February 8, 2011

9:10 AM

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

9:50 AM

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.

11:00 AM

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.

11:40 AM

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.

2:10 PM

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

2:45 PM

A deficit in intrinsic excitability of striatal neurons in a transgenic mouse model of HD

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.

3:35 PM

Targeting cognitive deficits in schizophrenia

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.

4:10 PM

Prodromal HD: Motor cortex excitability and plasticity assessed with transcranial magnetic stimulation

Michael Orth, MD, PhD – 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.

4:45 PM

Assessment of synaptic dysfunction in Huntington’s disease – CHDI’s strategy and programs

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.

5:20 PM

Unraveling the mode of action of pridopidine – From a brain circuitry to a molecular perspective and back

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.