CHDI’s 6th Annual HD Therapeutics Conference took place February 7-10, 2011, in Palm Springs, California. This unique conference series focuses on drug discovery and development for Huntington’s disease, and draws participants and speakers from the biotech and pharmaceutical sectors as well as academia and research institutions. The conference is intended as a forum where all participants can share ideas, learn about new disciplines, network with colleagues and build new collaborative partnerships. We are indebted to all of the conference speakers, and especially grateful to those who are able to make their presentations available here for a wider audience.
- Huntingtin Normal Function Scott Zeitlin, PhD
- Drug Development in SMA: Parallels from Another Orphan Disease Karen Chen, PhD
- Huntingtin Protein Bioassays for Drug Discovery, Translational Research and Clinical Trials Andreas Weiss, PhD
- Modulation of Huntingtin Levels as a Therapeutics Approach Douglas Macdonald, PhD
- Synaptic Dysfunction in Huntington’s Disease: Role of Mislocalization and Altered Signaling of NR2B-type NMDA Receptors Lynn Raymond, MD, PhD
- A Deficit in Intrinsic Excitability of Striatal Neurons in a Transgenic Mouse Model of HD Don Faber, PhD
- Targeting Cognitive Deficits in Schizophrenia David Gerber, PhD
- Prodromal HD: Motor Cortex Excitability and Plasticity Assessed with Transcranial Magnetic Stimulation Michael Orth, MD, PhD
- Assessment of Synaptic Dysfunction in Huntington’s Disease – CHDI’s Strategy and Programs Vahri Beaumont, PhD
- Unraveling the Mode of Action of Pridopidine – From a Brain Circuitry to a Molecular Perspective and Back Nicholas Waters, PhD
- Mitochondrial and Bioenergetic Dysfunction in Huntington’s Disease: Overview Timothy Greenamyre, MD, PhD
- Evaluating Regional Cerebral Dysfunction and Bioenergetics by Magnetic Resonance Spectroscopic Imaging Hoby Hetherington, MSc, PhD
- What Can Be Learned from Another Neurodegenerative Disease: Mitochondrial Dynamics Impairment in Parkinson’s Disease Sarah B. Berman, MD, PhD
- Optimization of Brain-penetrant Isozyme Selective PDHK Inhibitors as Potential Therapeutic Agents for HD Leticia Toledo-Sherman, PhD
- Molecular Approaches to Drug Receptors: Relevance to HD Solomon H. Snyder, MD
- Upstream and downstream of Trk receptors Moses Chao, PhD
- Jordi Alberch, MD
- AAV2-neurturin (CERE-120) Gene Transfer for Parkinson’s and Huntington’s Disease Joao Siffert, MD
- Staying on Trk’s: Pharmacological Intervention Strategies Alex Kiselyov, PhD
- Introduction – CHDI’s Internal Programs Robert Pacifici, PhD
- Kynurenine Pathway Modulation as a Potential Treatment for HD – Advances in the Development of a Selective Brain Penetrant KMO Inhibitor Ignacio Muñoz-Sanjuan, PhD
- Histone Deacetylase Class IIa (HDAC4) Inhibitors: Potential Therapeutics for Huntington’s Disease Celia Dominguez, PhD
Normal Huntingtin Function
Scott Zeitlin, PhD – University of Virginia School of Medicine
To evaluate the safety of therapeutic approaches that reduce mRNA levels encoding both normal and mutant huntingtin, an understanding of the consequences of lowering normal huntingtin expression is required. A number of laboratories have explored the role of huntingtin in development and in the postnatal brain using knock-out, and Cre/loxP conditional knock-out mouse models that eliminate huntingtin expression in the whole animal or selected cell populations. Results obtained from these mouse models suggest that huntingtin has critical developmental functions and is needed for neuronal function/survival. Recent results we have obtained with conditional knock-outs have also revealed that huntingtin is involved in neuronal protein homeostasis, as ubiquitinated protein aggregates appear in aging Nestin-cre;Hdh(flox/-) mutant brains. Additional information about huntingtin’s potential role in neuronal homeostasis can also obtained from knock-in mice expressing versions of huntingtin lacking the normal polyglutamine stretch (DQ-htt) or the adjacent proline-rich region in the context of a normal or expanded polyglutamine stretch (DPRR- and 140Q-DPRR-htt, respectively). However, despite what we have learned from these mouse models and from a variety of in vitro systems, we still do not know how low we can reduce huntingtin expression in the brain without deleterious consequences, and if there are preferred therapeutic windows in the adult where substantial reduction of huntingtin expression can be tolerated. New mouse models may be required to answer these questions, and potential experimental strategies will be discussed.
Supported by: NIH, HDF, CHDI
Drug Development in SMA: Parallels from Another Orphan Disease
Karen Chen, PhD – SMA Foundation
Proximal spinal muscular atrophy (SMA) is an autosomal recessive motor neuron disease that is the leading genetic killer of infants and toddlers, and in its most severe form causes death by the age of two years. SMA is caused by deletion or mutation of the survival motor neuron 1 (SMN1) gene resulting in insufficiency in the survival motor neuron (SMN) protein. SMN protein plays a role in assembly of the spliceosome and is required in all cells for survival. A “backup” gene, SMN2, produces less full-length SMN mRNA and protein than SMN1. In SMA patients, disease severity is inversely correlated with increased SMN2 gene copy number. These observations suggest that increasing expression of the SMN2 gene may provide a strategy for treatment. Currently pursued drug development strategies for SMA include induction of SMN2 gene expression, modulation of splicing of SMN2-derived transcripts, stabilization of SMN protein, neuroprotection of motor neurons, and SMN1 gene replacement; and utilize different types of therapeutics such as antisense oligonucleotides and small molecules. Clinical trials in this disease present a unique set of challenges, including the development of meaningful outcome measures and disease biomarkers, which include SMN-related measurements and non-SMN markers, as well as considerations of appropriate patient populations.
Huntingtin Protein Bioassays for Drug Discovery, Translational Research and Clinical Trials
Andreas Weiss, PhD – Novartis Institute for Biomedical Research
Mutant huntingtin protein is the culprit of Huntington’s Disease. Recent drug discovery efforts have therefore been focused on identifying treatments capable of lowering mutant huntingtin protein levels in the cell. In order to evaluate the potential success of these disease-modifying therapeutics in patients, accurate quantification of the protein as a pharmacodynamic marker in human tissue or body fluids is a necessity. In addition, to better understand the role of the full-length mutant huntingtin protein, its described proteolytic products and its conformational forms in the pathomechanism of the disease, it is essential to be able to measure and characterize these different protein manifestations in biological samples. To address these challenges, we have developed time-resolved FRET immunoassays for the quantification of wild-type and mutant huntingtin protein in a variety of biological samples including Huntington’s Disease patient-derived tissue samples and body fluids. The current use of these assays and their ongoing further development for drug discovery, translational research and clinical trial applications will be discussed.
Modulation of Huntingtin Levels as a Therapeutic Approach
Douglas Macdonald, PhD – CHDI
As a monogenic disorder caused by a mutation in the Huntingtin (Htt) gene, Huntington’s disease (HD) is a prime candidate for disease modifying therapies that target the suppression of mutant Htt expression. Ongoing late preclinical programs aim to intervene at the RNA level in an attempt to modulate Htt protein levels as a therapeutic approach. Antisense oligonucleotides and short interfering RNAs have been optimized to reduce Htt mRNA and protein levels in vitro and in vivo. Both non-selective and mutant-selective allelic lowering agents have been developed and the advantages and disadvantages of these strategies will be discussed. Additionally, the potential detrimental effect driven by a loss of wild-type Htt function below a certain threshold and other essential aspects for successful therapeutic intervention, such as the degree of suppression as well as spatial and temporal considerations, will be presented. Lastly, the identification of biomarkers sensitive to Htt dosage, an aspect of critical importance when planning long-term therapeutic trials with any Htt modulating agent, will be addressed. Despite these challenges, the Htt lowering approach holds great promise for disease modifying treatments that will delay or slow HD.
Synaptic Dysfunction in Huntington Disease: Role of Mislocalization and Altered Signaling of NR2B-type NMDA Receptors
Lynn Raymond, MD, PhD – University of British Columbia
Huntington disease (HD) is a dominantly inherited neurodegenerative disease that is characterized by striatal neuronal degeneration and caused by a polyglutamine (polyQ) expansion in the protein huntingtin (htt). Overactivation of N-methyl-D-aspartate-type glutamate receptors (NMDARs) causing excitotoxicity had been postulated to contribute to selective neurodegeneration because acute intrastriatal injection of NMDAR agonists reproduced many of the neuropathological and clinical features of HD in rodents and non-human primates. Recent studies in hippocampal or cortical neuronal cultures indicate that synaptic NMDARs signal neuronal survival whereas extrasynaptic NMDAR stimulation triggers cell death. Genetic mouse models of HD have facilitated testing the role of excitotoxicity in HD. In the yeast artificial chromosome (YAC) HD mouse model, we have shown increased expression and function of striatal extrasynaptic N-methyl-D-aspartate (NMDA) receptors (Ex-NMDARs) containing the NR2B subunit, associated with reduced activity of the pro-survival transcription factor cAMP Response Element Binding protein (CREB) and enhanced sensitivity to excitotoxicity. Moreover, we found a similar increase in Ex-NMDAR current in a knock-in HD mouse model. These changes occur prior to motor onset and may prove an important target for therapy aimed toward delaying disease onset. To determine mechanisms underlying accumulation of Ex-NMDARs, and the consequences of this shift in NMDAR distribution on downstream cell death vs. survival signaling, we compared striatal tissue and cultured striatal neurons from presymptomatic YAC128 mice (expressing full-length htt with 128 polyQ) to those from YAC18 and/or WT mice as controls. Our studies have focused on a role for calpains and Striatal-Enriched Phosphatase (STEP) because they are known to regulate NR2B-type NMDAR interactions with scaffolding proteins such as postsynaptic density 95 (PSD-95) and retention in synapses, and because these enzymes are activated by Ex-NMDAR stimulation. We have found enhanced activity of both enzymes in YAC128 striatum at baseline, and that treatment of acute cortico-striatal slices with inhibitors of calpain or STEP reduce Ex-NMDAR levels and increase synaptic NMDARs, respectively. Furthermore, Ex-NMDAR-stimulated activation of p38 MAPK has been shown previously to be regulated by calpain and STEP and to signal cell death in hippocampal neurons. Notably, we found p38 MAPK activity to be increased basally in YAC128 striatum while its inhibition reduced NMDA toxicity in striatal cultures from YAC128 but not WT mice. Together, our results suggest that manipulation of calpain and STEP activity, with consequences on Ex-NMDAR expression and p38 MAPK activity, may be a viable approach to reducing deleterious signaling by eNMDARs in HD.
Supported by: Canadian Institutes of Health Research and CHDI
A Deficit in Intrinsic Excitability of Striatal Neurons in a Transgenic Mouse Model of HD
Presentation not available
Don Faber, PhD – Albert Einstein College of Medicine
The proper formation and operation of neuronal circuits depends in part upon the appropriate interplay between different modes of neuronal plasticity, including those serving homeostatic functions. Although homeostasis is most often invoked at the synaptic level, it also pertains to intrinsic excitability, that is, to mechanisms that directly regulate neuronal responsiveness. We describe an activity-dependent homeostatic regulation of repetitive firing present in striatal output neurons (SONs) of Wild Type (WT) mice. Specifically, depolarizing stimuli that initially trigger sustained spike trains progressively become relatively ineffective when repeated at low frequency, due to a build up of accommodation. This is a negative feedback mechanism that scales excitability as a function of the recent level of activity of a neuron, and it is named immediate activity-dependent homeostasis of intrinsic excitability (iADH). Pharmacological and voltage clamp data indicate that this modulation enables increased voltage dependent activation of the M-current, i.e., of KCNQ2/3 channels. M-currents can be modulated via a Ca2+ -dependent phosphorylation pathway and by synthesis/hydrolysis of PIP2, which is Ca2+ -independent. Chelation of intracellular Ca2+ with BAPTA has no effect on iADH, suggesting it is mediated by the latter pathway. Strikingly, iADH is absent in SONs of the R6/2 transgenic mouse model of Huntington’s Disease (HD), at an age when motor or cognitive deficits are just beginning to appear, and the WT phenotype can be rescued in vitro by application of M-current activators. Absence of this homeostatic mechanism in HD might bias the development of striatal networks and facilitate neurodegenerative responses in adults.
Targeting Cognitive Deficits in Schizophrenia
Presentation not available
David Gerber, PhD – Galenea
Schizophrenia is a severe psychiatric disorder that affects approximately 0.7% of the global population. Symptoms of schizophrenia are grouped into three categories: positive symptoms, including hallucinations and delusions; negative symptoms, such as social isolation and flattened affect; and cognitive symptoms, including impaired attention and working memory. These cognitive deficits are a core component of the disease and often prevent schizophrenia patients from functioning in society. Current antipsychotic therapies are not effective at treating the cognitive symptoms of schizophrenia, which remain a major unmet medical need. A key reason for the lack of more effective treatments for schizophrenia is our poor understanding of the molecular mechanisms underlying the disease. Based upon functional and behavioral characterization of the forebrain-specific calcineurin knockout mouse, we have developed a novel disease hypothesis for the cognitive deficits of schizophrenia in which a disruption of synaptic vesicle cycling leads to impairments of the neuronal and network activities in the prefrontal cortex (PFC) that support working memory. To target this disease mechanism for drug discovery, we have developed a synaptic transmission technology platform including two critical components: 1) a high throughput screening technology to directly evaluate compounds for effects on synaptic vesicle cycling in primary neuronal cultures in 96-well plates (MANTRA – Multiwell Automated NeuroTRansmission Assay); and 2) a system for correlating PFC neuronal network activity with specific behavioral parameters using EEG recording in freely behaving mice. Our discovery approach is to define specific alterations in synaptic function associated with disease models, then identify and optimize small molecule modulators that restore normal synaptic function in disease-relevant assays using the MANTRA system and evaluate compound efficacy utilizing objective measures derived from the in vivo EEG system. We are currently applying this approach to target cognitive deficits in schizophrenia, and it can be utilized to address the multiple CNS disorders associated with alterations of synaptic function, including Huntington’s disease.
Prodromal HD: Motor Cortex Excitability and Plasticity Assessed with Transcranial Magnetic Stimulation
Michael Orth, MD, PhD, MD – University of Ulm
In Huntington disease (HD) there is good evidence to indicate early cortical involvement including the motor cortex. The motor cortex is also the main outflow of the motor basal ganglia. Transcranial magnetic stimulation (TMS), a safe, non-invasive and painless tool, examines the excitability of the motor cortex in vivo. This provides insight into the electrophysiological properties of corticospinal neurones and the trans-synaptic regulation of inhibitory and facilitatory circuits within the motor cortex. Three aspects of motor cortex function are of particular interest.
1. Excitability of corticospinal motor neurones. Prodromal and early manifest HD patients have higher resting and active motor cortex thresholds. At rest, recruitment of motor evoked potentials was more gradual in both HD groups. When active, recruitment and the duration of the cortical silent period were similar to normal. However, none of these electrophysiological parameters was associated with the severity of motor signs. Thus, motor-neurones and their modulation by inhibitory inter-neurones, i.e. the quality and shaping of the motor command, may not necessarily change as HD advances from the prodromal to the early manifest stage.
2. Sensory-motor integration. The electrophysiological measure of inhibitory interactions of sensory input and motor output, short-latency sensory afferent inhibition (SAI), was reduced in early manifest but not in prodromal HD. There was an inverse relationship to UHDRS motor scores in line with the known reduction in amplitude of somatosensory evoked potentials in manifest HD. Employing receiver operating characteristics (ROC) using pilot data and a cut-point chosen by hand based on the ROC graph with pi=0.5 (logistic regression) results in a sensitivity of 88% and a specificity of 88%. This suggests that SAI may serve as a biomarker to distinguish prodromal HD far from and near onset.
3. Motor cortex plasticity. This may be abnormal in prodromal and very early manifest HD in a similar way, and the amount of plasticity may not be associated with age, CAG repeat length or UHDRS motor score. This suggests that abnormal motor cortex plasticity is not closely linked to the development of motor signs of HD.
Assessment of Synaptic Dysfunction in Huntington’s Disease – CHDI’s Strategy and Programs
Presentation not yet available
Vahri Beaumont, PhD – CHDI
Aside from the large investment in dedicated programs to decrease mHtt, CHDI has recently aligned its internal strategy for Huntington’s disease modification to immediately focus into 3 key mechanisms thought to be central to Htt function and disease; 1) Autophagy, clearance and folding strategies, 2) Energetics, and 3) Synaptic and Neuronal dysfunction.
Our strategy for this latter mechanistic area will be presented. This encompasses an expansion in the coming years of our support of clinical studies in both premanifest and manifest HD patients, to better define our understanding of the earliest circuitry and neurochemical changes contributing to HD symptomatology, especially with a view to clinical biomarkers. Of equal weight, our parallel effort to further define the nature of synaptic dysfunction in HD models, and to develop context-relevant readouts and assays with higher translational predictive power will be presented, drawing from examples taken from our evaluations of relevant compounds classes within our own internal programs (GLT-1 upregulation, Phosphodiesterase inhibitors and P38 inhibitors), where we have queried whether these may improve synaptic or neuronal dysfunction in HD.
Unraveling the mode of action of pridopidine – From a brain circuitry to a molecular perspective and back
Presentation not available
Nicholas Waters, PhD – NeuroSearch
Given the understanding of motor and mental disorders, including HD, arising as a consequence of a dysfunctioning cortico-subcortical circuitry, it was recognised that a systems biology based drug discovery programme could be tailored to efficiently capture perturbations and pharmacological effects in this circuitry. The discovery of the dopaminergic stabilizer pridopidine stems from such an approach, relying on assessment of biochemical and behavioural response profiles in vivo. To detect potential effects on psychomotor hypo- and hyperfunction, effects on locomotor activity and quality were investigated in rodent models covering hypo- as well as hyperactivity. Neurochemical and gene expression markers were assessed in key parts of the cortico-subcortical circuitry. Pridopidine was found to have the unique properties of leaving the normal, healthy behaviours unaffected, while reducing excessive activity and involuntary movements in different settings of circuitry perturbation, but enhancing activity in a hypoactive state. The biomarker response profile was found to suggest dopaminergic systems as a major mediator of these effects, hence the term “dopaminergic stabilizer” was coined. At the molecular level more recent functional in vitro investigations have demonstrated that pridopidine acts as a competitive, full antagonist with fast dissociation kinetics at dopamine D2 receptors. Yet, this does not appear to fully explain the in vivo effect profile. Other compounds with similar in vitro profile do not generally display the stabilizer profile. Going back to the in vivo neuro-circuitry level, pridopidine has also been found to affect glutamatergic transmission, in a way that suggests that as a net effect it strengthens cortico-striatal connectivity and synaptic glutmatergic transmission.
Taken together, the balancing effects on motor function through the dopamine system and the putative strengthening of cortico-striatal synaptic glutamate mediated signalling suggest that pridopidine affects the core neuronal pathways disrupted in HD, providing a circuitry-level mechanistic rationale for the effects observed in clinical trials with pridopidine.
Mitochondrial and Bioenergetic Dysfunction in Huntington’s Disease: Overview
Timothy Greenamyre, MD, PhD – University of Pittsburgh Medical Center
Huntington’s disease (HD) is caused by an expansion of CAG triplet repeats in the gene encoding huntingtin protein; however, the mechanism by which the altered protein exerts its pleiotropic and deleterious effects remains elusive. Several lines of evidence support the notion that abnormal energy metabolism and mitochondrial dysfunction may be responsible for much of the observed HD pathophysiology. In this talk, I will present an overview of mitochondrial and bioenergetic abnormalities including altered calcium handling, changes in mitochondrial structure and dynamics, and transcriptional dysregulation, which are all thought to contribute to neurodegeneration and neuronal loss in in vitro and in vivo HD models. Furthermore, I will provide current evidence in support of metabolic disturbances in HD patients, and an overview of recent HD clinical trials using the bioenergetic agents. Understanding huntingtin-induced perturbations to energy homeostasis and free radical production should provide greater validation for this mechanism and a more defined set of targets for therapeutic intervention.
Evaluating Regional Cerebral Dysfunction and Bioenergetics by Magnetic Resonance Spectroscopic Imaging
Hoby Hetherington, MSc, PhD – Yale University, School of Medicine
Magnetic Resonance Spectroscopic Imaging (MRSI) is a valuable tool in evaluating neuronal injury and dysfunction in a variety of pathologies including epilepsy, multiple sclerosis and Alzheimer’s disease. Measurements of N-acetyl aspartate (NAA), by 1H MRSI have demonstrated substantial reductions in these patient groups. Through studies involving multiple research groups it is clear that: 1) NAA is primarily localized to neurons and synthesized in neuronal mitochondria; 2) reductions in NAA are not due solely to neuronal loss, but do reflect measures of neuronal injury, which occur prior to neuronal loss in animals models of epilepsy and are reversible MS and epilepsy; 3) NAA reductions do reflect true neuronal dysfunction and are correlated with reductions in cognitive performance in epilepsy, MS and AD; 4) in healthy control subjects NAA levels are linearly correlated with ADP suggesting that NAA levels are a response to energetic demands and 5) in mitochondrial preparations NAA and ATP synthesis rates are linearly correlated and sensitive to mitochondrial poisons. Taken together this supports the role of NAA as an early marker for neuronal bioenergetic defects. Although NAA provides a highly sensitive surrogate marker for the presence of impaired neuronal function and bioenergetics, it is an indirect measure of cerebral bioenergetics and has been hypothesized to be sensitive to other physiologic perturbations including osmotic perturbations. Direct measurements of cerebral bioenergetics can be obtained using 31P MRSI. Although its lower intrinsic sensitivity degrades the achievable spatial resolution (6cc v 0.5cc for 1H), measurements of phosphocreatine, ATP and inorganic phosphate have been used to demonstrate the presence of energetic impairment in epilepsy and its reversibility with effective medical interventions. These regional measures have been correlated with changes on a cellular level including glial proliferation, and a variety of measures of hyper excitability in resected epileptogenic tissue. Application of these methods to HD in animal models and patients is relatively straight forwards, with the caveat that spectral quality from regions about the basal ganglia are often degraded in the human brain due to susceptibility artifacts arising from air-tissue interfaces. Fortunately, a recent advance in shim hardware design has overcome this limitation.
What Can Be Learned from Another Neurodegenerative Disease: Mitochondrial Dynamics Impairment in Parkinson’s Disease
Sarah B. Berman, MD, PhD – University of Pittsburgh
Mitochondrial dysfunction is increasingly linked to both Huntington’s disease (HD) and Parkinson’s disease (PD). There are many similarities in links to impaired mitochondrial function in HD and PD, including evidence for a role of oxidative stress, bioenergetic dysfunction, alterations in mitochondrial biogenesis regulation, fission, fusion, and trafficking, all important components of mitochondrial homeostasis. I will present an overview of mitochondrial impairment in PD, including involvement of oxidative stress, changes in mitochondrial dynamics and quality control (fission, fusion, trafficking, biogenesis and degradation through mitophagy) and mitochondrial DNA damage. I will discuss our ongoing research into the role of mitochondrial homeostasis, dynamics and bioenergetics in Parkinson’s disease and how this might be relevant in furthering understanding of neuropathology and neuroprotection in HD.
Optimization of Brain-penetrant Isozyme Selective PDHK Inhibitors as Potential Therapeutic Agents for HD
Leticia Toledo-Sherman, PhD – CHDI
Huntington’s disease (HD) is associated with impaired energy metabolism. The activity of the pyruvate dehydrogenase complex (PDHC), a key component of the energetic-metabolic machinery of cells, has been found to be reduced in R6/2 mice at 12 weeks of age and in the caudate of human HD postmortem brains, with the most severe deficits correlating with disease progression. The PDHC is composed of three key enzymes, PDH (E12E12), E2 and E3, and an auxiliary binding protein, E3bp. This complex catalyses the conversion of pyruvate to acetyl coenzyme A, an essential molecule in many biological processes, including, fatty acid metabolism, cholesterol biosynthesis, histone acetylation, and the synthesis of the neurotransmitter acetylcholine. The PDHC is tightly regulated by two enzymes, pyruvate dehydrogenase kinase (PDHK), which down-regulates its activity via phosphorylation at any of three serine residues on the PDH component of the enzyme complex, and pyruvate dehydrogenase phosphatase, which dephosphorylates those residues resulting in activation of PDHC. Thus, inhibition of PDHK, and consequently up-regulation and restoration of PDHC activity, could be a promising therapeutic intervention in HD.
Evidence in support of this therapeutic strategy has been demonstrated with DCA, an inhibitor of PDHKs that stimulates the activity of the PDHC; treatment with DCA had beneficial effects in the R6/2 mice, showing significant increase in survival, improved motor function, delayed loss of body weight, and attenuation in the development of striatal neuron atrophy. However, given DCA’s poor drug profile, it is not a feasible molecule to prosecute as a therapeutic agent for HD. Fortuitously, there are safe PDHK inhibitors known, such as Nov3r and AZD7545, but these are not optimal CNS agents. Neither AZD7545 nor Nov3r are selective against PDHK1 and only Nov3r penetrates the brain. Thus, is desirable to obtain compounds which are not only brain penetrant, but also selective for PDHK2/3, the predominant neuronal isoforms of PDHK, and thus have a good safety-efficacy window. Taking these latter agents as benchmarks, CHDI in collaboration with AMRI has initiated a lead optimization program to deliver brain penetrant selective inhibitors of PDHK2/3 for proof-of-concept studies in HD. The team has developed, validated and recently implemented biochemical assays for PDHK1-4 isoforms, as well as a functional cell-based assay. To attain blood brain barrier permeability, ADME profiles and Pharmacokinetic analysis of the benchmark compounds has been used to identify the liabilities of these compounds and the physical properties that need to be optimized. Similarly, to attain selectivity, a thorough structural analysis of the crystal structures of all PDHK isosymes has been performed and molecular modeling methods are being applied to design novel compounds. Even though, this program is in early stages the team has already obtained novel compounds with inhibitory activities similar or better than the benchmark compounds that also display promising selectivity profiles. Progress on this program will be presented at the meeting.
Molecular Approaches to Drug Receptors: Relevance to HD
Solomon H. Snyder, MD – Johns Hopkins Medical School
Except for enzyme inhibitors, molecular strategies for drug development, especially in the neurosciences, were quite limited till the 1970s. Test tube monitoring of neurotransmitter uptake permitted advances, such as discriminating norepinephrine and serotonin uptake. Reversible ligand binding, exemplified in opiate receptors and then receptors for most neurotransmitters, led to an explication of many psychotropic drugs’ therapeutic and adverse actions. Similar approaches elucidated second messengers such as IP3 receptors. For HD, a major challenge has been to understand the selective striatal damage, despite the ubiquitous localizations of huntingtin. Binding of the small G protein Rhes, which is selectively enriched in the striatum, to mutant huntingtin (mHtt) mediates its sumoylation. Sumoylation disaggregates mHtt leading to augmented neurotoxicity. The Rhes-mHtt interaction may explain the striatal selectivity of HD neuropathology (Subramaniam et al Science 324: 1327, 2009). One might screen for drugs that selectively block Rhes-mHtt binding and thus could delay HD symptom onset. As Rhes is striatal selective, the risk of adverse effects for such drugs would be minimized. Rhes, like the G protein Rheb, activates mTOR and is the principal stimulus to striatal protein translation. Sequestration of Rhes by mHtt might diminish its access to mTOR, accounting for striatal shrinkage in HD.
Upstream and Downstream of Trk Receptors
Moses Chao, PhD – New York University School of Medicine
Neurotrophins are potent trophic factors that were first characterized for their ability to regulate neuronal growth, survival and differentiation. There are many lines of evidence indicating that neurotrophins play an important role in the pathophysiology of depression, anxiety and other psychiatric disorders, as well as a wide number of neurodegenerative disorders. BDNF is a key component in the etiology of Huntington’s diseases. We are interested in the role of the BDNF TrkB receptor signaling in dictating neuronal responsiveness following activity-dependent events. We have identified several new adaptor proteins and enzymatic activities downstream of Trk receptors that influence changes in axon and dendritic growth and synaptic transmission and plasticity. They include MAP kinase phosphatase, the Nedd4-2 E3 ligase, GIPC, Hrs/STAM and the scaffold protein, ARMS/Kidins220. We have also identified small molecule agonists that promote neurotrophin signaling through transactivation of Trk receptors. For example, activation of Trk tyrosine kinase receptors occurs after treatment with adenosine, which acts through the A2A adenosine receptor. This signaling mechanism offers the opportunity to identify biochemical events and pathways that promote the actions of neurotrophin signaling at a molecular and synaptic level. Because of numerous difficulties in using neurotrophins clinically due to problems in delivery and unwanted side effects, these approaches also raise the possibility that small molecules may be used to elicit neurotrophic effects for the treatment of psychiatric and neurodegenerative diseases.
New Strategies to Deliver BDNF in Huntington’s Disease Models
Jordi Alberch, MD, PhD – University of Barcelona
Brain-derived neurotrophic factor (BDNF) is an important candidate for neuroprotective therapeutic strategies for Huntington’s disease (HD), because of its trophic effects on cortical and striatal neurons. However, the administration system and the control over the dosage are still important problems to be solved. Conditional systems to deliver BDNF are necessary to avoid the adverse effects that can be produced by chronic high doses of BDNF. Here, we propose a conditional system that only releases BDNF under pathological conditions. We used transgenic mice overexpressing BDNF under the promoter of the glial fibrillary acidic protein (GFAP) (pGFAP-BDNF mice). These mice are viable and have a normal phenotype. However, intrastriatal administration of quinolinate increased the number of reactive astrocytes and enhanced the release of BDNF in pGFAP-BDNF mice compared with wild-type mice. This increase in BDNF made pGFAP-BDNF mice more resistant to quinolinate, suggesting a protective effect of astrocyte-derived BDNF.
On the other hand, pGFAP-BDNF mice can also be a good source to obtain astrocytes that release high levels of BDNF under pathological conditions. Mice grafted with pGFAP-BDNF astrocytes showed important and sustained behavioral improvements 30 days after quinolinate administration as compared with mice grafted with wild-type astrocytes.
We also examined whether BDNF release by the GFAP promoter could improve the behavioral and structural deficits in a transgenic mouse model of HD. Thus, we cross-mated pGFAP-BDNF mice with R6/2 mice. In these double mutant mice (R6/2:pG-B mice), we have performed a behavioral, morphological, biochemical and electrophysiological characterization. The levels of BDNF were higher in R6/2:pG-B mice respect to R6/2 mice at 12 weeks of age, when astrogliosis was significantly increased in these transgenic mice. The recovery of BDNF levels improved motor coordination in R6/2:pG-B mice respect to R6/2 measured by the Rotarod. The behavioral improvements observed in the R6/2:pG-B mice were associated with a rescue of neuronal function: increase in striatal and neuronal soma volumes and a lesser loss of dendritic spine density. Altogether indicates that the conditional administration of BDNF under the GFAP promoter could constitute a therapeutic strategy to release BDNF in a HD context.
Supported by: Ministerio de Ciencia e Innovacion and CIBERNED (Spain)
AAV2-neurturin (CERE-120) Gene Transfer for Parkinson’s and Huntington’s Disease
Joao Siffert, MD – Ceregene
CERE-120 is being developed as a potential therapy to improve neuronal function and delay disease progression in Parkinson’s and Huntington’s disease. It is comprised of an AAV2 vector that expresses only human neurturin (NRTN). NRTN exerts potent neurotrophic activity on nigral and striatal neurons that degenerate in PD and HD, respectively.
Over two-dozen nonclinical studies were performed to support CERE-120 as a novel treatment for PD and HD. Collectively, these studies demonstrate that: (1) CERE-120 expresses NRTN in an orderly, dose-response manner, (2) NRTN expression reaches an asymptote at ~1 month, with no further change thereafter; (3) NRTN is potently bioactive, enhancing the function and integrity of degenerating neurons in animal models of both PD and HD, and (3) a wide safety-margin exists, showing no side effects or evidence of toxicity at dose multiples 25-100-times higher than required for efficacy.
Intraputaminal CERE-120 was tested in 50 PD patients, involving an initial open-label Phase-1 and a double-blinded, controlled Phase-2 trial. Both studies supported the safety of CERE-120 (follow-up of 3+ to 5+ years) with some suggestion of efficacy (Marks et al, 2008; Marks et. al, 2010). The efficacy data from the controlled trial were inconsistent, but those results, coupled with insight gained from brain autopsy CERE-120-treated-subjects suggested impairment in retrograde-transport delayed and blunted the neurotrophic response to NRTN in PD (Bartus et al, 2010). This led to a new Phase 1/2b study, employing a new dosing-paradigm which administers CERE-120 to both the degenerating soma in the substantia nigra and their terminals in the putamen. Following confirmation of safety/feasibility in the new Phase 1, the Phase 2b portion is now underway.
Because CERE-120 was injected into the striatum for all PD-related animal and human studies, the extensive safety data provide evidence for safely dosing HD patients. The neuroprotection achieved with CERE-120 following striatal injections in the 3-nitroproprionic acid (3-NP) and the N171-82Q transgenic models of HD provides reason to believe CERE-120 may improve function and integrity of degenerating neurons in HD. Thus, we plan to test the safety of CERE-120 in HD in a Phase-1 clinical trial.
Staying on Trk’s: Pharmacological Intervention Strategies
Alex Kiselyov, PhD – CHDI
Both reduced brain-derived neurotrophic factor (BDNF) expression and trafficking are key factors in Huntington disease (HD) pathology including the degeneration of striatal neurons. BDNF binds to the tyrosine kinase and neurotrophin receptors (TrkB and p75NTR, respectively). Activation of TrkB signaling cascade promotes differentiation and cell survival, whereas p75NTR binding triggers pro-apoptotic message. In addition, modulation of adenosine A2A receptors (A2ARs) affects BDNF/TrkB pathway leading to receptor transactivation and enhanced synaptic transmission. At CHDI, we initiated a multifaceted approach to identify TrkB modulators with relevant in vitro and ex vivo activities that are capable of emulating BDNF activity in a Huntington’s disease model. Representative strategies include: i) targeting TrkBIg2 (receptor dimerization, TrkB-BDNF interface) and other extracellular domains; ii) stabilization of BDNF dimer at the receptor site; iii) inhibition of p75NTR, iv) BDNF-independent activation of TrkB signalling (ex., A2AR activation). We utilize a combination of screening, computational modeling, structural biology and chemistry tools to identify potent, selective and pharmacologically relevant modalities. In this presentation, we will describe our current progress including development of cellular and biophysical (Surface Plasmon Resonance and crystallography) assay platforms, specific pharmacological hypothesis, respective synthetic/medicinal chemistry efforts and summarize further steps towards achieving the goal of the program. A specific emphasis will be placed on identification of small molecule agents that modulate BDNF/TrkB signalling axis.
Introduction – CHDI’s Internal Programs
Robert Pacifici, PhD – CHDI
Kynurenine Pathway Modulation as a Potential Treatment for HD – Advances in the Development of a Selective Brain Penetrant KMO Inhibitor
Ignacio Muñoz-Sanjuan, PhD – CHDI
Dysregulation of the kynurenine pathway has been implicated in a variety of central nervous system disorders, including Huntington’s disease. Several metabolites of kynurenine catabolism are bioactive and can modulate both synaptic and inflammatory processes in adult tissues. In particular, kynurenic acid (KYNA) can act as a neuroprotective agent through its weak antagonistic activity at the NMDA receptor; in addition, KYNA has been shown to modulate synaptic glutamate release and cholinergic signaling, activities which might be of relevance to HD pathophysiology. Other kynurenine metabolites, 3-hydroxy-kynurenine (3HK), and quinolinic acid (QA), have been shown to be neurotoxic in vitro and in vivo. In early stage patients as well as in the R6/2 model of HD, 3HK levels are elevated in a region-specific manner. The enzyme which catalyzes the conversion of kynurenine to 3HK is kynurenine mono-oxygenase (KMO). CHDI, in collaboration with Evotec, has identified and optimized selective inhibitors of KMO. The presentation will cover the status of the program, and describe the challenges associated with the development of molecules with adequate pharmacokinetic properties for in vivo efficacy testing. In addition, CHDI and collaborators have characterized the phenotypes of animal models with decreased activity of KMO in an effort to extend our validation for this therapeutic approach. Finally, the clinical development strategies for the KMO inhibitor program will be discussed, with an emphasis on finding translational endpoints that would allow translation from rodents to humans in a clinical setting.
Histone Deacetylase Class IIa (HDAC4) Inhibitors: Potential Therapeutics for Huntington’s Disease
Celia Dominguez, PhD – CHDI
Histone deacytelases have been implicated as therapeutic targets in various neurodegenerative diseases, including Huntington’s disease. This involvement was largely predicated on observations made using non-selective compounds with poor potency and low brain exposure. CHDI’s strategy has been to confirm and further these intriguing findings by developing novel inhibitors with improved drug-like properties and with an isozyme selectivity profile that would maximize the beneficial effects and minimize untoward side effects. Of the eleven metal-dependent histone deacetylase (HDAC) isoforms our efforts have been trained on HDAC4 based on the finding that a murine genetic cross of a heterozygous knock out of the HDAC4 gene into R6/2 mice resulted in a partially restored phenotype displaying delayed disease progression (Professor Gill Bates, King’s College London). As HDAC4 may carry out multiple biological functions (including an adaptor/scaffolding role), a critical question is whether pharmacological blockage of its catalytic site will recapitulate the genetic observation. CHDI in collaboration with BioFocus, have established the necessary biochemical and cellular tools to support a medicinal chemistry program to develop potent and selective HDAC4 deacetylase inhibitors that exhibit pharmacokinetic profiles suitable for a proof-of-concept study in HD models. This presentation will provide the status of our current inhibitors, the challenges with regards to assessing HDAC4 deacetylase activity in vivo, and the path forward in our goal to identify a potential therapeutic for Huntington’s disease.