Tara M. Caffrey

LRRTM1 on chromosome 2p12 is a maternally suppressed gene that is associated paternally with handedness and schizophrenia

Left–right asymmetrical brain function underlies much of human cognition, behavior and emotion. Abnormalities of cerebral asymmetry are associated with schizophrenia and other neuropsychiatric disorders. The molecular, developmental and evolutionary origins of human brain asymmetry are unknown. We found significant association of a haplotype upstream of the gene LRRTM1 (Leucine-rich repeat transmembrane neuronal 1) with a quantitative measure of human handedness in a set of dyslexic siblings, when the haplotype was inherited paternally (P=0.00002). While we were unable to find this effect in an epidemiological set of twin-based sibships, we did find that the same haplotype is overtransmitted paternally to individuals with schizophrenia/schizoaffective disorder in a study of 1002 affected families (P=0.0014). We then found direct confirmatory evidence that LRRTM1 is an imprinted gene in humans that shows a variable pattern of maternal downregulation. We also showed that LRRTM1 is expressed during the development of specific forebrain structures, and thus could influence neuronal differentiation and connectivity. This is the first potential genetic influence on human handedness to be identified, and the first putative genetic effect on variability in human brain asymmetry. LRRTM1 is a candidate gene for involvement in several common neurodevelopmental disorders, and may have played a role in human cognitive and behavioral evolution.

Haplotype-specific expression of the N-terminal exons 2 and 3 at the human MAPT locus

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.

Functional MAPT haplotypes: bridging the gap between genotype and neuropathology.

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 Alzheimers disease and Parkinsons 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 approximately 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.

Induced pluripotent stem cell (iPSC)-derived dopaminergic models of Parkinson's disease.

iPSCs (induced pluripotent stem cells) are the newest tool used to model PD (Parkinsons disease). Fibroblasts from patients carrying pathogenic mutations that lead to PD have been reprogrammed into iPSCs, which can subsequently be differentiated into important cell types. Given the characteristic loss of dopaminergic neurons in the substantia nigra pars compacta of PD patients, iPSC-derived midbrain dopaminergic neurons have been generated to investigate pathogenic mechanisms in this important cell type as a means of modelling PD. iPSC-derived cultures studied so far have been made from patients carrying mutations in LRRK2 (leucine-rich repeat kinase 2), PINK1 [PTEN (phosphatase and tensin homologue deleted on chromosome 10)-induced putative kinase 1], PARK2 (encodes parkin) or GBA (β-glucocerebrosidase), in addition to those with SNCA (α-synuclein) multiplication and idiopathic PD. In some cases, isogenic control lines have been created to minimize inherent variability between lines from different individuals. Disruptions in autophagy, mitochondrial function and dopamine biology at the synapse have been described. Future applications for iPSC-derived models of PD beyond modelling include drug testing and the ability to investigate the genetic diversity of PD.

The role of MAPT sequence variation in mechanisms of disease susceptibility

The microtubule-associated protein tau (MAPT or tau) is of great interest in the field of neurodegeneration as there is a well-established genetic link between the MAPT gene locus and tauopathies, a diverse group of neurodegenerative dementias and movement disorders. The genomic architecture in the region spanning the MAPT locus contains a ~1.8 Mb block of linkage disequilibrium characterized by two major haplotypes: H1 and H2. Recent studies have established strong genetic association between the MAPT locus and neurodegenerative disease and uncovered haplotype-specific differences in expression and alternative splicing of MAPT transcripts. Integrating genetic association data and gene expression data to understand how non-coding genetic variation at a gene locus affects gene expression and leads to susceptibility to disease is a high priority in disease genetics, and the MAPT locus provides an excellent paradigm for this. In the absence of protein-coding changes caused by haplotype sequence variation, altered levels of protein expression or altered ratios of isoform expression are excellent candidate mechanisms to link the MAPT genetic disease association with biological function. The use of novel transgenic and endogenous genetic models are required to understand the role of MAPT sequence variation in mechanisms of disease susceptibility.

MAPT Genetic Variation and Neuronal Maturity Alter Isoform Expression Affecting Axonal Transport in iPSC-Derived Dopamine Neurons

Summary The H1 haplotype of the microtubule-associated protein tau (MAPT) locus is genetically associated with neurodegenerative diseases, including Parkinsons disease (PD), and affects gene expression and splicing. However, the functional impact on neurons of such expression differences has yet to be fully elucidated. Here, we employ extended maturation phases during differentiation of induced pluripotent stem cells (iPSCs) into mature dopaminergic neuronal cultures to obtain cultures expressing all six adult tau protein isoforms. After 6 months of maturation, levels of exon 3+ and exon 10+ transcripts approach those of adult brain. Mature dopaminergic neuronal cultures display haplotype differences in expression, with H1 expressing 22% higher levels of MAPT transcripts than H2 and H2 expressing 2-fold greater exon 3+ transcripts than H1. Furthermore, knocking down adult tau protein variants alters axonal transport velocities in mature iPSC-derived dopaminergic neuronal cultures. This work links haplotype-specific MAPT expression with a biologically functional outcome relevant for PD.

MAPT GENETIC VARIATION AND NEURONAL MATURITY ALTER ISOFORM EXPRESSION AFFECTING AXONAL TRANSPORT IN IPSC-DERIVED NEURONS

Background:The H1 Haplotype of the microtubule associated protein tau (MAPT) locus is genetically associated with neurodegenerative diseases, including Parkinson’s disease, progressive supranuclear palsy and and corticobasal degeneration (CBD), and affects gene expression and splicing. However the functional impact of haplotype-specific sequence expression in neurons has yet to be fully elucidated.Methods:Here, we differentiated dopaminergic neuronal cultures from induced pluripotent stem cells (iPSCs) heterozygous for the MAPT H1/H2 haplotypes from three individuals to assess the effect of MAPT haplotype on gene expression and the role of tau protein isoforms in dopamine neurons. We measured total tau expression as well as the expression of the alternatively spliced exon3 and 10 at time points up to six months maturation and developed allele-specific expression assays to quantify the expression from the H1 and H2 alleles within the heterozygous cultures. Short-hairpin RNAs were generated to knockdown total tau or specific isoforms in long term cultures to determine the functional effect of tau expression on axonal transport. Results:After six months of maturation, levels of expression of exon 3+ and exon 10+ transcripts approach adult levels with strong expression of 2N and 4R tau protein isoforms evident. Mature dopaminergic neuronal cultures display haplotype differences in expression with H1 expressing 22% higher levels of MAPT transcripts than H2, and H2 expressing 2-fold greater exon 3+ transcripts than H1. We also identified a novel variant in MAPTintron 10 that increased the inclusion of exon 10 by two-fold. Furthermore, altering tau protein expression altered axonal transport velocities in mature iPSCderived dopaminergic neuronal cultures. Conclusions: This model is suitable to study common genetic variation, displaying significant haplotype-specific differences in MAPT expression and splicing. This works links haplotype-specific MAPT expression with axonal transport function, a biologically functional outcome relevant for neurons. The physiological MAPT expression profile makes iPSC-derived dopaminergic neurons a highly suitable system to study the genetic regulation of tau expression in a tractable human neuronal culture model.