Disease-specific, neurosphere-derived cells as models for brain disorders

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

Familial Occurrence of Multiple Sclerosis with Thyroid Disease and Systemic Lupus Erythematosus

Multiple sclerosis (MS) has some features which suggest it is an autoimmune disease. Autoimmune diseases frequently occur in families, and patients and families often have more than one type of autoimmune disease. However, there are few reports of MS occurring in patients or families with other autoimmune conditions. It is difficult to make a separate diagnosis of MS in a patient who has a systemic autoimmune disease such as systemic lupus erythematosus (SLE) or Sjogren's syndrome, because these diseases can affect the nervous system directly. However, it is possible to make independent diagnoses of MS and an autoimmune disease confined to another single organ in the same patient, or diagnoses of MS and SLE (or other autoimmune diseases) in different family members. Here we describe clinically definite MS in 2 sisters, one of whom had Graves' disease, and the other of whom had a daughter with SLE and with a high titre of anti-thyroid antibodies. Other female family members over 4 generations had histories of thyroid disease, MS and Addison's disease. Available family members were HLA typed. The MS patients were positive for HLA DR2. All but one of the affected family members were related to the proband on the maternal side, and all of these affected females shared an HLA haplotype. However, this haplotype was also present in unaffected individuals. Thus HLA type alone cannot account for the familial occurrence of these disorders. We conclude that, in this family, MS, like autoimmune thyroid disease and SLE, may be an autoimmune disease developing in genetically predisposed individuals

Fluorescence properties of green fluorescent protein FRET pairs concatenated with the small G protein, Rac, and its interacting domain of the kinase, p21-activated kinase

Many diseases are caused by aberrant cell signalling controlled by intracellular protein–protein interactions. Inhibitors of such interactions thus have enormous potential as chemotherapeutic agents. It is advantageous to test for such inhibitors using cell-based screens in which modulation of the interaction gives a rapid response. Fluorescence resonance energy transfer (FRET) systems, based on interacting donor and acceptor green fluorescent proteins (GFPs), have potential in such screens. Here, we describe experiments aimed at using a FRET system to monitor the interaction between the small G protein Rac and a region of its binding partner, the Ser/Thr kinase, p21-activated kinase (PAK). Initial attempts to use a previously described construct, enhanced GFP-PAK-enhanced blue fluorescent protein, failed because of the difficulty of obtaining equal and high expression levels of both the fusion protein and Rac in mammalian cells. Here, three proteins in which Rac, PAK, and the two GFPs were concatenated in different combinations on a single protein were expressed and characterised. In each construct, however, intramolecular interaction of PAK and Rac was observed. As this was of extremely high affinity, presumably because of entropy effects from the interacting partners being tethered, these molecules were not suitable for detection of inhibitors of the interaction. Molecular modelling was used to investigate the way in which the concatenated constructs might form intramolecular interactions. As this explained key properties of these proteins, it is likely that this approach could be used to design constructs where the unwanted intramolecular protein–protein interactions are prevented, whilst allowing the desired intermolecular Rac/PAK interaction. This would provide constructs that are useable for drug discovery