Microglial targeted therapy relieves cognitive impairment caused by Cntnap4 deficiency.

Contactin-associated protein-like 4 (Cntnap4) is critical for GABAergic transmission in the brain. Impaired Cntnap4 function is implicated in neurological disorders, such as autism; however, the role of Cntnap4 on memory processing is poorly understood. Here, we demonstrate that hippocampal Cntnap4 deficiency in female mice manifests as impaired cognitive function and synaptic plasticity. The underlying mechanisms may involve effects on the pro-inflammatory response resulting in dysfunctional GABAergic transmission and activated tryptophan metabolism. To efficiently and accurately inhibit the pro-inflammatory reaction, we established a biomimetic microglial nanoparticle strategy to deliver FDA-approved PLX3397 (termed MNPs@PLX). We show MNPs@PLX successfully penetrates the blood brain barrier and facilitates microglial-targeted delivery of PLX3397. Furthermore, MNPs@PLX attenuates cognitive decline, dysfunctional synaptic plasticity, and pro-inflammatory response in female heterozygous Cntnap4 knockout mice. Together, our findings show loss of Cntnap4 causes pro-inflammatory cognitive decline that is effectively prevented by supplementation with microglia-specific inhibitors; thus validating the targeting of microglial function as a therapeutic intervention in neurocognitive disorders.

Caveolin-1 Expression in the Dorsal Striatum Drives Methamphetamine Addiction-Like Behavior.

Dopamine D1 receptor (D1R) function is regulated by membrane/lipid raft-resident protein caveolin-1 (Cav1). We examined whether altered expression of Cav1 in the dorsal striatum would affect self-administration of methamphetamine, an indirect agonist at the D1Rs. A lentiviral construct expressing Cav1 (LV-Cav1) or containing a short hairpin RNA against Cav1 (LV-shCav1) was used to overexpress or knock down Cav1 expression respectively, in the dorsal striatum. Under a fixed-ratio schedule, LV-Cav1 enhanced and LV-shCav1 reduced responding for methamphetamine in an extended access paradigm compared to LV-GFP controls. LV-Cav1 and LV-shCav1 also produced an upward and downward shift in a dose-response paradigm, generating a drug vulnerable/resistant phenotype. LV-Cav1 and LV-shCav1 did not alter responding for sucrose. Under a progressive-ratio schedule, LV-shCav1 generally reduced positive-reinforcing effects of methamphetamine and sucrose as seen by reduced breakpoints. Western blotting confirmed enhanced Cav1 expression in LV-Cav1 rats and reduced Cav1 expression in LV-shCav1 rats. Electrophysiological findings in LV-GFP rats demonstrated an absence of high-frequency stimulation (HFS)-induced long-term potentiation (LTP) in the dorsal striatum after extended access methamphetamine self-administration, indicating methamphetamine-induced occlusion of plasticity. LV-Cav1 prevented methamphetamine-induced plasticity via increasing phosphorylation of calcium calmodulin kinase II, suggesting a mechanism for addiction vulnerability. LV-shCav1 produced a marked deficit in the ability of HFS to produce LTP and, therefore, extended access methamphetamine was unable to alter striatal plasticity, indicating a mechanism for resistance to addiction-like behavior. Our results demonstrate that Cav1 expression and knockdown driven striatal plasticity assist with modulating addiction to drug and nondrug rewards, and inspire new strategies to reduce psychostimulant addiction.

Sexually dimorphic prelimbic cortex mechanisms play a role in alcohol dependence: protection by endostatin.

Angiogenesis or proliferation of endothelial cells plays a role in brain microenvironment homeostasis. Previously we have shown enhanced expression of markers of angiogenesis in the medial prefrontal cortex during abstinence in an animal model of ethanol dependence induced by chronic intermittent ethanol vapor (CIE) and ethanol drinking (ED) procedure. Here we report that systemic injections of the angiogenesis inhibitor endostatin reduced relapse to drinking behavior in female CIE-ED rats without affecting relapse to drinking in male CIE-ED rats, and female and male nondependent ED rats. Endostatin did not alter relapse to sucrose drinking in both sexes. Endostatin reduced expression of platelet endothelial cell adhesion molecule-1 (PECAM-1) in all groups; however, rescued expression of tight junction protein claudin-5 in the prelimbic cortex (PLC) of female CIE-ED rats. In both sexes, CIE-ED enhanced microglial activation in the PLC and this was selectively prevented by endostatin in female CIE-ED rats. Endostatin prevented CIE-ED-induced enhanced NF-kB activity and expression and Fos expression in females and did not alter reduced Fos expression in males. Analysis of synaptic processes within the PLC revealed sexually dimorphic adaptations, with CIE-ED reducing synaptic transmission and altering synaptic plasticity in the PLC in females, and increasing synaptic transmission in males. Endostatin prevented the neuroadaptations in the PLC in females via enhancing phosphorylation of CaMKII, without affecting the neuroadaptations in males. Our multifaceted approach is the first to link PLC endothelial cell damage to the behavioral, neuroimmune, and synaptic changes associated with relapse to ethanol drinking in female subjects, and provides a new therapeutic strategy to reduce relapse in dependent subjects.

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.

Initial Biphasic Fractional Anisotropy Response to Blast-Induced Mild Traumatic Brain Injury in a Mouse Model.

INTRODUCTION: Blast-induced mild traumatic brain injury was generated in a mouse model using a shock tube to investigate recovery and axonal injury from single blast. METHODS: A supersonic helium wave hit the head of anesthetized male young adult mice with a reflected pressure of 69 psi for 0.2 ms on Day 1. Subsequently, the mice were cardioperfused on Days 2, 5, or 12. The isolated brains were subjected to diffusion tensor imaging. Reduced fractional anisotropy (FA) indicated axonal injury. RESULTS: After single blast, FA showed a biphasic response in the corpus callosum with decrease on Days 2 and 12 and increase on Day 5. CONCLUSIONS: Blast-induced mild traumatic brain injury in a mouse model follows a biphasic FA response within 12 days after a single blast similar to that reported for human subjects.

Acute Ethanol Exposure Enhances Synaptic Plasticity in the Dorsal Striatum in Adult Male and Female Rats.

BACKGROUND: Acute (ex vivo) and chronic (in vivo) alcohol exposure induces neuroplastic changes in the dorsal striatum, a critical region implicated in instrumental learning. OBJECTIVE: Sex differences are evident in alcohol reward and reinforcement, with female rats consuming higher amount of alcohol in operant paradigms compared to male rats. However, sex differences in the neuroplastic changes produced by acute alcohol in the dorsal striatum have been unexplored. METHODS: Using electrophysiological recordings from dorsal striatal slices obtained from adult male and female rats, we investigated the effects of ex vivo ethanol exposure on synaptic transmission and synaptic plasticity. Ethanol (44 mM) enhanced basal synaptic transmission in both sexes. Ethanol also enhanced long-term potentiation in both sexes. Other measures of synaptic plasticity including paired-pulse ratio were unaltered by ethanol in both sexes. RESULTS: The results suggest that alterations in synaptic plasticity induced by acute ethanol, at a concentration associated with intoxication, could play an important role in alcohol-induced experience-dependent modification of corticostriatal circuits underlying the learning of goal-directed instrumental actions and formation of habits mediating alcohol seeking and taking. CONCLUSIONS: Taken together, understanding the mechanism(s) underlying alcohol induced changes in corticostriatal function may lead to the development of more effective therapeutic agents to reduce habitual drinking and seeking associated with alcohol use disorders.

TREM2 Is a Receptor for ß-Amyloid that Mediates Microglial Function.

Mutations in triggering receptor expressed on myeloid cells 2 (TREM2) have been linked to increased Alzheimer's disease (AD) risk. Neurobiological functions of TREM2 and its pathophysiological ligands remain elusive. Here we found that TREM2 directly binds to ß-amyloid (Aß) oligomers with nanomolar affinity, whereas AD-associated TREM2 mutations reduce Aß binding. TREM2 deficiency impairs Aß degradation in primary microglial culture and mouse brain. Aß-induced microglial depolarization, K+ inward current induction, cytokine expression and secretion, migration, proliferation, apoptosis, and morphological changes are dependent on TREM2. In addition, TREM2 interaction with its signaling adaptor DAP12 is enhanced by Aß, regulating downstream phosphorylation of SYK and GSK3ß. Our data demonstrate TREM2 as a microglial Aß receptor transducing physiological and AD-related pathological effects associated with Aß.

SORLA attenuates EphA4 signaling and amyloid ß-induced neurodegeneration.

Sortilin-related receptor with LDLR class A repeats (SORLA, SORL1, or LR11) is a genetic risk factor associated with Alzheimer's disease (AD). Although SORLA is known to regulate trafficking of the amyloid ß (Aß) precursor protein to decrease levels of proteotoxic Aß oligomers, whether SORLA can counteract synaptic dysfunction induced by Aß oligomers remains unclear. Here, we show that SORLA interacts with the EphA4 receptor tyrosine kinase and attenuates ephrinA1 ligand-induced EphA4 clustering and activation to limit downstream effects of EphA4 signaling in neurons. Consistent with these findings, SORLA transgenic mice, compared with WT mice, exhibit decreased EphA4 activation and redistribution to postsynaptic densities, with milder deficits in long-term potentiation and memory induced by Aß oligomers. Importantly, we detected elevated levels of active EphA4 in human AD brains, where EphA4 activation is inversely correlated with SORLA/EphA4 association. These results demonstrate a novel role for SORLA as a physiological and pathological EphA4 modulator, which attenuates synaptotoxic EphA4 activation and cognitive impairment associated with Aß-induced neurodegeneration in AD.

MEF2D haploinsufficiency downregulates the NRF2 pathway and renders photoreceptors susceptible to light-induced oxidative stress.

Gaining mechanistic insight into interaction between causative factors of complex multifactorial diseases involving photoreceptor damage might aid in devising effective therapies. Oxidative stress is one of the potential unifying mechanisms for interplay between genetic and environmental factors that contribute to photoreceptor pathology. Interestingly, the transcription factor myocyte enhancer factor 2d (MEF2D) is known to be important in photoreceptor survival, as knockout of this transcription factor results in loss of photoreceptors in mice. Here, using a mild light-induced retinal degeneration model, we show that the diminished MEF2D transcriptional activity in Mef2d+/- retina is further reduced under photostimulation-induced oxidative stress. Reactive oxygen species cause an aberrant redox modification on MEF2D, consequently inhibiting transcription of its downstream target, nuclear factor (erythroid-derived 2)-like 2 (NRF2). NRF2 is a master regulator of phase II antiinflammatory and antioxidant gene expression. In the Mef2d heterozygous mouse retina, NRF2 is not up-regulated to a normal degree in the face of light-induced oxidative stress, contributing to accelerated photoreceptor cell death. Furthermore, to combat this injury, we found that activation of the endogenous NRF2 pathway using proelectrophilic drugs rescues photoreceptors from photo-induced oxidative stress and may therefore represent a viable treatment for oxidative stress-induced photoreceptor degeneration, which is thought to contribute to some forms of retinitis pigmentosa and age-related macular degeneration.

Levetiracetam inhibits oligomeric Aß-induced glutamate release from human astrocytes.

A recently identified mechanism for oligomeric Aß-induced glutamate release from astrocytes involves intracellular Ca elevation, potentially by Ca-dependent vesicular release. Evidence suggests that levetiracetam (LEV; Keppra), an antiepileptic drug, can improve cognitive performance in both humans with mild cognitive impairment and animal models of Alzheimer disease. Because LEV acts by modulating neurotransmitter release from neurons by interaction with synaptic vesicles, we tested the effect of LEV on Aß-induced astrocytic release of glutamate. We used a fluorescence resonance energy transfer-based glutamate sensor (termed SuperGluSnFR), whose structure is based on the ligand-binding site of glutamate receptors, to monitor glutamate release from primary cultures of human astrocytes exposed to oligomeric amyloid-ß peptide 1-42 (Aß42). We found that LEV (10 µM) inhibited oligomeric Aß-induced astrocytic glutamate release. In addition, we show that this Aß-induced glutamate release from astrocytes is sensitive to tetanus neurotoxin, an inhibitor of the vesicle release machinery. Taken together, our evidence suggests that LEV inhibits Aß-induced vesicular glutamate release from astrocytes and thus may underlie, at least in part, the ability of LEV to reduce hyperexcitability in Alzheimer disease.

Astrocytes contribute to gamma oscillations and recognition memory.

Glial cells are an integral part of functional communication in the brain. Here we show that astrocytes contribute to the fast dynamics of neural circuits that underlie normal cognitive behaviors. In particular, we found that the selective expression of tetanus neurotoxin (TeNT) in astrocytes significantly reduced the duration of carbachol-induced gamma oscillations in hippocampal slices. These data prompted us to develop a novel transgenic mouse model, specifically with inducible tetanus toxin expression in astrocytes. In this in vivo model, we found evidence of a marked decrease in electroencephalographic (EEG) power in the gamma frequency range in awake-behaving mice, whereas neuronal synaptic activity remained intact. The reduction in cortical gamma oscillations was accompanied by impaired behavioral performance in the novel object recognition test, whereas other forms of memory, including working memory and fear conditioning, remained unchanged. These results support a key role for gamma oscillations in recognition memory. Both EEG alterations and behavioral deficits in novel object recognition were reversed by suppression of tetanus toxin expression. These data reveal an unexpected role for astrocytes as essential contributors to information processing and cognitive behavior.

Differential effects of synaptic and extrasynaptic NMDA receptors on Aß-induced nitric oxide production in cerebrocortical neurons.

Oligomerized amyloid-ß (Aß) peptide is thought to contribute to synaptic damage, resulting in dysfunctional neuronal networks in patients with Alzheimer's disease. It has been previously suggested that Aß may be detrimental to neuronal health, at least in part, by triggering oxidative/nitrosative stress. However, the mechanisms underlying this process remain to be elucidated. Here, using rat primary cerebrocortical cultures, we demonstrate that Aß1-42 oligomers trigger a dramatic increase in intracellular nitric oxide (NO) concentration via a process mediated by activation of NMDA-type glutamate receptors (NMDARs). Considering that synaptic NMDARs and extrasynaptic NMDARs (eNMDARs) can have opposite effects on neuronal viability, we explored their respective roles in Aß-induced increases in NO levels. Surprisingly, after pharmacological isolation of eNMDARs, we discovered that eNMDARs are primarily responsible for the increase in neuronal NO triggered by Aß oligomers. Moreover, we found that the eNMDAR-mediated increase in NO can produce S-nitrosylation of Drp1 (dynamin-related protein 1) and Cdk5 (cyclin-dependent kinase 5), targets known to contribute to Aß-induced synaptic damage. These results suggest that pharmacological intervention specifically aimed at eNMDARs may decrease Aß-induced nitrosative stress and thus ameliorate neurotoxic damage to synapses.

Aß induces astrocytic glutamate release, extrasynaptic NMDA receptor activation, and synaptic loss.

Synaptic loss is the cardinal feature linking neuropathology to cognitive decline in Alzheimer's disease (AD). However, the mechanism of synaptic damage remains incompletely understood. Here, using FRET-based glutamate sensor imaging, we show that amyloid-ß peptide (Aß) engages α7 nicotinic acetylcholine receptors to induce release of astrocytic glutamate, which in turn activates extrasynaptic NMDA receptors (eNMDARs) on neurons. In hippocampal autapses, this eNMDAR activity is followed by reduction in evoked and miniature excitatory postsynaptic currents (mEPSCs). Decreased mEPSC frequency may reflect early synaptic injury because of concurrent eNMDAR-mediated NO production, tau phosphorylation, and caspase-3 activation, each of which is implicated in spine loss. In hippocampal slices, oligomeric Aß induces eNMDAR-mediated synaptic depression. In AD-transgenic mice compared with wild type, whole-cell recordings revealed excessive tonic eNMDAR activity accompanied by eNMDAR-sensitive loss of mEPSCs. Importantly, the improved NMDAR antagonist NitroMemantine, which selectively inhibits extrasynaptic over physiological synaptic NMDAR activity, protects synapses from Aß-induced damage both in vitro and in vivo.

High-frequency hippocampal oscillations activated by optogenetic stimulation of transplanted human ESC-derived neurons.

After transplantation, individual stem cell-derived neurons can functionally integrate into the host CNS; however, evidence that neurons derived from transplanted human embryonic stem cells (hESCs) can drive endogenous neuronal network activity in CNS tissue is still lacking. Here, using multielectrode array recordings, we report activation of high-frequency oscillations in the ß and γ ranges (10-100 Hz) in the host hippocampal network via targeted optogenetic stimulation of transplanted hESC-derived neurons.

Excitatory glycine responses of CNS myelin mediated by NR1/NR3 "NMDA" receptor subunits.

NMDA receptors are typically excited by a combination of glutamate and glycine. Here we describe excitatory responses in CNS myelin that are gated by a glycine agonist alone and mediated by NR1/NR3 "NMDA" receptor subunits. Response properties include activation by d-serine, inhibition by the glycine-site antagonist CNQX, and insensitivity to the glutamate-site antagonist d-APV. d-Serine responses were abrogated in NR3A-deficient mice. Our results suggest the presence of functional NR1/NR3 receptors in CNS myelin.

Motor impairment and neuronal damage following hypothermia in tropical amphibians.

Although the induction of mild to moderate cerebral hypothermia in mammals can have neuroprotective activity, some deleterious effects have been described when inducing deep hypothermia during cooling of the brain. In the spinal cord, rapid deep cooling can induce seizure activity accompanied by release of the excitatory neurotransmitters, glutamate and aspartate. We used cold-sensitive tropical amphibians as a model to determine (a) the critical temperature inside the central nervous system necessary to induce seizures during rapid cooling; (b) the survival rate during slow deep cooling of the whole animal; and (c) whether deep cooling can cause neuronal cell damage. Seizures induced by deep rapid (or=30 min) deep cooling of the whole animal (12 h at 2-3 degrees C), around 70% of animals died. Spinal reflexes were enhanced when temperatures within the spinal cord reached between 9.0 degrees C and 11.6 degrees C. A fivefold increase in blood glucose level was observed during slow deep cooling. Recovery after slow deep cooling was accompanied by motor impairment and the main histological findings were condensation of the cytoplasm and nuclear pyknosis. Severe neuronal cell damage was characterized by swelling, vacuolated cytoplasm with distended neuronal bodies. These results indicate that deep cooling can easily induce neuronal cell damage in the central nervous system of cold-sensitive animals. They also warn us to the potential sequels associated with the use of deep brain cooling as a neuroprotective strategy.

Activity of common anticonvulsant drugs on spinal seizure-induced by sudden cooling.

Although some of the clinical signs associated with epilepsy have their origin in supraspinal structures, it is the spinal cord in the end, which is responsible for generating the typical pattern of tonic-clonic contractions associated with a convulsion. Indeed, the spinal cord isolated from influence of the brain is capable of convulsive and paroxysmal activity that exhibits the same stereotyped motor pattern seen in the intact animal. This motor pattern can be reproduced experimentally by cooling the isolated spinal cord of amphibians. The isolated spinal cord-hindleg preparation of toad was used. Convulsive activity was induced by placing the isolated spinal cord into a Ringer's bath kept at 7 degrees C. The characteristic phases of the convulsion and their intensity were assessed by recording tonic-clonic contractions of hindleg muscles. Two main endpoints were used to assess the anticonvulsive activity of the drugs tested: first, their ability to block only the tonic hind-limb extension (THE) and second, their ability to block all tonic-clonic activity. The ED50 values and its 95% confidence interval estimated for abolition of THE for each drugs was (mg/kg): carbamazepine 8.6 (6.8-10.8), phenytoin 13 (7.1-23.6), diazepam 0.007 (0.004-0.01), MK-801 3.4 (2.0-5.7), valproate 120 (40-400), phenobarbital 17.1 (12.2-23.9), pentobarbital 10 (6-16.4), mephenesin 2-5 and acetazolamide >500. The ability of some of these drugs to inhibit this kind of seizure activity at doses within therapeutic range suggests a potential use of this isolated preparation as a model in the study and testing of new anticonvulsive drugs.

Subtypes of NMDA receptors in new-born rat hippocampal granule cells.

To investigate the properties of NMDA receptors expressed in new-born rat hippocampal granule cells, recordings were made of single-channel currents produced by application of glutamate or NMDA to outside-out membrane patches. Outside-out patches displayed two distinct patterns of single-channel activity. In some patches only high conductance single-channel activity composed of 42 and 50 pS currents was observed while in others both high (42 and 50 pS) and low (17 and 33 pS) conductance single-channel currents occurred. An absence of direct transitions connecting the smallest (17 pS) and largest (50 pS) conductance unitary currents, as well as an absence of direct transitions connecting 17, 42 and 50 pS currents in sequence, suggested that high and low conductance single-channel activity may have been produced as a result of the activation of two distinct NMDA receptor populations. The NR2B subunit-selective NMDA receptor antagonist, ifenprodil, blocked the high conductance currents suggesting that these receptors contain the NR2B subunit while a clear asymmetry in the frequency of direct transitions between 17 and 42 pS conductance levels indicates the presence of NMDA receptors containing NR2D subunits. In patches containing both high and low conductance-channel activity, evidence for negative coupling between NR2B- and NR2D-like channel activity was observed, suggesting receptors containing these subunits do not gate independently or that both NR2B and NR2D subunits may be part of a single receptor molecule. We conclude that NMDA receptors in P0 hippocampal granule cells are likely to be a mixture of NR1/NR2B diheteromers and receptors of novel molecular composition that may be triheteromeric receptors composed of NR1, NR2B and NR2D subunits.