Marcus Semtner

Changes in neural network homeostasis trigger neuropsychiatric symptoms

The mechanisms that regulate the strength of synaptic transmission and intrinsic neuronal excitability are well characterized; however, the mechanisms that promote disease-causing neural network dysfunction are poorly defined. We generated mice with targeted neuron type-specific expression of a gain-of-function variant of the neurotransmitter receptor for glycine (GlyR) that is found in hippocampectomies from patients with temporal lobe epilepsy. In this mouse model, targeted expression of gain-of-function GlyR in terminals of glutamatergic cells or in parvalbumin-positive interneurons persistently altered neural network excitability. The increased network excitability associated with gain-of-function GlyR expression in glutamatergic neurons resulted in recurrent epileptiform discharge, which provoked cognitive dysfunction and memory deficits without affecting bidirectional synaptic plasticity. In contrast, decreased network excitability due to gain-of-function GlyR expression in parvalbumin-positive interneurons resulted in an anxiety phenotype, but did not affect cognitive performance or discriminative associative memory. Our animal model unveils neuron type-specific effects on cognition, formation of discriminative associative memory, and emotional behavior in vivo. Furthermore, our data identify a presynaptic disease-causing molecular mechanism that impairs homeostatic regulation of neural network excitability and triggers neuropsychiatric symptoms.

Palmitoylation of gephyrin controls receptor clustering and plasticity of GABAergic synapses.

Gephyrin, the principal scaffolding protein at inhibitory synapses, needs to be palmitoylated in order to cluster and to assemble functional synapses.

Rare variants in γ-aminobutyric acid type A receptor genes in rolandic epilepsy and related syndromes.

To test whether mutations in γ‐aminobutyric acid type A receptor (GABAA‐R) subunit genes contribute to the etiology of rolandic epilepsy (RE) or its atypical variants (ARE).

Human iPSC-Derived Neural Progenitors Are an Effective Drug Discovery Model for Neurological mtDNA Disorders

Mitochondrial DNA (mtDNA) mutations frequently cause neurological diseases. Modeling of these defects has been difficult because of the challenges associated with engineering mtDNA. We show here that neural progenitor cells (NPCs) derived from human induced pluripotent stem cells (iPSCs) retain the parental mtDNA profile and exhibit a metabolic switch toward oxidative phosphorylation. NPCs derived in this way from patients carrying a deleterious homoplasmic mutation in the mitochondrial gene MT-ATP6 (m.9185T>C) showed defective ATP production and abnormally high mitochondrial membrane potential (MMP), plus altered calcium homeostasis, which represents a potential cause of neural impairment. High-content screening of FDA-approved drugs using the MMP phenotype highlighted avanafil, which we found was able to partially rescue the calcium defect in patient NPCs and differentiated neurons. Overall, our results show that iPSC-derived NPCs provide an effective model for drug screening to target mtDNA disorders that affect the nervous system.

Presynaptic mechanisms of neuronal plasticity and their role in epilepsy

Synaptic communication requires constant adjustments of pre- and postsynaptic efficacies. In addition to synaptic long term plasticity, the presynaptic machinery underlies homeostatic regulations which prevent out of range transmitter release. In this minireview we will discuss the relevance of selected presynaptic mechanisms to epilepsy including voltage- and ligand-gated ion channels as well as cannabinoid and adenosine receptor signaling.

Identification of Parvalbumin Interneurons as Cellular Substrate of Fear Memory Persistence

Parvalbumin-positive (PV) basket cells provide perisomatic inhibition in the cortex and hippocampus and control generation of memory-related network activity patterns, such as sharp wave ripples (SPW-R). Deterioration of this class of fast-spiking interneurons has been observed in neuropsychiatric disorders and evidence from animal models suggests their involvement in the acquisition and extinction of fear memories. Here, we used mice with neuron type-targeted expression of the presynaptic gain-of-function glycine receptor RNA variant GlyR α3L185L to genetically enhance the network activity of PV interneurons. These mice showed reduced extinction of contextual fear memory but normal auditory cued fear memory. They furthermore displayed increase of SPW-R activity in area CA3 and CA1 and facilitated propagation of this particular network activity pattern, as determined in ventral hippocampal slice preparations. Individual freezing levels during extinction and SPW-R propagation were correlated across genotypes. The same was true for parvalbumin immunoreactivity in the ventral hippocampus, which was generally augmented in the GlyR mutant mice and correlated with individual freezing levels. Together, these results identify PV interneurons as critical cellular substrate of fear memory persistence and associated SPW-R activity in the hippocampus. Our findings may be relevant for the identification and characterization of physiological correlates for posttraumatic stress and anxiety disorders.

S-sulfocysteine/NMDA receptor–dependent signaling underlies neurodegeneration in molybdenum cofactor deficiency

Molybdenum cofactor deficiency (MoCD) is an autosomal recessive inborn error of metabolism characterized by neurodegeneration and death in early childhood. The rapid and progressive neurodegeneration in MoCD presents a major clinical challenge and may relate to the poor understanding of the molecular mechanisms involved. Recently, we reported that treating patients with cyclic pyranopterin monophosphate (cPMP) is a successful therapy for a subset of infants with MoCD and prevents irreversible brain damage. Here, we studied S-sulfocysteine (SSC), a structural analog of glutamate that accumulates in the plasma and urine of patients with MoCD, and demonstrated that it acts as an N-methyl D-aspartate receptor (NMDA-R) agonist, leading to calcium influx and downstream cell signaling events and neurotoxicity. SSC treatment activated the protease calpain, and calpain-dependent degradation of the inhibitory synaptic protein gephyrin subsequently exacerbated SSC-mediated excitotoxicity and promoted loss of GABAergic synapses. Pharmacological blockade of NMDA-R, calcium influx, or calpain activity abolished SSC and glutamate neurotoxicity in primary murine neurons. Finally, the NMDA-R antagonist memantine was protective against the manifestation of symptoms in a tungstate-induced MoCD mouse model. These findings demonstrate that SSC drives excitotoxic neurodegeneration in MoCD and introduce NMDA-R antagonists as potential therapeutics for this fatal disease.

Intracellular glycine receptor function facilitates glioma formation in vivo.

ABSTRACT The neuronal function of Cys-loop neurotransmitter receptors is established; however, their role in non-neuronal cells is poorly defined. As brain tumors are enriched in the neurotransmitter glycine, we studied the expression and function of glycine receptors (GlyRs) in glioma cells. Human brain tumor biopsies selectively expressed the GlyR &agr;1 and &agr;3 subunits, which have nuclear localization signals (NLSs). The mouse glioma cell line GL261 expressed GlyR &agr;1, and knockdown of GlyR &agr;1 protein expression impaired the self-renewal capacity and tumorigenicity of GL261 glioma cells, as shown by a neurosphere assay and GL261 cell inoculation in vivo, respectively. We furthermore showed that the pronounced tumorigenic effect of GlyR &agr;1 relies on a new intracellular signaling function that depends on the NLS region in the large cytosolic loop and impacts on GL261 glioma cell gene regulation. Stable expression of GlyR &agr;1 and &agr;3 loops rescued the self-renewal capacity of GlyR &agr;1 knockdown cells, which demonstrates their functional equivalence. The new intracellular signaling function identified here goes beyond the well-established role of GlyRs as neuronal ligand-gated ion channels and defines NLS-containing GlyRs as new potential targets for brain tumor therapies.

Changes in phagocytosis and potassium channel activity in microglia of 5xFAD mice indicate alterations in purinergic signaling in a mouse model of Alzheimer's disease

As the immunocompetent cells of the central nervous system, microglia accumulate at amyloid beta plaques in Alzheimers disease (AD) and acquire a morphological phenotype of activated microglia. Recent functional studies, however, indicate that in mouse models of amyloidosis and AD, these cells are rather dysfunctional indicated by a reduced phagocytic activity. Here, we report that this reduction in phagocytic activity is associated with perturbed purinergic receptor signaling, since phagocytosis could be stimulated by P2Y6 receptor activation in control, but not in 5xFAD transgenic animals, an animal model of amyloid deposition. Impaired phagocytosis is not innate, and develops only at later stages of amyloidosis. Furthermore, we show that membrane currents induced by uridine diphosphate, a ligand activating P2Y6 receptors, are altered in response rate and amplitude in microglia in close vicinity to plaques, but not in plaque-free areas of 5xFAD animals. These changes were accompanied by changes in membrane properties and potassium channel activity of plaque-associated microglia in early and late stages of amyloidosis. As a conclusion, the physiological properties of plaque-associated microglia are altered with a strong impact on purinergic signaling.

Electrophysiological Signature of Homomeric and Heteromeric Glycine Receptor Channels.

Glycine receptors are chloride-permeable, ligand-gated ion channels and contribute to the inhibition of neuronal firing in the central nervous system or to facilitation of neurotransmitter release if expressed at presynaptic sites. Recent structure-function studies have provided detailed insights into the mechanisms of channel gating, desensitization, and ion permeation. However, most of the work has focused only on comparing a few isoforms, and among studies, different cellular expression systems were used. Here, we performed a series of experiments using recombinantly expressed homomeric and heteromeric glycine receptor channels, including their splice variants, in the same cellular expression system to investigate and compare their electrophysiological properties. Our data show that the current-voltage relationships of homomeric channels formed by the α2 or α3 subunits change upon receptor desensitization from a linear to an inwardly rectifying shape, in contrast to their heteromeric counterparts. The results demonstrate that inward rectification depends on a single amino acid (Ala254) at the inner pore mouth of the channels and is closely linked to chloride permeation. We also show that the current-voltage relationships of glycine-evoked currents in primary hippocampal neurons are inwardly rectifying upon desensitization. Thus, the alanine residue Ala254 determines voltage-dependent rectification upon receptor desensitization and reveals a physio-molecular signature of homomeric glycine receptor channels, which provides unprecedented opportunities for the identification of these channels at the single cell level.