Misha Zilberter

Amyloid β-Induced Neuronal Hyperexcitability Triggers Progressive Epilepsy

Alzheimers disease is associated with an increased risk of unprovoked seizures. However, the underlying mechanisms of seizure induction remain elusive. Here, we performed video-EEG recordings in mice carrying mutant human APPswe and PS1dE9 genes (APdE9 mice) and their wild-type littermates to determine the prevalence of unprovoked seizures. In two recording episodes at the onset of amyloid β (Aβ) pathogenesis (3 and 4.5 months of age), at least one unprovoked seizure was detected in 65% of APdE9 mice, of which 46% had multiple seizures and 38% had a generalized seizure. None of the wild-type mice had seizures. In a subset of APdE9 mice, seizure phenotype was associated with a loss of calbindin-D28k immunoreactivity in dentate granular cells and ectopic expression of neuropeptide Y in mossy fibers. In APdE9 mice, persistently decreased resting membrane potential in neocortical layer 2/3 pyramidal cells and dentate granule cells underpinned increased network excitability as identified by patch-clamp electrophysiology. At stimulus strengths evoking single-component EPSPs in wild-type littermates, APdE9 mice exhibited decreased action potential threshold and burst firing of pyramidal cells. Bath application (1 h) of Aβ1–42 or Aβ25–35 (proto-)fibrils but not oligomers induced significant membrane depolarization of pyramidal cells and increased the activity of excitatory cell populations as measured by extracellular field recordings in the juvenile rodent brain, confirming the pathogenic significance of bath-applied Aβ (proto-)fibrils. Overall, these data identify fibrillar Aβ as a pathogenic entity powerfully altering neuronal membrane properties such that hyperexcitability of pyramidal cells culminates in epileptiform activity.

Molecular reorganization of endocannabinoid signalling in Alzheimer’s disease

Retrograde messengers adjust the precise timing of neurotransmitter release from the presynapse, thus modulating synaptic efficacy and neuronal activity. 2-Arachidonoyl glycerol, an endocannabinoid, is one such messenger produced in the postsynapse that inhibits neurotransmitter release upon activating presynaptic CB(1) cannabinoid receptors. Cognitive decline in Alzheimers disease is due to synaptic failure in hippocampal neuronal networks. We hypothesized that errant retrograde 2-arachidonoyl glycerol signalling impairs synaptic neurotransmission in Alzheimers disease. Comparative protein profiling and quantitative morphometry showed that overall CB(1) cannabinoid receptor protein levels in the hippocampi of patients with Alzheimers disease remain unchanged relative to age-matched controls, and CB(1) cannabinoid receptor-positive presynapses engulf amyloid-β-containing senile plaques. Hippocampal protein concentrations for the sn-1-diacylglycerol lipase α and β isoforms, synthesizing 2-arachidonoyl glycerol, significantly increased in definite Alzheimers (Braak stage VI), with ectopic sn-1-diacylglycerol lipase β expression found in microglia accumulating near senile plaques and apposing CB(1) cannabinoid receptor-positive presynapses. We found that microglia, expressing two 2-arachidonoyl glycerol-degrading enzymes, serine hydrolase α/β-hydrolase domain-containing 6 and monoacylglycerol lipase, begin to surround senile plaques in probable Alzheimers disease (Braak stage III). However, Alzheimers pathology differentially impacts serine hydrolase α/β-hydrolase domain-containing 6 and monoacylglycerol lipase in hippocampal neurons: serine hydrolase α/β-hydrolase domain-containing 6 expression ceases in neurofibrillary tangle-bearing pyramidal cells. In contrast, pyramidal cells containing hyperphosphorylated tau retain monoacylglycerol lipase expression, although at levels significantly lower than in neurons lacking neurofibrillary pathology. Here, monoacylglycerol lipase accumulates in CB(1) cannabinoid receptor-positive presynapses. Subcellular fractionation revealed impaired monoacylglycerol lipase recruitment to biological membranes in post-mortem Alzheimers tissues, suggesting that disease progression slows the termination of 2-arachidonoyl glycerol signalling. We have experimentally confirmed that altered 2-arachidonoyl glycerol signalling could contribute to synapse silencing in Alzheimers disease by demonstrating significantly prolonged depolarization-induced suppression of inhibition when superfusing mouse hippocampi with amyloid-β. We propose that the temporal dynamics and cellular specificity of molecular rearrangements impairing 2-arachidonoyl glycerol availability and actions may differ from those of anandamide. Thus, enhanced endocannabinoid signalling, particularly around senile plaques, can exacerbate synaptic failure in Alzheimers disease.

Non-fibrillar β-amyloid abates spike-timing-dependent synaptic potentiation at excitatory synapses in layer 2/3 of the neocortex by targeting postsynaptic AMPA receptors

Cognitive decline in Alzheimers disease (AD) stems from the progressive dysfunction of synaptic connections within cortical neuronal microcircuits. Recently, soluble amyloid β protein oligomers (Aβols) have been identified as critical triggers for early synaptic disorganization. However, it remains unknown whether a deficit of Hebbian‐related synaptic plasticity occurs during the early phase of AD. Therefore, we studied whether age‐dependent Aβ accumulation affects the induction of spike‐timing‐dependent synaptic potentiation at excitatory synapses on neocortical layer 2/3 (L2/3) pyramidal cells in the APPswe/PS1dE9 transgenic mouse model of AD. Synaptic potentiation at excitatory synapses onto L2/3 pyramidal cells was significantly reduced at the onset of Aβ pathology and was virtually absent in mice with advanced Aβ burden. A decreased α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionate (AMPA)/N‐methyl‐d‐aspartate (NMDA) receptor‐mediated current ratio implicated postsynaptic mechanisms underlying Aβ synaptotoxicity. The integral role of Aβols in these processes was verified by showing that pretreatment of cortical slices with Aβ(25−35)ols disrupted spike‐timing‐dependent synaptic potentiation at unitary connections between L2/3 pyramidal cells, and reduced the amplitude of miniature excitatory postsynaptic currents therein. A robust decrement of AMPA, but not NMDA, receptor‐mediated currents in nucleated patches from L2/3 pyramidal cells confirmed that Aβols perturb basal glutamatergic synaptic transmission by affecting postsynaptic AMPA receptors. Inhibition of AMPA receptor desensitization by cyclothiazide significantly increased the amplitude of excitatory postsynaptic potentials evoked by afferent stimulation, and rescued synaptic plasticity even in mice with pronounced Aβ pathology. We propose that soluble Aβols trigger the diminution of synaptic plasticity in neocortical pyramidal cell networks during early stages of AD pathogenesis by preferentially targeting postsynaptic AMPA receptors.

System A Transporter SAT2 Mediates Replenishment of Dendritic Glutamate Pools Controlling Retrograde Signaling by Glutamate

Glutamate mediates several modes of neurotransmission in the central nervous system including recently discovered retrograde signaling from neuronal dendrites. We have previously identified the system N transporter SN1 as being responsible for glutamine efflux from astroglia and proposed a system A transporter (SAT) in subsequent transport of glutamine into neurons for neurotransmitter regeneration. Here, we demonstrate that SAT2 expression is primarily confined to glutamatergic neurons in many brain regions with SAT2 being predominantly targeted to the somatodendritic compartments in these neurons. SAT2 containing dendrites accumulate high levels of glutamine. Upon electrical stimulation in vivo and depolarization in vitro, glutamine is readily converted to glutamate in activated dendritic subsegments, suggesting that glutamine sustains release of the excitatory neurotransmitter via exocytosis from dendrites. The system A inhibitor MeAIB (alpha-methylamino-iso-butyric acid) reduces neuronal uptake of glutamine with concomitant reduction in intracellular glutamate concentrations, indicating that SAT2-mediated glutamine uptake can be a prerequisite for the formation of glutamate. Furthermore, MeAIB inhibited retrograde signaling from pyramidal cells in layer 2/3 of the neocortex by suppressing inhibitory inputs from fast-spiking interneurons. In summary, we demonstrate that SAT2 maintains a key metabolic glutamine/glutamate balance underpinning retrograde signaling by dendritic release of the neurotransmitter glutamate.

Glycolysis and oxidative phosphorylation in neurons and astrocytes during network activity in hippocampal slices.

Network activation triggers a significant energy metabolism increase in both neurons and astrocytes. Questions of the primary neuronal energy substrate (e.g., glucose vs. lactate) as well as the relative contributions of glycolysis and oxidative phosphorylation and their cellular origin (neurons vs. astrocytes) are still a matter of debates. Using simultaneous measurements of electrophysiological and metabolic parameters during synaptic stimulation in hippocampal slices from mature mice, we show that neurons and astrocytes use both glycolysis and oxidative phosphorylation to meet their energy demands. Supplementation or replacement of glucose in artificial cerebrospinal fluid (ACSF) with pyruvate or lactate strongly modifies parameters related to network activity-triggered energy metabolism. These effects are not induced by changes in ATP content, pHi, [Ca2+]i or accumulation of reactive oxygen species. Our results suggest that during network activation, a significant fraction of NAD(P)H response (its overshoot phase) corresponds to glycolysis and the changes in cytosolic NAD(P)H and mitochondrial FAD are coupled. Our data do not support the hypothesis of a preferential utilization of astrocyte-released lactate by neurons during network activation in slices—instead, we show that during such activity glucose is an effective energy substrate for both neurons and astrocytes.

Secretagogin is a Ca2+-binding protein specifying subpopulations of telencephalic neurons

The Ca2+-binding proteins (CBPs) parvalbumin, calbindin, and calretinin are phenotypic markers of terminally differentiated neurons in the adult brain. Although subtle phylogenetic variations in the neuronal distribution of these CBPs may occur, morphologically and functionally diverse subclasses of interneurons harbor these proteins in olfactory and corticolimbic areas. Secretagogin (scgn) is a recently cloned CBP from pancreatic β and neuroendocrine cells. We hypothesized that scgn is expressed in the mammalian brain. We find that scgn is a marker of neuroblasts commuting in the rostral migratory stream. Terminally differentiated neurons in the olfactory bulb retain scgn expression, with scgn being present in periglomerular cells and granular layer interneurons. In the corticolimbic system, scgn identifies granule cells distributed along the dentate gyrus, indusium griseum, and anterior hippocampal continuation emphasizing the shared developmental origins, and cytoarchitectural and functional similarities of these neurons. We also uncover unexpected phylogenetic differences in scgn expression, since this CBP is restricted to primate cholinergic basal forebrain neurons. Overall, we characterize scgn as a neuron-specific CBP whose distribution identifies neuronal subtypes and hierarchical organizing principles in the mammalian brain.

Dietary energy substrates reverse early neuronal hyperactivity in a mouse model of Alzheimer's disease.

Deficient energy metabolism and network hyperactivity are the early symptoms of Alzheimers disease (AD). In this study, we show that administration of exogenous oxidative energy substrates (OES) corrects neuronal energy supply deficiency that reduces the amyloid‐beta‐induced abnormal neuronal activity in vitro and the epileptic phenotype in AD model in vivo. In vitro, acute application of protofibrillar amyloid‐β1–42 (Aβ1–42) induced aberrant network activity in wild‐type hippocampal slices that was underlain by depolarization of both the neuronal resting membrane potential and GABA‐mediated current reversal potential. Aβ1–42 also impaired synaptic function and long‐term potentiation. These changes were paralleled by clear indications of impaired energy metabolism, as indicated by abnormal NAD(P)H signaling induced by network activity. However, when glucose was supplemented with OES pyruvate and 3‐beta‐hydroxybutyrate, Aβ1–42 failed to induce detrimental changes in any of the above parameters. We administered the same OES as chronic supplementation to a standard diet to APPswe/PS1dE9 transgenic mice displaying AD‐related epilepsy phenotype. In the ex‐vivo slices, we found neuronal subpopulations with significantly depolarized resting and GABA‐mediated current reversal potentials, mirroring abnormalities we observed under acute Aβ1‐42 application. Ex‐vivo cortex of transgenic mice fed with standard diet displayed signs of impaired energy metabolism, such as abnormal NAD(P)H signaling and strongly reduced tolerance to hypoglycemia. Transgenic mice also possessed brain glycogen levels twofold lower than those of wild‐type mice. However, none of the above neuronal and metabolic dysfunctions were observed in transgenic mice fed with the OES‐enriched diet. In vivo, dietary OES supplementation abated neuronal hyperexcitability, as the frequency of both epileptiform discharges and spikes was strongly decreased in the APPswe/PS1dE9 mice placed on the diet. Altogether, our results suggest that early AD‐related neuronal malfunctions underlying hyperexcitability and energy metabolism deficiency can be prevented by dietary supplementation with native energy substrates.

Input specificity and dependence of spike timing-dependent plasticity on preceding postsynaptic activity at unitary connections between neocortical layer 2/3 pyramidal cells

Layer 2/3 (L2/3) pyramidal cells receive excitatory afferent input both from neighbouring pyramidal cells and from cortical and subcortical regions. The efficacy of these excitatory synaptic inputs is modulated by spike timing-dependent plasticity (STDP). Here we report that synaptic connections between L2/3 pyramidal cell pairs are located proximal to the soma, at sites overlapping those of excitatory inputs from other cortical layers. Nevertheless, STDP at L2/3 pyramidal to pyramidal cell connections showed fundamental differences from known STDP rules at these neighbouring contacts. Coincident low-frequency pre- and postsynaptic activation evoked only LTD, independent of the order of the pre- and postsynaptic cell firing. This symmetric anti-Hebbian STDP switched to a typical Hebbian learning rule if a postsynaptic action potential train occurred prior to the presynaptic stimulation. Receptor dependence of LTD and LTP induction and their pre- or postsynaptic loci also differed from those at other L2/3 pyramidal cell excitatory inputs. Overall, we demonstrate a novel means to switch between STDP rules dependent on the history of postsynaptic activity. We also highlight differences in STDP at excitatory synapses onto L2/3 pyramidal cells which allow for input specific modulation of synaptic gain.

Amyloid-β-induced action potential desynchronization and degradation of hippocampal gamma oscillations is prevented by interference with peptide conformation change and aggregation.

The amyloid-β hypothesis of Alzheimers Disease (AD) focuses on accumulation of amyloid-β peptide (Aβ) as the main culprit for the myriad physiological changes seen during development and progression of AD including desynchronization of neuronal action potentials, consequent development of aberrant brain rhythms relevant for cognition, and final emergence of cognitive deficits. The aim of this study was to elucidate the cellular and synaptic mechanisms underlying the Aβ-induced degradation of gamma oscillations in AD, to identify aggregation state(s) of Aβ that mediate the peptides neurotoxicity, and to test ways to prevent the neurotoxic Aβ effect. We show that Aβ1-42 in physiological concentrations acutely degrades mouse hippocampal gamma oscillations in a concentration- and time-dependent manner. The underlying cause is an Aβ-induced desynchronization of action potential generation in pyramidal cells and a shift of the excitatory/inhibitory equilibrium in the hippocampal network. Using purified preparations containing different aggregation states of Aβ, as well as a designed ligand and a BRICHOS chaperone domain, we provide evidence that the severity of Aβ neurotoxicity increases with increasing concentration of fibrillar over monomeric Aβ forms, and that Aβ-induced degradation of gamma oscillations and excitatory/inhibitory equilibrium is prevented by compounds that interfere with Aβ aggregation. Our study provides correlative evidence for a link between Aβ-induced effects on synaptic currents and AD-relevant neuronal network oscillations, identifies the responsible aggregation state of Aβ and proofs that strategies preventing peptide aggregation are able to prevent the deleterious action of Aβ on the excitatory/inhibitory equilibrium and on the gamma rhythm.

Chronic inhibition of brain glycolysis initiates epileptogenesis.

Metabolic abnormalities found in epileptogenic tissue provide considerable evidence of brain hypometabolism, while major risk factors for acquired epilepsy all share brain hypometabolism as one common outcome, suggesting that a breakdown of brain energy homeostasis may actually precede epileptogenesis. However, a causal link between deficient brain energy metabolism and epilepsy initiation has not been yet established. To address this issue we developed an in vivo model of chronic energy hypometabolism by daily intracerebroventricular (i.c.v.) injection of the nonmetabolizable glucose analog 2‐deoxy‐D‐glucose (2‐DG) and also investigated acute effects of 2‐DG on the cellular level. In hippocampal slices, acute glycolysis inhibition by 2‐DG (by about 35%) led to contrasting effects on the network: a downregulation of excitatory synaptic transmission together with a depolarization of neuronal resting potential and a decreased drive of inhibitory transmission. Therefore, the potential acute effect of 2‐DG on network excitability depends on the balance between these opposing pre‐ and postsynaptic changes. In vivo, we found that chronic 2‐DG i.c.v. application (estimated transient inhibition of brain glycolysis under 14%) for a period of 4 weeks induced epileptiform activity in initially healthy male rats. Our results suggest that chronic inhibition of brain energy metabolism, characteristics of the well‐established risk factors of acquired epilepsy, and specifically a reduction in glucose utilization (typically observed in epileptic patients) can initiate epileptogenesis.