Biochemical studies of pathological inclusions in α-synucleinopathies and frontotemporal lobar degeneration with ubiquitin-positive inclusions

The formation of pathological inclusions, composed of aggregates of misfolded proteins, is believed to be a key event in the neuronal dysfunction and death characteristic of various neurodegenerative diseases. A detailed understanding of the composition of these pathological inclusions and mechanisms by which they form may eventually enable the development of therapeutic interventions. This thesis describes work focused on ubiquitin-positive inclusions present in two types of neurodegenerative diseases: the α-synucleinopathies and frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U). Evidence presented shows that α-synuclein within pathological inclusions is ubiquitinated in α-synucleinopathies as well as in a transgenic α-synucleinopathy mouse model. Further work suggests, however, that ubiquitination of α-synuclein is not required for inclusion formation, but rather represents a later event in disease pathogenesis which occurs following the aggregation of α-synuclein. Unlike the ubiquitin-positive inclusions in α-synucleinopathies, the protein components of pathological inclusions in FTLD-U are unknown. Described in this thesis are biochemical approaches which were taken in attempt to identify proteins which are the building-blocks of inclusions in FTLD-U. These approaches included various biochemical fractionation methods aimed at enriching for the protein(s) within inclusions but were unsuccessful in identifying the major protein component(s) of the inclusions, perhaps due to inadequate sensitivity of the assays used to detect proteins in the biochemical fractions. An alternative approach, involving immunization of mice with high molecular mass insoluble material from an FTLD-U brain, however, resulted in the generation of novel monoclonal antibodies which immunostain pathological inclusions in FTLD-U brains. The antibodies detect FTLD-U ubiquitin-positive inclusions only in a subset of FTLD-U cases, but not in other FTLD-U cases nor in other neurodegenerative diseases. The antibodies, therefore, distinguish one pathological subtype of FTLD-U and provide evidence that FTLD-U is heterogeneous. In biochemical experiments, some of the antibodies immunolabel an acidic 24 kD band, which is hypothesized to be a main protein component of pathological inclusions in the FTLD-U subtype. Future experiments are aimed at purifying and identifying this 24 kD entity, which will stimulate further investigations into involvement of this protein in the molecular pathogenesis of FTLD-U