Misfolding at the synapse: A role in amyotrophic lateral sclerosis pathogenesis?

A growing wave of evidence has placed the concept of protein homeostasis at the center of the pathogenesis of amyotrophic lateral sclerosis (ALS). This is due primarily to the presence of pathological transactive response DNA-binding protein (TDP-43), fused in sarcoma (FUS) or superoxide dismutase-1 (SOD1) inclusions within motor neurons of ALS postmortem tissue. However, the earliest pathological alterations associated with ALS occur to the structure and function of the synapse, prior to motor neuron loss. Recent evidence demonstrates the pathological accumulation of ALS-associated proteins (TDP-43, FUS, C9orf72-associated di-peptide repeats and SOD1) within the axo-synaptic compartment of motor neurons. In this review, we discuss this recent evidence and how axo-synaptic proteome dyshomeostasis may contribute to synaptic dysfunction in ALS.

Cells Overexpressing ALS-Associated SOD1 Variants Are Differentially Susceptible to CuATSM-Associated Toxicity.

CuATSM has repeatedly demonstrated to be therapeutically effective in SOD1 mouse models of amyotrophic lateral sclerosis (ALS), leading to current clinical trials. CuATSM acts to stabilize ALS-associated mutant SOD1 protein by supplying copper. However, in vitro work has demonstrated that CuATSM is only therapeutic for wild-type-like SOD1 mutants, not metal-binding-region mutants, suggesting that CuATSM may have genotype-specific effects. Furthermore, relatively high doses of CuATSM have been shown to produce adverse events in humans and mice. Here, we investigated the genotype-specific therapeutic window of CuATSM. NSC-34 cells transiently expressing copper-binding or non-binding mutations of SOD1 were treated with a broad range of CuATSM concentrations and examined for survival via time-lapse microscopy. Determination of the no-observed-adverse-effect level and the LC50 suggest that CuATSM-associated toxicity is dependent on the amount of copper-depleted SOD1 available as well as the mutant's ability to bind copper. Our results suggest that the particular variant of SOD1 mutant is crucial in not only determining the level of efficacy achieved but also potential adverse events.

An Optimized Direct Lysis Gene Expression Microplate Assay and Applications for Disease, Differentiation, and Pharmacological Cell-Based Studies.

Routine cell culture reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) gene expression analysis is limited in scalability due to minimum sample requirement and multistep isolation procedures. In this study, we aimed to optimize and apply a cost-effective and rapid protocol for directly sampling gene expression data from microplate cell cultures. The optimized protocol involves direct lysis of microplate well population followed by a reduced thermocycler reaction time one-step RT-qPCR assay. In applications for inflammation and stress-induced cell-based models, the direct lysis RT-qPCR microplate assay was utilized to detect IFN1 and PPP1R15A expression by poly(I:C) treated primary fibroblast cultures, IL6 expression by poly(I:C) iPSC-derived astrocytes, and differential PPP1R15A expression by ER-stressed vanishing white-matter disease patient induced pluripotent stem cell (iPSC)-derived astrocytes. In application for neural differentiation medium recipe optimizations, conditions were screened for SYN1 and VGLUT1 in neuronal cultures, and S100B, GFAP and EAAT1 in astrocyte cultures. The protocol provides microplate gene expression results from cell lysate to readout within ~35 min, with comparable cost to routine RT-qPCR, and it may be utilized to support laboratory cell-based assays in basic and applied scientific and medical fields of research including stem-cell differentiation, cell physiology, and drug mechanism studies.

Vulnerability of the spinal motor neuron presynaptic terminal sub-proteome in ALS.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder, characterised by the loss of motor neurons and subsequent paralysis. Evidence indicates that synaptic alterations are associated with the early stages of ALS pathogenesis. A hallmark of ALS postmortem tissue is the presence of proteinaceous inclusions, indicative of disturbed protein homeostasis, particularly in spinal cord motor neurons. We recently demonstrated that spinal cord motor neurons contain a supersaturated proteome, as they possess proteins at concentrations that exceed their solubility limits, resulting in a metastable proteome conducive to protein misfolding and aggregation. Recent evidence indicates metastable sub-proteomes within neuronal compartments, such as the synapse, may be particularly vulnerable and underlie their involvement in the initial stages of neurodegenerative diseases. To investigate if the motor neuron presynaptic terminal possesses a metastable sub-proteome, we used human and mouse spinal cord motor neuron expression data to calculate supersaturation scores. Here, we found that both the human and mouse presynaptic terminal sub-proteomes have higher supersaturation scores than the entire motor neuron proteome. In addition, we observed that proteins down-regulated in ALS were over-represented in the synapse. These results provide support for the notion that the metastability of the sub-proteome within the motor neuron presynaptic terminal may be particularly susceptible to protein homeostasis disturbances in ALS, and may contribute to explaining the observed synaptic dysfunction in ALS.

CuATSM improves motor function and extends survival but is not tolerated at a high dose in SOD1G93A mice with a C57BL/6 background.

The synthetic copper-containing compound, CuATSM, has emerged as one of the most promising drug candidates developed for the treatment of amyotrophic lateral sclerosis (ALS). Multiple studies have reported CuATSM treatment provides therapeutic efficacy in various mouse models of ALS without any observable adverse effects. Moreover, recent results from an open label clinical study suggested that daily oral dosing with CuATSM slows disease progression in patients with both sporadic and familial ALS, providing encouraging support for CuATSM in the treatment of ALS. Here, we assessed CuATSM in high copy SOD1G93A mice on the congenic C57BL/6 background, treating at 100 mg/kg/day by gavage, starting at 70 days of age. This dose in this specific model has not been assessed previously. Unexpectedly, we report a subset of mice initially administered CuATSM exhibited signs of clinical toxicity, that necessitated euthanasia in extremis after 3-51 days of treatment. Following a 1-week washout period, the remaining mice resumed treatment at the reduced dose of 60 mg/kg/day. At this revised dose, treatment with CuATSM slowed disease progression and increased survival relative to vehicle-treated littermates. This work provides the first evidence that CuATSM produces positive disease-modifying outcomes in high copy SOD1G93A mice on a congenic C57BL/6 background. Furthermore, results from the 100 mg/kg/day phase of the study support dose escalation determination of tolerability as a prudent step when assessing treatments in previously unassessed models or genetic backgrounds.

Prenatal methadone exposure impairs adolescent cognition and GABAergic neurodevelopment in a novel rat model of maternal methadone treatment.

Methadone maintenance treatment (MMT) is the most common treatment for opioid-dependent pregnant women worldwide. Despite its widespread use, MMT is associated with a variety of adverse neurodevelopmental outcomes in exposed offspring, particularly cognitive impairments. The neurobiological abnormalities underlying these cognitive impairments are, however, poorly understood. This is, in part, due to a lack of animal models that represents the standard of care that methadone is administered in the clinic, with inconsistencies in the timing, doses and durations of treatment. Here we describe the characterisation of a clinically relevant rat model of MMT in which the long-term behavioural and neurobiological effects of prenatal methadone exposure can be assessed in adolescent offspring. Female Sprague-Dawley rats were treated orally with an ascending methadone dosage schedule (5, 10, 15, 20, 25 and 30 mg/kg/day), self-administered in drinking water prior to conception, throughout gestation and lactation. Pregnancy success, maternal gestational weight gain, litter survival and size were not significantly altered in methadone-exposed animals. Methadone-exposed offspring body and brain weights were significantly lower at birth. Novel object recognition tests performed at adolescence revealed methadone-exposed offspring had impaired recognition memory. Furthermore, the rewarded T-maze alternation task demonstrated that methadone-exposed female, but not male, offspring also exhibit working memory and learning deficits. Immunoblots of the adolescent prefrontal cortex and hippocampus showed methadone-exposed offspring displayed reduced levels of mature BDNF, in addition to the GABAergic proteins, GAD67 and parvalbumin, in a sex- and brain region-specific fashion. This rat model closely emulates the clinical scenario in which methadone is administered to opioid-dependent pregnant woman and provides evidence MMT can cause cognitive impairments in adolescent offspring that may be underlined by perturbed neurodevelopment of the GABAergic system.

The effects of perinatal fluoxetine exposure on emotionality behaviours and cortical and hippocampal glutamatergic receptors in female Sprague-Dawley and Wistar-Kyoto rats.

RATIONALE: There is increasing concern regarding the use of selective serotonin reuptake inhibitors (SSRIs) in pregnancy. Animal studies repeatedly show increased anxiety- and depressive-like behaviours in offspring exposed perinatally to SSRIs, however much of this research is in male offspring. OBJECTIVES: The primary aim of this study was to investigate the effects of perinatal SSRI exposure on emotionality-related behaviours in female offspring and associated glutamatergic markers, in Sprague-Dawley (SD) rats and in the Wistar-Kyoto (WKY) rat model of depression. Secondly, we sought to investigate the glutamatergic profile of female WKY rats that may underlie their depressive- and anxiety-like phenotype. METHODS: WKY and SD rat dams were treated with the SSRI, fluoxetine (FLX; 10 mg/kg/day), or vehicle, throughout gestation and lactation (5 weeks total). Female adolescent offspring underwent behaviour testing followed by quantitative immunoblot of glutamatergic markers in the prefrontal cortex and ventral hippocampus. RESULTS: Naïve female WKY offspring displayed an anxiety-like and depressive-like phenotype as well as reductions in NMDA and AMPA receptor subunits and PSD-95 in both ventral hippocampus and prefrontal cortex, compared to SD controls. Perinatal FLX treatment increased anxiety-like and forced swim immobility behaviours in SD offspring but did not influence behaviour in female WKY offspring using these tests. Perinatal FLX exposure did not influence NMDA or AMPA receptor subunit expression in female WKY or SD offspring; it did however have restricted effects on group I mGluR expression in SD and WKY offspring and reduce the glutamatergic synaptic scaffold, PSD-95. CONCLUSION: These findings suggest female offspring of the WKY strain display deficits in glutamatergic markers which may be related to their depressive- and anxiety-like phenotype. While FLX exposed SD offspring displayed increases in anxiety-like and depressive-like behaviours, further studies are needed to assess the potential impact of developmental FLX exposure on the behavioural phenotype of female WKY rats.

Sex differences in the neurochemistry of frontal cortex: Impact of early life stress.

Traumatic events during early life have been linked with later life psychopathology. To understand this risk factor, researchers have studied the effects of prenatal and postnatal early life stress on neurochemical changes. Here we review the rodent literature on sex differences and sex-specific impact of early life stress on frontal cortex neurochemistry. This region is implicated in regulating motivation and emotion, which are often disrupted in psychological disorders. The prefrontal cortex (PFC) in particular is one of the last brain regions to develop, and there are sex differences in the rate of this development. To draw direct comparisons between sexes, our review of the literature was restricted to studies where the effects of prenatal or postnatal stress had been described in male and female littermates. This literature included research describing glutamate, γ-amino butyric acid (GABA), corticosteroids, monoamines, and cannabinoids. We found that sex-dependent effects of stress are mediated by the age at which stress is experienced, age at test, and type of stress endured. More research is required, particularly into the effects of adolescent stress on male and female littermates. We hope that a greater understanding of sex-specific susceptibilities in response to stress across development will help to uncover risk factors for psychological disorders in vulnerable populations.

Amyotrophic Lateral Sclerosis: Proteins, Proteostasis, Prions, and Promises.

Amyotrophic lateral sclerosis (ALS) is characterized by the progressive degeneration of the motor neurons that innervate muscle, resulting in gradual paralysis and culminating in the inability to breathe or swallow. This neuronal degeneration occurs in a spatiotemporal manner from a point of onset in the central nervous system (CNS), suggesting that there is a molecule that spreads from cell-to-cell. There is strong evidence that the onset and progression of ALS pathology is a consequence of protein misfolding and aggregation. In line with this, a hallmark pathology of ALS is protein deposition and inclusion formation within motor neurons and surrounding glia of the proteins TAR DNA-binding protein 43, superoxide dismutase-1, or fused in sarcoma. Collectively, the observed protein aggregation, in conjunction with the spatiotemporal spread of symptoms, strongly suggests a prion-like propagation of protein aggregation occurs in ALS. In this review, we discuss the role of protein aggregation in ALS concerning protein homeostasis (proteostasis) mechanisms and prion-like propagation. Furthermore, we examine the experimental models used to investigate these processes, including in vitro assays, cultured cells, invertebrate models, and murine models. Finally, we evaluate the therapeutics that may best prevent the onset or spread of pathology in ALS and discuss what lies on the horizon for treating this currently incurable disease.

Ubiquitin Homeostasis Is Disrupted in TDP-43 and FUS Cell Models of ALS.

A major feature of amyotrophic lateral sclerosis (ALS) pathology is the accumulation of ubiquitin (Ub) into intracellular inclusions. This sequestration of Ub may reduce the availability of free Ub, disrupting Ub homeostasis and ultimately compromising cellular function and survival. We previously reported significant disturbance of Ub homeostasis in neuronal-like cells expressing mutant SOD1. Here, we show that Ub homeostasis is also perturbed in neuronal-like cells expressing either TDP-43 or FUS. The expression of mutant TDP-43 and mutant FUS led to UPS dysfunction, which was associated with a redistribution of Ub and depletion of the free Ub pool. Redistribution of Ub is also a feature of sporadic ALS, with an increase in Ub signal associated with inclusions and no compensatory increase in Ub expression. Together, these findings suggest that alterations to Ub homeostasis caused by the misfolding and aggregation of ALS-associated proteins play an important role in the pathogenesis of ALS.

Supplement of microbiota-accessible carbohydrates prevents neuroinflammation and cognitive decline by improving the gut microbiota-brain axis in diet-induced obese mice.

BACKGROUND: Western pattern diets induce neuroinflammation and impair cognitive behavior in humans and animals. Neuroinflammation and cognitive impairment have been associated with microbiota dysbiosis, through the gut-brain axis. Furthermore, microbiota-accessible carbohydrates (MACs) found in dietary fiber are important in shaping the microbial ecosystem and have the potential to improve the gut-brain-axis. However, the effects of MACs on neuroinflammation and cognition in an obese condition have not yet been investigated. The present study aimed to evaluate the effect of MACs on the microbiota-gut-brain axis and cognitive function in obese mice induced by a high-fat and fiber deficient (HF-FD) diet. METHODS: C57Bl/6 J male mice were fed with either a control HF-FD or a HF-MAC diet for 15 weeks. Moreover, an additional group was fed with the HF-MAC diet in combination with an antibiotic cocktail (HF-MAC + AB). Following the 15-week treatment, cognitive behavior was investigated; blood, cecum content, colon, and brain samples were collected to determine metabolic parameters, endotoxin, gut microbiota, colon, and brain pathology. RESULTS: We report MACs supplementation prevented HF-FD-induced cognitive impairment in nesting building and temporal order memory tests. MACs prevented gut microbiota dysbiosis, including increasing richness, α-diversity and composition shift, especially in Bacteroidetes and its lower taxa. Furthermore, MACs increased colonic mucus thickness, tight junction protein expression, reduced endotoxemia, and decreased colonic and systemic inflammation. In the hippocampus, MACs suppressed HF-FD-induced neuroglia activation and inflammation, improved insulin IRS-pAKT-pGSK3ß-pTau synapse signaling, in addition to the synaptic ultrastructure and associated proteins. Furthermore, MACs' effects on improving colon-cognitive parameters were eliminated by wide spectrum antibiotic microbiota ablation. CONCLUSIONS: These results suggest that MACs improve cognitive impairments via the gut microbiota-brain axis induced by the consumption of an HF-FD. Supplemental MACs to combat obesity-related gut and brain dysfunction offer a promising approach to prevent neurodegenerative diseases associated with Westernized dietary patterns and obesity.

Cannabidiol improves behavioural and neurochemical deficits in adult female offspring of the maternal immune activation (poly I:C) model of neurodevelopmental disorders.

Cognitive impairment is a major source of disability in schizophrenia and current antipsychotic drugs (APDs) have minimal efficacy for this symptom domain. Cannabidiol (CBD), the major non-intoxicating component of Cannabis sativa L., exhibits antipsychotic and neuroprotective properties. We recently reported the effects of CBD on cognition in male offspring of a maternal immune activation (polyinosinic-polycytidilic acid (poly I:C)) model relevant to the aetiology of schizophrenia; however, the effects of CBD treatment in females are unknown. Sex differences are observed in the onset of schizophrenia symptoms and response to APD treatment. Furthermore, the endogenous cannabinoid system, a direct target of CBD, is sexually dimorphic in humans and rodents. Therefore, the present work aimed to assess the therapeutic impact of CBD treatment on behaviour and neurochemical signalling markers in female poly I:C offspring. Time-mated pregnant Sprague-Dawley rats (n = 16) were administered poly I:C (4 mg/kg; i.v.) or saline (control) on gestational day 15. From postnatal day 56, female offspring received CBD (10 mg/kg, i.p.) or vehicle treatment for approximately 3 weeks. Following 2 weeks of CBD treatment, offspring underwent behavioural testing, including the novel object recognition, rewarded alternation T-maze and social interaction tests to assess recognition memory, working memory and sociability, respectively. After 3 weeks of CBD treatment, the prefrontal cortex (PFC) and hippocampus (HPC) were collected to assess effects on endocannabinoid, glutamatergic and gamma-aminobutyric acid (GABA) signalling markers. CBD attenuated poly I:C-induced deficits in recognition memory, social interaction and glutamatergic N-methyl-d-aspartate receptor (NMDAR) binding in the PFC of poly I:C offspring. Working memory performance was similar between treatment groups. CBD also increased glutamate decarboxylase 67, the rate-limiting enzyme that converts glutamate to GABA, and parvalbumin protein levels in the HPC. In contrast to the CBD treatment effects observed in poly I:C offspring, CBD administration to control rats reduced social interaction, cannabinoid CB1 receptor and NMDAR binding density in the PFC, suggesting that CBD administration to healthy rats may have negative consequences on social behaviour and brain maturation in adulthood. Overall, the findings of this study support the therapeutic benefits of CBD on recognition memory and sociability in female poly I:C offspring, and provide insight into the neurochemical changes that may underlie the therapeutic benefits of CBD in the poly I:C model.

Effect of cannabidiol on endocannabinoid, glutamatergic and GABAergic signalling markers in male offspring of a maternal immune activation (poly I:C) model relevant to schizophrenia.

The mainstay treatment for schizophrenia is antipsychotic drugs (APDs), which are mostly effective against the positive symptoms (e.g. hallucinations), but provide minimal benefits for the negative symptoms (e.g. social withdrawal) and cognitive deficits. We have recently shown that treatment with the non-intoxicating phytocannabinoid, cannabidiol (CBD), can improve cognition and social interaction deficits in a maternal immune activation (MIA) model relevant to the aetiology of schizophrenia, however, the mechanisms underlying this effect are unknown. An imbalance in the main excitatory (glutamate) and inhibitory (GABA) neurotransmitter systems in the brain plays a role in the pathophysiology of schizophrenia. Therefore, the endocannabinoid system could represent a therapeutic target for schizophrenia as a regulator of glutamate and GABA release via the CB1 receptor (CB1R). This study investigated the effects of chronic CBD treatment on markers of glutamatergic, GABAergic and endocannabinoid signalling in brain regions implicated in social behaviour and cognitive function, including the prefrontal cortex (PFC) and hippocampus (HPC). Time-mated pregnant Sprague-Dawley rats (n = 16) were administered poly I:C (4 mg/kg, i.v.) or saline (control) on gestational day 15. Male offspring were injected with CBD (10 mg/kg, i.p.) or vehicle twice daily from postnatal day 56 for 3 weeks. The prefrontal cortex (PFC) and hippocampus (HPC) were collected for post-mortem receptor binding and Western blot analyses (n = 8 per group). CBD treatment attenuated poly I:C-induced deficits in cannabinoid CB1 receptor binding in the PFC and glutamate decarboxylase 67, the enzyme that converts glutamate to GABA, in the HPC. CBD treatment increased parvalbumin levels in the HPC, regardless of whether offspring were exposed to poly I:C in utero. Conversely, CBD did not affect N-methyl-d-aspartate receptor and gamma-aminobutyric acid (GABA) A receptor binding or protein levels of fatty acid amide hydrolase, the enzyme that degrades the endocannabinoid, anandamide. Overall, these findings show that CBD can restore cannabinoid/GABAergic signalling deficits in regions of the brain implicated in schizophrenia pathophysiology following maternal poly I:C exposure. These findings provide novel evidence for the potential mechanisms underlying the therapeutic effects of CBD treatment in the poly I:C model.

Perinatal exposure to fluoxetine increases anxiety- and depressive-like behaviours and alters glutamatergic markers in the prefrontal cortex and hippocampus of male adolescent rats: A comparison between Sprague-Dawley rats and the Wistar-Kyoto rat model of depression.

BACKGROUND: With approximately 10% of pregnant women prescribed antidepressant drugs for the treatment of depressive disorders, there is growing concern regarding the potential long-term effects of this exposure on offspring. Research is needed in clinically relevant models to determine the effects on offspring behaviour and associated neurobiological systems. AIM: The aim of this study was to determine the effects of maternal fluoxetine treatment on anxiety-like and depressive-like behaviours in adolescent offspring as well as associated glutamatergic markers, using a clinically relevant rodent model of depression. METHODS: Wistar-Kyoto (model of innate depression) and Sprague-Dawley rats were treated with fluoxetine (10 mg/kg) from gestational day 0 to postnatal day 14. Male offspring underwent behavioural testing (open field, elevated plus maze, forced swim test) at adolescence followed by quantitative immuno-detection of glutamatergic markers in the prefrontal cortex and ventral hippocampus. RESULTS: Perinatal fluoxetine exposure exacerbated the anxiety-like and depressive-like phenotype in Wistar-Kyoto offspring and induced an anxiety-like and depressive-like phenotype in Sprague-Dawley offspring. Wistar-Kyoto offspring showed reductions in NMDA receptor NR1, NR2A and NR2B subunits, as well as post-synaptic density 95 (PSD-95) and metabotropic glutamate receptor subtype 1 (mGluR1) in the prefrontal cortex; perinatal fluoxetine exposure further reduced NR1, NR2A, PSD-95 and mGluR1 expression in Wistar-Kyoto as well as Sprague-Dawley offspring. In the ventral hippocampus perinatal fluoxetine exposure reduced PSD-95 and increased metabotropic glutamate receptor subtype 5 (mGluR5) and Homer1b/c in both Sprague-Dawley and Wistar-Kyoto strains. CONCLUSION: These findings suggest that maternal fluoxetine treatment exacerbates effects of underlying maternal depression on offspring behaviour, which may be mediated through alterations in the glutamatergic system. Further research investigating how to minimise these effects, whilst ensuring optimal treatment for mothers, is essential to move the field forward.

Chronic Adolescent CDPPB Treatment Alters Short-Term, but not Long-Term, Glutamatergic Receptor Expression.

Dysfunction of the glutamatergic system is believed to underlie many neurodevelopmental disorders including autism, Rett syndrome and schizophrenia. Metabotropic glutamate receptor (mGluR5) positive allosteric modulators (PAM) potentiate glutamatergic signaling, particularly indirectly via the NMDA receptor. Preclinical studies report mGluR5 PAMs can improve schizophrenia-relevant behaviours. Furthermore, adolescent administration has shown to prevent cognitive induced deficits in adult rodents. However, there is limited understanding of the short- and long-term neurochemical effects of mGluR5 PAMs, which may underlie their therapeutic effects. We examined the effect of 7-day adolescent (PN28-34) treatment with the mGluR5 PAM, CDDPB (30 mg/kg), on glutamatergic receptor expression at adolescence (PN35) and adulthood (PN96). Immunoblot analysis revealed that 7-day adolescent CDPPB treatment increased protein expression of glutamatergic receptors including the NMDA receptor subunits, NR1 and NR2A and the AMPA subunits (GluA1 and GluA2) in the adolescent hippocampus, changes that did not extend to adulthood. In contrast, there were no changes in the adolescent frontal cortex, however elevated mGluR5 protein expression was observed at adulthood following adolescent CDPPB treatment. The present study indicates adolescent CDPPB treatment may cause brain region dependent effects on the glutamatergic system, which do not persist into adulthood. These findings may have implications for the preclinical development of mGluR5 PAMs for the treatment of neurodevelopmental disorders.

A postmortem analysis of NMDA ionotropic and group 1 metabotropic glutamate receptors in the nucleus accumbens in schizophrenia.

BACKGROUND: The nucleus accumbens (NAcc) has been implicated in the pathology and treatment of schizophrenia. Recent postmortem evidence suggests a hyperglutamatergic state in the NAcc. With the present study we aimed to explore possible glutamatergic alterations in the NAcc of a large schizophrenia cohort. METHODS: We performed immunoblots on postmortem NAcc samples from 30 individuals who had schizophrenia and 30 matched controls. We examined the protein expression of primary glutamatergic receptors, including the N-methyl-D-aspartate (NMDA) receptor (NR1, NR2A and NR2B subunits) and the group 1 metabotropic glutamate receptor (mGluR1 and mGluR5; dimeric and monomeric forms). In addition, we measured the group 1 mGluR endogenous regulators, neurochondrin and Homer1b/c, which have recently been implicated in the pathophysiology of schizophrenia. RESULTS: Protein levels of glutamatergic receptors and endogenous regulators were not significantly different between the controls and individuals who had schizophrenia. Furthermore, mGluR5, but not mGluR1, showed a positive association with NMDA receptor subunits, suggesting differential interactions between these receptors in this brain region. LIMITATIONS: Investigation of these proteins in antipsychotic-naive individuals, in addition to the subregions of the NAcc and subcellular fractions, will strengthen future studies. CONCLUSION: The present study does not provide evidence for glutamatergic abnormalities within the NAcc of individuals with schizophrenia. Taken together with the results of previous studies, these findings suggest NMDA receptors and group 1 mGluRs are altered in a brain region-dependent manner in individuals with schizophrenia. The differential associations between mGluR1, mGluR5 and NMDA receptors observed in this study warrant further research into the interactions of these proteins and the implications for the therapeutic and adverse effect profile of glutamatergic-based novel therapeutics.

Effects of short- and long-term aripiprazole treatment on Group I mGluRs in the nucleus accumbens: Comparison with haloperidol.

The D2 receptor partial agonist, aripiprazole, has shown increased therapeutic efficacy for schizophrenia, autism and Tourette's syndrome compared to traditional antipsychotics such as the D2 receptor antagonist, haloperidol. Recent evidence suggests this superior profile may be associated with downstream effects on glutamatergic synapses. Group 1 metabotropic glutamate receptors (mGluRs) and their endogenous modulators, Norbin and Homer1, are regulated by D2 receptor activity, particularly within the nucleus accumbens (NAc), a target region of aripiprazole and haloperidol. This study sought to evaluate the effects of aripiprazole on Group 1 mGluRs, Norbin and Homer1 in the NAc, in comparison to haloperidol. Sprague-Dawley rats were orally administered daily doses of aripiprazole (2.25mg/kg), haloperidol (0.3mg/kg) or vehicle for 1 or 10-weeks. Immunoblot analyses revealed Group 1 mGluR protein levels were not altered following 1-week and 10-week aripiprazole or haloperidol treatment, compared to vehicle treated rodents. However, 1-week aripiprazole and haloperidol treatment significantly elevated Homer1a and Norbin protein expression, respectively. After 10 weeks of treatment, aripiprazole, but not haloperidol, significantly increased Norbin expression. These findings indicate the antipsychotics, aripiprazole and haloperidol, exert differential temporal effects on Norbin and Homer1 expression that may have consequences on synaptic glutamatergic transmission underlying their therapeutic profile.

First-in-Human Study With the Inhaled TLR9 Oligonucleotide Agonist AZD1419 Results in Interferon Responses in the Lung, and Is Safe and Well-Tolerated.

Current asthma treatments address symptoms rather than the underlying disease pathophysiology, a better understanding of which has led to the identification of the Th2 high endotype. The activation of Toll-like receptors to induce Type I interferons directly in the lungs represents a novel therapeutic approach to reset this underlying Th2 pathophysiology with the potential to provide long-term disease modification. We present the nonclinical data and phase I clinical profile of an inhaled TLR9 agonist, AZD1419, a C-type CpG designed to induce interferon in the lung. In healthy volunteers, AZD1419 was found to be safe and well-tolerated. Target engagement in the lung was demonstrated at all dose levels tested. No evidence of tolerization or amplification of responses was evident on repeated dosing and 15.4 mg was defined as the maximum tolerated dose. AZD1419 clinical data supports its continued development as a potentially disease-modifying therapeutic in asthma.