[Show abstract][Hide abstract] ABSTRACT: We have applied the CRISPR/Cas9 system to Drosophila S2 cells to generate targeted genetic mutations in more than 85% of alleles. By targeting a constitutive exon of the AGO1 gene, we demonstrate homozygous mutation in up to 82% of cells, thereby allowing the study of genetic knockouts in a Drosophila cell line for the first time. We have shown that homologous gene targeting is possible at 1-4% efficiency using this system, allowing for the construction of defined insertions and deletions. We demonstrate that a 1 kb homology arm length is optimal for integration by homologous gene targeting, and demonstrate its efficacy by tagging the endogenous AGO1 protein. This technology enables controlled genetic manipulation in Drosophila cell lines, and its simplicity offers the opportunity to study cellular phenotypes genome-wide.
[Show abstract][Hide abstract] ABSTRACT: iPSCs (induced pluripotent stem cells) are the newest tool used to model PD (Parkinson's 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.
Biochemical Society Transactions 12/2013; 41(6):1503-1508.
[Show abstract][Hide abstract] ABSTRACT: Angiogenesis is thought to decrease stroke size and improve behavioral outcomes and therefore several clinical trials are seeking to augment it. Galectin-3 (Gal-3) expression increases after middle cerebral artery occlusion (MCAO) and has been proposed to limit damage 3days after stroke. We carried out mild MCAO that damages the striatum but spares the cerebral cortex and SVZ. Gal-3 gene deletion prevented vascular endothelial growth factor (VEGF) upregulation after MCAO. This inhibited post-MCAO increases in endothelial proliferation and angiogenesis in the striatum allowing us to uniquely address the function of angiogenesis in this model of stroke. Apoptosis and infarct size were unchanged in Gal-3(-/-) mice 7 and 14days after MCAO, suggesting angiogenesis does not affect lesion size. Microglial and astrocyte activation/proliferation after MCAO were similar in wild type and Gal-3(-/-) mice. In addition, openfield activity, motor hemiparesis, proprioception, reflex, tremors and grooming behaviors were essentially identical between WT and Gal-3(-/-) mice at 1, 3, 7, 10 and 14days after MCAO, suggesting penumbral angiogenesis has limited impact on behavioral recovery. In addition to angiogenesis, increased adult subventricular zone (SVZ) neurogenesis is thought to provide neuroprotection after stroke in animal models. SVZ neurogenesis and migration to lesion was overall unaffected by the loss of Gal-3, suggesting no compensation for the lack of angiogenesis in Gal-3(-/-) mice. Because angiogenesis and neurogenesis are usually coordinately regulated, identifying their individual effects on stroke has hitherto been difficult. These results show that Gal-3 is necessary for angiogenesis in stroke in a VEGF-dependant manner, but suggest that angiogenesis may be dispensable for post-stroke endogenous repair, therefore drawing into question the clinical utility of augmenting angiogenesis.
[Show abstract][Hide abstract] ABSTRACT: Autism spectrum disorder (ASD) is a highly heritable complex neurodevelopmental condition characterized by impairments in social interaction and communication and restricted and repetitive behaviors. Although roles for both de novo and familial genetic variation have been documented, the underlying disease mechanisms remain poorly elucidated. In this study, we defined and explored distinct etiologies of genetic variants that affect genes regulated by Fragile-X mental retardation protein (FMRP), thought to play a key role in neuroplasticity and neuronal translation, in ASD-affected individuals. In particular, we developed the Trend test, a pathway-association test that is able to robustly detect multiple-hit etiologies and is more powerful than existing approaches. Exploiting detailed spatiotemporal maps of gene expression within the human brain, we identified four discrete FMRP-target subpopulations that exhibit distinct functional biases and contribute to ASD via different types of genetic variation. We also demonstrated that FMRP target genes are more likely than other genes with similar expression patterns to contribute to disease. We developed the hypothesis that FMRP targets contribute to ASD via two distinct etiologies: (1) ultra-rare and highly penetrant single disruptions of embryonically upregulated FMRP targets ("single-hit etiology") or (2) the combination of multiple less penetrant disruptions of nonembryonic, synaptic FMRP targets ("multiple-hit etiology"). The Trend test provides rigorous support for a multiple-hit genetic etiology in a subset of autism cases and is easily extendible to combining information from multiple types of genetic variation (i.e., copy-number and exome variants), increasing its value to next-generation sequencing approaches.
The American Journal of Human Genetics 11/2013; 93(5):825-39.
[Show abstract][Hide abstract] ABSTRACT: Objective: Obtaining new details for rotational motion of left ventricular (LV) segments using velocity encoding cardiac MR and correlating the regional motion patterns to LV insertion sites. Methods: Cardiac MR examinations were performed on 14 healthy volunteers aged between 19 and 26 years. Peak rotational velocities and circumferential velocity curves were obtained for 16 ventricular segments. Results: Reduced peak clockwise velocities of anteroseptal segments (i.e. Segments 2 and 8) and peak counterclockwise velocities of inferoseptal segments (i.e. Segments 3 and 9) were the most prominent findings. The observations can be attributed to the LV insertion sites into the right ventricle, limiting the clockwise rotation of anteroseptal LV segments and the counterclockwise rotation of inferoseptal segments as viewed from the apex. Relatively lower clockwise velocities of Segment 5 and counterclockwise velocities of Segment 6 were also noted, suggesting a cardiac fixation point between these two segments, which is in close proximity to the lateral LV wall. Conclusion: Apart from showing different rotational patterns of LV base, mid ventricle and apex, the study showed significant differences in the rotational velocities of individual LV segments. Correlating regional wall motion with known orientation of myocardial aggregates has also provided new insights into the mechanisms of LV rotational motions during a cardiac cycle. Advances in knowledge: LV insertion into the right ventricle limits the clockwise rotation of anteroseptal LV segments and the counterclockwise rotation of inferoseptal segments adjacent to the ventricular insertion sites. The pattern should be differentiated from wall motion abnormalities in cardiac pathology.
The British journal of radiology 11/2013; 86(1031):20130326.
[Show abstract][Hide abstract] ABSTRACT: Identifying behaviorally relevant sounds in the presence of background noise is one of the most important and poorly understood challenges faced by the auditory system. An elegant solution to this problem would be for the auditory system to represent sounds in a noise-invariant fashion. Since a major effect of background noise is to alter the statistics of the sounds reaching the ear, noise-invariant representations could be promoted by neurons adapting to stimulus statistics. Here we investigated the extent of neuronal adaptation to the mean and contrast of auditory stimulation as one ascends the auditory pathway. We measured these forms of adaptation by presenting complex synthetic and natural sounds, recording neuronal responses in the inferior colliculus and primary fields of the auditory cortex of anaesthetized ferrets, and comparing these responses with a sophisticated model of the auditory nerve. We find that the strength of both forms of adaptation increases as one ascends the auditory pathway. To investigate whether this adaptation to stimulus statistics contributes to the construction of noise-invariant sound representations, we also presented complex, natural sounds embedded in stationary noise, and used a decoding approach to assess the noise tolerance of the neuronal population code. We find that the code for complex sounds in the periphery is affected more by the addition of noise than the cortical code. We also find that noise tolerance is correlated with adaptation to stimulus statistics, so that populations that show the strongest adaptation to stimulus statistics are also the most noise-tolerant. This suggests that the increase in adaptation to sound statistics from auditory nerve to midbrain to cortex is an important stage in the construction of noise-invariant sound representations in the higher auditory brain.
[Show abstract][Hide abstract] ABSTRACT: VEGF (vascular endothelial growth factor) is an endothelial cell mitogen responsible for physiological and pathological angiogenesis. Abnormal regulation of VEGF expression in anterior pituitary folliculostellate (FS) cells has been implicated in pituitary tumour progression. FS and endocrine cells express VEGF which is thought to be secreted by the constitutive pathway. The present study investigated the mechanism of VEGF secretion in TtT/GF cells, a mouse FS cell line. TtT/GF cells were shown to express VEGF164 , the most potent and bioavailable isoform of VEGF. Immunofluorescence and immunogold electron microscopy localised VEGF to the cytoplasm and small electron-lucent vesicles. PACAP, a well-documented stimulant of VEGF secretion, caused a robust increase in VEGF secretion over 24h. Glyburide, an ABCA1 and KATP channel blocker, also caused an increase in VEGF secretion when applied alone, and amplified the response to PACAP. Other ABCA1 transport blockers did not affect VEGF secretion. Exposure of TtT/GF cells to cycloheximide with PACAP or glyburide inhibited the increased secretion of VEGF, consistent with control of secretion at the transcription level. The SUR2B/Kir6.1 form of KATP channels was shown to be expressed by TtT/GF cells. Diazoxide, a KATP activator, inhibited PACAP-and PACAP+glyburide-stimulated VEGF secretion, but not that of glyburide alone. These data suggest that KATP channels are expressed by FS cells and play a significant role in the control of VEGF secretion and that activation of KATP channels inhibits the secretion of VEGF at the level of transcription. This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: For over a century, the duplex theory has guided our understanding of human sound localization in the horizontal plane. According to this theory, the auditory system uses interaural time differences (ITDs) and interaural level differences (ILDs) to localize low-frequency and high-frequency sounds, respectively. Whilst this theory successfully accounts for the localization of tones by humans, some species show very different behaviour. Ferrets are widely used for studying both clinical and fundamental aspects of spatial hearing, but it is not known whether the duplex theory applies to this species or, if so, to what extent the frequency range over which each binaural cue is used depends on acoustical or neurophysiological factors. To address these issues, we trained ferrets to lateralize tones presented over earphones and found that the frequency dependence of ITD and ILD sensitivity broadly paralleled that observed in humans. Compared with humans, however, the transition between ITD and ILD sensitivity was shifted toward higher frequencies. We found that the frequency dependence of ITD sensitivity in ferrets can partially be accounted for by acoustical factors, although neurophysiological mechanisms are also likely to be involved. Moreover, we show that binaural cue sensitivity can be shaped by experience, as training ferrets on a 1-kHz ILD task resulted in significant improvements in thresholds that were specific to the trained cue and frequency. Our results provide new insights into the factors limiting the use of different sound localization cues and highlight the importance of sensory experience in shaping the underlying neural mechanisms.
European Journal of Neuroscience 10/2013;
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