Systematic assessment of the replicability and generalizability of preclinical findings: Impact of protocol harmonization across laboratory sites.

The influence of protocol standardization between laboratories on their replicability of preclinical results has not been addressed in a systematic way. While standardization is considered good research practice as a means to control for undesired external noise (i.e., highly variable results), some reports suggest that standardized protocols may lead to idiosyncratic results, thus undermining replicability. Through the EQIPD consortium, a multi-lab collaboration between academic and industry partners, we aimed to elucidate parameters that impact the replicability of preclinical animal studies. To this end, 3 experimental protocols were implemented across 7 laboratories. The replicability of results was determined using the distance travelled in an open field after administration of pharmacological compounds known to modulate locomotor activity (MK-801, diazepam, and clozapine) in C57BL/6 mice as a worked example. The goal was to determine whether harmonization of study protocols across laboratories improves the replicability of the results and whether replicability can be further improved by systematic variation (heterogenization) of 2 environmental factors (time of testing and light intensity during testing) within laboratories. Protocols were tested in 3 consecutive stages and differed in the extent of harmonization across laboratories and standardization within laboratories: stage 1, minimally aligned across sites (local protocol); stage 2, fully aligned across sites (harmonized protocol) with and without systematic variation (standardized and heterogenized cohort); and stage 3, fully aligned across sites (standardized protocol) with a different compound. All protocols resulted in consistent treatment effects across laboratories, which were also replicated within laboratories across the different stages. Harmonization of protocols across laboratories reduced between-lab variability substantially compared to each lab using their local protocol. In contrast, the environmental factors chosen to introduce systematic variation within laboratories did not affect the behavioral outcome. Therefore, heterogenization did not reduce between-lab variability further compared to the harmonization of the standardized protocol. Altogether, these findings demonstrate that subtle variations between lab-specific study protocols may introduce variation across independent replicate studies even after protocol harmonization and that systematic heterogenization of environmental factors may not be sufficient to account for such between-lab variation. Differences in replicability of results within and between laboratories highlight the ubiquity of study-specific variation due to between-lab variability, the importance of transparent and fine-grained reporting of methodologies and research protocols, and the importance of independent study replication.

A potent and selective Sirtuin 1 inhibitor alleviates pathology in multiple animal and cell models of Huntington's disease.

Protein acetylation, which is central to transcriptional control as well as other cellular processes, is disrupted in Huntington's disease (HD). Treatments that restore global acetylation levels, such as inhibiting histone deacetylases (HDACs), are effective in suppressing HD pathology in model organisms. However, agents that selectively target the disease-relevant HDACs have not been available. SirT1 (Sir2 in Drosophila melanogaster) deacetylates histones and other proteins including transcription factors. Genetically reducing, but not eliminating, Sir2 has been shown to suppress HD pathology in model organisms. To date, small molecule inhibitors of sirtuins have exhibited low potency and unattractive pharmacological and biopharmaceutical properties. Here, we show that highly selective pharmacological inhibition of Drosophila Sir2 and mammalian SirT1 using the novel inhibitor selisistat (selisistat; 6-chloro-2,3,4,9-tetrahydro-1H-carbazole-1-carboxamide) can suppress HD pathology caused by mutant huntingtin exon 1 fragments in Drosophila, mammalian cells and mice. We have validated Sir2 as the in vivo target of selisistat by showing that genetic elimination of Sir2 eradicates the effect of this inhibitor in Drosophila. The specificity of selisistat is shown by its effect on recombinant sirtuins in mammalian cells. Reduction of HD pathology by selisistat in Drosophila, mammalian cells and mouse models of HD suggests that this inhibitor has potential as an effective therapeutic treatment for human disease and may also serve as a tool to better understand the downstream pathways of SirT1/Sir2 that may be critical for HD.

Phenotypic characterization of recessive gene knockout rat models of Parkinson's disease.

Recessively inherited loss-of-function mutations in the PTEN-induced putative kinase 1(Pink1), DJ-1 (Park7) and Parkin (Park2) genes are linked to familial cases of early-onset Parkinson's disease (PD). As part of its strategy to provide more tools for the research community, The Michael J. Fox Foundation for Parkinson's Research (MJFF) funded the generation of novel rat models with targeted disruption ofPink1, DJ-1 or Parkin genes and determined if the loss of these proteins would result in a progressive PD-like phenotype. Pathological, neurochemical and behavioral outcome measures were collected at 4, 6 and 8months of age in homozygous KO rats and compared to wild-type (WT) rats. Both Pink1 and DJ-1 KO rats showed progressive nigral neurodegeneration with about 50% dopaminergic cell loss observed at 8 months of age. ThePink1 KO and DJ-1 KO rats also showed a two to three fold increase in striatal dopamine and serotonin content at 8 months of age. Both Pink1 KO and DJ-1 KO rats exhibited significant motor deficits starting at 4months of age. However, Parkin KO rats displayed normal behaviors with no neurochemical or pathological changes. These results demonstrate that inactivation of the Pink1 or DJ-1 genes in the rat produces progressive neurodegeneration and early behavioral deficits, suggesting that these recessive genes may be essential for the survival of dopaminergic neurons in the substantia nigra (SN). These MJFF-generated novel rat models will assist the research community to elucidate the mechanisms by which these recessive genes produce PD pathology and potentially aid in therapeutic development.

Characterization of behavioral and neuromuscular junction phenotypes in a novel allelic series of SMA mouse models.

A number of mouse models for spinal muscular atrophy (SMA) have been genetically engineered to recapitulate the severity of human SMA by using a targeted null mutation at the mouse Smn1 locus coupled with the transgenic addition of varying copy numbers of human SMN2 genes. Although this approach has been useful in modeling severe SMA and very mild SMA, a mouse model of the intermediate form of the disease would provide an additional research tool amenable for drug discovery. In addition, many of the previously engineered SMA strains are multi-allelic by design, containing a combination of transgenes and targeted mutations in the homozygous state, making further genetic manipulation difficult. A new genetic engineering approach was developed whereby variable numbers of SMN2 sequences were incorporated directly into the murine Smn1 locus. Using combinations of these alleles, we generated an allelic series of SMA mouse strains harboring no, one, two, three, four, five, six or eight copies of SMN2. We report here the characterization of SMA mutants in this series that displayed a range in disease severity from embryonic lethal to viable with mild neuromuscular deficits.

Caspase-6 activity in a BACHD mouse modulates steady-state levels of mutant huntingtin protein but is not necessary for production of a 586 amino acid proteolytic fragment.

Huntington's disease (HD) is caused by a mutation in the huntingtin (htt) gene encoding an expansion of glutamine repeats at the N terminus of the Htt protein. Proteolysis of Htt has been identified as a critical pathological event in HD models. In particular, it has been postulated that proteolysis of Htt at the putative caspase-6 cleavage site (at amino acid Asp-586) plays a critical role in disease progression and pathogenesis. However, whether caspase-6 is indeed the essential enzyme that cleaves Htt at this site in vivo has not been determined. To evaluate, we crossed the BACHD mouse model with a caspase-6 knock-out mouse (Casp6(-/-)). Western blot and immunocytochemistry confirmed the lack of caspase-6 protein in Casp6(-/-) mice, regardless of HD genotype. We predicted the Casp6(-/-) mouse would have reduced levels of caspase-6 Htt fragments and increased levels of full-length Htt protein. In contrast, we found a significant reduction of full-length mutant Htt (mHtt) and fragments in the striatum of BACHD Casp6(-/-) mice. Importantly, we detected the presence of Htt fragments consistent with cleavage at amino acid Asp-586 of Htt in the BACHD Casp6(-/-) mouse, indicating that caspase-6 activity cannot fully account for the generation of the Htt 586 fragment in vivo. Our data are not consistent with the hypothesis that caspase-6 activity is critical in generating a potentially toxic 586 aa Htt fragment in vivo. However, our studies do suggest a role for caspase-6 activity in clearance pathways for mHtt protein.

Enrichment analysis of phenotypic data for drug repurposing in rare diseases.

Drug-induced Behavioral Signature Analysis (DBSA), is a machine learning (ML) method for in silico screening of compounds, inspired by analytical methods quantifying gene enrichment in genomic analyses. When applied to behavioral data it can identify drugs that can potentially reverse in vivo behavioral symptoms in animal models of human disease and suggest new hypotheses for drug discovery and repurposing. We present a proof-of-concept study aiming to assess Drug-induced Behavioral Signature Analysis (DBSA) as a systematic approach for drug discovery for rare disorders. We applied Drug-induced Behavioral Signature Analysis to high-content behavioral data obtained with SmartCube®, an automated in vivo phenotyping platform. The therapeutic potential of several dozen approved drugs was assessed for phenotypic reversal of the behavioral profile of a Huntington's Disease (HD) murine model, the Q175 heterozygous knock-in mice. The in silico Drug-induced Behavioral Signature Analysis predictions were enriched for drugs known to be effective in the symptomatic treatment of Huntington's Disease, including bupropion, modafinil, methylphenidate, and several SSRIs, as well as the atypical antidepressant tianeptine. To validate the method, we tested acute and chronic effects of tianeptine (20 mg/kg, i. p.) in vivo, using Q175 mice and wild type controls. In both experiments, tianeptine significantly rescued the behavioral phenotype assessed with the SmartCube® platform. Our target-agnostic method thus showed promise for identification of symptomatic relief treatments for rare disorders, providing an alternative method for hypothesis generation and drug discovery for disorders with huge disease burden and unmet medical needs.

The GBA1 D409V mutation exacerbates synuclein pathology to differing extents in two alpha-synuclein models.

Heterozygous mutations in the GBA1 gene - encoding lysosomal glucocerebrosidase (GCase) - are the most common genetic risk factors for Parkinson's disease (PD). Experimental evidence suggests a correlation between decreased GCase activity and accumulation of alpha-synuclein (aSyn). To enable a better understanding of the relationship between aSyn and GCase activity, we developed and characterized two mouse models that investigate aSyn pathology in the context of reduced GCase activity. The first model used constitutive overexpression of wild-type human aSyn in the context of the homozygous GCase activity-reducing D409V mutant form of GBA1. Although increased aSyn pathology and grip strength reductions were observed in this model, the nigrostriatal system remained largely intact. The second model involved injection of aSyn preformed fibrils (PFFs) into the striatum of the homozygous GBA1 D409V knock-in mouse model. The GBA1 D409V mutation did not exacerbate the pathology induced by aSyn PFF injection. This study sheds light on the relationship between aSyn and GCase in mouse models, highlighting the impact of model design on the ability to model a relationship between these proteins in PD-related pathology.

Benefits of global mutant huntingtin lowering diminish over time in a Huntington's disease mouse model.

We have developed an inducible Huntington's disease (HD) mouse model that allows temporal control of whole-body allele-specific mutant huntingtin (mHtt) expression. We asked whether moderate global lowering of mHtt (~50%) was sufficient for long-term amelioration of HD-related deficits and, if so, whether early mHtt lowering (before measurable deficits) was required. Both early and late mHtt lowering delayed behavioral dysfunction and mHTT protein aggregation, as measured biochemically. However, long-term follow-up revealed that the benefits, in all mHtt-lowering groups, attenuated by 12 months of age. While early mHtt lowering attenuated cortical and striatal transcriptional dysregulation evaluated at 6 months of age, the benefits diminished by 12 months of age, and late mHtt lowering did not ameliorate striatal transcriptional dysregulation at 12 months of age. Only early mHtt lowering delayed the elevation in cerebrospinal fluid neurofilament light chain that we observed in our model starting at 9 months of age. As small-molecule HTT-lowering therapeutics progress to the clinic, our findings suggest that moderate mHtt lowering allows disease progression to continue, albeit at a slower rate, and could be relevant to the degree of mHTT lowering required to sustain long-term benefits in humans.

Transcriptional Assessment of Striatal mRNAs as Valid Biomarkers of Disease Progression in Three Mouse Models of Huntington's Disease.

BACKGROUND: Huntington's disease (HD) is a progressive neurodegenerative disorder that prominently affects the basal ganglia, leading to affective, cognitive, behavioral, and motor decline. The primary site of neuron loss in HD is the striatal part of the basal ganglia, with GABAergic medium size spiny neurons (MSNs) being nearly completely lost in advanced HD. OBJECTIVE: Based on the hypothesis that mutant huntingtin (mHTT) protein injures neurons via transcriptional dysregulation, we set out to establish a transcriptional profile of HD disease progression in the well characterized transgenic mouse model, R6/2, and two Knock-in models (KI); zQ175KI (expressing mutant mouse/human chimeric Htt protein) and HdhQ200 HET KI (carrying one allele of expanded mouse CAG repeats). METHODS: In this study, we used quantitative PCR (qPCR) to evaluate striatal mRNA levels of markers of neurotransmission, neuroinflammation, and energy metabolism. RESULTS: After analyzing and comparing transcripts from pre-symptomatic and symptomatic stages, markers expressed in the basal ganglia MSNs, which are typically involved in maintaining normal neurotransmission, showed a genotype-specific decrease in mRNA expression in a pattern consistent with human studies. In contrast, transcripts associated with neuroinflammation and energy metabolism were mostly unaffected in these animal models of HD. CONCLUSION: Our results show that transcripts linked to neurotransmission are significantly reduced and are consistent with disease progression in both zQ175KI and R6/2 transgenic mouse models.

Allele-selective transcriptional repression of mutant HTT for the treatment of Huntington's disease.

Huntington's disease (HD) is a dominantly inherited neurodegenerative disorder caused by a CAG trinucleotide expansion in the huntingtin gene (HTT), which codes for the pathologic mutant HTT (mHTT) protein. Since normal HTT is thought to be important for brain function, we engineered zinc finger protein transcription factors (ZFP-TFs) to target the pathogenic CAG repeat and selectively lower mHTT as a therapeutic strategy. Using patient-derived fibroblasts and neurons, we demonstrate that ZFP-TFs selectively repress >99% of HD-causing alleles over a wide dose range while preserving expression of >86% of normal alleles. Other CAG-containing genes are minimally affected, and virally delivered ZFP-TFs are active and well tolerated in HD neurons beyond 100 days in culture and for at least nine months in the mouse brain. Using three HD mouse models, we demonstrate improvements in a range of molecular, histopathological, electrophysiological and functional endpoints. Our findings support the continued development of an allele-selective ZFP-TF for the treatment of HD.

Large-scale phenome analysis defines a behavioral signature for Huntington's disease genotype in mice.

Rapid technological advances for the frequent monitoring of health parameters have raised the intriguing possibility that an individual's genotype could be predicted from phenotypic data alone. Here we used a machine learning approach to analyze the phenotypic effects of polymorphic mutations in a mouse model of Huntington's disease that determine disease presentation and age of onset. The resulting model correlated variation across 3,086 behavioral traits with seven different CAG-repeat lengths in the huntingtin gene (Htt). We selected behavioral signatures for age and CAG-repeat length that most robustly distinguished between mouse lines and validated the model by correctly predicting the repeat length of a blinded mouse line. Sufficient discriminatory power to accurately predict genotype required combined analysis of >200 phenotypic features. Our results suggest that autosomal dominant disease-causing mutations could be predicted through the use of subtle behavioral signatures that emerge in large-scale, combinatorial analyses. Our work provides an open data platform that we now share with the research community to aid efforts focused on understanding the pathways that link behavioral consequences to genetic variation in Huntington's disease.

Non-invasive imaging of GFAP expression after neuronal damage in mice.

Up-regulation of glial fibrillary acidic protein (GFAP) expression is often used as a surrogate marker of neuronal damage. We have created a transgenic mouse line that carries the luciferase gene under the transcriptional control of the mouse GFAP promoter. Biophotonic imaging was used to non-invasively detect the increase in GFAP expression after kainic acid induced neuronal cell death. We demonstrate that after kainic acid treatment, strong biophotonic signals were detected from the brain area. This correlated with both endogenous GFAP and luciferase RNA levels as well as with hippocampal cell death observed histologically. The transgenic mouse line will provide a powerful tool to dynamically monitor neuronal cell death in the living animal and will aid in the discovery and development of drugs to treat damage due to stroke and other neurodegenerative diseases.

Cognitive deficits in transgenic and knock-in HTT mice parallel those in Huntington's disease.

BACKGROUND: Huntington's disease (HD) is characterized not only by severe motor deficits but also by early cognitive dysfunction that significantly increases the burden of the disease for patients and caregivers. Considerable efforts have concentrated, therefore, on the assessment of cognitive deficits in some HD mouse models. However, many of these models that exhibit cognitive deficits also have contemporaneous serious motor deficits, confounding interpretation of cognitive decline. OBJECTIVE: The BACHD and zQ175 mouse models present a more slowly progressing disease phenotype in both motor and cognitive domains, and might therefore offer a better opportunity to measure cognitive decline over a longer timeframe; such models could be useful in screening therapeutic compounds. In order to better define the cognitive impairments evident in BACHD and zQ175 HD mice, both were tested in an instrumental touchscreen visual discrimination assay designed to assess discrimination learning and cognitive flexibility. METHODS: BACHD and zQ175 mice, as well as their WT controls were tested for their ability to discriminate two complex visual stimuli. Following this discrimination phase, the reinforcement contingencies were reversed and the previously incorrect stimulus became the correct stimulus. In a final, third phase of testing, two novel stimuli were introduced and mice were required to undergo a second round of discrimination testing with these stimuli. RESULTS: Our results show that learning during the discrimination phase was similar between the WT and BACHD mice. In contrast, the zQ175 at 26 weeks of age showed decreased accuracy over the last 10 days of discrimination, compared to WT controls. During subsequent reversal and novel stimuli phases, both BACHD and zQ175 mice exhibited significant deficits compared to WT controls. CONCLUSIONS: Our results suggest that the BACHD, and for the first time, zQ175 HD models exhibit cognitive inflexibility and psychomotor slowing, a phenotype that is consistent with cognitive symptoms described in HD patients.

The PDE1/5 Inhibitor SCH-51866 Does Not Modify Disease Progression in the R6/2 Mouse Model of Huntington's Disease.

Huntington's disease is a neurodegenerative disorder caused by mutations in the CAG tract of huntingtin. Several studies in HD cellular and rodent systems have identified disturbances in cyclic nucleotide signaling, which might be relevant to pathogenesis and therapeutic intervention. To investigate whether selective phosphodiesterase (PDE) inhibitors can improve some aspects of disease pathogenesis in HD models, we have systematically evaluated the effects of a variety of cAMP and cGMP selective PDE inhibitors in various HD models. Here we present the lack of effect in a variety of endpoints of the PDE subtype selective inhibitor SCH-51866, a PDE1/5 inhibitor, in the R6/2 mouse model of HD, after chronic oral dosing.

Genetic deletion of transglutaminase 2 does not rescue the phenotypic deficits observed in R6/2 and zQ175 mouse models of Huntington's disease.

Huntington's disease (HD) is an autosomal dominant, progressive neurodegenerative disorder caused by expansion of CAG repeats in the huntingtin gene. Tissue transglutaminase 2 (TG2), a multi-functional enzyme, was found to be increased both in HD patients and in mouse models of the disease. Furthermore, beneficial effects have been reported from the genetic ablation of TG2 in R6/2 and R6/1 mouse lines. To further evaluate the validity of this target for the treatment of HD, we examined the effects of TG2 deletion in two genetic mouse models of HD: R6/2 CAG 240 and zQ175 knock in (KI). Contrary to previous reports, under rigorous experimental conditions we found that TG2 ablation had no effect on either motor or cognitive deficits, or on the weight loss. In addition, under optimal husbandry conditions, TG2 ablation did not extend R6/2 lifespan. Moreover, TG2 deletion did not change the huntingtin aggregate load in cortex or striatum and did not decrease the brain atrophy observed in either mouse line. Finally, no amelioration of the dysregulation of striatal and cortical gene markers was detected. We conclude that TG2 is not a valid therapeutic target for the treatment of HD.

Deficits in a Simple Visual Go/No-go Discrimination Task in Two Mouse Models of Huntington's Disease.

Huntington's disease (HD), a devastating neurodegenerative disorder caused by a CAG repeat expansion on the HTT gene located on chromosome 4, is associated with a characteristic pattern of progressive cognitive dysfunction known to involve early deficits in executive function. A modified Go/No-go successive discrimination task was designed to assess the type of online response control/executive function known to be disrupted in patients with HD. The present studies show that this simple discrimination assay revealed early and robust deficits in two mouse models of HD, the zQ175 KI mouse (deficits from 28 weeks of age) and the R6/2 mouse, carrying ~240 CAG repeats (deficits from 9 weeks of age). These deficits are not due to gross motor dysfunction in the test animals, but instead appear to measure some inability to inhibit responding in the HD mouse models, suggesting this assay may measure deficits in underlying attentional and/or behavioral inhibition processes. Accordingly, this assay may be well suited to evaluation of simple deficits in cognitive function in mouse HD models, providing a potential platform for preclinical screening.

Increased Body Weight of the BAC HD Transgenic Mouse Model of Huntington's Disease Accounts for Some but Not All of the Observed HD-like Motor Deficits.

The genome of the Bacterial Artificial Chromosome (BAC) transgenic mouse model of Huntington's Disease (BAC HD) contains the 170 kb human HTT locus modified by the addition of exon 1 with 97 mixed CAA-CAG repeats. BAC HD mice present robust behavioral deficits in both the open field and the accelerating rotarod tests, two standard behavioral assays of motor function. BAC HD mice, however, also typically present significantly increased body weights relative to wildtype littermate controls (WT) which potentially confounds the interpretation of any motor deficits associated directly with the effects of mutant huntingtin. In order to evaluate this possible confound of body weight, we directly compared the performance of BAC HD and WT female mice under food restricted versus free feeding conditions in both the open field and rotarod tasks to test the hypothesis that some of the motor deficits observed in this HTT-transgenic mouse line results solely from increased body weight. Our results suggest that the rotarod deficit exhibited by BAC HD mice is modulated by both body weight and non-body weight factors resulting from overexpression of full length mutant Htt. When body weights of WT and BAC HD transgenic mice were normalized using restricted feeding, the deficits exhibited by BAC HD mice on the rotarod task were less marked, but were still significant. Since the rotarod deficit between WT and BAC HD mice is attenuated when body weight is normalized by food restriction, utilization of this task in BAC HD mice during pre-clinical evaluation must be powered accordingly and results carefully considered as therapeutic benefit can result from decreased overall body weight and or motoric improvement that may not be related to body mass. Furthermore, after controlling for body weight differences, the hypoactive phenotype displayed by ad libitum fed BAC HD mice in the open field assay was not observed in the BAC HD mice undergoing food restriction. These findings suggest that assessment of spontaneous locomotor activity, as measured in the open field test, may not be the appropriate behavioral endpoint to evaluate the BAC HD mouse during preclinical evaluation since it appears that the apparent hypoactive phenotype in this model is driven primarily by body weight differences.

High-Throughput Automated Phenotyping of Two Genetic Mouse Models of Huntington's Disease.

Phenotyping with traditional behavioral assays constitutes a major bottleneck in the primary screening, characterization, and validation of genetic mouse models of disease, leading to downstream delays in drug discovery efforts. We present a novel and comprehensive one-stop approach to phenotyping, the PhenoCube™. This system simultaneously captures the cognitive performance, motor activity, and circadian patterns of group-housed mice by use of home-cage operant conditioning modules (IntelliCage) and custom-built computer vision software. We evaluated two different mouse models of Huntington's Disease (HD), the R6/2 and the BACHD in the PhenoCube™ system. Our results demonstrated that this system can efficiently capture and track alterations in both cognitive performance and locomotor activity patterns associated with these disease models. This work extends our prior demonstration that PhenoCube™ can characterize circadian dysfunction in BACHD mice and shows that this system, with the experimental protocols used, is a sensitive and efficient tool for a first pass high-throughput screening of mouse disease models in general and mouse models of neurodegeneration in particular.

A mixed fixed ratio/progressive ratio procedure reveals an apathy phenotype in the BAC HD and the z_Q175 KI mouse models of Huntington's disease.

Apathy, characterized by generally reduced interest in and likelihood to perform goal-directed actions, is a recognized symptom of Huntington's disease (HD), a devastating neurological disorder caused by a CAG repeat expansion of the Htt gene located on chromosome 4. The present experiments used a modified progressive ratio task that incorporated a fixed-ratio schedule of reinforcement component to assess consummatory behavior, and a progressive-ratio schedule component that required increasing numbers of lever-presses for successive reinforcers (0.01 ml of evaporated milk). The studies revealed an apathetic phenotype in two mouse models of HD, with decreased response rates either overall or only at higher ratio requirements in the progressive-ratio component relative to wild-type controls. Based on the procedure used (within-session fixed- and progressive-ratio components), it is proposed that an observed phenotype can be ascribed either specifically to reduced motivation to work for food reinforcement or more generally to deficits in consummatory behavior. This procedure provides a simple means to assess this type of phenotype in rodents, with issues in consummatory vs. incentive motivation reflected in general alterations in fixed- versus progressive alterations on an escalating-ratio schedules respectively, providing translational measures of the amotivation/apathy construct of the human realm to the homologous construct of incentive motivation in preclinical models of human disease.

Identification of a Maleimide-Based Glycogen Synthase Kinase-3 (GSK-3) Inhibitor, BIP-135, that Prolongs the Median Survival Time of Δ7 SMA KO Mouse Model of Spinal Muscular Atrophy.

The discovery of upregulated glycogen synthase kinase-3 (GSK-3) in various pathological conditions has led to the development of a host of chemically diverse small molecule GSK-3 inhibitors, such as BIP-135. GSK-3 inhibition emerged as an alternative therapeutic target for treating spinal muscular atrophy (SMA) when a number of GSK-3 inhibitors were shown to elevate survival motor neuron (SMN) levels in vitro and to rescue motor neurons when their intrinsic SMN level was diminished by SMN-specific short hairpin RNA (shRNA). Despite their cellular potency, the in vivo efficacy of GSK-3 inhibitors has yet to be evaluated in an animal model of SMA. Herein, we disclose that a potent and reasonably selective GSK-3 inhibitor, namely BIP-135, was tested in a transgenic Δ7 SMA KO mouse model of SMA, and found to prolong the median survival of these animals. In addition, this compound was shown to elevate the SMN protein level in SMA patient-derived fibroblast cells as determined by western blot, and was neuroprotective in a cell-based, SMA-related model of oxidative stress-induced neurodegeneration.