This grant builds upon the research from a prior grant: CSF Lysosomal Hydrolases' Activity as Possible Marker of Parkinson's Disease
Promising Outcomes of Original Grant:
To date, there is no accepted diagnostic test based on biochemical analysis of blood or cerebrospinal fluid (CSF) for clinical diagnosis of PD. Recent studies have supported clinical, neuropathological and genetic associations between Gaucher’s disease, a lysosomal storage disease caused by deficiency of the enzyme β-glucocerebrosidase, and PD. Since alpha-synuclein is degraded via a lysosomal degradation pathway and the lysosomal system is altered in PD, we hypothesized that measurement of lysosomal hydrolases activity in CSF could be a promising diagnostic marker. We investigated the activity of a series of lysosomal enzymes in CSF of 71 PD patients and 50 controls. β-glucocerebrosidase, α-mannosidase, β-hexosaminidase, α-fucosidase and cathepsin D activities were significantly different in PD versus controls, and their combination with the ratio between total synuclein and synuclein oligomers gave the best discriminative performance.
Objectives for Supplemental Investigation:
In order to definitely ascertain the reliability of our results, we will carry out a systematic series of experiments aimed at controlling for different pre-analytical and analytical factors potentially influencing the lysosomal enzymes activity measured in CSF. In particular, we will check for the influence of (i) total protein concentration in CSF; (ii) freezing conditions (flash freezing in liquid nitrogen vs freezing at –80°C) and effects of freeze-thaw cycles on detection of enzyme activities; (iii) length of storage (one- and two weeks; one – two - four - eight - ten months) at different temperatures (-20°C vs – 80°C); (iv) intra-assay (10 triplicate aliquots from the same CSF sample) and inter-assay precision (10 runs on triplicate aliquots run in different days) by calculating the coefficients of variation.
The verification of all these parameters is of utmost important in order to subsequently propose this type of biochemical assay for extended use in clinical practice.
Importance of This Research for the Development of a New PD Therapy:
Recent studies demonstrated that the combination of CSF biomarkers reflecting different etiopathogenetic mechanisms might be of support for an earlier PD diagnosis. We demonstrated that the combination of some lysosomal hydrolases (namely, β-glucocerebrosidase, α-mannosidase, β-hexosaminidase, α-fucosidase and cathepsin D) with the ratio between total synuclein and synuclein oligomers allows us to distinguish PD from neurological controls. The accurate knowledge of pre-analytical and analytical factors influencing the reliability of these determinations is necessary for their subsequent use in clinical practice. Early and accurate clinical diagnosis is mandatory in order to achieve a timely and focused pharmacological treatment.