Sequenzierung der Antigen-bindenden Region von natürlich vorkommenden alpha-Synuklein-reaktiven Autoantikörpern

Bei dem idiopathischen Parkinson-Syndrom handelt es sich nach der Alzheimer-Erkrankung um die zweithäufigste neurodegenerative Erkrankung, deren Prävalenz aufgrund der immer älter werdenden Bevölkerung noch weiter zunehmen wird. Rigor, Tremor und Bradykinese sind die Hauptsymptome dieser Erkrankung,...

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Bibliographic Details
Main Author: Jördens, Marit
Contributors: Dodel, Richard (Prof. Dr. med.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2023
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The idiopathic Parkinson's syndrome is the second most common neurodegenerative disease after Alzheimer's disease and its prevalence is predicted to increase even further due to demographic changes. The main clinical symptoms of the idiopathic Parkinson's syndrome, which was first described in 1817 by James Parkinson, are rigor, resting tremor and bradykinesia. These motor symptoms can be linked to a progressing decline of the dopaminergic neurons within the substantia nigra, which belongs to the basal ganglia in the midbrain and inter alia participates in motoric functions, learning processes, coordination and cognition. Pathologically, intraneural deposits of proteins, commonly known as Lewy-bodies, are characteristic of idiopathic Parkinson's syndrome. These inclusions mainly consist of aggregated and misfolded α-Syn. Lewy-bodies damage dopaminergic neurons in the substantia nigra through a yet unknown mechanism. The current therapy of the idiopathic Parkinson's syndrome mainly aims at compensating the dopaminergic deficit and thereby controlling the motoric symptoms. At present there is no disease modifying or even curative therapy available. Nevertheless, passive immunization with alpha-synuclein-reactive antibodies are promising therapeutic approaches. In this context alpha-synuclein-reactive naturally occurring autoantibodies were identified, which are able to recognize pathologically aggregated proteins and inhibit their accumulation by promoting the microglial uptake. Specific alpha-synuclein-reactive naturally occurring autoantibodies can be isolated from commercially acquired intravenous immunoglobulins of the subtype IgG by affinity chromatography. However, IVIG is a limited and expensive resource, as its preparation is dependent on blood donations. Therefore, the establishment of an intravenous immuno-globulins of the subtype IgG-independent method of producing alpha-synuclein-reactive naturally occurring autoantibodies would be beneficial. Naturally occurring autoantibodies are a component of the human antibody pool and they are secreted by B1-lymphocytes. These B1-cells can be distinguished from other B-lymphocytes by characteristic clusters of differentiation on their cell surface. As the aim of this thesis was to establish a protocol that allows the isolation of alpha-synuclein-reactive naturally occurring autoantibody-producing B1-lymphocytes and grants the sequencing of their antigen-binding domain. The basis of this approach were blood samples from clinically healthy donors. After the purification from the blood by means of density gradient centrifugation, a B-cell enrichment was performed. Fluorescent-activated cell sorting allowed the characterization of the B1-lymphocyte population which secretes alpha-synuclein-reactive naturally occurring autoantibodies. Subsequently, a sin-gle cell sorting of the desired B1-lymphocytes was executed. Antibody-coding base sequences of the individual cells were obtained by single-cell-reverse-transcription-polymerase-chain-reaction and with the help of a genetic database transcribed into amino acid sequences. Apart from that, a phylogenetic analysis of the variable regions of the antibodies’ light and heavy chain sequences was performed, which results were consistent with previous studies regarding the germinal gen usage of tau-reactive naturally occurring autoantibodies. However, a detailed investigation of the alpha-synuclein-reactive naturally occurring autoantibodies’ origin is less important for their potential therapeutic use. In conclusion, we were able to isolate the alpha-synuclein-reactive naturally occurring autoantibody-producing B1-lymphocytes from the blood of healthy donors. It was also possible to gain the antigen-binding regions from the specific B1-lymphocytes. For the generation of a monoclonal antibody based on the genetic information of these naturally circulating antibodies, the sequences, which were identified as the most promising in the analysis, from the cells 20160422_B3, 20160426_B2 und 20160427_C3, shall be cloned in a suitable expression vector. Prospectively human embryonal kidney cells can be transfected with this construct. After that the monoclonal antibodies which were obtained in this way, must be reviewed concerning their binding characteristics towards alpha-synuclein, their therapeutic efficiency and probable side effects. Hopefully, it will be possible prospectively to use a monoclonal antibody based on the genetic information of an alpha-synuclein-reactive naturally occurring autoantibody as a passive immunization approach, whilst binding misfolded alpha-synuclein and thereby inhibiting oligomerization and the formation of Lewy-bodies, which would enable the constraint of the disease progression.