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.
Nicastrin; Haplotype variation; Functional genomics; Alzheimer's disease; γ-Secretase complex
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.
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.
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.
cocaine; dopamine; knockout mice; Parkinson’s disease; synuclein; voltammetry
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.
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.
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.
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.
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.
LRRK2; dardarin; alpha-synuclein; Parkinson's disease; brainstem; Lewy body
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.
MAPT; Tau; Tauopathies; Transgenic mouse models; Knock-out mouse models; Alzheimer's disease; Progressive supranuclear palsy
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.
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.
MAPT; H1 haplotype; progressive supranuclear palsy; tauopathy; splicing; gene expression; functional polymorphisms; susceptibility mechanisms
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.
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.
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.
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.