mGlu5 positive allosteric modulation normalizes synaptic plasticity defects and motor phenotypes in a mouse model of Rett syndrome.

Rett syndrome (RS) is a neurodevelopmental disorder that shares many symptomatic and pathological commonalities with idiopathic autism. Alterations in protein synthesis-dependent synaptic plasticity (PSDSP) are a hallmark of a number of syndromic forms of autism; in the present work, we explore the consequences of disruption and rescue of PSDSP in a mouse model of RS. We report that expression of a key regulator of synaptic protein synthesis, the metabotropic glutamate receptor 5 (mGlu5) protein, is significantly reduced in both the brains of RS model mice and in the motor cortex of human RS autopsy samples. Furthermore, we demonstrate that reduced mGlu5 expression correlates with attenuated DHPG-induced long-term depression in the hippocampus of RS model mice, and that administration of a novel mGlu5 positive allosteric modulator (PAM), termed VU0462807, can rescue synaptic plasticity defects. Additionally, treatment of Mecp2-deficient mice with VU0462807 improves motor performance (open-field behavior and gait dynamics), corrects repetitive clasping behavior, as well as normalizes cued fear-conditioning defects. Importantly, due to the rationale drug discovery approach used in its development, our novel mGlu5 PAM improves RS phenotypes and synaptic plasticity defects without evoking the overt adverse effects commonly associated with potentiation of mGlu5 signaling (i.e. seizures), or affecting cardiorespiratory defects in RS model mice. These findings provide strong support for the continued development of mGlu5 PAMs as potential therapeutic agents for use in RS, and, more broadly, for utility in idiopathic autism.

Unilateral whisker trimming in newborn rats alters neuronal coincident discharge among mature barrel cortex neurons.

It is known that sensory deprivation, including postnatal whisker trimming, can lead to severe deficits in the firing rate properties of cortical neurons. Recent results indicate that development of synchronous discharge among cortical neurons is also activity influenced, and that correlated discharge is significantly impaired following loss of bilateral sensory input in rats. Here we investigate whether unilateral whisker trimming (unilateral deprivation or UD) after birth interferes in the same way with the development of synchronous discharge in cortex. We measured the coincidence of spikes among pairs of neurons recorded under urethane anesthesia in one whisker barrel field deprived by trimming all contralateral whiskers for 60 days after birth (UD), and in untrimmed controls (CON). In the septal columns around barrels, UD significantly increased the coincident discharge among cortical neurons compared with CON, most notably in layers II/III. In contrast, synchronous discharge was normal between layer IV UD barrel neurons: i.e., not different from CON. Thus, while bilateral whisker deprivation (BD) produced a global deficit in the development of synchrony in layer IV, UD did not block the development of synchrony between neurons in layer IV barrels and increased synchrony within septal circuits. We conclude that changes in synchronous discharge after UD are unexpectedly different from those recorded after BD, and we speculate that this effect may be due to the driven activity from active commissural inputs arising from the contralateral hemisphere that received normal activity levels during postnatal development.

TAAR1 agonist ulotaront modulates striatal and hippocampal glutamate function in a state-dependent manner.

Aberrant dopaminergic and glutamatergic function, particularly within the striatum and hippocampus, has repeatedly been associated with the pathophysiology of schizophrenia. Supported by preclinical and recent clinical data, trace amine-associated receptor 1 (TAAR1) agonism has emerged as a potential new treatment approach for schizophrenia. While current evidence implicates TAAR1-mediated regulation of dopaminergic tone as the primary circuit mechanism, little is known about the effects of TAAR1 agonists on the glutamatergic system and excitation-inhibition balance. Here we assessed the impact of ulotaront (SEP-363856), a TAAR1 agonist in Phase III clinical development for schizophrenia, on glutamate function in the mouse striatum and hippocampus. Ulotaront reduced spontaneous glutamatergic synaptic transmission and neuronal firing in striatal and hippocampal brain slices, respectively. Interestingly, ulotaront potentiated electrically-evoked excitatory synaptic transmission in both brain regions, suggesting the ability to modulate glutamatergic signaling in a state-dependent manner. Similar striatal effects were also observed with the TAAR1 agonist, RO5166017. Furthermore, we show that ulotaront regulates excitation-inhibition balance in the striatum by specifically modulating glutamatergic, but not GABAergic, spontaneous synaptic events. These findings expand the mechanistic circuit hypothesis of ulotaront and TAAR1 agonists, which may be uniquely positioned to normalize both the excessive dopaminergic tone and regulate abnormal glutamatergic function associated with schizophrenia.

Protective role for kidney TREM2high macrophages in obesity- and diabetes-induced kidney injury.

Diabetic kidney disease (DKD), the most common cause of kidney failure, is a frequent complication of diabetes and obesity, and yet to date, treatments to halt its progression are lacking. We analyze kidney single-cell transcriptomic profiles from DKD patients and two DKD mouse models at multiple time points along disease progression-high-fat diet (HFD)-fed mice aged to 90-100 weeks and BTBR ob/ob mice (a genetic model)-and report an expanding population of macrophages with high expression of triggering receptor expressed on myeloid cells 2 (TREM2) in HFD-fed mice. TREM2high macrophages are enriched in obese and diabetic patients, in contrast to hypertensive patients or healthy controls in an independent validation cohort. Trem2 knockout mice on an HFD have worsening kidney filter damage and increased tubular epithelial cell injury, all signs of worsening DKD. Together, our studies suggest that strategies to enhance kidney TREM2high macrophages may provide therapeutic benefits for DKD.

Cross-sensory modulation of primary sensory cortex is developmentally regulated by early sensory experience.

The presence of cross-sensory influences on neuronal responses in primary sensory cortex has been observed previously using several different methods. To test this idea in rat S1 barrel cortex, we hypothesized that auditory stimuli combined with whisker stimulation ("cross-sensory" stimuli) may modify response levels to whisker stimulation. Since the brain has been shown to have a remarkable capacity to be modified by early postnatal sensory activity, manipulating postnatal sensory experiences would be predicted to alter the degree of cross-sensory interactions. To test these ideas, we raised rats with or without whisker deprivation and with or without postnatal exposure to repeated auditory clicks. We recorded extracellular responses under urethane anesthesia from barrel cortex neurons in response to principal whisker stimulation alone, to auditory click stimulation alone, or to a cross-sensory stimulus. The responses were compared statistically across different stimulus conditions and across different rearing groups. Barrel neurons did not generate action potentials in response to auditory click stimuli alone in any rearing group. However, in cross-sensory stimulus conditions the response magnitude was facilitated in the 0-15 ms post-whisker-stimulus epoch in all rearing conditions, whereas modulation of response magnitude in a later 15-30 ms post-whisker-stimulus epoch was significantly different in each rearing condition. The most significant cross-sensory effect occurred in rats that were simultaneously whisker deprived and click reared. We conclude that there is a modulatory type of cross-sensory auditory influence on normal S1 barrel cortex, which can be enhanced by early postnatal experiences.

Early bilateral sensory deprivation blocks the development of coincident discharge in rat barrel cortex.

Several theories have proposed a functional role for synchronous neuronal firing in generating the neural code of a sensory perception. Synchronous neural activity develops during a critical postnatal period of cortical maturation, and severely reducing neural activity in a sensory pathway during this period could interfere with the development of coincident discharge among cortical neurons. Loss of such synchrony could provide a fundamental mechanism for the degradation of acuity shown in behavioral studies. We tested the hypothesis that synchronous discharge of barrel cortex neurons would fail to develop after sensory deprivation produced by bilateral whisker trimming from birth to postnatal day 60. By studying the correlated discharge of cortical neuron pairs, we found evidence for strong correlated firing in control animals, and this synchrony was almost absent among pairs of cortical barrel neurons in deprived animals. The degree of synchrony impairment was different in subregions of rat barrel cortex. The model that best fits the data is that cortical neurons receiving direct inputs from the primary sensory (lemniscal) pathway show the greatest decrement in synchrony following sensory deprivation, while neurons with diverse inputs from other areas of thalamus and cortex are relatively less affected in this dimension of cortical function.

Effects of a patient-derived de novo coding alteration of CACNA1I in mice connect a schizophrenia risk gene with sleep spindle deficits.

CACNA1I, a schizophrenia risk gene, encodes a subtype of voltage-gated T-type calcium channel CaV3.3. We previously reported that a patient-derived missense de novo mutation (R1346H) of CACNA1I impaired CaV3.3 channel function. Here, we generated CaV3.3-RH knock-in animals, along with mice lacking CaV3.3, to investigate the biological impact of R1346H (RH) variation. We found that RH mutation altered cellular excitability in the thalamic reticular nucleus (TRN), where CaV3.3 is abundantly expressed. Moreover, RH mutation produced marked deficits in sleep spindle occurrence and morphology throughout non-rapid eye movement (NREM) sleep, while CaV3.3 haploinsufficiency gave rise to largely normal spindles. Therefore, mice harboring the RH mutation provide a patient derived genetic model not only to dissect the spindle biology but also to evaluate the effects of pharmacological reagents in normalizing sleep spindle deficits. Importantly, our analyses highlighted the significance of characterizing individual spindles and strengthen the inferences we can make across species over sleep spindles. In conclusion, this study established a translational link between a genetic allele and spindle deficits during NREM observed in schizophrenia patients, representing a key step toward testing the hypothesis that normalizing spindles may be beneficial for schizophrenia patients.

mGlu5 Positive Allosteric Modulators Facilitate Long-Term Potentiation via Disinhibition Mediated by mGlu5-Endocannabinoid Signaling.

Metabotropic glutamate (mGlu) receptor type 5 (mGlu5) positive allosteric modulators (PAMs) enhance hippocampal long-term potentiation (LTP) and have cognition-enhancing effects in animal models. These effects were initially thought to be mediated by potentiation of mGlu5 modulation of N-methyl-d-aspartate receptor (NMDAR) currents. However, a biased mGlu5 PAM that potentiates Gαq-dependent mGlu5 signaling, but not mGlu5 modulation of NMDAR currents, retains cognition-enhancing effects in animal models, suggesting that potentiation of NMDAR currents is not required for these in vivo effects of mGlu5 PAMs. However, it is not clear whether the potentiation of NMDAR currents is critical for the ability of mGlu5 PAMs to enhance hippocampal LTP. We now report the characterization of effects of two structurally distinct mGlu5 PAMs, VU-29 and VU0092273, on NMDAR currents and hippocampal LTP. As with other mGlu5 PAMs that do not display observable bias for potentiation of NMDAR currents, VU0092273 enhanced both mGlu5 modulation of NMDAR currents and induction of LTP at the hippocampal Schaffer collateral (SC)-CA1 synapse. In contrast, VU-29 did not potentiate mGlu5 modulation of NMDAR currents but induced robust potentiation of hippocampal LTP. Interestingly, both VU-29 and VU0092273 suppressed evoked inhibitory postsynaptic currents (eIPSCs) in CA1 pyramidal cells, and this effect was blocked by the cannabinoid receptor type 1 (CB1) antagonist AM251. Furthermore, AM251 blocked the ability of both mGlu5 PAMs to enhance LTP. Finally, both PAMs failed to enhance LTP in mice with the restricted genetic deletion of mGlu5 in CA1 pyramidal cells. Taken together with previous findings, these results suggest that enhancement of LTP by mGlu5 PAMs does not depend on mGlu5 modulation of NMDAR currents but is mediated by a previously established mechanism in which mGlu5 in CA1 pyramidal cells induces endocannabinoid release and CB1-dependent disinhibition.

Effects of 3D culturing conditions on the transcriptomic profile of stem-cell-derived neurons.

Understanding neurological diseases requires tractable genetic systems. Engineered 3D neural tissues are an attractive choice, but how the cellular transcriptomic profiles in these tissues are affected by the encapsulating materials and are related to the human-brain transcriptome is not well understood. Here, we report the characterization of the effects of culturing conditions on the transcriptomic profiles of induced neuronal cells, as well as a method for the rapid generation of 3D co-cultures of neuronal and astrocytic cells from the same pool of human embryonic stem cells. By comparing the gene-expression profiles of neuronal cells in culture conditions relevant to the developing human brain, we found that modifying the degree of crosslinking of composite hydrogels can tune expression patterns so they correlate with those of specific brain regions and developmental stages. Moreover, by using single-cell sequencing, we show that our engineered tissues recapitulate transcriptional patterns of cell types in the human brain. The analysis of culturing conditions will inform the development of 3D neural tissues for use as tractable models of brain diseases.

Role of mGlu5 Receptors and Inhibitory Neurotransmission in M1 Dependent Muscarinic LTD in the Prefrontal Cortex: Implications in Schizophrenia.

Selective potentiation of the mGlu5 subtype of metabotropic glutamate (mGlu) receptor using positive allosteric modulators (PAMs) has robust cognition-enhancing effects in rodent models that are relevant for schizophrenia. Until recently, these effects were thought to be due to potentiation of mGlu5-induced modulation of NMDA receptor (NMDAR) currents and NMDAR-dependent synaptic plasticity. However, "biased" mGlu5 PAMs that do not potentiate mGlu5 effects on NMDAR currents show efficacy that is similar to that of prototypical mGlu5 PAMs, suggesting that NMDAR-independent mechanisms must be involved in these actions. We now report that synaptic activation of mGlu5 is required for a form of long-term depression (mLTD) in mouse prefrontal cortex (PFC) that is induced by activation of M1 muscarinic acetylcholine (mAChR) receptors, which was previously thought to be independent of mGlu5 activation. Interestingly, a biased mGlu5 PAM, VU0409551, that does not potentiate mGlu5 modulation of NMDAR currents, potentiated induction of mLTD. Furthermore, coactivation of mGlu5 and M1 receptors increased GABAA-dependent inhibitory tone in the PFC pyramidal neurons, which likely contributes to the observed mLTD. Finally, systemic administration of the biased mGlu5 PAM reversed deficits in mLTD and associated cognitive deficits in a model of cortical disruption caused by repeated phencyclidine exposure that is relevant for schizophrenia and was previously shown to be responsive to selective M1 muscarinic receptor PAMs. These studies provide exciting new insights into a novel mechanism by which mGlu5 PAMs can reverse deficits in PFC function and cognition that is independent of modulation of NMDAR currents.

The Role of mGlu Receptors in Hippocampal Plasticity Deficits in Neurological and Psychiatric Disorders: Implications for Allosteric Modulators as Novel Therapeutic Strategies.

Long-term potentiation (LTP) and long-term depression (LTD) are two distinct forms of synaptic plasticity that have been extensively characterized at the Schaffer collateral-CA1 (SCCA1) synapse and the mossy fiber (MF)-CA3 synapse within the hippocampus, and are postulated to be the molecular underpinning for several cognitive functions. Deficits in LTP and LTD have been implicated in the pathophysiology of several neurological and psychiatric disorders. Therefore, there has been a large effort focused on developing an understanding of the mechanisms underlying these forms of plasticity and novel therapeutic strategies that improve or rescue these plasticity deficits. Among many other targets, the metabotropic glutamate (mGlu) receptors show promise as novel therapeutic candidates for the treatment of these disorders. Among the eight distinct mGlu receptor subtypes (mGlu1-8), the mGlu1,2,3,5,7 subtypes are expressed throughout the hippocampus and have been shown to play important roles in the regulation of synaptic plasticity in this brain area. However, development of therapeutic agents that target these mGlu receptors has been hampered by a lack of subtype-selective compounds. Recently, discovery of allosteric modulators of mGlu receptors has provided novel ligands that are highly selective for individual mGlu receptor subtypes. The mGlu receptors modulate the multiple forms of synaptic plasticity at both SC-CA1 and MF synapses and allosteric modulators of mGlu receptors have emerged as potential therapeutic agents that may rescue plasticity deficits and improve cognitive function in patients suffering from multiple neurological and psychiatric disorders.

The hippocampo-prefrontal pathway: a possible therapeutic target for negative and cognitive symptoms of schizophrenia.

The hippocampo-prefrontal (H-PFC) pathway has been linked to cognitive and emotional disturbances in several psychiatric disorders including schizophrenia. Preclinical evidence from the NMDA receptor antagonism rodent model of schizophrenia shows severe pathology selective to the H-PFC pathway. It is speculated that there is an increased excitatory drive from the hippocampus to the prefrontal cortex due to dysfunctions in the H-PFC plasticity, which may serve as the basis for the behavioral consequences observed in this rodent model. Thus, the H-PFC pathway is currently emerging as a promising therapeutic target for the negative and cognitive symptom clusters of schizophrenia. Here, we have reviewed the physiological, pharmacological and functional characteristics of the H-PFC pathway and we propose that allosteric activation of glutamatergic and cholinergic neurotransmission can serve as a plausible therapeutic approach.

Biased mGlu5-Positive Allosteric Modulators Provide In Vivo Efficacy without Potentiating mGlu5 Modulation of NMDAR Currents.

Schizophrenia is associated with disruptions in N-methyl-D-aspartate glutamate receptor subtype (NMDAR)-mediated excitatory synaptic signaling. The metabotropic glutamate receptor subtype 5 (mGlu5) is a closely associated signaling partner with NMDARs and regulates NMDAR function in forebrain regions implicated in the pathology of schizophrenia. Efficacy of mGlu5 positive allosteric modulators (PAMs) in animal models of psychosis and cognition was previously attributed to potentiation of NMDAR function. To directly test this hypothesis, we identified VU0409551 as a novel mGlu5 PAM that exhibits distinct stimulus bias and selectively potentiates mGlu5 coupling to Gαq-mediated signaling but not mGlu5 modulation of NMDAR currents or NMDAR-dependent synaptic plasticity in the rat hippocampus. Interestingly, VU0409551 produced robust antipsychotic-like and cognition-enhancing activity in animal models. These data provide surprising new mechanistic insights into the actions of mGlu5 PAMs and suggest that modulation of NMDAR currents is not critical for in vivo efficacy. VIDEO ABSTRACT.

Abrogated inflammatory response promotes neurogenesis in a murine model of Japanese encephalitis.

BACKGROUND: Japanese encephalitis virus (JEV) induces neuroinflammation with typical features of viral encephalitis, including inflammatory cell infiltration, activation of microglia, and neuronal degeneration. The detrimental effects of inflammation on neurogenesis have been reported in various models of acute and chronic inflammation. We investigated whether JEV-induced inflammation has similar adverse effects on neurogenesis and whether those effects can be reversed using an anti-inflammatory compound minocycline. METHODOLOGY/PRINCIPAL FINDINGS: Here, using in vitro studies and mouse models, we observed that an acute inflammatory milieu is created in the subventricular neurogenic niche following Japanese encephalitis (JE) and a resultant impairment in neurogenesis occurs, which can be reversed with minocycline treatment. Immunohistological studies showed that proliferating cells were replenished and the population of migrating neuroblasts was restored in the niche following minocycline treatment. In vitro, we checked for the efficacy of minocycline as an anti-inflammatory compound and cytokine bead array showed that production of cyto/chemokines decreased in JEV-activated BV2 cells. Furthermore, mouse neurospheres grown in the conditioned media from JEV-activated microglia exhibit arrest in both proliferation and differentiation of the spheres compared to conditioned media from control microglia. These effects were completely reversed when conditioned media from JEV-activated and minocycline treated microglia was used. CONCLUSION/SIGNIFICANCE: This study provides conclusive evidence that JEV-activated microglia and the resultant inflammatory molecules are anti-proliferative and anti-neurogenic for NSPCs growth and development, and therefore contribute to the viral neuropathogenesis. The role of minocycline in restoring neurogenesis may implicate enhanced neuronal repair and attenuation of the neuropsychiatric sequelae in JE survivors.

Proinflammatory mediators released by activated microglia induces neuronal death in Japanese encephalitis.

While a number of studies have documented the importance of microglia in central nervous system (CNS) response to injury, infection and disease, little is known regarding its role in viral encephalitis. We therefore, exploited an experimental model of Japanese Encephalitis, to better understand the role played by microglia in Japanese Encephalitis Virus (JEV) infection. Lectin staining performed to assess microglial activation indicated a robust increase in reactive microglia following infection. A difference in the topographic distribution of activated, resting, and phagocytic microglia was also observed. The levels of various proinflammatory mediators, such as inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (Cox-2), IL-6, IL-1beta, TNF-alpha, and MCP-1 that have been implicated in microglial response to an activational state was significantly elevated following infection. These cytokines exhibited region selective expression in the brains of infected animals, with the highest expression observed in the hippocampus. Moreover, the expression of neuronal specific nuclear protein NeuN was markedly downregulated during progressive infection indicating neuronal loss. In vitro studies further confirmed that microglial activation and subsequent release of various proinflammatory mediators induces neuronal death following JEV infection. Although initiation of immune responses by microglial cells is an important protective mechanism in the CNS, unrestrained inflammatory responses may result in irreparable brain damage. Our findings suggest that the increased microglial activation following JEV infection influences the outcome of viral pathogenesis. It is likely that the increased microglial activation triggers bystander damage, as the animals eventually succumb to infection.