PCSK9 Affects Astrocyte Cholesterol Metabolism and Reduces Neuron Cholesterol Supplying In Vitro: Potential Implications in Alzheimer’s Disease

Abstract

The Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) involvement in Alzheimer’s disease (AD) is poorly investigated. We evaluated the in vitro PCSK9 modulation of astrocyte cholesterol metabolism and neuronal cholesterol supplying, which is fundamental for neuronal functions. Moreover, we investigated PCSK9 neurotoxic effects. In human astrocytoma cells, PCSK9 reduced cholesterol content (−20%; p < 0.05), with a greater effect in presence of beta amyloid peptide (Aβ) (−37%; p < 0.01). PCSK9 increased cholesterol synthesis and reduced the uptake of apoE-HDL-derived cholesterol (−36%; p < 0.0001), as well as the LDL receptor (LDLR) and the apoE receptor 2 (ApoER2) expression (−66% and −31%, respectively; p < 0.01). PCSK9 did not modulate ABCA1- and ABCG1-cholesterol efflux, ABCA1 levels, or membrane cholesterol. Conversely, ABCA1 expression and activity, as well as membrane cholesterol, were reduced by Aβ (p < 0.05). In human neuronal cells, PCSK9 reduced apoE-HDL-derived cholesterol uptake (−41%; p < 0.001) and LDLR/apoER2 expression (p < 0.05). Reduced cholesterol internalization occurred also in PCSK9-overexpressing neurons exposed to an astrocyte-conditioned medium (−39%; p < 0.001). PCSK9 reduced neuronal cholesterol content overall (−29%; p < 0.05) and increased the Aβ-induced neurotoxicity (p < 0.0001). Our data revealed an interfering effect of PCSK9, in cooperation with Aβ, on brain cholesterol metabolism leading to neuronal cholesterol reduction, a potentially deleterious effect. PCSK9 also exerted a neurotoxic effect, and thus represents a potential pharmacological target in AD.

Plasma and cerebrospinal fluid cholesterol esterification is hampered in Alzheimer’s disease

“…CSF-CEC was measured in human glioblastoma astrocytoma U373-MG cell line, a standard surrogate model for human astrocytes, with a radio-isotopic assay as previously described [ 27 ]. The cells were kindly donated by Prof. Ovidio Bussolati from the Department of Medicine and Surgery of the University of Parma (Italy) and authenticated through the Cell Line Authentication (CLA) service (Eurofins Genomic, Ebersberg, Germany).…”

“…After seeding, cells were radiolabeled for 24 h with 2 µCi/mL of [1,2- 3 H(N)]-cholesterol (PerkinElmer, Waltham, MA, USA) in the presence of 2 µg/mL of an Acyl CoA: Cholesterol O-acyl transferase (ACAT) inhibitor compound (Sandoz; Sigma-Aldrich, Milano, Italy), to ensure all cholesterol present in the unesterified form. Subsequently, cells were treated for 18 h with DMEM supplemented with 0.2% bovine serum albumin (Sigma-Aldrich, Milano, Italy), the ACAT inhibitor, and the LXR/RXR agonists 22-hydroxycholesterol and 9-cis retinoic acid (5 µg/mL and 10 µM, respectively) to induce the expression of the active transporters ABCA1 and ABCG1 [ 27 ]. Finally, cells were exposed to CSF (30% v:v in DMEM) from AD patients and control subjects for 6 h. Cholesterol efflux was calculated as the percentage of radioactivity released into the medium over the total radioactivity incorporated by the cells.…”

Cellular cholesterol loss by DHCR24 knockdown leads to Aβ production by changing APP intracellular localization

“…Most notably, our previous and other studies used human neuronal cell line called SH-SY5Y and have also made a similar conclusion of DHCR24’s effects on cholesterol and Aβ metabolism ( 11 , 28 ). Additionally, it has been reported that upregulating PCSK9 expression in SH-SY5Y cell line inhibited neuronal cholesterol uptake and then increased Aβ neuronal toxicity ( 49 ). To sum up, the above results supported that cellular cholesterol deficiency induced Aβ generation or Aβ toxicity.…”

PCSK9 deficiency alters brain lipid composition without affecting brain development and function

“…The regulation of PCSK9 in plasma and CSF appears to be different and, unlike in plasma, PCSK9 levels in CSF are not affected by diurnal rhythm ( Chen et al, 2014 ). The role of PCSK9 in the brain still remains unclear but previous studies suggest that it may be involved in neuronal differentiation and apoptosis ( Rashid et al, 2005 ; Poirier et al, 2006 ), and influence cholesterol metabolism in cultured neuronal cells ( Papotti et al, 2022 ). In addition, pretreatment with a PCSK9 inhibitory peptide was found to be protective against brain damage in a rat model of cardiac ischemia/reperfusion injury ( Apaijai et al, 2019 ).…”