A team of Italian investigators previously observed dysregulation of autophagy in HHV-6A- and HHV-6B-infected HSB-2 cells, MOLT-3 cells, and monocytes from healthy donors (Romeo 2018, Romeo 2019). Building on this prior work, the team has produced new data indicating that HHV-6A is also able to induce dysregulation of this process in neurons and astrocytoma cells. Moreover, HHV-6A-induced autophagy was associated with increased beta-amyloid (Aβ) production and tau protein hyper-phosphorylation, phenomena that are linked to Alzheimer’s disease (AD), suggesting that these cellular processes may represent a mechanism by which HHV-6A could contribute to AD.
The process of autophagy is necessary for adequate clearance of toxic and unnecessary compounds in a cell, and this is especially important for cells that do not undergo replication, which could otherwise reduce the load of these compounds on any given cell through cell division. As post-mitotic cells, neurons are consequently at risk for such a buildup of unwanted materials and subsequent neurodegeneration, as is seen in AD.
The team used HHV-6A-infected and mock-infected neurons and U373 (a glioblastoma-astrocytoma cell line) cells in their experiments. By measuring the expression levels of LC3I/II and p62, markers of autophagy, reduced autophagy was observed in the astrocytoma cells after 72 hours of infection compared to the mock-infected cells. In cells that were simply exposed to UV-inactivated virus, binding of the inactivated virus to cells was not found to be sufficient for the reduction in autophagy, and cross-linking of the CD46 receptor did not affect autophagy either.
Lysosomal storage disorders, characterized by lysosomal dysfunction that results in accumulation of cellular waste, can be associated with dementia in young and middle-aged adults (Sun 2018). Impaired lysosomal activity is also seen in cells from patients with Down Syndrome (trisomy 21), who suffer from early-onset AD (Jiang 2019).
To determine whether alteration of lysosomal pH might be involved in the HHV-6A-mediated reduction of autophagy, the investigators stained infected and mock-infected cells with acridine orange, which changes color based on pH, and found that infected cells demonstrated reduced lysosomal acidification, which could result in intracellular accumulation of abnormal proteins such as Aβ, and its release into the extracellular space. However, expression of the lysosomal protease cathepsin D was found to be higher in infected cells, which the authors postulated could be a compensatory product of the cell’s attempts to cope with the altered lysosomal pH and reinitiate autophagy. Aβ production was also higher in infected cells than mock-infected cells.
Heightened cathepsin D expression has been observed in AD brains (Chai 2019). Cathepsin S, which is involved in inhibiting autophagy in glioblastoma cells and has been considered a potential target in the treatment of AD (Schechter 2011, Zhang 2014), has also been found to be upregulated in gene expression analysis of HHV-6A-infected cultured astrocytes (Shao 2016).
ER stress can result in unfolded protein response (UPR) activation, which promotes autophagy to reduce cellular stress via negative feedback. UPR activation was observed in infected cells, with slight up-regulation of the Ire1 alpha sensor and clear upregulation of ATF4, CHOP, and eIF2 alpha phosphorylation. BIP was also up-regulated, which pointed to activation of both pro-death and pro-survival UPR molecules. Tau phosphorylation at several serine residues, which can promote neurofibrillary tangle formation, was increased in infected cells. As reducing ER stress/UPR through use of 4-PBA or BSK2606414 reduced tau phosphorylation, ER stress/UPR was considered to trigger the phosphorylation in the HHV-6A+ cells.
Mean fold increase in Aβ secretion as evaluated by ELISA assay after 72 hours of culture or 24 hours of treatment with chloroquine. Source: BBA, Molecular Basis of Disease, March 2020
Administration of chloroquine, which is known to block autophagy and impair lysosomal acidification, affected lysosomal pH and Aβ production similarly to HHV-6A infection in U373 cells. In addition, chloroquine’s block of autophagy activated UPR and increased tau phosphorylation. Together, the findings suggested that the impaired autophagy brought about by HHV-6A infection may result in increased Aβ production, greater tau phosphorylation, and heightened ER stress and UPR in astrocytoma cells.
In a similar fashion to what was observed in astrocytoma cells, HHV-6A-infected neurons were found to have reduced autophagy. Tau phosphorylation was also increased compared to mock-infected neurons, as was Aβ production. In contrast to astrocytoma cells, neurons showed a strong cytopathic effect following infection and up-regulation of the pro-death UPR molecule CHOP without simultaneous up-regulation of the pro-survival molecule BIP.
Find the full paper here: Romeo 2020.