In previous studies, passive administration of A and -synuclein antibodies to cell and animal models have indicated antibody-directed protein clearance via the endosomal/lysosomal pathway[20],[23]In agreement with these findings, we showed that this levels of -synuclein were decreased both in cell lysate and conditioned media after antibody treatment, pointing to an increased degradation of the targeted proteins (Fig. suggesting increased protein turnover. Taken together, our results propose that extracellular administration of monoclonal antibodies can change or inhibit early actions in the aggregation process of -synuclein, thus providing further support for passive immunization against diseases with -synuclein pathology. == Introduction == Parkinson’s disease, dementia with Lewy body and multiple system atrophy are neurodegenerative disorders characterized by the loss of neurons in the brain along with the presence of large intracellular protein inclusions known as Lewy body[1],[2]. The major protein component of Lewy body is usually -synuclein, a 140 amino acid long protein with a partially unfolded structure[3]. Although -synuclein has a largely unknown function, recent findings suggest it to be involved in neurotransmitter regulation. For example, -synuclein may regulate the reuptake of dopamine into striatum of transgenic mice[4]or be more generally involved in synaptic release by promoting SNARE complex assembly[5]. The aggregation cascade of -synuclein is usually believed to begin with the formation of dimers and smaller oligomers before the appearance of larger oligomers or protofibrils[6]. Such soluble pre-aggregated species have been demonstrated to have toxic properties and may thus play a central role in the pathogenesis[7],[8],[9],[10],[11]. In addition, the disease associated mutations in the gene encoding for -synuclein have been found to increase the formation of oligomers/protofibrils, further supporting the pathogenic significance of such species[12],[13],[14]. Alpha-synuclein aggregation has been widely analyzed in cell culture models. By overexpressing -synuclein, intracellular inclusions can be induced in a wide range of cell types via numerous aggregation-promoting conditions[15],[16]. Early stages of protein aggregation can be assessed with protein-fragment complementation techniques[17]. One such method, the bimolecular fluorescence complementation (BiFC) assay, has previously been adopted for the study of -synuclein aggregation[7]. By fusing DNA encoding either the RO4987655 C-terminal or N-terminal halves of GFP to the entire -synuclein sequence, two forms of -synuclein hemi:GFP constructs are generated. Upon double transfection of cells with these constructs, fluorescence occurs only when the fragments are brought together, i.e. after dimerization/oligomerization of -synuclein. In the last decade, immunotherapy has emerged as a encouraging tool to target and clear protein pathology in neurodegenerative diseases. With active immunization of transgenic amyloid-beta precursor protein (APP) mice, using fibrils of the amyloid beta peptide (A), a distinct reduction of A pathology could be seen[18]. In addition, A immunization has been found to alleviate memory impairment in transgenic animal models[19]. Instead of vaccination in Alzheimer’s disease, focus has now been set on passive treatment with antibodies against A. Such an approach has proven to be equally efficient in both cell and animal models and is likely to be a safer therapeutic option, as T-cell mediated side effects can be avoided[20],[21]. Immunotherapy has now also begun to be evaluated as an approach to treat -synuclein pathology. In RO4987655 one study, active immunization with -synuclein on transgenic mice showed that this pathology was less pronounced in vaccinated mice as compared to placebo[22]. As for passive immunotherapy against HSP90AA1 -synuclein pathology, a recent study described reduced behavioral deficit as well as decreased accumulation of -synuclein aggregates in an -synuclein transgenic mouse model[23]. Here, we explored the use RO4987655 of monoclonal -synuclein antibodies to target dimerization/oligomerization on a cell culture model, using BiFC. == Materials and Methods == == Alpha-synuclein constructs == The G-N-155–syn and -syn-G-156-C constructs used for the BiFC assay were generated as described earlier[7]. For all those transfection experiments, an empty pcDNA3.1 expression vector (Invitrogen, Carlsbad, CA) was used as control. == Cell culture == Human H4 neuroglioma cells were a kind gift of Dr. Bradley T. Hyman (Massachusetts General Hospital, Charlestown, MA). Cells were cultured at 37C and 5% CO2in OPTI-MEM (Invitrogen) and supplemented with 10% fetal bovine serum (FBS) (Invitrogen) and 4 mM Glutamine (Invitrogen). == Antibodies == The following -synuclein monoclonal antibodies (mAb) were used for cell culture treatment: mAb211 (Santa Cruz Biotechnology, Santa Cruz, CA), mAb5C2 (Santa Cruz Biotechnology) and the oligomer-selective antibody mAb49/G (BioArctic Neuroscience, Stockholm, Sweden). The monoclonal GAPDH antibody 9484 (Abcam, Cambridge, UK) was used as a negative treatment control. All antibodies used for cellular treatment were diluted RO4987655 in TBS to reach a final concentration of 1 1 g/ml in the extracellular media. For the sandwich ELISA, the Syn-1 (BD Biosciences, Franklin Lakes, NJ) and FL-140 (Santa Cruz Biotechnology) -synuclein antibodies were used for capture and detection, respectively. For immunocytochemistry experiments, anti-mouse Cy3 or Alexa594 conjugated secondary antibodies (Invitrogen) were used. == Generation of.