Prolonged exposure to lactate causes TREK1 channel clustering in rat hippocampal astrocytes.

Increased concentrations of lactate (15-30 mM) are associated with and found to be neuroprotective in various brain pathophysiology. In our earlier studies we showed that high levels of lactate can increase TREK1 channel activity and expression within 1 h. TREK1 channels are two pore domain leak potassium ion channels that are upregulated during cerebral ischemia, epilepsy and other brain pathologies. They play a prominent neuroprotective role against excitotoxicity. Although it has been previously shown that chronic application of lactate (6 h) causes increased gene transcription and protein expression, we observe clustering of TREK1 channels that is dependent on time of exposure (3-6 h) and concentration of lactate (15-30 mM). Using immunofluorescence techniques and image analysis, we show that the clustering of TREK1 channels is dependent on the actin cytoskeletal network of the astrocytes. Clustering of TREK1 channels can augment astrocytic functions during pathophysiological conditions and have significant implications in lactate mediated neuroprotection.

Aberrant protein S-nitrosylation contributes to hyperexcitability-induced synaptic damage in Alzheimer's disease: Mechanistic insights and potential therapies.

Alzheimer's disease (AD) is arguably the most common cause of dementia in the elderly and is marked by progressive synaptic degeneration, which in turn leads to cognitive decline. Studies in patients and in various AD models have shown that one of the early signatures of AD is neuronal hyperactivity. This excessive electrical activity contributes to dysregulated neural network function and synaptic damage. Mechanistically, evidence suggests that hyperexcitability accelerates production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) that contribute to neural network impairment and synapse loss. This review focuses on the pathways and molecular changes that cause hyperexcitability and how RNS-dependent posttranslational modifications, represented predominantly by protein S-nitrosylation, mediate, at least in part, the deleterious effects of hyperexcitability on single neurons and the neural network, resulting in synaptic loss in AD.

Targeted protein S-nitrosylation of ACE2 inhibits SARS-CoV-2 infection.

Prevention of infection and propagation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a high priority in the Coronavirus Disease 2019 (COVID-19) pandemic. Here we describe S-nitrosylation of multiple proteins involved in SARS-CoV-2 infection, including angiotensin-converting enzyme 2 (ACE2), the receptor for viral entry. This reaction prevents binding of ACE2 to the SARS-CoV-2 spike protein, thereby inhibiting viral entry, infectivity and cytotoxicity. Aminoadamantane compounds also inhibit coronavirus ion channels formed by envelope (E) protein. Accordingly, we developed dual-mechanism aminoadamantane nitrate compounds that inhibit viral entry and, thus, the spread of infection by S-nitrosylating ACE2 via targeted delivery of the drug after E protein channel blockade. These non-toxic compounds are active in vitro and in vivo in the Syrian hamster COVID-19 model and, thus, provide a novel avenue to pursue therapy.

Targeted protein S-nitrosylation of ACE2 as potential treatment to prevent spread of SARS-CoV-2 infection.

Prevention of infection and propagation of SARS-CoV-2 is of high priority in the COVID-19 pandemic. Here, we describe S-nitrosylation of multiple proteins involved in SARS-CoV-2 infection, including angiotensin converting enzyme 2 (ACE2), the receptor for viral entry. This reaction prevents binding of ACE2 to the SARS-CoV-2 Spike protein, thereby inhibiting viral entry, infectivity, and cytotoxicity. Aminoadamantane compounds also inhibit coronavirus ion channels formed by envelope (E) protein. Accordingly, we developed dual-mechanism aminoadamantane nitrate compounds that inhibit viral entry and thus spread of infection by S-nitrosylating ACE2 via targeted delivery of the drug after E-protein channel blockade. These non-toxic compounds are active in vitro and in vivo in the Syrian hamster COVID-19 model, and thus provide a novel avenue for therapy.

Emerging hiPSC Models for Drug Discovery in Neurodegenerative Diseases.

Neurodegenerative diseases affect millions of people worldwide and are characterized by the chronic and progressive deterioration of neural function. Neurodegenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and Huntington's disease (HD), represent a huge social and economic burden due to increasing prevalence in our aging society, severity of symptoms, and lack of effective disease-modifying therapies. This lack of effective treatments is partly due to a lack of reliable models. Modeling neurodegenerative diseases is difficult because of poor access to human samples (restricted in general to postmortem tissue) and limited knowledge of disease mechanisms in a human context. Animal models play an instrumental role in understanding these diseases but fail to comprehensively represent the full extent of disease due to critical differences between humans and other mammals. The advent of human-induced pluripotent stem cell (hiPSC) technology presents an advantageous system that complements animal models of neurodegenerative diseases. Coupled with advances in gene-editing technologies, hiPSC-derived neural cells from patients and healthy donors now allow disease modeling using human samples that can be used for drug discovery.

S-nitrosylated TDP-43 triggers aggregation, cell-to-cell spread, and neurotoxicity in hiPSCs and in vivo models of ALS/FTD.

Rare genetic mutations result in aggregation and spreading of cognate proteins in neurodegenerative disorders; however, in the absence of mutation (i.e., in the vast majority of "sporadic" cases), mechanisms for protein misfolding/aggregation remain largely unknown. Here, we show environmentally induced nitrosative stress triggers protein aggregation and cell-to-cell spread. In patient brains with amyotrophic lateral sclerosis (ALS)/frontotemporal dementia (FTD), aggregation of the RNA-binding protein TDP-43 constitutes a major component of aberrant cytoplasmic inclusions. We identify a pathological signaling cascade whereby reactive nitrogen species cause S-nitrosylation of TDP-43 (forming SNO-TDP-43) to facilitate disulfide linkage and consequent TDP-43 aggregation. Similar pathological SNO-TDP-43 levels occur in postmortem human FTD/ALS brains and in cell-based models, including human-induced pluripotent stem cell (hiPSC)-derived neurons. Aggregated TDP-43 triggers additional nitrosative stress, representing positive feed forward leading to further SNO-TDP-43 formation and disulfide-linked oligomerization/aggregation. Critically, we show that these redox reactions facilitate cell spreading in vivo and interfere with the TDP-43 RNA-binding activity, affecting SNMT1 and phospho-(p)CREB levels, thus contributing to neuronal damage in ALS/FTD disorders.

α-Synuclein Oligomers Induce Glutamate Release from Astrocytes and Excessive Extrasynaptic NMDAR Activity in Neurons, Thus Contributing to Synapse Loss.

Synaptic and neuronal loss are major neuropathological characteristics of Parkinson's disease. Misfolded protein aggregates in the form of Lewy bodies, comprised mainly of α-synuclein (αSyn), are associated with disease progression, and have also been linked to other neurodegenerative diseases, including Lewy body dementia, Alzheimer's disease, and frontotemporal dementia. However, the effects of αSyn and its mechanism of synaptic damage remain incompletely understood. Here, we show that αSyn oligomers induce Ca2+-dependent release of glutamate from astrocytes obtained from male and female mice, and that mice overexpressing αSyn manifest increased tonic release of glutamate in vivo In turn, this extracellular glutamate activates glutamate receptors, including extrasynaptic NMDARs (eNMDARs), on neurons both in culture and in hippocampal slices of αSyn-overexpressing mice. Additionally, in patch-clamp recording from outside-out patches, we found that oligomerized αSyn can directly activate eNMDARs. In organotypic slices, oligomeric αSyn induces eNMDAR-mediated synaptic loss, which can be reversed by the drug NitroSynapsin. When we expose human induced pluripotent stem cell-derived cerebrocortical neurons to αSyn, we find similar effects. Importantly, the improved NMDAR antagonist NitroSynapsin, which selectively inhibits extrasynaptic over physiological synaptic NMDAR activity, protects synapses from oligomeric αSyn-induced damage in our model systems, thus meriting further study for its therapeutic potential.SIGNIFICANCE STATEMENT Loss of synaptic function and ensuing neuronal loss are associated with disease progression in Parkinson's disease (PD), Lewy body dementia (LBD), and other neurodegenerative diseases. However, the mechanism of synaptic damage remains incompletely understood. α-Synuclein (αSyn) misfolds in PD/LBD, forming Lewy bodies and contributing to disease pathogenesis. Here, we found that misfolded/oligomeric αSyn releases excessive astrocytic glutamate, in turn activating neuronal extrasynaptic NMDA receptors (eNMDARs), thereby contributing to synaptic damage. Additionally, αSyn oligomers directly activate eNMDARs, further contributing to damage. While the FDA-approved drug memantine has been reported to offer some benefit in PD/LBD (Hershey and Coleman-Jackson, 2019), we find that the improved eNMDAR antagonist NitroSynapsin ameliorates αSyn-induced synaptic spine loss, providing potential disease-modifying intervention in PD/LBD.

Novel Therapeutic Approach for Excitatory/Inhibitory Imbalance in Neurodevelopmental and Neurodegenerative Diseases.

Excitatory/inhibitory (E/I) balance, defined as the balance between excitation and inhibition of synaptic activity in a neuronal network, accounts in part for the normal functioning of the brain, controlling, for example, normal spike rate. In many pathological conditions, this fine balance is perturbed, leading to excessive or diminished excitation relative to inhibition, termed E/I imbalance, reflected in network dysfunction. E/I imbalance has emerged as a contributor to neurological disorders that occur particularly at the extremes of life, including autism spectrum disorder and Alzheimer's disease, pointing to the vulnerability of neuronal networks at these critical life stages. Hence, it is important to develop approaches to rebalance neural networks. In this review, we describe emerging therapies that can normalize the E/I ratio or the underlying abnormality that contributes to the imbalance in electrical activity, thus improving neurological function in these maladies.

NitroSynapsin ameliorates hypersynchronous neural network activity in Alzheimer hiPSC models.

Beginning at early stages, human Alzheimer's disease (AD) brains manifest hyperexcitability, contributing to subsequent extensive synapse loss, which has been linked to cognitive dysfunction. No current therapy for AD is disease-modifying. Part of the problem with AD drug discovery is that transgenic mouse models have been poor predictors of potential human treatment. While it is undoubtedly important to test drugs in these animal models, additional evidence for drug efficacy in a human context might improve our chances of success. Accordingly, in order to test drugs in a human context, we have developed a platform of physiological assays using patch-clamp electrophysiology, calcium imaging, and multielectrode array (MEA) experiments on human (h)iPSC-derived 2D cortical neuronal cultures and 3D cerebral organoids. We compare hiPSCs bearing familial AD mutations vs. their wild-type (WT) isogenic controls in order to characterize the aberrant electrical activity in such a human context. Here, we show that these AD neuronal cultures and organoids manifest increased spontaneous action potentials, slow oscillatory events (~1 Hz), and hypersynchronous network activity. Importantly, the dual-allosteric NMDAR antagonist NitroSynapsin, but not the FDA-approved drug memantine, abrogated this hyperactivity. We propose a novel model of synaptic plasticity in which aberrant neural networks are rebalanced by NitroSynapsin. We propose that hiPSC models may be useful for screening drugs to treat hyperexcitability and related synaptic damage in AD.

Mechanisms of hyperexcitability in Alzheimer's disease hiPSC-derived neurons and cerebral organoids vs isogenic controls.

Human Alzheimer's disease (AD) brains and transgenic AD mouse models manifest hyperexcitability. This aberrant electrical activity is caused by synaptic dysfunction that represents the major pathophysiological correlate of cognitive decline. However, the underlying mechanism for this excessive excitability remains incompletely understood. To investigate the basis for the hyperactivity, we performed electrophysiological and immunofluorescence studies on hiPSC-derived cerebrocortical neuronal cultures and cerebral organoids bearing AD-related mutations in presenilin-1 or amyloid precursor protein vs. isogenic gene corrected controls. In the AD hiPSC-derived neurons/organoids, we found increased excitatory bursting activity, which could be explained in part by a decrease in neurite length. AD hiPSC-derived neurons also displayed increased sodium current density and increased excitatory and decreased inhibitory synaptic activity. Our findings establish hiPSC-derived AD neuronal cultures and organoids as a relevant model of early AD pathophysiology and provide mechanistic insight into the observed hyperexcitability.

Parkinson's disease: what the model systems have taught us so far.

Parkinson's disease (PD) is a debilitating neurodegenerative disorder, for which people above the age of 60 show an increased risk. Although there has been great advancement in understanding the disease-related abnormalities in brain circuitry and development of symptomatic treatments, a cure for PD remains elusive. The discovery of PD associated gene mutations and environmental toxins have yielded animal models of the disease. These models could recapitulate several key aspects of PD, and provide more insights into the disease pathogenesis. They have also revealed novel aspects of the disease mechanism including noncell autonomous events and spreading of pathogenic protein species across the brain. Nevertheless, none of these models so far can comprehensively represent all aspects of the human disease. While the field is still searching for the perfect model system, recent developments in stem cell biology have provided a new dimension to modelling PD, especially doing it in a patient-specific manner. In the current review, we attempt to summarize the key findings in the areas discussed above, and highlight how the core PD pathology distinguishes itself from other neurodegenerative disorders while also resembling them in many aspects.

l-Lactate mediates neuroprotection against ischaemia by increasing TREK1 channel expression in rat hippocampal astrocytes in vitro.

Brain ischaemia is a highly debilitating condition where shortage of oxygen and glucose leads to profuse cell death. Lactate is a neuroprotective metabolite whose concentrations increase up to 15-30 mmol/L during ischaemia and TREK1 is a neuroprotective potassium channel which is upregulated during ischaemia. The aim of this study was to investigate the effect of l-lactate on TREK1 expression and to evaluate the role of l-lactate-TREK1 interaction in conferring neuroprotection in ischaemia-prone hippocampus. We show that 15-30 mmol/L l-lactate increases functional TREK1 protein expression by 1.5-3-fold in hippocampal astrocytes using immunostaining and electrophysiology. Studies with transcription blocker actinomycin-D and quantitative PCR indicate that the increase in TREK1 expression is due to enhanced TREK1 mRNA transcription. We further report that l-lactate-mediated increase in TREK1 expression is via protein kinase A (PKA)-dependent pathway. This is the first report of an ischaemic metabolite affecting functional expression of an ion channel. Our studies in an in vitro model of ischaemia using oxygen glucose deprivation show that 30 mmol/L l-lactate fails to reduce cell death in rat hippocampal slices treated with TREK1 blockers, PKA inhibitors and gliotoxin. The above effects were specific to l-lactate as pyruvate failed to increase TREK1 expression and reduce cell death. l-Lactate-induced TREK1 upregulation is a novel finding of physiological significance as TREK1 channels contribute to neuroprotection by enhancing potassium buffering and glutamate clearance capacity of astrocytes. We propose that l-lactate promotes neuronal survival in hippocampus by increasing TREK1 channel expression via PKA pathway in astrocytes during ischaemia. Insufficient blood supply to the brain leads to cerebral ischaemia and increase in extracellular lactate concentrations. We incubated hippocampal astrocytes in lactate and observed increase in TREK1 channel expression via protein kinase A (PKA). Inhibition of TREK1, PKA and metabolic impairment of astrocytes prevented lactate from reducing cell death in ischaemic hippocampus. This pathway serves as an alternate mechanism of neuroprotection. Cover image for this issue: doi: 10.1111/jnc.13326.

Lactate modulates the intracellular pH sensitivity of human TREK1 channels.

Tissue acidosis and high lactate concentrations are associated with cerebral ischaemia. The degree of acidosis is dependent on circulating glucose concentration, hyperglycaemia being associated with increased acidosis. Among other agents, lactate and protons have been shown to activate the leak potassium channel; TREK1 (TWIK related potassium channel 1) from the intracellular side and its increased activity is implicated in tolerance towards ischaemic cell damage. In the present study, we show that ischaemic concentrations of lactate (30 mM) at pH 7.0 and 6.5, commonly observed during ischemia, cause robust potentiation of human TREK1 (hTREK1) activity at single-channel level in cell-free inside-out membrane patches, while 30 mM lactate at pH 6.0 to 5.5, commonly observed during hyperglycaemic ischemia, reduces hTREK1 channel activity significantly. The biphasic effect of 30 mM lactate (ischaemic concentrations) on modulation of hTREK1 by varying pH conditions is specific since basal concentrations of lactate (3 mM) and 30 mM pyruvate at pH 7.0 and 5.5 failed to show similar effect as lactate. Experiments with deletion and point mutants of hTREK1 channel suggest that lactate changes the pH modulation of hTREK1 by interacting differently with the histidine residue at 328th position (H328) above and below its pKa (∼6.0) in the intracellular carboxyl-terminal domain of TREK1. This lactate-induced pH modulation of hTREK1 is absent in C-terminal deletion mutant, CTDΔ100, and is similar in E321A-hTREK1 mutant as in wild-type hTREK1 suggesting that it is independent of pH-sensitive glutamate residue at 321st position. Such a differential pH-dependent effect of lactate on an ion channel function has not been reported earlier and has important implications in different stages of ischaemia.

Ischaemic concentrations of lactate increase TREK1 channel activity by interacting with a single histidine residue in the carboxy terminal domain.

KEY POINTS: The physiological metabolite, lactate and the two-pore domain leak potassium channel, TREK1 are known neuroprotectants against cerebral ischaemia. However, it is not known whether lactate interacts with TREK1 channel to provide neuroprotection. In this study we show that lactate increases TREK1 channel activity and hyperpolarizes CA1 stratum radiatum astrocytes in hippocampal slices. Lactate increases open probability and decreases longer close time of the human (h)TREK1 channel in a concentration dependent manner. Lactate interacts with histidine 328 (H328) in the carboxy terminal domain of hTREK1 channel to decrease its dwell time in the longer closed state. This interaction was dependent on the charge on H328. Lactate-insensitive mutant H328A hTREK1 showed pH sensitivity similar to wild-type hTREK1, indicating that the effect of lactate on hTREK1 is independent of pH change. A rise in lactate concentration and the leak potassium channel TREK1 have been independently associated with cerebral ischaemia. Recent literature suggests lactate to be neuroprotective and TREK1 knockout mice show an increased sensitivity to brain and spinal cord ischaemia; however, the connecting link between the two is missing. Therefore we hypothesized that lactate might interact with TREK1 channels. In the present study, we show that lactate at ischaemic concentrations (15-30 mm) at pH 7.4 increases TREK1 current in CA1 stratum radiatum astrocytes and causes membrane hyperpolarization. We confirm the intracellular action of lactate on TREK1 in hippocampal slices using monocarboxylate transporter blockers and at single channel level in cell-free inside-out membrane patches. The intracellular effect of lactate on TREK1 is specific since other monocarboxylates such as pyruvate and acetate at pH 7.4 failed to increase TREK1 current. Deletion and point mutation experiments suggest that lactate decreases the longer close dwell time incrementally with increase in lactate concentration by interacting with the histidine residue at position 328 (H328) in the carboxy terminal domain of the TREK1 channel. The interaction of lactate with H328 is dependent on the charge on the histidine residue since isosteric mutation of H328 to glutamine did not show an increase in TREK1 channel activity with lactate. This is the first demonstration of a direct effect of lactate on ion channel activity. The action of lactate on the TREK1 channel signifies a separate neuroprotective mechanism in ischaemia since it was found to be independent of the effect of acidic pH on channel activity.