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Neuronal trafficking dynamics and regulators
Author(s) -
Feole Monica,
Devoto Victorio Martin Pozo,
Carna Maria,
Stokin Gorazd Bernard
Publication year - 2020
Publication title -
alzheimer's and dementia
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 6.713
H-Index - 118
eISSN - 1552-5279
pISSN - 1552-5260
DOI - 10.1002/alz.038474
Subject(s) - axoplasmic transport , neuroscience , neurodegeneration , biology , immunocytochemistry , microbiology and biotechnology , amyloid precursor protein , alzheimer's disease , pathology , medicine , disease , endocrinology
Background Neurons are highly polarized cells continuously relying on intracellular transport, through which they communicate with the external world. Among the somatodendritic and axonal compartments many proteins with different functions are sorted to reach long distances. The relevance of neuronal transport in ensuring cell development and homeostasis maintenance raises a big interest on this topic. Moreover, several neurodegenerative diseases, such as Alzheimer’s or Amyotrophic Lateral Sclerosis, exhibit significant axonal pathology, where transport impairment seems to have a crucial influence. However, the role of this process in neurodegeneration, remains poorly understood. In this study we aim to characterize neuronal transport dynamics of specific proteins, and the molecular machinery regulating this process. Furthermore, we aim to explore transport from the pathological point of view, evaluating the effect that mutant proteins (e.g. TDP43‐related) may have on it. Method Human Neural Stem Cells derived neurons were differentiated for 30 DIV. Plasmids encoding for different GFP‐coupled proteins were designed to express those who are involved in main neuronal functions (e.g. synaptic activity) and transfected in neurons. Time‐lapse movies of cargoes were acquired to detect changes in movement. Data analysis was performed using object segmentation and tracking algorithms. Raw data were processed to describe the main motion parameters: average velocity, directionality, track length, and others. Post‐imaging Immunocytochemistry was performed to assess cargoes localization and discriminate between axonal and dendritic transport. Result Amyloid Precursor Protein (APP) cargoes with higher velocity in anterograde direction were observed compared with the retrograde one; meanwhile for Synaptophysin, a major population of stationary particles was found. Moreover, sorted data for axonal and dendritic transport description shown faster APP particles in axonal anterograde movement. Relying on the movement dynamics description, we decided to screen for protein‐protein interactions, using GFP‐trap and CoIP approaches followed by Mass Spectrometry analysis, which can shed light on possible partners involved in the transport regulation. Conclusion Our work describes an in vitro model for neuronal transport study and highlights the different behavior of proteins with various physiological functions. A complete view will elucidate details about transport regulation and will open a way to understand its role in neuropathological processes.