RESUMO
Progressive development of pathology is one of the major characteristic features of neurodegenerative diseases. Alzheimer's disease (AD) is the most prevalent among them. Extracellular amyloid-ß (Aß) plaques and intracellular tau neurofibrillary tangles are the pathological phenotypes of AD. However, cellular and animal studies implicate tau as a secondary pathology in developing AD while Aß aggregates is considered as a trigger point. Interaction of Aß peptides with plasma membrane (PM) seems to be a promising site of involvement in the events that lead to AD. Aß binding to the lipid membranes initiates formation of oligomers of Aß species, and these oligomers are known as primary toxic agents for neuronal toxicities. Once initiated, neuropathological toxicities spread in a "prion-like" fashion probably through the mechanism of intercellular transfer of pathogenic aggregates. In the last two decades, several studies have demonstrated neuron-to-neuron transfer of neurodegenerative proteins including Aß and tau via exosomes and tunneling nanotubes (TNTs), the two modes of long-range intercellular transfer. Emerging pieces of evidence indicate that molecular pathways related to the biogenesis of exosomes and TNTs interface with endo-lysosomal pathways and cellular signaling in connection to vesicle recycling-imposed PM and actin remodulation. In this review, we discuss interactions of Aß aggregates at the membrane level and its implications in intercellular spread of pathogenic aggregates. Furthermore, we hypothesize how spread of pathogenic aggregates contributes to complex molecular events that could regulate pathological and synaptic changes related to AD.
RESUMO
Recent discoveries have revealed that cells perform direct, long-range, intercellular transfer via nano-scale, actin-membrane conduits, namely "tunneling nanotubes" (TNTs). TNTs are defined as open-ended, lipid bilayer-encircled membrane extensions that mediate continuity between neighboring cells of diameters ranging between 50 nm and 1 µm. TNTs were demonstrated initially in neuronal cells, but successive studies have revealed the existence of TNTs in several cell types and diseases, such as neurodegenerative diseases, viral infections, and cancer. Several studies have referred to close-ended, electrically coupled membrane nanostructures between neighboring cells as TNTs or TNT-like structures. The elucidation of ultrastructure in terms of membrane continuity at the endpoint is technically challenging. In addition, studies on cell-cell communication are challenging in terms of the characterization of TNTs using conventional methods due to the lack of specific markers. TNTs are primarily defined as F-actin-based, open-ended membrane protrusions. However, one major limitation is that F-actin is present in all types of protrusions, making it challenging to differentiate TNTs from other protrusions. One of the notable characteristics of F-actin-based TNTs is that these structures hover between two cells without touching the substratum. Therefore, distinct F-actin-stained TNTs can conveniently be distinguished from other protrusions such as filopodia and neurites based on their hovering between cells. We have recently shown that the internalization of oligomeric amyloid-ß1-42 (oAß) via actin-dependent endocytosis stimulates activated p21-activated kinase-1 (PAK1), which mediates the formation of F-actin-containing TNTs coexpressed with phospho-PAK1 between SH-SY5Y neuronal cells. This protocol outlines a 3D volume analysis method to identify and characterize TNTs from the captured z-stack images of F-actin- and phospho-PAK1-immunostained membrane protrusions in oAß-treated neuronal cells. Further, TNTs are distinguished from developing neurites and neuronal outgrowths based on F-actin- and ß-III tubulin-immunostained membrane conduits.
