SynGAP isoforms exert opposing effects on synaptic strength

Peer reviewedPublisher PD

Mutant induced pluripotent stem cell lines recapitulate aspects of TDP-43 proteinopathies and reveal cell-specific vulnerability

Transactive response DNA-binding (TDP-43) protein is the dominant disease protein in amyotrophic lateral sclerosis (ALS) and a subgroup of frontotemporal lobar degeneration (FTLD-TDP). Identification of mutations in the gene encoding TDP-43 (TARDBP) in familial ALS confirms a mechanistic link between misaccumulation of TDP-43 and neurodegeneration and provides an opportunity to study TDP-43 proteinopathies in human neurons generated from patient fibroblasts by using induced pluripotent stem cells (iPSCs). Here, we report the generation of iPSCs that carry the TDP-43 M337V mutation and their differentiation into neurons and functional motor neurons. Mutant neurons had elevated levels of soluble and detergent-resistant TDP-43 protein, decreased survival in longitudinal studies, and increased vulnerability to antagonism of the PI3K pathway. We conclude that expression of physiological levels of TDP-43 in human neurons is sufficient to reveal a mutation-specific cell-autonomous phenotype and strongly supports this approach for the study of disease mechanisms and for drug screening

Characterizing Ligand-Gated Ion Channel Receptors with Genetically Encoded Ca++ Sensors

We present a cell based system and experimental approach to characterize agonist and antagonist selectivity for ligand-gated ion channels (LGIC) by developing sensor cells stably expressing a Ca2+ permeable LGIC and a genetically encoded Förster (or fluorescence) resonance energy transfer (FRET)-based calcium sensor. In particular, we describe separate lines with human α7 and human α4β2 nicotinic acetylcholine receptors, mouse 5-HT3A serotonin receptors and a chimera of human α7/mouse 5-HT3A receptors. Complete concentration-response curves for agonists and Schild plots of antagonists were generated from these sensors and the results validate known pharmacology of the receptors tested. Concentration-response relations can be generated from either the initial rate or maximal amplitudes of FRET-signal. Although assaying at a medium throughput level, this pharmacological fluorescence detection technique employs a clonal line for stability and has versatility for screening laboratory generated congeners as agonists or antagonists on multiple subtypes of ligand-gated ion channels. The clonal sensor lines are also compatible with in vivo usage to measure indirectly receptor activation by endogenous neurotransmitters

Neuronal homeostasis: Time for a change?

Homeostatic processes that regulate electrical activity in neurones are now an established aspect of physiology and rest on a large body of experimental evidence that points to roles in development, learning and memory, and disease. However, the concepts underlying homeostasis are too often summarized in ways that restrict their explanatory power and obviate important subtleties. Here, we present a review of the underlying theory of homeostasis - control theory - in an attempt to reconcile some existing conceptual problems in the context of neuronal physiology. In addition to clarifying the underlying theory, this review highlights the remaining challenges posed when analysing homeostatic phenomena that underlie the regulation of neuronal excitability. Moreover, we suggest approaches for future experimental and computational work that will further our understanding of neuronal homeostasis and the fundamental neurophysiological functions it serves. © 2011 The Authors. Journal compilation © 2011 The Physiological Society

Single-channel properties of N-methyl-D-aspartate receptors containing chimaeric GluN2A/GluN2D subunits

Subtypes of NMDARs (N-methyl-d-aspartate receptors) display differences in their pharmacological and biophysical properties. The differences are, to a large extent, determined by the identities of the GluN2 (glutamate-binding) NMDAR subunits that are co-expressed with GluN1 (glycine-binding) subunits, which form the final tetrameric NMDAR assembly. Of the four GluN2 subunits that exist (termed A-D), NMDARs composed of GluN1/GluN2A and GluN1/GluN2D subunits display the greatest differences in their sensitivities to a variety of agonists, antagonists and channel blockers as well as showing marked differences in their single-channel conductances and deactivation kinetics. Here, we describe a series of experiments where we have generated and studied two chimaeric GluN2A/GluN2D subunits. The first chimaera, referred to as GluN2A(2D-M1M2M3), replaces the membrane-associated regions M1, M2 and M3 of the GluN2A subunit with the corresponding regions found in the GluN2D subunit. The second chimaera, GluN2A(2D-S1M1M2M3S2), replaces the same three membrane-associated regions of the GluN2A subunit plus the LBD (ligand-binding domain) with the corresponding regions of the GluN2D subunit. Our results show that the identity of the GluN2 LBD not only controls glutamate potency, but also influences the potency of the NMDAR co-agonist glycine, whereas the single-channel conductance and the duration of single activations of ion channels can be predicted by the identities of the M1-M3 regions and the LBD. © The Authors Journal compilation © 2009 Biochemical Society

Homeostasis of intrinsic excitability in hippocampal neurones: Dynamics and mechanism of the response to chronic depolarization

In order to maintain stable functionality in the face of continually changing input, neurones in the CNS must dynamically modulate their electrical characteristics. It has been hypothesized that in order to retain stable network function, neurones possess homeostatic mechanisms which integrate activity levels and alter network and cellular properties in such a way as to counter long-term perturbations. Here we describe a simple model system where we investigate the effects of sustained neuronal depolarization, lasting up to several days, by exposing cultures of primary hippocampal pyramidal neurones to elevated concentrations (10-30 m. m) of KCl. Following exposure to KCl, neurones exhibit lower input resistances and resting potentials, and require more current to be injected to evoke action potentials. This results in a rightward shift in the frequency-input current (FI) curve which is explained by a simple linear model of the subthreshold I-V relationship. No changes are observed in action potential profiles, nor in the membrane potential at which action potentials are evoked. Furthermore, following depolarization, an increase in subthreshold potassium conductance is observed which is accounted for within a biophysical model of the subthreshold I-V characteristics of neuronal membranes. The FI curve shift was blocked by the presence of the L-type Ca2+ channel blocker nifedipine, whilst antagonism of NMDA receptors did not interfere with the effect. Finally, changes in the intrinsic properties of neurones are reversible following removal of the depolarizing stimulus. We suggest that this experimental system provides a convenient model of homeostatic regulation of intrinsic excitability, and permits the study of temporal characteristics of homeostasis and its dependence on stimulus magnitude. © 2010 The Authors. Journal compilation © 2010 The Physiological Society

Inhibition of rat recombinant GluN1/GluN2A and GluN1/GluN2B NMDA receptors by ethanol at concentrations based on the US/UK drink-drive limit

Many studies examine the actions of ethanol on N-methyl-D-aspartate (NMDA) receptors using concentrations that are highly toxic (≥ 100 mM). This study re-assesses the actions of ethanol at concentrations based around the US/UK 'drink-drive' limit (17 mM). Using two-electrode voltage-clamp recordings we examined the actions of ethanol on recombinant GluN1/GluN2A and GluN1/GluN2B NMDA receptors expressed in Xenopus laevis oocytes. We also investigated its actions on NMDA receptors containing GluN2A subunits with truncated or deleted carboxy terminal domains. Ethanol inhibition was voltage-independent and for GluN1/GluN2A NMDA receptors mean inhibition (20 mM at - 60 mV) was 9.5 ± 0.8% (n = 33) while corresponding values for GluN1/GluN2B NMDA receptors were 6.5 ± 0.8% (n = 21). EC50 values for glutamate at GluN1/GluN2A and glutamate and glycine at GluN1/GluN2B NMDA receptors were unaffected by the presence of ethanol. We did however observe a small increase in glycine potency, in the presence of ethanol, at GluN1/GluN2A NMDA receptors. Neither voltage-dependent Mg2+ block nor memantine block was affected by ethanol. Reduced ethanol inhibition was observed however at NMDA receptors containing GluN2A subunits with mutated carboxy terminal domains. We conclude that the levels of inhibition seen with ethanol concentrations near to the US/UK drink-driving limit are very modest and even at higher (intoxicating) concentrations do not alter characteristic NMDA receptor properties. © 2009 Elsevier B.V. All rights reserved

SynGAP isoforms exert opposing effects on synaptic strength.

Alternative promoter usage and alternative splicing enable diversification of the transcriptome. Here we demonstrate that the function of Synaptic GTPase-Activating Protein (SynGAP), a key synaptic protein, is determined by the combination of its amino-terminal sequence with its carboxy-terminal sequence. 5' rapid amplification of cDNA ends and primer extension show that different N-terminal protein sequences arise through alternative promoter usage that are regulated by synaptic activity and postnatal age. Heterogeneity in C-terminal protein sequence arises through alternative splicing. Overexpression of SynGAP α1 versus α2 C-termini-containing proteins in hippocampal neurons has opposing effects on synaptic strength, decreasing and increasing miniature excitatory synaptic currents amplitude/frequency, respectively. The magnitude of this C-terminal-dependent effect is modulated by the N-terminal peptide sequence. This is the first demonstration that activity-dependent alternative promoter usage can change the function of a synaptic protein at excitatory synapses. Furthermore, the direction and degree of synaptic modulation exerted by different protein isoforms from a single gene locus is dependent on the combination of differential promoter usage and alternative splicing

Comment on "Drug screening for ALS using patient-specific induced pluripotent stem cells".

Egawa et al. recently showed the value of patient-specific induced pluripotent stem cells (iPSCs) for modeling amyotrophic lateral sclerosis in vitro. Their study and our work highlight the need for complementary assays to detect small, but potentially important, phenotypic differences between control iPSC lines and those carrying disease mutations