Characterization of ApoJ-reconstituted high-density lipoprotein (rHDL) nanodisc for the potential treatment of cerebral ß-amyloidosis.

Cerebral ß-amyloidosis is a major feature of Alzheimer's disease (AD), characterized by the accumulation of ß-amyloid protein (Aß) in the brain. Several studies have implicated lipid/lipoprotein metabolism in the regulation of ß-amyloidosis. In this regard, HDL (High Density Lipoprotein)-based therapies could ameliorate pathological features associated with AD. As apolipoprotein J (ApoJ) is a natural chaperone that interacts with Aß, avoiding its aggregation and toxicity, in this study we propose to prepare reconstituted rHDL-rApoJ nanoparticles by assembling phospholipids with recombinant human ApoJ (rApoJ). Hence, rHDL particles were prepared using the cholate dialysis method and characterized by N-PAGE, dynamic light scattering, circular dichroism and electron transmission microscopy. The preparation of rHDL particles showed two-sized populations with discoidal shape. Functionally, rHDL-rApoJ maintained the ability to prevent the Aß fibrillization and mediated a higher cholesterol efflux from cultured macrophages. Fluorescently-labelled rHDL-rApoJ nanoparticles were intravenously administrated in mice and their distribution over time was determined using an IVIS Xenogen® imager. It was confirmed that rHDL-rApoJ accumulated in the cranial region, especially in old transgenic mice presenting a high cerebral Aß load. In conclusion, we have standardized a reproducible protocol to produce rHDL-rApoJ nanoparticles, which may be potentially considered as a therapeutic option for ß-amyloid-related pathologies.

Intravenous treatment with human recombinant ApoA-I Milano reduces beta amyloid cerebral deposition in the APP23-transgenic mouse model of Alzheimer's disease.

Beyond the crucial role of apolipoprotein A-I (ApoA-I) on peripheral cholesterol metabolism, this apolipoprotein has also been implicated in beta amyloid (Aß)-related neuropathologies. ApoA-I-Milano (M) is a mutated variant, which showed increased vasoprotective properties compared to ApoA-I-wild type in models of atherosclerosis and cardiovascular damage. We speculated that ApoA-I-M may also protect Aß-affected vasculature and reverse some of the pathological features associated with Alzheimer's disease (AD). For this purpose, we produced and characterized human recombinant ApoA-I-wild type and ApoA-I-M proteins. Both of them were able to avoid the aggregation of Aß in vitro, even though recombinant ApoA-I-M was significantly more effective in protecting endothelial cells from Aß(1-42)-toxicity. Next, we determined the effect of chronic intravenous administration of rApoA-I-M in the APP23-transgenic mouse model of AD. We found reduced cerebral Aß levels in mice that received rApoA-I-M, which were accompanied by a lower expression of astrocyte and microglia neuroinflammatory markers. Our results suggest an applicability of this molecule as a therapeutic candidate for protecting the brain in AD.

Modulation of Amyloid-ß1-40 Transport by ApoA1 and ApoJ Across an in vitro Model of the Blood-Brain Barrier.

Amyloid-ß (Aß) accumulation in Alzheimer's disease (AD) and cerebral amyloid angiopathy (CAA) is likely caused by the impairment of its brain clearance that partly occurs through the blood-brain barrier (BBB). In this context, an in vitro BBB model is a valuable tool for studying the molecular mechanisms that regulate this process. This study assessed brain Aß elimination across the BBB and its modulation by the natural chaperones Apolipoprotein A1 (ApoA1) and Apolipoprotein J/Clusterin (ApoJ). The model was based on primary cerebral endothelial cells that were cultured on Matrigel-coated Transwells and treated with fluorescently labeled-Aß1-40 to track its efflux across the BBB, which corresponds to trafficking from the basolateral (brain) to apical (blood) compartments. We observed that the transport of basolateral Aß1-40 was enhanced when it was complexed to rApoJ, whereas the complex formed with rApoA1 did not influence Aß1-40 efflux. However, the presence of rApoA1 in the apical compartment was able to mobilize Aß1-40 from the basolateral side. We also observed that both rApoA1 and rApoJ moderately crossed the monolayer (from blood to brain) through a mechanism involving the LDL receptor-related protein family. In contrast to the increased rApoJ efflux when complexed to Aß1-40, rApoA1 trafficking was restricted when it was bound to the Aß peptide. In summary, the present study highlights the role of ApoJ and ApoA1 in the in vitro modulation of Aß elimination across the BBB.

ApoA1, ApoJ and ApoE Plasma Levels and Genotype Frequencies in Cerebral Amyloid Angiopathy.

The involvement of apolipoproteins, such as the ApoE4 isoform, in Alzheimer's disease (AD) and cerebral amyloid angiopathy (CAA) highlights the fact that certain lipid carriers may participate in soluble ß-amyloid (Aß) transport. Our general aim was to characterize the soluble levels of the apolipoproteins apoE, apoA1 and apoJ/clusterin and their genotype status in patients with CAA. We analyzed the genotypes frequency of APOA1 (rs5069, rs670), CLU (rs11136000, rs1532278, rs7012010, rs9331888) and APOE (rs429358, rs7412) in a cohort of patients with CAA-associated intracerebral hemorrhage (ICH) (n = 59) and compared the results with those from hypertension-associated ICH (n = 42), AD patients (n = 73) and controls (n = 88). In a subgroup of patients, we also determined the plasma concentrations of apoE, apoA1 and apoJ/clusterin. We found increased plasma apoJ/clusterin levels in CAA patients compared to AD patients or controls after adjusting for sex and age (CAA vs. controls, p = 0.033; CAA vs. AD, p = 0.013). ApoA1 levels were not altered between groups, although a strong correlation was observed between plasma Aß(1-40) and apoA1 among CAA patients (r = 0.583, p = 0.007). Regarding plasma apoE concentration, a robust association between circulating levels and genotype status was confirmed (p < 0.001). Whereas the APOE4 frequency was higher in AD (p < 0.001) and CAA (p = 0.013), the APOA1 and CLU genotypes were not different among groups. In the CAA cohort, the risk-linked CLU variant (C) rs11136000 was associated with white matter hyperintensities (p = 0.045) and the presence of lobar microbleeds (p = 0.023) on MRI. In summary, our findings suggest that apoA1 may act as a physiological transporter of Aß(1-40) and that apoJ/clusterin appears to be a chaperone related to distinctive lesions in CAA brains.

Matrix Metalloproteinases in Alzheimer's Disease and Concurrent Cerebral Microbleeds.

Matrix metalloproteinases (MMPs) are a family of enzymes able to degrade components of the extracellular matrix, which is important for normal blood-brain barrier function. Their function is regulated by tissue inhibitors of matrix metalloproteinases (TIMPs). We investigated whether MMPs and TIMPs in cerebrospinal fluid (CSF) and plasma were altered in Alzheimer's disease (AD) and vascular dementia (VaD), and whether this effect was modified by presence of cerebral micro-bleeds in AD patients. In addition, we assessed associations of MMPs and TIMPs with CSF amyloid-ß(1-42) (Aß42), tau, and tau phosphorylated at threonine-181 (p-tau). We measured MMP2, MMP9, and MMP10, and TIMP1 and TIMP2 in CSF and plasma of 52 AD patients, 26 matched controls, and 24 VaD patients. AD patients showed higher plasma MMP2 levels compared to VaD patients (p <  0.05), and higher CSF MMP10 levels compared to controls (p <  0.05). Microbleeds in AD were associated with lower CSF TIMP1, TIMP2 and MMP9 in a dose-response relation. In addition, CSF MMP2 was associated with p-tau (St.B 0.23, p <  0.05), and CSF MMP10 with tau (St.B 0.38, p <  0.001) and p-tau (St.B 0.40, p <  0.001). Our findings suggest involvement of MMP2 and MMP10 in AD pathology. Lower levels of TIMPs in AD patients with microbleeds suggest less MMP inhibition in patients with concurrent cerebral microbleeds, which may hypothetically lead to a more vulnerable blood-brain barrier in these patients.