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1.  A GAA repeat expansion reporter model of Friedreich's ataxia recapitulates the genomic context and allows rapid screening of therapeutic compounds 
Human Molecular Genetics  2013;22(25):5173-5187.
Friedreich's ataxia (FRDA) is caused by large GAA expansions in intron 1 of the frataxin gene (FXN), which lead to reduced FXN expression through a mechanism not fully understood. Understanding such mechanism is essential for the identification of novel therapies for FRDA and this can be accelerated by the development of cell models which recapitulate the genomic context of the FXN locus and allow direct comparison of normal and expanded FXN loci with rapid detection of frataxin levels. Here we describe the development of the first GAA-expanded FXN genomic DNA reporter model of FRDA. We modified BAC vectors carrying the whole FXN genomic DNA locus by inserting the luciferase gene in exon 5a of the FXN gene (pBAC-FXN-Luc) and replacing the six GAA repeats present in the vector with an ∼310 GAA repeat expansion (pBAC-FXN-GAA-Luc). We generated human clonal cell lines carrying the two vectors using site-specific integration to allow direct comparison of normal and expanded FXN loci. We demonstrate that the presence of expanded GAA repeats recapitulates the epigenetic modifications and repression of gene expression seen in FRDA. We applied the GAA-expanded reporter model to the screening of a library of novel small molecules and identified one molecule which up-regulates FXN expression in FRDA patient primary cells and restores normal histone acetylation around the GAA repeats. These results suggest the potential use of genomic reporter cell models for the study of FRDA and the identification of novel therapies, combining physiologically relevant expression with the advantages of quantitative reporter gene expression.
PMCID: PMC3842177  PMID: 23943791
2.  R-loops Associated with Triplet Repeat Expansions Promote Gene Silencing in Friedreich Ataxia and Fragile X Syndrome 
PLoS Genetics  2014;10(5):e1004318.
Friedreich ataxia (FRDA) and Fragile X syndrome (FXS) are among 40 diseases associated with expansion of repeated sequences (TREDs). Although their molecular pathology is not well understood, formation of repressive chromatin and unusual DNA structures over repeat regions were proposed to play a role. Our study now shows that RNA/DNA hybrids (R-loops) form in patient cells on expanded repeats of endogenous FXN and FMR1 genes, associated with FRDA and FXS. These transcription-dependent R-loops are stable, co-localise with repressive H3K9me2 chromatin mark and impede RNA Polymerase II transcription in patient cells. We investigated the interplay between repressive chromatin marks and R-loops on the FXN gene. We show that decrease in repressive H3K9me2 chromatin mark has no effect on R-loop levels. Importantly, increasing R-loop levels by treatment with DNA topoisomerase inhibitor camptothecin leads to up-regulation of repressive chromatin marks, resulting in FXN transcriptional silencing. This provides a direct molecular link between R-loops and the pathology of TREDs, suggesting that R-loops act as an initial trigger to promote FXN and FMR1 silencing. Thus R-loops represent a common feature of nucleotide expansion disorders and provide a new target for therapeutic interventions.
Author Summary
Friedreich ataxia and Fragile X syndrome are among 40 human diseases associated with expansion of repeated sequences. In both disorders repeat expansion leads to gene silencing, the molecular mechanism of which is not well understood, impeding the development of specific therapies to treat these disorders. It is proposed that formation of unusual DNA structures (RNA/DNA hybrids, or R-loops) over repeat regions may play a role, but their molecular function has not been investigated in vivo. We show that R-loops form on expanded repeats of FXN and FMR1 genes in cells from FRDA and FXS patients. These R-loops are stable, correlate with repressive chromatin marks and hinder FXN transcription in patient cells. We studied the relationship between repressive chromatin and R-loops. Decrease in the amount of repressive chromatin has no effect on R-loop levels. In contrast, increase in the R-loops leads to transcriptional silencing of FXN gene and formation of repressive chromatin, providing a direct molecular link between R-loops and pathology of expansion diseases. This discovery is important for understanding the basic molecular mechanism underlying the pathology of expansion diseases. The ability of R-loops to trigger transcriptional silencing makes them an attractive target for future therapeutic approaches to treat these diseases.
PMCID: PMC4006715  PMID: 24787137
3.  Physiological Characterisation of Human iPS-Derived Dopaminergic Neurons 
PLoS ONE  2014;9(2):e87388.
Human induced pluripotent stem cells (hiPSCs) offer the potential to study otherwise inaccessible cell types. Critical to this is the directed differentiation of hiPSCs into functional cell lineages. This is of particular relevance to research into neurological disease, such as Parkinson’s disease (PD), in which midbrain dopaminergic neurons degenerate during disease progression but are unobtainable until post-mortem. Here we report a detailed study into the physiological maturation over time of human dopaminergic neurons in vitro. We first generated and differentiated hiPSC lines into midbrain dopaminergic neurons and performed a comprehensive characterisation to confirm dopaminergic functionality by demonstrating dopamine synthesis, release, and re-uptake. The neuronal cultures include cells positive for both tyrosine hydroxylase (TH) and G protein-activated inward rectifier potassium channel 2 (Kir3.2, henceforth referred to as GIRK2), representative of the A9 population of substantia nigra pars compacta (SNc) neurons vulnerable in PD. We observed for the first time the maturation of the slow autonomous pace-making (<10 Hz) and spontaneous synaptic activity typical of mature SNc dopaminergic neurons using a combination of calcium imaging and electrophysiology. hiPSC-derived neurons exhibited inositol tri-phosphate (IP3) receptor-dependent release of intracellular calcium from the endoplasmic reticulum in neuronal processes as calcium waves propagating from apical and distal dendrites, and in the soma. Finally, neurons were susceptible to the dopamine neuron-specific toxin 1-methyl-4-phenylpyridinium (MPP+) which reduced mitochondrial membrane potential and altered mitochondrial morphology. Mature hiPSC-derived dopaminergic neurons provide a neurophysiologically-defined model of previously inaccessible vulnerable SNc dopaminergic neurons to bridge the gap between clinical PD and animal models.
PMCID: PMC3931621  PMID: 24586273
4.  Region-specific deficits in dopamine, but not norepinephrine, signaling in a novel A30P α-synuclein BAC transgenic mouse☆ 
Neurobiology of Disease  2014;62(100):193-207.
Parkinson's disease (PD) is a neurodegenerative disorder classically characterized by the death of dopamine (DA) neurons in the substantia nigra pars compacta and by intracellular Lewy bodies composed largely of α-synuclein. Approximately 5–10% of PD patients have a familial form of Parkinsonism, including mutations in α-synuclein. To better understand the cell-type specific role of α-synuclein on DA neurotransmission, and the effects of the disease-associated A30P mutation, we generated and studied a novel transgenic model of PD. We expressed the A30P mutant form of human α-synuclein in a spatially-relevant manner from the 111 kb SNCA genomic DNA locus on a bacterial artificial chromosome (BAC) insert on a mouse null (Snca −/−) background. The BAC transgenic mice expressed α-synuclein in tyrosine hydroxylase-positive neurons and expression of either A30P α-synuclein or wildtype α-synuclein restored the sensitivity of DA neurons to MPTP in resistant Snca −/− animals. A30P α-synuclein mice showed no Lewy body-like aggregation, and did not lose catecholamine neurons in substantia nigra or locus coeruleus. However, using cyclic voltammetry at carbon-fiber microelectrodes we identified a deficit in evoked DA release in the caudate putamen, but not in the nucleus accumbens, of SNCA-A30P Snca −/− mice but no changes to release of another catecholamine, norepinephrine (NE), in the NE-rich ventral bed nucleus of stria terminalis. SNCA-A30P Snca −/− mice had no overt behavioral impairments but exhibited a mild increase in wheel-running. In summary, this refined PD mouse model shows that A30P α-synuclein preferentially perturbs the dopaminergic system in the dorsal striatum, reflecting the region-specific change seen in PD.
•SNCA A30P BAC transgenic mice recapitulate endogenous α-synuclein expression pattern•SNCA A30P BAC transgenic mice demonstrate a region specific deficit in evoked DA, but not NE, release.•Expression of A30P or WT α-syn restored the sensitivity of DA neurons to MPTP.•A30P BAC mice had no Lewy body-like aggregation or neuronal loss in SNpc or LC.•Early changes in DA neurotransmission in the absence of aggregation
PMCID: PMC3898275  PMID: 24121116
Parkinson's disease; α-Synuclein; Dopamine; Norepinephrine; Voltammetry; Behavior
5.  Tau protein is required for amyloid β-induced impairment of hippocampal long-term potentiation 
Amyloid beta (Aβ) and tau protein are both implicated in memory impairment in mild cognitive impairment (MCI) and early Alzheimer’s disease (AD), but whether and how they interact is unknown. Consequently, here we asked if tau protein is required for the robust phenomenon of Aβ-induced impairment of hippocampal long-term potentiation (LTP), a widely accepted cellular model of memory. We used wild-type mice and mice with a genetic knockout of tau protein and recorded field potentials in an acute slice preparation. We demonstrate that the absence of tau protein prevents Aβ-induced impairment of LTP. Moreover, we show that Aβ increases tau phosphorylation and that a specific inhibitor of the tau kinase, glycogen synthase kinase 3 (GSK-3), blocks the increased tau phosphorylation induced by Aβ and prevents Aβ-induced impairment of LTP in wild-type mice. Together, these findings show that tau protein is required for Aβ to impair synaptic plasticity in the hippocampus and suggest that the Aβ-induced impairment of LTP is mediated by tau phosphorylation. We conclude that preventing the interaction between Aβ and tau could be a promising strategy for treating cognitive impairment in MCI and early AD.
PMCID: PMC3836238  PMID: 21289177
Alzheimer’s disease; beta amyloid; tau protein; LTP; hippocampus; mouse
6.  Functional and genetic analysis of haplotypic sequence variation at the nicastrin genomic locus 
Neurobiology of aging  2012;33(8):1848.e1-1848.13.
Nicastrin (NCSTN) is a component of the γ-secretase complex and therefore potentially a candidate risk gene for Alzheimer's disease. Here, we have developed a novel functional genomics methodology to express common locus haplotypes to assess functional differences. DNA recombination was used to engineer 5 bacterial artificial chromosomes (BACs) to each express a different haplotype of the NCSTN locus. Each NCSTN-BAC was delivered to knockout nicastrin (Ncstn−/−) cells and clonal NCSTN-BAC+/Ncstn−/− cell lines were created for functional analyses. We showed that all NCSTN-BAC haplotypes expressed nicastrin protein and rescued γ-secretase activity and amyloid beta (Aβ) production in NCSTN-BAC+/Ncstn−/− lines. We then showed that genetic variation at the NCSTN locus affected alternative splicing in human postmortem brain tissue. However, there was no robust functional difference between clonal cell lines rescued by each of the 5 different haplotypes. Finally, there was no statistically significant association of NCSTN with disease risk in the 4 cohorts. We therefore conclude that it is unlikely that common variation at the NCSTN locus is a risk factor for Alzheimer's disease.
PMCID: PMC3683320  PMID: 22405046
Nicastrin; Haplotype variation; Functional genomics; Alzheimer's disease; γ-Secretase complex
7.  α-Synuclein and dopamine at the crossroads of Parkinson’s disease 
Trends in neurosciences  2010;33(12):559-568.
α-Synuclein is central to the Lewy body neuropathology of Parkinson’s disease (PD), a devastating neurodegenerative disorder characterized by numerous motor and non-motor manifestations. The cardinal motor symptoms are linked to death of dopaminergic neurons in the nigrostriatal pathway. Here we ask why these neurons are preferentially susceptible to neurodegeneration in PD and how α-synuclein is involved. To address these questions we bring together recent findings from genome-wide association studies, which reveal the involvement of α-synuclein gene variants in sporadic PD, with recent studies highlighting important roles for α-synuclein in synaptic transmission and dopaminergic neuron physiology. These latest advances add to our understanding of PD etiology and provide a central link between the genetic findings and neurodegeneration observed in sporadic PD.
PMCID: PMC3631137  PMID: 20961626
8.  Combining M-FISH and Quantum Dot technology for fast chromosomal assignment of transgenic insertions 
BMC Biotechnology  2011;11:121.
Physical mapping of transgenic insertions by Fluorescence in situ Hybridization (FISH) is a reliable and cost-effective technique. Chromosomal assignment is commonly achieved either by concurrent G-banding or by a multi-color FISH approach consisting of iteratively co-hybridizing the transgenic sequence of interest with one or more chromosome-specific probes at a time, until the location of the transgenic insertion is identified.
Here we report a technical development for fast chromosomal assignment of transgenic insertions at the single cell level in mouse and rat models. This comprises a simplified 'single denaturation mixed hybridization' procedure that combines multi-color karyotyping by Multiplex FISH (M-FISH), for simultaneous and unambiguous identification of all chromosomes at once, and the use of a Quantum Dot (QD) conjugate for the transgene detection.
Although the exploitation of the unique optical properties of QD nanocrystals, such as photo-stability and brightness, to improve FISH performance generally has been previously investigated, to our knowledge this is the first report of a purpose-designed molecular cytogenetic protocol in which the combined use of QDs and standard organic fluorophores is specifically tailored to assist gene transfer technology.
PMCID: PMC3266222  PMID: 22165957
9.  Functional alterations to the nigrostriatal system in mice lacking all three members of the synuclein family 
The synucleins (α, β and γ) are highly homologous proteins thought to play a role in regulating neurotransmission and are found abundantly in presynaptic terminals. To overcome functional overlap between synuclein proteins and to understand their role in presynaptic signalling from mesostriatal dopaminergic neurons, we produced mice lacking all three members of the synuclein family. The effect on the mesostriatal system was assessed in adult (4-14 month old) animals using a combination of behavioural, biochemical, histological and electrochemical techniques. Adult triple synuclein null (TKO) mice displayed no overt phenotype, and no change in the number of midbrain dopaminergic neurons. TKO mice were hyperactive in novel environments and exhibited elevated evoked release of dopamine in the striatum detected with fast-scan cyclic voltammetry. Elevated dopamine release was specific to the dorsal not ventral striatum and was accompanied by a decrease of dopamine tissue content. We confirmed a normal synaptic ultrastructure and a normal abundance of SNARE protein complexes in the dorsal striatum. Treatment of TKO animals with drugs affecting dopamine metabolism revealed normal rate of synthesis, enhanced turnover and reduced presynaptic striatal dopamine stores. Our data uniquely reveal the importance of the synuclein proteins in regulating neurotransmitter release from specific populations of midbrain dopamine neurons through mechanisms which differ from those reported in other neurons. The finding that the complete loss of synucleins leads to changes in dopamine handling by presynaptic terminals specifically in those regions preferentially vulnerable in Parkinson’s disease (PD) may ultimately inform on the selectivity of the disease process.
PMCID: PMC3154647  PMID: 21593311
10.  Increased striatal dopamine release and hyperdopaminergic-like behaviour in mice lacking both alpha-synuclein and gamma-synuclein 
Alpha-synuclein is intimately involved in the pathogenesis of Parkinson’s disease, and has been implicated in the regulation of synthesis, release and reuptake of dopamine. However, mice lacking members of the synuclein family have been reported to display no overt behavioural phenotype. This may be a result of compensatory upregulation of other synucleins during development. Here we report on behaviour and dopamine synapse function of alpha-synuclein null, gamma-synuclein null and alpha-gamma-synuclein double-null knockout mice. Double-null mice were hyperactive in a novel environment and alternated at a lower rate in a T-maze spontaneous alternation task, a phenotype reminiscent of mice expressing reduced levels of the dopamine transporter. To investigate a possible hyperdopaminergic phenotype in alpha-gamma-synuclein double-null mice, we used fast-scan cyclic voltammetry at carbon-fiber microelectrodes to assess dopamine release and reuptake in striatal slices from wild-type, alpha-null, gamma-null and double-null mice in real time. Double-null mice were found to have a two-fold increase in the extracellular concentration of dopamine detected after discrete electrical stimuli in the striatum. By measuring the rate of reuptake of dopamine and tissue dopamine content in these animals, we showed that the observed increase in size of striatal dopamine transients was not attributable to a decrease in reuptake of dopamine via the dopamine transporter, and can not be attributed to an increase in tissue dopamine levels in the striatum. Rather, we propose that loss of both alpha and gamma-synuclein causes an increase in release probability from dopaminergic synapses.
PMCID: PMC3145106  PMID: 18333965
cocaine; dopamine; knockout mice; Parkinson’s disease; synuclein; voltammetry
11.  The effect of α-synuclein knockdown on MPP+ toxicity in models of human neurons 
The European journal of neuroscience  2008;28(12):2459-2473.
The protein α-synuclein is central to the pathophysiology of Parkinson’s disease (PD) but its role in the development of neurodegeneration remains unclear. α-synuclein knockout mice develop without gross abnormality and are resistant to MPTP, a mitochondrial inhibitor widely used to model parkinsonism. Here we show that differentiated human dopaminergic neuron-like cells also have increased resistance to MPP+, the active metabolite of MPTP, when α-synuclein is knocked down using RNA interference. In attempting to understand how this occurred we found that lowering α-synuclein levels caused changes to intracellular vesicles, DAT and VMAT2, each of which is known to be important components of the early events leading to MPP+ toxicity. Knockdown of α-synuclein reduced the availability of DAT on the neuronal surface by 50%, decreased the total number of intracellular vesicles by 37% but increased the density of vesicular monoamine transporter (VMAT2) molecules per vesicle by 2.8-fold. However, these changes were not associated with any reduction in MPP+-induced superoxide production suggesting that α-synuclein knockdown may have other downstream effects which are important. We then showed that α-synuclein knockdown prevented MPP+-induced activation of nitric oxide synthase (NOS). Activation of NOS is an essential step in MPTP toxicity and increasing evidence points to nitrosative stress as being important in neurodegeneration. Overall, these results show that as well as having a number of effects on cellular events upstream of mitochondrial dysfunction α-synuclein affects pathways downstream of superoxide production, possibly involving regulation of NOS activity.
PMCID: PMC3132457  PMID: 19032594
12.  LRRK2 regulates autophagic activity and localizes to specific membrane microdomains in a novel human genomic reporter cellular model 
Human Molecular Genetics  2009;18(21):4022-4034.
Leucine rich repeat kinase 2 (LRRK2) mutations are the most common genetic cause of Parkinson's disease (PD) although LRRK2 function remains unclear. We report a new role for LRRK2 in regulating autophagy and describe the recruitment of LRRK2 to the endosomal–autophagic pathway and specific membrane subdomains. Using a novel human genomic reporter cellular model, we found LRRK2 to locate to membrane microdomains such as the neck of caveolae, microvilli/filopodia and intraluminal vesicles of multivesicular bodies (MVBs). In human brain and in cultured human cells LRRK2 was present in cytoplasmic puncta corresponding to MVBs and autophagic vacuoles (AVs). Expression of the common R1441C mutation from a genomic DNA construct caused impaired autophagic balance evident by the accumulation of MVBs and large AVs containing incompletely degraded material and increased levels of p62. Furthermore, the R1441C mutation induced the formation of skein-like abnormal MVBs. Conversely, LRRK2 siRNA knockdown increased autophagic activity and prevented cell death caused by inhibition of autophagy in starvation conditions. The work necessitated developing a new, more efficient recombineering strategy, which we termed Sequential insertion of Target with ovErlapping Primers (STEP) to seamlessly fuse the green fluorescent protein-derivative YPet to the human LRRK2 protein in the LRRK2 genomic locus carried by a bacterial artificial chromosome. Taken together our data demonstrate the functional involvement of LRRK2 in the endosomal–autophagic pathway and the recruitment to specific membrane microdomains in a physiological human gene expression model suggesting a novel function for this important PD-related protein.
PMCID: PMC2758136  PMID: 19640926
13.  A high throughput pharmaceutical screen identifies compounds with specific toxicity against BRCA2-deficient tumors 
Hereditary breast cancer is partly explained by germline mutations in BRCA1 and BRCA2. While patients carry heterozygous mutations, their tumors have typically lost the remaining wild-type allele. Selectively targeting BRCA-deficiency may therefore constitute an important therapeutic approach. Clinical trials applying this principle are underway, but it is unknown whether the compounds tested are optimal. It is therefore important to identify alternative compounds that specifically target BRCA-deficiency and to test new combination therapies to establish optimal treatment strategies.
Experimental Design:
We performed a high-throughput pharmaceutical screen on BRCA2-deficient mouse mammary tumor cells and isogenic controls with restored BRCA2 function. Subsequently, we validated positive hits in vitro and in vivo using mice carrying BRCA2-deficient mammary tumors.
Three alkylators – chlorambucil, melphalan and nimustine – displayed strong and specific toxicity against BRCA2-deficient cells. In vivo, these showed heterogeneous but generally strong BRCA2-deficient antitumor activity, with melphalan and nimustine outperforming cisplatin and the poly-(ADP-ribose)-polymerase (PARP) inhibitor olaparib (AZD2281) in this small study. In vitro drug combination experiments showed synergistic interactions between the alkylators and olaparib. Tumor intervention studies combining nimustine and olaparib resulted in recurrence-free survival exceeding 330 days in 3 out of 5 animals tested.
We generated and validated a platform for identification of compounds with specific activity against BRCA2-deficient cells that translates well to the preclinical setting. Our data call for the re-evaluation of alkylators – especially melphalan and nimustine – alone or in combination with PARP inhibitors for the treatment of breast cancers with a defective BRCA pathway.
PMCID: PMC2802735  PMID: 20008842
14.  LRRK2 is a component of granular alpha-synuclein pathology in the brainstem of Parkinson's disease 
Classical Parkinson's disease (PD) is characterized by the appearance of Lewy bodies (LBs) in affected brain regions, showing mostly compact alpha-synuclein deposition, in contrast with punctate or granular deposition, hypothesized to represent early stages of aggregation. Leucine-rich repeat kinase 2 (LRRK2) is the commonest mutated gene in inherited and idiopathic PD. LRRK2 mutation carriers display a diverse neuropathology, including alpha-synuclein and tau inclusions, suggesting an upstream role for LRRK2 in protein aggregation. We studied LRRK2 expression throughout the normal human brain with three different antibodies. We also examined the pattern of LRRK2 expression in relation to alpha-synuclein aggregation and LB formation in the brainstem of sporadic LB disease. Physiological LRRK2 expression was not restricted to regions preferentially affected in PD and LRRK2 often localised to the nuclear envelope in addition to the known cytoplasmic expression. In PD, we were able to consistently detect LRRK2 in the halo of a minority (~10%) of nigral LBs using three different antibodies. Only one antibody detected LRRK2 in the core of ~80% of classic LBs. In the lower brainstem, most notably in the dorsal motor nucleus of the vagus, we found previously unrecognised LRRK2 labelling of complex globular lesions, filled with LB-like-matter showing a punctate or granular staining for alpha-synuclein. This was often accompanied by strong LRRK2 expression within dystrophic neurites. Our findings confirm widespread physiological LRRK2 expression in the human brain and suggest an association of LRRK2 with possible early-stage alpha-synuclein pathology in the brainstem of PD.
PMCID: PMC2833010  PMID: 17971075
LRRK2; dardarin; alpha-synuclein; Parkinson's disease; brainstem; Lewy body
15.  Knock-out and transgenic mouse models of tauopathies 
Neurobiology of aging  2007;30(1):1-13.
Tauopathies, characterized by the dysfunction and aggregation of the microtubule-associated protein tau (MAPT), represent some of the most devastating neurodegenerative disorders afflicting the elderly, including Alzheimer's disease and progressive supranuclear palsy. Here we review the range of Mapt knock-out and MAPT transgenic mouse models which have proven successful at providing insights into the molecular mechanisms of neurodegenerative disease. In this overview we highlight several themes, including the insights such models provide into the cellular and molecular mechanisms of tauopathy, the direct relationship between neuropathology and behaviour, and the use of mouse models to help provide a platform for testing novel therapies. Mouse models have helped clarify the relationship between pathological forms of tau, cell death, and the emergence of disease, as well as the interaction between tau and other disease-associated molecules, such as the Aβ peptide. Finally, we discuss potential future MAPT genomic DNA models to investigate the importance of alternative splicing of the MAPT locus and its role in sporadic tauopathies.
PMCID: PMC2806682  PMID: 17590238
MAPT; Tau; Tauopathies; Transgenic mouse models; Knock-out mouse models; Alzheimer's disease; Progressive supranuclear palsy
16.  Haplotype-specific expression of the N-terminal exons 2 and 3 at the human MAPT locus 
Neurobiology of aging  2007;29(12):1923-1929.
The microtubule associated protein tau (MAPT) H1 haplotype shows a strong association to the sporadic neurodegenerative diseases progressive supranuclear palsy and corticobasal degeneration. The functional biological mechanisms behind the genetic association have started to emerge with differences recently shown in haplotype splicing of the neuropathologically relevant exon 10. Here we investigate the hypothesis that expression of the alternatively spliced N-terminal exons also differs between the two MAPT haplotypes. We performed allele-specific gene expression analysis on a H1/H2 heterozygous human neuronal cell line model and 14 H1/H2 heterozygous human post-mortem brain tissues from two brain regions. In both cell culture and post-mortem brain tissue, we show that the protective MAPT H2 haplotype significantly expresses two-fold more 2N (exons 2+ 3+) MAPT transcripts than the disease-associated H1 haplotype. We suggest that inclusion of exon 3 in MAPT transcripts may contribute to protecting H2 carries from neurodegeneration.
PMCID: PMC2801070  PMID: 17602795
17.  Functional MAPT haplotypes: Bridging the gap between genotype and neuropathology 
Neurobiology of disease  2007;27(1):1-10.
The microtubule associated protein tau (MAPT) locus has long been associated with sporadic neurodegenerative disease, notably progressive supranuclear palsy and corticobasal degeneration, and more recently with Alzheimer’s disease and Parkinson’s disease. However, the functional biological mechanisms behind the genetic association have only now started to emerge. The genomic architecture in the region spanning MAPT is highly complex, and includes a ~1.8 Mb block of linkage disequilibrium (LD). The region is divided into two major haplotypes, H1 and H2, defined by numerous single nucleotide polymorphisms and a 900 kb inversion which suppresses recombination. Fine mapping of the MAPT region has identified sub-clades of the MAPT H1 haplotype which are specifically associated with neurodegenerative disease. Here we briefly review the role of MAPT in sporadic and familial neurodegenerative disease, and then discuss recent work which, for the first time, proposes functional mechanisms to link MAPT haplotypes with the neuropathology seen in patients.
PMCID: PMC2801069  PMID: 17555970
MAPT; H1 haplotype; progressive supranuclear palsy; tauopathy; splicing; gene expression; functional polymorphisms; susceptibility mechanisms
18.  A Common Variant Associated with Dyslexia Reduces Expression of the KIAA0319 Gene 
PLoS Genetics  2009;5(3):e1000436.
Numerous genetic association studies have implicated the KIAA0319 gene on human chromosome 6p22 in dyslexia susceptibility. The causative variant(s) remains unknown but may modulate gene expression, given that (1) a dyslexia-associated haplotype has been implicated in the reduced expression of KIAA0319, and (2) the strongest association has been found for the region spanning exon 1 of KIAA0319. Here, we test the hypothesis that variant(s) responsible for reduced KIAA0319 expression resides on the risk haplotype close to the gene's transcription start site. We identified seven single-nucleotide polymorphisms on the risk haplotype immediately upstream of KIAA0319 and determined that three of these are strongly associated with multiple reading-related traits. Using luciferase-expressing constructs containing the KIAA0319 upstream region, we characterized the minimal promoter and additional putative transcriptional regulator regions. This revealed that the minor allele of rs9461045, which shows the strongest association with dyslexia in our sample (max p-value = 0.0001), confers reduced luciferase expression in both neuronal and non-neuronal cell lines. Additionally, we found that the presence of this rs9461045 dyslexia-associated allele creates a nuclear protein-binding site, likely for the transcriptional silencer OCT-1. Knocking down OCT-1 expression in the neuronal cell line SHSY5Y using an siRNA restores KIAA0319 expression from the risk haplotype to nearly that seen from the non-risk haplotype. Our study thus pinpoints a common variant as altering the function of a dyslexia candidate gene and provides an illustrative example of the strategic approach needed to dissect the molecular basis of complex genetic traits.
Author Summary
Dyslexia, or reading disability, is a common disorder caused by both genetic and environmental factors. Genetic studies have implicated a number of genes as candidates for playing a role in dyslexia. We functionally characterized one such gene (KIAA0319) to identify variant(s) that might affect gene expression and contribute to the disorder. We discovered a variant residing outside of the protein-coding region of KIAA0319 that reduces expression of the gene. This variant creates a binding site for the transcription factor OCT-1. Previous studies have shown that OCT-1 binding to a specific DNA sequence upstream of a gene can reduce the expression of that gene. In this case, reduced KIAA0319 expression could lead to improper development of regions of the brain involved in reading ability. This is the first study to identify a functional variant implicated in dyslexia. More broadly, our study illustrates the steps that can be utilized for identifying mutations causing other complex genetic disorders.
PMCID: PMC2653637  PMID: 19325871
19.  An S/MAR-based infectious episomal genomic DNA expression vector provides long-term regulated functional complementation of LDLR deficiency 
Nucleic Acids Research  2007;35(15):e98.
Episomal gene expression vectors offer a safe and attractive alternative to integrating vectors. Here we describe the development of a high capacity episomal vector system exploiting human episomal retention sequences to provide efficient vector maintenance and regulated gene expression through the delivery of a genomic DNA locus. The iBAC-S/MAR vector is capable of the infectious delivery and retention of large genomic DNA transgenes by exploiting the high transgene capacity of herpes simplex virus type 1 (HSV-1) and the episomal retention properties of the scaffold/matrix attachment region (S/MAR). The iBAC-S/MAR vector was used to deliver and maintain a 135 kb genomic DNA insert carrying the human low density lipoprotein receptor (LDLR) genomic DNA locus at high efficiency in CHO ldlr−/− a7 cells. Long-term studies on CHO ldlr−/− a7 clonal cell lines carrying iBAC-S/MAR-LDLR demonstrated low copy episomal stability of the vector for >100 cell generations without selection. Expression studies demonstrated that iBAC-S/MAR-LDLR completely restored LDLR function in CHO ldlr−/− a7 cells to physiological levels and that this expression can be repressed by ∼70% by high sterol levels, recapitulating the same feedback regulation seen at the endogenous LDLR locus. This vector overcomes the major problems of vector integration and unregulated transgene expression.
PMCID: PMC1976449  PMID: 17675302
20.  Sequences Adjacent to oriP Improve the Persistence of Epstein-Barr Virus-Based Episomes in B Cells 
Journal of Virology  2001;75(22):11249-11252.
Epstein-Barr virus (EBV) oriP and the EBV nuclear antigen 1 (EBNA-1) protein allow persistence of EBV-based episomes. A nuclear matrix attachment region (MAR) spans oriP and the adjacent region of the EBV genome containing the EBV-expressed RNAs. Here, we show that episomes with the MAR are retained significantly more efficiently in EBV-positive B cells than episomes containing oriP alone.
PMCID: PMC114707  PMID: 11602767

Results 1-20 (20)