APOE4-promoted gliosis and degeneration in tauopathy are ameliorated by pharmacological inhibition of HMGB1 release.

Apolipoprotein E4 (APOE4) is an important driver of Tau pathology, gliosis, and degeneration in Alzheimer's disease (AD). Still, the mechanisms underlying these APOE4-driven pathological effects remain elusive. Here, we report in a tauopathy mouse model that APOE4 promoted the nucleocytoplasmic translocation and release of high-mobility group box 1 (HMGB1) from hippocampal neurons, which correlated with the severity of hippocampal microgliosis and degeneration. Injection of HMGB1 into the hippocampus of young APOE4-tauopathy mice induced considerable and persistent gliosis. Selective removal of neuronal APOE4 reduced HMGB1 translocation and release. Treatment of APOE4-tauopathy mice with HMGB1 inhibitors effectively blocked the intraneuronal translocation and release of HMGB1 and ameliorated the development of APOE4-driven gliosis, Tau pathology, neurodegeneration, and myelin deficits. Single-nucleus RNA sequencing revealed that treatment with HMGB1 inhibitors diminished disease-associated and enriched disease-protective subpopulations of neurons, microglia, and astrocytes in APOE4-tauopathy mice. Thus, HMGB1 inhibitors represent a promising approach for treating APOE4-related AD.

NOX-induced oxidative stress is a primary trigger of major neurodegenerative disorders.

Neurodegenerative diseases (NDDs) causing cognitive impairment and dementia are difficult to treat due to the lack of understanding of primary initiating factors. Meanwhile, major sporadic NDDs share many risk factors and exhibit similar pathologies in their early stages, indicating the existence of common initiation pathways. Glucose hypometabolism associated with oxidative stress is one such primary, early and shared pathology, and a likely major cause of detrimental disease-associated cascades; targeting this common pathology may therefore be an effective preventative strategy for most sporadic NDDs. However, its exact cause and trigger remain unclear. Recent research suggests that early oxidative stress caused by NADPH oxidase (NOX) activation is a shared initiating mechanism among major sporadic NDDs and could prove to be the long-sought ubiquitous NDD trigger. We focus on two major NDDs - Alzheimer's disease (AD) and Parkinson's disease (PD), as well as on acquired epilepsy which is an increasingly recognized comorbidity in NDDs. We also discuss available data suggesting the relevance of the proposed mechanisms to other NDDs. We delve into the commonalities among these NDDs in neuroinflammation and NOX involvement to identify potential therapeutic targets and gain a deeper understanding of the underlying causes of NDDs.

Neuronal apoE4 induces early hyperexcitability in select populations of hippocampal neurons by altering Nell2 expression.

The impact of apolipoprotein E4 (apoE4), the strongest genetic risk factor for Alzheimer's disease (AD), on neuronal function remains unclear. We investigated this by examining excitatory neurons in the hippocampus of young and aged human apoE4 knock-in (apoE4-KI) and apoE3-KI mice using electrophysiology and single-nucleus RNA-sequencing (snRNA-seq). In young apoE4-KI mice, we identified region-specific subpopulations of excitatory neurons with hyperexcitability underlain by reduced cell size, which were eliminated by selective removal of neuronal apoE4. Aged apoE4-KI mice showed an increased fraction of hyperexcitable granule cells, a pronounced inhibitory deficit, and E/I imbalance in the dentate gyrus, contributing to network dysfunction. snRNA-seq analysis revealed neuron type-specific and age-dependent transcriptomic changes, identifying Nell2 overexpression in apoE4-KI mice. Reducing Nell2 expression in specific neuronal types of apoE4-KI mice with CRISPRi rescued their morphological and excitability phenotypes, supporting Nell2 overexpression as a cause for apoE4-induced neuronal dysfunction. Our findings highlight the early transcriptomic and morpho-electric alterations behind the apoE4-induced neuronal dysfunction in AD. HIGHLIGHTS: ApoE4 causes hyperexcitability of select hippocampal neurons in young apoE4 mice.ApoE4 causes dentate hyperexcitability and inhibitory deficit in aged apoE4 mice.snRNA-seq reveals apoE genotype-, cell type-, and age-dependent transcriptomic changes.Nell2 overexpression identified as a cause of apoE4-induced neuronal hyperexcitability.

Neurons require glucose uptake and glycolysis in vivo.

Neurons require large amounts of energy, but whether they can perform glycolysis or require glycolysis to maintain energy remains unclear. Using metabolomics, we show that human neurons do metabolize glucose through glycolysis and can rely on glycolysis to supply tricarboxylic acid (TCA) cycle metabolites. To investigate the requirement for glycolysis, we generated mice with postnatal deletion of either the dominant neuronal glucose transporter (GLUT3cKO) or the neuronal-enriched pyruvate kinase isoform (PKM1cKO) in CA1 and other hippocampal neurons. GLUT3cKO and PKM1cKO mice show age-dependent learning and memory deficits. Hyperpolarized magnetic resonance spectroscopic (MRS) imaging shows that female PKM1cKO mice have increased pyruvate-to-lactate conversion, whereas female GLUT3cKO mice have decreased conversion, body weight, and brain volume. GLUT3KO neurons also have decreased cytosolic glucose and ATP at nerve terminals, with spatial genomics and metabolomics revealing compensatory changes in mitochondrial bioenergetics and galactose metabolism. Therefore, neurons metabolize glucose through glycolysis in vivo and require glycolysis for normal function.

Neuronal APOE4 removal protects against tau-mediated gliosis, neurodegeneration and myelin deficits.

Apolipoprotein E4 (APOE4) is the strongest known genetic risk factor for late-onset Alzheimer's disease (AD). Conditions of stress or injury induce APOE expression within neurons, but the role of neuronal APOE4 in AD pathogenesis is still unclear. Here we report the characterization of neuronal APOE4 effects on AD-related pathologies in an APOE4-expressing tauopathy mouse model. The selective genetic removal of APOE4 from neurons led to a significant reduction in tau pathology, gliosis, neurodegeneration, neuronal hyperexcitability and myelin deficits. Single-nucleus RNA-sequencing revealed that the removal of neuronal APOE4 greatly diminished neurodegenerative disease-associated subpopulations of neurons, oligodendrocytes, astrocytes and microglia whose accumulation correlated to the severity of tau pathology, neurodegeneration and myelin deficits. Thus, neuronal APOE4 plays a central role in promoting the development of major AD pathologies and its removal can mitigate the progressive cellular and tissue alterations occurring in this model of APOE4-driven tauopathy.

Unifying mechanism behind the onset of acquired epilepsy.

Acquired epilepsy (AE) can result from a number of brain insults and neurological diseases with wide etiological diversity sharing one common outcome of brain epileptiform activity. This implies that despite their disparity, all these initiating pathologies affect the same fundamental brain functions underlying network excitability. Identifying such mechanisms and their availability as therapeutic targets would help develop an effective strategy for epileptogenesis prevention. In this opinion article, we propose that the vicious cycle of NADPH oxidase (NOX)-mediated oxidative stress and glucose hypometabolism is the underlying cause of AE, as available data reveal a critical role for both pathologies in epileptogenesis and the process of seizure initiation. Altogether, here we present a novel view on the mechanisms behind the onset of AE and identify therapeutic targets for potential clinical applications.

Dentate gyrus and CA3 GABAergic interneurons bidirectionally modulate signatures of internal and external drive to CA1.

Specific classes of GABAergic neurons play specific roles in regulating information processing in the brain. In the hippocampus, two major classes, parvalbumin-expressing (PV+) and somatostatin-expressing (SST+), differentially regulate endogenous firing patterns and target subcellular compartments of principal cells. How these classes regulate the flow of information throughout the hippocampus is poorly understood. We hypothesize that PV+ and SST+ interneurons in the dentate gyrus (DG) and CA3 differentially modulate CA3 patterns of output, thereby altering the influence of CA3 on CA1. We find that while suppressing either interneuron class increases DG and CA3 output, the effects on CA1 were very different. Suppressing PV+ interneurons increases local field potential signatures of coupling from CA3 to CA1 and decreases signatures of coupling from entorhinal cortex to CA1; suppressing SST+ interneurons has the opposite effect. Thus, DG and CA3 PV+ and SST+ interneurons bidirectionally modulate the flow of information through the hippocampal circuit.

Aß initiates brain hypometabolism, network dysfunction and behavioral abnormalities via NOX2-induced oxidative stress in mice.

A predominant trigger and driver of sporadic Alzheimer's disease (AD) is the synergy of brain oxidative stress and glucose hypometabolism starting at early preclinical stages. Oxidative stress damages macromolecules, while glucose hypometabolism impairs cellular energy supply and antioxidant defense. However, the exact cause of AD-associated glucose hypometabolism and its network consequences have remained unknown. Here we report NADPH oxidase 2 (NOX2) activation as the main initiating mechanism behind Aß1-42-related glucose hypometabolism and network dysfunction. We utilize a combination of electrophysiology with real-time recordings of metabolic transients both ex- and in-vivo to show that Aß1-42 induces oxidative stress and acutely reduces cellular glucose consumption followed by long-lasting network hyperactivity and abnormalities in the animal behavioral profile. Critically, all of these pathological changes were prevented by the novel bioavailable NOX2 antagonist GSK2795039. Our data provide direct experimental evidence for causes and consequences of AD-related brain glucose hypometabolism, and suggest that targeting NOX2-mediated oxidative stress is a promising approach to both the prevention and treatment of AD.

Adult trkB Signaling in Parvalbumin Interneurons is Essential to Prefrontal Network Dynamics.

Inhibitory interneurons expressing parvalbumin (PV) are central to cortical network dynamics, generation of γ oscillations, and cognition. Dysfunction of PV interneurons disrupts cortical information processing and cognitive behavior. Brain-derived neurotrophic factor (BDNF)/tyrosine receptor kinase B (trkB) signaling regulates the maturation of cortical PV interneurons but is also implicated in their adult multidimensional functions. Using a novel viral strategy for cell-type-specific and spatially restricted expression of a dominant-negative trkB (trkB.DN), we show that BDNF/trkB signaling is essential to the integrity and maintenance of prefrontal PV interneurons in adult male and female mice. Reduced BDNF/trkB signaling in PV interneurons in the medial prefrontal cortex (mPFC) resulted in deficient PV inhibition and increased baseline local field potential (LFP) activity in a broad frequency band. The altered network activity was particularly pronounced during increased activation of the prefrontal network and was associated with changed dynamics of local excitatory neurons, as well as decreased modulation of the LFP, abnormalities that appeared to generalize across stimuli and brain states. In addition, our findings link reduced BDNF/trkB signaling in prefrontal PV interneurons to increased aggression. Together our investigations demonstrate that BDNF/trkB signaling in PV interneurons in the adult mPFC is essential to local network dynamics and cognitive behavior. Our data provide direct support for the suggested association between decreased trkB signaling, deficient PV inhibition, and altered prefrontal circuitry.SIGNIFICANCE STATEMENT Brain-derived neurotrophic factor (BDNF)/tyrosine receptor kinase B (trkB) signaling promotes the maturation of inhibitory parvalbumin (PV) interneurons, neurons central to local cortical dynamics, γ rhythms, and cognition. Here, we used a novel viral approach for reduced BDNF/trkB signaling in PV interneurons in the medial prefrontal cortex (mPFC) to establish the role of BDNF/trkB signaling in adult prefrontal network activities. Reduced BDNF/trkB signaling caused pronounced morphologic alterations, reduced PV inhibition, and deficient prefrontal network dynamics. The altered network activity appeared to manifest across stimuli and brain states and was associated with aberrant local field potential (LFP) activities and increased aggression. The results demonstrate that adult BDNF/trkB signaling is essential to PV inhibition and prefrontal circuit function and directly links BDNF/trkB signaling to network integrity in the adult brain.

Experimental and real-world evidence supporting the computational repurposing of bumetanide for APOE4-related Alzheimer's disease.

The evident genetic, pathological, and clinical heterogeneity of Alzheimer's disease (AD) poses challenges for traditional drug development. We conducted a computational drug repurposing screen for drugs to treat apolipoprotein (apo) E4-related AD. We first established apoE-genotype-dependent transcriptomic signatures of AD by analyzing publicly-available human brain database. We then queried these signatures against the Connectivity Map database containing transcriptomic perturbations of >1300 drugs to identify those that best reverse apoE-genotype-specific AD signatures. Bumetanide was identified as a top drug for apoE4 AD. Bumetanide treatment of apoE4 mice without or with Aß accumulation rescued electrophysiological, pathological, or cognitive deficits. Single-nucleus RNA-sequencing revealed transcriptomic reversal of AD signatures in specific cell types in these mice, a finding confirmed in apoE4-iPSC-derived neurons. In humans, bumetanide exposure was associated with a significantly lower AD prevalence in individuals over the age of 65 in two electronic health record databases, suggesting effectiveness of bumetanide in preventing AD.

In Vivo Chimeric Alzheimer's Disease Modeling of Apolipoprotein E4 Toxicity in Human Neurons.

Despite its clear impact on Alzheimer's disease (AD) risk, apolipoprotein (apo) E4's contributions to AD etiology remain poorly understood. Progress in answering this and other questions in AD research has been limited by an inability to model human-specific phenotypes in an in vivo environment. Here we transplant human induced pluripotent stem cell (hiPSC)-derived neurons carrying normal apoE3 or pathogenic apoE4 into human apoE3 or apoE4 knockin mouse hippocampi, enabling us to disentangle the effects of apoE4 produced in human neurons and in the brain environment. Using single-nucleus RNA sequencing (snRNA-seq), we identify key transcriptional changes specific to human neuron subtypes in response to endogenous or exogenous apoE4. We also find that Aß from transplanted human neurons forms plaque-like aggregates, with differences in localization and interaction with microglia depending on the transplant and host apoE genotype. These findings highlight the power of in vivo chimeric disease modeling for studying AD.

HCN Channel Activity Balances Quiescence and Proliferation in Neural Stem Cells and Is a Selective Target for Neuroprotection During Cancer Treatment.

Children suffering from neurologic cancers undergoing chemotherapy and radiotherapy are at high risk of reduced neurocognitive abilities likely via damage to proliferating neural stem cells (NSC). Therefore, strategies to protect NSCs are needed. We argue that induced cell-cycle arrest/quiescence in NSCs during cancer treatment can represent such a strategy. Here, we show that hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels are dynamically expressed over the cell cycle in NSCs, depolarize the membrane potential, underlie spontaneous calcium oscillations and are required to maintain NSCs in the actively proliferating pool. Hyperpolarizing pharmacologic inhibition of HCN channels during exposure to ionizing radiation protects NSCs cells in neurogenic brain regions of young mice. In contrast, brain tumor-initiating cells, which also express HCN channels, remain proliferative during HCN inhibition. IMPLICATIONS: Our finding that NSCs can be selectively rescued while cancer cells remain sensitive to the treatment, provide a foundation for reduction of cognitive impairment in children with neurologic cancers.

Activation of nicotinamide adenine dinucleotide phosphate oxidase is the primary trigger of epileptic seizures in rodent models.

OBJECTIVE: Despite decades of epilepsy research, 30% of focal epilepsies remain resistant to antiseizure drugs, with effective drug development impeded by lack of understanding on how seizures are initiated. Here, we report the mechanism of seizure onset relevant to most seizures that are characteristic of focal epilepsies. METHODS: Electric and metabolic network parameters were measured using several seizure models in mouse hippocampal slices and acutely induced seizures in rats in vivo to determine metabolic events occurring at seizure onset. RESULTS: We show that seizure onset is associated with a rapid release of H2 O2 resulting from N-methyl-D-aspartate (NMDA) receptor-mediated activation of nicotinamide adenine dinucleotide phosphate oxidase (NOX). NOX blockade prevented the fast H2 O2 release as well as the direct current shift and seizurelike event induction in slices. Similarly, intracerebroventricular injection of NOX antagonists prevented acutely induced seizures in rats. INTERPRETATION: Our results show that seizures are initiated by NMDA receptor-mediated NOX-induced oxidative stress and can be arrested by NOX inhibition. We introduce a novel use for blood-brain barrier-permeable NOX inhibitor with a significant potential to become the first seizure-specific medication. Thus, targeting NOX may provide a breakthrough treatment for focal epilepsies. ANN NEUROL 2019;85:907-920.

Seizure-induced reduction in glucose utilization promotes brain hypometabolism during epileptogenesis.

Brain glucose hypometabolism is an early symptom of acquired epilepsy, its causative mechanism yet unclear. We suggest that a bidirectional positive feedback linking seizures and hypometabolism (hypometabolism induces seizures while seizures disrupt glucose metabolism) may be a primary cause for acquired epileptogenesis. We reported recently that chronic partial inhibition of brain glycolysis triggers epileptogenesis in healthy rats. Here, by monitoring dynamic electrical and multiple metabolic parameters before and following seizure generation in mouse hippocampal slices using the 4-aminopyridine model of epileptiform activity, we show that in turn seizures are followed by a long-lasting glucose hypometabolism, indicating possible existence of a positive feedback in the mechanism of epileptogenesis. Seizures were associated with acute oxidative stress that may contribute to the subsequent glucose metabolism impairment, since exogenous application of H2O2 replicated the post-seizure metabolic effects. Exogenous pyruvate, the principal mitochondrial energy substrate with a broad spectrum of neuroprotective properties, effectively normalized the post-seizure glucose consumption. We have shown recently that pyruvate exhibited a strong antiepileptic action in three rodent chronic epilepsy models, while in the present study we find that pyruvate effectively normalizes impaired glucose metabolism following seizures. Together, our results provide the mechanistic basis for the metabolic concept of acquired epileptogenesis and an efficient treatment strategy.

The vicious circle of hypometabolism in neurodegenerative diseases: Ways and mechanisms of metabolic correction.

Hypometabolism, characterized by decreased brain glucose consumption, is a common feature of many neurodegenerative diseases. Initial hypometabolic brain state, created by characteristic risk factors, may predispose the brain to acquired epilepsy and sporadic Alzheimer's and Parkinson's diseases, which are the focus of this review. Analysis of available data suggests that deficient glucose metabolism is likely a primary initiating factor for these diseases, and that resulting neuronal dysfunction further promotes the metabolic imbalance, establishing an effective positive feedback loop and a downward spiral of disease progression. Therefore, metabolic correction leading to the normalization of abnormalities in glucose metabolism may be an efficient tool to treat the neurological disorders by counteracting their primary pathological mechanisms. Published and preliminary experimental results on this approach for treating Alzheimer's disease and epilepsy models support the efficacy of metabolic correction, confirming the highly promising nature of the strategy. © 2017 Wiley Periodicals, Inc.

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. © 2017 Wiley Periodicals, Inc.

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

The amyloid-ß hypothesis of Alzheimer's Disease (AD) focuses on accumulation of amyloid-ß peptide (Aß) 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 Aß-induced degradation of gamma oscillations in AD, to identify aggregation state(s) of Aß that mediate the peptides neurotoxicity, and to test ways to prevent the neurotoxic Aß effect. We show that Aß(1-42) in physiological concentrations acutely degrades mouse hippocampal gamma oscillations in a concentration- and time-dependent manner. The underlying cause is an Aß-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 Aß, as well as a designed ligand and a BRICHOS chaperone domain, we provide evidence that the severity of Aß neurotoxicity increases with increasing concentration of fibrillar over monomeric Aß forms, and that Aß-induced degradation of gamma oscillations and excitatory/inhibitory equilibrium is prevented by compounds that interfere with Aß aggregation. Our study provides correlative evidence for a link between Aß-induced effects on synaptic currents and AD-relevant neuronal network oscillations, identifies the responsible aggregation state of Aß and proofs that strategies preventing peptide aggregation are able to prevent the deleterious action of Aß on the excitatory/inhibitory equilibrium and on the gamma rhythm.