👤 Stefano Zanotti

Increasing evidence highlights the importance of novel players in Alzheimer's disease (AD) pathophysiology, including alterations of lipid metabolism and neuroinflammation. Indeed, a potential involvement of Proprotein convertase subtilisin/kexin type 9 (PCSK9) in AD has been recently postulated. Here, we first investigated the effects of PCSK9 on neuroinflammation in vitro. Then, we examined the impact of a genetic ablation of PCSK9 on cognitive performance in a severe mouse model of AD. Finally, in the same animals we evaluated the effect of PCSK9 loss on Aβ pathology, neuroinflammation, and brain lipids. For in vitro studies, U373 human astrocytoma cells were treated with Aβ fibrils and human recombinant PCSK9. mRNA expression of the proinflammatory cytokines and inflammasome-related genes were evaluated by q-PCR, while MCP-1 secretion was measured by ELISA. For in vivo studies, the cognitive performance of a newly generated mouse line - obtained by crossing 5XFAD In vitro, PCSK9 significantly increased IL6, IL1B and TNFΑ mRNA levels in Aβ fibrils-treated U373 cells, without influencing inflammasome gene expression, except for an increase in NLRC4 mRNA levels. In vivo, PCSK9 ablation in 5XFAD mice significantly improved the performance at the Morris water maze test; these changes were accompanied by a reduced corticohippocampal Aβ burden without affecting plaque spatial/regional distribution and composition or global BACE1 expression. Furthermore, PCSK9 loss in 5XFAD mice induced decreased microgliosis and astrocyte reactivity in several brain regions. Conversely, knocking out PCSK9 had minimal impact on brain cholesterol and hydroxysterol levels. In vitro studies showed a pro-inflammatory effect of PCSK9. Consistently, in vivo data indicated a protective role of PCSK9 ablation against cognitive impairments, associated with improved Aβ pathology and attenuated neuroinflammation in a severe mouse model of AD. PCSK9 may thus be considered a novel pharmacological target for the treatment of AD. Show less

The reduction of plasma apolipoprotein B (apoB) containing lipoproteins has long been pursued as the main modifiable risk factor for the development of cardiovascular disease (CVD). This has led to an intense search for strategies aiming at reducing plasma apoB-lipoproteins, culminating in reduction of overall CV risk. Despite 3 decades of progress, CVD remains the leading cause of morbidity and mortality worldwide and, as such, new therapeutic targets are still warranted. Clinical and preclinical research has moved forward from the original concept, under which some lipids must be accumulated and other removed to achieve the ideal condition in disease prevention, into the concept that mechanisms that orchestrate lipid movement between lipoproteins, cells and organelles is equally involved in CVD. As such, this review scrutinizes potentially atherogenic changes in lipid trafficking and assesses the molecular mechanisms behind it. New developments in risk assessment and new targets for the mitigation of residual CVD risk are also addressed. Show less

Glucocorticoids in excess suppress osteoblast function and cause osteoporosis. We demonstrated that cortisol induces the expression of selected Notch receptors in osteoblasts, revealing a potential mechanism for the skeletal effects of glucocorticoids. However, it remains to be determined whether increased expression of Notch receptors results into enhanced signaling. Following activation of Notch, its intracellular domain (NICD) binds to the DNA-associated protein recombination signal binding protein for immunoglobulin kappa-J region (RBPJ) and induces the expression of target genes such as Hey1, Hey2, and HeyL. To determine whether glucocorticoids modulate Notch signaling in the skeleton, 1 month old wild-type mice were administered prednisolone or placebo and sacrificed after 72 h, and gene expression was analyzed in femoral bone. Prednisolone induced Tsc22d3, a glucocorticoid target gene, and suppressed Hey1 and HeyL expression, which is indicative of inhibited Notch receptor activity or direct Hey downregulation. To determine the mechanisms of Hey suppression, wild-type osteoblast-enriched cells were seeded on the Notch cognate ligand Delta-like (DLL)1 or transfected with constructs expressing the NOTCH1 NICD fragment and exposed to either cortisol or vehicle. Cortisol opposed the induction of mRNA and heterogeneous nuclear RNA for Hey1, Hey2, and HeyL by DLL1, but had no effect on mRNA stability, indicating that glucocorticoids inhibit Hey expression by transcriptional mechanisms. Transactivation studies and electrophoretic mobility shift assays revealed that cortisol did not oppose RBPJ-mediated transcription or RBPJ/DNA interactions, respectively. In conclusion, glucocorticoids suppress expression of Hey1, Hey2, and HeyL in osteoblasts by RBPJ-independent transcriptional mechanisms. Show less

Parathyroid hormone (PTH) and Notch receptors regulate bone formation by governing the function of osteoblastic cells. To determine whether PTH interacts with Notch signaling as a way to control osteoblast function, we tested the effects of PTH on Notch activity in osteoblast- and osteocyte-enriched cultures. Notch signaling was activated in osteoblast-enriched cells from wild-type C57BL/6J mice following exposure to the Notch ligand Delta-like (Dll)1 or by the transient transfection of the Notch intracellular domain (NICD), the transcriptionally active fragment of Notch1. To induce Notch signaling in osteocyte-enriched cultures, a murine model of Notch2 gain-of-function was used. PTH opposed the stimulatory effects of Dll1 on Hey1, Hey2 and HeyL mRNA levels in osteoblast-enriched cells and suppressed the expression of selected Notch target genes in osteocyte-enriched cultures, either under basal conditions or in the context of Notch2 gain-of-function. Induction of Notch signaling in osteocytes did not alter the inhibitory effect of PTH on Sost expression, but reduced the stimulation of Tnfsf11 mRNA levels by PTH. In agreement with these in vitro observations, male mice administered with PTH displayed suppressed Hey1 and HeyL expression in parietal bones. Transactivation experiments with a Notch reporter construct and electrophoretic mobility shift assays in osteoblast-enriched cells suggest that PTH acts by decreasing the capacity of Rbpjκ to bind to DNA. In conclusion, downregulation of Notch in osteoblasts and osteocytes may represent a mechanism contributing to the anabolic effects of PTH in bone. Show less

Notch induces Hairy Enhancer of Split (Hes)1 and Hes-related with YRPW motif (Hey) Hey1, Hey2 and Hey-like (HeyL) expression in osteoblasts, but it is not known whether any of these target genes mediates the effect of Notch in the skeleton. We demonstrated that Notch1 activation in osteoblasts/osteocytes induces Hes1, Hey1, Hey2, and HeyL, but HeyL was induced to a greater extent than other target genes. To characterize HeyL null mice for their skeletal phenotype, microcomputed tomography (µCT) and histomorphometric analysis of HeyL null and sex-matched littermate controls was performed. µCT demonstrated modest cancellous bone osteopenia in 1 month old male mice and normal microarchitecture in 3 month old male HeyL null mice. Female HeyL null mice were not different from controls at either 1 or 3 months of age. Bone histomorphometry did not demonstrate differences between HeyL null mice of either sex and littermate controls. In conclusion, HeyL null mice do not exhibit an obvious skeletal phenotype demonstrating that HeyL is dispensable for skeletal homeostasis. J. Cell. Biochem. 118: 1819-1826, 2017. © 2016 Wiley Periodicals, Inc. Show less

Connective tissue growth factor (Ctgf) or CCN2 is a protein synthesized by osteoblasts necessary for skeletal homeostasis, although its overexpression inhibits osteogenic signals and bone formation. Ctgf is induced by bone morphogenetic proteins, transforming growth factor β and Wnt; and in the present studies, we explored whether Notch regulated Ctgf expression in osteoblasts. We employed Rosa(Notch) mice, where the Notch intracellular domain (NICD) is expressed following the excision of a STOP cassette, placed between the Rosa26 promoter and NICD. Notch was activated by transduction of adenoviral vectors expressing Cre recombinase (Ad-CMV-Cre). Notch induced Ctgf mRNA levels in a time dependent manner and increased Ctgf heterogeneous nuclear RNA. Notch also destabilized Ctgf mRNA shortening its half-life from 13h to 3h. The effect of Notch on Ctgf expression was lost following Rbpjκ downregulation, demonstrating that it was mediated by Notch canonical signaling. However, downregulation of the classic Notch target genes Hes1, Hey1 and Hey2 did not modify the effect of Notch on Ctgf expression. Wild type osteoblasts exposed to immobilized Delta-like 1 displayed enhanced Notch signaling and increased Ctgf expression. In addition to the effects of Notch in vitro, Notch induced Ctgf in vivo, and calvariae and femurs from Rosa(Notch) mice mated with transgenics expressing the Cre recombinase in cells of the osteoblastic lineage exhibited increased expression of Ctgf. In conclusion, Ctgf is a target of Notch canonical signaling in osteoblasts, and may act in concert with Notch to regulate skeletal homeostasis. Show less

Notch induces Hairy and Enhancer of Split-related with YRPW motif (Hey)1, Hey2, and HeyL expression in osteoblasts, but the contributions of these genes to the skeletal effects of Notch are not fully understood. HEY1 misexpression has limited skeletal impact, female HeyL null mice display increased bone mass, and Hey2 inactivation is developmentally lethal. To inactivate Hey2 in immature or mature osteoblasts, Hey2(loxP/loxP) mice were crossed with transgenics expressing CRE under the control of the osterix (Osx-Cre) or osteocalcin (Oc-Cre) promoters to generate Osx-Cre(+/-);Hey2(Δ/Δ) or Oc-Cre(+/-);Hey2(Δ/Δ) mice. Trabecular bone volume increased in 3-month-old Osx-Cre(+/-);Hey2(Δ/Δ) and Oc-Cre(+/-);Hey2(Δ/Δ) male mice and in 1-month-old Oc-Cre(+/-);Hey2(Δ/Δ) female mice, although 3-month-old Oc-Cre(+/-);Hey2(Δ/Δ) females developed osteopenia. Alkaline phosphatase liver/bone/kidney (ALPL) expression and activity were suppressed in osteoblasts from Oc-Cre(+/-);Hey2(Δ/Δ) mice of both sexes. To overexpress HEY2 in osteoblasts, transgenic mice where a 3.6-kb fragment of the rat collagen type-I α1 promoter directs HEY2 expression were created. Three-month-old Hey2 transgenic males exhibited decreased osteoblast activity and increased bone resorption and developed osteopenia at 6 months of age. Hey2 transgenic females exhibited reduced osteoblast number and function, but no changes in bone resorption. HEY2 overexpression in osteoblasts from mice of both sexes inhibited ALPL expression and activity and suppressed osteocalcin transcripts in cells from male mice only. HEY2 overexpression in osteoblasts from male mice enhanced bone resorption by co-cultured splenocytes and induced interleukin-6, a molecule that promotes osteoclastogenesis. In conclusion, HEY2 decreases skeletal mass and regulates bone remodeling in male mice. Show less

Notch receptors regulate osteoblastogenesis, and Notch activation induces cleavage and nuclear translocation of the Notch intracellular domain (NICD), which associates with Epstein-Barr virus latency C-promoter binding factor-1/suppressor of hairless/lag-1 (CSL) and induces transcription of Notch target genes, such as hairy enhancer of split-related with YRPW motif (Hey)1 and Hey2. Nuclear factors of activated T-cells (NFAT) are transcription factors that regulate osteoclastogenesis, but their function in osteoblasts is not clear. Notch inhibits NFATc1 transcription, but interactions between Notch and NFAT are understood poorly. To determine the regulation of NFAT expression by Notch, osteoblasts from Rosa(Notch) mice, where NICD is transcribed following excision of a loxP flanked STOP cassette, were used. Alternatively, wild-type C57BL/6 osteoblasts were exposed to the Notch ligand Delta-like (Dll)1 to induce Notch signaling or to bovine serum albumin as control. In Rosa(Notch) osteoblasts, Notch suppressed NFATc1 expression, increased Nfatc2 mRNA by post-transcriptional mechanisms, and had no effect on NFATc3 and NFATc4 transcripts. Induction of Nfatc2 transcripts by Notch was confirmed in C57BL/6 osteoblasts exposed to Dll1. To investigate NFATc2 function in osteoblasts, constitutively active NFATc2 was overexpressed in Rosa(Notch) osteoblasts. NFATc2 suppressed Notch transactivation and expression of Hey genes. Electrophoretic mobility shift assays revealed that NFATc2 and CSL bind to similar DNA sequences, and chromatin immunoprecipitation indicated that NFATc2 displaced CSL from the Hey2 promoter. The effects of NICD and NFATc2 in Rosa(Notch) osteoblasts were assessed, and both proteins inhibited osteoblast function. In conclusion, Notch stabilizes Nfatc2 transcripts, NFATc2 suppresses Notch signaling, and both proteins inhibit osteoblast function. Show less