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Simple Summary: The present study questions whether cells from glioblastoma multiforme (GBM), which overexpress α-synuclein (α-syn), may alter neighboring non-tumoral astrocyte cell lines. The occurrence of α-syn in GBM correlates with the expression of the stem cell marker nestin. When astrocytes are co-cultured with GBM cells in a trans-well apparatus the occurrence of α-syn and nestin rises remarkably. The increase in α-syn in co-cultured astrocytes is more pronounced at the plasma membrane, which mimics the placement of α-syn in GBM cells. When the mTOR inhibitor rapamycin is administered, GBM-induced expression of α-syn and nestin within co-cultured astrocytes is occluded, and morphological alterations are reverted. In the presence of rapamycin the sub-cellular placement of α-syn is modified being allocated within whorls and vacuoles instead of the plasma membrane. The effects induced by rapamycin occur both in baseline GBM cells and within astrocytes primed by co-cultured GBM cells. Evidence has been recently provided showing that, in baseline conditions, GBM cells feature high levels of α-syn which are way in excess compared with α-syn levels measured within control astrocytes. These findings are consistent along various techniques. In fact, they are replicated by using antibody-based protein detection, such as immuno-fluorescence, immuno-peroxidase, immunoblotting and ultrastructural stoichiometry as well as by measuring α-syn transcript levels at RT-PCR. The present manuscript further questions whether such a high amount of α-syn may be induced within astrocytes, which are co-cultured with GBM cells in a trans-well system. In astrocytes co-cultured with GBM cells, α-syn overexpression is documented. Such an increase is concomitant with increased expression of the stem cell marker nestin, along with GBM-like shifting in cell morphology. This concerns general cell morphology, subcellular compartments and profuse convolutions at the plasma membrane. Transmission electron microscopy (TEM) allows us to assess the authentic amount and sub-cellular compartmentalization of α-syn and nestin within baseline GBM cells and the amount, which is induced within co-cultured astrocytes, as well as the shifting of ultrastructure, which is reminiscent of GBM cells. These phenomena are mitigated by rapamycin administration, which reverts nestin- and α-syn-related overexpression and phenotypic shifting within co-cultured astrocytes towards baseline conditions of naïve astrocytes. The present study indicates that: (i) α-syn increases in astrocyte co-cultured with GBM cells; (ii) α-syn increases in astrocytes along with the stem cell marker nestin; (iii) α-syn increases along with a GBM-like shift of cell morphology; (iv) all these changes are replicated in different GBM cell lines and are reverted by the mTOR inhibitor rapamycin. The present findings indicate that α-syn does occur in high amount within GBM cells and may transmit to neighboring astrocytes as much as a stem cell phenotype. This suggests a mode of tumor progression for GBM cells, which may transform, rather than merely substitute, surrounding tissue; such a phenomenon is sensitive to mTOR inhibition. [ABSTRACT FROM AUTHOR]

Simple Summary: The accumulation of alpha-synuclein (α-syn) is considered a pathological hallmark of the neurodegenerative disorders known as synucleinopathies. The clearance of α-syn depends on autophagy activity, which is inhibited by the mechanistic target of rapamycin (mTOR). Thus, it is likely that α-syn accumulation may occur whenever mTOR is overactive and autophagy is suppressed. In fact, the lack of effective autophagy increases the amount of α-syn and may produce protein aggregation. Therefore, in the present study, we questioned whether cells from glioblastoma multiforme (GBM), a lethal brain neoplasm, wherein mTOR is upregulated and autophagy is suppressed, may overexpress α-syn. In fact, a large quantity of α-syn is measured in GBM cells compared with astrocytes, which includes proteinase K-resistant α-syn. Rapamycin, while inhibiting mTOR activity, significantly reduces the amount of α-syn and allocates α-syn within autophagy-like vacuoles. Alpha-synuclein (α-syn) is a protein considered to be detrimental in a number of degenerative disorders (synucleinopathies) of which α-syn aggregates are considered a pathological hallmark. The clearance of α-syn strongly depends on autophagy, which can be stimulated by inhibiting the mechanistic target of rapamycin (mTOR). Thus, the overexpression of mTOR and severe autophagy suppression may produce α-syn accumulation, including the proteinase K-resistant protein isoform. Glioblastoma multiforme (GBM) is a lethal brain tumor that features mTOR overexpression and severe autophagy inhibition. Cell pathology in GBM is reminiscent of a fast, progressive degenerative disorder. Therefore, the present work questions whether, as is analogous to neurons during degenerative disorders, an overexpression of α-syn occurs within GBM cells. A high amount of α-syn was documented in GBM cells via real-time PCR (RT-PCR), Western blotting, immunohistochemistry, immuno-fluorescence, and ultrastructural stoichiometry, compared with the amount of β- and γ-synucleins and compared with the amount of α-syn counted within astrocytes. The present study indicates that (i) α-syn is overexpressed in GBM cells, (ii) α-syn expression includes a proteinase-K resistant isoform, (iii) α-syn is dispersed from autophagy-like vacuoles to the cytosol, (iv) α-syn overexpression and cytosol dispersion are mitigated by rapamycin, and (v) the α-syn-related GBM-like phenotype is mitigated by silencing the SNCA gene. [ABSTRACT FROM AUTHOR]