Accumulation of amyloid beta 1–42 (Aβ42) peptide in the extracellular space in the brain is a major observation in Alzheimer’s Disease (AD)-related pathology. Astroc... More
Accumulation of amyloid beta 1–42 (Aβ42) peptide in the extracellular space in the brain is a major observation in Alzheimer’s Disease (AD)-related pathology. Astrocytes are known to play pivotal role in clearing the extracellular aβ peptide from the brain, and the underlying mechanism of Aβ42 peptide clearance remains underappreciated. Like other cell types in the brain, astrocytes have primary cilia, a nonmotile microtubule-based organelle. Aβ42 peptide is reported to affect cilia length or structure in multiple cell types including neurons and inhibit ciliary p75 neurotrophin receptor (p75NTR). To date, the relationship between the extracellular Aβ42 and the astrocytic cilia has not been established. In this work, using primary human hippocampal astrocytes and post-mortem brain specimens obtained from AD patients, we performed molecular, flow cytometry and imaging approaches to investigate the relationship of astrocytic cilia and extracellular Aβ42 peptide. Our data demonstrate that the exogenous Aβ42 peptide treatment in vitro, induces expression of p75NTR in astrocyte cilia in a dose-dependent fashion. We also observed the enrichment of exogenous Aβ42 peptide in the astrocyte cilia and the plasma membrane of astrocytes. In exogenous Aβ42 peptide-treated groups, we observed aberrant proliferation and cell cycle, increased oxidative stress and apoptosis. Interestingly, we observed an enrichment of astrocytic p75NTR expression in the human post-mortem AD-brain. Silencing RNA (siRNA)-mediated knockdown of p75NTR gene significantly minimized the enrichment of exogenous Aβ peptide and the oxidative stress in primary hippocampal astrocytes in vitro. These studies unravel a molecular signaling mechanism that involves Aβ42 peptide-induced p75NTR-mediated oxidative stress that affects overall astrocyte health in AD-associated pathology. Less
The cryopreservation of primary human brain vascular pericytes (HBVPs) has facilitated their commercial availability as an essential component of the blood–brain barrie... More
The cryopreservation of primary human brain vascular pericytes (HBVPs) has facilitated their commercial availability as an essential component of the blood–brain barrier. Among the commercial suppliers of HBVPs in suspension, only one provides information regarding the cryoprotectant and vehicle used, i.e., 5% dimethyl sulfoxide (Me2SO) in complete medium, with no cryopreservation procedure given. Published protocols for the cryopreservation of brain vascular pericytes derived from human-induced pluripotent stem cells use traditional 10% Me2SO. In this study, we characterized the cryobiological response of HBVPs using graded freezing, both in the absence of cryoprotectants, which identifies their susceptibility to the main types of cryoinjury, and in the presence of several different cryoprotectants with the aim of quantifying post-thaw outcome. We first validated our graded freezing technique using human cerebral microvascular endothelial cell/D3 clone (hCMEC/D3) by comparing the membrane integrity results in this study in the absence and presence of 5% Me2SO plus 6% hydroxyethyl starch (HES) in hCMEC/D3 complete medium, with those previously published by our group. We then assessed the cryobiological response of HBVPs to controlled slow cooling at 1 °C/min in the absence and presence of various cryoprotectants, namely 5% Me2SO, 10% Me2SO, 5% Me2SO plus 6% HES, or 10% glycerol, all in HBVP complete medium. We found that all cryoprotectants tested yielded immediate post-thaw membrane integrity >93%. Cryopreservation with 10% Me2SO resulted in 95.3 ± 0.7% membrane integrity; using a lower concentration of Me2SO (5%) alone, or in the presence of 6% HES, resulted in membrane integrities of 93.3 ± 0.8% and 94.2 ± 0.8%, respectively. For applications where avoiding Me2SO is preferred, we showed that HBVPs can be cryopreserved in 10% glycerol (post-thaw membrane integrity of 95.7 ± 0.5%). The AlamarBlue reduction assay with 3 h incubation time indicated that HBVPs cryopreserved with either 5% Me2SO, 10% Me2SO, 5% Me2SO plus 6% HES, or 10% glycerol exhibited post-thaw metabolic activities which were not statistically different from those of unfrozen controls. Our study presents various application-specific options for cryopreservation of primary HBVPs with validated post-thaw membrane integrity and metabolic activity.
Keywords:
Human brain vascular pericytes, Graded freezing, Membrane integrity, Metabolic activity, Dimethyl sulfoxide, Hydroxyethyl starch, Glycerol, Fluorescence microscopy, Cryopreservation Less
ˆCCL24 is a pro-fibrotic, pro-inflammatory chemokine expressed in several chronic fibrotic diseases. In the liver, CCL24 plays a role in fibrosis and inflammation, and b... More
ˆCCL24 is a pro-fibrotic, pro-inflammatory chemokine expressed in several chronic fibrotic diseases. In the liver, CCL24 plays a role in fibrosis and inflammation, and blocking CCL24 led to reduced liver injury in experimental models. We studied the role of CCL24 in primary sclerosing cholangitis (PSC) and evaluated the potential therapeutic effect of blocking CCL24 in this disease. Multidrug resistance gene 2–knockout (Mdr2–/–) mice demonstrated CCL24 expression in liver macrophages and were used as a relevant experimental PSC model. CCL24-neutralizing monoclonal antibody, CM-101, significantly improved inflammation, fibrosis, and cholestasis-related markers in the biliary area. Moreover, using spatial transcriptomics, we observed reduced proliferation and senescence of cholangiocytes following CCL24 neutralization. Next, we demonstrated that CCL24 expression was elevated under pro-fibrotic conditions in primary human cholangiocytes and macrophages, and it induced proliferation of primary human hepatic stellate cells and cholangiocytes, which was attenuated following CCL24 inhibition. Correspondingly, CCL24 was found to be highly expressed in liver biopsies of patients with PSC. CCL24 serum levels correlated with Enhanced Liver Fibrosis score, most notably in patients with high alkaline phosphatase levels. These results suggest that blocking CCL24 may have a therapeutic effect in patients with PSC by reducing liver inflammation, fibrosis, and cholestasis. Less
