Jeremiah K. Britt

Discovery of a Proneurogenic, Neuroprotective Chemical

An in vivo screen was performed in search of chemicals capable of enhancing neuron formation in the hippocampus of adult mice. Eight of 1000 small molecules tested enhanced neuron formation in the subgranular zone of the dentate gyrus. Among these was an aminopropyl carbazole, designated P7C3, endowed with favorable pharmacological properties. In vivo studies gave evidence that P7C3 exerts its proneurogenic activity by protecting newborn neurons from apoptosis. Mice missing the gene encoding neuronal PAS domain protein 3 (NPAS3) are devoid of hippocampal neurogenesis and display malformation and electrophysiological dysfunction of the dentate gyrus. Prolonged administration of P7C3 to npas3(-/-) mice corrected these deficits by normalizing levels of apoptosis of newborn hippocampal neurons. Prolonged administration of P7C3 to aged rats also enhanced neurogenesis in the dentate gyrus, impeded neuron death, and preserved cognitive capacity as a function of terminal aging. PAPERCLIP:

Neuroprotective efficacy of aminopropyl carbazoles in a mouse model of Parkinson disease

We previously reported the discovery of P7C3, an aminopropyl carbazole having proneurogenic and neuroprotective properties in newborn neural precursor cells of the dentate gyrus. Here, we provide evidence that P7C3 also protects mature neurons in brain regions outside of the hippocampus. P7C3 blocks 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-mediated cell death of dopaminergic neurons in the substantia nigra of adult mice, a model of Parkinson disease (PD). Dose–response studies show that the P7C3 analog P7C3A20 blocks cell death with even greater potency and efficacy, which parallels the relative potency and efficacy of these agents in blocking apoptosis of newborn neural precursor cells of the dentate gyrus. P7C3 and P7C3A20 display similar relative effects in blocking 1-methyl-4-phenylpyridinium (MPP+)-mediated death of dopaminergic neurons in Caenorhabditis elegans, as well as in preserving C. elegans mobility following MPP+ exposure. Dimebon, an antihistaminergic drug that is weakly proneurogenic and neuroprotective in the dentate gyrus, confers no protection in either the mouse or the worm models of PD. We further demonstrate that the hippocampal proneurogenic efficacy of eight additional analogs of P7C3 correlates with their protective efficacy in MPTP-mediated neurotoxicity. In vivo screening of P7C3 analogs for proneurogenic efficacy in the hippocampus may thus provide a reliable means of predicting neuroprotective efficacy. We propose that the chemical scaffold represented by P7C3 and P7C3A20 provides a basis for optimizing and advancing pharmacologic agents for the treatment of patients with PD.

Wild-type microglia do not reverse pathology in mouse models of Rett syndrome.

arising from N. C. Derecki et al. 484, 105–109 (2012); doi:10.1038/nature10907Rett syndrome is a severe neurodevelopmental disorder caused by mutations in the X chromosomal gene MECP2 (ref. 1), and its treatment so far is symptomatic. Mecp2 disruption in mice phenocopies major features of the syndrome that can be reversed after Mecp2 re-expression. Recently, Derecki et al. reported that transplantation of wild-type bone marrow into lethally irradiated Mecp2-null (Mecp2tm1.1Jae/y) mice prevented neurological decline and early death by restoring microglial phagocytic activity against apoptotic targets, and clinical trials of bone marrow transplantation (BMT) for patients with Rett syndrome have thus been initiated. We aimed to replicate and extend the BMT experiments in three different Rett syndrome mouse models, but found that despite robust microglial engraftment, BMT from wild-type donors did not prevent early death or ameliorate neurological deficits. Furthermore, early and specific Mecp2 genetic expression in microglia did not rescue Mecp2-deficient mice.

P7C3 Neuroprotective Chemicals Block Axonal Degeneration and Preserve Function after Traumatic Brain Injury

The P7C3 class of neuroprotective aminopropyl carbazoles has been shown to block neuronal cell death in models of neurodegeneration. We now show that P7C3 molecules additionally preserve axonal integrity after injury, before neuronal cell death occurs, in a rodent model of blast-mediated traumatic brain injury (TBI). This protective quality may be linked to the ability of P7C3 molecules to activate nicotinamide phosphoribosyltransferase, the rate-limiting enzyme in nicotinamide adenine dinucleotide salvage. Initiation of daily treatment with our recently reported lead agent, P7C3-S243, 1 day after blast-mediated TBI blocks axonal degeneration and preserves normal synaptic activity, learning and memory, and motor coordination in mice. We additionally report persistent neurologic deficits and acquisition of an anxiety-like phenotype in untreated animals 8 months after blast exposure. Optimized variants of P7C3 thus offer hope for identifying neuroprotective agents for conditions involving axonal damage, neuronal cell death, or both, such as occurs in TBI.

Forebrain elimination of cacna1c mediates anxiety-like behavior in mice.

The CACNA1C gene encoding the Cav1.2 subunit of the L-type calcium channel has emerged as a new candidate gene for neuropsychiatric disease, including bipolar disorder, major depression, schizophrenia and autism.1, 2, 3 We report that global haploinsufficiency, forebrain-specific elimination and prefrontal cortex (PFC)-specific knockdown of cacna1c all increase anxiety-related behavior in mice, a prominent component of the forms of neuropsychiatric disease in which aberrations in CACNA1C have been implicated, without affecting compulsive behavior. Constitutive cacna1c heterozygous mice (HET) were evaluated in three behavioral assays related to anxiety: open field test, light–dark conflict test and elevated plus maze (EPM). HETs displayed anxiety-like behavior in the EPM (Figure 1a), spending significantly less time exploring the open arms compared with wild-type littermate controls (WT; F1,19=6.437; P<0.05). However, no differences were observed between HETs and WTs in the open field and light–dark conflict test, (Figures 1a and b, Supplementary Material). We also observed a similar statistically significant effect of increased anxiety-like behavior compared with WTs in EPM in adult female HETs (Figure 1d, Supplementary Material) and adolescent male HETs (Figure 1e, Supplementary Material). To more specifically investigate the function of cacna1c in the brain, we generated forebrain-specific conditional cacna1c-deficient mice (forebrain-cacna1c cKO) by crossing cacna1c-floxed mice with mice harboring alphaCaM Kinase II promoter-driven expression of Cre recombinase.4 Relative to WTs, this strategy achieved ∼70% elimination of cacna1c mRNA in the hippocampus, PFC, basolateral amygdala, striatum and nucleus accumbens, as assessed by quantitative PCR (Table 1, Supplementary Material). Cacna1c mRNA levels were unaffected in the ventral tegmental area and cerebellum. With this greater reduction in cacna1c in forebrain than could be achieved in HETs, significantly increased anxiety-like behavior was observed across all three behavioral assays. In EPM, forebrain-cacna1c cKO mice spent significantly less time exploring the open arms compared with WTs (Figure 1b, F1,16=68.587; P<0.0001 and Figure 2c, Supplementary Material). In the open field test, forebrain-cacna1c cKO mice spent less time exploring the center of the chamber compared with WTs (Figures 2a and 3a, Supplementary Material). In the light-dark conflict test, forebrain-cacna1c cKO mice spent significantly less time in the brightly lit side compared with WTs (Figures 2b and 3b, Supplementary Material). Figure 1 Anxiety-like behavior as measured in the elevated plus maze (EPM) assay is shown for (a) cacna1c haplosufficient (cacna1c HET; n=10) and wild-type (WT; n=11) littermates, (b) forebrain-specific cacna1c knockout (forebrain-cacna1c cKO; n=8) and WT controls ... Clinically, anxiety is often accompanied by compulsive behavior, such as in obsessive-compulsive disorder (OCD), in which patients seek alleviation from recurrent bouts of anxiety-inducing intrusive thoughts by engaging in compulsively repetitive behaviors. Experimental models for OCD, such as SAPAP3-5 or SLITRK5-deficient6 mice, display pathologically high compulsive grooming that is readily quantified by the spray-induced grooming test. Compared with respective WTs, we did not observe elevated grooming in either HETs or forebrain-cacna1c cKO mice (Figures 1c and 3c, Supplementary Material). Thus, the form of anxiety associated with cacna1c function is distinct from that associated with OCD spectrum illnesses. Some genetic variations in CACNA1C have been associated with altered PFC function7, 8, 9 in neuropsychiatric disease, so we next generated focal elimination of cacna1c in the PFC with adeno-associated viral (AAV) vector-expressing Cre recombinase (AAV-Cre).10 AAV-Cre was stereotaxically delivered bilaterally into the PFC of floxed cacna1c mice, and regional elimination of Cav1.2 was immunohistochemically confirmed (Figures 1d and e). Following elimination of cacna1c in the PFC, mice showed no differences in basal locomotor activity compared with AAV-GFP control injected mice (Figure 4, Supplementary Material). However, selective elimination of cacna1c in the PFC was associated with less time spent exploring open arms of the EPM, compared with control AAV-GFP injected mice (Figure 1c, F1,16=5.477; P<0.05). To evaluate the specificity of PFC cacna1c knockdown in mediating anxiety, we used AAV-expressing cacna1d siRNA10 to selectively eliminate cacna1d in the PFC, the other L-type Ca2+ channel isoform expressed in brain. Selective knockdown of cacna1d in the PFC had no effect on locomotor behavior (Figure 5a, Supplementary Material) or time spent in open arms in the EPM (Figure 5b, Supplementary Material). In summary, we report here the first direct evidence for a role of forebrain cacna1c in regulating anxiety. Mice harboring forebrain-specific elimination of cacna1c may thus provide a useful tool for studying the pathophysiology of anxiety in forms of neuropsychiatric diseases in which CACNA1C is implicated.

PRICKLE1 Interaction with SYNAPSIN I Reveals a Role in Autism Spectrum Disorders

The frequent comorbidity of Autism Spectrum Disorders (ASDs) with epilepsy suggests a shared underlying genetic susceptibility; several genes, when mutated, can contribute to both disorders. Recently, PRICKLE1 missense mutations were found to segregate with ASD. However, the mechanism by which mutations in this gene might contribute to ASD is unknown. To elucidate the role of PRICKLE1 in ASDs, we carried out studies in Prickle1+/− mice and Drosophila, yeast, and neuronal cell lines. We show that mice with Prickle1 mutations exhibit ASD-like behaviors. To find proteins that interact with PRICKLE1 in the central nervous system, we performed a yeast two-hybrid screen with a human brain cDNA library and isolated a peptide with homology to SYNAPSIN I (SYN1), a protein involved in synaptogenesis, synaptic vesicle formation, and regulation of neurotransmitter release. Endogenous Prickle1 and Syn1 co-localize in neurons and physically interact via the SYN1 region mutated in ASD and epilepsy. Finally, a mutation in PRICKLE1 disrupts its ability to increase the size of dense-core vesicles in PC12 cells. Taken together, these findings suggest PRICKLE1 mutations contribute to ASD by disrupting the interaction with SYN1 and regulation of synaptic vesicles.

Double deletion of melanocortin 4 receptors and SAPAP3 corrects compulsive behavior and obesity in mice

Compulsive behavior is a debilitating clinical feature of many forms of neuropsychiatric disease, including Tourette syndrome, obsessive-compulsive spectrum disorders, eating disorders, and autism. Although several studies link striatal dysfunction to compulsivity, the pathophysiology remains poorly understood. Here, we show that both constitutive and induced genetic deletion of the gene encoding the melanocortin 4 receptor (MC4R), as well as pharmacologic inhibition of MC4R signaling, normalize compulsive grooming and striatal electrophysiologic impairments in synapse-associated protein 90/postsynaptic density protein 95-associated protein 3 (SAPAP3)-null mice, a model of human obsessive-compulsive disorder. Unexpectedly, genetic deletion of SAPAP3 restores normal weight and metabolic features of MC4R-null mice, a model of human obesity. Our findings offer insights into the pathophysiology and treatment of both compulsive behavior and eating disorders.

The Neuropsychiatric Disease-Associated Gene cacna1c Mediates Survival of Young Hippocampal Neurons.

Visual Overview Genetic variations in CACNA1C, which encodes the Cav1.2 subunit of L-type calcium channels (LTCCs), are associated with multiple forms of neuropsychiatric disease that manifest high anxiety in patients. Genetic variations in CACNA1C, which encodes the Cav1.2 subunit of L-type calcium channels (LTCCs), are associated with multiple forms of neuropsychiatric disease that manifest high anxiety in patients. In parallel, mice harboring forebrain-specific conditional knockout of cacna1c (forebrain-Cav1.2 cKO) display unusually high anxiety-like behavior. LTCCs in general, including the Cav1.3 subunit, have been shown to mediate differentiation of neural precursor cells (NPCs). However, it has not previously been determined whether Cav1.2 affects postnatal hippocampal neurogenesis in vivo. Here, we show that forebrain-Cav1.2 cKO mice exhibit enhanced cell death of young hippocampal neurons, with no change in NPC proliferation, hippocampal size, dentate gyrus thickness, or corticosterone levels compared with wild-type littermates. These mice also exhibit deficits in brain levels of brain-derived neurotrophic factor (BDNF), and Cre recombinase-mediated knockdown of adult hippocampal Cav1.2 recapitulates the deficit in young hippocampal neurons survival. Treatment of forebrain-Cav1.2 cKO mice with the neuroprotective agent P7C3-A20 restored the net magnitude of postnatal hippocampal neurogenesis to wild-type levels without ameliorating their deficit in BDNF expression. The role of Cav1.2 in young hippocampal neurons survival may provide new approaches for understanding and treating neuropsychiatric disease associated with aberrations in CACNA1C. Visual Abstract

Protective efficacy of P7C3-S243 in the 6-hydroxydopamine model of Parkinson's disease

Background:There are currently no therapeutic options for patients with Parkinsons disease that prevent or slow the death of dopaminergic neurons. We have recently identified the novel P7C3 class of neuroprotective molecules that blocks neuron cell death.AIMS:The aim of this study was to determine whether treatment with highly active members of the P7C3 series blocks dopaminergic neuron cell death and associated behavioral and neurochemical deficits in the rat 6-hydroxydopamine (6-OHDA) model of Parkinsons disease.Methods:After unilateral injection of 6-OHDA into the median forebrain bundle, rats were assessed for behavioral function in the open field, cylinder test, and amphetamine-induced circling test. Thereafter, their brains were subjected to neurochemical and immunohistochemical analysis of dopaminergic neuron survival. Analysis was conducted as a function of treatment with P7C3 compounds, with administration initiated either before or after 6-OHDA exposure.Results:Animals administered P7C3-A20 or P7C3-S243, two of the most advanced agents in the P7C3 series of neuroprotective compounds, both before and after 6-OHDA exposure showed evidence of protective efficacy in all measures. When P7C3-S243 administration was initiated after 6-OHDA exposure, rats also showed protective efficacy in all measures, which included blocking dopaminergic neuron cell death in ipsilateral substantia nigra pars compacta, preservation of dopamine and its metabolites in ipsilateral striatum, and preservation of normal motor behavior.Conclusions:The P7C3 series of compounds may form the basis for developing new therapeutic agents for slowing or preventing progression of Parkinsons disease.

Repeated mild traumatic brain injury produces neuroinflammation, anxiety-like behaviour and impaired spatial memory in mice

ABSTRACT Primary Objective: Repeated traumatic brain injuries (rmTBI) are frequently associated with debilitating neuropsychiatric conditions such as cognitive impairment, mood disorders, and post-traumatic stress disorder. We tested the hypothesis that repeated mild traumatic brain injury impairs spatial memory and enhances anxiety-like behaviour. Research Design: We used a between groups design using single (smTBI) or repeated (rmTBI) controlled cranial closed skull impacts to mice, compared to a control group. Methods and Procedures: We assessed the effects of smTBI and rmTBI using measures of motor performance (Rotarod Test [RT]), anxiety-like behaviour (Elevated Plus Maze [EPM] and Open Field [OF] tests), and spatial memory (Morris Water Maze [MWM]) within 12 days of the final injury. In separate groups of mice, astrocytosis and microglial activation were assessed 24 hours after the final injury using GFAP and IBA-1 immunohistochemistry. Main Outcomes and Results: RmTBI impaired spatial memory in the MWM and increased anxiety-like behaviour in the EPM and OFT. In addition, rmTBI elevated GFAP and IBA-1 immunohistochemistry throughout the mouse brain. RmTBI produced astrocytosis and microglial activation, and elicited impaired spatial memory and anxiety-like behaviour. Conclusions: rmTBI produces acute cognitive and anxiety-like disturbances associated with inflammatory changes in brain regions involved in spatial memory and anxiety.