Huntington's disease modeling and treatment:from primary neuronal cultures to rodents

Huntington's disease (HD) is a mid-life-onset neurodegenerative disorder characterized by involuntary movements, personality changes and dementia. It progresses to death within 10-20 years after onset. There is currently no cure to treat this fatal disease. In HD patients, the protein huntingtin contains an abnormal expansion of a polyglutamine tract, which leads to the selective death of striatal neurons. The functions of huntingtin, as well as the dysfunctions induced by the mutation are still poorly understood. The first chapter of this thesis describes the state of the art in the study of Huntington's disease: huntingtin (htt), the protein which induces the disease; the hypothesis of toxic pathways induced by the mutant htt and the proposed therapeutic strategies to interfere with them; the available cellular and animal models and finally; the promise of gene therapy for neurodegenerative diseases such as Huntington's disease. The second chapter presents a new cellular model of the developed by infecting primary cultures with lentiviral vectors expressing a mutant htt fragment. This model is characterized by generalized neuronal transgene chronic pathology, expression, neuronal dysfunction and finally cell death. The slow progression in this model allows the study of the cascade of events leading to cell death. These unique characteristics allow the investigation of the pathological events induced by htt expression with analytical techniques on the entire neuronal population. The chapter ends with the description of an experiment showing the neuroprotective effects of ciliary neurotrophic factor (CNTF) and brain-derived neurotrophic factor (BDNF) on thiese neurons. The third chapter describes the delivery of CNTF in transgenic HD mice which express the human full-length htt (YAC72). CNTF was delivered directly to the affected neurons by striatal lentiviral injection. The sustained one-year delivery was not associated with side effects; it was correlated to reduced hyperactivity and a reduction in degenerating neurons. However, a neuroprotective effect was difficult to establish, due to the very mild and subtle pathology occurring in these animals. The fourth chapter considers delivery of BDNF in two genetic rodent HD models: in the mild phenotypic HD mice (YAC128) and a more severe rat model based on the lentiviral delivery of mutant htt in the striatum. No neuroprotective effect of BDNF could be detected in either animal model. The conclusions of this thesis discuss the potential of lentiviral vectors in modeling neurodegenerative diseases in vitro and in vivo, as well as their potential role in the treatment of such diseases. The perspective gained by the developed cellular model toward the understanding of the molecular events induced by mutant huntingtin is discussed, as well as the difficulties in testing neuroprotective approaches on animal models of HD

Genetic and pharmacological inhibition of calcineurin corrects the BDNF transport defect in Huntington's disease

<p>Abstract</p> <p>Background</p> <p>Huntington's disease (HD) is an inherited neurogenerative disease caused by an abnormal expansion of glutamine repeats in the huntingtin protein. There is currently no treatment to prevent the neurodegeneration caused by this devastating disorder. Huntingtin has been shown to be a positive regulator of vesicular transport, particularly for neurotrophins such as brain-derived neurotrophic factor (BDNF). This function is lost in patients with HD, resulting in a decrease in neurotrophic support and subsequent neuronal death. One promising line of treatment is therefore the restoration of huntingtin function in BDNF transport.</p> <p>Results</p> <p>The phosphorylation of huntingtin at serine 421 (S421) restores its function in axonal transport. We therefore investigated whether inhibition of calcineurin, the <it>bona fide </it>huntingtin S421 phosphatase, restored the transport defects observed in HD. We found that pharmacological inhibition of calcineurin by FK506 led to sustained phosphorylation of mutant huntingtin at S421. FK506 restored BDNF transport in two complementary models: rat primary neuronal cultures expressing mutant huntingtin and mouse cortical neurons from <it>Hdh</it>^Q111/Q111HD knock-in mice. This effect was the result of specific calcineurin inhibition, as calcineurin silencing restored both anterograde and retrograde transport in neurons from <it>Hdh</it>^Q111/Q111mice. We also observed a specific increase in calcineurin activity in the brain of <it>Hdh</it>^Q111/Q111mice potentially accounting for the selective loss of huntingtin phosphorylation and contributing to neuronal cell death in HD.</p> <p>Conclusion</p> <p>Our results validate calcineurin as a target for the treatment of HD and provide the first demonstration of the restoration of huntingtin function by an FDA-approved compound.</p

Location of care for people with serious mental illness (LOCAPE) : implications for service use and costs using a mixed-methods approach

BackgroundThis study focuses on health care received by people with serious mental illness (SMI). The aim is to examine the economic implications of different locations of management of care and the views of service users and staff regarding services set up as alternatives to secondary care.ObjectivesSpecific objectives are to (1) identify people with SMI managed in primary or secondary care; (2) identify those who could be potentially transferred to primary care; (3) compare the characteristics of these groups; (4) compare service use and costs; (5) generate models to estimate cost changes following transfer between settings; (6) identify characteristics associated with time to transition to secondary care; (7) investigate experiences of patients receiving support from community-based interventions; and (8) assess the economic impact of interventions to facilitate transfer of care management.Methods(1) Quantitative component – using linked primary and secondary care data we examined differences between those discharged to primary care (n = 1410) and those still in secondary care (n = 1629). Service use and costs were compared and predictors of costs were identified using regression models. (2) Qualitative component – interviews following a topic guide were conducted at two time points with 31 people using and 10 people working in services set up as alternatives to secondary care. (3) Economic modelling – an analysis of the health-care costs of the above services compared with usual care was conducted using decision modelling. Data were obtained from local services where possible, and the time horizon was 12 months.Results(1) Quantitative component – characteristics of those discharged to primary care (n = 1410) were similar to those still in secondary care (n = 1629). Costs for those discharged to primary care were 48% lower than for those remaining in secondary care. Other variables strongly associated with costs were a history of violence and a diagnosis of schizophrenia or bipolar disorder. Few patients in secondary care had a high probability of primary care management and, therefore, excess costs were only around £150,000 across the sample. (2) Qualitative component – service users’ views about a community options team and a primary care support service were positive and compared favourably to services used previously. Views about peer support were slightly less consistent. Staff had concerns with regard to caseload sizes and staff turnover. (3) Economic modelling – services to help transition had costs that were 40% of those for standard care. The results of this showed that triaging patients into these services would save £1578 over a 1-year period and that the results were robust to changes in most parameters.LimitationsAnalysis was hindered by the extent to which data were available. Qualitative analyses were limited by the fact that most of the participants did not have a SMI as usually defined and that many had been out of contact with secondary services for a long period of time.ConclusionsCosts are substantially lower in primary care than secondary care, even after controlling for service-user patient differences. Generally, there is satisfaction with services to help facilitate primary care provision and these appear to be cost saving. Future work should continue the analysis of linked data and involve a more comprehensive evaluation of the specific services investigated here.FundingThe National Institute for Health Research Health Services and Delivery Research programme

Huntingtin’s Function in Axonal Transport Is Conserved in <i>Drosophila melanogaster</i>

<div><p>Huntington’s disease (HD) is a devastating dominantly inherited neurodegenerative disorder caused by an abnormal polyglutamine expansion in the N-terminal part of the huntingtin (HTT) protein. HTT is a large scaffold protein that interacts with more than a hundred proteins and is probably involved in several cellular functions. The mutation is dominant, and is thought to confer new and toxic functions to the protein. However, there is emerging evidence that the mutation also alters HTT’s normal functions. Therefore, HD models need to recapitulate this duality if they are to be relevant. <i>Drosophila melanogaster</i> is a useful <i>in vivo</i> model, widely used to study HD through the overexpression of full-length or N-terminal fragments of mutant human HTT. However, it is unclear whether <i>Drosophila</i> huntingtin (DmHTT) shares functions similar to the mammalian HTT. Here, we used various complementary approaches to analyze the function of DmHTT in fast axonal transport. We show that DmHTT interacts with the molecular motor dynein, associates with vesicles and co-sediments with microtubules. DmHTT co-localizes with Brain-derived neurotrophic factor (BDNF)-containing vesicles in rat cortical neurons and partially replaces mammalian HTT in a fast axonal transport assay. DmHTT-KO flies show a reduced fast axonal transport of synaptotagmin vesicles in motoneurons <i>in vivo</i>. These results suggest that the function of HTT in axonal transport is conserved between flies and mammals. Our study therefore validates <i>Drosophila melanogaster</i> as a model to study HTT function, and its dysfunction associated with HD.</p> </div

<i>Drosophila</i> Huntingtin is dynamic and rescues loss of mammalian huntingtin in axonal transport.

<p>(A) Dm620HTT-GFP is dynamic. Image within a microchannel, of an axon from a rat cortical neuron expressing Dm620HTT-GFP. The generated kymograph below shows the vesicular trajectories and the paths that were analyzed (green for anterograde moving vesicles, red for retrograde moving vesicles and blue for stationary vesicles). (B) Two-colors kymograph obtained from neurons co-transfected with BDNF-mCherry and Dm620HTT-GFP shows co-transport of DmHTT and BDNF-mCherry in axons. (C) Western blotting shows silencing of endogenous rat <i>Htt</i> gene and its replacement by fly <i>htt</i> in cortical neurons. (D) The generated kymograph shows the vesicular trajectory in the three conditions and the paths that were analyzed. (E) The mean velocity of anterograde and retrograde moving vesicles was calculated. (Mean+S.E.M, p* <0.05, p**<0.01 and p***<0.001).</p

Fast axonal transport is impaired in <i>Drosophila</i> larvae knock out for HTT.

<p>(A) Anti DmHTT-S50 antibody recognizes Dm620HTT-GFP but not GFP when expressed in HEK 293 cells. (B) DmHTT-S50 recognizes a band of about 400 kDa in <i>Drosophila</i> L3 larvae brain extracts (see star). This band is absent in HTT-KO fly extracts. (C) Typical kymographs of wild-type and HTT-KO larvae with the analyzed trajectories for synaptotagmin-GFP vesicles. (D) Quantification of the mean velocity of axonal synaptotagmin-GFP vesicles in wild-type and HTT-KO larvae. (E) Distribution of synaptotagmin-GFP vesicles velocities shows a reduced number of fast-moving vesicles in HTT-KO larvae compared with wild-type flies.</p

The dynein interacting domain of human HTT is conserved in <i>Drosophila melanogaster</i>.

<p>(A) Alignment of hHTT[600–698] and DmHTT[381–549]. The red box highlights the high homology region. (B) Western blot analyses of the co-immunoprecipitation of Dm620HTT-GFP with endogenous DIC on transfected HEK 293 cells: Dm620HTT-GFP but not control GFP co-precipitates DIC using an anti-GFP antibody. Anti-HA antibody was used as negative control and showed no Dm620HTT-GFP or DIC signal.</p