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The serotonin transporter (SERT) removes synaptic serotonin and is the target of anti-depressant drugs. SERT adopts three conformations: outward-open, occluded, and inward-open. All known inhibitors target the outward-open state except ibogaine, which has unusual anti-depressant and substance-withdrawal effects, and stabilizes the inward-open conformation. Unfortunately, ibogaine's promiscuity and cardiotoxicity limit the understanding of inward-open state ligands. We docked over 200 million small molecules against the inward-open state of the SERT. Thirty-six top-ranking compounds were synthesized, and thirteen inhibited; further structure-based optimization led to the selection of two potent (low nanomolar) inhibitors. These stabilized an outward-closed state of the SERT with little activity against common off-targets. A cryo-EM structure of one of these bound to the SERT confirmed the predicted geometry. In mouse behavioral assays, both compounds had anxiolytic- and anti-depressant-like activity, with potencies up to 200-fold better than fluoxetine (Prozac), and one substantially reversed morphine withdrawal effects.

Glutaric aciduria type I (GA-1) is an inborn error of metabolism with a severe neurological phenotype caused by the deficiency of glutaryl–coenzyme A dehydrogenase (GCDH), the last enzyme of lysine catabolism. Current literature suggests that toxic catabolites in the brain are produced locally and do not cross the blood-brain barrier. In a series of experiments using knockout mice of the lysine catabolic pathway and liver cell transplantation, we uncovered that toxic GA-1 catabolites in the brain originated from the liver. Moreover, the characteristic brain and lethal phenotype of the GA-1 mouse model was rescued by two different liver-directed gene therapy approaches: Using an adeno-associated virus, we replaced the defective Gcdh gene or we prevented flux through the lysine degradation pathway by CRISPR deletion of the aminoadipate-semialdehyde synthase ( Aass ) gene. Our findings question the current pathophysiological understanding of GA-1 and reveal a targeted therapy for this devastating disorder.

Supplemental Figure 2: Neurocognitive analysis of mice by Morris watermaze (A and B), novel object test (C and D), and fear conditioning (E and F). Tests were performed as described (29). (G and H) Distance and velocity during the novel object training and test periods and overall averages.

Supplemental Figure 3: Effects of radiation and MnTnBuOE-2-PyP5+ on tumor cells. (A) LN-18 and (B) LN-229 glioblastoma cell lines treated with or without 50 nM MnTnBuOE-2-PyP5+ in clonogenic survival assays prior to various doses of radiation.

Supplemental Figure 4: Model of differential actions of MnTnBuOE-2-PyP5+ in causing the apoptosis of tumor during radiotherapy, while suppressing radation injury to surrounding normal tissue. Such effects are due to the differential redox environment of tumor vs normal cell, in particular much lower ability of cancer cell to remove cytotoxic hydrogen peroxide. Consequently, much higher levels of hydrogen peroxide already exist in tumor cell, and are greatly enhanced upon radiation. In normal cells and tissues, porphyrins suppress cycling inflammatory responses that may result from radiation also.

We have developed and characterized a novel D2R antagonist with exceptional GPCR selectivity – ML321. In functional profiling screens of 168 different GPCRs, ML321 showed little activity beyond potent inhibition of the D2R, and to a lesser extent the D3R, demonstrating excellent receptor selectivity. The D2R selectivity of ML321 may be related to the fact that, unlike other monoaminergic ligands, ML321 lacks a positively charged amine group and adopts a unique binding pose within the orthosteric binding site of the D2R. PET imaging studies in non-human primates demonstrated that ML321 penetrates the CNS and occupies the D2R in a dose-dependent manner. Behavioral paradigms in rats demonstrate that ML321 can selectively antagonize a D2R-mediated response (hypothermia) while not affecting a D3R-mediated response (yawning) using the same dose of drug, thus indicating exceptionalin vivoselectivity. We also investigated the effects of ML321 in animal models that are predictive of antipsychotic efficacy in humans. We found that ML321 attenuates both amphetamine- and phencyclidine-induced locomotor activity and restored pre-pulse inhibition (PPI) of acoustic startle in a dose-dependent manner. Surprisingly, using doses that were maximally effective in both the locomotor and PPI studies, ML321 was relatively ineffective in promoting catalepsy. Kinetic studies revealed that ML321 exhibits slow-on and fast-off receptor binding rates, similar to those observed with atypical antipsychotics with reduced extrapyramidal side effects. Taken together, these observations suggest that ML321, or a derivative thereof, may exhibit “atypical” antipsychotic activity in humans with significantly fewer side effects than observed with currently FDA-approved D2R antagonists.

ProSAAS is a neuroendocrine protein that is cleaved by neuropeptide-processing enzymes into more than a dozen products including the bigLEN and PEN peptides, which bind and activate the receptors GPR171 and GPR83, respectively. Previous studies have suggested that proSAAS-derived peptides are involved in physiological functions that include body weight regulation, circadian rhythms and anxiety-like behavior. In the present study, we find that proSAAS knockout mice display robust anxiety-like behaviors in the open field, light-dark emergence and elevated zero maze tests. These mutant mice also show a reduction in cued fear and an impairment in fear-potentiated startle, indicating an important role for proSAAS-derived peptides in emotional behaviors. ProSAAS knockout mice exhibit reduced water consumption and urine production relative to wild-type controls. No differences in food consumption and overall energy expenditure were observed between the genotypes. However, the respiratory exchange ratio was elevated in the mutants during the light portion of the light-dark cycle, indicating decreased fat metabolism during this period. While proSAAS knockout mice show normal circadian patterns of activity, even upon long-term exposure to constant darkness, they were unable to shift their circadian clock upon exposure to a light pulse. Taken together, these results show that proSAAS-derived peptides modulate a wide range of behaviors including emotion, metabolism and the regulation of the circadian clock.

SUMMARYA role for macronutrient metabolism in learning and memory is supported by numerous epidemiological studies. The Ppm1k gene encodes the branched-chain keto acid dehydrogenase (BCKDH) phosphatase that promotes the metabolism of branched-chain amino acids (BCAA). Here we show that nervous system deletion of Ppm1k in mice increases BCAA levels in brain tissue but not in plasma. These mice have significant impairments in working memory accompanied by a robust accumulation of DCX+/NeuroD1+ immature neurons within the dentate gyrus granule cell layer. Through single cell RNA sequencing and pathway analysis we identified substantial increases in transit-amplifying cells and immature neurons along with activated hedgehog signaling in Ppm1k deficient primary neural stem cells (NSCs). Inhibition of mTOR signaling reversed the effects of Ppm1k deletion on neuronal progenitor gene activation in primary NSCs. Together our findings uncover a new molecular link between BCAA metabolism, hippocampal neurogenesis, and cognitive performance.

SUMMARYThe serotonin transporter (SERT) removes synaptic serotonin and is the target of anti-depressant drugs. SERT adopts three conformations: outward-open, occluded, and inward-open. All known inhibitors target the outward-open state except ibogaine, which has an unusual anti-depressant profile and stabilizes the inward-open conformation. Unfortunately, ibogaine is promiscuous and cardiotoxic, limiting understanding of inward-open state ligands. We computationally docked over 200 million small molecules against the ibogaine stabilized inward-open state of SERT. Thirty-six top-ranking compounds were synthesized and thirteen inhibited with potencies ranging from 29 to 5000 nM. Structure-based optimization led to two novel inhibitors with Ki values down to 3 nM. The new molecules stabilized an outward-closed state of the transporter and had little activity against off-targets. A cryo-EM structure of one of these bound to SERT confirmed the predicted geometry. In mouse behavioral assays, both had anxiolytic and anti-depressant activity, with potencies up to 200 better than fluoxetine.

The neuronal primary cilium and centriolar satellites have functions in neurogenesis, but little is known about their roles in the postnatal brain. We show that ablation of pericentriolar material 1 in the mouse leads to progressive ciliary, anatomical, psychomotor, and cognitive abnormalities. RNAseq reveals changes in amine- and G-protein coupled receptor pathways. The physiological relevance of this phenotype is supported by decreased available dopamine D2 receptor (D2R) levels and the failure of antipsychotic drugs to rescue adult behavioral defects. Immunoprecipitations show an association with Pcm1 and D2Rs. Finally, we sequence PCM1 in two human cohorts with severe schizophrenia. Systematic modeling of all discovered rare alleles by zebrafish in vivo complementation reveals an enrichment for pathogenic alleles. Our data emphasize a role for the pericentriolar material in the postnatal brain, with progressive degenerative ciliary and behavioral phenotypes; and they support a contributory role for PCM1 in some individuals diagnosed with schizophrenia., The role of ciliary/centriolar components in the postnatal brain is unclear. Here, the authors show via ablation of Pcm1 in mice that degenerative ciliary/centriolar phenotypes induce neuroanatomical and behavioral changes. Sequencing of PCM1 in human cohorts and zebrafish in vivo complementation suggests PCM1 mutations can contribute to schizophrenia.

Small ubiquitin-like modifier (SUMO1–3) conjugation (SUMOylation), a post-translational modification, modulates almost all major cellular processes. Mounting evidence indicates that SUMOylation plays a crucial role in maintaining and regulating neural function, and importantly its dysfunction is implicated in cognitive impairment in humans. We have previously shown that simultaneously silencing SUMO1–3 expression in neurons negatively affects cognitive function. However, the roles of the individual SUMOs in modulating cognition and the mechanisms that link SUMOylation to cognitive processes remain unknown. To address these questions, in this study, we have focused on SUMO2 and generated a new conditional Sumo2 knockout mouse line. We found that conditional deletion of Sumo2 predominantly in forebrain neurons resulted in marked impairments in various cognitive tests, including episodic and fear memory. Our data further suggest that these abnormalities are attributable neither to constitutive changes in gene expression nor to alterations in neuronal morphology, but they involve impairment in dynamic SUMOylation processes associated with synaptic plasticity. Finally, we provide evidence that dysfunction on hippocampal-based cognitive tasks was associated with a significant deficit in the maintenance of hippocampal long-term potentiation in Sumo2 knockout mice. Collectively, these data demonstrate that protein conjugation by SUMO2 is critically involved in cognitive processes.

Objective The present work evaluates the relationship between postoperative immune and neurovascular changes and the pathogenesis of surgery-induced delirium superimposed on dementia. Background and rationale Postoperative delirium is a common complication in many older adults and in patients with dementia including Alzheimer's disease (AD). The course of delirium can be particularly debilitating, while its pathophysiology remains poorly defined. Historical evolution As of 2019, an estimated 5.8 million people of all ages have been diagnosed with AD, 97% of whom are >65 years of age. Each year, many of these patients require surgery. However, anesthesia and surgery can increase the risk for further cognitive decline. Surgery triggers neuroinflammation both in animal models and in humans, and a failure to resolve this inflammatory state may contribute to perioperative neurocognitive disorders as well as neurodegenerative pathology. Updated hypothesis We propose an immunovascular hypothesis whereby dysregulated innate immunity negatively affects the blood-brain interface, which triggers delirium and thereby exacerbates AD neuropathology. Early experimental data We have developed a translational model to study delirium superimposed on dementia in APPSwDI/mNos2-/- AD mice (CVN-AD) after orthopedic surgery. At 12 months of age, CVN-AD showed distinct neuroimmune and vascular impairments after surgery, including acute microgliosis and amyloid-β deposition. These changes correlated with attention deficits, a core feature of delirium-like behavior. Future experiments and validation studies Future research should determine the extent to which prevention of surgery-induced microgliosis and/or neurovascular unit dysfunction can prevent or ameliorate postoperative memory and attention deficits in animal models. Translational human studies should evaluate perioperative indices of innate immunity and neurovascular integrity and assess their potential link to perioperative neurocognitive disorders. Major challenges for the hypothesis Understanding the complex relationships between delirium and dementia will require mechanistic studies aimed at evaluating the role of postoperative neuroinflammation and blood-brain barrier changes in the setting of pre-existing neurodegenerative and/or aging-related pathology. Linkage to other major theories Non-resolving inflammation with vascular disease that leads to cognitive impairments and dementia is increasingly important in risk stratification for AD in the aging population. The interdependence of these factors with surgery-induced neuroinflammation and cognitive dysfunction is also becoming apparent, providing a strong platform for assessing the relationship between postoperative delirium and longer term cognitive dysfunction in older adults.

There is considerable interest in screening ultralarge chemical libraries for ligand discovery, both empirically and computationally

Functionally selective G protein-coupled receptor ligands are valuable tools for deciphering the roles of downstream signaling pathways that potentially contribute to therapeutic effects versus side effects. Recently, we discovered both Gi/o-biased and β-arrestin2-biased D2 receptor agonists based on the Food and Drug Administration (FDA)-approved drug aripiprazole. In this work, based on another FDA-approved drug, cariprazine, we conducted a structure-functional selectivity relationship study and discovered compound 38 (MS1768) as a potent partial agonist that selectively activates the Gi/o pathway over β-arrestin2. Unlike the dual D2R/D3R partial agonist cariprazine, compound 38 showed selective agonist activity for D2R over D3R. In fact, compound 38 exhibited potent antagonism of dopamine-stimulated β-arrestin2 recruitment. In our docking studies, compound 38 directly interacts with S1935.42 on TM5 but has no interactions with extracellular loop 2, which appears to be in contrast to the binding poses of D2R β-arrestin2-biased ligands. In in vivo studies, compound 38 showed high D2R receptor occupancy in mice and effectively inhibited phencyclidine-induced hyperlocomotion.

The 16p11.2 BP4-BP5 deletion and duplication syndromes are associated with a complex spectrum of neurodevelopmental phenotypes that includes developmental delay and autism spectrum disorder, with a reciprocal effect on head circumference, brain structure and body mass index. Mouse models of the 16p11.2 copy number variant have recapitulated some of the patient phenotypes, while studies in flies and zebrafish have uncovered several candidate contributory genes within the region, as well as complex genetic interactions. We evaluated one of these loci, KCTD13, by modeling haploinsufficiency and complete knockout in mice. In contrast to the zebrafish model, and in agreement with recent data, we found normal brain structure in heterozygous and homozygous mutants. However, recapitulating previously observed genetic interactions, we discovered sex-specific brain volumetric alterations in double heterozygous Kctd13xMvp and Kctd13xLat mice. Behavioral testing revealed a significant deficit in novel object recognition, novel location recognition and social transmission of food preference in Kctd13 mutants. These phenotypes were concomitant with a reduction in density of mature spines in the hippocampus, but potentially independent of RhoA abundance, which was unperturbed postnatally in our mutants. Furthermore, transcriptome analyses from cortex and hippocampus highlighted the dysregulation of pathways important in neurodevelopment, the most significant of which was synaptic formation. Together, these data suggest that KCTD13 contributes to the neurocognitive aspects of patients with the BP4-BP5 deletion, likely through genetic interactions with other loci.

Recent evidence suggests that psychedelic drugs can exert beneficial effects on anxiety, depression, and ethanol and nicotine abuse in humans. However, their hallucinogenic side-effects often preclude their clinical use. Lysergic acid diethylamide (LSD) is a prototypical hallucinogen and its psychedelic actions are exerted through the 5-HT2A serotonin receptor (5-HT2AR). 5-HT2AR activation stimulates Gq- and β-arrestin- (βArr) mediated signaling. To separate these signaling modalities, we have used βArr1 and βArr2 mice. We find that LSD stimulates motor activities to similar extents in WT and βArr1-KO mice, with non-significant effects in βArr2-KOs. LSD robustly stimulates many surrogates of psychedelic drug actions including head twitches, grooming, retrograde walking, and nose-poking in WT and βArr1-KO animals. By contrast, LSD slightly stimulates head twitches in βArr2-KO mice, without effects on other surrogates. The 5-HT2AR antagonist MDL100907 (MDL) blocks these LSD effects. LSD also disrupts prepulse inhibition (PPI) in WT and βArr1-KOs; PPI is unaffected in βArr2-KOs. MDL restores PPI in WT mice, but this antagonist is without effect and haloperidol is required in βArr1-KOs. Collectively, these results reveal that LSD’s psychedelic drug-like actions appear to require βArr2.

Recent evidence suggests that psychedelic drugs can exert beneficial effects on anxiety, depression, and ethanol and nicotine abuse in humans. However, the hallucinogenic side-effects of psychedelics often preclude their clinical use. Lysergic acid diethylamide (LSD) is a prototypical hallucinogen and its psychedelic actions are exerted through the 5-HT2A serotonin receptor (5-HT2AR). 5-HT2AR activation stimulates Gq- and β-arrestin-(βArr) mediated signaling. To separate effects of these signaling modes, we have used βArr1 and βArr2 mice. We find that LSD stimulates motor activities to similar extents in WT and βArr1-KO mice, with non-significant effects in βArr2-KOs. LSD robustly stimulates many surrogates of psychedelic drug actions including head twitches, grooming, retrograde walking, and nose poking in WT and βArr1-KO animals. In contrast, LSD only slightly stimulates head twitches in βArr2-KO mice, without effects on retrograde walking or nose poking. The 5-HT2AR antagonist MDL100907 (MDL) blocks these LSD effects. LSD also disrupts prepulse inhibition (PPI) in WT and βArr1-KOs; PPI is unaffected in βArr2-KOs. MDL restores PPI in WT mice, but this antagonist is without effect and haloperidol is required in βArr1-KOs. LSD produces a biphasic body-temperature response in WT mice, a monophasic response in βArr1-KOs, and is without effect in βArr2 mutants. Both MDL and the 5-HT1AR antagonist, WAY 100635 (WAY), block the effects of LSD on body temperatures in WT mice, whereas WAY is effective in βArr1-KOs. Collectively, these results reveal that LSD produces diverse behavioral effects through βArr1 and βArr2, and that LSD’s psychedelic drug-like actions appear to require βArr2.

Neuroinflammation is a growing hallmark of perioperative neurocognitive disorders (PNDs), including delirium and longer-lasting cognitive deficits. We have developed a clinically-relevant orthopedic mouse model to study the impact of a common surgical procedure on the vulnerable brain. The mechanism underlying PNDs remain unknown. Here we evaluated the impact of surgical trauma on the NLRP3 inflammasome signaling, including the expression of apoptosis-associated speck-like protein containing a CARD (ASC), caspase-1, and IL-1β in the hippocampus of C57BL6/J male mice, adult (3-months) and aged (>18-months). Surgery triggered ASC specks formation in CA1 hippocampal microglia, but without inducing significant morphological changes in NLRP3 and ASC knockout mice. Since no therapies are currently available to treat PNDs, we assessed the neuroprotective effects of a biomimetic peptide derived from the endogenous inflammation-ending molecule, Annexin-A1 (ANXA1). We tested the hypothesis that this peptide (ANXA1sp) inhibits NLRP3 inflammasome activation, thus preventing microglial activation and hippocampal-dependent memory deficits. Together these results uncover a previously underrecognized role of the NLRP3 inflammasome in triggering postoperative neuroinflammation and offer a new target for advancing treatment of PNDs through resolution of inflammation.

Human mutations in the dystroglycan complex (DGC) result in not only muscular dystrophy but also cognitive impairments. However, the molecular architecture critical for the synaptic organization of the DGC in neurons remains elusive. Here, we report Inhibitory Synaptic protein 1 (InSyn1) is a critical component of the DGC whose loss alters the composition of the GABAergic synapses, excitatory/inhibitory balance in vitro and in vivo, and cognitive behavior. Association of InSyn1 with DGC subunits is required for InSyn1 synaptic localization. InSyn1 null neurons also show a significant reduction in DGC and GABA receptor distribution as well as abnormal neuronal network activity. Moreover, InSyn1 null mice exhibit elevated neuronal firing patterns in the hippocampus and deficits in fear conditioning memory. Our results support the dysregulation of the DGC at inhibitory synapses and altered neuronal network activity and specific cognitive tasks via loss of a novel component, InSyn1.

All FDA-approved antipsychotic drugs (APDs) target primarily dopamine D2 or serotonin (5-HT2A) receptors, or both; however, these medications are not universally effective, they may produce undesirable side effects, and provide only partial amelioration of negative and cognitive symptoms. The heterogeneity of pharmacological responses in schizophrenic patients suggests that additional drug targets may be effective in improving aspects of this syndrome. Recent evidence suggests that 5-HT2C receptors may be a promising target for schizophrenia since their activation reduces mesolimbic nigrostriatal dopamine release (which conveys antipsychotic action), they are expressed almost exclusively in CNS, and have weight-loss-promoting capabilities. A difficulty in developing 5-HT2C agonists is that most ligands also possess 5-HT2B and/or 5-HT2A activities. We have developed selective 5-HT2C ligands and herein describe their preclinical effectiveness for treating schizophrenia-like behaviors. JJ-3-45, JJ-3-42, and JJ-5-34 reduced amphetamine-stimulated hyperlocomotion, restored amphetamine-disrupted prepulse inhibition, improved social behavior, and novel object recognition memory in NMDA receptor hypofunctioning NR1-knockdown mice, and were essentially devoid of catalepsy. However, they decreased motivation in a breakpoint assay and did not promote reversal learning in MK-801-treated mice. Somewhat similar effects were observed with lorcaserin, a 5-HT2C agonist with potent 5-HT2B and 5-HT2A agonist activities, which is approved for treating obesity. Microdialysis studies revealed that both JJ-3-42 and lorcaserin reduced dopamine efflux in the infralimbic cortex, while only JJ-3-42 decreased it in striatum. Collectively, these results provide additional evidence that 5-HT2C receptors are suitable drug targets with fewer side effects, greater therapeutic selectivity, and enhanced efficacy for treating schizophrenia and related disorders than current APDs.

Bone remodeling of the auditory ossicles and the otic capsule is highly restricted and tightly controlled by the osteoprotegerin (OPG) / receptor activator of nuclear factor kappa-B ligand (RANKL) / receptor activator of nuclear factor kappa-B (RANK) system. In these bony structures, a pathological decrease in OPG expression stimulates osteoclast differentiation and excessive resorption followed by accrual of sclerotic bone, ultimately resulting in the development of otosclerosis, a leading cause of deafness in adults. Understanding the signaling pathways involved in maintaining OPG expression in the ear would shed light on the pathophysiology of otosclerosis and other ear bone-related diseases. We and others previously demonstrated that Ca(2+) signaling through the L-type Ca(V)1.2 Ca(2+) channel positively regulates OPG expression and secretion in long bone osteoblasts and their precursor cells in vitro and in vivo. Whether Ca(V)1.2 regulates OPG expression in ear bones has not been investigated. We drove expression of a gain-of-function Ca(V)1.2 mutant channel (Ca(V)1.2(TS)) using Col2a1-Cre, which we found to target osteochondral/osteoblast progenitors in the auditory ossicles and the otic capsule. Col2a1-Cre;Ca(V)1.2(TS) mice displayed osteopetrosis of these bones shown by μCT 3D reconstruction, histological analysis, and lack of bone sculpting, findings similar to phenotypes seen in mice with an osteoclast defect. Consistent with those observations, we found that Col2a1-Cre;Ca(V)1.2(TS) mutant mice showed reduced osteoclasts in the otic capsule, upregulated mRNA expression of Opg and Opg/Rankl ratio, and increased mRNA expression of osteoblast differentiation marker genes in the otic capsule, suggesting both an anti-catabolic and anabolic effect of Ca(V)1.2(TS) mutant channel contributed to the observed morphological changes of the ear bones. Further, we found that Col2a1-Cre;Ca(V)1.2(TS) mice experienced hearing loss and displayed defects of body balance in behavior tests, confirming that the Ca(V)1.2-dependent Ca(2+) influx affects bone structure in the ear and consequent hearing and vestibular functions. Together, these data support our hypothesis that Ca(2+) influx through Ca(V)1.2(TS) promotes OPG expression from osteoblasts, thereby affecting bone modeling/remodeling in the auditory ossicles and the otic capsule. These data provide insight into potential pathological mechanisms underlying perturbed OPG expression and otosclerosis.

Functionally selective G protein-coupled receptor ligands are valuable tools for deciphering the roles of downstream signaling pathways that potentially contribute to therapeutic effects versus side effects. Recently, we discovered both G(i/o)-biased and β-arrestin2-biased D(2) receptor agonists based on the Food and Drug Administration (FDA)-approved drug aripiprazole. In this work, based on another FDA-approved drug, cariprazine, we conducted a structure–functional selectivity relationship study and discovered compound 38 (MS1768) as a potent partial agonist that selectively activates the G(i/o) pathway over β-arrestin2. Unlike the dual D(2)R/D(3)R partial agonist cariprazine, compound 38 showed selective agonist activity for D(2)R over D(3)R. In fact, compound 38 exhibited potent antagonism of dopamine-stimulated β-arrestin2 recruitment. In our docking studies, compound 38 directly interacts with S193(5.42) on TM5 but has no interactions with extracellular loop 2, which appears to be in contrast to the binding poses of D(2)R β-arrestin2-biased ligands. In in vivo studies, compound 38 showed high D(2)R receptor occupancy in mice and effectively inhibited phencyclidine-induced hyperlocomotion.

Background Patients with pre-existing neurodegenerative disease commonly experience fractures that require orthopedic surgery. Perioperative neurocognitive disorders (PND), including delirium and postoperative cognitive dysfunction, are serious complications that can result in increased 1-year mortality when superimposed on dementia. Importantly, there are no disease-modifying therapeutic options for PND. Our lab developed the “broad spectrum” mixed-lineage kinase 3 inhibitor URMC-099 to inhibit pathological innate immune responses that underlie neuroinflammation-associated cognitive dysfunction. Here, we test the hypothesis that URMC-099 can prevent surgery-induced neuroinflammation and cognitive impairment. Methods Orthopedic surgery was performed by fracturing the tibia of the left hindlimb with intramedullary fixation under general anesthesia and analgesia. In a pilot experiment, 9-month-old mice were treated five times with URMC-099 (10 mg/kg, i.p.), spaced 12 h apart, with three doses prior to surgery and two doses following surgery. In this experiment, microgliosis was evaluated using unbiased stereology and blood-brain barrier (BBB) permeability was assessed using immunoglobulin G (IgG) immunostaining. In follow-up experiments, 3-month-old mice were treated only three times with URMC-099 (10 mg/kg, i.p.), spaced 12 h apart, prior to orthopedic surgery. Two-photon scanning laser microscopy and CLARITY with light-sheet microscopy were used to define surgery-induced changes in microglial dynamics and morphology, respectively. Surgery-induced memory impairment was assessed using the “What-Where-When” and Memory Load Object Discrimination tasks. The acute peripheral immune response to surgery was assessed by cytokine/chemokine profiling and flow cytometry. Finally, long-term fracture healing was assessed in fracture callouses using micro-computerized tomography (microCT) and histomorphometry analyses. Results Orthopedic surgery induced BBB disruption and microglial activation, but had no effect on microglial process motility. Surgically treated mice exhibited impaired object place and identity discrimination in the “What-Where-When” and Memory Load Object Discrimination tasks. Both URMC-099 dosing paradigms prevented the neuroinflammatory sequelae that accompanied orthopedic surgery. URMC-099 prophylaxis had no effect on the mobilization of the peripheral innate immune response and fracture healing. Conclusions These findings show that prophylactic URMC-099 treatment is sufficient to prevent surgery-induced microgliosis and cognitive impairment without affecting fracture healing. Together, these findings provide compelling evidence for the advancement of URMC-099 as a therapeutic option for PND.

As discussion of stress and stress-related disorders rapidly extends beyond the brain, gut microbiota have emerged as a promising contributor to individual differences in the risk of illness, disease course, and treatment response. Here, we employed chronic mild social defeat stress and 16S rRNA gene metagenomic sequencing to investigate the role of microbial composition in mediating anxiety- and depressive-like behavior. In socially defeated animals, we found significant reductions in the overall diversity and relative abundances of numerous bacterial genera, including Akkermansia spp., that positively correlated with behavioral metrics of both anxiety and depression. Functional analyses predicted a reduced frequency of signaling molecule pathways, including G-protein-coupled receptors, in defeated animals. Collectively, our data suggest that shifts in microbial composition may play a role in the pathogenesis of anxiety and depression.

G protein-coupled receptor kinase-interacting protein 2 (GIT2) and GIT1 are highly similar, sharing the same domain structure and many binding partners. The most important GIT partners are the p21-activated protein kinase-interacting exchange factor (PIX) proteins, since through homomeric and heteromeric interactions, GIT and PIX proteins form oligomeric GIT/PIX complexes. Oligomeric GIT/PIX complexes function both as regulators of small GTP-binding proteins and as scaffolds for signalling molecules, including p21-activated protein kinases (PAKs). Deficits in learning and memory have been demonstrated in GIT1 knockout mice, and it has been assumed that GIT2 also would affect learning and memory. Unexpectedly, we find that GIT2-deficient mice respond normally in multiple tests of learning and memory, and have normal hippocampal long-term potentiation. Further, we find no evidence that GIT2 regulates ADHD-like phenotypes. To investigate why GIT2 and GIT1 differ so markedly in the brain, we identified the major isoform of GIT2 in the brain as a previously uncharacterized splice variant, GIT2(ΔBCE). This variant cannot dimerize or form oligomeric complexes with PIX proteins, and is thus incapable of regulating PAK in synapses, compared to oligomeric GIT1/PIX complexes. Because localized activation of PAK in synapses is required for structural plasticity underlying cognitive performance, loss of monomeric GIT2(ΔBCE) in the brain does not influence these responses.

Rare de novo mutations in genes associated with inherited Mendelian disorders are potential contributors to sporadic disease. DYT1 dystonia is an autosomal dominant, early-onset, generalized dystonia associated with an in-frame, trinucleotide deletion (n. delGAG, p. ΔE 302/303) in the Tor1a gene. Here we examine the significance of a rare missense variant in the Tor1a gene (c. 613T > A, p. F205I), previously identified in a patient with sporadic late-onset focal dystonia, by modeling it in mice. Homozygous F205I mice have motor impairment, reduced steady-state levels of TorsinA, altered corticostriatal synaptic plasticity, and prominent brain imaging abnormalities in areas associated with motor function. Thus, the F205I variant causes abnormalities in domains affected in people and/or mouse models with the DYT1 Tor1a mutation (ΔE). Our findings establish the pathological significance of the F205I Tor1a variant and provide a model with both etiological and phenotypic relevance to further investigate dystonia mechanisms.

Combinations of radiotherapy (RT) and chemotherapy have shown efficacy toward brain tumors. However, therapy-induced oxidative stress can damage normal brain tissue, resulting in both progressive neurocognitive loss and diminished quality of life. We have recently shown that MnTnBuOE-2-PyP(5+) (Mn(III)meso-tetrakis(N-n-butoxyethylpyridinium -2-yl)porphyrin) rescued RT-induced white matter damage in cranially-irradiated mice. Radiotherapy is not used in isolation for treatment of brain tumors; temozolomide is the standard-of-care for adult glioblastoma, whereas cisplatin is often used for treatment of pediatric brain tumors. Therefore, we evaluated the brain radiation mitigation ability of MnTnBuOE-2-PyP(5+) after either temozolomide or cisplatin was used singly or in combination with 10 Gy RT. MnTnBuOE-2-PyP(5+) accumulated in brains at low nanomolar levels. Histological and neurobehavioral testing showed a drastic decrease (1) of axon density in the corpus callosum and (2) rotorod and running wheel performance in the RT only treatment group, respectively. MnTnBuOE-2-PyP(5+) completely rescued this phenotype in irradiated animals. In the temozolomide groups, temozolomide/ RT treatment resulted in further decreased rotorod responses over RT alone. Again, MnTnBuOE-2-PyP(5+) treatment rescued the negative effects of both temozolomide ± RT on rotorod performance. While the cisplatin-treated groups did not give similar results as the temozolomide groups, inclusion of MnTnBuOE-2-PyP(5+) did not negatively affect rotorod performance. Additionally, MnTnBuOE-2-PyP(5+) sensitized glioblastomas to either RT ± temozolomide in flank tumor models. Mice treated with both MnTnBuOE-2-PyP(5+) and radio-/chemo-therapy herein demonstrated brain radiation mitigation. MnTnBuOE-2-PyP(5+) may well serve as a normal tissue radio-/chemo-mitigator adjuvant therapy to standard brain cancer treatment regimens. Environ. Mol. Mutagen. 57:372-381, 2016. © 2016 Wiley Periodicals, Inc.

D2C7-(scdsFv)-PE38KDEL (D2C7-IT) is a novel immunotoxin that reacts with wild-type epidermal growth factor receptor (EGFRwt) and mutant EGFRvIII proteins overexpressed in glioblastomas. This study assessed the toxicity of intracerebral administration of D2C7-IT to support an initial Food and Drug Administration Investigational New Drug application. After the optimization of the formulation and administration, two cohorts (an acute and chronic cohort necropsied on study days 5 and 34) of Sprague-Dawley (SD) rats (four groups of 5 males and 5 females) were infused with the D2C7-IT formulation at total doses of 0, 0.05, 0.1, 0.4 µg (the acute cohort) and 0, 0.05, 0.1, 0.35 µg (the chronic cohort) for approximately 72 hours by intracerebral convection-enhanced delivery using osmotic pumps. Mortality was observed in the 0.40 µg (5/10 rats) and 0.35 µg (4/10 rats) high-dose groups of each cohort. Body weight loss and abnormal behavior were only revealed in the rats treated with high doses of D2C7-IT. No dose-related effects were observed in clinical laboratory tests in either cohort. A gross pathologic examination of systemic tissues from the high-dose and control groups in both cohorts exhibited no dose-related or drug-related pathologic findings. Brain histopathology revealed the frequent occurrence of dose-related encephalomalacia, edema, and demyelination in the high-dose groups of both cohorts. In this study, the maximum tolerated dose of D2C7-IT was determined to be between 0.10 and 0.35 µg, and the no observed adverse effect level was 0.05 µg in SD rats. Both parameters were utilized to design the Phase I/II D2C7-IT clinical trial.

Background The vagus nerve is involved in regulating immunity and resolving inflammation. Current strategies aimed at modulating neuroinflammation and cognitive decline, in many cases, are limited and ineffective. Objective We sought to develop a minimally invasive, targeted, vagus nerve stimulation approach (pVNS), and we tested its efficacy with respect to microglial activation and amelioration of cognitive dysfunction following lipopolysaccharide (LPS) endotoxemia in mice. Methods We stimulated the cervical vagus nerve in mice using an ultrasound-guided needle electrode under sevoflurane anesthesia. The concentric bipolar needle electrode was percutaneously placed adjacent to the carotid sheath and stimulation was verified in real-time using bradycardia as a biomarker. Activation of vagal fibers was confirmed with immunostaining in relevant brainstem structures, including the dorsal motor nucleus and nucleus tractus solitarius. Efficacy of pVNS was evaluated following administration of LPS and analyses of changes in inflammation and behavior. Results pVNS enabled stimulation of the vagus nerve as demonstrated by changes in bradycardia and histological evaluation of c-Fos and choline acetyltransferase expression in brainstem nuclei. Following LPS administration, pVNS significantly reduced plasma levels of tumor necrosis factor-α at 3 h post-injection. pVNS prevented LPS-induced hippocampal microglial activation as analyzed by changes in Iba-1 immunoreactivity, including cell body enlargement and shortened ramifications. Cognitive dysfunction following endotoxemia was also restored by pVNS. Conclusion Targeted cervical VNS using this novel percutaneous approach reduced LPS-induced systemic and brain inflammation and significantly improved cognitive responses. These results provide a novel therapeutic approach using bioelectronic medicine to modulate neuro-immune interactions that affect cognition.

We previously reported a new line of Shank3 mutant mice which led to a complete loss of Shank3 by deleting exons 4−22 (Δe4−22) globally. Δe4−22 mice display robust ASD-like behaviors including impaired social interaction and communication, increased stereotypical behavior and excessive grooming, and a profound deficit in instrumental learning. However, the anatomical and neural circuitry underlying these behaviors are unknown. We generated mice with Shank3 selectively deleted in forebrain, striatum, and striatal D1 and D2 cells. These mice were used to interrogate the circuit/brain-region and cell-type specific role of Shank3 in the expression of autism-related behaviors. Whole-cell patch recording and biochemical analyses were used to study the synaptic function and molecular changes in specific brain regions. We found perseverative exploratory behaviors in mice with deletion of Shank3 in striatal inhibitory neurons. Conversely, self-grooming induced lesions were observed in mice with deletion of Shank3 in excitatory neurons of forebrain. However, social, communicative, and instrumental learning behaviors were largely unaffected in these mice, unlike what is seen in global Δe4−22 mice. We discovered unique patterns of change for the biochemical and electrophysiological findings in respective brain regions that reflect the complex nature of transcriptional regulation of Shank3. Reductions in Homer1b/c and membrane hyper-excitability were observed in striatal loss of Shank3. By comparison, Shank3 deletion in hippocampal neurons resulted in increased NMDAR-currents and GluN2B-containing NMDARs. These results together suggest that Shank3 may differentially regulate neural circuits that control behavior. Our study supports a dissociation of Shank3 functions in cortical and striatal neurons in ASD-related behaviors, and it illustrates the complexity of neural circuit mechanisms underlying these behaviors.

Current antipsychotic drugs (APDs) show efficacy with positive symptoms, but are limited in treating negative or cognitive features of schizophrenia. Whereas all currently FDA-approved medications target primarily the dopamine D2 receptor (D2R) to inhibit G(i/o)-mediated adenylyl cyclase, a recent study has shown that many APDs affect not only G(i/o)- but they can also influence β-arrestin- (βArr)-mediated signaling. The ability of ligands to differentially affect signaling through these pathways is termed functional selectivity. We have developed ligands that are devoid of D2R-mediated G(i/o) protein signaling, but are simultaneously partial agonists for D2R/βArr interactions. The purpose of this study was to test the effectiveness of UNC9975 or UNC9994 on schizophrenia-like behaviors in phencyclidine-treated or NR1-knockdown hypoglutamatergic mice. We have found the UNC compounds reduce hyperlocomotion in the open field, restore PPI, improve novel object recognition memory, partially normalize social behavior, decrease conditioned avoidance responding, and elicit a much lower level of catalepsy than haloperidol. These preclinical results suggest that exploitation of functional selectivity may provide unique opportunities to develop drugs with fewer side effects, greater therapeutic selectivity, and enhanced efficacy for treating schizophrenia and related conditions than medications that are currently available.

Cranial irradiation is a standard therapy for primary and metastatic brain tumors. A major drawback of radiotherapy (RT), however, is long-term cognitive loss that affects quality of life. Radiation-induced oxidative stress in normal brain tissue is thought to contribute to cognitive decline. We evaluated the effectiveness of a novel mimic of superoxide dismutase enzyme (SOD), MnTnBuOE-2-PyP5+(Mn(III) meso-tetrakis(N-n-butoxyethylpyridinium-2-yl)porphyrin), to provide long-term neuroprotection following 8 Gy of whole brain irradiation. Long-term RT damage can only be assessed by brain imaging and neurocognitive studies. C57BL/6J mice were treated with MnTnBuOE-2-PyP5+ before and after RT and evaluated three months later. At this time point, drug concentration in the brain was 25 nmol/L. Mice treated with MnTnBuOE-2-PyP5+/RT exhibited MRI evidence for myelin preservation in the corpus callosum compared with saline/RT treatment. Corpus callosum histology demonstrated a significant loss of axons in the saline/RT group that was rescued in the MnTnBuOE-2-PyP5+/RT group. In addition, the saline/RT groups exhibited deficits in motor proficiency as assessed by the rotorod test and running wheel tests. These deficits were ameliorated in groups treated with MnTnBuOE-2-PyP5+/RT. Our data demonstrate that MnTnBuOE-2-PyP5+ is neuroprotective for oxidative stress damage caused by radiation exposure. In addition, glioblastoma cells were not protected by MnTnBuOE-2-PyP5+ combination with radiation in vitro. Likewise, the combination of MnTnBuOE-2-PyP5+ with radiation inhibited tumor growth more than RT alone in flank tumors. In summary, MnTnBuOE-2-PyP5+ has dual activity as a neuroprotector and a tumor radiosensitizer. Thus, it is an attractive candidate for adjuvant therapy with RT in future studies with patients with brain cancer. Mol Cancer Ther; 14(1); 70–79. ©2014 AACR.

Summary Objectives Mutations in the ATP1α3 subunit of the neuronal Na+/K+-ATPase are thought to be responsible for seizures, hemiplegias, and other symptoms of alternating hemiplegia of childhood (AHC). However, the mechanisms through which ATP1A3 mutations mediate their pathophysiologic consequences are not yet understood. The following hypotheses were investigated: (1) Our novel knock-in mouse carrying the most common heterozygous mutation causing AHC (D801N) will exhibit the manifestations of the human condition and display predisposition to seizures; and (2) the underlying pathophysiology in this mouse model involves increased excitability in response to electrical stimulation of Schaffer collaterals and abnormal predisposition to spreading depression (SD). Methods We generated the D801N mutant mouse (Mashlool, Mashl+/−) and compared mutant and wild-type (WT) littermates. Behavioral tests, amygdala kindling, flurothyl-induced seizure threshold, spontaneous recurrent seizures (SRS), and other paroxysmal activities were compared between groups. In vitro electrophysiologic slice experiments on hippocampus were performed to assess predisposition to hyperexcitability and SD. Results Mutant mice manifested a distinctive phenotype similar to that of humans with AHC. They had abnormal impulsivity, memory, gait, motor coordination, tremor, motor control, endogenous nociceptive response, paroxysmal hemiplegias, diplegias, dystonias, and SRS, as well as predisposition to kindling, to flurothyl-induced seizures, and to sudden unexpected death. Hippocampal slices of mutants, in contrast to WT animals, showed hyperexcitable responses to 1 Hz pulse-trains of electrical stimuli delivered to the Schaffer collaterals and had significantly longer duration of K+-induced SD responses. Significance Our model reproduces the major characteristics of human AHC, and indicates that ATP1α3 dysfunction results in abnormal short-term plasticity with increased excitability (potential mechanism for seizures) and a predisposition to more severe SD responses (potential mechanism for hemiplegias). This model of the human condition should help in understanding the molecular pathways underlying these phenotypes and may lead to identification of novel therapeutic strategies of ATP1α3 related disorders and seizures.