Automated quantification of neuronal swellings in a preclinical rodent model of Parkinson's disease detects region-specific changes in pathology.

BACKGROUND: The development of axonal pathology is a key characteristic of many neurodegenerative disease such as Parkinson's disease and Alzheimer's disease. With advanced disease progression, affected axons do display several signs of pathology such as swelling and fragmentation. In the AAV vector-mediated alpha-synuclein overexpression model of Parkinson's disease, large (> 20 µm2) pathological swellings are prominent characteristics in cortical and subcortical structures. NEW METHOD: This report describes a novel, macro-based workflow to quantify axonal pathology in the form of axonal swellings in the AAV vector-based alpha-synuclein overexpression model. Specifically, the approach is using background correction and thresholding before quantification of structures in 3D throughout a tissue stack. RESULTS: The method was used to quantify TH and aSYN axonal swellings in the prefrontal cortex, striatum, and hippocampus. Regional differences in volume and number of axonal swellings were observed for both in TH and aSYN, with the striatum displaying the greatest signs of pathology. COMPARISON WITH EXISTING METHODS: Existing methods for the quantification of axonal pathology do either rely on proprietary software or are based on manual quantification. The ImageJ workflow described here provides a method to objectively quantify axonal swellings both in volume and number. CONCLUSION: The method described can readily assess axonal pathology in preclinical rodent models of Parkinson's disease and can be easily adapted to other model systems and/or markers.

Autoantibodies in Pandemrix®-induced narcolepsy: Nine candidate autoantigens fail the conformational autoantibody test.

Study objectives: Narcolepsy type 1 (NT1) is a chronic sleep disorder characterized by loss of hypocretin-producing neurons. Increased NT1 incidence was observed in Sweden following mass-vaccination with Pandemrix®. Genetic association to HLA DQB1*06:02 implies an autoimmune origin, but target autoantigen remains unknown. Candidate autoantigens for NT1 have previously been identified in solid-phase immunoassays, while autoantibodies against conformation-dependent epitopes are better detected in radiobinding assays. The aims are to determine autoantibody levels against nine candidate autoantigens representing (1) proteins of the hypocretin transmitter system; Preprohypocretin (ppHypocretin), Hypocretin peptides 1 and 2 (HCRT1 and HCRT2) and Hypocretin receptor 2 (HCRTR2); (2) proteins previously associated with NT1; Tribbles homologue 2 (TRIB2), Pro-opiomelanocortin/alpha-melanocyte-stimulating-hormone (POMC/α-MSH) and Prostaglandin D2 Receptor DP1 (DP1); (3) proteins suggested as autoantigens for multiple sclerosis (another HLA DQB1*06:02-associated neurological disease); ATP-dependent Inwardly Rectifying Potassium Channel Kir4.1 (KIR4.1) and Calcium-activated chloride channel Anoctamin 2 (ANO2). Methods: Serum from post-Pandemrix® NT1 patients (n = 31) and their healthy first-degree relatives (n = 66) were tested for autoantibody levels in radiobinding assays separating autoantibody bound from free labelled antigen with Protein A-Sepharose. 125I-labelled HCRT1 and HCRT2 were commercially available while 35S-methionine-labelled ppHypocretin, HCRTR2, TRIB2, α-MSH/POMC, DP1, KIR4.1 or ANO2 was prepared by in vitro transcription translation of respective cDNA. In-house standards were used to express data in arbitrary Units/ml (U/ml). Results: All radiolabelled autoantigens were detected in a concentration-dependent manner by respective standard sera. Levels of autoantibodies in the NT1 patients did not differ from healthy first-degree relatives in any of the nine candidate autoantigens. Conclusions: None of the nine labelled proteins proposed to be autoantigens were detected in the radiobinding assays for conformation-dependent autoantibodies. The results emphasise the need of further studies to identify autoantigen(s) and clarify the mechanisms in Pandemrix®-induced NT1.