-
[show abstract]
[hide abstract]
ABSTRACT: BACKGROUND: Evidence from genetic association studies implicate genes involved in neural migration associated with schizophrenia risk. Neural stem/progenitor cell cultures (neurosphere-derived cells) from olfactory mucosa of schizophrenia patients have significantly dysregulated expression of genes in focal adhesion kinase (FAK) signaling, a key pathway regulating cell adhesion and migration. The aim of this study was to investigate whether olfactory neurosphere-derived cells from schizophrenia patients have altered cell adhesion, cell motility, and focal adhesion dynamics. METHODS: Olfactory neurosphere-derived cells from nine male schizophrenia patients and nine male healthy control subjects were used. Cells were assayed for cell adhesion and cell motility and analyzed for integrins and FAK proteins. Focal adhesions were counted and measured in fixed cells, and time-lapse imaging was used to assess cell motility and focal adhesion dynamics. RESULTS: Patient-derived cells were less adhesive and more motile than cells derived from healthy control subjects, and their motility was reduced to control cell levels by integrin-blocking antibodies and by inhibition of FAK. Vinculin-stained focal adhesion complexes were significantly smaller and fewer in patient cells. Time-lapse imaging of cells expressing FAK tagged with green fluorescent protein revealed that the disassembly of focal adhesions was significantly faster in patient cells. CONCLUSIONS: The evidence for altered motility and focal adhesion dynamics in patient-derived cells is consistent with dysregulated gene expression in the FAK signaling pathway in these cells. Alterations in cell adhesion dynamics and cell motility could bias the trajectory of brain development in schizophrenia.
Biological psychiatry 03/2013; · 8.93 Impact Factor
-
Greger Abrahamsen,
Yongjun Fan,
Nicholas Matigian,
Gautam Wali,
Bernadette Bellette,
Ratneswary Sutharsan,
Jyothy Raju,
Stephen A Wood,
David Veivers,
Carolyn M Sue,
Alan Mackay-Sim
[show abstract]
[hide abstract]
ABSTRACT: Hereditary Spastic Paraplegia (HSP) leads to progressive gait disturbances with lower limb muscle weakness and spasticity. Mutations in SPAST are a major cause of adult-onset, autosomal-dominant HSP. Spastin, the protein encoded by SPAST, is a microtubule-severing protein that is enriched in the distal axon of corticospinal motor neurons which degenerate in HSP patients. Animal and cell models have identified functions of spastin and mutated spastin but these models lack the gene dosage, mutation variability and genetic background that characterize patients with the disease. In this study, this genetic variability is encompassed by comparing neural stem/progenitor cells derived from biopsies of the olfactory mucosa of HSP patients with SPAST mutations and healthy controls, in order to identify cell functions altered in HSP. Patient-derived cells were similar to control-derived cells in proliferation and multiple metabolic functions but had major dysregulation of gene expression, with 57% of all mRNA transcripts affected, including many associated with microtubule dynamics. Compared to Control cells, Patient-derived cells had 50% spastin, 50% acetylated α-tubulin and 150% stathmin, a microtubule destabilising enzyme. Patient-derived cells were smaller than control cells. They had altered intracellular distributions of peroxisomes and mitochondria and they had slower moving peroxisomes. These results suggest that patient-derived cells may compensate for reduced spastin but their increased stathmin expression reduced stabilised microtubules and altered organelle trafficking. Sub-nanomolar concentrations of the microtubule-binding drugs, paclitaxel and vinblastine, increased acetylated α-tubulin levels in patient cells to control levels, indicating the utility of this cell model for screening other candidate compounds for drug therapies.
Disease Models and Mechanisms 12/2012; · 4.94 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The olfactory mucosa, the organ of smell in the nose, is a neural tissue that regenerates new sensory neurons throughout adult life. Based on this tissue, we previously demonstrated increased mitosis in olfactory biopsy cultures from schizophrenia patients compared with healthy control subjects. In addition, neural stem/progenitor cell cultures (neurosphere-derived cells) from nasal biopsies from individuals with schizophrenia show significantly altered gene and protein expression in key cell cycle control pathways.
The aim of this study was to investigate cell cycle dynamics in olfactory neurosphere-derived cells from nine male schizophrenia patients and nine male healthy control subjects. Cell cycles were arrested by serum deprivation after which cell population doubling time, proliferation fraction, and cell cycle period were calculated from cell counts over 96 hours. Cell cycle phase was investigated using flow cytometry. Cell lysates were analyzed for expression of cyclin proteins.
Cell population proliferation rate was increased in schizophrenia through a larger pool of proliferating progenitors and a reduced cell cycle period. All phases of the cell cycle were phase-shifted by 2 hours in the schizophrenia-derived cells, which expressed higher levels of the cyclins D1, E, and A2.
Our observations indicate that schizophrenia is associated with subtle alterations in cell cycle dynamics, shortening of the cell cycle period, and increased expression of G1/S phase cyclins. We speculate that this underlying diathesis could alter the temporal and spatial cascade of brain development and contribute to an altered neurodevelopmental trajectory in schizophrenia.
Biological psychiatry 11/2011; 71(2):129-35. · 8.93 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Labelling and identifying proliferating cells is central to understanding neurogenesis and neural lineages in vivo and in vitro. We present here a novel thymidine analogue, ethynyl deoxyuridine (EdU) for labelling dividing cells, detected with a fluorescent azide which forms a covalent bond via the "click" chemistry reaction (the Huisgen 1,3-dipolar cycloaddition reaction of an organic azide to a terminal acetylene). Unlike the commonly used BrdU, EdU detection requires no heat or acid treatment. It is quick and easy and compatible with multiple probes for fluorescence immunochemistry, facilitating the characterisation of proliferating cells at high resolution.
Journal of Neuroscience Methods 11/2008; 177(1):122-30. · 1.98 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Labelling and identifying proliferating cells is central to understanding neurogenesis and neural lineages in vivo and in vitro .The standard method of labelling proliferating cells uses the thymidine analogue, bromodeoxyuridine (BrdU), which incorporates into the DNA during S-phase of the cell cycle. A disadvantage of this method is that the immunochemical processing requires pre-treatment of the cells and tissue with heat or acid to reveal the antigen. This pre-treatment reduces reliability of the method and degrades the specimen, reducing the ability for multiple immuno-fluorescence labelling at high resolution. We report here the utility of a novel thymidine analogue, ethynyl deoxyuridine (EdU), detected with a fluorescent azide via the “click” chemistry reaction (the Huisgen 1,3-dipolar cycloaddition reaction of an organic azide to a terminal acetylene). The detection of EdU requires no heat or acid treatment and the incorporated EdU is covalently conjugated to a fluorescent probe, using a copper-catalysed chemical reaction. The reaction is quick and compatible with fluorescence immunochemistry and other fluorescent probes. We show here that EdU efficiently labels proliferating cells of embryos and adult animals. It effectively labels cells during neurogenesis and the progeny may be identified at least 30 days later, thus allowing the tracking and quantification of proliferating cells in multiple neurogenic regions including the olfactory neuroepithelium1. We demonstrate its utility, superseding BrdU as a cell proliferation marker, as it markedly improves the detection of proliferating cells and allows concurrent high resolution fluorescence immunochemistry. 1 Chehrehasa et al 2008, J Neuroscience Methods, In Press. No Yes
-
Nicholas Matigian, Greger Abrahamsen,
Ratneswary Sutharsan,
Anthony Cook,
Alejandra Mariel Vitale,
Amanda Nouwens,
Bernadette Bellette,
Jay An,
Matthew James Anderson,
Anthony Gordon Beckhouse, [......],
Jyothy Raju,
Sugandha Ravishankar,
Peter Allen Silburn,
Greg Trevor Sutherland,
Stephen Mahler,
George Mellick,
Stephen Andrew Wood,
Carolyn M Sue,
Christine Wells,
Alan Mackay-Sim
[show abstract]
[hide abstract]
ABSTRACT: There is a pressing need for patient-derived cell models of brain diseases that are relevant and robust enough to produce the large quantities of cells required for molecular and functional analyses. We describe here a new cell model based on patient-derived cells from the human olfactory mucosa, the organ of smell, which regenerates throughout life from neural stem cells. Olfactory mucosa biopsies were obtained from healthy controls and patients with either schizophrenia, a neurodevelopmental psychiatric disorder, or Parkinson's disease, a neurodegenerative disease. Biopsies were dissociated and grown as neurospheres in defined medium. Neurosphere-derived cell lines were grown in serum-containing medium as adherent monolayers and stored frozen. By comparing 42 patient and control cell lines we demonstrated significant disease-specific alterations in gene expression, protein expression and cell function, including dysregulated neurodevelopmental pathways in schizophrenia and dysregulated mitochondrial function, oxidative stress and xenobiotic metabolism in Parkinson's disease. The study has identified new candidate genes and cell pathways for future investigation. Fibroblasts from schizophrenia patients did not show these differences. Olfactory neurosphere-derived cells have many advantages over embryonic stem cells and induced pluripotent stem cells as models for brain diseases. They do not require genetic reprogramming and they can be obtained from adults with complex genetic diseases. They will be useful for understanding disease aetiology, for diagnostics and for drug discovery. Yes Yes
-
Nicholas Matigian, Greger Abrahamsen,
Ratneswary Sutharsan,
Anthony L Cook,
Alejandra M Vitale,
Amanda Nouwens,
Bernadette Bellette,
Jiyuan An,
Matthew Anderson,
Anthony G Beckhouse, [......],
Jyothy Raju,
Sugandha Ravishankar,
Peter A Silburn,
Greg T Sutherland,
Stephen Mahler,
George D Mellick,
Stephen A Wood,
Carolyn M Sue,
Christine A Wells,
Alan Mackay-Sim
[show abstract]
[hide abstract]
ABSTRACT: There is a pressing need for patient-derived cell models of brain diseases that are relevant and robust enough to produce the large quantities of cells required for molecular and functional analyses. We describe here a new cell model based on patient-derived cells from the human olfactory mucosa, the organ of smell, which regenerates throughout life from neural stem cells. Olfactory mucosa biopsies were obtained from healthy controls and patients with either schizophrenia, a neurodevelopmental psychiatric disorder, or Parkinson's disease, a neurodegenerative disease. Biopsies were dissociated and grown as neurospheres in defined medium. Neurosphere-derived cell lines were grown in serum-containing medium as adherent monolayers and stored frozen. By comparing 42 patient and control cell lines we demonstrated significant disease-specific alterations in gene expression, protein expression and cell function, including dysregulated neurodevelopmental pathways in schizophrenia and dysregulated mitochondrial function, oxidative stress and xenobiotic metabolism in Parkinson's disease. The study has identified new candidate genes and cell pathways for future investigation. Fibroblasts from schizophrenia patients did not show these differences. Olfactory neurosphere-derived cells have many advantages over embryonic stem cells and induced pluripotent stem cells as models for brain diseases. They do not require genetic reprogramming and they can be obtained from adults with complex genetic diseases. They will be useful for understanding disease aetiology, for diagnostics and for drug discovery.
Disease Models and Mechanisms 3(11-12):785-98. · 4.94 Impact Factor
