CHDI’s 9th Annual HD Therapeutics Conference took place February 24 – 27, 2014, 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.
- Mouse Genetic Approaches to Discover and Validate Molecular Targets for Huntington’s Disease X. William Yang, MD, PhD
- A Network View of the Pathways Altered by Mutant Huntingtin Ernest Fraenkel, PhD
- Creating a Coherent Huntington’s Disease Dataset for Systems Biology Modeling Jim Rosinski, PhD
- Disease Modification in Huntington’s Disease Due to Naturally Occurring Genetic Variants Jim Gusella, PhD
- A Prospective Pilot Trial for Pallidal Deep Brain Stimulation in Huntington’s Disease Jan Vesper, MD
- Myostatin Inhibition as a Novel Approach to Targeting the Peripheral Pathology of HD Gill Bates, PhD
- Circadian Disruptions in Huntington’s Disease: Mechanisms and Possible Treatment Options Chris Colwell, PhD
- Dysregulation of Classical Complement Cascade in HD: Molecular Mechanisms and Therapeutic Strategies Beth Stevens, PhD
- Huntington’s Disease Model Mice Exhibit Astrocyte Kir4.1 Ion Channel Dysfunction that Contributes to Medium Spiny Neuron Excitability Baljit S. Khakh, PhD
- Effects of Huntingtin Levels and Mutation on Synaptic Function in a Cortical-striatal Co-culture System Lynn Raymond, MD, PhD
- Disrupted Autonomous Activity of Subthalamic Nucleus Neurons: A Biomarker of Excessive NMDA Receptor Activation in Mouse Models of Huntington’s and Parkinson’s Disease? Mark Bevan, PhD
- Basal Ganglia Circuitry and Huntington’s Disease Anton Reiner, PhD
- Corticostriatal Circuit Dysfunction in Behaving Transgenic Mouse Models of Huntington’s Disease George Rebec, PhD
- Mutant Huntingtin Quantification in Human Body Fluids Andreas Weiss, PhD
- Translational Kinetic Biomarkers to Enable the Development of HTT-lowering Therapies Patrizia Fanara, PhD
- The EEG is a Sensitive Biomarker for the Presence and Progression of Disease in HD Mouse Models Stephen Morairty, PhD
- Diagnosing Dysfunction: NAD Redox Reveals Mitochondrial Changes in HD in vivo Kevin Conley, PhD
- The Use of Multi-modal Imaging to Discovery Sensitive Neuorimaging Biomarkers for Clinical Trials in Huntington’s Disease: The Melbourne Based IMAGE-HD Study Nellie Georgiou-Karistianis, PhD
- Positron Emission Tomography Imaging of Phosphodiesterase 10A (PDE10A) Enzyme and Dopamine D2 Receptors in Huntington’s Disease Gene Expansion Carriers Andrea Varrone, MD, PhD
- Sleep and Metabolic Abnormalities in Pre-manifest Patients with Huntington’s Disease Alpar Lazar, PhD
- Neuroendocrine Study of Huntington’s Disease Tom Warner, PhD
Mouse Genetic Approaches to Discover and Validate Molecular Targets for Huntington’s Disease
Presentation not yet available
X. William Yang, MD, PhD – University of California, Los Angeles
To address the complex question of how a polyglutamine expansion near the N-terminus of human mutant Huntingtin (mHTT) elicits age-dependent behavioral deficits and selective cortical and striatal neurodegeneration in Huntington’s disease (HD), we have developed a series of conditional Bacterial Artificial Chromosome transgenic mouse models of HD (BACHD) that express human mHTT under its own genomic regulation. BACHD mice exhibit progressive behavioral deficits with late-onset selective cortical and striatal atrophy. Using these novel HD mouse models, we have investigated both neuronal and molecular targets that are either correlated with, or causally linked to, diseases process in vivo. In particular, I will highlight our studies demonstrating the pivotal disease-modifying roles of the N-terminus 17 amino acid domain (N17) of mHTT and new evidence suggesting the targeting of DNA damage signaling pathway for the amelioration of mHTT toxicities in HD neurons and mice.
A Network View of the Pathways Altered by Mutant Huntingtin
Presentation not yet available
Ernest Fraenkel, PhD – Massachusetts Institute of Technology
We recently demonstrated that mutant huntingtin induces large changes in the epigenome, including alterations in DNA methylation and in methylation of histone H3 lysine 4. Importantly, we found that the changes were gene-specific, suggesting a regulated process that causes gain or loss of specific epigenomic modifiers at targeted loci. To search for therapeutic strategies, we are using computational and experimental methods to identify potential regulators. Together with collaborators, we have determined that reducing the levels of the H3K4 demethylase SMCX/Jarid1c in primary neurons can reverse some of the HD-induced expression changes. We have also identified transcriptional regulatory proteins that bind near the sites of epigenetic modifications. Through network modeling, we are able to link epigenomic changes to other molecular processes detected through complementary “omic” methods.
Creating a Coherent Huntington’s Disease Dataset for Systems Biology Modeling
Jim Rosinski, PhD – CHDI
Over the past two years, CHDI has undertaken the building of a comprehensive, coherent, genomic dataset to examine the relationship between CAG repeat length and the motoric age of onset of Huntington’s disease. This dataset, based on a series of mouse knock-in models covering a range of CAG lengths (Q20, 50, 80, 92, 111, 140, and 175), will include RNA, proteomic, epigenomic, electrophysiology and behavioral profiling of the animals across a large number of tissues at two, six and ten months of age. The RNA data has now become available and our analysis of it has progressed to the point that we look to open up the discussion to the HD community. Our hope is that the datasets, as they grow, will be a key piece in untangling the molecular mechanisms underlying the CAG and age dependence of the disease, and thereby highlight effective therapeutic avenues.
We will show the initial results and findings of our mRNA, micro RNA and behavioral profiling of the six month old animals. We hope these results will convince the audience that this dataset is capturing key known elements of HD molecular biology as well as uncovering novel mechanisms and relationships. We will also discuss the planned timeline going forward for completing the profiling work and beginning the computational modeling of the dataset. Finally, we will describe our plan for disseminating this dataset to the global HD community.
The dataset may be found at www.chdifoundation.org/datasets
Disease Modification in Huntington’s Disease Due to Naturally Occurring Genetic Variants
Presentation not yet available
Jim Gusella, PhD – Massachusetts General Hospital
In Huntington’s disease (HD), the primary determinant of the rate of the pathogenic process that leads to clinical diagnosis is the length of the CAG expansion mutation in HTT. However, while the effect of the genetic defect predominates, it does not explain all of the variation that HD subjects display in either age at clinical diagnosis or in other disease-associated phenotypes. Some of the remaining variation is likely to be due to naturally occurring genetic variations in the human genome that, through their effects on genes other than HTT, act to modify disease expression. Identifying these variants could yield evidence for mechanisms capable of altering the course of HD in human patients. Consequently, the GeM Consortium has pursued an unbiased strategy to locate chromosomal regions that harbor genetic modifiers of HD. By genotyping ~3500 HD subjects from the collections of the MGH HD research team and from the European Huntington’s Disease Network’s Registry with 2.5 million single-nucleotide polymorphisms (SNPs), the GeM Consortium has carried out a genome-wide association study (GWAS) of age at motor diagnosis. The results of this analysis are being combined with two smaller independent HD GWAS datasets to provide the most powerful and rigorous investigation of HD genetic modifiers performed to date. Genetic variants that show statistically significant association with the residual of age of motor onset (i.e., with the difference between observed onset age and expected onset age based upon the individual’s CAG repeat length) provide robust evidence that a chromosomal region contains functional DNA sequence variation. In those who possess it, the impact of the functional variant(s) alters the rate of HD pathogenesis that leads to motor onset, through altering the regulation or expression of one or more genes in the vicinity or by some other mechanism. Consequently, bona fide genetic modifiers of HD, identified from the information provided by HD patients and their genomes can point to genes, and to the biochemical pathways in which they participate, as capable of disease modification and therefore as validated for potential development of rational therapies for HD.
A Prospective Pilot Trial for Pallidal Deep Brain Stimulation in Huntington´s Disease
Jan Vesper, MD – University of Dusseldorf
Severe forms of movement disorders in Huntington´s disease are often medially refractive. Case reports suggest deep brain stimulation as treatment option, but the exact target for stimulation is under debate. We assessed procedure safety, equality of internal and external pallidal stimulation and efficacy in patients with Huntington´s disease followed up for 6 months in a prospective pilot trial.
We studied 6 Huntington´s Disease patients (4 chorea-dominant, 2 Westphal variant) with stable predominant movement disorder. After surgery, patients were randomly assigned either the sequence of 6 week internal/6 week external pallidum stimulation or vice versa followed by 6 months chronic pallidal stimulation. Primary endpoints were changes in the Unified Huntington´s Disease Rating Scale (UHDRS) Motor Score, Chorea Subscore and Total Motor Score 4 (TMS-4) comparing internal versus external pallidum stimulation and 6 month versus baseline. Secondary endpoints assessed scores on dystonia, hypokinesia, cognition, mood, functionability/disability and quality of life. Scores were rated double-blinded using standardized video recordings. Data was analyzed on intention-to-treat basis. The trial is registered with ClinicalTrials.gov (NCT00902889).
Intention to treat analysis included all 6 patients with n=3 in each treatment sequence. Internal and external pallidal stimulation was equally in terms of efficacy. Chorea sub score decreased significantly within 6 months (-5.3 (60.2%), p=0.037). Effects on dystonia were not significant over the group due to three responders (>50% improvement) and three non-responders. Westphal patients did not improve. Cognition was stable over 6 months. Mood and several functionability/disability and quality of life scores improved significantly. Eight adverse events and two additional serious adverse events were recorded. Most of them were stimulation related and under internal pallidum stimulation with bradykinesia, gait impairment, hyperkinesia and hyperthermia as the most common ones. All adverse events resolved without sequelae. No procedure related complication occurred.
Internal as well external pallidal deep brain stimulation occurred as safe treatment option for reduction of chorea in Huntington´s Disease. Effects on chorea, dystonia and long-term effect on quality of life should be examined in further larger trials.
Myostatin Inhibition as a Novel Approach to Targeting the Peripheral Pathology of HD
Presentation not yet available
Gill Bates, PhD – King’s College London
Muscle atrophy is a well-documented symptom of Huntington’s disease (HD) and there are multiple lines of evidence to support a muscle-based pathology in HD patients and in mouse models of HD. Inhibition of the myostatin signaling pathway has been shown to increase muscle mass and therapeutic approaches, which include the use of an ActRIIB receptor decoy and antibodies directed at the myostatin ligand, are in clinical development for a number of indications.
We have used an ActRIIB receptor decoy to test the effects of myostatin inhibition in the R6/2 mouse model of HD. We found that weekly administration from five weeks of age completely rescued the body weight loss, reduction in muscle mass (quadriceps, tibialis and gastrocnemius) and grip strength impairment during the expected life span of an R6/2 mouse and delayed end-stage disease by approximately 2 weeks. Treatment had no effect on rotarod performance or open field deficits suggesting that these behavioral phenotypes arise through centrally located pathogenic mechanisms. We have assessed neuromuscular function by determining the maximum twitch and tetanic force in the tibialis anterior and extensor digitorum longus (EDL) muscles and shown that treatment with the receptor decoy results in considerable restoration of the corresponding impairments. Similarly, the loss of motor units was completely restored in EDL muscles.
The R6/2 and HdhQ150 knock-in mice both develop a skeletal muscle-based aggregate pathology in the form of nuclear inclusions. Analysis by the Seprion ELISA indicates that the aggregate load is greater in tibialis as compared to quadriceps or gastrocnemius, and that this is reduced by approximately 50% at 12 weeks of age in all three muscle types in treated mice. We have quantified the expression level of a wide range of transcripts and whilst in the case of many genes, the level of dysregulation is less pronounced in the treated as compared to untreated mice, the pattern of dysregulation is complex and can differ between muscle types. Taken together, these molecular data suggest that ActRIIB receptor decoy treatment may have had disease modifying effects in skeletal muscle.
Knowledge of the safety and tolerability of the various myostatin inhibition modalities that are in clinical trials will drive future preclinical work to evaluate this as a potential therapeutic target for HD.
This work was funded by the CHDI Foundation.
Circadian Disruptions in Huntington’s Disease: Mechanisms and Possible Treatment Options
Chris Colwell, PhD – University of California, Los Angeles
Sleep and circadian disruptions are commonly reported by patients with neurodegenerative diseases, suggesting they are an endophenotype for degenerative diseases of the nervous system. For Huntington’s disease (HD) patients, sleep disruptions occur early in disease progression and are recapitulated in several mouse models, including the R6/2, Q175, and BACHD lines. To better understand these changes, we used an integrative approach to describe age-related dysfunction in the circadian timing system of the Q175 and BACHD lines. Consistent with impaired rhythms in autonomic tone, we observed that heart-rates and blood pressure in a subset of BACHD mice failed to appropriately decrease during sleep. In humans, this non-dipping hypertension is associated with poorer health outcomes and increased mortality. Cells in the suprachiasmatic nucleus (SCN) generate circadian rhythms in physiology throughout the body, including rhythmic regulation of autonomic nervous system function. The hallmark feature of the SCN network is that this population of neurons exhibits a robust, resilient, and precise rhythm of electrical activity. In the BACHD line, we see clear evidence for the loss of SCN neural activity rhythms and are presently working on identifying the underlying ionic mechanisms. In summary, we’ve found clear evidence of pathophysiology in the BACHD line, but as of yet, have not found evidence of pathology or alterations in the circadian gene expression rhythms in the SCN, although our search is ongoing.
Weakened output from the SCN would be expected to alter the timing of circadian clocks throughout the body. Previous work has shown disruptions and misalignment of the circadian system impact cognition, cardiovascular function, metabolism, and the immune system. HD patients commonly experience this symptom cluster, raising the possibility that stabilizing their circadian system may improve mental/physical health and potentially even delay disease progression. Currently we are examining the potential therapeutic impact of scheduled exposure to blue-light, exercise and feeding to enhance the temporal structure of a HD day. These simple means could potentially counteract the effects of declining SCN output to improve quality of life and reduce health care costs for the HD population, and potentially more broadly for a host of neurodegenerative disease patients.
Dysregulation of Classical Complement Cascade in HD: Molecular Mechanisms and Therapeutic Strategies
Presentation not yet available
Beth Stevens, PhD – Boston Children’s Hospital
Growing evidence suggests that a hallmark of age-dependent neurodegenerative diseases, including HD, is early neuronal dysfunction and loss of CNS synapses, followed by progressive neurodegeneration. Our recent research supports an unexpected role of the complement system in synaptic pruning during development and disease. During development complement proteins C1q and C3 localize to synapses and mediate synapse elimination. Our recent studies support a model in which “weaker” or less active synapses are “tagged” by complement and then eliminated by microglia, the primary phagocytic immune cells in CNS that express phagocytic complement receptors (i.e. CR3/Cd11b). Complement up-regulation and microglial activation have been observed in human HD brain tissue and several HD mouse models. We propose that similar complement and microglia-dependent mechanisms contribute to synapse loss, which in turn is crucial to pathogenesis of synaptic and behavioral deficits and selective neurodegeneration in HD. Our preliminary data reveal complement factors are significantly up-regulated and target cortico-striatal in the vulnerable brain regions (i.e. striatum) with known synapse loss in two HD genetic mouse models. I will present emerging evidence that genetic ablation of specific classical complement cascade components protect against neuronal and synapse loss in several disease models. Elucidating the mechanisms underlying and validating the disease impact of complement factors in independent HD mouse models a providing key insight into development of novel therapeutic strategies for HD and related age-dependent neurodegenerative disorders.
Huntington’s Disease Model Mice Exhibit Astrocyte Kir4.1 Ion Channel Dysfunction that Contributes to Medium Spiny Neuron Excitability
Baljit S. Khakh, PhD – University of California, Los Angeles
Huntington’s disease (HD) is characterized by striatal medium spiny neuron (MSN) dysfunction, but underlying mechanisms remain unclear. We explored roles for astrocytes, which display mutant huntingtin in HD patients and mouse models. We found that symptom onset in R6/2 and Q175 HD mouse models is not associated with classical astrogliosis, but is associated with decreased Kir4.1 K+ channel functional expression, leading to elevated in vivo levels of striatal extracellular K+, which increased MSN excitability in vitro. Viral delivery of Kir4.1 channels to striatal astrocytes restored Kir4.1 function, normalized extracellular K+, recovered aspects of MSN dysfunction, prolonged survival and attenuated some motor phenotypes in R6/2 mice. These findings indicate that components of altered MSN excitability in HD may be caused by heretofore unknown disturbances of astrocyte-mediated K+ homeostasis, revealing Kir4.1 channels as potentially novel therapeutic targets.
Effects of Huntingtin Levels and Mutation on Synaptic Function in a Cortical-striatal Co-culture System
Lynn Raymond, MD, PhD – University of British Columbia
Several animal models of Huntington’s disease (HD) show altered cortical-striatal (C-S) presynaptic glutamate release, glutamate uptake and trafficking/signaling of postsynaptic glutamate receptors. Synaptic changes precede the HD-like motor phenotype, and synaptic proteins form interaction hubs with wild-type huntingtin (Htt). Therapy to ameliorate functional deficits in neuronal circuitry may therefore delay HD onset and provide neuroprotection. Here, we aimed to characterize the “signature” changes in C-S synaptic transmission and signaling to the prosurvival transcriptional regulator – phospho- cyclic-AMP response element-binding protein (pCREB) – associated with mutant Htt expression in a reduced model system, the C-S co-culture, with the goal of using the model as a platform for testing therapeutics. Moreover, since the first-line strategy for treating HD may be Htt lowering therapies, we also investigated the impact of Htt expression levels on C-S synaptic function. We compared recordings in wild-type (FVB/N), YAC18 and YAC128 embryonic mouse C-S cultures. Alterations in synaptic function were tracked over the course of synaptic development and maturation in neuronal co-culture from day in vitro (DIV) 7 to DIV 21, using whole-cell patch clamp recording from GFP-labeled striatal neurons. We found no difference in basic membrane properties or inhibitory synaptic activity among the genotypes up to DIV 21. In addition, the amplitude of synaptic AMPA- and NMDA-type glutamate receptor-mediated current was similar across the genotypes. However, the current mediated by extrasynaptic NMDA receptors was increased in YAC128 striatal neurons as early as DIV 14, in agreement with studies in acute cortical-striatal brain slice from 4-week old YAC128 mice. Strikingly, although excitatory synaptic development progressed normally from DIV 7 through 14, the frequency of miniature excitatory postsynaptic currents (mEPSCs) was profoundly reduced at DIV 21 in YAC128 striatal neurons. Furthermore, pilot experiments suggest a distinct pattern for each genotype of nuclear CREB activation in response to chemical synaptic stimulation or BDNF. Finally, we found that increasing or decreasing levels of wild-type Htt resulted in an increase or decrease, respectively, of synaptic PSD-95 – a key synaptic scaffold for glutamate receptors and a hub for Htt protein-protein interactions in the postsynaptic compartment. Together, these results reveal the earliest synaptic changes, intrinsic to cortical-striatal synapses in the absence of other modulatory neurotransmitters or circuitry, that are mediated by mutant Htt expression or altered wild-type Htt levels. This simple model system offers a novel platform for testing therapy to ameliorate synaptic dysfunction.
Supported by CHDI and Canadian Institutes of Health Research
Disrupted Autonomous Activity of Subthalamic Nucleus Neurons: A Biomarker of Excessive NMDA Receptor Activation in Mouse Models of Huntington’s and Parkinson’s Disease?
Presentation not yet available
Mark Bevan, PhD – Northwestern University
The subthalamic nucleus (STN) is a key element of cortico-basal ganglia thalamocortical circuitry but its properties have not been thoroughly characterized in Huntington’s disease (HD) mouse models. Thus, the intrinsic and synaptic properties of STN neurons in BACHD and wild type (WT) mice were compared at pre-symptomatic (ex vivo brain slices.
At 6 months (but not at ATP channel blockade rescued firing in BACHD but had little effect in WT. Disrupted firing was caused by membrane hyperpolarization consistent with a K+ channel conductance increase. However, the responses of STN neurons to depolarizing/hyperpolarizing current injection in BACHD resembled WT, implying a similar complement of voltage-dependent ion channels. The frequency of mEPSCs in BACHD was significantly greater at
Given evidence of augmented NMDAR activation in BACHD, WT STN slices were pre-incubated in NMDA prior to recording. NMDA pre-incubation produced a persistent KATP channel-dependent disruption of autonomous activity. Preliminary observations suggest that nitric oxide (NO)-guanylate cyclase signaling is necessary but not sufficient for disrupted firing: a) inhibition of NO synthase rescued autonomous activity in BACHD; b) application of a NO donor inhibited autonomous activity in WT in a KATP channel-dependent (but relatively transient) manner. Interestingly, chronic dopamine depletion in the unilateral 6-hydroxydopamine mouse model of Parkinson’s disease (PD) also produced a KATP channel-dependent disruption of autonomous STN activity, which was prevented by knockout of STN NMDARs in vivo. Together our data suggest that disrupted autonomous firing may result from excessive NMDAR activation in the STN in both HD and PD models.
Basal Ganglia Circuitry and Huntington’s Disease
Anton Reiner, PhD – University of Tennessee Health Science Center
Huntington’s disease symptoms are in large part a consequence of degenerative changes in the cortex, thalamus, and striatum, and in all likelihood in cortical and thalamic input to striatum. The cortex accounts for about 65% of the input to spines of striatal projection neurons, and the thalamus for about 35%. We have found that indirect pathway striatal projection neurons (iSPNs) are more vulnerable in human HD than are direct pathway striatal projection neurons (dSPNs), which helps explain the early hyperkinesia and late hypokinesia in HD. Our studies in R6/2 mice with the mGluR2/3 agonist LY379268 have shown the benefit of presynaptic modulation of corticostriatal terminals in slowing pathogenesis. In particular, daily LY379268 restored corticostriatal BDNF, which particularly benefitted iSPN survival. In our work in presymptomatic Q140 HD knock-in mice, we found that loss of thalamostriatal and corticostriatal terminals precedes SPN loss. Given the extensive cortical and striatal white matter loss in presymptomatic HD, it seems likely that loss of thalamostriatal and corticostriatal terminals also occurs in human HD and contributes to the slight deficits seen in presymptomatic HD. Although thalamostriatal terminal loss occurs equally from dSPN and iSPN spines in Q140 mice, corticostriatal terminal loss from dSPN spines greatly exceeds that from iSPN spines. We have found that the corticostriatal loss from dSPNs is in smaller axospinous terminals, which arise from intratelencephalically projecting (IT) type cortical neurons of upper layer 5 involved in motor planning, and from which dSPNs preferentially get their cortical input. The pruning of IT-type input from dSPNs is correlated with a slight hypokinesia we see in these mice. Moreover, the lowered potential for excitotoxic injury attending loss of IT-type cortical input may contribute to the lesser vulnerability of dSPNs in HD. Conversely, a dependence of iSPNs on BDNF from their preferential cortical input from pyramidal tract (PT) neurons of deep layer 5 may contribute to their early vulnerability in HD. Finally, we have recently shown that fast-spiking GABAergic interneurons, whose feedforward inhibition of SPNs is important for refining striatal motor output, are lost in mid-HD, which may contribute to the dystonia that becomes evident in mid-HD.
Corticostriatal Circuit Dysfunction in Behaving Transgenic Mouse Models of Huntington’s Disease
George Rebec, PhD – Indiana University
Across a wide range of transgenic mouse models of Huntington’s disease (HD), early signs of neuronal dysfunction occur in both cerebral cortex and striatum. These signs appear even before the onset of a robust behavioral phenotype and in the absence of overt movement. Growing evidence suggests that a failure of communication between cortical and striatal neurons sets the stage for subsequent neuropathology and may involve dysregulation of glutamate transmission mediated in part by an action of the mutant huntingtin gene (mhtt) on the mechanisms underlying glutamate transport. Here, we assessed whether abnormalities in behavioral modulation of striatal signaling can be explained by the presence of mhtt in striatal neurons alone, a cell-autonomous process, or by the interaction of mhtt in both cortical and striatal neurons. We used the BAC-Emx1Cre (BE) conditional HD transgenic mouse model, in which mhtt is suppressed in forebrain glutamate projecting neurons including the cortical pyramidal neurons that project to striatum. We recorded neuronal activity simultaneously in primary motor cortex (M1) and dorsal striatum, which receives M1 input, while the animals behaved in an open-field arena and plus maze. Neurobehavioral data also were obtained from BACHD mice, a transgenic model that expresses full-length mhtt, and wild-type strain controls. Animals ranged in age between 15 and 60 weeks, which for BACHD mice includes both pre-symptomatic and symptomatic periods. In striatum, relative to wild-type, BACHD mice showed changes in neural activity similar to those reported for other HD models including changes in the firing pattern of individual neurons and in the power spectral density of local field potentials. These striatal changes were accompanied by signs of behavioral inflexibility such as increased open-field grooming and decreased turning probability in the plus maze. In contrast, BE mice typically showed electrophysiological and behavioral changes similar to wild-type and often significantly different from BACHD. Surprisingly, electrophysiological abnormalities were enhanced in M1 of BE mice relative to BACHD, suggesting a possible compensatory response to cortical inputs containing mhtt. Collectively, our results argue against the cell-autonomous model of HD and support a role for cell interactions in the dysfunction of corticostriatal circuits in HD mice.
Mutant Huntingtin Quantification in Human Body Fluids
Andreas Weiss, PhD – IRBM Promidis S.r.l.
Lowering of mutant huntingtin by antisense, siRNA and gene therapy approaches has become the leading approach for drug discovery in Huntington’s disease. These approaches have shown clear beneficial effects in animal models and are close to first applications in humans. However, translating these findings into the clinic is challenging as proof of mechanism, dosing optimization and treatment duration should ideally be guided by quantification of huntingtin in brain, peripheral tissues and body fluids. Today, no analytical method exists to quantify huntingtin in the brain and body fluids of patients, in particular in CSF, which is considered as a window into the brain.
To address these shortcomings, we have developed a novel ultra-sensitive mutant huntingtin detection assay that allows for the detection of zeptomol amounts of mutant huntingtin protein in biological matrices. This presentation will give an overview of the assay characteristics and will present the successful quantification of mutant huntingtin protein in plasma and CSF samples from Huntington’s disease patients.
Translational Kinetic Biomarkers to Enable the Development of HTT-lowering Therapies
Presentation not yet available
Patrizia Fanara, PhD – KineMed Inc.
The absence of validated diagnostic biomarkers of neurodegeneration is a major obstacle for developing drugs that slow progression of Huntington’s disease. KineMed’s translatable kinetic biomarker platform reveals the dynamics of disease-modifying processes (i.e., causal pathways in disease) and can help to ensure that a therapeutic agent reached its intended disease target and that the desired biological and biochemical responses are being obtained.
The presentation will describe in vivo dynamic measures of hyperdynamic microtubules and axonal transport deficits in degenerating brain cells of Huntington’s preclinical models and how these kinetic measurements provide evidence of treatment efficacy of huntingtin (HTT) lowering therapies. By virtue of detecting either slowing or reversal of neurodegeneration in Huntington’s disease, this is a translational diagnostic test that could prove crucial to the advancement of disease-modifying treatments.
The talk will further explore how the power of KineMed’s neurodegeneration biomarker of axonal transport deficit can be used to track disease progression and assess efficacy of HTT-lowering strategies in the clinic.
The EEG is a Sensitive Biomarker for the Presence and Progression of Disease in HD Mouse Models
Stephen Morairty, PhD – SRI International
In Huntington’s disease (HD), neurodegeneration begins to occur long before the onset of the major behavioral, cognitive and psychiatric symptoms. By the time symptoms of HD are manifest, the neurodegeneration is extensive and there is currently little hope of reversing such damage. Therefore, early interventions are needed to slow or stop disease progress effectively preventing neurodegeneration. However, development of therapeutics has been hampered by the lack of a biomarker showing progression of the disease prior to the onset of major symptoms. The electroencephalogram (EEG) is an electrical signal of the brain that is an easily obtainable from both patients and animal models of HD. A recent study by Hunter et al. (2010) reported EEG spectral patterns in premanifest HD patients which differed from non-HD, age matched controls. Further, the magnitude of these HD-related EEG patterns increased in patients that were closer to the onset of major symptoms and continued to progress in mildly manifest to severely manifest patients. These results suggest the EEG might be a predictive biomarker for the progression of HD.
We have investigated mouse models of HD to determine whether the EEG is a sensitive biomarker for the presence and progression of disease in these preclinical models. We have performed longitudinal recordings in the R6/2 and zQ175 mouse models starting at ~8 wk of age. The R6/2 mice show very large changes in EEG patterns at the earliest recordings and prior to degradations in motor function, circadian rhythms and sleep patterns. zQ175 mice also show changes in EEG patterns from our first recordings and long before the onset of behavioral symptoms. Our results support our hypothesis that the EEG is a sensitive biomarker for early detection of neuronal changes in the progression of HD and could be useful for monitoring the efficacy of potential therapies during the prodromal phase.
Diagnosing Dysfunction: NAD Redox Reveals Mitochondrial Changes in HD in vivo
Presentation not yet available
Kevin Conley, PhD – University of Washington Medical Center
Mitochondrial dysfunction and loss play key roles in tissue degeneration that underlies the pathology of HD. Here we present a non-invasive approach that opens a new window on how mitochondria change with HD in human tissue in vivo. Our breakthrough is the ability to measure the redox states of a key metabolite in oxidative phosphorylation and mitochondrial biogenesis, nicotinamide adenine dinucleotide (NAD). We can resolve the NAD redox states in the cell and mitochondrion in muscle, heart and brain using an MRI. The redox states are linked to two key mitochondrial changes associated with degeneration: mitochondrial dysfunction (NADH) and mitochondrial loss (NAD+). We verify and calibrate these links using validated non-invasive metabolic tools in vivo and show that redox states are very sensitive to metabolism and treatment. These qualities of non-invasive measurement, links to mitochondrial properties and sensitivity to intervention in human subjects make NAD redox an important tool for identifying mechanisms of mitochondrial dysfunction and loss in HD and for identifying treatments that reverse this dysfunction and loss.
We present data from HD individuals in context of metabolic diseases and degenerative conditions to demonstrate that NAD redox provides insight into mitochondrial changes with HD disease progression. We show that mitochondrial NADH paired with the signal for oxidative phosphorylation – ADP – identifies individuals with mitochondrial dysfunction. We also show that NAD+ varies with mitochondrial capacity to provide a measure of the mitochondrial content changes with HD. Finally, we show parallel changes in mitochondrial NADH mitochondrial dysfunction and in NAD+ and mitochondrial capacity with acute and chronic treatment indicating that these redox indices are sensitive to the impact of an intervention for HD. Thus NAD redox holds the promise of opening a new window on how mitochondria change in HD, starting well before the onset of symptoms, and revealing the impact of interventions to improve the mitochondrial dysfunction and degeneration that are thought to lead to cell death and tissue wasting characteristic of HD.
The Use of Multi-modal Imaging to Discover Sensitive Neuroimaging Biomarkers for Clinical Trials in Huntington’s Disease: The Melbourne Based IMAGE-HD Study
Nellie Georgiou-Karistianis, PhD – Monash University
A considerable effort is currently underway to establish sensitive and reliable biomarkers of disease onset and progression in Huntington’s disease (HD), and neuroimaging measures are an important area for biomarker development. When conducting neuroimaging studies however there are a number of important issues that need to be considered. Firstly, different neuroimaging measures may be sensitive to neurodegenerative change at different points in the disease trajectory. Secondly, these changes may progress at different rates, being visible within different neural structures at different points in time. In addition, we need to consider the practical utility of these techniques in large multi-site studies, as well as their ability to detect treatment induced improvements. Large-scale multi-site studies (e.g., TRACK-HD, PREDICT-HD) have used structural (MRI) and microstructural (DTI) imaging methods (along with other neurocognitive/behavioural measures) to document sensitivity and reliability of various measures in tracking progressive changes. Moreover, TRACK-ON has been recently extended to investigate functional changes in the brain. Volumetric changes have been shown throughout the course of the disease and are seen prior to robust changes in clinical phenotype. Although there is some evidence to suggest that functional deficits in multiple cortical and subcortical regions extend well beyond the volumetric abnormalities seen in HD, we are still some way from understanding whether functional changes reflect pathology or compensation, or in determining the utility of functional markers for clinical trials. IMAGE-HD is a biomarker development study that adopts a multi-modal approach with consideration of MRI, DTI, functional MRI (fMRI) and susceptibility weighted imaging (SWI). IMAGE-HD has shown 30 month functional changes during the premanifest stages, which suggests a dynamic course of brain reorganisation very early during the disease. This presentation will present multi-modal data from IMAGE-HD, will comment on the preparedness of imaging markers for therapeutic trials, and what should be our next steps.
Positron Emission Tomography Imaging of Phosphodiesterase 10A (PDE10A) Enzyme and Dopamine D2 Receptors in Huntington´s Disease Gene Expansion Carriers
Andrea Varrone, MD, PhD – Karolinska Institutet
PDE10A is an enzyme involved in the regulation of cyclic AMP/GMP levels, highly enriched in striatal medium spiny neurons. PDE10A and D2 receptors are interesting targets for studying the neurodegenerative process in Huntington´s disease (HD), given their striatal co-localization. The aim of this study was to examine both PDE10A enzyme and D2 receptor levels in the living human brain of HD gene expansion carriers (HDGECs) and control subjects with positron emission tomography (PET).
Five controls (4M/1F, age:53.2±11.4y) and 5 stage 1 HDGECs (4M/1F, age:53.6±9.3y) were examined with [18F]MNI-659 and [11C]raclopride, selective radioligands for PDE10A and D2 receptors, respectively. HDGECs were recruited from the European REGISTRY study. PET measurements were performed using the high-resolution research tomograph. Volumes of interest were delineated automatically on 3T MRI images using FreeSurfer. Outcome measures were the total distribution volume (VT) for [18F]MNI-659 and the binding potential (BPND) for [18F]MNI-659 and [11C]raclopride. [18F]MNI-659 data were analyzed with Logan graphical analysis and data of both radioligands with the simplified reference tissue model using the cerebellum as reference region.
In stage 1 HDGECs vs. controls, [18F]MNI-659 VT was ~50% lower in caudate (0.72±0.22 vs. 1.44±0.32; p=0.003), putamen (1.07±0.25 vs. 2.15±0.43; p=0.001), and globus pallidus (1.10±0.25 vs. 1.99±0.36; p=0.002). No significant differences in VT were seen for the cerebellum. In stage 1 HDGECs, [18F]MNI-659 BPND was lower than in controls by 75% in caudate (0.65±0.40 vs. 2.60±0.49; p<0.001), 64% in putamen (1.48±0.61 vs. 4.12±0.53; p<0.001), and 56% in globus pallidus (1.48±0.47 vs. 3.38±0.42; p<0.001). [11C]raclopride BPND in stage 1 HDGECs was lower than in controls by 63% in caudate, 44% in putamen and 29% in globus pallidus.
[18F]MNI-659 is a suitable radioligand for PDE10A imaging in the striatum. Initial results suggest significantly lower availability of PDE10A enzyme and D2 receptors in Stage 1 HDGECs compared with controls. Pre-manifest HDGECs are currently under examination as next study cohort. Integrated examination of PDE10A and D2 receptors across different disease stages will provide key findings to help clinical development plans for assessment of PDE10A inhibitors in HD.
EHDN recruiting sites
Research support: CHDI Foundation, Inc, Princeton, USA.
Sleep and Metabolic Abnormalities in Pre-manifest Patients with Huntington’s Disease
Alpar Lazar, PhD – University of Cambridge
32 Pre-manifest HD (Pre-HD) participants and 25 age and gender balanced healthy controls (mean±SD, age: 44.3±12.7 years, disease burden score: 247.2±65, UHDRS motor score: 1.3±1.4.) participated in multiple sleep and metabolic studies. This included polysomnography, double-labelled water and whole body indirect calorimetry studies including body composition measurements. Assessments were repeated three times at 20 months intervals.
At baseline the Pre-HD patients tended to go to bed earlier and stay in bed longer compared to controls as measured by sleep diary and actigraphy. Although there were no subjective complaints of sleep quality the objective measures of sleep yielded multiple genotype dependent abnormalities and associations with estimated time to disease onset. The Pre-HD group showed decreased sleep quality marked by frequent awakenings, and rapid and large scale fluctuation between sleep stages. We also found an atypical spectral profile characterized by increased high frequency activity in the Pre-HD group mainly present in NREM sleep. Most sleep structural and spectral abnormalities affected primarily the first half of the sleep period. The amplitude of the individually detected slow oscillations during NREM sleep failed to show the expected decreasing trajectory during sleep period linked to the dissipation of sleep pressure, which may indicate an abnormality in the sleep dependent brain recovery in the Pre-HD group. Lastly, we found that interhemispheric EEG phase coherence during sleep was significantly greater in the Pre-HD group compared to healthy controls indicating altered sleep dependent brain connectivity.
There were also multiple indications for metabolic abnormalities in the Pre-manifest group. The bone mineral density and the physical activity related energy expenditure were significantly lower in patients closer to estimated disease onset. Although food intake was standardized for body mass the satiety after the main meal was significantly higher in the Pre-HD group.
This is the first study showing abnormalities in objectively measured sleep quality and sleep dependent oscillatory brain activity in Pre-HD participants, especially in those closest to estimated time of disease onset. Such an abnormality may not only help track disease progression in the pre-manifest stage but may also help explain some of the early abnormalities in HD as it presents clinically.
Neuroendocrine study of Huntington’s disease
Presentation not yet available
Tom Warner, PhD, FRCP – University College London
Classical features of Huntington’s disease (HD) include motor manifestations, cognitive and psychiatric symptoms. However, these are not the sole manifestations in HD, and disruption of circadian rhythms, alterations in sleep patterns, altered glucose homeostasis, muscle atrophy and weight loss have a great impact on the quality of life of the patients and often precede motor symptoms by many years. Extensive research in animal models and humans suggests that these symptoms could be linked to progressive hypothalamic pathology and changes in the neuroendocrine systems.
A significant confounder in comparing a number of previous studies of neuroendocrine factors are that they are based on samples from patient cohorts attending clinics that cannot be standardized for time of sampling and food intake, concomitant medication and other factors such as sleep pattern, smoking and alcohol intake. In the Neuroendocrine Study of HD we attempted to standardize these variables and allow comparison across cohorts by admitting participants for 24 hours study and using rigorous inclusion and exclusion criteria. We analysed a comprehensive panel of endocrine and metabolic factors in cohorts of pre-manifest HD gene carriers, moderate (stage II/III) HD patients and a control group.
This presentation will show that we were unable to replicate previously reported associations of HD with abnormalities of corticosteroids, growth hormone and IGF-1, testosterone, leptin and branched chain amino-acids. The most striking finding was of decreased plasma melatonin concentration in premanifest and manifest HD gene carriers, which may represent a potential state biomarker and also directly relate to sleep disorders in HD. There was also evidence implicating abnormal FT3 and ghrelin, glucose metabolism (OGTT) and growth hormone. These data will be discussed in relation to the previous studies and their relevance to HD phenotype and therapeutics.