Design, Synthesis, Pharmacology, and In Silico Studies of (1S,2S,3S)-2-((S)-Amino(carboxy)methyl)-3-(carboxymethyl)cyclopropane-1-carboxylic Acid (LBG30300): A Picomolar Potency Subtype-Selective mGlu2 Receptor Agonist.

Metabotropic glutamate (Glu) receptors (mGlu receptors) play a key role in modulating excitatory neurotransmission in the central nervous system (CNS). In this study, we report the structure-based design and pharmacological evaluation of densely functionalized, conformationally restricted glutamate analogue (1S,2S,3S)-2-((S)-amino(carboxy)methyl)-3-(carboxymethyl)cyclopropane-1-carboxylic acid (LBG30300). LBG30300 was synthesized in a stereocontrolled fashion in nine steps from a commercially available optically active epoxide. Functional characterization of all eight mGlu receptor subtypes showed that LBG30300 is a picomolar agonist at mGlu2 with excellent selectivity over mGlu3 and the other six mGlu receptor subtypes. Bioavailability studies on mice (IV administration) confirm CNS exposure, and an in silico study predicts a binding mode of LBG30300 which induces a flipping of Tyr144 to allow for a salt bridge interaction of the acetate group with Arg271. The Tyr144 residue now prevents Arg271 from interacting with Asp146, which is a residue of differentiation between mGlu2 and mGlu3 and thus could explain the observed subtype selectivity.

The role of glutamate NMDA receptors of the mediodorsal thalamus in scopolamine-induced amnesia in rats.

The current study was designed to examine the role of glutamate NMDA receptors of the mediodorsal thalamus (MD) in scopolamine-induced memory impairment. Adult male rats were bilaterally cannulated into the MD. According to the results, intraperitoneal (i.p.) administration of scopolamine (1.5 mg/kg) immediately after the training phase (post-training) impaired memory consolidation. Bilateral microinjection of the glutamate NMDA receptors agonist, N-Methyl-D-aspartic acid (NMDA; 0.05 µg/rat), into the MD significantly improved scopolamine-induced memory consolidation impairment. Co-administration of D-AP5, a glutamate NMDA receptor antagonist (0.001-0.005 µg/rat, intra-MD) potentiated the response of an ineffective dose of scopolamine (0.5 mg/kg, i.p.) to impair memory consolidation, mimicking the response of a higher dose of scopolamine. Noteworthy, post-training intra-MD microinjections of the same doses of NMDA or D-AP5 alone had no effect on memory consolidation. Moreover, the blockade of the glutamate NMDA receptors by 0.003 ng/rat of D-AP5 prevented the improving effect of NMDA on scopolamine-induced amnesia. Thus, it can be concluded that the MD glutamatergic system may be involved in scopolamine-induced memory impairment via the NMDA receptor signaling pathway.

Pluripotential GluN1 (NMDA NR1): Functional Significance in Cellular Nuclei in Pain/Nociception.

The N-methyl-D-aspartate (NMDA) glutamate receptors function as plasma membrane ionic channels and take part in very tightly controlled cellular processes activating neurogenic and inflammatory pathways. In particular, the NR1 subunit (new terminology: GluN1) is required for many neuronal and non-neuronal cell functions, including plasticity, survival, and differentiation. Physiologic levels of glutamate agonists and NMDA receptor activation are required for normal neuronal functions such as neuronal development, learning, and memory. When glutamate receptor agonists are present in excess, binding to NMDA receptors produces neuronal/CNS/PNS long-term potentiation, conditions of acute pain, ongoing severe intractable pain, and potential excitotoxicity and pathology. The GluNR1 subunit (116 kD) is necessary as the anchor component directing ion channel heterodimer formation, cellular trafficking, and the nuclear localization that directs functionally specific heterodimer formation, cellular trafficking, and nuclear functions. Emerging studies report the relevance of GluN1 subunit composition and specifically that nuclear GluN1 has major physiologic potential in tissue and/or subnuclear functioning assignments. The shift of the GluN1 subunit from a surface cell membrane to nuclear localization assigns the GluN1 promoter immediate early gene behavior with access to nuclear and potentially nucleolar functions. The present narrative review addresses the nuclear translocation of GluN1, focusing particularly on examples of the role of GluN1 in nociceptive processes.

Possible Compensatory Role of ASICs in Glutamatergic Synapses.

Proton-gated channels of the ASIC family are widely distributed in central neurons, suggesting their role in common neurophysiological functions. They are involved in glutamatergic neurotransmission and synaptic plasticity; however, the exact function of these channels remains unclear. One problem is that acidification of the synaptic cleft due to the acidic content of synaptic vesicles has opposite effects on ionotropic glutamate receptors and ASICs. Thus, the pH values required to activate ASICs strongly inhibit AMPA receptors and almost completely inhibit NMDA receptors. This, in turn, suggests that ASICs can provide compensation for post-synaptic responses in the case of significant acidifications. We tested this hypothesis by patch-clamp recordings of rat brain neuron responses to acidifications and glutamate receptor agonists at different pH values. Hippocampal pyramidal neurons have much lower ASICs than glutamate receptor responses, whereas striatal interneurons show the opposite ratio. Cortical pyramidal neurons and hippocampal interneurons show similar amplitudes in their responses to acidification and glutamate. Consequently, the total response to glutamate agonists at different pH levels remains rather stable up to pH 6.2. Besides these pH effects, the relationship between the responses mediated by glutamate receptors and ASICs depends on the presence of Mg2+ and the membrane voltage. Together, these factors create a complex picture that provides a framework for understanding the role of ASICs in synaptic transmission and synaptic plasticity.

The D-amino acid oxidase inhibitor luvadaxistat improves mismatch negativity in patients with schizophrenia in a randomized trial.

Several attempts have been made to enhance N-methyl-D-aspartate (NMDA) receptor function in schizophrenia, but they have yielded mixed results. Luvadaxistat, a D-amino acid oxidase (DAAO) inhibitor that increases the glutamate co-agonist D-serine levels, is being developed for the treatment of cognitive impairment associated with schizophrenia. We conducted a biomarker study in patients, assessing several endpoints related to physiological outcomes of NMDA receptor modulation to determine whether luvadaxistat affects neural circuitry biomarkers relevant to NMDA receptor function and schizophrenia. This was a randomized, placebo-controlled, double-blind, two-period crossover phase 2a study assessing luvadaxistat 50 mg and 500 mg for 8 days in 31 patients with schizophrenia. There were no treatment effects of luvadaxistat at either dose in eyeblink conditioning, a cerebellar-dependent learning measure, compared with placebo. We observed a nominally significant improvement in mismatch negativity (MMN) and a statistical trend to improvement for auditory steady-state response at 40 Hz, in both cases with 50 mg, but not with 500 mg, compared with placebo. Although the data should be interpreted cautiously owing to the small sample size, they suggest that luvadaxistat can improve an illness-related circuitry biomarker at doses associated with partial DAAO inhibition. These results are consistent with 50 mg, but not higher doses, showing a signal of efficacy in cognitive endpoints in a larger phase 2, 12-week study conducted in parallel. Thus, MMN responses after a short treatment period may predict cognitive function improvement. MMN and ASSR should be considered as biomarkers in early trials addressing NMDA receptor hypofunction.

Dose-Dependent Augmentation of Neuroplasticity-Based Auditory Learning in Schizophrenia: A Double-Blind, Placebo-Controlled, Randomized, Target Engagement Clinical Trial of the NMDA Glutamate Receptor Agonist d-serine.

BACKGROUND: Patients with schizophrenia show reduced NMDA glutamate receptor-dependent auditory plasticity, which is rate limiting for auditory cognitive remediation (AudRem). We evaluate the utility of behavioral and neurophysiological pharmacodynamic target engagement biomarkers, using a d-serine+AudRem combination. METHODS: Forty-five participants with schizophrenia or schizoaffective disorder were randomized to 3 once-weekly AudRem visits + double-blind d-serine (80, 100, or 120 mg/kg) or placebo in 3 dose cohorts of 12 d-serine and 3 placebo-treated participants each. In AudRem, participants indicated which paired tone was higher in pitch. The primary outcome was plasticity improvement, operationalized as change in pitch threshold between AudRem tones [(test tone Hz - reference tone Hz)/reference tone Hz] between the initial plateau pitch threshold (mean of trials 20-30 of treatment visit 1) to pitch threshold at the end of visit(s). Target engagement was assessed by electroencephalography outcomes, including mismatch negativity (pitch primary). RESULTS: There was a significant overall treatment effect for plasticity improvement (p = .014). Plasticity improvement was largest within the 80 and 100 mg/kg groups (p < .001, d > 0.67), while 120 mg/kg and placebo-treated participants showed nonsignificant within-group changes. Plasticity improvement was seen after a single treatment and was sustained on subsequent treatments. Target engagement was demonstrated by significantly larger mismatch negativity (p = .049, d = 1.0) for the 100 mg/kg dose versus placebo. CONCLUSIONS: Our results demonstrate sufficient proof of principle for continued development of both the d-serine+AudRem combination and our target engagement methodology. The ultimate utility is dependent on the results of an ongoing larger, longer study of the combination for clinically relevant outcomes.

Purinergic ionotropic P2X7 and metabotropic glutamate mGlu5 receptors crosstalk influences pro-inflammatory conditions in microglia.

Microglia represent the resident immune system in the brain. They mediate neuroinflammatory processes and have been described as important regulators of homeostasis in the central nervous system (CNS). Among several players and mechanisms contributing to microglial function in inflammation, ATP and glutamate have been shown to be involved in microgliosis. In this study, we focused on receptor subtypes that respond to these neurotransmitters, purinergic ionotropic P2X7 receptor and metabotropic glutamate mGlu5 receptor. We found that both receptors are functionally expressed in a murine microglia cell line, BV2 cells, and we performed patch-clamp experiments to measure purinergic ionotropic P2X7 receptor ion flux in control condition and after metabotropic glutamate mGlu5 receptor activation. The selective purinergic ionotropic P2X7 receptor agonist, 2'(3')-O-(4-benzoylbenzoyl)adenosine-5'-triphosphate (BzATP, 100 µM), elicited a robust current that was prevented by the selective purinergic ionotropic P2X7 receptor antagonist A438079 (10 µM). When BV2 cells were acutely stimulated with the selective metabotropic glutamate mGlu5 agonist, (RS)-2-chloro-5-hydroxyphenylglycine (CHPG, 200 µM), purinergic ionotropic P2X7 receptor current was increased. This positive modulation was prevented by the selective metabotropic glutamate mGlu5 receptor antagonist 3-((2-Methyl-4-thiazolyl)ethynyl)pyridine (MTEP, 1 µM). Moreover, nitric oxide synthesis elicited by purinergic ionotropic P2X7 receptor activation was enhanced by metabotropic glutamate mGlu5 receptor co-stimulation. Taken together, our results suggest an important crosstalk between ATP and glutamate in inflammation. Pro-inflammatory effects mediated by purinergic ionotropic P2X7 receptor might be exacerbated by simultaneous exposure of microglia to ATP and glutamate, suggesting new pharmacological targets to modulate neuroinflammation.

Role of interaction of mGlu2 and 5-HT2A receptors in antipsychotic effects.

The serotonergic and glutamatergic neurotransmitter systems have been implicated in the pathophysiology of schizophrenia, and increasing evidence shows that they interact functionally. Of note, the Gq/11-coupled serotonin 5-HT2A (5-HT2A) and the Gi/o-coupled metabotropic glutamate type 2 (mGlu2) receptors have been demonstrated to assemble into a functional heteromeric complex that modulates the function of each individual receptor. For conformation of the heteromeric complex, corresponding transmembrane-4 segment of 5-HT2A and mGlu2 are required. The 5-HT2A/mGlu2 heteromeric complex is necessary for the activation of Gq/11 proteins and for the subsequent increase in the levels of the intracellular messenger Ca2+. Furthermore, signaling via the heteromeric complex is dysregulated in the post-mortem brains of patients with schizophrenia, and could be linked to altered cortical function. From a behavioral perspective, this complex contributes to the hallucinatory and antipsychotic behaviors associated with 5-HT2A and mGlu2/3 agonists, respectively. Synaptic and epigenetic mechanisms have also been found to be significantly associated with the mGlu2/5-HT2A heteromeric complex. This review summarizes the role of crosstalk between mGlu2 and 5-HT2A in the mechanism of antipsychotic effects and introduces recent key advancements on this topic.

Glutamate can act as a signaling molecule in mouse preimplantation embryos†.

Free amino acids are present in the natural environment of the preimplantation embryo, and their availability can influence early embryo development. Glutamic acid is one of the amino acids with the highest concentrations in female reproductive fluids, and we investigated whether glutamic acid/glutamate can affect preimplantation embryo development by acting through cell membrane receptors. Using reverse transcription-polymerase chain reaction, we detected 15 ionotropic glutamate receptor transcripts and 8 metabotropic glutamate receptor transcripts in mouse ovulated oocytes and/or in vivo developed blastocysts. Using immunohistochemistry, we detected the expression of two α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits, three kainate receptor subunits, and member 5 metabotropic glutamate receptor protein in blastocysts. Extracellular concentrations of glutamic acid starting at 5 mM impaired mouse blastocyst development, and this fact may be of great practical importance since glutamic acid and its salts (mainly monosodium glutamate) are widely used as food additives. Experiments with glutamate receptor agonists (in combination with gene expression analysis) revealed that specific AMPA receptors (formed from glutamate receptor, ionotropic, AMPA3 [GRIA3] and/or glutamate receptor, ionotropic, AMPA4 [GRIA4] subunits), kainate receptors (formed from glutamate receptor, ionotropic, kainate 3 [GRIK3] and glutamate receptor, ionotropic, kainate 4 [GRIK4] or glutamate receptor, ionotropic, kainate 5 [GRIK5] subunits), and member 5 metabotropic glutamate receptor (GRM5) were involved in this effect. The glutamic acid-induced effects were prevented or reduced by pretreatment of blastocysts with AMPA, kainate, and GRM5 receptor antagonists, further confirming the involvement of these receptor types. Our results show that glutamic acid can act as a signaling molecule in preimplantation embryos, exerting its effects through the activation of cell membrane receptors.

Piperine Provides Neuroprotection against Kainic Acid-Induced Neurotoxicity via Maintaining NGF Signalling Pathway.

The neuroprotective properties of piperine, the major alkaloid extracted from black pepper, have been under investigation, but its mechanism of action in excitotoxicity is still poorly understood. This study aimed to evaluate the protective effects of piperine with a focus on nerve growth factor (NGF) signalling in a kainic acid (KA) rat model of excitotoxicity. Rats were administered intraperitoneally (i.p.) piperine (10 or 50 mg/kg) before KA injection (15 mg/kg, i.p.). Our results show that KA exposure in rats caused seizure behaviour, intrinsic neuronal hyperactivity, glutamate elevation, hippocampal neuronal damage, and cognitive impairment. These KA-induced alterations could be restored to the normal state by piperine treatment. In addition, piperine decreased the expression of the NGF precursor proNGF and NGF-degrading protease matrix metalloproteinase 9, whereas it increased the expression of proNGF processing enzyme matrix metalloproteinase 7, NGF, and NGF-activated receptor TrkA in the hippocampus of KA-treated rats. Furthermore, KA decreased phosphorylation of the protein kinase B (Akt) and glycogen synthase kinase 3ß (GSK3ß) in the hippocampus, and piperine reversed these changes. Our data suggest that piperine protects hippocampal neurons against KA-induced excitotoxicity by upregulating the NGF/TrkA/Akt/GSK3ß signalling pathways.

Derivatives of (R)-3-(5-Furanyl)carboxamido-2-aminopropanoic Acid as Potent NMDA Receptor Glycine Site Agonists with GluN2 Subunit-Specific Activity.

NMDA receptors mediate glutamatergic neurotransmission and are therapeutic targets due to their involvement in a variety of psychiatric and neurological disorders. Here, we describe the design and synthesis of a series of (R)-3-(5-furanyl)carboxamido-2-aminopropanoic acid analogues 8a-s as agonists at the glycine (Gly) binding site in the GluN1 subunit, but not GluN3 subunits, of NMDA receptors. These novel analogues display highly variable potencies and agonist efficacies among the NMDA receptor subtypes (GluN1/2A-D) in a manner dependent on the GluN2 subunit. Notably, compound 8p is identified as a potent partial agonist at GluN1/2C (EC50 = 0.074 µM) with an agonist efficacy of 28% relative to activation by Gly and virtually no agonist activity at GluN1/2A, GluN1/2B, and GluN1/2D. Thus, these novel agonists can modulate the activity of specific NMDA receptor subtypes by replacing the full endogenous agonists Gly or d-serine (d-Ser), thereby providing new opportunities in the development of novel therapeutic agents.

TIGAR plays neuroprotective roles in KA-induced excitotoxicity through reducing neuroinflammation and improving mitochondrial function.

Excitotoxicity refers to the ability of excessive extracellular excitatory amino acids to damage neurons via receptor activation. It is a crucial pathogenetic process in neurodegenerative diseases. TP53 is confirmed to be involved in excitotoxicity. It is demonstrated that TP53 induced glycolysis and apoptotic regulator (TIGAR)-regulated metabolic pathway can protect against neuronal injury. However, the role of TIGAR in excitotoxicity and specific mechanisms is still unknown. In this study, an in vivo excitotoxicity model was constructed via stereotypical kainic acid (KA) injection into the striatum of mice. KA reduced TIGAR expression levels, neuroinflammatory responses and mitochondrial dysfunction. TIGAR overexpression could reverse KA-induced neuronal injury by reducing neuroinflammation and improving mitochondrial function, thereby exerting neuroprotective effects. Therefore, this study could provide a potential therapeutic target for neurodegenerative diseases.

Neuronal activity reorganization in motor cortex for successful locomotion after a lesion in the ventrolateral thalamus.

Thalamic stroke leads to ataxia if the cerebellum-receiving ventrolateral thalamus (VL) is affected. The compensation mechanisms for this deficit are not well understood, particularly the roles that single neurons and specific neuronal subpopulations outside the thalamus play in recovery. The goal of this study was to clarify neuronal mechanisms of the motor cortex involved in mitigation of ataxia during locomotion when part of the VL is inactivated or lesioned. In freely ambulating cats, we recorded the activity of neurons in layer V of the motor cortex as the cats walked on a flat surface and horizontally placed ladder. We first reversibly inactivated ∼10% of the VL unilaterally using glutamatergic transmission antagonist CNQX and analyzed how the activity of motor cortex reorganized to support successful locomotion. We next lesioned 50%-75% of the VL bilaterally using kainic acid and analyzed how the activity of motor cortex reorganized when locomotion recovered. When a small part of the VL was inactivated, the discharge rates of motor cortex neurons decreased, but otherwise the activity was near normal, and the cats walked fairly well. Individual neurons retained their ability to respond to the demand for accuracy during ladder locomotion; however, most changed their response. When the VL was lesioned, the cat walked normally on the flat surface but was ataxic on the ladder for several days after lesion. When ladder locomotion normalized, neuronal discharge rates on the ladder were normal, and the shoulder-related group was preferentially active during the stride's swing phase.NEW & NOTEWORTHY This is the first analysis of reorganization of the activity of single neurons and subpopulations of neurons related to the shoulder, elbow, or wrist, as well as fast- and slow-conducting pyramidal tract neurons in the motor cortex of animals walking before and after inactivation or lesion in the thalamus. The results offer unique insights into the mechanisms of spontaneous recovery after thalamic stroke, potentially providing guidance for new strategies to alleviate locomotor deficits after stroke.

Synaptic protein interaction networks encode experience by assuming stimulus-specific and brain-region-specific states.

A core network of widely expressed proteins within the glutamatergic post-synapse mediates activity-dependent synaptic plasticity throughout the brain, but the specific proteomic composition of synapses differs between brain regions. Here, we address the question, how does proteomic composition affect activity-dependent protein-protein interaction networks (PINs) downstream of synaptic activity? Using quantitative multiplex co-immunoprecipitation, we compare the PIN response of in vivo or ex vivo neurons derived from different brain regions to activation by different agonists or different forms of eyeblink conditioning. We report that PINs discriminate between incoming stimuli using differential kinetics of overlapping and non-overlapping PIN parameters. Further, these "molecular logic rules" differ by brain region. We conclude that although the PIN of the glutamatergic post-synapse is expressed widely throughout the brain, its activity-dependent dynamics show remarkable stimulus-specific and brain-region-specific diversity. This diversity may help explain the challenges in developing molecule-specific drug therapies for neurological disorders.

The Protective Role of E-64d in Hippocampal Excitotoxic Neuronal Injury Induced by Glutamate in HT22 Hippocampal Neuronal Cells.

Epilepsy is the most common childhood neurologic disorder. Status epilepticus (SE), which refers to continuous epileptic seizures, occurs more frequently in children than in adults, and approximately 40-50% of all cases occur in children under 2 years of age. Conventional antiepileptic drugs currently used in clinical practice have a number of adverse side effects. Drug-resistant epilepsy (DRE) can progressively develop in children with persistent SE, necessitating the development of novel therapeutic drugs. During SE, the persistent activation of neurons leads to decreased glutamate clearance with corresponding glutamate accumulation in the synaptic extracellular space, increasing the chance of neuronal excitotoxicity. Our previous study demonstrated that after developmental seizures in rats, E-64d exerts a neuroprotective effect on the seizure-induced brain damage by modulating lipid metabolism enzymes, especially ApoE and ApoJ/clusterin. In this study, we investigated the impact and mechanisms of E-64d administration on neuronal excitotoxicity. To test our hypothesis that E-64d confers neuroprotective effects by regulating autophagy and mitochondrial pathway activity, we simulated neuronal excitotoxicity in vitro using an immortalized hippocampal neuron cell line (HT22). We found that E-64d improved cell viability while reducing oxidative stress and neuronal apoptosis. In addition, E-64d treatment regulated mitochondrial pathway activity and inhibited chaperone-mediated autophagy in HT22 cells. Our findings indicate that E-64d may alleviate glutamate-induced damage via regulation of mitochondrial fission and apoptosis, as well as inhibition of chaperone-mediated autophagy. Thus, E-64d may be a promising therapeutic treatment for hippocampal injury associated with SE.

mGlu1 potentiation enhances prelimbic somatostatin interneuron activity to rescue schizophrenia-like physiological and cognitive deficits.

Evidence for prefrontal cortical (PFC) GABAergic dysfunction is one of the most consistent findings in schizophrenia and may contribute to cognitive deficits. Recent studies suggest that the mGlu1 subtype of metabotropic glutamate receptor regulates cortical inhibition; however, understanding the mechanisms through which mGlu1 positive allosteric modulators (PAMs) regulate PFC microcircuit function and cognition is essential for advancing these potential therapeutics toward the clinic. We report a series of electrophysiology, optogenetic, pharmacological magnetic resonance imaging, and animal behavior studies demonstrating that activation of mGlu1 receptors increases inhibitory transmission in the prelimbic PFC by selective excitation of somatostatin-expressing interneurons (SST-INs). An mGlu1 PAM reverses cortical hyperactivity and concomitant cognitive deficits induced by N-methyl-d-aspartate (NMDA) receptor antagonists. Using in vivo optogenetics, we show that prelimbic SST-INs are necessary for mGlu1 PAM efficacy. Collectively, these findings suggest that mGlu1 PAMs could reverse cortical GABAergic deficits and exhibit efficacy in treating cognitive dysfunction in schizophrenia.

Distinct regulation of tonic GABAergic inhibition by NMDA receptor subtypes.

Tonic inhibition mediated by extrasynaptic GABAARs regulates various brain functions. However, the mechanisms that regulate tonic inhibition remain largely unclear. Here, we report distinct actions of GluN2A- and GluN2B-NMDA receptors (NMDARs) on tonic inhibition in hippocampal neurons under basal and high activity conditions. Specifically, overexpression of GluN2B, but not GluN2A, reduces α5-GABAAR surface expression and tonic currents. Additionally, knockout of GluN2A and GluN2B decreases and increases tonic currents, respectively. Mechanistically, GluN2A-NMDARs inhibit and GluN2B-NMDARs promote α5-GABAAR internalization, resulting in increased and decreased surface α5-GABAAR expression, respectively. Furthermore, GluN2A-NMDARs, but not GluN2B-NMDARs, are required for homeostatic potentiation of tonic inhibition induced by prolonged increase of neuronal activity. Last, tonic inhibition decreases during acute seizures, whereas it increases 24 h later, involving GluN2-NMDAR-dependent signaling. Collectively, these data reveal an NMDAR subunit-specific regulation of tonic inhibition in physiological and pathological conditions and provide mechanistic insight into activity-dependent modulation of tonic inhibition.

BMAA, Methylmercury, and Mechanisms of Neurodegeneration in Dolphins: A Natural Model of Toxin Exposure.

Dolphins are well-regarded sentinels for toxin exposure and can bioaccumulate a cyanotoxin called ß-N-methylamino-l-alanine (BMAA) that has been linked to human neurodegenerative disease. The same dolphins also possessed hallmarks of Alzheimer's disease (AD), suggesting a possible association between toxin exposure and neuropathology. However, the mechanisms of neurodegeneration in dolphins and the impact cyanotoxins have on these processes are unknown. Here, we evaluate BMAA exposure by investigating transcription signatures using PCR for dolphin genes homologous to those implicated in AD and related dementias: APP, PSEN1, PSEN2, MAPT, GRN, TARDBP, and C9orf72. Immunohistochemistry and Sevier Münger silver staining were used to validate neuropathology. Methylmercury (MeHg), a synergistic neurotoxicant with BMAA, was also measured using PT-GC-AFS. We report that dolphins have up to a three-fold increase in gene transcription related to Aß+ plaques, neurofibrillary tangles, neuritic plaques, and TDP-43+ intracytoplasmic inclusions. The upregulation of gene transcription in our dolphin cohort paralleled increasing BMAA concentration. In addition, dolphins with BMAA exposures equivalent to those reported in AD patients displayed up to a 14-fold increase in AD-type neuropathology. MeHg was detected (0.16-0.41 µg/g) and toxicity associated with exposure was also observed in the brain. These results demonstrate that dolphins develop neuropathology associated with AD and exposure to BMAA and MeHg may augment these processes.

Fundamentally different roles of neuronal TNF receptors in CNS pathology: TNFR1 and IKKß promote microglial responses and tissue injury in demyelination while TNFR2 protects against excitotoxicity in mice.

BACKGROUND: During inflammatory demyelination, TNF receptor 1 (TNFR1) mediates detrimental proinflammatory effects of soluble TNF (solTNF), whereas TNFR2 mediates beneficial effects of transmembrane TNF (tmTNF) through oligodendroglia, microglia, and possibly other cell types. This model supports the use of selective inhibitors of solTNF/TNFR1 as anti-inflammatory drugs for central nervous system (CNS) diseases. A potential obstacle is the neuroprotective effect of solTNF pretreatment described in cultured neurons, but the relevance in vivo is unknown. METHODS: To address this question, we generated mice with neuron-specific depletion of TNFR1, TNFR2, or inhibitor of NF-κB kinase subunit ß (IKKß), a main downstream mediator of TNFR signaling, and applied experimental models of inflammatory demyelination and acute and preconditioning glutamate excitotoxicity. We also investigated the molecular and cellular requirements of solTNF neuroprotection by generating astrocyte-neuron co-cultures with different combinations of wild-type (WT) and TNF and TNFR knockout cells and measuring N-methyl-D-aspartate (NMDA) excitotoxicity in vitro. RESULTS: Neither neuronal TNFR1 nor TNFR2 protected mice during inflammatory demyelination. In fact, both neuronal TNFR1 and neuronal IKKß promoted microglial responses and tissue injury, and TNFR1 was further required for oligodendrocyte loss and axonal damage in cuprizone-induced demyelination. In contrast, neuronal TNFR2 increased preconditioning protection in a kainic acid (KA) excitotoxicity model in mice and limited hippocampal neuron death. The protective effects of neuronal TNFR2 observed in vivo were further investigated in vitro. As previously described, pretreatment of astrocyte-neuron co-cultures with solTNF (and therefore TNFR1) protected them against NMDA excitotoxicity. However, protection was dependent on astrocyte, not neuronal TNFR1, on astrocyte tmTNF-neuronal TNFR2 interactions, and was reproduced by a TNFR2 agonist. CONCLUSIONS: These results demonstrate that neuronal TNF receptors perform fundamentally different roles in CNS pathology in vivo, with neuronal TNFR1 and IKKß promoting microglial inflammation and neurotoxicity in demyelination, and neuronal TNFR2 mediating neuroprotection in excitotoxicity. They also reveal that previously described neuroprotective effects of solTNF against glutamate excitotoxicity in vitro are indirect and mediated via astrocyte tmTNF-neuron TNFR2 interactions. These results consolidate the concept that selective inhibition of solTNF/TNFR1 with maintenance of TNFR2 function would have combined anti-inflammatory and neuroprotective properties required for safe treatment of CNS diseases.

Altered cardiac structure and function is related to seizure frequency in a rat model of chronic acquired temporal lobe epilepsy.

OBJECTIVE: This study aimed to prospectively examine cardiac structure and function in the kainic acid-induced post-status epilepticus (post-KA SE) model of chronic acquired temporal lobe epilepsy (TLE), specifically to examine for changes between the pre-epileptic, early epileptogenesis and the chronic epilepsy stages. We also aimed to examine whether any changes related to the seizure frequency in individual animals. METHODS: Four hours of SE was induced in 9 male Wistar rats at 10 weeks of age, with 8 saline treated matched control rats. Echocardiography was performed prior to the induction of SE, two- and 10-weeks post-SE. Two weeks of continuous video-EEG and simultaneous ECG recordings were acquired for two weeks from 11 weeks post-KA SE. The video-EEG recordings were analyzed blindly to quantify the number and severity of spontaneous seizures, and the ECG recordings analyzed for measures of heart rate variability (HRV). PicroSirius red histology was performed to assess cardiac fibrosis, and intracellular Ca2+ levels and cell contractility were measured by microfluorimetry. RESULTS: All 9 post-KA SE rats were demonstrated to have spontaneous recurrent seizures on the two-week video-EEG recording acquired from 11 weeks SE (seizure frequency ranging from 0.3 to 10.6 seizures/day with the seizure durations from 11 to 62 s), and none of the 8 control rats. Left ventricular wall thickness was thinner, left ventricular internal dimension was shorter, and ejection fraction was significantly decreased in chronically epileptic rats, and was negatively correlated to seizure frequency in individual rats. Diastolic dysfunction was evident in chronically epileptic rats by a decrease in mitral valve deceleration time and an increase in E/E` ratio. Measures of HRV were reduced in the chronically epileptic rats, indicating abnormalities of cardiac autonomic function. Cardiac fibrosis was significantly increased in epileptic rats, positively correlated to seizure frequency, and negatively correlated to ejection fraction. The cardiac fibrosis was not a consequence of direct effect of KA toxicity, as it was not seen in the 6/10 rats from separate cohort that received similar doses of KA but did not go into SE. Cardiomyocyte length, width, volume, and rate of cell lengthening and shortening were significantly reduced in epileptic rats. SIGNIFICANCE: The results from this study demonstrate that chronic epilepsy in the post-KA SE rat model of TLE is associated with a progressive deterioration in cardiac structure and function, with a restrictive cardiomyopathy associated with myocardial fibrosis. Positive correlations between seizure frequency and the severity of the cardiac changes were identified. These results provide new insights into the pathophysiology of cardiac disease in chronic epilepsy, and may have relevance for the heterogeneous mechanisms that place these people at risk of sudden unexplained death.