CSF biomarkers for ageing: proteomics and in vivo function

The cerebrospinal fluid (CSF) is a specialised fluid that protects the brain. It is made by cells in the centre of the brain and flows out over the surface, draining into the blood. The CSF protects the delicate brain tissues in a number of ways. It acts as a shock absorber physically protecting the brain within the skull; it carries vitamins and hormones needed to keep the brain healthy; it acts as a waste removal system carrying away toxins from brain tissue as it drains into the blood. As we get older, less CSF is made and it takes longer to flow around the brain, so removal of toxins like amyloid-beta present in Alzheimer's disease is slowed down. There are also less of the CSF proteins that carry thyroid hormone and Vitamin A around the brain. In this study we will be examining whether any other proteins are changed in CSF with ageing and whether the changes are to do with genes. This will help us understand why brain functions decline with age. We will study more closely what happens to amyloid-beta in old CSF and how it enters the brain. For this we will use a genetically altered mouse model that does not make the CSF protein (transthyretin) known to protect the brain from amyloid damage. The transgenic mouse will be compared with normal mice, and both populations of mice will be studied when they are young, middle-aged and old. This work will help us understand the 'normal' changes to CSF that accompany old age, whether these are risk factors for late life diseases and whether replacing particular proteins may help prevent this.

Age-related changes to the cerebrospinal fluid (CSF)-ventricular system are not in themselves a disease state, but several links have been made between the aging system and pathologies. Late-life changes in the protein profile of CSF may predispose to risk of later life neurodegeneration, because levels of some protective proteins and peptides decline (such as transthyretin, clusterin and Apo E) and potentially deleterious proteins increase (such as amyloid -beta peptides). Transthyretin is of particular interest since it has several roles within CSF including chaperone protein for thyroxine (T4) and retinoic acid via binding to retinol binding protein (RBP). TTR also prevents amyloid -beta protein fibril formation and hence neurotoxicity, and reduced CSF TTR is associated with Alzheimer's disease (AD) and depression. The importance of T4 in maintaining mature neuronal metabolism (including neurogenesis), and of amyloid in the pathogenesis of AD, has led to the suggestion that lack of sufficient TTR contributes directly to age-related cognitive decline. Previous studies have shown that amyloid -beta clearance from CSF and brain is impaired with age, and that the capacity for thyroxine uptake at the CP is reduced. In this study we will look at four main areas: 1. Changes in CSF protein profile with increasing age (potential ageing biomarkers) Much of the current proteomic analysis of CSF has been focussed on diseases, and there is no information on what happens to the entire protein profile of proteins with healthy ageing. Using CSF samples already collected from a previous project using sheep, we will undertake rapid screening using 2D gel electrophoresis (2DE), covering the whole adult lifecourse, to help identify potential biomarkers for ageing, and which may help explain age-related CNS alterations. CSF will also be collected from aged mice, which are also the subject of this study, for cross-species comparison. Such proteomic analysis of CSF proteins can select candidates for further functional study. 2. Age-related changes in CP protein expression Any changes in CSF proteins will be a consequence of many factors (such as rates of CSF secretion and turnover, protein degradation and synthesis), but a fundamental step is the rate of protein synthesis. De novo protein synthesis will be assessed by measuring the rate of incorporation of isotopically labelled amino acids into proteins in vivo, in mouse brain and CP, and in situ in sheep perfused CP, followed by autoradiography. 3. CP mRNA expression with increasing age Following identification of specific proteins declining in CSF, Real-time PCR can be used to assess mRNA expression for those of CP origin in the young and old sheep, and brain and CP origin in the mouse. 4. The functional role of TTR in ageing will be investigated in vivo using the uptake and CNS distribution of its radio-labelled ligands; thyroxine, Vitamin A, amyloid -beta 1-40. Uptake into, and efflux from CSF can be assessed. Specifically CP, CSF, brain and blood distributions will be compared between young, middle-aged and old mice. Similar comparisons will be made with TTR-null mice to determine what the effect of loss of TTR is on these parameters in young animals what the effect of ageing 'per se' is on the CNS uptake and removal of T4, amyloid -beta 1-40 and Vitamin A.