Neuronal-exosomes biomarkers from plasma of neurodegenerative disease patients
by Leticia Camila Fernández Flores
Date of Examination:2025-02-17
Date of issue:2025-01-21
Advisor:Prof. Dr. Inga Zerr
Referee:Prof. Dr. Inga Zerr
Referee:PD Dr. Niels Hansen
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Abstract
English
The dementia group of diseases share some features such as the degeneration of neurons, which leads to progressive and irreversible loss of cognitive functions. Since almost 10 years, dementia syndromes were declared a common public health problem because over 131 million patients will be diagnosed with dementia by 2050. The aetiology of these pathologies is based on protein misfolding/aggregates, hence, protein misfolding has recently emerged as a new field, in which the prion protein, Tau, α-synuclein, and Aβ are studied. Furthermore, some researchers are considering “prion-like” pathologies, i.e. Alzheimer's disease, Parkinson’s disease, both as the most common ones, even other disorders like Huntington’s Disease (HD) and Amyotrophic Lateral Sclerosis (ALS) as prion-like pathologies. The illness duration of Alzheimer’s disease and/or Parkinson’s disease is approximately 5-10 years. There is another group of neurodegenerative disorders, in which the disease duration is just weeks or a few months, these are known as rapidly progressive dementias. One of the first differential diagnostics is made on prion diseases, which Sporadic Creutzfeldt-Jakob disease is the most prevalent form affecting approximately 90% of the cases. A difficult challenge for the physicians is to distinguish between the different rapidly progressive dementias. Even though, the implementation of new diagnostic tools and technologies in the recent years have allowed physicians to perform a more accurate differential diagnosis, the lack of diagnostic tools is still an issue. Extracellular vesicles are defined by the International Society of Extracellular Vesicles as the vesicles released from the cell. There are three types of them: Apoptotic bodies, Microvesicles/Ectosomes and exosomes. Exosomes are the smallest ones among them, with a size of ∼40–160 nm. The fact that exosomes are released from many different types of cells and found in almost all body fluids, make them approachable for diagnostic purposes. In neurodegenerative disease, neuronal-derived exosomes can be collected from blood, CSF, saliva and urine and several potential biomarkers were discovered. However, there is a lack of an effective, rapid method to isolate neuronal-derived exosomes. In the present study, we proposed a sensitive, reliable, and reproducible method for isolation of neuronal exosomes from blood samples. This method can also be a proficient and appropriate method for diagnostic laboratories working on neurodegenerative diseases. Our aim was to optimize a sensitive and reliable method to isolate neuronal exosomes from plasma sample, in special from healthy controls, Alzheimer's disease and Creutzfeldt-Jakob disease patients. At the end, we were able to evaluate suitability of this new protocol for diagnostic purposes in terms of time and cost. Our cohort included 98 healthy control samples for the protocol optimization and 40 patients for the validation of neurodegenerative biomarkers. To optimize the final nanowire-based protocol, several attempts were done to obtain the best yield. Techniques such as immunoprecipitation and commercial kit SmartSECTM were also performed to collect neuronal exosomes. Since we needed to validate this nanowire-based protocol according to the International Society of Extracellular Vesicles, the exosomes were measured by nanoparticle tracking analysis and characterized by electron transmission microscopy. Additionally, we proved our eluates against different exosome markers; 4 internal exosome markers (Alix, Annexin V, Flotillin 1 & TSG101) and 3 surface markers (CD9, CD63 & CD81) were found by immunoblotting. After we collected the neuronal derived exosomes from plasma, we performed ELISA of degenerative CSF biomarkers. We measured the levels of P-Tau (181P), Total Tau, Aβ 1-40, Aβ 1-42, SFPQ and also performed a preliminary mass-spectrometry analysis with these neuronal exosomes. Total-Tau, P-Tau (181P-S199), SFPQ, PrP, Aβ 1-40, were successfully measured by this method. The preliminary mass-spectrometry analysis showed a proteomic profile with 1250 identified proteins, in which 319 proteins showed statistical significance. The proteins with most statistical significance were Ras-related protein Rab-10 (RAB10), Heterogeneous nuclear ribonucleoprotein R (HNRNPR), and Cell division control protein 42 homolog (CDC42). To conclude this new nanowire-based protocol targeting neuronal exosomes is an accurate, rapid, effective, standardized, and reproducible method. Nanowires must be coated with two neuronal markers (L1CAM and NCAM) and one exosomal surface marker (CD81). Our next steps are: To increase the number of samples and measure them by ELISA, optimize downstream analysis such as RT-QuIC to detect aggregated-prone proteins and to confirm new possible biomarkers discovered from the mass spectrometry analysis by ELISA.
Keywords: plasma; protocol; Alzheimer's disease; nanowires; Exosomes
Schlagwörter: plasma; protocol; Alzheimer's disease; nanowires; Exosomes