animals , Basic Helix-Loop-Helix Transcription Factors/metabolism , Cognition/physiology , Cognitive Dysfunction/genetics , Disks Large Homolog 4 Protein/genetics , Disks Large Homolog 4 Protein/metabolism , Guanylate Kinases/genetics , Guanylate Kinases/metabolism , Hippocampus/metabolism , Male , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Mice, Inbred C57BL , Neurons/metabolism , Signal Transduction , Synapses/genetics , Synaptic TransmissionABSTRACT
Annexin VI is a widely expressed calcium- and phospholipid-binding protein that lacks a clear physiological role. We now report that A431 cells expressing annexin VI are defective in their ability to sustain elevated levels of cytosolic Ca(2+) following stimulation with EGF. Other aspects of EGF receptor signaling, such as protein tyrosine phosphorylation and induction of c-fos are normal in these cells. However, EGF-mediated membrane hyperpolarization is attenuated and Ca(2+) entry abolished in cells expressing annexin VI. This effect of annexin VI was only observed for the larger of the two annexin VI splice forms, the smaller splice variant had no discernable effect on either cellular phenotype or growth rate. Inhibition of Ca(2+) influx was specific for the EGF-induced pathway; capacitative Ca(2+) influx initiated by emptying of intracellular stores was unaffected. These results provide the first evidence that the two splice forms of annexin VI have different functions.
Subject(s)
Annexin A6/pharmacology , Calcium/metabolism , Epidermal Growth Factor/antagonists & inhibitors , Calcium Signaling/drug effects , Cell Division/drug effects , Cell Line , Epidermal Growth Factor/pharmacology , ErbB Receptors/metabolism , Ion Transport , Membrane Potentials/drug effectsABSTRACT
N1E-115 mouse neuroblastoma cells were injected with a calcium buffer/indicator solution to allow both ratiometric measurement of free calcium concentration and the release of calcium ions upon UV flash. The solution contained sulforhodamine, a marker dye used to estimate the volume injected; fluo-3, a calcium indicator, and NP-EGTA, a high affinity calcium-selective buffer that is converted by UV flash to products with negligible calcium affinity. The calcium increase recorded upon UV irradiation (Delta[Ca2+]i) was small for small injection volumes, increased with larger injection volumes, but approached a plateau at the largest injection volumes. From this relation we estimate the buffering capacity of the cytosol as 1700 ions bound per ion free.
