David F. Fischer

Histological and Functional Benefit Following Transplantation of Motor Neuron Progenitors to the Injured Rat Spinal Cord

Background Motor neuron loss is characteristic of cervical spinal cord injury (SCI) and contributes to functional deficit. Methodology/Principal Findings In order to investigate the amenability of the injured adult spinal cord to motor neuron differentiation, we transplanted spinal cord injured animals with a high purity population of human motor neuron progenitors (hMNP) derived from human embryonic stem cells (hESCs). In vitro, hMNPs displayed characteristic motor neuron-specific markers, a typical electrophysiological profile, functionally innervated human or rodent muscle, and secreted physiologically active growth factors that caused neurite branching and neuronal survival. hMNP transplantation into cervical SCI sites in adult rats resulted in suppression of intracellular signaling pathways associated with SCI pathogenesis, which correlated with greater endogenous neuronal survival and neurite branching. These neurotrophic effects were accompanied by significantly enhanced performance on all parameters of the balance beam task, as compared to controls. Interestingly, hMNP transplantation resulted in survival, differentiation, and site-specific integration of hMNPs distal to the SCI site within ventral horns, but hMNPs near the SCI site reverted to a neuronal progenitor state, suggesting an environmental deficiency for neuronal maturation associated with SCI. Conclusions/Significance These findings underscore the barriers imposed on neuronal differentiation of transplanted cells by the gliogenic nature of the injured spinal cord, and the physiological relevance of transplant-derived neurotrophic support to functional recovery.

Targeting ATM ameliorates mutant Huntingtin toxicity in cell and animal models of Huntington’s disease

Reducing ATM signaling is neuroprotective in cell and animal models of Huntington’s disease. Cashing in with ATM Age-related neurodegenerative disorders, including Alzheimer’s disease and Huntington’s disease (HD), consistently show elevated markers of DNA damage. It remains unclear, however, whether such defects are mere consequences of or actively contribute to disease processes. In a new study, Lu et al. show that ataxia-telangiectasia mutated (ATM), a pivotal signaling molecule in the DNA damage response pathway, can modify the toxicity of the mutant protein that causes HD. ATM signaling activity was aberrantly increased in HD cells, animal models of HD, and postmortem brain tissue from HD patients. Reducing ATM signaling by genetic manipulation or using small-molecule inhibitors of ATM consistently reduced HD protein toxicities in cellular and animal models. Age-related neurodegenerative disorders including Alzheimer’s disease and Huntington’s disease (HD) consistently show elevated DNA damage, but the relevant molecular pathways in disease pathogenesis remain unclear. One attractive gene is that encoding the ataxia-telangiectasia mutated (ATM) protein, a kinase involved in the DNA damage response, apoptosis, and cellular homeostasis. Loss-of-function mutations in both alleles of ATM cause ataxia-telangiectasia in children, but heterozygous mutation carriers are disease-free. Persistently elevated ATM signaling has been demonstrated in Alzheimer’s disease and in mouse models of other neurodegenerative diseases. We show that ATM signaling was consistently elevated in cells derived from HD mice and in brain tissue from HD mice and patients. ATM knockdown protected from toxicities induced by mutant Huntingtin (mHTT) fragments in mammalian cells and in transgenic Drosophila models. By crossing the murine Atm heterozygous null allele onto BACHD mice expressing full-length human mHTT, we show that genetic reduction of Atm gene dosage by one copy ameliorated multiple behavioral deficits and partially improved neuropathology. Small-molecule ATM inhibitors reduced mHTT-induced death of rat striatal neurons and induced pluripotent stem cells derived from HD patients. Our study provides converging genetic and pharmacological evidence that reduction of ATM signaling could ameliorate mHTT toxicity in cellular and animal models of HD, suggesting that ATM may be a useful therapeutic target for HD.

A Monoclonal Antibody TrkB Receptor Agonist as a Potential Therapeutic for Huntington’s Disease

Huntington’s disease (HD) is a devastating, genetic neurodegenerative disease caused by a tri-nucleotide expansion in exon 1 of the huntingtin gene. HD is clinically characterized by chorea, emotional and psychiatric disturbances and cognitive deficits with later symptoms including rigidity and dementia. Pathologically, the cortico-striatal pathway is severely dysfunctional as reflected by striatal and cortical atrophy in late-stage disease. Brain-derived neurotrophic factor (BDNF) is a neuroprotective, secreted protein that binds with high affinity to the extracellular domain of the tropomyosin-receptor kinase B (TrkB) receptor promoting neuronal cell survival by activating the receptor and down-stream signaling proteins. Reduced cortical BDNF production and transport to the striatum have been implicated in HD pathogenesis; the ability to enhance TrkB signaling using a BDNF mimetic might be beneficial in disease progression, so we explored this as a therapeutic strategy for HD. Using recombinant and native assay formats, we report here the evaluation of TrkB antibodies and a panel of reported small molecule TrkB agonists, and identify the best candidate, from those tested, for in vivo proof of concept studies in transgenic HD models.

Quantification Assays for Total and Polyglutamine-Expanded Huntingtin Proteins

The expansion of a CAG trinucleotide repeat in the huntingtin gene, which produces huntingtin protein with an expanded polyglutamine tract, is the cause of Huntingtons disease (HD). Recent studies have reported that RNAi suppression of polyglutamine-expanded huntingtin (mutant HTT) in HD animal models can ameliorate disease phenotypes. A key requirement for such preclinical studies, as well as eventual clinical trials, aimed to reduce mutant HTT exposure is a robust method to measure HTT protein levels in select tissues. We have developed several sensitive and selective assays that measure either total human HTT or polyglutamine-expanded human HTT proteins on the electrochemiluminescence Meso Scale Discovery detection platform with an increased dynamic range over other methods. In addition, we have developed an assay to detect endogenous mouse and rat HTT proteins in pre-clinical models of HD to monitor effects on the wild type protein of both allele selective and non-selective interventions. We demonstrate the application of these assays to measure HTT protein in several HD in vitro cellular and in vivo animal model systems as well as in HD patient biosamples. Furthermore, we used purified recombinant HTT proteins as standards to quantitate the absolute amount of HTT protein in such biosamples.

A small molecule mitigates hearing loss in a mouse model of Usher syndrome III

Usher syndrome type III (USH3) characterized by progressive deafness, variable balance disorder, and blindness is caused by destabilizing mutations in the gene encoding the clarin-1 protein (CLRN1). Here we report a novel strategy to mitigate hearing loss associated with a common USH3 mutation CLRN1N48K that involved a cell-based high-throughput screening of small molecules capable of stabilizing CLRN1N48K, a secondary screening to eliminate general proteasome inhibitors, and finally an iterative process to optimize structure activity relationships. This resulted in the identification of BF844. To test the efficacy of BF844, a mouse model was developed that mimicked the progressive hearing loss of USH3. BF844 effectively attenuated progressive hearing loss and prevented deafness in this model. Because the human CLRN1N48K mutation causes both hearing and vision loss, BF844 could in principle prevent both sensory deficiencies in USH3. Moreover, the strategy described here could help identify drugs for other protein-destabilizing monogenic disorders.

Quantification of huntingtin protein species in Huntington’s disease patient leukocytes using optimised electrochemiluminescence immunoassays

Background Huntington’s disease (HD) is an autosomal dominant neurodegenerative condition caused by an expanded CAG repeat in the gene encoding huntingtin (HTT). Optimizing peripheral quantification of huntingtin throughout the course of HD is valuable not only to illuminate the natural history and pathogenesis of disease, but also to detect peripheral effects of drugs in clinical trial. Rationale We previously demonstrated that mutant HTT (mHTT) was significantly elevated in purified HD patient leukocytes compared with controls and that these levels track disease progression. Our present study investigates whether the same result can be achieved with a simpler and more scalable collection technique that is more suitable for clinical trials. Methods We collected whole blood at 133 patient visits in two sample sets and generated peripheral blood mononuclear cells (PBMCs). Levels of mHTT, as well as N-, and C-terminal and mid-region huntingtin were measured in the PBMCs using ELISA-based Meso Scale Discovery (MSD) electrochemiluminescence immunoassay platforms, and we evaluated the relationship between different HTT species, disease stage, and brain atrophy on magnetic resonance imaging. Conclusions The assays were sensitive and accurate. We confirm our previous findings that mHTT increases with advancing disease stage in patient PBMCs, this time using a simple collection protocol and scalable assay.

Assessment of p.Phe508del-CFTR functional restoration in pediatric primary cystic fibrosis airway epithelial cells

Background Mutations in the cystic fibrosis transmembrane regulator (CFTR) gene can reduce function of the CFTR ion channel activity and impair cellular chloride secretion. The gold standard method to assess CFTR function of ion transport using the Ussing chamber requires a high number of airway epithelial cells grown at air-liquid interface, limiting the application of this method for high throughput screening of potential therapeutic compounds in primary airway epithelial cells (pAECs) featuring less common CFTR mutations. This study assessed an alternative approach, using a small scale halide assay that can be adapted for a personalized high throughput setting to analyze CFTR function of pAEC. Methods Pediatric pAECs derived from children with CF (pAECCF) were established and expanded as monolayer cultures, before seeding into 96-well plates for the halide assay. Cells were then transduced with an adenoviral construct containing yellow fluorescent protein (eYFP) reporter gene, alone or in combination with either wild-type CFTR (WT-CFTR) or p.Phe508del CFTR. Four days post transduction, cells were stimulated with forskolin and genistein, and assessed for quenching of the eYFP signal following injection of iodide solution into the assay media. Results Data showed that pAECCF can express eYFP at high efficiency following transduction with the eYFP construct. The halide assay was able to discriminate functional restoration of CFTR in pAECCF treated with either WT-CFTR construct or the positive controls syntaxin 8 and B-cell receptor-associated protein 31 shRNAs. Significance The current study demonstrates that the halide assay can be adapted for pediatric pAECCF to evaluate restoration of CFTR function. With the ongoing development of small molecules to modulate the folding and/or activity of various mutated CFTR proteins, this halide assay presents a small-scale personalized screening platform that could assess therapeutic potential of molecules across a broad range of CFTR mutations.

L19 Optimisation of potent, selective and brain penetrant atm inhibitors as potential therapeutic agents for HD

Emerging evidence shows that the ATM signalling pathway is dysregulated in neurodegenerative disorders including Huntington’s disease (HD). ATM signalling has been shown to be elevated in cells derived from HD patients and mouse models of HD. Genetic and pharmacological evidence from cellular and animal models of HD suggests that reduction of ATM signalling can ameliorate mHTT toxicity. Thus inhibitor of ATM kinase presents a promising therapeutic intervention strategy for the treatment of HD. We present data from our medicinal chemistry program which aims to develop potent, selective and brain-penetrant ATM inhibitors. Chemical optimisation of CNS-compliant physical chemical properties led to the identification of compounds suitable for a proof-of-concept study in HD models. Our lead compound, CHDI-00485194, displayed excellent oral bioavailability and pharmacokinetics. PO administration to mice showed distribution into brain (brain:plasma ratio of 1.3) and linear pharmacokinetics in a dose escalation study. An acute PK/PD biomarker study to evaluate the effect of CHDI-00485194 on irradiation induced DNA damage biomarkers showed a significant dose-dependent inhibition of KAP1 phosphorylation by CHDI-00485194 at 30 and 60 minutes post-irradiation.

Towards Small Molecules as Therapies for Alzheimer’s Disease and Other Neurodegenerative Disorders

Abstract Neurodegenerative diseases caused by hereditary or idiosyncratic neuronal dysfunction share some phenotypic commonalities. Intracellular aggregation of proteins, metal dyshomeostasis, generic loss of synaptic connectivity all lead to gradual decline of cognitive or motor neuronal function as patients descend into a clinically symptomatic state. Though significant progress has been made in our understanding of neurological disorders in the past decade, it has yet to translate into therapeutic advancements in disease treatment. We have chosen to focus this review on Alzheimer’s disease (AD) to highlight the main disease modifying mechanisms shared in common with the Huntington’s (HD) and Parkinson’s disease (PD) phenotypes, specifically, the aggregation of amyloid-β (Aβ) phospho-tau (p-tau), mutant huntingtin (mHtt) and α -synuclein (α-syn) proteins, respectively. We highlight a number of approaches used in pre-clinical drug discovery to identify clinical tools. In addition, we describe a number of less explored alternative hypotheses which have demonstrated good (pre)clinical evidence for a potential therapeutic intervention. In particular, for AD, we will review the main concepts which have driven drug discovery research in the recent past and for each molecular target, we summarize a rationale and available validation data with commentary on relevant chemical matter and structural biology, then discuss advanced pre-clinical and clinical compounds.