A novel glucosylceramide synthase inhibitor attenuates alpha synuclein pathology and lysosomal dysfunction in preclinical models of synucleinopathy.

Mutations in the lysosomal enzyme glucocerebrosidase (GCase, GBA1 gene) are the most common genetic risk factor for developing Parkinson's disease (PD). GCase metabolizes the glycosphingolipids glucosylceramide (GlcCer) and glucosylsphingosine (GlcSph). Mutations in GBA1 reduce enzyme activity and the resulting accumulation of glycosphingolipids may contribute to the underlying pathology of PD, possibly via altering lysosomal function. While reduction of GCase activity exacerbates α-synuclein (α-syn) aggregation, it has not been determined that this effect is the result of altered glycosphingolipid levels and lysosome function or some other effect of altering GCase. The glycosphingolipid GlcCer is synthesized by a single enzyme, glucosylceramide synthase (GCS), and small molecule inhibitors (GCSi) reduce cellular glycosphingolipid levels. In the present studies, we utilize a preformed fibril (PFF) rodent primary neuron in vitro model of α-syn pathology to investigate the relationship between glycosphingolipid levels, α-syn pathology, and lysosomal function. In primary cultures, pharmacological inhibition of GCase and D409V GBA1 mutation enhanced accumulation of glycosphingolipids and insoluble phosphorylated α-syn. Administration of a novel small molecule GCSi, benzoxazole 1 (BZ1), significantly decreased glycosphingolipid concentrations in rodent primary neurons and reduced α-syn pathology. BZ1 rescued lysosomal deficits associated with the D409V GBA1 mutation and α-syn PFF administration, and attenuated α-syn induced neurodegeneration of dopamine neurons. In vivo studies revealed BZ1 had pharmacological activity and reduced glycosphingolipids in the mouse brain to a similar extent observed in neuronal cultures. These data support the hypothesis that reduction of glycosphingolipids through GCS inhibition may impact progression of synucleinopathy and BZ1 is useful tool to further examine this important biology.

Intracranial administration of alpha-synuclein fibrils in A30P-synuclein transgenic mice causes robust synucleinopathy and microglial induction.

Synucleinopathies are neurodegenerative disorders involving pathological alpha-synuclein (αSyn) protein, including dementia with Lewy bodies, multiple system atrophy and Parkinson's disease (PD). Current in vivo models of synucleinopathy include transgenic mice overexpressing αSyn variants and methods based on administration of aggregated, exogenous αSyn. Combining these techniques offers the ability to study consequences of introducing pathological αSyn into primed neuronal environments likely to develop synucleinopathy. Herein, we characterize the impacts pre-formed fibrils (PFFs) of recombinant, human αSyn have in mice overexpressing human A30P αSyn, a mutation associated with autosomal dominant PD. A30P mouse brain contains detergent insoluble αSyn biochemically similar to PD brain, and these mice develop Lewy-like synucleinopathy with age. Administration of PFFs in A30P mice resulted in regionally-specific accumulations of phosphorylated synuclein, microglial induction and a motor phenotype that differed from PFF-induced effects in wildtype mice. Surprisingly, PFF-induced losses of tyrosine hydroxylase were similar in A30P and wildtype mice. Thus, the PFF-A30P model recapitulates key aspects of synucleinopathy with induction of microglia, creating an appropriate system for evaluating neurodegenerative therapeutics.

Functionalization of the TMEM175 p.M393T variant as a risk factor for Parkinson disease.

Multiple genome-wide association studies (GWAS) in Parkinson disease (PD) have identified a signal at chromosome 4p16.3; however, the causal variant has not been established for this locus. Deep investigation of the region resulted in one identified variant, the rs34311866 missense SNP (p.M393T) in TMEM175, which is 20 orders of magnitude more significant than any other SNP in the region. Because TMEM175 is a lysosomal gene that has been shown to influence α-synuclein phosphorylation and autophagy, the p.M393T variant is an attractive candidate, and we have examined its effect on TMEM175 protein and PD-related biology. After knocking down each of the genes located under the GWAS peak via multiple shRNAs, only TMEM175 was found to consistently influence accumulation of phosphorylated α-synuclein (p-α-syn). Examination of the p.M393T variant showed effects on TMEM175 function that were intermediate between the wild-type (WT) and knockout phenotypes, with reduced regulation of lysosomal pH in response to starvation and minor changes in clearance of autophagy substrates, reduced lysosomal localization, and increased accumulation of p-α-syn. Finally, overexpression of WT TMEM175 protein reduced p-α-syn, while overexpression of the p.M393T variant resulted in no change in α-synuclein phosphorylation. These results suggest that the main signal in the chromosome 4p16.3 PD risk locus is driven by the TMEM175 p.M393T variant. Modulation of TMEM175 may impact α-synuclein biology and therefore may be a rational therapeutic strategy for PD.

Indole acids as a novel PDE2 inhibitor chemotype that demonstrate pro-cognitive activity in multiple species.

An internal HTS effort identified a novel PDE2 inhibitor series that was subsequently optimized for improved PDE2 activity and off-target selectivity. The optimized lead, compound 4, improved cognitive performance in a rodent novel object recognition task as well as a non-human primate object retrieval task. In addition, co-crystallization studies of close analog of 4 in the PDE2 active site revealed unique binding interactions influencing the high PDE isoform selectivity.

TMEM175 deficiency impairs lysosomal and mitochondrial function and increases α-synuclein aggregation.

Parkinson disease (PD) is a neurodegenerative disorder pathologically characterized by nigrostriatal dopamine neuron loss and the postmortem presence of Lewy bodies, depositions of insoluble α-synuclein, and other proteins that likely contribute to cellular toxicity and death during the disease. Genetic and biochemical studies have implicated impaired lysosomal and mitochondrial function in the pathogenesis of PD. Transmembrane protein 175 (TMEM175), the lysosomal K+ channel, is centered under a major genome-wide association studies peak for PD, making it a potential candidate risk factor for the disease. To address the possibility that variation in TMEM175 could play a role in PD pathogenesis, TMEM175 function was investigated in a neuronal model system. Studies confirmed that TMEM175 deficiency results in unstable lysosomal pH, which led to decreased lysosomal catalytic activity, decreased glucocerebrosidase activity, impaired autophagosome clearance by the lysosome, and decreased mitochondrial respiration. Moreover, TMEM175 deficiency in rat primary neurons resulted in increased susceptibility to exogenous α-synuclein fibrils. Following α-synuclein fibril treatment, neurons deficient in TMEM175 were found to have increased phosphorylated and detergent-insoluble α-synuclein deposits. Taken together, data from these studies suggest that TMEM175 plays a direct and critical role in lysosomal and mitochondrial function and PD pathogenesis and highlight this ion channel as a potential therapeutic target for treating PD.

The PDE10A inhibitor MP-10 and haloperidol produce distinct gene expression profiles in the striatum and influence cataleptic behavior in rodents.

Phosphodiesterase 10A (PDE10A) has garnered attention as a potential therapeutic target for schizophrenia due to its prominent striatal expression and ability to modulate striatal signaling. The present study used the selective PDE10A inhibitor MP-10 and the dopamine D2 antagonist haloperidol to compare effects of PDE10A inhibition and dopamine D2 blockade on striatopallidal (D2) and striatonigral (D1) pathway activation. Our studies confirmed that administration of MP-10 significantly elevates expression of the immediate early genes (IEG) c-fos, egr-1, and arc in rat striatum. Furthermore, we demonstrated that MP-10 induced egr-1 expression was distributed evenly between enkephalin-containing D2-neurons and substance P-containing D1-neurons. In contrast, haloperidol (3 mg/kg) selectively activated egr-1 expression in enkephalin neurons. Co-administration of MP-10 and haloperidol (0.5 mg/kg) increased IEG expression to a greater extent than either compound alone. Similarly, in a rat catalepsy assay, administration of haloperidol (0.5 mg/kg) or MP-10 (3-30 mg/kg) did not produce cataleptic behavior when dosed alone, but co-administration of haloperidol with MP-10 (3 and 10 mg/kg) induced cataleptic behaviors. Interestingly, co-administration of haloperidol with a high dose of MP-10 (30 mg/kg) failed to produce cataleptic behavior. These findings are important for understanding the neural circuits involved in catalepsy and suggest that the behavioral effects produced by PDE10A inhibitors may be influenced by concomitant medication and the level of PDE10A inhibition achieved by the dose of the inhibitor.

Genetic deletion and pharmacological inhibition of phosphodiesterase 10A protects mice from diet-induced obesity and insulin resistance.

Phosphodiesterase 10A (PDE10A) is a novel therapeutic target for the treatment of schizophrenia. Here we report a novel role of PDE10A in the regulation of caloric intake and energy homeostasis. PDE10A-deficient mice are resistant to diet-induced obesity (DIO) and associated metabolic disturbances. Inhibition of weight gain is due to hypophagia after mice are fed a highly palatable diet rich in fats and sugar but not a standard diet. PDE10A deficiency produces a decrease in caloric intake without affecting meal frequency, daytime versus nighttime feeding behavior, or locomotor activity. We tested THPP-6, a small molecule PDE10A inhibitor, in DIO mice. THPP-6 treatment resulted in decreased food intake, body weight loss, and reduced adiposity at doses that produced antipsychotic efficacy in behavioral models. We show that PDE10A inhibition increased whole-body energy expenditure in DIO mice fed a Western-style diet, achieving weight loss and reducing adiposity beyond the extent seen with food restriction alone. Therefore, chronic THPP-6 treatment conferred improved insulin sensitivity and reversed hyperinsulinemia. These data demonstrate that PDE10A inhibition represents a novel antipsychotic target that may have additional metabolic benefits over current medications for schizophrenia by suppressing food intake, alleviating weight gain, and reducing the risk for the development of diabetes.

The novel phosphodiesterase 10A inhibitor THPP-1 has antipsychotic-like effects in rat and improves cognition in rat and rhesus monkey.

Phosphodiesterase 10A (PDE10A) is a novel target for the treatment of schizophrenia that may address multiple symptomatic domains associated with this disorder. PDE10A is highly expressed in the brain and functions to metabolically inactivate the important second messengers cAMP and cGMP. Here we describe effects of a potent and orally bioavailable PDE10A inhibitor [2-(6-chloropyridin-3-yl)-4-(2-methoxyethoxy)-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl](imidazo[1,5-a]pyridin-1-yl)methanone] (THPP-1) on striatal signaling pathways, in behavioral tests that predict antipsychotic potential, and assays that measure episodic-like memory in rat and executive function in rhesus monkey. THPP-1 exhibits nanomolar potency on the PDE10A enzyme, demonstrates excellent pharmacokinetic properties in multiple preclinical animal species, and is selective for PDE10A over other PDE families of enzymes. THPP-1 significantly increased phosphorylation of proteins in the striatum involved in synaptic plasticity, including the a-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid receptor (AMPA) GluR1 subunit, extracellular receptor kinase (ERK), and cAMP-response element binding protein (CREB). THPP-1 produced dose-dependent effects in preclinical assays predictive of antipsychotic activity including attenuation of MK-801-induced psychomotor activation and condition avoidance responding in rats. At similar plasma exposures, THPP-1 significantly increased object recognition memory in rat and attenuated a ketamine-induced deficit in the object retrieval detour task in rhesus monkey. These findings suggest that PDE10A inhibitors have the potential to impact multiple symptomatic domains of schizophrenia including positive symptoms and cognitive impairment. This article is part of a Special Issue entitled 'Cognitive Enhancers'.

cSSMD: assessing collective activity for addressing off-target effects in genome-scale RNA interference screens.

MOTIVATION: Off-target activity commonly exists in RNA interference (RNAi) screens and often generates false positives. Existing analytic methods for addressing the off-target effects are demonstrably inadequate in RNAi confirmatory screens. RESULTS: Here, we present an analytic method assessing the collective activity of multiple short interfering RNAs (siRNAs) targeting a gene. Using this method, we can not only reduce the impact of off-target activities, but also evaluate the specific effect of an siRNA, thus providing information about potential off-target effects. Using in-house RNAi screens, we demonstrate that our method obtains more reasonable and sensible results than current methods such as the redundant siRNA activity (RSA) method, the RNAi gene enrichment ranking (RIGER) method, the frequency approach and the t-test. CONTACT: xiaohua_zhang@merck.com SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

High throughput monitoring of amyloid-ß(42) assembly into soluble oligomers achieved by sensitive conformation state-dependent immunoassays.

Accumulation of small soluble assemblies of amyloid-ß (Aß)(42) in the brain is thought to play a key role in the pathogenesis of Alzheimer's disease. As a result, there has been much interest in finding small molecules that inhibit the formation of synaptotoxic Aß(42) oligomers that necessitates sensitive methods for detecting the initial steps in the oligomerization of Aß(42). Modeling suggests that oligomerized Aß(42) adopts a conformation in which the C-terminus is embedded in the center, whereas the N-terminus is exposed at the periphery of the oligomer. Here we report that an inverse change in Aß(42) C-terminal and N-terminal epitope accessibility provides the basis of a sensitive method for assessing early steps in Aß(42) oligomerization. Using ELISA and AlphaLISA, we found that Aß(42) C-terminal immunoreactivity decreased in a time- and concentration-dependent manner under conditions favoring oligomerization. This reduction was accompanied by an increase in the N-terminal immunoreactivity, suggesting that assemblies with multiple exposed N-terminal epitopes were detected. Importantly the assay generates a robust window between monomers and oligomers at as low as 1 nM Aß(42). Using this assay, known oligomerization inhibitors produced a dose-dependent unmasking of the Aß(42) C-terminal epitope. After automation, the assay proved to be highly reproducible and effective for high throughput screening of small molecules that inhibit Aß(42) oligomerization.

Induction of Alzheimer's-like changes in brain of mice expressing mutant APP fed excess methionine.

Elevated plasma homocysteine, a risk factor for Alzheimer's disease, could result from increased production from methionine or by inefficient clearance by folate- and B-vitamin-dependent pathways. Understanding the relative contributions of these processes to pathogenesis is important for therapeutic strategies designed to lower homocysteine. To assess these alternatives, we elevated plasma homocysteine by feeding mutant amyloid precursor protein (APP)-expressing mice diets with either high methionine (HM) or deficient in B-vitamins and folate (B Def). Mutant APP mice fed HM demonstrated increased brain beta amyloid. Interestingly, this increase was not observed in mutant APP mice fed B Def diet, nor was it observed in C57Bl6 or YAC-APP mice fed HM. Furthermore, HM, but not B Def, produced a prolonged increase in brain homocysteine only in mutant APP mice but not wild-type mice. These changes were time-dependent over 10 weeks. Further, by 10 weeks HM increased brain cholesterol and phosphorylated tau in mutant APP mice. Transcriptional profiling experiments revealed robust differences in RNA expression between C57Bl6 and mutant APP mice. The HM diet in C57Bl6 mice transiently induced a transcriptional profile similar to mutant APP cortex, peaking at 2 weeks , following a time course comparable to brain homocysteine changes. Together, these data suggest a link between APP and methionine metabolism.

The use of SSMD-based false discovery and false nondiscovery rates in genome-scale RNAi screens.

In genome-scale RNA interference (RNAi) screens, it is critical to control false positives and false negatives statistically. Traditional statistical methods for controlling false discovery and false nondiscovery rates are inappropriate for hit selection in RNAi screens because the major goal in RNAi screens is to control both the proportion of short interfering RNAs (siRNAs) with a small effect among selected hits and the proportion of siRNAs with a large effect among declared nonhits. An effective method based on strictly standardized mean difference (SSMD) has been proposed for statistically controlling false discovery rate (FDR) and false nondiscovery rate (FNDR) appropriate for RNAi screens. In this article, the authors explore the utility of the SSMD-based method for hit selection in RNAi screens. As demonstrated in 2 genome-scale RNAi screens, the SSMD-based method addresses the unmet need of controlling for the proportion of siRNAs with a small effect among selected hits, as well as controlling for the proportion of siRNAs with a large effect among declared nonhits. Furthermore, the SSMD-based method results in reasonably low FDR and FNDR for selecting inhibition or activation hits. This method works effectively and should have a broad utility for hit selection in RNAi screens with replicates.

A conformational constraint improves a beta-secretase inhibitor but for an unexpected reason.

During our ongoing efforts to develop a small molecule inhibitor targeting the beta-amyloid cleaving enzyme (BACE-1), we discovered a class of compounds bearing an aminoimidazole motif. Initial optimization led to potent compounds that have high Pgp efflux ratios. Crystal structure-aided design furnished conformationally constrained compounds that are both potent and have relatively low Pgp efflux ratios. Computational studies performed after these optimizations suggest that the introduction of the constraint enhances potency via additional hydrophobic interactions rather than conformational restriction.