Biomedical subjects

Frederick J Livesey

Publications and source records attributed to Frederick J Livesey.

9 recordsLinked to original sources

PubMed2025

Tau uptake by human neurons depends on receptor LRP1 and kinase LRRK2.

Extracellular release and uptake of pathogenic forms of the microtubule-associated protein tau contribute to the pathogenesis of several neurodegenerative diseases, including Alzheimer's disease. Defining the cellular mechanisms and pathways for tau entry to human neurons is essential to understanding tauopathy pathogenesis and enabling the rational design of disease-modifying therapeutics. Here, whole-genome, loss-of-function CRISPR screens in human iPSC-derived excitatory neurons, the major neuronal cell type affected in these diseases, provide insights into the different cellular pathways for uptake of extracellular monomeric and fibrillar tau. Monomeric and fibrillar tau are both taken up by human neurons by receptor-mediated endocytosis, but involve different routes of entry at the neuronal surface: the low-density lipoprotein LRP1 is the primary receptor for monomeric tau, but contributes less to fibrillar tau entry. Similarly, endocytosis of monomeric tau is dependent on the familial Parkinson's disease gene LRRK2, but not required for endocytosis of fibrillar tau. These findings implicate LRP1 and LRRK2 in the pathogenesis of tauopathies and Parkinson's disease, and identify LRRK2 as a potential therapeutic target for altering progression of these diseases.

Humans↗

PubMed2006

A role for the transcriptional repressor REST in maintaining the phenotype of neurosecretory-deficient PC12 cells.

The rat PC12 variant cell line, A35C, lacks regulated secretory organelles due to a selective transcriptional block. Hence, A35C may provide clues about the mechanisms that underlie control of neurosecretion. We used mRNA microarray profiling to examine gene expression in A35C. Genes for regulated secretory proteins were down-regulated, while other membrane trafficking pathways were unaffected. A subset of genes repressed in A35C contain binding sites for the neuronal transcriptional repressor, RE1-silencing transcription factor (REST), and REST is expressed in A35C but not normal PC12 cells. Blocking the activity of REST in A35C using a dominant-negative construct induced the reappearance of mRNAs for synaptophysin, chromogranin A, synaptotagmin IV and the beta3 subunit of the voltage-gated sodium channel (Scn3b), all of which contain RE1 sites in their genes. In the case of Scn3b, the corresponding protein was also re-expressed. Granule and synaptic vesicle proteins were not re-expressed at the protein level, despite reactivation of their mRNA, suggesting the existence of additional post-transcriptional control for these proteins. Our work identifies one of the mechanisms underlying the phenotype of neurosecretory-deficient neuroendocrine cells, and begins to define the critical components that determine a key aspect of the neuroendocrine phenotype.

Animals↗

PubMed2006

A role for Dicer in immune regulation.

Micro RNAs (miRNAs) regulate gene expression at the posttranscriptional level. Here we show that regulatory T (T reg) cells have a miRNA profile distinct from conventional CD4 T cells. A partial T reg cell-like miRNA profile is conferred by the enforced expression of Foxp3 and, surprisingly, by the activation of conventional CD4 T cells. Depleting miRNAs by eliminating Dicer, the RNAse III enzyme that generates functional miRNAs, reduces T reg cell numbers and results in immune pathology. Dicer facilitates, in a cell-autonomous fashion, the development of T reg cells in the thymus and the efficient induction of Foxp3 by transforming growth factor beta. These results suggest that T reg cell development involves Dicer-generated RNAs.

Animals↗

PubMed2006

Electrophysiological and gene expression profiling of neuronal cell types in mammalian neocortex.

It is a challenging question to understand how different neuronal types are organized into a complex architecture in the cortex, an architecture which is also adapted in different regions to subserve very different functions. Recent developments in genetic and molecular techniques have opened up the possibility of using gene expression profiling for neuronal cell typing, with the aim of uncovering novel cell types and the underlying mechanisms which generate and maintain neuronal heterogeneity in the cortex. This review introduces some current ideas about neuronal cell types in the cortex and describes recent approaches to expression profiling for defining cortical neuronal cell types.

Animals↗

PubMed2006

Comparative evaluation of linear and exponential amplification techniques for expression profiling at the single-cell level.

BACKGROUND: Single-cell microarray expression profiling requires 108-109-fold amplification of the picogram amounts of total RNA typically found in eukaryotic cells. Several methods for RNA amplification are in general use, but little consideration has been given to the comparative analysis of those methods in terms of the overall validity of the data generated when amplifying from single-cell amounts of RNA, rather than their empirical performance in single studies. RESULTS: We tested the performance of three methods for amplifying single-cell amounts of RNA under ideal conditions: T7-based in vitro transcription; switching mechanism at 5' end of RNA template (SMART) PCR amplification; and global PCR amplification. All methods introduced amplification-dependent noise when mRNA was amplified 108-fold, compared with data from unamplified cDNA. PCR-amplified cDNA demonstrated the smallest number of differences between two parallel replicate samples and the best correlation between independent amplifications from the same cell type, with SMART outperforming global PCR amplification. SMART had the highest true-positive rate and the lowest false-positive rate when comparing expression between two different cell types, but had the lowest absolute discovery rate of all three methods. Direct comparison of the performance of SMART and global PCR amplification on single-cell amounts of total RNA and on single neural stem cells confirmed these findings. CONCLUSION: Under the conditions tested, PCR amplification was more reliable than linear amplification for detecting true expression differences between samples. SMART amplification had a higher true-positive rate than global amplification, but at the expense of a considerably lower absolute discovery rate and a systematic compression of observed expression ratios.

3T3 Cells↗

PubMed2005

Genomic characterisation of a Fgf-regulated gradient-based neocortical protomap.

Recent findings support a model for neocortical area formation in which neocortical progenitor cells become patterned by extracellular signals to generate a protomap of progenitor cell areas that in turn generate area-specific neurons. The protomap is thought to be underpinned by spatial differences in progenitor cell identity that are reflected at the transcriptional level. We systematically investigated the nature and composition of the protomap by genomic analyses of spatial and temporal neocortical progenitor cell gene expression. We did not find gene expression evidence for progenitor cell organisation into domains or compartments, instead finding rostrocaudal gradients of gene expression across the entire neocortex. Given the role of Fgf signalling in rostrocaudal neocortical patterning, we carried out an in vivo global analysis of cortical gene expression in Fgfr1 mutant mice, identifying consistent alterations in the expression of candidate protomap elements. One such gene, Mest, was predicted by those studies to be a direct target of Fgf8 signalling and to be involved in setting up, rather than implementing, the progenitor cell protomap. In support of this, we confirmed Mest as a direct transcriptional target of Fgf8-regulated signalling in vitro. Functional studies demonstrated that this gene has a role in establishing patterned gene expression in the developing neocortex, potentially by acting as a negative regulator of the Fgf8-controlled patterning system.

Animals↗

PubMed2004

A dynamic switch in the replication timing of key regulator genes in embryonic stem cells upon neural induction.

Mammalian embryonic stem (ES) cells can either self-renew or generate progenitor cells that have a more restricted developmental potential. This provides an important model system to ask how pluripotency, cell commitment and differentiation are regulated at the level of chromatin-based changes that distinguish stem cells from their differentiated progeny. Here we show that the differentiation of ES cells to neural progenitors results in dynamic changes in the epigenetic status of multiple genes that encode transcription factors critical for early embryonic development or lineage specification. In particular, we demonstrate that DNA replication at a subset of neural-associated genes including Pax3, Pax6, Irx3, Nkx2.9 and Mash1 is advanced upon neural induction, consistent with increased locus accessibility. Conversely, many ES-associated genes including Oct4, Nanog, Utf1, Foxd3, Cripto and Rex1 that replicate early in ES cells switch their replication timing to later in S-phase in response to differentiation. Detailed analysis of the Rex1 locus reveals that delayed replication extends to a 2.8 Mb region surrounding the gene and is associated with substantial reductions in the level of histone H3K9 and H4 acetylation at the promoter. These results show that loss of pluripotency (and lineage choice) is associated with extensive and predictable changes in the replication timing of key regulator genes.

Animals↗

PubMed2004

Molecular organization of the ferret visual thalamus.

The visual system encodes and deciphers information using parallel, anatomically segregated, processing streams. To reveal patterns of gene expression in the visual thalamus correlated with physiological processing streams, we designed a custom ferret cDNA microarray. By isolating specific subregions and layers of the thalamus, we identified a set of transcription factors, including Zic2, Islet1, and Six3, the unique distribution profiles of which differentiated the lateral geniculate nucleus (LGN) from the associated perigeniculate nucleus. Within the LGN, odd homeobox1 differentiated the A layers, which contain X cells and Y cells, from the C layers. One neuron-specific protein, Purkinje cell protein 4 (PCP4), was strongly expressed in Y cells in the ferret LGN and in the magnocellular layers of the primate LGN. In the ferret LGN, PCP4 expression began as early as postnatal day 7 (P7), suggesting that Y cells are already specified by P7. These results reveal a rich molecular repertoire that correlates with functional divisions of the LGN.

Animals↗

PubMed2003

Prox1 function controls progenitor cell proliferation and horizontal cell genesis in the mammalian retina.

Retinal progenitor cells regulate their proliferation during development so that the correct number of each cell type is made at the appropriate time. We found that the homeodomain protein Prox1 regulates the exit of progenitor cells from the cell cycle in the embryonic mouse retina. Cells lacking Prox1 are less likely to stop dividing, and ectopic expression of Prox1 forces progenitor cells to exit the cell cycle. During retinogenesis, Prox1 can be detected in differentiating horizontal, bipolar and AII amacrine cells. Horizontal cells are absent in retinae of Prox1-/- mice and misexpression of Prox1 in postnatal progenitor cells promotes horizontal-cell formation. Thus, Prox1 activity is both necessary and sufficient for progenitor-cell proliferation and cell-fate determination in the vertebrate retina.

Animals↗