Discovery of Small-Molecule CD33 Pre-mRNA Splicing Modulators.

CD33/Siglec 3 is a myeloid lineage cell surface receptor that is known to regulate microglia activity. Multiple genome-wide association studies (GWAS) have identified genetic variants in the CD33 gene that convey protection from late-onset Alzheimer's disease. Furthermore, mechanistic studies into GWAS-linked variants suggest that disease protection is attributed to the alternative splicing of exon 2 of the CD33 pre-mRNA. Using a phenomimetic screen, a series of compounds were found to enhance the exclusion of CD33 exon 2, acting as a chemomimetic of the GWAS-linked gene variants. Additional studies confirmed that meyloid lineage cells treated with several of these compounds have a reduced full-length V-domain containing CD33 protein, while targeted RNA-seq concordantly demonstrated that compound 1 increases exon 2 skipping in cellular mRNA pools. These studies demonstrate how pharmacological interventions can be used to manipulate disease-relevant pre-mRNA splicing and provide a starting point for future efforts to identify small molecules that alter neuroimmune function that is rooted in the human biology of neurodegenerative disease.

Development of a one-step RT-ddPCR method to determine the expression and potency of AAV vectors.

Robust assays to quantify adeno-associated virus (AAV) vector expression and potency are essential for gene therapy development. These assays inform the efficacy, safety, and pharmacodynamic profiles of AAV development candidates. Additionally, for gene downregulation strategies such as RNAi, knockdown of endogenous genes reflects the mechanism of action of such development candidates. Therefore, a method to quantify target mRNA repression is necessary for measuring vector potency both in vitro and in vivo. Here, we report the development of a one-step reverse-transcription droplet digital PCR (RT-ddPCR) method to analyze expression of AAV vectors and the potency of AAV-RNAi vectors. This one-step RT-ddPCR method simplifies the workflow, allows for duplexing reactions, and enables absolute quantification of transcripts without standard materials. With a gene augmentation vector, we demonstrate the application of RT-ddPCR in quantifying vector expression in vitro and in non-human primate (NHP) samples. This novel method is demonstrated to be precise and linear within the range of 0.05-25 ng of RNA input. Using an AAV-RNAi vector, we further demonstrate the utility of this RT-ddPCR method in quantifying potency. Orthogonal potency assays, including ELISA and functional readout, correlate well with RT-ddPCR results. Therefore, one-step RT-ddPCR can be implemented in the analytical and pharmacological characterization of AAV vectors.

The Parkinson's disease-associated gene ITPKB protects against α-synuclein aggregation by regulating ER-to-mitochondria calcium release.

Inositol-1,4,5-triphosphate (IP3) kinase B (ITPKB) is a ubiquitously expressed lipid kinase that inactivates IP3, a secondary messenger that stimulates calcium release from the endoplasmic reticulum (ER). Genome-wide association studies have identified common variants in the ITPKB gene locus associated with reduced risk of sporadic Parkinson's disease (PD). Here, we investigate whether ITPKB activity or expression level impacts PD phenotypes in cellular and animal models. In primary neurons, knockdown or pharmacological inhibition of ITPKB increased levels of phosphorylated, insoluble α-synuclein pathology following treatment with α-synuclein preformed fibrils (PFFs). Conversely, ITPKB overexpression reduced PFF-induced α-synuclein aggregation. We also demonstrate that ITPKB inhibition or knockdown increases intracellular calcium levels in neurons, leading to an accumulation of calcium in mitochondria that increases respiration and inhibits the initiation of autophagy, suggesting that ITPKB regulates α-synuclein pathology by inhibiting ER-to-mitochondria calcium transport. Furthermore, the effects of ITPKB on mitochondrial calcium and respiration were prevented by pretreatment with pharmacological inhibitors of the mitochondrial calcium uniporter complex, which was also sufficient to reduce α-synuclein pathology in PFF-treated neurons. Taken together, these results identify ITPKB as a negative regulator of α-synuclein aggregation and highlight modulation of ER-to-mitochondria calcium flux as a therapeutic strategy for the treatment of sporadic PD.

Hyperactivity and Hypermotivation Associated With Increased Striatal mGluR1 Signaling in a Shank2 Rat Model of Autism.

Mutations in the SHANK family of genes have been consistently identified in genetic and genomic screens of autism spectrum disorder (ASD). The functional overlap of SHANK with several other ASD-associated genes suggests synaptic dysfunction as a convergent mechanism of pathophysiology in ASD. Although many ASD-related mutations result in alterations to synaptic function, the nature of those dysfunctions and the consequential behavioral manifestations are highly variable when expressed in genetic mouse models. To investigate the phylogenetic conservation of phenotypes resultant of Shank2 loss-of-function in a translationally relevant animal model, we generated and characterized a novel transgenic rat with a targeted mutation of the Shank2 gene, enabling an evaluation of gene-associated phenotypes, the elucidation of complex behavioral phenotypes, and the characterization of potential translational biomarkers. The Shank2 loss-of-function mutation resulted in a notable phenotype of hyperactivity encompassing hypermotivation, increased locomotion, and repetitive behaviors. Mutant rats also expressed deficits in social behavior throughout development and in the acquisition of operant tasks. The hyperactive phenotype was associated with an upregulation of mGluR1 expression, increased dendritic branching, and enhanced long-term depression (LTD) in the striatum but opposing morphological and cellular alterations in the hippocampus (HP). Administration of the mGluR1 antagonist JNJ16259685 selectively normalized the expression of striatally mediated repetitive behaviors and physiology but had no effect on social deficits. Finally, Shank2 mutant animals also exhibited alterations in electroencephalography (EEG) spectral power and event-related potentials, which may serve as translatable EEG biomarkers of synaptopathic alterations. Our results show a novel hypermotivation phenotype that is unique to the rat model of Shank2 dysfunction, in addition to the traditional hyperactive and repetitive behaviors observed in mouse models. The hypermotivated and hyperactive phenotype is associated with striatal dysfunction, which should be explored further as a targetable mechanism for impairment in ASD.

Impaired ß-arrestin recruitment and reduced desensitization by non-catechol agonists of the D1 dopamine receptor.

Selective activation of dopamine D1 receptors (D1Rs) has been pursued for 40 years as a therapeutic strategy for neurologic and psychiatric diseases due to the fundamental role of D1Rs in motor function, reward processing, and cognition. All known D1R-selective agonists are catechols, which are rapidly metabolized and desensitize the D1R after prolonged exposure, reducing agonist response. As such, drug-like selective D1R agonists have remained elusive. Here we report a novel series of selective, potent non-catechol D1R agonists with promising in vivo pharmacokinetic properties. These ligands stimulate adenylyl cyclase signaling and are efficacious in a rodent model of Parkinson's disease after oral administration. They exhibit distinct binding to the D1R orthosteric site and a novel functional profile including minimal receptor desensitization, reduced recruitment of ß-arrestin, and sustained in vivo efficacy. These results reveal a novel class of D1 agonists with favorable drug-like properties, and define the molecular basis for catechol-specific recruitment of ß-arrestin to D1Rs.

Inhibition of monoacylglycerol lipase terminates diazepam-resistant status epilepticus in mice and its effects are potentiated by a ketogenic diet.

OBJECTIVE: Status epilepticus (SE) is a life-threatening and commonly drug-refractory condition. Novel therapies are needed to rapidly terminate seizures to prevent mortality and morbidity. Monoacylglycerol lipase (MAGL) is the key enzyme responsible for the hydrolysis of the endocannabinoid 2-arachidonoylglycerol (2-AG) and a major contributor to the brain pool of arachidonic acid (AA). Inhibiting of monoacylglycerol lipase modulates synaptic activity and neuroinflammation, 2 mediators of excessive neuronal activation underlying seizures. We studied the effect of a potent and selective irreversible MAGL inhibitor, CPD-4645, on SE that was refractory to diazepam, its neuropathologic sequelae, and the mechanism underlying the drug's effects. METHODS: Diazepam-resistant SE was induced in adult mice fed with standard or ketogenic diet or in cannabinoid receptor type 1 (CB1) receptor knock-out mice. CPD-4645 (10 mg/kg, subcutaneously) or vehicle was dosed 1 and 7 h after status epilepticus onset in video-electroencephalography (EEG) recorded mice. At the end of SE, mice were examined in the novel object recognition test followed by neuronal cellloss analysis. RESULTS: CPD-4645 maximal plasma and brain concentrations were attained 0.5 h postinjection (half-life = 3.7 h) and elevated brain 2-AG levels by approximately 4-fold. CPD-4645 administered to standard diet-fed mice progressively reduced spike frequency during 3 h postinjection, thereby shortening SE duration by 47%. The drug immediately abrogated SE in ketogenic diet-fed mice. CPD-4645 rescued neuronal cell loss and cognitive deficit and reduced interleukin (IL)-1ß and cyclooxygenase 2 (COX-2) brain expression resulting from SE. The CPD-4645 effect on SE was similar in mice lacking CB1 receptors. SIGNIFICANCE: MAGL represents a novel therapeutic target for treating status epilepticus and improving its sequelae. CPD-4645 therapeutic effects appear to be predominantly mediated by modulation of neuroinflammation.

Systematic Evaluation of Bioorthogonal Reactions in Live Cells with Clickable HaloTag Ligands: Implications for Intracellular Imaging.

Bioorthogonal reactions, including the strain-promoted azide-alkyne cycloaddition (SPAAC) and inverse electron demand Diels-Alder (iEDDA) reactions, have become increasingly popular for live-cell imaging applications. However, the stability and reactivity of reagents has never been systematically explored in the context of a living cell. Here we report a universal, organelle-targetable system based on HaloTag protein technology for directly comparing bioorthogonal reagent reactivity, specificity, and stability using clickable HaloTag ligands in various subcellular compartments. This system enabled a detailed comparison of the bioorthogonal reactions in live cells and informed the selection of optimal reagents and conditions for live-cell imaging studies. We found that the reaction of sTCO with monosubstituted tetrazines is the fastest reaction in cells; however, both reagents have stability issues. To address this, we introduced a new variant of sTCO, Ag-sTCO, which has much improved stability and can be used directly in cells for rapid bioorthogonal reactions with tetrazines. Utilization of Ag complexes of conformationally strained trans-cyclooctenes should greatly expand their usefulness especially when paired with less reactive, more stable tetrazines.

Dopamine D1 receptor-agonist interactions: A mutagenesis and homology modeling study.

The dopamine D1 receptor is a G protein-coupled receptor that regulates intracellular signaling via agonist activation. Although the number of solved GPCR X-ray structures has been steadily increasing, still no structure of the D1 receptor exists. We have used site-directed mutagenesis of 12 orthosteric vicinity residues of possible importance to G protein-coupled activation to examine the function of prototypical orthosteric D1 agonists and partial agonists. We find that residues from four different regions of the D1 receptor make significant contributions to agonist function. All compounds studied, which are catechol-amines, are found to interact with the previously identified residues: the conserved D103(3.32), as well as the trans-membrane V serine residues. Additional key interactions are found for trans-membrane VI residues F288(6.51), F289(6.52) and N292(6.55), as well as the extra-cellular loop residue L190(ECL2). Molecular dynamics simulations of a D1 homology model have been used to help put the ligand-residue interactions into context. Finally, we considered the rescaling of fold-shift data as a method to account for the change in the size of the mutated side-chain and found that this rescaling helps to relate the calculated ligand-residue energies with observed experimental fold-shifts.

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

BACKGROUND: Austism 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. METHODS: Transcript 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. RESULTS: Quantification 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. CONCLUSIONS: This 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.