Mitochondrial Regulation of the Hippocampal Firing Rate Set Point and Seizure Susceptibility.

Maintaining average activity within a set-point range constitutes a fundamental property of central neural circuits. However, whether and how activity set points are regulated remains unknown. Integrating genome-scale metabolic modeling and experimental study of neuronal homeostasis, we identified mitochondrial dihydroorotate dehydrogenase (DHODH) as a regulator of activity set points in hippocampal networks. The DHODH inhibitor teriflunomide stably suppressed mean firing rates via synaptic and intrinsic excitability mechanisms by modulating mitochondrial Ca2+ buffering and spare respiratory capacity. Bi-directional activity perturbations under DHODH blockade triggered firing rate compensation, while stabilizing firing to the lower level, indicating a change in the firing rate set point. In vivo, teriflunomide decreased CA3-CA1 synaptic transmission and CA1 mean firing rate and attenuated susceptibility to seizures, even in the intractable Dravet syndrome epilepsy model. Our results uncover mitochondria as a key regulator of activity set points, demonstrate the differential regulation of set points and compensatory mechanisms, and propose a new strategy to treat epilepsy.

Secreted amyloid-ß precursor protein functions as a GABABR1a ligand to modulate synaptic transmission.

Amyloid-ß precursor protein (APP) is central to the pathogenesis of Alzheimer's disease, yet its physiological function remains unresolved. Accumulating evidence suggests that APP has a synaptic function mediated by an unidentified receptor for secreted APP (sAPP). Here we show that the sAPP extension domain directly bound the sushi 1 domain specific to the γ-aminobutyric acid type B receptor subunit 1a (GABABR1a). sAPP-GABABR1a binding suppressed synaptic transmission and enhanced short-term facilitation in mouse hippocampal synapses via inhibition of synaptic vesicle release. A 17-amino acid peptide corresponding to the GABABR1a binding region within APP suppressed in vivo spontaneous neuronal activity in the hippocampus of anesthetized Thy1-GCaMP6s mice. Our findings identify GABABR1a as a synaptic receptor for sAPP and reveal a physiological role for sAPP in regulating GABABR1a function to modulate synaptic transmission.

Alzheimer's Disease: From Firing Instability to Homeostasis Network Collapse.

Alzheimer's disease (AD) starts from pure cognitive impairments and gradually progresses into degeneration of specific brain circuits. Although numerous factors initiating AD have been extensively studied, the common principles underlying the transition from cognitive deficits to neuronal loss remain unknown. Here we describe an evolutionarily conserved, integrated homeostatic network (IHN) that enables functional stability of central neural circuits and safeguards from neurodegeneration. We identify the critical modules comprising the IHN and propose a central role of neural firing in controlling the complex homeostatic network at different spatial scales. We hypothesize that firing instability and impaired synaptic plasticity at early AD stages trigger a vicious cycle, leading to dysregulation of the whole IHN. According to this hypothesis, the IHN collapse represents the major driving force of the transition from early memory impairments to neurodegeneration. Understanding the core elements of homeostatic control machinery, the reciprocal connections between distinct IHN modules, and the role of firing homeostasis in this hierarchy has important implications for physiology and should offer novel conceptual approaches for AD and other neurodegenerative disorders.

Multiporous Supramolecular Microspheres for Artificial Photosynthesis.

Artificial photosynthesis shows a promising potential for sustainable supply of nutritional ingredients. While most studies focus on the assembly of the light-sensitive chromophores to 1-D architectures in an artificial photosynthesis system, other supramolecular morphologies, especially bioinspired ones, which may have more efficient light-harvesting properties, have been far less studied. Here, MCpP-FF, a bioinspired building block fabricated by conjugating porphyrin and diphenylalanine, was designed to self-assemble into nanofibers-based multiporous microspheres. The highly organized aromatic moieties result in extensive excitation red-shifts and notable electron transfer, thus leading to a remarkable attenuated fluorescence decay and broad-spectrum light sensitivity of the microspheres. Moreover, the enhanced photoelectron production and transfer capability of the microspheres are demonstrated, making them ideal candidates for sunlight-sensitive antennas in artificial photosynthesis. These properties induce a high turnover frequency of NADH, which can be used to produce bioproducts in biocatalytic reactions. In addition, the direct electron transfer makes external mediators unnecessary, and the insolubility of the microspheres in water allows their easy retrieval for sustainable applications. Our findings demonstrate an alternative to design new platforms for artificial photosynthesis, as well as a new type of bioinspired, supramolecular multiporous materials.

Sensory Deprivation Triggers Synaptic and Intrinsic Plasticity in the Hippocampus.

Hippocampus, a temporal lobe structure involved in learning and memory, receives information from all sensory modalities. Despite extensive research on the role of sensory experience in cortical map plasticity, little is known about whether and how sensory experience regulates functioning of the hippocampal circuits. Here, we show that 9 ± 2 days of whisker deprivation during early mouse development depresses activity of CA3 pyramidal neurons by several principal mechanisms: decrease in release probability, increase in the fraction of silent synapses, and reduction in intrinsic excitability. As a result of deprivation-induced presynaptic inhibition, CA3-CA1 synaptic facilitation was augmented at high frequencies, shifting filtering properties of synapses. The changes in the AMPA-mediated synaptic transmission were accompanied by an increase in NR2B-containing NMDA receptors and a reduction in the AMPA/NMDA ratio. The observed reconfiguration of the CA3-CA1 connections may represent a homeostatic adaptation to augmentation in synaptic activity during the initial deprivation phase. In adult mice, tactile disuse diminished intrinsic excitability without altering synaptic facilitation. We suggest that sensory experience regulates computations performed by the hippocampus by tuning its synaptic and intrinsic characteristics.

Alzheimer's disease-causing proline substitutions lead to presenilin 1 aggregation and malfunction.

Do different neurodegenerative maladies emanate from the failure of a mutual protein folding mechanism? We have addressed this question by comparing mutational patterns that are linked to the manifestation of distinct neurodegenerative disorders and identified similar neurodegeneration-linked proline substitutions in the prion protein and in presenilin 1 that underlie the development of a prion disorder and of familial Alzheimer's disease (fAD), respectively. These substitutions were found to prevent the endoplasmic reticulum (ER)-resident chaperone, cyclophilin B, from assisting presenilin 1 to fold properly, leading to its aggregation, deposition in the ER, reduction of γ-secretase activity, and impaired mitochondrial distribution and function. Similarly, reduced quantities of the processed, active presenilin 1 were observed in brains of cyclophilin B knockout mice. These discoveries imply that reduced cyclophilin activity contributes to the development of distinct neurodegenerative disorders, propose a novel mechanism for the development of certain fAD cases, and support the emerging theme that this disorder can stem from aberrant presenilin 1 function. This study also points at ER chaperones as targets for the development of counter-neurodegeneration therapies.

APP homodimers transduce an amyloid-ß-mediated increase in release probability at excitatory synapses.

Accumulation of amyloid-ß peptides (Aß), the proteolytic products of the amyloid precursor protein (APP), induces a variety of synaptic dysfunctions ranging from hyperactivity to depression that are thought to cause cognitive decline in Alzheimer's disease. While depression of synaptic transmission has been extensively studied, the mechanisms underlying synaptic hyperactivity remain unknown. Here, we show that Aß40 monomers and dimers augment release probability through local fine-tuning of APP-APP interactions at excitatory hippocampal boutons. Aß40 binds to the APP, increases the APP homodimer fraction at the plasma membrane, and promotes APP-APP interactions. The APP activation induces structural rearrangements in the APP/Gi/o-protein complex, boosting presynaptic calcium flux and vesicle release. The APP growth-factor-like domain (GFLD) mediates APP-APP conformational changes and presynaptic enhancement. Thus, the APP homodimer constitutes a presynaptic receptor that transduces signal from Aß40 to glutamate release. Excessive APP activation may initiate a positive feedback loop, contributing to hippocampal hyperactivity in Alzheimer's disease.

Spatially resolved recording of transient fluorescence-lifetime effects by line-scanning TCSPC.

We present a technique that records transient changes in the fluorescence lifetime of a sample with spatial resolution along a one-dimensional scan. The technique is based on scanning the sample with a high-frequency pulsed laser beam, detecting single photons of the fluorescence light, and building up a photon distribution over the distance along the scan, the arrival times of the photons after the excitation pulses and the time after a stimulation of the sample. The maximum resolution at which lifetime changes can be recorded is given by the line scan period. Transient lifetime effects can thus be resolved at a resolution of about one millisecond. We demonstrate the technique for recording photochemical and nonphotochemical chlorophyll transients in plants and transient changes in free Ca(2+) in cultured neurons.

Role of phosphoinositides at the neuronal synapse.

Synaptic transmission is amongst the most sophisticated and tightly controlled biological phenomena in higher eukaryotes. In the past few decades, tremendous progress has been made in our understanding of the molecular mechanisms underlying multiple facets of neurotransmission, both pre- and postsynaptically. Brought under the spotlight by pioneer studies in the areas of secretion and signal transduction, phosphoinositides and their metabolizing enzymes have been increasingly recognized as key protagonists in fundamental aspects of neurotransmission. Not surprisingly, dysregulation of phosphoinositide metabolism has also been implicated in synaptic malfunction associated with a variety of brain disorders. In the present chapter, we summarize current knowledge on the role of phosphoinositides at the neuronal synapse and highlight some of the outstanding questions in this research field.

Acute manipulation of phosphoinositide levels in cells.

Phosphoinositides are membrane-bound signaling phospholipids that function in a myriad of cellular processes, including membrane trafficking, cytoskeletal dynamics, ion channel and transporter function, and signal transduction. In order to better understand the role of phosphoinositides in cellular processes, different approaches to study the effects of the presence or absence of these lipids must be devised. Conventional approaches of manipulating phosphoinositide levels such as over-expression or genetic ablation of lipid enzymes cause prolonged exposure of the cells to changes in lipid levels that could result in compensatory actions by the cell or downstream alterations in cell physiology. In this chapter we present an approach used recently by various laboratories, including our own, to acutely manipulate phosphoinositide levels at target locations using chemically induced dimerization (CID) that can be spatially and temporally controlled. We discuss considerations when designing expression constructs for targeting specific cellular compartment membranes and present examples from the literature on different ways of perturbing phosphoinositide levels at particular organelle membranes using CID. In addition, we provide details on image acquisition, data collection, and data interpretation. CID technology can be applied to many lipid enzymes to broaden the understanding of the role lipid signaling plays in cell physiology.

Synaptojanin 1-mediated PI(4,5)P2 hydrolysis is modulated by membrane curvature and facilitates membrane fission.

Phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] plays a fundamental role in clathrin-mediated endocytosis. However, precisely how PI(4,5)P2 metabolism is spatially and temporally regulated during membrane internalization and the functional consequences of endocytosis-coupled PI(4,5)P2 dephosphorylation remain to be explored. Using cell-free assays with liposomes of varying diameters, we show that the major synaptic phosphoinositide phosphatase, synaptojanin 1 (Synj1), acts with membrane curvature generators/sensors, such as the BAR protein endophilin, to preferentially remove PI(4,5)P2 from curved membranes as opposed to relatively flat ones. Moreover, in vivo recruitment of Synj1's inositol 5-phosphatase domain to endophilin-induced membrane tubules results in fragmentation and condensation of these structures largely in a dynamin-dependent fashion. Our study raises the possibility that geometry-based mechanisms may contribute to spatially restricting PI(4,5)P2 elimination during membrane internalization and suggests that the PI(4,5)P2-to-PI4P conversion achieved by Synj1 at sites of high curvature may cooperate with dynamin to achieve membrane fission.

Synaptojanin 1-linked phosphoinositide dyshomeostasis and cognitive deficits in mouse models of Down's syndrome.

Phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P(2)] is a signaling phospholipid implicated in a wide variety of cellular functions. At synapses, where normal PtdIns(4,5)P(2) balance is required for proper neurotransmission, the phosphoinositide phosphatase synaptojanin 1 is a key regulator of its metabolism. The underlying gene, SYNJ1, maps to human chromosome 21 and is thus a candidate for involvement in Down's syndrome (DS), a complex disorder resulting from the overexpression of trisomic genes. Here, we show that PtdIns(4,5)P(2) metabolism is altered in the brain of Ts65Dn mice, the most commonly used model of DS. This defect is rescued by restoring Synj1 to disomy in Ts65Dn mice and is recapitulated in transgenic mice overexpressing Synj1 from BAC constructs. These transgenic mice also exhibit deficits in performance of the Morris water maze task, suggesting that PtdIns(4,5)P(2) dyshomeostasis caused by gene dosage imbalance for Synj1 may contribute to brain dysfunction and cognitive disabilities in DS.

Functional stabilization of weakened thalamic pacemaker channel regulation in rat absence epilepsy.

Aberrant function of pacemaker currents (Ih), carried by hyperpolarization-activated cation non-selective (HCN) channels, affects neuronal excitability and accompanies epilepsy, but its distinct roles in epileptogenesis and chronic epilepsy are unclear. We probed Ih function and subunit composition during both pre- and chronically epileptic stages in thalamocortical (TC) neurones of the Genetic Absence Epilepsy Rat from Strasbourg (GAERS). Voltage gating of Ih was unaltered in mature somatosensory TC cells, both in vivo and in vitro. However, the enhancement of Ih by phasic, near-physiological, cAMP pulses was diminished by approximately 40% and the half-maximal cAMP concentration increased by approximately 5-fold. This decreased responsiveness of Ih to its major cellular modulator preceded epilepsy onset in GAERS, persisted throughout the chronic state, and was accompanied by an enhanced expression of the cAMP-insensitive HCN1 channel mRNA (> 50%), without changes in the mRNA levels of HCN2 and HCN4. To assess for alterations in TC cell excitability, we monitored the slow up-regulation of Ih that is induced by Ca2+-triggered cAMP synthesis and important for terminating in vitro synchronized oscillations. Remarkably, repetitive rebound Ca2+ spikes evoked normal slow Ih up-regulation in mature GAERS neurones; that sufficed to attenuate spontaneous rhythmic burst discharges. These adaptive mechanisms occurred upstream of cAMP turnover and involved enhanced intracellular Ca2+ accumulation upon repetitive low-threshold Ca2+ discharges. Therefore, HCN channels appear to play a dual role in epilepsy. Weakened cAMP binding to HCN channels precedes, and likely promotes, epileptogenesis in GAERS, whereas compensatory mechanisms stabilizing Ih function contribute to the termination of spike-and-wave discharges in chronic epilepsy.

Selective GABAergic control of higher-order thalamic relays.

GABAergic signaling is central to the function of the thalamus and has been traditionally attributed primarily to the nucleus reticularis thalami (nRT). Here we present a GABAergic pathway, distinct from the nRT, that exerts a powerful inhibitory effect selectively in higher-order thalamic relays of the rat. Axons originating in the anterior pretectal nucleus (APT) innervated the proximal dendrites of relay cells via large GABAergic terminals with multiple release sites. Stimulation of the APT in an in vitro slice preparation revealed a GABA(A) receptor-mediated, monosynaptic IPSC in relay cells. Activation of presumed single APT fibers induced rebound burst firing in relay cells. Different APT neurons recorded in vivo displayed fast bursting, tonic, or rhythmic firing. Our data suggest that selective extrareticular GABAergic control of relay cell activity will result in effective, state-dependent gating of thalamocortical information transfer in higher-order but not in first-order relays.

Pacemaker channels in mouse thalamocortical neurones are regulated by distinct pathways of cAMP synthesis.

A crucial aspect of pacemaker current (Ih) function is the regulation by cyclic nucleotides. To assess the endogenous mechanisms controlling cAMP levels in the vicinity of pacemaker channels, Ih regulation by G-protein-coupled neurotransmitter receptors was studied in mouse thalamocortical neurones. Activation of beta-adrenergic receptors with (-)-isoproterenol (Iso) led to a small steady enhancement of Ih amplitude, whereas activation of GABAB receptors with (+/-)-Baclofen (Bac) reduced Ih, consistent with an up- and down-regulation of basal cAMP levels, respectively. In contrast, a transient (taudecay, approximately 200 s), supralinear up-regulation of Ih was observed upon coapplication of Iso and Bac that was larger than that observed with Iso alone. This up-regulation appeared to involve a cAMP synthesis pathway distinct from that recruited by Iso, as it was associated with a reversible acceleration in Ih activation kinetics and an occlusion of modulation by photolytically released cAMP, yet showed an 11 mV as opposed to a 6 mV positive shift in the activation curve and an at least seven-fold increase in duration. GABA, in the presence of the GABAA antagonist picrotoxin, mimicked, whereas N-ethylmaleimide, an inhibitor of Gi-proteins, blocked the up-regulation, supporting a requirement for GABAB receptor activation in the potentiation. Activation of synaptic GABAB responses via stimulation of inhibitory afferents from the nucleus reticularis potentiated Iso-induced increments in Ih, suggesting that synaptically located receptors couple positively to cAMP synthesis induced by beta-adrenergic receptors. These findings indicate that distinct pathways of cAMP synthesis target the pacemaker current and the recruitment of these may be controlled by GABAergic activity within thalamic networks.

Regulation of recombinant and native hyperpolarization-activated cation channels.

Ionic currents generated by hyperpolarization-activated cation-nonselective (HCN) channels have been principally known as pacemaker h-currents (Ih), because they allow cardiac and neuronal cells to be rhythmically active over precise intervals of time. Presently, these currents are implicated in numerous additional cellular functions, including neuronal integration, synaptic transmission, and sensory reception. These roles are accomplished by virtue of the regulation of Ih by both voltage and ligands. The article summarizes recent developments on the properties and allosteric interactions of these two regulatory pathways in cloned and native channels. Additionally, it discusses how the expression and properties of native channels may be controlled via regulation of the transcription of the HCN channel gene family and the assembly of channel subunits. Recently, several cardiac and neurological diseases were found to be intimately associated with a dysregulation of HCN gene transcription, suggesting that HCN-mediated currents may be involved in the pathophysiology of excitable systems. As a starting point, we briefly review the general characteristics of Ih and the regulatory mechanisms identified in heterologously expressed HCN channels.