Wild-type Slices Research Articles

Interference with glutamate antiporter system xc - enables post-hypoxic long-term potentiation in hippocampus.

Our group previously showed that genetic or pharmacological inhibition of the cystine/glutamate antiporter, system xc -, mitigates excitotoxicity after anoxia by increasing latency to anoxic depolarization, thus attenuating the ischaemic core. Hypoxia, however, which prevails in the ischaemic penumbra, is a condition where neurotransmission is altered, but excitotoxicity is not triggered. The present study employed mild hypoxia to further probe ischaemia-induced changes in neuronal responsiveness from wild-type and xCT KO (xCT-/-) mice. Synaptic transmission was monitored in hippocampal slices from both genotypes before, during and after a hypoxic episode. Although wild-type and xCT-/- slices showed equal suppression of synaptic transmission during hypoxia, mutant slices exhibited a persistent potentiation upon re-oxygenation, an effect we termed 'post-hypoxic long-term potentiation (LTP)'. Blocking synaptic suppression during hypoxia by antagonizing adenosine A1 receptors did not preclude post-hypoxic LTP. Further examination of the induction and expression mechanisms of this plasticity revealed that post-hypoxic LTP was driven by NMDA receptor activation, as well as increased calcium influx, with no change in paired-pulse facilitation. Hence, the observed phenomenon engaged similar mechanisms as classical LTP. This was a remarkable finding as theta-burst stimulation-induced LTP was equivalent between genotypes. Importantly, post-hypoxic LTP was generated in wild-type slices pretreated with system xc - inhibitor, S-4-carboxyphenylglycine, thereby confirming the antiporter's role in this phenomenon. Collectively, these data indicate that system xc - interference enables neuroplasticity in response to mild hypoxia, and, together with its regulation of cellular damage in the ischaemic core, suggest a role for the antiporter in post-ischaemic recovery of the penumbra.

Misprogramming of glucose metabolism impairs recovery of hippocampal slices from neuronal GLT-1 knockout mice and contributes to excitotoxic injury through mitochondrial superoxide production.

We have previously reported a failure of recovery of synaptic function in the CA1 region of acute hippocampal slices from mice with a conditional neuronal knockout (KO) of GLT-1 (EAAT2, Slc1A2) driven by synapsin-Cre (synGLT-1 KO). The failure of recovery of synaptic function is due to excitotoxic injury. We hypothesized that changes in mitochondrial metabolism contribute to the heightened vulnerability to excitotoxicity in the synGLT-1 KO mice. We found impaired flux of carbon from 13C-glucose into the tricarboxylic acid cycle in synGLT-1 KO cortical and hippocampal slices compared with wild-type (WT) slices. In addition, we found downregulation of the neuronal glucose transporter GLUT3 in both genotypes. Flux of carbon from [1,2-13C]acetate, thought to be astrocyte-specific, was increased in the synGLT-KO hippocampal slices but not cortical slices. Glycogen stores, predominantly localized to astrocytes, are rapidly depleted in slices after cutting, and are replenished during exvivo incubation. In the synGLT-1 KO, replenishment of glycogen stores during exvivo incubation was compromised. These results suggest both neuronal and astrocytic metabolic perturbations in the synGLT-1 KO slices. Supplementing incubation medium during recovery with 20 mM D-glucose normalized glycogen replenishment but had no effect on recovery of synaptic function. In contrast, 20 mM non-metabolizable L-glucose substantially improved recovery of synaptic function, suggesting that D-glucose metabolism contributes to the excitotoxic injury in the synGLT-1 KO slices. L-lactate substitution for D-glucose did not promote recovery of synaptic function, implicating mitochondrial metabolism. Consistent with this hypothesis, phosphorylation of pyruvate dehydrogenase, which decreases enzyme activity, was increased in WT slices during the recovery period, but not in synGLT-1 KO slices. Since metabolism of glucose by the mitochondrial electron transport chain is associated with superoxide production, we tested the effect of drugs that scavenge and prevent superoxide production. The superoxide dismutase/catalase mimic EUK-134 conferred complete protection and full recovery of synaptic function. A site-specific inhibitor of complex III superoxide production, S3QEL-2, was also protective, but inhibitors of NADPH oxidase were not. In summary, we find that the failure of recovery of synaptic function in hippocampal slices from the synGLT-1 KO mouse, previously shown to be due to excitotoxic injury, is caused by production of superoxide by mitochondrial metabolism.

Long-Term-But Not Short-Term-Plasticity at the Mossy Fiber-CA3 Pyramidal Cell Synapse in Hippocampus Is Altered in M1/M3 Muscarinic Acetylcholine Receptor Double Knockout Mice.

Muscarinic acetylcholine receptors are well-known for their crucial involvement in hippocampus-dependent learning and memory, but the exact roles of the various receptor subtypes (M1-M5) are still not fully understood. Here, we studied how M1 and M3 receptors affect plasticity at the mossy fiber (MF)-CA3 pyramidal cell synapse. In hippocampal slices from M1/M3 receptor double knockout (M1/M3-dKO) mice, the signature short-term plasticity of the MF-CA3 synapse was not significantly affected. However, the rather unique NMDA receptor-independent and presynaptic form of long-term potentiation (LTP) of this synapse was much larger in M1/M3-deficient slices compared to wild-type slices in both field potential and whole-cell recordings. Consistent with its presynaptic origin, induction of MF-LTP strongly enhanced the excitatory drive onto single CA3 pyramidal cells, with the effect being more pronounced in M1/M3-dKO cells. In an earlier study, we found that the deletion of M2 receptors in mice disinhibits MF-LTP in a similar fashion, suggesting that endogenous acetylcholine employs both M1/M3 and M2 receptors to constrain MF-LTP. Importantly, such synergism was not observed for MF long-term depression (LTD). Low-frequency stimulation, which reliably induced LTD of MF synapses in control slices, failed to do so in M1/M3-dKO slices and gave rise to LTP instead. In striking contrast, loss of M2 receptors augmented LTD when compared to control slices. Taken together, our data demonstrate convergence of M1/M3 and M2 receptors on MF-LTP, but functional divergence on MF-LTD, with the net effect resulting in a well-balanced bidirectional plasticity of the MF-CA3 pyramidal cell synapse.

Organotypic Hippocampal Slice Cultures from Adult Tauopathy Mice and Theragnostic Evaluation of Nanomaterial Phospho-TAU Antibody-Conjugates.

Organotypic slice culture models surpass conventional in vitro methods in many aspects. They retain all tissue-resident cell types and tissue hierarchy. For studying multifactorial neurodegenerative diseases such as tauopathies, it is crucial to maintain cellular crosstalk in an accessible model system. Organotypic slice cultures from postnatal tissue are an established research tool, but adult tissue-originating systems are missing, yet necessary, as young tissue-originating systems cannot fully model adult or senescent brains. To establish an adult-originating slice culture system for tauopathy studies, we made hippocampal slice cultures from transgenic 5-month-old hTau.P301S mice. In addition to the comprehensive characterization, we set out to test a novel antibody for hyperphosphorylated TAU (pTAU, B6), with and without a nanomaterial conjugate. Adult hippocampal slices retained intact hippocampal layers, astrocytes, and functional microglia during culturing. The P301S-slice neurons expressed pTAU throughout the granular cell layer and secreted pTAU to the culture medium, whereas the wildtype slices did not. Additionally, cytotoxicity and inflammation-related determinants were increased in the P301S slices. Using fluorescence microscopy, we showed target engagement of the B6 antibody to pTAU-expressing neurons and a subtle but consistent decrease in intracellular pTAU with the B6 treatment. Collectively, this tauopathy slice culture model enables measuring the extracellular and intracellular effects of different mechanistic or therapeutic manipulations on TAU pathology in adult tissue without the hindrance of the blood-brain barrier.

Temporary alteration of neuronal network communication is a protective response to redox imbalance that requires GPI-anchored prion protein

Cellular prion protein (PrPC) protects neurons against oxidative stress damage. This role is lost upon its misfolding into insoluble prions in prion diseases, and correlated with cytoskeletal breakdown and neurophysiological deficits. Here we used mouse neuronal models to assess how PrPC protects the neuronal cytoskeleton, and its role in network communication, from oxidative stress damage. Oxidative stress was induced extrinsically by potassium superoxide (KO2) or intrinsically by Mito-Paraquat (MtPQ), targeting the mitochondria. In mouse neural lineage cells, KO2 was damaging to the cytoskeleton, with cells lacking PrPC (PrP-/-) damaged more than wild-type (WT) cells. In hippocampal slices, KO2 acutely inhibited neuronal communication in WT controls without damaging the cytoskeleton. This inhibition was not observed in PrP-/- slices. Neuronal communication and the cytoskeleton of PrP-/- slices became progressively disrupted and degenerated post-recovery, whereas the dysfunction in WT slices recovered in 5 days. This suggests that the acute inhibition of neuronal activity in WT slices in response to KO2 was a neuroprotective role of PrPC, which PrP-/- slices lacked. Heterozygous expression of PrPC was sufficient for this neuroprotection. Further, hippocampal slices from mice expressing PrPC without its GPI anchor (PrPGPI-/-) displayed acute inhibition of neuronal activity by KO2. However, they failed to restore normal activity and cytoskeletal formation post-recovery. This suggests that PrPC facilitates the depressive response to KO2 and its GPI anchoring is required to restore KO2-induced damages. Immuno spin-trapping showed increased radicals formed on the filamentous actin of PrP-/- and PrPGPI-/- slices, but not WT and PrP+/- slices, post-recovery suggesting ongoing dysregulation of redox balance in the slices lacking GPI-anchored PrPC. The MtPQ treatment of hippocampal slices temporarily inhibited neuronal communication independent of PrPC expression. Overall, GPI-anchored PrPC alters synapses and neurotransmission to protect and repair the neuronal cytoskeleton, and neuronal communication, from extrinsically induced oxidative stress damages.

Centrally derived gasotransmitter modulation of hypoglossal motoneuron activity

Airway obstruction during inspiration is a hallmark trait of sleep apnea (SA). In mice, genetic elimination of the CO producing enzyme, heme oxygenase-2 (HO-2), leads to a SA phenotype predominately associated with recurrent airway obstruction. This phenotype is mitigated by either inhibition or genetic ablation of the H2S producing enzyme, cystathionine g-lysase (CSE). While these observations indicate that mutual interactions between HO-2/CO and CSE/H2S are important for regulating airway clearance, the extent of contribution from these interaction in the central nervous system to airway tone are unclear. Here, we determine how HO-2/CO and CSE/H2S impact inspiratory output from the hypoglossal nucleus (XIIn) in the isolated rhythmic brainstem slice preparation. We hypothesize that these enzymes and their respective gasotransmitter products locally interact in the inspiratory circuit to modulate the input-output (I/O) relationship between preBötC and XIIn. Electrophysiological studies conducted in brainstem slices from mice (P5-12) showed that HO-2 dysregulation, using either in wildtype slices exposed to Cr (III) Mesoporphyrin IX chloride or in slices from HO-2 null mice, increased transmission failure from preBötC to XIIn and altered the I/O relationship between the rhythm generating network and XIIn. HO-2 dysregulation also increased subnetwork activity in the preBötC that was associated with a decrease in amplitude regularity. While many transmission failures to the XIIn were associated with preBötC subnetwork activity, simultaneous recordings from the preBötC, premotor area, and the XIIn revealed that preBötC subnetwork activity was reliably transmitted to the premotor field but not to the XIIn. Dual extracellular and intracellular recordings from the preBötC and individual XIIn neurons under disinhibited conditions revealed that HO-2 dysregulation reduced inspiratory drive currents received by XIIn neurons, decreased the intrinsic excitability of XIIn neurons while also reducing the amount of preBötC subnetwork activity. Additionally, targeted inhibition of CSE/H2S activity using L-PAG or by genetic elimination of CSE in HO-2 null mice mitigated these effects of HO-2 dysregulation. Thus, the mutual interactions HO-2/CSE and CSE/H2S in the medullary brainstem appear to modulate XIIn output by: (1) causing a E/I imbalance that promotes preBötC subnetwork activity; (2) suppressing inspiratory drive received in XIIn; and (3) suppressing intrinsic excitability of XIIn neurons. These findings may be key to understanding the action by which central gasotransmitters actions contribute to upper airway muscle regulation. This work was supported by NIH: P01-HL-44454; R01NS107421. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

A requirement for astrocyte IP3R2 signaling for whisker experience-dependent depression and homeostatic upregulation in the mouse barrel cortex.

Changes to sensory experience result in plasticity of synapses in the cortex. This experience-dependent plasticity (EDP) is a fundamental property of the brain. Yet, while much is known about neuronal roles in EDP, very little is known about the role of astrocytes. To address this issue, we used the well-described mouse whiskers-to-barrel cortex system, which expresses a number of forms of EDP. We found that all-whisker deprivation induced characteristic experience-dependent Hebbian depression (EDHD) followed by homeostatic upregulation in L2/3 barrel cortex of wild type mice. However, these changes were not seen in mutant animals (IP3R2–/–) that lack the astrocyte-expressed IP3 receptor subtype. A separate paradigm, the single-whisker experience, induced potentiation of whisker-induced response in both wild-type (WT) mice and IP3R2–/– mice. Recordings in ex vivo barrel cortex slices reflected the in vivo results so that long-term depression (LTD) could not be elicited in slices from IP3R2–/– mice, but long-term potentiation (LTP) could. Interestingly, 1 Hz stimulation inducing LTD in WT paradoxically resulted in NMDAR-dependent LTP in slices from IP3R2–/– animals. The LTD to LTP switch was mimicked by acute buffering astrocytic [Ca2+]i in WT slices. Both WT LTD and IP3R2–/– 1 Hz LTP were mediated by non-ionotropic NMDAR signaling, but only WT LTD was P38 MAPK dependent, indicating an underlying mechanistic switch. These results demonstrate a critical role for astrocytic [Ca2+]i in several EDP mechanisms in neocortex.

TRAIL protects the immature lung from hyperoxic injury

The hyperoxia-induced pro-inflammatory response and tissue damage constitute pivotal steps leading to bronchopulmonary dysplasia (BPD) in the immature lung. The pro-inflammatory cytokines are considered attractive candidates for a directed intervention but the complex interplay between inflammatory and developmental signaling pathways requires a comprehensive evaluation before introduction into clinical trials as studied here for the death inducing ligand TRAIL. At birth and during prolonged exposure to oxygen and mechanical ventilation, levels of TRAIL were lower in tracheal aspirates of preterm infants <29 weeks of gestation which developed moderate/severe BPD. These findings were reproduced in the newborn mouse model of hyperoxic injury. The loss of TRAIL was associated with increased inflammation, apoptosis induction and more pronounced lung structural simplification after hyperoxia exposure for 7 days while activation of NFκB signaling during exposure to hyperoxia was abrogated. Pretreatment with recombinant TRAIL rescued the developmental distortions in precision cut lung slices of both wildtype and TRAIL−/− mice exposed to hyperoxia. Of importance, TRAIL preserved alveolar type II cells, mesenchymal progenitor cells and vascular endothelial cells. In the situation of TRAIL depletion, our data ascribe oxygen toxicity a more injurious impact on structural lung development. These data are not surprising taking into account the diverse functions of TRAIL and its stimulatory effects on NFκB signaling as central driver of survival and development. TRAIL exerts a protective role in the immature lung as observed for the death inducing ligand TNF-α before.

Osteoprotegerin Expression in Liver is Induced by IL13 through TGFβ.

Osteoprotegerin (OPG) is a profibrotic mediator produced by myofibro-blasts under influence of transforming growth factor β (TGFβ). Its expression in experimental models of liver fibrosis correlates well with disease severity and treatment responses. The regulation of OPG in liver tissue is largely unknown and we therefore set out to elucidate which growth factors/interleukins associated with fibrosis induce OPG and through which pathways. Precision-cut liver slices of wild type and STAT6-deficient mice and 3T3 fibroblasts were used to investigate the effects of TGFβ, interleukin (IL) 13 (IL13), IL1β, and platelet-derived growth factor BB (PDGF-BB) on expression of OPG. OPG protein was measure by ELISA, whereas OPG mRNA and expression of other relevant genes was measured by qPCR. In addition to TGFβ, only IL13 and not PDGF-BB or IL1β could induce OPG expression in 3T3 fibroblasts and liver slices. This IL13-dependent induction was not shown in liver slices of STAT6-deficient mice and when wild type slices were cotreated with TGFβ receptor 1 kinase inhibitor galunisertib, STAT6 inhibitor AS1517499, or AP1 inhibitor T5224. This suggests that the OPG-inducing effect of IL13 is mediated through IL13 receptor α1-activation and subsequent STAT6-dependent upregulation of IL13 receptor α2, which in turn activates AP1 and induces production of TGFβ and subsequent production of OPG. We have shown that IL13 induces OPG release by liver tissue through a TGFβ-dependent pathway involving both the α1 and the α2 receptor of IL13 and transcription factors STAT6 and AP1. OPG may therefore be a novel target for the treatment liver fibrosis as it is mechanistically linked to two important regulators of fibrosis in liver, namely IL13 and TGFβ1.

Activity‐dependent dysfunctional gene expression patterns are normalized by in vivo treatment of late‐stage Aβ pathology mice with a TrkB/C small molecule ligand

AbstractBackgroundNeurotrophin signaling via TrkB and TrkC receptors participates in long‐term potentiation (LTP), a biological substrate of memory formation that is disrupted by amyloid‐β (Aβ) in APP mice. In this study, we ask whether there are alterations in activity‐dependent gene expression signatures in APP mice, and if so, could elements of these alterations be restored by prior treatment with a small molecule TrkB/C partial agonist, PTX‐BD10‐2.MethodWe administered PTX‐BD10‐2 (50 mg/kg) or vehicle (VEH) orally once daily to male wild type (WT) and APP mice expressing London and Swedish mutations (APP‐L/S) for 3 months starting at 13 months of age. Paired‐pulse ratios (PPR) and Theta Burst Stimulation (TBS) were applied to acute hippocampal slices, in absence of drug, to induce short and long‐term synaptic plasticity. Upon completion of recordings, we examined whole tissue transcriptomic profiles for differential gene expression and enrichment of ontological terms and pathways.ResultPronounced deficits in PPR (at 10 and 20 ms) and LTP were found in APP‐L/S relative to WT slices, and entirely restored in slices from APP‐L/S mice treated with PTX‐BD10‐2 (Fig. 1). To identify candidate mechanisms of this pre/post synaptic plasticity rescue, we examined activity (TBS)‐dependent transcriptomic signatures. Analysis of TBS‐only slices demonstrated alterations in gene expression between WT_VEH_TBS and APP‐L/S_VEH_TBS mice. In particular, significant downregulation of genes in pathways known to be important for synapse function: axoneme, glutamatergic/GABAergic synaptic signaling, neurotransmitter release/transmission and neuronal action potential; and upregulation of gene pathways that are candidates contributing to degeneration in AD and in APP‐L/S_VEH_TBS mice: immune system processes, inflammatory responses, aging and neuron death; when compared to WT_VEH_TBS. Remarkably, the altered expression levels of most of the genes from these pathways were reverted to WT pattern in APP‐L/S_BD10‐2_TBS mice (Fig. 2), thus revealing candidate mechanisms to restore PPR and LTP deficits in APP‐L/S mice.ConclusionPTX‐BD10‐2 treatment mitigates Aβ‐related synapse dysfunction and alterations in synapse‐relevant gene expression profiles in APP‐L/S mice in the context of stimulation. These findings point to therapeutic potential of targeting TrkB/C, and moreover, provide a cellular model to identify candidate signaling modules for understanding synaptic impairment and revealing novel therapeutic targets.

Abstract 2885: Connexin 43-dependent miRNA transfer drives perivascular glioma invasion through dysregulation of astrocytes

Abstract Background: Glioblastoma multiforme (GBM) invasion occurs along vasculature, and intercellular communication between glioma cells and the astrocyte endfoot may manipulate astrocytes to repress cell-matrix adhesion through transfer of miRNAs. Intercellular communication, specifically through Connexin 43 (Cx43) gap junction (GJ) channels, has been confoundingly demonstrated to both limit and facilitate GBM invasion. In solid tumors, Cx43 between tumor cells is correlated with a low invasion and the GJs have long been postulated to be required for growth control. By contrast, functional Cx43 channels between GBM and non-cancerous cells is correlated with high invasiveness, and GJs have been proposed to provide the pathway through which GBM drives surrounding astrocytes into a tumor-permissive state. How GJs participate in the transfer of genetic regulatory molecules from GBM to astrocytes is incompletely understood and might involve direct diffusion through Cx43 channels, GJ-facilitated exosomal transfer, and biased GJ endocytosis. In this study we examine astrocyte miRNA expression after contact with GBM and used an ex vivo assay to assess Cx43-dependent invasion. Methods: Immortalized wild-type (WT) and connexin 43 (Cx43) knockout (KO) murine astrocytes were co-cultured for 24 hours with U87 GBM cells expressing cytosolic GFP. Cells were separated by FACS and collected for miRNAseq. For ex vivo invasion model, 350µm whole brain coronal slices (from WT and GFAP-Cre Cx43-/- C57BL/6J mice) were obtained via vibratome in high-sucrose ACSF and cultured for &amp;gt;12 hours in 20% serum recovery medium. At that time, 250-350µm U87 spheroids (4-8 per slice) expressing cytosolic mCherry (either WT or CRISPR-generated Cx43 KO U87 cell lines) were injected into the cerebral cortex. Slices were cultured for up to 72 hours, fixed, and cleared by SeeDB protocol. Invasion was imaged by confocal microscopy at 10x. Extent of perivascular invasion by the injected tumor spheroids was quantified using Sholl analysis to compare number and lengths of branches. Results: miRNASeq identified a cohort of &amp;gt;10 miRNAs that were upregulated in WT astrocytes, but not KO astrocytes, co-cultured with GBM. Some of these miRNAs are known to have elevated expression in GBM and have predicted cell adhesion protein targets. GBM pervascular invasion was substantially lower for WT-U87 spheroids in GFAP-Cre Cx43-/- slice when compared to WT-U87 in WT slice or Cx43 KO U87 in Cx43-/- slice. Conclusions: Specific miRNAs are transferred via Cx43-mediated mechanisms from GBM to astrocytes and may play a role in astrocyte-matrix adhesion at the endfoot, creating an opening for GBM invasion. Initial slice culture data indicate that functional GBM-GBM GJs inhibit peritumoral outgrowth, whereas astrocyte-GBM GJs are critical for invasion, presumably facilitating remodeling of host tissue by the miRNAs to accommodate invading GBM. Citation Format: Sean McCutcheon, David C. Spray. Connexin 43-dependent miRNA transfer drives perivascular glioma invasion through dysregulation of astrocytes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2885.

Neuronal activity modulates alpha-synuclein aggregation and spreading in organotypic brain slice cultures and in vivo.

Alpha-synuclein (αSyn) preformed fibrils (PFF) induce endogenous αSyn aggregation leading to reduced synaptic transmission. Neuronal activity modulates release of αSyn; however, whether neuronal activity regulates the spreading of αSyn pathology remains elusive. Here, we established a hippocampal slice culture system from wild-type (WT) mice and found that both Ca2+ influx and the uptake of αSyn PFF were higher in the CA3 than in the CA1 sub-region. Pharmacologically enhancing neuronal activity substantially increased αSyn pathology in αSyn PFF-treated hippocampal or midbrain slice cultures and accelerated dopaminergic neuron degeneration. Consistently, neuronal hyperactivity promoted PFF trafficking along axons/dendrites within microfluidic chambers. Unexpectedly, enhancing neuronal activity in LRRK2 G2019S mutant slice cultures further increased αSyn pathology, especially with more Lewy body (LB) forming than in WT slice cultures. Finally, following injection of αSyn PFF and chemogenetic modulators into the dorsal striatum of WT mice, both motor behavior and αSyn pathology were exacerbated likely by enhancing neuronal activity, since they were ameliorated by reducing neuronal activity. Thus, a greater understanding of the impact of neuronal activity on αSyn aggregation and spreading, as well as dopaminergic neuronal vulnerability, may provide new therapeutic strategies for patients with LB disease (LBD).

Familial Alzheimer's disease mutations at position 22 of the amyloid β-peptide sequence differentially affect synaptic loss, tau phosphorylation and neuronal cell death in an ex vivo system.

Familial forms of Alzheimer’s disease (AD) are caused by mutations in the presenilin genes or in the gene encoding for the amyloid precursor protein (APP). Proteolytic cleavage of APP generates the β-amyloid peptide (Aβ), which aggregates into amyloid plaques, one of the major hallmarks of AD. APP mutations within the Aβ sequence, so-called intra-Aβ mutations, cluster around position E693 of APP, which corresponds to position E22 in the Aβ sequence. One of these mutations is the Osaka mutation, E693Δ, which has unique aggregation properties with patients showing unusually low brain amyloid levels on amyloid PET scans. Despite intense research on the pathomechanisms of different intra-Aβ mutants, our knowledge is limited due to controversial findings in various studies. Here, we investigated in an ex vivo experimental system the neuro- and synaptotoxic properties of two intra-Aβ mutants with different intrinsic aggregation propensities, the Osaka mutation E22Δ and the Arctic mutation E22G, and compared them to wild-type (wt) Aβ. Experiments in hippocampal slice cultures from transgenic mice were complemented by treating wild-type slices with recombinantly produced Aβ40 or Aβ42 containing the respective intra-Aβ mutations. Our analyses revealed that wt Aβ and E22G Aβ, both recombinant and transgenic, caused a loss of dendritic spines along with an increase in tau phosphorylation and tau-dependent neurodegeneration. In all experiments, the 42-residue variants of wt and E22G Aβ showed stronger effects than the respective Aβ40 isoforms. In contrast, E22Δ Aβ neither reduced dendritic spine density nor resulted in increased tau phosphorylation or neuronal cell death in our ex vivo system. Our findings suggest that the previously reported major differences in the aggregation kinetics between E22G and E22Δ Aβ are likely reflected in different disease pathomechanisms.

Nitric Oxide Critically Regulates Purkinje Neuron Dendritic Development Through a Metabotropic Glutamate Receptor Type 1-Mediated Mechanism.

Nitric oxide (NO), specifically derived from neuronal nitric oxide synthase (nNOS), is a well-established regulator of synaptic transmission in Purkinje neurons (PNs), governing fundamental processes such as motor learning and coordination. Previous phenotypic analyses showed similar cerebellar structures between neuronal nitric oxide null (nNOS-/-) and wild-type (WT) adult male mice, despite prominent ataxic behavior within nNOS-/- mice. However, a study has yet to characterize PN molecular structure and their excitatory inputs during development in nNOS-/- mice. This study is the first to explore morphological abnormalities within the cerebellum of nNOS-/- mice, using immunohistochemistry and immunoblotting. This study sought to examine PN dendritic morphology and the expression of metabotropic glutamate receptor type 1 (mGluR1), vesicular glutamate transporter type 1 and 2 (vGluT1 and vGluT2), stromal interaction molecule 1 (STIM1), and calpain-1 within PNs of WT and nNOS-/- mice at postnatal day 7 (PD7), 2weeks (2W), and 7weeks (7W) of age. Results showed a decrease in PN dendritic branching at PD7 in nNOS-/- cerebella, while aberrant dendritic spine formation was noted in adult ages. Total protein expression of mGluR1 was decreased in nNOS-/- cerebella across development, while vGluT2, STIM1, and calpain-1 were significantly increased. Ex vivo treatment of WT slices with NOS inhibitor L-NAME increased calpain-1 expression, whereas treating nNOS-/- cerebellar slices with NO donor NOC-18 decreased calpain-1. Moreover, mGluR1 agonist DHPG increased calpain-1 in WT, but not in nNOS-/- slices. Together, these results indicate a novel role for nNOS/NO signaling in PN development, particularly by regulating an mGluR1-initiated calcium signaling mechanism.

Microglial ROCK is essential for chronic methylmercury-induced neurodegeneration.

Methylmercury (MeHg), an environmental pollutant, causes serious damage to many organs. Effects on the CNS were initially thought to arise from MeHg acting directly on neurons, but it also has significant effects on non-neuronal cells such as microglia. Microglia, which are very sensitive to changes in the brain environment, show various phenotypes. We previously reported that upon short exposure to MeHg (MeHgshort ) at low concentration, microglia exhibited a neuroprotective phenotype; whereas, long-term exposure (MeHglong ) induced a neurotoxic phenotype of microglia. However, contributions of microglia to MeHg-induced CNS damage remain unknown. Even at very low concentrations, MeHglong but not MeHgshort caused significant neuronal damage associated with an increased number of reactive microglia in cortical slices from wild-type (WT) mice. Two-photon imaging of cortical slices from Iba1-GFP mice revealed that microglia in control conditions exhibited elongated and complex processes with high motility. MeHglong caused a significant reduction in process motility, retraction of processes, and hypertrophic cell bodies, indicating activated microglia. Moreover, MeHglong -treated microglia upregulated pro-inflammatory molecues, suggesting a change into a neurotoxic phenotype of microglia. As a molecular target, Rho-kinase (ROCK) was found to be key for controlling microglial reactivity and neurotoxicity. Expression level of ROCK was increased by MeHglong in WT slices, which was abolished by minocycline or Y-27632. We confirmed that MeHg directly activates microglial ROCK pathways prepared from WT mice. In addition, MeHg-evoked damage of primary neurons was significantly enhanced by the presence of microglia from WT mice, but offset by minocycline or Y-27632. Taken together, our data demonstrate that MeHg causes neurodegeneration by inducing a neurotoxic microglia phenotype via a ROCK-mediated mechanism.

P2X7 Receptor Indirectly Regulates the JAM-A Protein Content via Modulation of GSK-3β.

The alveolar epithelial cells represent an important part of the alveolar barrier, which is maintained by tight junction proteins, particularly JAM-A, occludin, and claudin-18, which regulate paracellular permeability. In this study, we report on a strong increase in epithelial JAM-A expression in P2X7 receptor knockout mice when compared to the wildtype. Precision-cut lung slices of wildtype and knockout lungs and immortal epithelial lung E10 cells were treated with bleomycin, the P2X7 receptor inhibitor oxATP, and the agonist BzATP, respectively, to evaluate early changes in JAM-A expression. Biochemical and immunohistochemical data showed evidence for P2X7 receptor-dependent JAM-A expression in vitro. Inhibition of the P2X7 receptor using oxATP increased JAM-A, whereas activation of the receptor decreased the JAM-A protein level. In order to examine the role of GSK-3β in the expression of JAM-A in alveolar epithelial cells, we used lithium chloride for GSK-3β inhibiting experiments, which showed a modulating effect on bleomycin-induced alterations in JAM-A levels. Our data suggest that an increased constitutive JAM-A protein level in P2X7 receptor knockout mice may have a protective effect against bleomycin-induced lung injury. Bleomycin-treated precision-cut lung slices from P2X7 receptor knockout mice responded with a lower increase in mRNA expression of JAM-A than bleomycin-treated precision-cut lung slices from wildtype mice.

Coherency of circadian rhythms in the SCN is governed by the interplay of two coupling factors.

Circadian clocks are autonomous oscillators driving daily rhythms in physiology and behavior. In mammals, a network of coupled neurons in the suprachiasmatic nucleus (SCN) is entrained to environmental light-dark cycles and orchestrates the timing of peripheral organs. In each neuron, transcriptional feedbacks generate noisy oscillations. Coupling mediated by neuropeptides such as VIP and AVP lends precision and robustness to circadian rhythms. The detailed coupling mechanisms between SCN neurons are debated. We analyze organotypic SCN slices from neonatal and adult mice in wild-type and multiple knockout conditions. Different degrees of rhythmicity are quantified by pixel-level analysis of bioluminescence data. We use empirical orthogonal functions (EOFs) to characterize spatio-temporal patterns. Simulations of coupled stochastic single cell oscillators can reproduce the diversity of observed patterns. Our combination of data analysis and modeling provides deeper insight into the enormous complexity of the data: (1) Neonatal slices are typically stronger oscillators than adult slices pointing to developmental changes of coupling. (2) Wild-type slices are completely synchronized and exhibit specific spatio-temporal patterns of phases. (3) Some slices of Cry double knockouts obey impaired synchrony that can lead to co–existing rhythms (“splitting”). (4) The loss of VIP-coupling leads to desynchronized rhythms with few residual local clusters. Additional information was extracted from co–culturing slices with rhythmic neonatal wild-type SCNs. These co–culturing experiments were simulated using external forcing terms representing VIP and AVP signaling. The rescue of rhythmicity via co–culturing lead to surprising results, since a cocktail of AVP-antagonists improved synchrony. Our modeling suggests that these counter-intuitive observations are pointing to an antagonistic action of VIP and AVP coupling. Our systematic theoretical and experimental study shows that dual coupling mechanisms can explain the astonishing complexity of spatio-temporal patterns in SCN slices.

Modulation of Hyperpolarization-Activated Inward Current and Thalamic Activity Modes by Different Cyclic Nucleotides.

The hyperpolarization-activated inward current, Ih, plays a key role in the generation of rhythmic activities in thalamocortical (TC) relay neurons. Cyclic nucleotides, like 3′,5′-cyclic adenosine monophosphate (cAMP), facilitate voltage-dependent activation of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels by shifting the activation curve of Ih to more positive values and thereby terminating the rhythmic burst activity. The role of 3′,5′-cyclic guanosine monophosphate (cGMP) in modulation of Ih is not well understood. To determine the possible role of the nitric oxide (NO)-sensitive cGMP-forming guanylyl cyclase 2 (NO-GC2) in controlling the thalamic Ih, the voltage-dependency and cGMP/cAMP-sensitivity of Ih was analyzed in TC neurons of the dorsal part of the lateral geniculate nucleus (dLGN) in wild type (WT) and NO-GC2-deficit (NO-GC2−/−) mice. Whole cell voltage clamp recordings in brain slices revealed a more hyperpolarized half maximal activation (V1/2) of Ih in NO-GC2−/− TC neurons compared to WT. Different concentrations of 8-Br-cAMP/8-Br-cGMP induced dose-dependent positive shifts of V1/2 in both strains. Treatment of WT slices with lyase enzyme (adenylyl and guanylyl cyclases) inhibitors (SQ22536 and ODQ) resulted in further hyperpolarized V1/2. Under current clamp conditions NO-GC2−/− neurons exhibited a reduction in the Ih-dependent voltage sag and reduced action potential firing with hyperpolarizing and depolarizing current steps, respectively. Intrathalamic rhythmic bursting activity in brain slices and in a simplified mathematical model of the thalamic network was reduced in the absence of NO-GC2. In freely behaving NO-GC2−/− mice, delta and theta band activity was enhanced during active wakefulness (AW) as well as rapid eye movement (REM) sleep in cortical local field potential (LFP) in comparison to WT. These findings indicate that cGMP facilitates Ih activation and contributes to a tonic activity in TC neurons. On the network level basal cGMP production supports fast rhythmic activity in the cortex.

CA1 Neurons Acquire Rett Syndrome Phenotype After Brief Activation of Glutamatergic Receptors: Specific Role of mGluR1/5.

Rett syndrome (RTT) is a neurological disorder caused by the mutation of the X-linked MECP2 gene. The neurophysiological hallmark of the RTT phenotype is the hyperexcitability of neurons made responsible for frequent epileptic attacks in the patients. Increased excitability in RTT might stem from impaired glutamate handling in RTT and its long-term consequences that has not been examined quantitatively. We recently reported (Balakrishnan and Mironov, 2018a,b) that the RTT hippocampus consistently demonstrates repetitive glutamate transients that parallel the burst firing in the CA1 neurons. We aimed to examine how brief stimulation of specific types of ionotropic and metabotropic glutamate receptors (GluR) can modulate the neuronal phenotype. We imaged glutamate with a fluorescence sensor (iGluSnFr) expressed in CA1 neurons in hippocampal organotypic slices from wild-type (WT) and Mecp2-/y mice (RTT). The neuronal and synaptic activities were assessed by patch-clamp and calcium imaging. In both WT and RTT slices, activation of AMPA, kainate, and NMDA receptors for 30 s first enhanced neuronal activity that induced a global release of glutamate. After transient augmentation of excitability and ambient glutamate, they subsided. After wash out of the agonists for 10 min, WT slices recovered and demonstrated repetitive glutamate transients, whose pattern resembled those observed in naïve RTT slices. Hyperpolarization-activated (HCN) decreased and voltage-sensitive calcium channel (VSCC) currents increased. The effects were long-lasting and bigger in WT. We examined the role of mGluR1/5 in more detail. The effects of the agonist (S)-3,5-dihydroxyphenylglycine (DHPG) were the same as AMPA and NMDA and occluded by mGluR1/5 antagonists. Further modifications were examined using a non-stationary noise analysis of postsynaptic currents. The mean single channel current and their number at postsynapse increased after DHPG. We identified new channels as calcium-permeable AMPARs (CP-AMPAR). We then examined back-propagating potentials (bAPs) as a measure of postsynaptic integration. After bAPs, spontaneous afterdischarges were observed that lasted for ∼2 min and were potentiated by DHPG. The effects were occluded by intracellular CP-AMPAR blocker and did not change after NMDAR blockade. We propose that brief elevations in ambient glutamate (through brief excitation with GluR agonists) specifically activate mGluR1/5. This modifies CP-AMPAR, HCN, and calcium conductances and makes neurons hyperexcitable. Induced changes can be further supported by repetitive glutamate transients established and serve to persistently maintain the aberrant neuronal RTT phenotype in the hippocampus.

Activation of Serotonin 5-HT7 Receptors Modulates Hippocampal Synaptic Plasticity by Stimulation of Adenylate Cyclases and Rescues Learning and Behavior in a Mouse Model of Fragile X Syndrome.

We have previously demonstrated that activation of serotonin 5-HT7 receptors (5-HT7R) reverses metabotropic glutamate receptor-mediated long term depression (mGluR-LTD) in the hippocampus of wild-type (WT) and Fmr1 Knockout (KO) mice, a model of Fragile X Syndrome (FXS) in which mGluR-LTD is abnormally enhanced. Here, we have investigated intracellular mechanisms underlying the effect of 5-HT7R activation using patch clamp on hippocampal slices. Furthermore, we have tested whether in vivo administration of LP-211, a selective 5-HT7R agonist, can rescue learning and behavior in Fmr1 KO mice. In the presence of an adenylate cyclase blocker, mGluR-LTD was slightly enhanced in WT and therefore the difference between mGluR-LTD in WT and Fmr1 KO slices was no longer present. Conversely, activation of adenylate cyclase by either forskolin or Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) completely reversed mGluR-LTD in WT and Fmr1 KO. 5-HT7R activation reversed mGluR-LTD in WT and corrected exaggerated mGluR-LTD in Fmr1 KO; this effect was abolished by blockade of either adenylate cyclase or protein kinase A (PKA). Exposure of hippocampal slices to LP-211 caused an increased phosphorylation of extracellular signal regulated kinase (ERK), an intracellular effector involved in mGluR-LTD, in WT mice. Conversely, this effect was barely detectable in Fmr1 KO mice, suggesting that 5-HT7R-mediated reversal of mGluR-LTD does not require ERK stimulation. Finally, an acute in vivo administration of LP-211 improved novel object recognition (NOR) performance in WT and Fmr1 KO mice and reduced stereotyped behavior in Fmr1 KO mice. Our results indicate that mGluR-LTD in WT and Fmr1 KO slices is bidirectionally modulated in conditions of either reduced or enhanced cAMP formation. Activation of 5-HT7 receptors reverses mGluR-LTD by activation of the cAMP/PKA intracellular pathway. Importantly, a systemic administration of a 5-HT7R agonist to Fmr1 KO mice corrected learning deficits and repetitive behavior. We suggest that selective 5-HT7R agonists might become novel pharmacological tools for FXS therapy.