Circulating extracellular proteasome in the cerebrospinal fluid: a study on concentration and proteolytic activity

Alterations of the intracellular ubiquitin-proteasome pathway are found in neurodegenerative and inflammatory disorders of the central nervous system, as well as in its malignancies. Inhibitory substrates of the proteasomes represent promising approaches to control autoimmune inflammations and induction of apoptosis in cancer cells. Extracellular circulating proteasomes are positively correlated to outcome prognosis in hematogenic neoplasias and the outcome in critically ill patients. Previously, we reported raised levels of proteolytic active 20S proteasomes in the extracellular alveolar space in patients with acute respiratory distress syndrome (ARDS). For the cerebrospinal fluid, we assumed that extracellular circulating proteasomes with enzymatic activity can be found, too. Cerebrospinal fluid (CSF) samples of twenty-six patients (14 females, 12 males), who underwent diagnostic spinal myelography, were analyzed for leukocyte cell count, total protein content, lactate and interleukine-6 (Il-6) concentrations. CSF samples were analyzed for concentration and enzymatic activity of extracellular 20S proteasomes (fluorescenic substrate cleavage; femtokatal). Blood samples were analyzed with respect to concentration of extracellular circulating proteasomes. Choroidal plexus was harvested at autopsies and examined with immunoelectron microscopy (EM) for identification of possible transportation mechanisms. Statistical analysis was performed using SPSS (18.0.3). In all patients, extracellular proteasome was found in the CSF. The mean concentration was 24.6 ng/ml. Enzymatic activity of the 20S subunits of proteasomes was positively identified by the fluorescenic subtrate cleavage at a mean of 8.5 fkat/ml. Concentrations of extracellular proteasomes in the CSF, total protein content and Il-6 were uncorrelated. Immunoelectron microscopy revealed merging vesicles of proteasomes with the outer cell membrane suggestive of an exozytic transport mechanism. For the first time, extracellular circulating 20S proteasome in the CSF of healthy individuals is identified and its enzymatic activity detected. A possible exozytic vesicle-bond transportation mechanism is suggested by immunoelectron microscopy. The present study raises more questions on the function of extracellular proteasome in the CSF and encourages further studies on the role of extracellular protesomes in pathological conditions of the central nervous system (tumor lesions and inflammatory processes).