Publications by authors named "Gillian P Bates"

115 Publications

Development of novel bioassays to detect soluble and aggregated Huntingtin proteins on three technology platforms.

Brain Commun 2021 5;3(1):fcaa231. Epub 2021 Jan 5.

Department of Neurodegenerative Disease, Huntington's Disease Centre, UK Dementia Research Institute at UCL, Queen Square Institute of Neurology, University College London, London, UK.

Huntington's disease is caused by a CAG / polyglutamine repeat expansion. Mutated CAG repeats undergo somatic instability, resulting in tracts of several hundred CAGs in the brain; and genetic modifiers of Huntington's disease have indicated that somatic instability is a major driver of age of onset and disease progression. As the CAG repeat expands, the likelihood that exon 1 does not splice to exon 2 increases, resulting in two transcripts that encode full-length huntingtin protein, as well as the highly pathogenic and aggregation-prone exon 1 huntingtin protein. Strategies that target the huntingtin gene or transcripts are a major focus of therapeutic development. It is essential that the levels of all isoforms of huntingtin protein can be tracked, to better understand the molecular pathogenesis, and to assess the impact of huntingtin protein-lowering approaches in preclinical studies and clinical trials. Huntingtin protein bioassays for soluble and aggregated forms of huntingtin protein are in widespread use on the homogeneous time-resolved fluorescence and Meso Scale Discovery platforms, but these do not distinguish between exon 1 huntingtin protein and full-length huntingtin protein. In addition, they are frequently used to quantify huntingtin protein levels in the context of highly expanded polyglutamine tracts, for which appropriate protein standards do not currently exist. Here, we set out to develop novel huntingtin protein bioassays to ensure that all soluble huntingtin protein isoforms could be distinguished. We utilized the zQ175 Huntington's disease mouse model that has ∼190 CAGs, a CAG repeat size for which protein standards are not available. Initially, 30 combinations of six antibodies were tested on three technology platforms: homogeneous time-resolved fluorescence, amplified luminescent proximity homogeneous assay and Meso Scale Discovery, and a triage strategy was employed to select the best assays. We found that, without a polyglutamine-length-matched standard, the vast majority of soluble mutant huntingtin protein assays cannot be used for quantitative purposes, as the highly expanded polyglutamine tract decreased assay performance. The combination of our novel assays, with those already in existence, provides a tool-kit to track: total soluble mutant huntingtin protein, soluble exon 1 huntingtin protein, soluble mutant huntingtin protein (excluding the exon 1 huntingtin protein) and total soluble full-length huntingtin protein (mutant and wild type). Several novel aggregation assays were also developed that track with disease progression. These selected assays can be used to compare the levels of huntingtin protein isoforms in a wide variety of mouse models of Huntington's disease and to determine how these change in response to genetic or therapeutic manipulations.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/braincomms/fcaa231DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7878250PMC
January 2021

Subcellular Localization And Formation Of Huntingtin Aggregates Correlates With Symptom Onset And Progression In A Huntington'S Disease Model.

Brain Commun 2020 3;2(2):fcaa066. Epub 2020 Aug 3.

Huntington's Disease Centre, Department of Neurodegenerative Disease and UK Dementia Research Institute at UCL, Queen Square Institute of Neurology, UCL, Queen Square, WC1N 3BG, UK.

Huntington's disease is caused by the expansion of a CAG repeat within exon 1 of the gene, which is unstable, leading to further expansion, the extent of which is brain region and peripheral tissue specific. The identification of DNA repair genes as genetic modifiers of Huntington's disease, that were known to abrogate somatic instability in Huntington's disease mouse models, demonstrated that somatic CAG expansion is central to disease pathogenesis, and that the CAG repeat threshold for pathogenesis in specific brain cells might not be known. We have previously shown that the gene is incompletely spliced generating a small transcript that encodes the highly pathogenic exon 1 HTT protein. The longer the CAG repeat, the more of this toxic fragment is generated, providing a pathogenic consequence for somatic expansion. Here, we have used the R6/2 mouse model to investigate the molecular and behavioural consequences of expressing exon 1 with 90 CAGs, a mutation that causes juvenile Huntington's disease, compared to R6/2 mice carrying ∼200 CAGs, a repeat expansion of a size rarely found in Huntington's disease patient's blood, but which has been detected in post-mortem brains as a consequence of somatic CAG repeat expansion. We show that nuclear aggregation occurred earlier in R6/2(CAG) mice and that this correlated with the onset of transcriptional dysregulation. Whereas in R6/2(CAG) mice, cytoplasmic aggregates accumulated rapidly and closely tracked with the progression of behavioural phenotypes and with end-stage disease. We find that aggregate species formed in the R6/2(CAG) brains have different properties to those in the R6/2(CAG) mice. Within the nucleus, they retain a diffuse punctate appearance throughout the course of the disease, can be partially solubilized by detergents and have a greater seeding potential in young mice. In contrast, aggregates from R6/2(CAG) brains polymerize into larger structures that appear as inclusion bodies. These data emphasize that a subcellular analysis, using multiple complementary approaches, must be undertaken in order to draw any conclusions about the relationship between HTT aggregation and the onset and progression of disease phenotypes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/braincomms/fcaa066DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7425396PMC
August 2020

Silencing Srsf6 does not modulate incomplete splicing of the huntingtin gene in Huntington's disease models.

Sci Rep 2020 08 20;10(1):14057. Epub 2020 Aug 20.

Huntington's Disease Centre, Department of Neurodegenerative Disease and UK Dementia Research Institute at UCL, Queen Square Institute of Neurology, University College London, London, WC1N 3BG, UK.

We have previously shown that the incomplete splicing of exon 1 to exon 2 of the HTT gene results in the production of a small polyadenylated transcript (Httexon1) that encodes the highly pathogenic exon 1 HTT protein. There is evidence to suggest that the splicing factor SRSF6 is involved in the mechanism that underlies this aberrant splicing event. Therefore, we set out to test this hypothesis, by manipulating SRSF6 levels in Huntington's disease models in which an expanded CAG repeat had been knocked in to the endogenous Htt gene. We began by generating mice that were knocked out for Srsf6, and demonstrated that reduction of SRSF6 to 50% of wild type levels had no effect on incomplete splicing in zQ175 knockin mice. We found that nullizygosity for Srsf6 was embryonic lethal, and therefore, to decrease SRSF6 levels further, we established mouse embryonic fibroblasts (MEFs) from wild type, zQ175, and zQ175::Srsf6 mice and transfected them with an Srsf6 siRNA. The incomplete splicing of Htt was recapitulated in the MEFs and we demonstrated that ablation of SRSF6 did not modulate the levels of the Httexon1 transcript. We conclude that SRSF6 is not required for the incomplete splicing of HTT in Huntington's disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-020-71111-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7441155PMC
August 2020

TBK1 phosphorylates mutant Huntingtin and suppresses its aggregation and toxicity in Huntington's disease models.

EMBO J 2020 Sep 5;39(17):e104671. Epub 2020 Aug 5.

Laboratory of Molecular and Chemical Biology of Neurodegeneration, Brain Mind Institute, École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland.

Phosphorylation of the N-terminal domain of the huntingtin (HTT) protein has emerged as an important regulator of its localization, structure, aggregation, clearance and toxicity. However, validation of the effect of bona fide phosphorylation in vivo and assessing the therapeutic potential of targeting phosphorylation for the treatment of Huntington's disease (HD) require the identification of the enzymes that regulate HTT phosphorylation. Herein, we report the discovery and validation of a kinase, TANK-binding kinase 1 (TBK1), that efficiently phosphorylates full-length and N-terminal HTT fragments in vitro (at S13/S16), in cells (at S13) and in vivo. TBK1 expression in HD models (cells, primary neurons, and Caenorhabditis elegans) increases mutant HTT exon 1 phosphorylation and reduces its aggregation and cytotoxicity. We demonstrate that the TBK1-mediated neuroprotective effects are due to phosphorylation-dependent inhibition of mutant HTT exon 1 aggregation and an increase in autophagic clearance of mutant HTT. These findings suggest that upregulation and/or activation of TBK1 represents a viable strategy for the treatment of HD by simultaneously lowering mutant HTT levels and blocking its aggregation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.15252/embj.2020104671DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7459410PMC
September 2020

Expression of mutant exon 1 huntingtin fragments in human neural stem cells and neurons causes inclusion formation and mitochondrial dysfunction.

FASEB J 2020 06 23;34(6):8139-8154. Epub 2020 Apr 23.

Huntington's Disease Centre, Department of Neurodegenerative Disease, UCL Queen Square Institute of Neurology, University College London, London, UK.

Robust cellular models are key in determining pathological mechanisms that lead to neurotoxicity in Huntington's disease (HD) and for high throughput pre-clinical screening of potential therapeutic compounds. Such models exist but mostly comprise non-human or non-neuronal cells that may not recapitulate the correct biochemical milieu involved in pathology. We have developed a new human neuronal cell model of HD, using neural stem cells (ReNcell VM NSCs) stably transduced to express exon 1 huntingtin (HTT) fragments with variable length polyglutamine (polyQ) tracts. Using a system with matched expression levels of exon 1 HTT fragments, we investigated the effect of increasing polyQ repeat length on HTT inclusion formation, location, neuronal survival, and mitochondrial function with a view to creating an in vitro screening platform for therapeutic screening. We found that expression of exon 1 HTT fragments with longer polyQ tracts led to the formation of intra-nuclear inclusions in a polyQ length-dependent manner during neurogenesis. There was no overt effect on neuronal viability, but defects of mitochondrial function were found in the pathogenic lines. Thus, we have a human neuronal cell model of HD that may recapitulate some of the earliest stages of HD pathogenesis, namely inclusion formation and mitochondrial dysfunction.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1096/fj.201902277RRDOI Listing
June 2020

MEF2 impairment underlies skeletal muscle atrophy in polyglutamine disease.

Acta Neuropathol 2020 07 18;140(1):63-80. Epub 2020 Apr 18.

Department of Pathology, University of Michigan Medical School, Ann Arbor, MI, 48109, USA.

Polyglutamine (polyQ) tract expansion leads to proteotoxic misfolding and drives a family of nine diseases. We study spinal and bulbar muscular atrophy (SBMA), a progressive degenerative disorder of the neuromuscular system caused by the polyQ androgen receptor (AR). Using a knock-in mouse model of SBMA, AR113Q mice, we show that E3 ubiquitin ligases which are a hallmark of the canonical muscle atrophy machinery are not induced in AR113Q muscle. Similarly, we find no evidence to suggest dysfunction of signaling pathways that trigger muscle hypertrophy or impairment of the muscle stem cell niche. Instead, we find that skeletal muscle atrophy is characterized by diminished function of the transcriptional regulator Myocyte Enhancer Factor 2 (MEF2), a regulator of myofiber homeostasis. Decreased expression of MEF2 target genes is age- and glutamine tract length-dependent, occurs due to polyQ AR proteotoxicity, and is associated with sequestration of MEF2 into intranuclear inclusions in muscle. Skeletal muscle from R6/2 mice, a model of Huntington disease which develops progressive atrophy, also sequesters MEF2 into inclusions and displays age-dependent loss of MEF2 target genes. Similarly, SBMA patient muscle shows loss of MEF2 target gene expression, and restoring MEF2 activity in AR113Q muscle rescues fiber size and MEF2-regulated gene expression. This work establishes MEF2 impairment as a novel mechanism of skeletal muscle atrophy downstream of toxic polyglutamine proteins and as a therapeutic target for muscle atrophy in these disorders.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00401-020-02156-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7166004PMC
July 2020

Extensive Expression Analysis of Htt Transcripts in Brain Regions from the zQ175 HD Mouse Model Using a QuantiGene Multiplex Assay.

Sci Rep 2019 11 6;9(1):16137. Epub 2019 Nov 6.

Huntington's Disease Centre, Department of Neurodegenerative Disease and UK Dementia Research Institute at UCL, Queen Square Institute of Neurology, University College London, London, WC1N 3BG, UK.

Huntington's disease (HD) is an inherited neurodegenerative disorder caused by a CAG repeat expansion within exon 1 of the huntingtin (HTT) gene. HTT mRNA contains 67 exons and does not always splice between exon 1 and exon 2 leading to the production of a small polyadenylated HTTexon1 transcript, and the full-length HTT mRNA has three 3'UTR isoforms. We have developed a QuantiGene multiplex panel for the simultaneous detection of all of these mouse Htt transcripts directly from tissue lysates and demonstrate that this can replace the more work-intensive Taqman qPCR assays. We have applied this to the analysis of brain regions from the zQ175 HD mouse model and wild type littermates at two months of age. We show that the incomplete splicing of Htt occurs throughout the brain and confirm that this originates from the mutant and not endogenous Htt allele. Given that HTTexon1 encodes the highly pathogenic exon 1 HTT protein, it is essential that the levels of all Htt transcripts can be monitored when evaluating HTT lowering approaches. Our QuantiGene panel will allow the rapid comparative assessment of all Htt transcripts in cell lysates and mouse tissues without the need to first extract RNA.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-019-52411-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6834638PMC
November 2019

Genetic deletion of S6k1 does not rescue the phenotypic deficits observed in the R6/2 mouse model of Huntington's disease.

Sci Rep 2019 11 6;9(1):16133. Epub 2019 Nov 6.

MRC London Institute of Medical Sciences, Imperial College London, Hammersmith Campus, London, W12 0NN, UK.

Huntington's disease (HD) is a fatal inherited autosomal dominant neurodegenerative disorder caused by an expansion in the number of CAG trinucleotide repeats in the huntingtin gene. The disease is characterized by motor, behavioural and cognitive symptoms for which at present there are no disease altering treatments. It has been shown that manipulating the mTOR (mammalian target of rapamycin) pathway using rapamycin or its analogue CCI-779 can improve the cellular and behavioural phenotypes of HD models. Ribosomal protein S6 kinase 1 (S6K1) is a major downstream signalling molecule of mTOR, and its activity is reduced by rapamycin suggesting that deregulation of S6K1 activity may be beneficial in HD. Furthermore, S6k1 knockout mice have increased lifespan and improvement in age-related phenotypes. To evalute the potential benefit of S6k1 loss on HD-related phenotypes, we crossed the R6/2 HD model with the long-lived S6k1 knockout mouse line. We found that S6k1 knockout does not ameliorate behavioural or physiological phenotypes in the R6/2 mouse model. Additionally, no improvements were seen in brain mass reduction or mutant huntingtin protein aggregate levels. Therefore, these results suggest that while a reduction in S6K1 signalling has beneficial effects on ageing it is unlikely to be a therapeutic strategy for HD patients.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-019-52391-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6834565PMC
November 2019

Phenotype onset in Huntington's disease knock-in mice is correlated with the incomplete splicing of the mutant huntingtin gene.

J Neurosci Res 2019 12 7;97(12):1590-1605. Epub 2019 Jul 7.

Department of Neurology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California.

Huntington's disease (HD) is a progressive neurodegenerative disorder caused by an expanded CAG repeat within the huntingtin (HTT) gene. The Q140 and HdhQ150 knock-in HD mouse models were generated such that HdhQ150 mice have an expanded CAG repeat inserted into the mouse Htt gene, whereas in the Q140s, mouse exon 1 Htt was replaced with a mutated version of human exon 1. By standardizing mouse strain background, breeding to homozygosity and employing sensitive behavioral tests, we demonstrate that the onset of behavioral phenotypes occurs earlier in the Q140 than the HdhQ150 knock-in mouse models and that huntingtin (HTT) aggregation appears earlier in the striata of Q140 mice. We have previously found that the incomplete splicing of mutant HTT from exon 1 to exon 2 results in the production of a small polyadenylated transcript that encodes the highly pathogenic mutant HTT exon 1 protein. In this report, we have identified a functional consequence of the sequence differences between these two models at the RNA level, in that the level of incomplete splicing, and of the mutant exon 1 HTT protein, are greater in the brains of Q140 mice. While differences in the human and mouse exon 1 HTT proteins (e.g., proline rich sequences) could also contribute to the phenotypic differences, our data indicate that the incomplete splicing of HTT and approaches to lower the levels of the exon 1 HTT transcript should be pursued as therapeutic targets.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/jnr.24493DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6801054PMC
December 2019

Inhibition of tumour necrosis factor alpha in the R6/2 mouse model of Huntington's disease by etanercept treatment.

Sci Rep 2019 05 10;9(1):7202. Epub 2019 May 10.

Huntington's Disease Centre, Department of Neurodegenerative Disease and UK Dementia Research Institute, UCL Queen Square Institute of Neurology, University College London, London, WC1N 3BG, UK.

Huntington's disease (HD) is an inherited neurodegenerative disorder caused by the expansion of the CAG repeat in exon 1 of the huntingtin (HTT) gene, which results in a mutant protein with an extended polyglutamine tract. Inflammation occurs in both the brain and the periphery of HD patients and mouse models, with increases in brain and/or plasma levels of neurotoxic TNFα and several other proinflammatory cytokines. TNFα promotes the generation of many of these cytokines, such as IL6, which raises the possibility that TNFα is central to the inflammatory milieu associated with HD. A number of mouse studies have reported that the suppression of chronic immune activation during HD has beneficial consequences. Here, we investigated whether TNFα contributes to the peripheral inflammation that occurs in the R6/2 mouse model, and whether the in vivo blockade of TNFα, via etanercept treatment, can modify disease progression. We found that etanercept treatment normalised the elevated plasma levels of some cytokines. This did not modify the progression of certain behavioural measures, but slightly ameliorated brain weight loss, possibly related to a reduction in the elevated striatal level of soluble TNFα.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-019-43627-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6510744PMC
May 2019

Meso scale discovery-based assays for the detection of aggregated huntingtin.

PLoS One 2019 26;14(3):e0213521. Epub 2019 Mar 26.

CHDI Management/CHDI Foundation, Los Angeles, CA, United States of America.

Huntington's disease (HD) is a monogenic neurodegenerative disorder caused by an expansion of the CAG trinucleotide repeat domain in the huntingtin (HTT) gene, leading to an expanded poly-glutamine (polyQ) stretch in the HTT protein. This mutant HTT (mHTT) protein is highly prone to intracellular aggregation, causing significant damage and cellular loss in the striatal, cortical, and other regions of the brain. Therefore, modulation of mHTT levels in these brain regions in order to reduce intracellular mHTT and aggregate levels represents a direct approach in the development of HD therapeutics. To this end, assays that can be used to detect changes in HTT levels in biological samples are invaluable tools to assess target engagement and guide dose selection in clinical trials. The Meso Scale Discovery (MSD) ELISA-based assay platform is a robust and sensitive method previously employed for the quantification of HTT. However, the currently available MSD assays for HTT are primarily detecting the monomeric soluble form of the protein, but not aggregated species. In this study, we describe the development of novel MSD assays preferentially detecting mHTT in an aggregated form. Recombinant monomeric HTT(1-97)-Q46, which forms aggregates in a time-dependent manner, was used to characterize the ability of each established assay to distinguish between HTT monomers and HTT in a higher assembly state. Further validation of these assays was performed using brain lysates from R6/2, zQ175 knock-in, and BACHD mouse models, to replicate a previously well-characterized age-dependent increase in brain aggregate signals, as well as a significant reduction of aggregate levels in the striatum following mHTT knockdown with a CAG-directed allele-specific zinc-finger repressor protein (ZFP). Lastly, size exclusion chromatography was used to separate and characterize HTT species from brain tissue lysates to demonstrate specificity of the assays for the fractions containing aggregated HTT. In summary, we demonstrate that the newly developed assays preferentially detect aggregated HTT with improved performance in comparison to previous assay technologies. These assays complement the existing MSD platform assays specific for soluble HTT monomers, allowing for a more comprehensive analysis of disease-relevant HTT species in preclinical models of HD.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0213521PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6435127PMC
December 2019

Live-cell super-resolution microscopy reveals a primary role for diffusion in polyglutamine-driven aggresome assembly.

J Biol Chem 2019 01 6;294(1):257-268. Epub 2018 Nov 6.

From the Cambridge Infinitus Research Center, Department of Chemical Engineering and Biotechnology and

The mechanisms leading to self-assembly of misfolded proteins into amyloid aggregates have been studied extensively in the test tube under well-controlled conditions. However, to what extent these processes are representative of those in the cellular environment remains unclear. Using super-resolution imaging of live cells, we show here that an amyloidogenic polyglutamine-containing protein first forms small, amorphous aggregate clusters in the cytosol, chiefly by diffusion. Dynamic interactions among these clusters limited their elongation and led to structures with a branched morphology, differing from the predominantly linear fibrils observed Some of these clusters then assembled via active transport at the microtubule-organizing center and thereby initiated the formation of perinuclear aggresomes. Although it is widely believed that aggresome formation is entirely governed by active transport along microtubules, here we demonstrate, using a combined approach of advanced imaging and mathematical modeling, that diffusion is the principal mechanism driving aggresome expansion. We found that the increasing surface area of the expanding aggresome increases the rate of accretion caused by diffusion of cytosolic aggregates and that this pathway soon dominates aggresome assembly. Our findings lead to a different view of aggresome formation than that proposed previously. We also show that aggresomes mature over time, becoming more compacted as the structure grows. The presence of large perinuclear aggregates profoundly affects the behavior and health of the cell, and our super-resolution imaging results indicate that aggresome formation and development are governed by highly dynamic processes that could be important for the design of potential therapeutic strategies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.RA118.003500DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6322900PMC
January 2019

Regulatory mechanisms of incomplete huntingtin mRNA splicing.

Nat Commun 2018 09 27;9(1):3955. Epub 2018 Sep 27.

UCL Huntington's Disease Centre, Department of Neurodegenerative Disease and Dementia Research Institute, UCL Institute of Neurology, University College London, London, WC1N 3BG, UK.

Huntington's disease is caused by a CAG repeat expansion in exon 1 of the HTT gene. We have previously shown that exon 1 HTT does not always splice to exon 2 producing a small transcript (HTTexon1) that encodes the highly pathogenic exon 1 HTT protein. The mechanisms by which this incomplete splicing occurs are unknown. Here, we have generated a minigene system that recapitulates the CAG repeat-length dependence of HTTexon1 production, and has allowed us to define the regions of intron 1 necessary for incomplete splicing. We show that manipulation of the expression levels of the splicing factor SRSF6, predicted to bind CAG repeats, modulates this aberrant splicing event and also demonstrate that RNA polymerase II transcription speed regulates the levels of HTTexon1 production. Understanding the mechanisms by which this pathogenic exon 1 HTT is generated may provide the basis for the development of strategies to prevent its production.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-018-06281-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6160442PMC
September 2018

mHTT Seeding Activity: A Marker of Disease Progression and Neurotoxicity in Models of Huntington's Disease.

Mol Cell 2018 09;71(5):675-688.e6

Neuroproteomics, Max Delbrueck Center for Molecular Medicine, 13125 Berlin, Germany; Berlin Institute of Health (BIH), 10178 Berlin, Germany. Electronic address:

Self-propagating, amyloidogenic mutant huntingtin (mHTT) aggregates may drive progression of Huntington's disease (HD). Here, we report the development of a FRET-based mHTT aggregate seeding (FRASE) assay that enables the quantification of mHTT seeding activity (HSA) in complex biosamples from HD patients and disease models. Application of the FRASE assay revealed HSA in brain homogenates of presymptomatic HD transgenic and knockin mice and its progressive increase with phenotypic changes, suggesting that HSA quantitatively tracks disease progression. Biochemical investigations of mouse brain homogenates demonstrated that small, rather than large, mHTT structures are responsible for the HSA measured in FRASE assays. Finally, we assessed the neurotoxicity of mHTT seeds in an inducible Drosophila model transgenic for HTTex1. We found a strong correlation between the HSA measured in adult neurons and the increased mortality of transgenic HD flies, indicating that FRASE assays detect disease-relevant, neurotoxic, mHTT structures with severe phenotypic consequences in vivo.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molcel.2018.07.032DOI Listing
September 2018

In vivo neutralization of the protagonist role of macrophages during the chronic inflammatory stage of Huntington's disease.

Sci Rep 2018 07 30;8(1):11447. Epub 2018 Jul 30.

Huntington's Disease Centre, Department of Neurodegenerative Disease and Dementia Research Institute, UCL Institute of Neurology, University College London, London, WC1N 3BG, UK.

Neurodegenerative diseases, characterised by the progressive and selective neuronal death in the central nervous system, are frequently accompanied by an activated immune system. In Huntington's disease (HD), clinical and animal studies show evidence of immune activity, along with hyper-reactive monocyte/macrophage responses, while application of immunosuppressive regimens have imparted beneficial effects to HD mice. These findings suggest a contributory role of the immune system in HD pathology, with immune-based interventions offering a potential therapeutic strategy. Herein, we show that peripheral and CNS immune system activity increased with disease progression in HD mouse models and defined the phenotype of the immune response. Additionally, the depletion of monocytes and macrophages in vivo, via clodronate liposome treatment, revealed a major contributory role of these innate immune cells to the chronic inflammatory milieu observed during the course of the disease. This suggests that peripheral immunomodulatory strategies targeting monocytes and macrophages could be relevant for HD.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-018-29792-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6065433PMC
July 2018

Mouse Models of Huntington's Disease.

Methods Mol Biol 2018 ;1780:97-120

Department of Neurodegenerative Disease, Huntington's Disease Centre and Dementia Research Institute, University College London Institute of Neurology, London, WC1N 3BG, UK.

The identification of the mutation causing Huntington's disease (HD) has led to the generation of a large number of mouse models. These models are used to further enhance our understanding of the mechanisms underlying the disease, as well as investigating and identifying therapeutic targets for this disorder. Here we review the transgenic, knock-in mice commonly used to model HD, as well those that have been generated to study specific disease mechanisms. We then provide a brief overview of the importance of standardizing the use of HD mice and describe brief protocols used for genotyping the mouse models used within the Bates Laboratory.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-1-4939-7825-0_6DOI Listing
February 2019

RNA Related Pathology in Huntington's Disease.

Adv Exp Med Biol 2018 ;1049:85-101

Sobell Department of Motor Neuroscience, UCL Institute of Neurology, Queen Square, London, WC1N 3BG, UK.

This chapter summarises research investigating the expression of huntingtin sense and anti-sense transcripts, the effect of the mutation on huntingtin processing as well as the more global effect of the mutation on the coding and non-coding transcriptomes. The huntingtin gene is ubiquitously expressed, although expression levels vary between tissues and cell types. A SNP that affects NF-ĸB binding in the huntingtin promoter modulates the expression level of huntingtin transcripts and is associated with the age of disease onset. Incomplete splicing between exon 1 and exon 2 has been shown to result in the expression of a small polyadenylated mRNA that encodes the highly pathogenic exon 1 huntingtin protein. This occurs in a CAG-repeat length dependent manner in all full-length mouse models of HD as well as HD patient post-mortem brains and fibroblasts. An antisense transcript to huntingtin is generated that contains a CUG repeat that is expanded in HD patients. In myotonic dystrophy, expanded CUG repeats form RNA foci in cell nuclei that bind specific proteins (e.g. MBL1). Short, pure CAG RNAs of approximately 21 nucleotides that have been processed by DICER can inhibit the translation of other CAG repeat containing mRNAs. The HD mutation affects the transcriptome at the level of mRNA expression, splicing and the expression of non-coding RNAs. Finally, expanded repetitive stretched of nucleotides can lead to RAN translation, in which the ribosome translates from the expanded repeat in all possible reading frames, producing proteins with various poly-amino acid tracts. The extent to which these events contribute to HD pathogenesis is largely unknown.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-3-319-71779-1_4DOI Listing
July 2018

Stall in Canonical Autophagy-Lysosome Pathways Prompts Nucleophagy-Based Nuclear Breakdown in Neurodegeneration.

Curr Biol 2017 Dec 22;27(23):3626-3642.e6. Epub 2017 Nov 22.

Department of Basic and Clinical Neuroscience, King's College London, 125 Coldharbour Lane, SE5 9NU London, UK. Electronic address:

The terminal stages of neuronal degeneration and death in neurodegenerative diseases remain elusive. Autophagy is an essential catabolic process frequently failing in neurodegeneration. Selective autophagy routes have recently emerged, including nucleophagy, defined as degradation of nuclear components by autophagy. Here, we show that, in a mouse model for the polyglutamine disease dentatorubral-pallidoluysian atrophy (DRPLA), progressive acquirement of an ataxic phenotype is linked to severe cerebellar cellular pathology, characterized by nuclear degeneration through nucleophagy-based LaminB1 degradation and excretion. We find that canonical autophagy is stalled in DRPLA mice and in human fibroblasts from patients of DRPLA. This is evidenced by accumulation of p62 and downregulation of LC3-I/II conversion as well as reduced Tfeb expression. Chronic autophagy blockage in several conditions, including DRPLA and Vici syndrome, an early-onset autolysosomal pathology, leads to the activation of alternative clearance pathways including Golgi membrane-associated and nucleophagy-based LaminB1 degradation and excretion. The combination of these alternative pathways and canonical autophagy blockade, results in dramatic nuclear pathology with disruption of the nuclear organization, bringing about terminal cell atrophy and degeneration. Thus, our findings identify a novel progressive mechanism for the terminal phases of neuronal cell degeneration and death in human neurodegenerative diseases and provide a link between autophagy block, activation of alternative pathways for degradation, and excretion of cellular components.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cub.2017.10.054DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5723708PMC
December 2017

Myostatin inhibition prevents skeletal muscle pathophysiology in Huntington's disease mice.

Sci Rep 2017 10 27;7(1):14275. Epub 2017 Oct 27.

Sobell Department of Motor Neuroscience and Movement Disorders, University College London Institute of Neurology, London, WC1N 3BG, UK.

Huntington's disease (HD) is an inherited neurodegenerative disorder of which skeletal muscle atrophy is a common feature, and multiple lines of evidence support a muscle-based pathophysiology in HD mouse models. Inhibition of myostatin signaling increases muscle mass, and therapeutic approaches based on this are in clinical development. We have used a soluble ActRIIB decoy receptor (ACVR2B/Fc) to test the effects of myostatin/activin A inhibition in the R6/2 mouse model of HD. Weekly administration from 5 to 11 weeks of age prevented body weight loss, skeletal muscle atrophy, muscle weakness, contractile abnormalities, the loss of functional motor units in EDL muscles and delayed end-stage disease. Inhibition of myostatin/activin A signaling activated transcriptional profiles to increase muscle mass in wild type and R6/2 mice but did little to modulate the extensive Huntington's disease-associated transcriptional dysregulation, consistent with treatment having little impact on HTT aggregation levels. Modalities that inhibit myostatin signaling are currently in clinical trials for a variety of indications, the outcomes of which will present the opportunity to assess the potential benefits of targeting this pathway in HD patients.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-017-14290-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5660167PMC
October 2017

HSF1-dependent and -independent regulation of the mammalian in vivo heat shock response and its impairment in Huntington's disease mouse models.

Sci Rep 2017 10 2;7(1):12556. Epub 2017 Oct 2.

UCL Huntington's Disease Centre, Sobell Department of Motor Neuroscience, UCL Institute of Neurology, University College London, London, United Kingdom.

The heat shock response (HSR) is a mechanism to cope with proteotoxic stress by inducing the expression of molecular chaperones and other heat shock response genes. The HSR is evolutionarily well conserved and has been widely studied in bacteria, cell lines and lower eukaryotic model organisms. However, mechanistic insights into the HSR in higher eukaryotes, in particular in mammals, are limited. We have developed an in vivo heat shock protocol to analyze the HSR in mice and dissected heat shock factor 1 (HSF1)-dependent and -independent pathways. Whilst the induction of proteostasis-related genes was dependent on HSF1, the regulation of circadian function related genes, indicating that the circadian clock oscillators have been reset, was independent of its presence. Furthermore, we demonstrate that the in vivo HSR is impaired in mouse models of Huntington's disease but we were unable to corroborate the general repression of transcription that follows a heat shock in lower eukaryotes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-017-12897-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5624871PMC
October 2017

Disruption to schizophrenia-associated gene Fez1 in the hippocampus of HDAC11 knockout mice.

Sci Rep 2017 09 19;7(1):11900. Epub 2017 Sep 19.

UCL Huntington's Disease Centre, Sobell Department of Motor Neuroscience, UCL Institute of Neurology, University College London, London, United Kingdom.

Histone Deacetylase 11 (HDAC11) is highly expressed in the central nervous system where it has been reported to have roles in neural differentiation. In contrast with previous studies showing nuclear and cytoplasmic localisation, we observed synaptic enrichment of HDAC11. Knockout mouse models for HDACs 1-9 have been important for guiding the development of isoform specific HDAC inhibitors as effective therapeutics. Given the close relationship between HDAC11 and neural cells in vitro, we examined neural tissue in a previously uncharacterised Hdac11 knockout mouse (Hdac11 ). Loss of HDAC11 had no obvious impact on brain morphology and neural stem/precursor cells isolated from Hdac11 mice had comparable proliferation and differentiation characteristics. However, in differentiating neural cells we observed decreased expression of schizophrenia-associated gene Fez1 (fasciculation and elongation protein zeta 1), a gene previously reported to be regulated by HDAC11 activity. FEZ1 has been associated with the dendritic growth of neurons and risk of schizophrenia via its interaction with DISC1 (disrupted in schizophrenia 1). Examination of cortical, cerebellar and hippocampal tissue reveal decreased Fez1 expression specifically in the hippocampus of adult mice. The results of this study demonstrate that loss of HDAC11 has age dependent and brain-region specific consequences.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-017-11630-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5605701PMC
September 2017

The pathogenic exon 1 HTT protein is produced by incomplete splicing in Huntington's disease patients.

Sci Rep 2017 05 2;7(1):1307. Epub 2017 May 2.

UCL Huntington's Disease Centre, Sobell Department of Motor Neuroscience, UCL Institute of Neurology, University College London, London, United Kingdom.

We have previously shown that exon 1 of the huntingtin gene does not always splice to exon 2 resulting in the production of a small polyadenylated mRNA (HTTexon1) that encodes the highly pathogenic exon 1 HTT protein. The level of this read-through product is proportional to CAG repeat length and is present in all knock-in mouse models of Huntington's disease (HD) with CAG lengths of 50 and above and in the YAC128 and BACHD mouse models, both of which express a copy of the human HTT gene. We have now developed specific protocols for the quantitative analysis of the transcript levels of HTTexon1 in human tissue and applied these to a series of fibroblast lines and post-mortem brain samples from individuals with either adult-onset or juvenile-onset HD. We found that the HTTexon1 mRNA is present in fibroblasts from juvenile HD patients and can also be readily detected in the sensory motor cortex, hippocampus and cerebellum of post-mortem brains from HD individuals, particularly in those with early onset disease. This finding will have important implications for strategies to lower mutant HTT levels in patients and the design of future therapeutics.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-017-01510-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5431000PMC
May 2017

Correlations of Behavioral Deficits with Brain Pathology Assessed through Longitudinal MRI and Histopathology in the HdhQ150/Q150 Mouse Model of Huntington's Disease.

PLoS One 2017 18;12(1):e0168556. Epub 2017 Jan 18.

King's College London, Institute of Psychiatry, Department of Neuroscience, London, United Kingdom.

A variety of mouse models have been developed that express mutant huntingtin (mHTT) leading to aggregates and inclusions that model the molecular pathology observed in Huntington's disease. Here we show that although homozygous HdhQ150 knock-in mice developed motor impairments (rotarod, locomotor activity, grip strength) by 36 weeks of age, cognitive dysfunction (swimming T maze, fear conditioning, odor discrimination, social interaction) was not evident by 94 weeks. Concomitant to behavioral assessments, T2-weighted MRI volume measurements indicated a slower striatal growth with a significant difference between wild type (WT) and HdhQ150 mice being present even at 15 weeks. Indeed, MRI indicated significant volumetric changes prior to the emergence of the "clinical horizon" of motor impairments at 36 weeks of age. A striatal decrease of 27% was observed over 94 weeks with cortex (12%) and hippocampus (21%) also indicating significant atrophy. A hypothesis-free analysis using tensor-based morphometry highlighted further regions undergoing atrophy by contrasting brain growth and regional neurodegeneration. Histology revealed the widespread presence of mHTT aggregates and cellular inclusions. However, there was little evidence of correlations between these outcome measures, potentially indicating that other factors are important in the causal cascade linking the molecular pathology to the emergence of behavioral impairments. In conclusion, the HdhQ150 mouse model replicates many aspects of the human condition, including an extended pre-manifest period prior to the emergence of motor impairments.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0168556PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5242535PMC
August 2017

Embryonic Mutant Huntingtin Aggregate Formation in Mouse Models of Huntington's Disease.

J Huntingtons Dis 2016 12;5(4):343-346

Sobell Department of Motor Neuroscience and Movement Disorders, UCL Institute of Neurology, London, UK.

The role of aggregate formation in the pathophysiology of Huntington's disease (HD) remains uncertain. However, the temporal appearance of aggregates tends to correlate with the onset of symptoms and the numbers of neuropil aggregates correlate with the progression of clinical disease. Using highly sensitive immunohistochemical methods we have detected the appearance of diffuse aggregates during embryonic development in the R6/2 and YAC128 mouse models of HD. These are initially seen in developing axonal tracts and appear to spread throughout the cerebrum in the early neonate.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3233/JHD-160217DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5181660PMC
December 2016

Frequency of nuclear mutant huntingtin inclusion formation in neurons and glia is cell-type-specific.

Glia 2017 01 12;65(1):50-61. Epub 2016 Sep 12.

Department of Cell biology & Histology, Academic Medical Center, Amsterdam, The Netherlands.

Huntington's disease (HD) is an autosomal dominant inherited neurodegenerative disorder that is caused by a CAG expansion in the Huntingtin (HTT) gene, leading to HTT inclusion formation in the brain. The mutant huntingtin protein (mHTT) is ubiquitously expressed and therefore nuclear inclusions could be present in all brain cells. The effects of nuclear inclusion formation have been mainly studied in neurons, while the effect on glia has been comparatively disregarded. Astrocytes, microglia, and oligodendrocytes are glial cells that are essential for normal brain function and are implicated in several neurological diseases. Here we examined the number of nuclear mHTT inclusions in both neurons and various types of glia in the two brain areas that are the most affected in HD, frontal cortex, and striatum. We compared nuclear mHTT inclusion body formation in three HD mouse models that express either full-length HTT or an N-terminal exon1 fragment of mHTT, and we observed nuclear inclusions in neurons, astrocytes, oligodendrocytes, and microglia. When studying the frequency of cells with nuclear inclusions in mice, we found that half of the population of neurons contained nuclear inclusions at the disease end stage, whereas the proportion of GFAP-positive astrocytes and oligodendrocytes having a nuclear inclusion was much lower, while microglia hardly showed any nuclear inclusions. Nuclear inclusions were also present in neurons and all studied glial cell types in human patient material. This is the first report to compare nuclear mHTT inclusions in glia and neurons in different HD mouse models and HD patient brains. GLIA 2016;65:50-61.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/glia.23050DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5129569PMC
January 2017

UBQLN2 Mediates Autophagy-Independent Protein Aggregate Clearance by the Proteasome.

Cell 2016 Aug 28;166(4):935-949. Epub 2016 Jul 28.

Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, Davidson Building, Henry Wellcome Lab of Cell Biology, University of Glasgow, G12 8QQ Glasgow, UK; The MRC Protein Phosphorylation and Ubiquitylation Unit, The Sir James Black Centre, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland. Electronic address:

Clearance of misfolded and aggregated proteins is central to cell survival. Here, we describe a new pathway for maintaining protein homeostasis mediated by the proteasome shuttle factor UBQLN2. The 26S proteasome degrades polyubiquitylated substrates by recognizing them through stoichiometrically bound ubiquitin receptors, but substrates are also delivered by reversibly bound shuttles. We aimed to determine why these parallel delivery mechanisms exist and found that UBQLN2 acts with the HSP70-HSP110 disaggregase machinery to clear protein aggregates via the 26S proteasome. UBQLN2 recognizes client-bound HSP70 and links it to the proteasome to allow for the degradation of aggregated and misfolded proteins. We further show that this process is active in the cell nucleus, where another system for aggregate clearance, autophagy, does not act. Finally, we found that mutations in UBQLN2, which lead to neurodegeneration in humans, are defective in chaperone binding, impair aggregate clearance, and cause cognitive deficits in mice.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cell.2016.07.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5003816PMC
August 2016

The S/T-Rich Motif in the DNAJB6 Chaperone Delays Polyglutamine Aggregation and the Onset of Disease in a Mouse Model.

Mol Cell 2016 Apr 14;62(2):272-283. Epub 2016 Apr 14.

Department of Cell Biology, University Medical Center Groningen, University of Groningen, 9713 GZ Groningen, the Netherlands. Electronic address:

Expanded CAG repeats lead to debilitating neurodegenerative disorders characterized by aggregation of proteins with expanded polyglutamine (polyQ) tracts. The mechanism of aggregation involves primary and secondary nucleation steps. We show how a noncanonical member of the DNAJ-chaperone family, DNAJB6, inhibits the conversion of soluble polyQ peptides into amyloid fibrils, in particular by suppressing primary nucleation. This inhibition is mediated by a serine/threonine-rich region that provides an array of surface-exposed hydroxyl groups that bind to polyQ peptides and may disrupt the formation of the H bonds essential for the stability of amyloid fibrils. Early prevention of polyQ aggregation by DNAJB6 occurs also in cells and leads to delayed neurite retraction even before aggregates are visible. In a mouse model, brain-specific coexpression of DNAJB6 delays polyQ aggregation, relieves symptoms, and prolongs lifespan, pointing to DNAJB6 as a potential target for disease therapy and tool for unraveling early events in the onset of polyQ diseases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molcel.2016.03.017DOI Listing
April 2016

SIRT1 Activity Is Linked to Its Brain Region-Specific Phosphorylation and Is Impaired in Huntington's Disease Mice.

PLoS One 2016 27;11(1):e0145425. Epub 2016 Jan 27.

Department of Medical and Molecular Genetics, King's College London, London, United Kingdom.

Huntington's disease (HD) is a neurodegenerative disorder for which there are no disease-modifying treatments. SIRT1 is a NAD+-dependent protein deacetylase that is implicated in maintaining neuronal health during development, differentiation and ageing. Previous studies suggested that the modulation of SIRT1 activity is neuroprotective in HD mouse models, however, the mechanisms controlling SIRT1 activity are unknown. We have identified a striatum-specific phosphorylation-dependent regulatory mechanism of SIRT1 induction under normal physiological conditions, which is impaired in HD. We demonstrate that SIRT1 activity is down-regulated in the brains of two complementary HD mouse models, which correlated with altered SIRT1 phosphorylation levels. This SIRT1 impairment could not be rescued by the ablation of DBC1, a negative regulator of SIRT1, but was linked to changes in the sub-cellular distribution of AMPK-α1, a positive regulator of SIRT1 function. This work provides insights into the regulation of SIRT1 activity with the potential for the development of novel therapeutic strategies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0145425PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4731418PMC
July 2016

Characterization of Gastric Mucosa Biopsies Reveals Alterations in Huntington's Disease.

PLoS Curr 2015 Jun 26;7. Epub 2015 Jun 26.

Department of Clinical Genetics, Sheffield Children's Hospital, Sheffield Children's NHS foundation Trust, Sheffield, South Yorkshire, UK.

Weight loss is an important complication of Huntington's disease (HD), however the mechanism for weight loss in HD is not entirely understood. Mutant huntingtin is expressed in the gastrointestinal (GI) tract and, in HD mice, mutant huntingtin inclusions are found within the enteric nervous system along the GI tract. A reduction of neuropeptides, decreased mucosal thickness and villus length, as well as gut motility impairment, have also been shown in HD mice. We therefore set out to study gastric mucosa of patients with HD, looking for abnormalities of mucosal cells using immunohistochemistry. In order to investigate possible histological differences related to gastric acid production, we evaluated the cell density of acid producing parietal cells, as well as gastrin producing cells (the endocrine cell controlling parietal cell function). In addition, we looked at chief cells and somatostatin-containing cells. In gastric mucosa from HD subjects, compared to control subject biopsies, a reduced expression of gastrin (a marker of G cells) was found. This is in line with previous HD mouse studies showing reduction of GI tract neuropeptides.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/currents.hd.858b4cc7f235df068387e9c20c436a79DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4549855PMC
June 2015