NEW EVIDENCES OF TWO DIFFERENT KEY PATHOGENIC MECHANISMS IN HUNTINGTON'S DISEASE

Huntington’s disease (HD) is an adult-onset neurodegenerative disorder characterized by several alterations in critical molecular and cellular pathways. Although genetic and experimental evidence has provided insights into HD pathogenesis, and many hypotheses concerning its underlying mechanisms have been proposed, a treatment that delays disease onset or slows its progression is still missing (Zuccato et al., 2010). During my PhD, my research activity was focused on the investigation of new aspects of two key pathogenic mechanisms of HD: i) the reduction of Brain-Derived Neurotrophic Factor (BDNF) and ii) the cholesterol dysfunction. Reduced BDNF levels have been described in a number of patho-physiological conditions, most notably, in HD. Since BDNF is also produced in blood, we have undertaken the measurement of its peripheral levels in the attempt to identify a possible link with HD prognosis and/or its progression. In the first part of my PhD, I evaluated BDNF level in 398 blood samples including 138 controls, 56 preHD, and 204 HD subjects. I found that BDNF protein levels were not reliably different between groups, whether measured in plasma (52 controls, 26 preHD, 105 HD) or serum (39 controls, 5 preHD, 29 HD). The analysis of the literature highlighted that intra-group variability and methodological aspects affect this measurement, especially in serum. Moreover during my PhD, I set up a novel strategy for mRNA normalization in quantitative real-time PCR that is based on expressed Alu repeat amplification as a measure for the mRNA fraction. I demonstrated that expressed Alu repeat amplification is a fast, accurate normalization tool that can be successfully used for quantification of selected mRNA in the human transcriptome. This result is particularly important for clinical diagnosis and biomarker validation studies based on mRNA detection in human blood. Based on this new normalization method, I measured BDNF mRNA levels in blood samples from 47 controls, 25 preHD, and 70 HD subjects, and found no differences among the groups. These results indicated that levels of BDNF in human blood were not informative (mRNA levels or plasma protein level) nor reliable (serum protein levels) as HD biomarkers. Brain cholesterol, which is synthesized locally, is a major component of myelin and cell membranes and participates in several neuronal activities, such as neurite outgrowth and synaptogenesis. In post-natal life, the cholesterol-dependent activities of neurons mainly rely on the transport of cholesterol from astrocytes on ApoE-containing lipoproteins. Mounting evidence indicates that reduced cholesterol biosynthesis occurs in the brain of several models of Huntington’s disease (HD) and is manifest in astrocytes (Valenza et al., J. Neurosci 2010). However, how mutation in huntingtin elicits changes in cholesterol biosynthesis pathway is still unknown. In the second part of my thesis, I explored the cross-talk between neurons and astrocytes to determine whether reduced cholesterol biosynthesis/secretion by HD astrocytes influences cholesterol-dependent properties in wt (and HD) neurons, such as neurite outgrowth and synaptic vesicles formation/maintenance. Primary neurons and astrocytes expressing normal and mutant huntingtin have been used in different experimental settings, in order to evaluate i) the influence of cell non-autonomous mechanisms involving defective cholesterol synthesis/secretion by astrocytes bearing mutant htt and how this dysfunction affects networks and cholesterol-dependent properties of neurons that undergo degeneration in HD; ii) exploring the underlying molecular mechanism of the cholesterol biosynthesis dysfunction in HD astrocytes to identify potential targets of intervention from therapeutic standpoints. I show that glial-derived medium from wt astrocytes (GCMwt) and cholesterol administration improve neurite outgrowth and synaptic properties in wt and HD neurons. The same parameters are not increased after administration of GCM from primary HD astrocytes (GCMHD) or GCMwt depleted of lipoproteins. These findings support the hypothesis that non-cell autonomous mechanisms related to cholesterol and involving astrocytes may contribute to HD pathogenesis. Strategies aimed at selectively modulating brain cholesterol metabolism might be of impact in HD.