Thomas A. Lanz

MicroRNA-132 dysregulation in schizophrenia has implications for both neurodevelopment and adult brain function

Schizophrenia is characterized by affective, cognitive, neuromorphological, and molecular abnormalities that may have a neurodevelopmental origin. MicroRNAs (miRNAs) are small noncoding RNA sequences critical to neurodevelopment and adult neuronal processes by coordinating the activity of multiple genes within biological networks. We examined the expression of 854 miRNAs in prefrontal cortical tissue from 100 control, schizophrenic, and bipolar subjects. The cyclic AMP-responsive element binding- and NMDA-regulated microRNA miR-132 was significantly down-regulated in both the schizophrenic discovery cohort and a second, independent set of schizophrenic subjects. Analysis of miR-132 target gene expression in schizophrenia gene-expression microarrays identified 26 genes up-regulated in schizophrenia subjects. Consistent with NMDA-mediated hypofunction observed in schizophrenic subjects, administration of an NMDA antagonist to adult mice results in miR-132 down-regulation in the prefrontal cortex. Furthermore, miR-132 expression in the murine prefrontal cortex exhibits significant developmental regulation and overlaps with critical neurodevelopmental processes during adolescence. Adult prefrontal expression of miR-132 can be down-regulated by pharmacologic inhibition of NMDA receptor signaling during a brief postnatal period. Several key genes, including DNMT3A, GATA2, and DPYSL3, are regulated by miR-132 and exhibited altered expression either during normal neurodevelopment or in tissue from adult schizophrenic subjects. Our data suggest miR-132 dysregulation and subsequent abnormal expression of miR-132 target genes contribute to the neurodevelopmental and neuromorphological pathologies present in schizophrenia.

Concentration-Dependent Modulation of Amyloid-β in Vivo and in Vitro Using the γ-Secretase Inhibitor, LY-450139

LY-450139 is a γ-secretase inhibitor shown to have efficacy in multiple cellular and animal models. Paradoxically, robust elevations of plasma amyloid-β (Aβ) have been reported in dogs and humans after administration of subefficacious doses. The present study sought to further evaluate Aβ responses to LY-450139 in the guinea pig, a nontransgenic model that has an Aβ sequence identical to that of human. Male guinea pigs were treated with LY-450139 (0.2–60 mg/kg), and brain, cerebrospinal fluid, and plasma Aβ levels were characterized at 1, 3, 6, 9, and 14 h postdose. Low doses significantly elevated plasma Aβ levels at early time points, with return to baseline within hours. Higher doses inhibited Aβ levels in all compartments at early time points, but elevated plasma Aβ levels at later time points. To determine whether this phenomenon occurs under steady-state drug exposure, guinea pigs were implanted with subcutaneous minipumps delivering LY-450139 (0.3–30 mg/kg/day) for 5 days. Plasma Aβ was significantly inhibited at 10–30 mg/kg/day, but significantly elevated at 1 mg/kg/day. To further understand the mechanism of Aβ elevation by LY-450139, H4 cells overexpressing the Swedish mutant of amyloid-precursor protein and a mouse embryonic stem cell-derived neuronal cell line were studied. In both cellular models, elevated levels of secreted Aβ were observed at subefficacious concentrations, whereas dose-responsive inhibition was observed at higher concentrations. These results suggest that LY-450139 modulates the γ-secretase complex, eliciting Aβ lowering at high concentrations but Aβ elevation at low concentrations.

Chronic Suppression of Phosphodiesterase 10A Alters Striatal Expression of Genes Responsible for Neurotransmitter Synthesis, Neurotransmission, and Signaling Pathways Implicated in Huntington's Disease

Inhibition of phosphodiesterase 10A (PDE10A) promotes cyclic nucleotide signaling, increases striatal activation, and decreases behavioral activity. Enhanced cyclic nucleotide signaling is a well established route to producing changes in gene expression. We hypothesized that chronic suppression of PDE10A activity would have significant effects on gene expression in the striatum. A comparison of the expression profile of PDE10A knockout (KO) mice and wild-type mice after chronic PDE10A inhibition revealed altered expression of 19 overlapping genes with few significant changes outside the striatum or after administration of a PDE10A inhibitor to KO animals. Chronic inhibition of PDE10A produced up-regulation of mRNAs encoding genes that included prodynorphin, synaptotagmin10, phosphodiesterase 1C, glutamate decarboxylase 1, and diacylglycerol O-acyltransferase and a down-regulation of mRNAs encoding choline acetyltransferase and Kv1.6, suggesting long-term suppression of the PDE10A enzyme is consistent with altered striatal excitability and potential utility as a antipsychotic therapy. In addition, up-regulation of mRNAs encoding histone 3 (H3) and down-regulation of histone deacetylase 4, follistatin, and claspin mRNAs suggests activation of molecular cascades capable of neuroprotection. We used lentiviral delivery of cAMP response element (CRE)-luciferase reporter constructs into the striatum and live animal imaging of 2-{4-[-pyridin-4-yl-1-(2,2,2-trifluoro-ethyl)-1H-pyrazol-3-yl]-phenoxymethyl}-quinoline succinic acid (TP-10)-induced luciferase activity to further demonstrate PDE10 inhibition results in CRE-mediated transcription. Consistent with potential neuroprotective cascades, we also demonstrate phosphorylation of mitogen- and stress-activated kinase 1 and H3 in vivo after TP-10 treatment. The observed changes in signaling and gene expression are predicted to provide neuroprotective effects in models of Huntingtons disease.

Structural basis of C-terminal β-amyloid peptide binding by the antibody ponezumab for the treatment of Alzheimer's disease.

Alzheimers disease, the most common cause of dementia in the elderly and characterized by the deposition and accumulation of plaques, is composed in part of β-amyloid (Aβ) peptides, loss of neurons, and the accumulation of neurofibrillary tangles. Here, we describe ponezumab, a humanized monoclonal antibody, and show how it binds specifically to the carboxyl (C)-terminus of Aβ40. Ponezumab can label Aβ that is deposited in brain parenchyma found in sections from Alzheimers disease casualties and in transgenic mouse models that overexpress Aβ. Importantly, ponezumab does not label full-length, non-cleaved amyloid precursor protein on the cell surface. The C-terminal epitope of the soluble Aβ present in the circulation appears to be available for ponezumab binding because systemic administration of ponezumab greatly elevates plasma Aβ40 levels in a dose-dependent fashion after administration to a mouse model that overexpress human Aβ. Administration of ponezumab to transgenic mice also led to a dose-dependent reduction in hippocampal amyloid load. To further explore the nature of ponezumab binding to Aβ40, we determined the X-ray crystal structure of ponezumab in complex with Aβ40 and found that the Aβ40 carboxyl moiety makes extensive contacts with ponezumab. Furthermore, the structure-function analysis supported this critical requirement for carboxy group of AβV40 in the Aβ-ponezumab interaction. These findings provide novel structural insights into the in vivo conformation of the C-terminus of Aβ40 and the brain Aβ-lowering efficacy that we observed following administration of ponezumab in transgenic mouse models.

Evaluation of TrkB and BDNF transcripts in prefrontal cortex, hippocampus, and striatum from subjects with schizophrenia, bipolar disorder, and major depressive disorder.

Brain-derived neurotrophic factor (BDNF) signaling is integral to a range of neural functions, including synaptic plasticity and exhibits activity-dependent regulation of expression. As altered BDNF signaling has been implicated in multiple psychiatric diseases, here we report a quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis of mRNAs encoding TrkB, total BDNF, and the four most abundant BDNF transcripts (I, IIc, IV, and VI) in postmortem tissue from matched tetrads of subjects with schizophrenia, bipolar disorder, or major depressive disorder (MDD) and healthy comparison subjects. In all three regions examined, dorsolateral prefrontal cortex (DLPFC), associative striatum and hippocampus, total BDNF mRNA levels did not differ in any disease state. In DLPFC, BDNF IIc was significantly lower in schizophrenia relative to healthy comparison subjects. In hippocampus, BDNF I, IIc, and VI were lower in subjects with both schizophrenia and bipolar disorder relative to comparison subjects. In striatum, TrkB mRNA was lower in bipolar disorder and MDD, while BDNF IIc was elevated in MDD, relative to comparison subjects. These data highlight potential alterations in BDNF signaling in the corticohippocampal circuit in schizophrenia, and within the striatum in mood disorders. Novel therapies aimed at improving BDNF-TrkB signaling may therefore have potential to impact on a range of psychiatric disorders.

Demonstration of a common artifact in immunosorbent assays of brain extracts: Development of a solid-phase extraction protocol to enable measurement of amyloid-β from wild-type rodent brain

In the process of developing species-specific, immunosorbent assays for brain amyloid-beta (Abeta) in non-transgenic animals, we have demonstrated an artifact that impedes accurate quantitation of Abeta in this assay format. Using synthetic peptides, cerebrospinal fluid (CSF), or plasma samples, no nonspecific binding or cross-species immunoreactivity was detected in human or rodent Abeta assays. However, extracts of guinea pig brain (human Abeta sequence) or rat brain (rodent Abeta sequence) demonstrated immunoreactivity regardless of which capture antibody, detection antibody, or reporter method (colorimetric or fluorescent) was used. This immunoreactivity remained even in the absence of a capture antibody. Various blocking conditions failed to resolve the nonspecific binding of detection antibodies in the presence of brain extracts. Fractionation of DEA-extracted guinea pig brain over Sephadex G-50 demonstrated the feasibility of separating specific from nonspecific binding components in the brain extracts. Thus, a solid phase extraction method, compatible with multiple extraction buffers, has been developed to isolate and concentrate Abeta from brain extracts. This isolation method eliminates non-specific binding components from brain extracts and allows for accurate quantitation and robust detection of multiple Abeta peptides in extracts from wild-type animals.

Transcriptomic analysis of genetically defined autism candidate genes reveals common mechanisms of action.

BackgroundAustism spectrum disorder (ASD) is a heterogeneous behavioral disorder or condition characterized by severe impairment of social engagement and the presence of repetitive activities. The molecular etiology of ASD is still largely unknown despite a strong genetic component. Part of the difficulty in turning genetics into disease mechanisms and potentially new therapeutics is the sheer number and diversity of the genes that have been associated with ASD and ASD symptoms. The goal of this work is to use shRNA-generated models of genetic defects proposed as causative for ASD to identify the common pathways that might explain how they produce a core clinical disability.MethodsTranscript levels of Mecp2, Mef2a, Mef2d, Fmr1, Nlgn1, Nlgn3, Pten, and Shank3 were knocked-down in mouse primary neuron cultures using shRNA constructs. Whole genome expression analysis was conducted for each of the knockdown cultures as well as a mock-transduced culture and a culture exposed to a lentivirus expressing an anti-luciferase shRNA. Gene set enrichment and a causal reasoning engine was employed to identify pathway level perturbations generated by the transcript knockdown.ResultsQuantification of the shRNA targets confirmed the successful knockdown at the transcript and protein levels of at least 75% for each of the genes. After subtracting out potential artifacts caused by viral infection, gene set enrichment and causal reasoning engine analysis showed that a significant number of gene expression changes mapped to pathways associated with neurogenesis, long-term potentiation, and synaptic activity.ConclusionsThis work demonstrates that despite the complex genetic nature of ASD, there are common molecular mechanisms that connect many of the best established autism candidate genes. By identifying the key regulatory checkpoints in the interlinking transcriptional networks underlying autism, we are better able to discover the ideal points of intervention that provide the broadest efficacy across the diverse population of autism patients.

Pharmacodynamics and Pharmacokinetics of the γ-Secretase Inhibitor PF-3084014

PF-3084014 [(S)-2-((S)-5,7-difluoro-1,2,3,4-tetrahydronaphthalen-3-ylamino)-N-(1-(2-methyl-1-(neopentylamino)propan-2-yl)-1H-imidazol-4-yl)pentanamide] is a novel γ-secretase inhibitor that reduces amyloid-β (Aβ) production with an in vitro IC50 of 1.2 nM (whole-cell assay) to 6.2 nM (cell-free assay). This compound inhibits Notch-related T- and B-cell maturation in an in vitro thymocyte assay with an EC50 of 2.1 μM. A single acute dose showed dose-dependent reduction in brain, cerebrospinal fluid (CSF), and plasma Aβ in Tg2576 mice as measured by enzyme-linked immunosorbent assay and immunoprecipitation (IP)/mass spectrometry (MS). Guinea pigs were dosed with PF-3084014 for 5 days via osmotic minipump at 0.03 to 3 mg/kg/day and exhibited dose-dependent reduction in brain, CSF, and plasma Aβ. To further characterize Aβ dynamics in brain, CSF, and plasma in relation to drug exposure and Notch-related toxicities, guinea pigs were dosed with 0.03 to 10 mg/kg PF-3084014, and tissues were collected at regular intervals from 0.75 to 30 h after dose. Brain, CSF, and plasma all exhibited dose-dependent reductions in Aβ, and the magnitude and duration of Aβ lowering exceeded those of the reductions in B-cell endpoints. Other γ-secretase inhibitors have shown high potency at elevating Aβ in the conditioned media of whole cells and the plasma of multiple animal models and humans. Such potentiation was not observed with PF-3084014. IP/MS analysis, however, revealed dose-dependent increases in Aβ11-40 and Aβ1-43 at doses that potently inhibited Aβ1-40 and Aβ1-42. PF-3084014, like previously described γ-secretase inhibitors, preferentially reduced Aβ1-40 relative to Aβ1-42. Potency at Aβ relative to Notch-related endpoints in vitro and in vivo suggests that a therapeutic index can be achieved with this compound.

Solid-phase extraction enhances detection of beta-amyloid peptides in plasma and enables Aβ quantification following passive immunization with Aβ antibodies

Abstract We have previously developed a solid-phase extraction (SPE) procedure to enable the detection of beta-amyloid (Aβ) peptides in brain tissue from non-transgenic animals. We have now adapted these methods to enrich the Aβ fraction in cerebrospinal fluid (CSF) and plasma. Human CSF and plasma and Tg2576 mouse plasma were subjected to guanidine denaturation followed by SPE in 96-well cassettes. The resulting eluates could be concentrated significantly to enhance detection of low-abundance Aβ peptides by immunoassay. The concentrated eluates diluted in a linear fashion with consistent recovery between SPE columns. This technique was therefore used to facilitate quantification of Aβ1-X, 1-40, 1-42, and 1-38 peptides in normal human CSF and plasma samples. SPE sample preparation was also applied to the plasma of mice dosed peripherally with a monoclonal antibody raised against Aβ. When such samples were assayed directly, the presence of the systemically administered antibody interfered with the subsequent immunoassay, by preventing detection of antibody-bound Aβ. After subjecting plasma from antibody-treated animals to denaturation and SPE, the antibody–antigen complex was disrupted, and the Aβ fraction could be isolated from the antibody-containing fraction. Application of this method allowed for detection of a 100-fold increase in plasma Aβ1-40 following treatment of Tg2576 mice or wild type littermate control mice with Aβ40-specific monoclonal antibody 9TL. Given the availability of a variety of SPE matrices, we hypothesize that these methods could facilitate plasma antigen retrieval using multiple therapeutic antibody approaches.

Transcriptional Profiling of a Selective CREB Binding Protein Bromodomain Inhibitor Highlights Therapeutic Opportunities.

Bromodomains are involved in transcriptional regulation through the recognition of acetyl lysine modifications on diverse proteins. Selective pharmacological modulators of bromodomains are lacking, although the largely hydrophobic nature of the pocket makes these modules attractive targets for small-molecule inhibitors. This work describes the structure-based design of a highly selective inhibitor of the CREB binding protein (CBP) bromodomain and its use in cell-based transcriptional profiling experiments. The inhibitor downregulated a number of inflammatory genes in macrophages that were not affected by a selective BET bromodomain inhibitor. In addition, the CBP bromodomain inhibitor modulated the mRNA level of the regulator of G-protein signaling 4 (RGS4) gene in neurons, suggesting a potential therapeutic opportunity for CBP inhibitors in the treatment of neurological disorders.