Biology subjects

Forrest, M.

Publications and source records attributed to Forrest, M..

3 recordsLinked to original sources

bioRxiv2019

CNTNAP2 ectodomain, detected in neuronal and CSF sheddomes, modulates Ca2+ dynamics and network synchrony

SUMMARYWhile many neuronal membrane-anchored proteins undergo proteolytic cleavage, little is known about the biological significance of neuronal ectodomain shedding. Using mass spectrometry (MS)-based proteomics, we showed that the neuronal sheddome mirrors human cerebrospinal fluid (hCSF). Among shed synaptic proteins in hCSF was the ectodomain of CNTNAP2 (CNTNAP2-ecto), a risk factor for neurodevelopmental disorders (NDD). Using structured-illumination microscopy (SIM), we mapped the spatial organization of neuronal CNTNAP2-ecto shedding. Using affinity chromatography followed by MS, we identified the ATP2B/PMCA Ca2+ extrusion pumps as novel CNTNAP2-ecto binding partners. CNTNAP2-ecto coimmunoprecipitates with PMCA2, a known autism risk factor, and enhances its activity, thereby modulating neuronal Ca2+ levels. Finally, we showed that CNTNAP2-ecto regulates neuronal network synchrony in primary cultures and brain slices. These data provide new insights into the biology of synaptic ectodomain shedding and reveal a novel mechanism of regulation of Ca2+ homeostasis and neuronal network synchrony.

neuroscience↗

bioRxiv2019

CNTNAP2 is targeted to endosomes by the polarity protein Par3

A decade of genetic studies has established Contactin-associated protein-like 2 (CNTNAP2) as a prominent susceptibility gene associated with multiple neurodevelopmental disorders. The development and characterization of Cntnap2 knockout models in multiple species have bolstered this claim by establishing clear connections with certain endophenotypes. Despite these remarkable in vivo findings, CNTNAP2s molecular functions are relatively unexplored, highlighting the need to identify novel protein partners. Here, we characterized an interaction between CNTNAP2 and Partitioning-defective 3 (Par3) - a polarity molecule we isolated in a yeast-two hybrid screen with CNTNAP2s C-terminus. We provide evidence that the two proteins interact via PDZ domain-mediated binding, that CNTNAP2+/Par3+ complexes are largely associated with clathrin-coated endocytic vesicles, and that Par3 causes an enlargement of these structures. Live imaging and fluorescence recovery after photobleaching (FRAP) reveals that Par3 limits the mobility of CNTNAP2 at endosomes, thus stabilizing it at that location. Finally, expression of Par3 but not Par3{Delta}PDZ can cluster endogenous CNTNAP2 in primary neurons. Collectively, we conclude that Par3 regulates CNTNAP2 spatial localization to endocytic compartments.

neuroscience↗

bioRxiv2019

Impaired M-current in KCNQ2 Encephalopathy Evokes Dyshomeostatic Modulation of Excitability

Mutations in KCNQ2, which encodes a pore-forming K+ channel subunit responsible for neuronal M-current, cause neonatal epileptic encephalopathy, a complex disorder presenting with severe early-onset seizures and impaired neurodevelopment. The condition is exceptionally difficult to treat, partially because the effects of KCNQ2 mutations on the development and function of human neurons are unknown. Here, we used induced pluripotent stem cells and gene editing to establish a disease model, and measured the functional properties of patient-derived neurons using electrophysiological and optical approaches. We find that while patient-derived excitatory neurons exhibit reduced M-current early, they develop intrinsic and network hyperexcitability progressively. This hyperexcitability is associated with faster action potential repolarization, larger afterhyperpolarization, and a functional enhancement of large conductance Ca2+-activated K+ (BK) channels. These properties facilitate a burst-suppression firing pattern that is reminiscent of the interictal electroencephalography pattern in patients. Importantly, we were able to phenocopy these excitability features in control neurons only by chronic but not acute pharmacological inhibition of M-current. Our findings suggest that dyshomeostatic mechanisms compound KCNQ2 loss-of-function and lead to alterations in the neurodevelopmental trajectory of patient-derived neurons. Our work has therapeutic implications in explaining why KCNQ2 agonists are not beneficial unless started at an early disease stage.

neuroscience↗