CD81 Promotes a Migratory Phenotype in Neuronal-Like Cells.

Tetraspanins, such as CD81, can form lateral associations with each other and with other transmembrane proteins. These interactions may underlie CD81 functions in multiple cellular processes, such as adhesion, morphology, migration, and differentiation. Since CD81's role in neuronal cells' migration has not been established, we here evaluated effects of CD81 on the migratory phenotype of SH-SY5Y neuroblastoma cells. CD81 was found enriched at SH-SY5Y cell's membrane, co-localizing with its interactor filamentous-actin (F-actin) in migratory relevant structures of the leading edge (filopodia, stress fibers, and adhesion sites). CD81 overexpression increased the number of cells with a migratory phenotype, in a potentially phosphatidylinositol 3 kinase (PI3K)-Ak strain transforming (AKT) mediated manner. Indeed, CD81 also co-localized with AKT, a CD81-interactor and actin remodeling agent, at the inner leaflet of the plasma membrane. Pharmacologic inhibition of PI3K, the canonical AKT activator, led both to a decrease in the acquisition of a migratory phenotype and to a redistribution of intracellular CD81 and F-actin into cytoplasmic agglomerates. These findings suggest that in neuronal-like cells CD81 bridges active AKT and actin, promoting the actin remodeling that leads to a motile cell morphology. Further studies on this CD81-mediated mechanism will improve our knowledge on important physiological and pathological processes such as cell migration and differentiation, and tumor metastasis.

NeuronRead, an open source semi-automated tool for morphometric analysis of phase contrast and fluorescence neuronal images.

Neurons are specialized cells of the Central Nervous System whose function is intricately related to the neuritic network they develop to transmit information. Morphological evaluation of this network and other neuronal structures is required to establish relationships between neuronal morphology and function, and may allow monitoring physiological and pathophysiologic alterations. Fluorescence-based microphotographs are the most widely used in cellular bioimaging, but phase contrast (PhC) microphotographs are easier to obtain, more affordable, and do not require invasive, complicated and disruptive techniques. Despite the various freeware tools available for fluorescence-based images analysis, few exist that can tackle the more elusive and harder-to-analyze PhC images. To surpass this, an interactive semi-automated image processing workflow was developed to easily extract relevant information (e.g. total neuritic length, average cell body area) from both PhC and fluorescence neuronal images. This workflow, named 'NeuronRead', was developed in the form of an ImageJ macro. Its robustness and adaptability were tested and validated on rat cortical primary neurons under control and differentiation inhibitory conditions. Validation included a comparison to manual determinations and to a golden standard freeware tool for fluorescence image analysis. NeuronRead was subsequently applied to PhC images of neurons at distinct differentiation days and exposed or not to DAPT, a pharmacological inhibitor of the γ-secretase enzyme, which cleaves the well-known Alzheimer's amyloid precursor protein (APP) and the Notch receptor. Data obtained confirms a neuritogenic regulatory role for γ-secretase products and validates NeuronRead as a time- and cost-effective useful monitoring tool.

Gi/o-Protein Coupled Receptors in the Aging Brain.

Cells translate extracellular signals to regulate processes such as differentiation, metabolism and proliferation, via transmembranar receptors. G protein-coupled receptors (GPCRs) belong to the largest family of transmembrane receptors, with over 800 members in the human species. Given the variety of key physiological functions regulated by GPCRs, these are main targets of existing drugs. During normal aging, alterations in the expression and activity of GPCRs have been observed. The central nervous system (CNS) is particularly affected by these alterations, which results in decreased brain functions, impaired neuroregeneration, and increased vulnerability to neuropathologies, such as Alzheimer's and Parkinson diseases. GPCRs signal via heterotrimeric G proteins, such as Go, the most abundant heterotrimeric G protein in CNS. We here review age-induced effects of GPCR signaling via the Gi/o subfamily at the CNS. During the aging process, a reduction in protein density is observed for almost half of the Gi/o-coupled GPCRs, particularly in age-vulnerable regions such as the frontal cortex, hippocampus, substantia nigra and striatum. Gi/o levels also tend to decrease with aging, particularly in regions such as the frontal cortex. Alterations in the expression and activity of GPCRs and coupled G proteins result from altered proteostasis, peroxidation of membranar lipids and age-associated neuronal degeneration and death, and have impact on aging hallmarks and age-related neuropathologies. Further, due to oligomerization of GPCRs at the membrane and their cooperative signaling, down-regulation of a specific Gi/o-coupled GPCR may affect signaling and drug targeting of other types/subtypes of GPCRs with which it dimerizes. Gi/o-coupled GPCRs receptorsomes are thus the focus of more effective therapeutic drugs aiming to prevent or revert the decline in brain functions and increased risk of neuropathologies at advanced ages.