Igarashi, S. | Search bioRxiv

bioRxiv2021

Small Vessel Diseases: 3D Characteristics of the Vasculature and White Matter

Cerebral small vessel disease (SVD) is associated with white matter hyperintensities (WMHs), thereby contributing to vascular dementia and movement disorder. However, the pathomechanisms responsible for WMH-related small vessel degeneration remain poorly understood due to the technical limitations of current methods. The aim of this study was to clarify the 2- and 3-dimensional (2D and 3D) pathological features of small vessels and white matter (WM) in the brains of patients with cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), HTRA1-autosomal dominant disease (HTRA1-AD) and sporadic SVD (sSVD). From a cohort of 86 consecutive autopsied patients with SVDs, we retrieved those with genetically confirmed CADASIL and HTRA1-AD (three and three, respectively), and four with sSVD. We quantitatively evaluated WM and vascular changes in the frontal portion of the centrum semiovale and temporal lobe using conventional 2D and chemically cleared 3D analytical methods with light-sheet fluorescence microscopy. Quantitatively, the WM pathology, including the density of myelin, axons and gliosis, was most severe in CADASIL, but unexpectedly sSVD was second in order of severity, followed by HTRA1-AD. The density of clasmatodendrocytes, known to be irreversibly injured astrocytes, was considerably highest in HTRA1-AD. The vascular pathology, including arteriole and capillary sclerosis and the extent of the perivascular space, was most severe in CADASIL, whereas the density of smooth muscle actin (SMA) positivity was most decreased in HTRA1-AD. 3D immunohistochemistry for SMA demonstrated two distinct patterns of SMA loss within the vessels: (1) CADASIL and sSVD: diffuse loss, being prominent in small branches, (2) HTRA1-AD: selective loss in main branches. Overall, the extent of WM and vascular degeneration is most severe in CADASIL, whereas SMA loss is most evident in HTRA1-AD. These differences in the size and distribution of affected vessels may be related to the heterogeneous WM pathology and underlying pathomechanisms of SVD.

pathology↗

bioRxiv2019

EGR1 transcriptional control of human cytomegalovirus latency

Sustained phosphotinositide3-kinase (PI3K) signaling is critical to the maintenance of herpesvirus latency. We have previously shown that the beta-herpesvirus, human cytomegalovirus (CMV), regulates epidermal growth factor receptor (EGFR), upstream of PI3K, to control states of latency and reactivation. Inhibition of EGFR signaling enhances CMV reactivation from latency and increases viral replication, but the mechanisms by which EGFR impacts replication and latency is not known. We demonstrate that HCMV downregulates MEK/ERK and AKT phosphorylation, but not STAT3 or PLC{gamma} for productive replication. Similarly, inhibition of either MEK/ERK or PI3K/AKT, but not STAT or PLC{gamma}, pathways increases viral reactivation from latently infected CD34+ hematopoietic progenitor cells (HPCs), defining a role for these pathways in latency. We hypothesized that CMV modulation of EGFR signaling might impact viral transcription. Indeed, EGF-stimulation increased expression of the UL138 latency gene, but not immediate early or early viral genes, suggesting that EGFR signaling promotes latent gene expression. The early growth response-1 (EGR1) transcription factor is induced downstream of EGFR signaling through both PI3K/AKT and MEK/ERK pathways. EGR1 expression is important for the maintenance of HPC stemness and its downregulation drives HPC differentiation and mobilization. We demonstrate that EGR1 binds upstream of UL138 and is sufficient to promote UL138 expression. Further, disruption of EGR1 binding upstream of UL138 prevented CMV from establishing a latent infection in CD34+ HPCs. Our results indicate a model whereby UL138 modulation of EGFR signaling feeds back to promote UL138 expression and suppression of replication to establish or maintain viral quiescence.\n\nAUTHOR SUMMARYCMV regulates EGFR signaling to balance states of viral latency and replication. CMV blocks downstream PI3K/AKT and MEK/ERK signaling pathways through down-regulation of EGFR at the plasma membrane. PI3K/AKT and MEK/ERK signaling increases expression of the EGR1 transcription factor that is necessary for the maintenance of stem cell stemness. A decrease in EGR1 expression promotes HPC mobilization to the periphery and differentiation, a known stimulus for CMV reactivation. We identified functional EGR1 binding sites upstream of the UL138 gene and EGR-1 binding stimulates UL138 expression. Additionally, down-regulation of EGR1 by CMV miR-US22 decreases UL138 expression emphasizing the importance of this transcription factor in expression of this latency gene. Lastly, we demonstrate that a CMV mutant virus lacking an upstream EGR1 binding site is unable to establish latency in CD34+ HPCs. This study defines one mechanism by which EGFR signaling impacts viral gene expression to promote CMV latency.

molecular biology↗