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Background The aim of this study is to explore the quantity profiles of amyloid signature proteins (serum amyloid P component, SAP; apolipoprotein E, ApoE; apolipoprotein A-IV) in common types of renal amyloidosis by mass spectrometry and immunostaining methods. Methods Twenty-one patients with renal amyloidosis of different types evaluated at the Renal Pathological Center of Peking University First Hospital from 2000 to 2021 were enrolled. Immunohistochemistry (IHC) and laser microdissection combining with mass spectrometry (LMD-MS) were applied to investigate the localization and quantity profiles of signature proteins in renal amyloidosis. The co-localization relationships among signature proteins and amyloid fibrils, as well as the ultrastructural localization of SAP were examined by laser scanning confocal microscopy (LSCM) and immuno-electron microscopy (IEM), respectively. Results By MS-based proteomic analysis, large spectra numbers of ApoE and its higher abundance were noted in four types of amyloidosis when compared with SAP, and ApoA-IV was absent in ALECT2 amyloidosis. LSCM showed ApoE and SAP co-localized with amyloid fibrils in renal AL-κ, AL-λ and ALECT2 amyloidosis. ApoA-IV co-localized with amyloid fibrils in AL-κ and AL-λ amyloidosis, but was not found in ALECT2 amyloidosis. By semi-quantitative analysis based on LSCM and IEM, the quantity levels of signature proteins in AL-κ appeared to be lower than that in AL-λ ( P  < 0.05) or ALECT2 ( P  < 0.05), while there was no significant difference between AL-λ and ALECT2 amyloidosis. Conclusion Both of SAP and ApoE were the ubiquitous signature components of renal amyloidosis (AL, AA, ALECT2), as well as ApoA-IV in AL and AA, but not in ALECT2. ApoE was the key signature protein in renal amyloidosis. The quantity levels of signature proteins investigated through LCSM/IEM demonstrated variability among different types, with AL-κ amyloidosis appeared to have a lower level. Clinical trial number Not applicable.

Despite the important role of apolipoprotein E (apoE) secretion from astrocytes in brain lipid metabolism and the strong association of apoE4, one of the human apoE isoforms, with sporadic and late onset forms of Alzheimer's disease (AD) little is known about the regulation of astrocytic apoE. Utilizing annotated chemical libraries and a phenotypic screening strategy that measured apoE secretion from a human astrocytoma cell line, inhibition of pan class I histone deacetylases (HDACs) was identified as a mechanism to increase apoE secretion. Knocking down select HDAC family members alone or in combination revealed that inhibition of the class I HDAC family was responsible for enhancing apoE secretion. Knocking down LXRα and LXRβ genes revealed that the increase in astrocytic apoE in response to HDAC inhibition occurred via an LXR-independent pathway. Collectively, these data suggest that pan class I HDAC inhibition is a novel pathway for regulating astrocytic apoE secretion.

In the adult brain, neurons require local cholesterol production, which is supplied by astrocytes through apoE-containing lipoproteins. In Huntington’s disease (HD), such cholesterol biosynthesis in the brain is severely reduced. Here we show that this defect, occurring in astrocytes, is detrimental for HD neurons. Astrocytes bearing the huntingtin protein containing increasing CAG repeats secreted less apoE-lipoprotein-bound cholesterol in the medium. Conditioned media from HD astrocytes and lipoprotein-depleted conditioned media from wild-type (wt) astrocytes were equally detrimental in a neurite outgrowth assay and did not support synaptic activity in HD neurons, compared with conditions of cholesterol supplementation or conditioned media from wt astrocytes. Molecular perturbation of cholesterol biosynthesis and efflux in astrocytes caused similarly altered astrocyte–neuron cross talk, whereas enhancement of glial SREBP2 and ABCA1 function reversed the aspects of neuronal dysfunction in HD. These findings indicate that astrocyte-mediated cholesterol homeostasis could be a potential therapeutic target to ameliorate neuronal dysfunction in HD.

Purpose Amyloid PET tracers have been developed for in vivo detection of brain fibrillar amyloid deposition in Alzheimer’s disease (AD). To serve as an early biomarker in AD the amyloid PET tracers need to be analysed in multicentre clinical studies. Methods In this study 238 [ 11 C]Pittsburgh compound-B (PIB) datasets from five different European centres were pooled. Of these 238 datasets, 18 were excluded, leaving [ 11 C]PIB datasets from 97 patients with clinically diagnosed AD (mean age 69 ± 8 years), 72 patients with mild cognitive impairment (MCI; mean age 67.5 ± 8 years) and 51 healthy controls (mean age 67.4 ± 6 years) available for analysis. Of the MCI patients, 64 were longitudinally followed for 28 ± 15 months. Most participants (175 out of 220) were also tested for apolipoprotein E (ApoE) genotype. Results [ 11 C]PIB retention in the neocortical and subcortical brain regions was significantly higher in AD patients than in age-matched controls. Intermediate [ 11 C]PIB retention was observed in MCI patients, with a bimodal distribution (64 % MCI PIB-positive and 36 % MCI PIB-negative), which was significantly different the pattern in both the AD patients and controls. Higher [ 11 C]PIB retention was observed in MCI ApoE ε4 carriers compared to non-ApoE ε4 carriers ( p  < 0.005). Of the MCI PIB-positive patients, 67 % had converted to AD at follow-up while none of the MCI PIB-negative patients converted. Conclusion This study demonstrated the robustness of [ 11 C]PIB PET as a marker of neocortical fibrillar amyloid deposition in brain when assessed in a multicentre setting. MCI PIB-positive patients showed more severe memory impairment than MCI PIB-negative patients and progressed to AD at an estimated rate of 25 % per year. None of the MCI PIB-negative patients converted to AD, and thus PIB negativity had a 100 % negative predictive value for progression to AD. This supports the notion that PIB-positive scans in MCI patients are an indicator of prodromal AD.

Background The functionality of high-density lipoproteins (HDL) is a better cardiovascular risk predictor than HDL concentrations. One of the key elements of HDL functionality is its apolipoprotein composition. Lecithin-cholesterol acyl transferase (LCAT) and cholesterol-ester transfer protein (CETP) are enzymes involved in HDL-mediated reverse cholesterol transport. This study assessed the concentration and activity of LCAT and CETP in HDL subspecies defined by their content of apolipoproteins E (apoE) and C-III (apoC-III) in humans. Methods Eighteen adults (ten women and eight men, mean age 55.6, BMI 26.9 Kg/m 2 , HbA1c 5.4%) were studied. HDL from each participant were isolated and divided into four subspecies containing respectively: No apoE and no apoC-III (E-C-), apoE but not apoC-III (E + C-), apoC-III but no apoE (E-C+) and both apoE and apoC-III (E + C+). The concentration and enzymatic activity of LCAT and CETP were measured within each HDL subspecies using immunoenzymatic and fluorometric methods. Additionally, the size distribution of HDL in each apolipoprotein-defined fraction was determined using non-denaturing electrophoresis and anti-apoA-I western blotting. Results HDL without apoE or apoC-III was the predominant HDL subtype. The size distribution of HDL was very similar in all the four apolipoprotein-defined subtypes. LCAT was most abundant in E-C- HDL (3.58 mg/mL, 59.6% of plasma LCAT mass), while HDL with apoE or apoC-III had much less LCAT (19.8, 12.2 and 8.37% of plasma LCAT respectively for E + C-, E-C+ and E + C+). LCAT mass was lower in E + C- HDL relative to E-C- HDL, but LCAT activity was similar in both fractions, signaling a greater activity-to-mass ratio associated with the presence of apoE. Both CETP mass and CETP activity showed only slight variations across HDL subspecies. There was an inverse correlation between plasma LCAT activity and concentrations of both E-C+ pre-beta HDL ( r  = − 0.55, P  = 0.017) and E-C- alpha 1 HDL ( r  = − 0.49, P  = 0.041). Conversely, there was a direct correlation between plasma CETP activity and concentrations of E-C+ alpha 1 HDL ( r  = 0.52, P  = 0.025). Conclusions The presence of apoE in small HDL is correlated with increased LCAT activity and esterification of plasma cholesterol. These results favor an interpretation that LCAT and apoE interact to enhance anti-atherogenic pathways of HDL.

Background The APOE4 allele variant is the strongest known genetic risk factor for developing late-onset Alzheimer’s disease. The link between apolipoprotein E (apoE) and Alzheimer’s disease is likely due in large part to the impact of apoE on the metabolism of amyloid β (Aβ) within the brain. Manipulation of apoE levels and lipidation within the brain has been proposed as a therapeutic target for the treatment of Alzheimer’s disease. However, we know little about the dynamic regulation of apoE levels and lipidation within the central nervous system. We have developed an assay to measure apoE levels in the brain interstitial fluid of awake and freely moving mice using large molecular weight cut-off microdialysis probes. Results We were able to recover apoE using microdialysis from human cerebrospinal fluid (CSF) in vitro and mouse brain parenchyma in vivo . Microdialysis probes were inserted into the hippocampus of wild-type mice and interstitial fluid was collected for 36 hours. Levels of apoE within the microdialysis samples were determined by ELISA. The levels of apoE were found to be relatively stable over 36 hours. No apoE was detected in microdialysis samples from apoE KO mice. Administration of the RXR agonist bexarotene increased ISF apoE levels while ISF Aβ levels were decreased. Extrapolation to zero-flow analysis allowed us to determine the absolute recoverable concentration of apoE3 in the brain ISF of apoE3 KI mice. Furthermore, analysis of microdialysis samples by non-denaturing gel electrophoresis determined lipidated apoE particles in microdialysis samples were consistent in size with apoE particles from CSF. Finally, we found that the concentration of apoE in the brain ISF was dependent upon apoE isoform in human apoE KI mice, following the pattern apoE2>apoE3>apoE4. Conclusions We are able to collect lipidated apoE from the brain of awake and freely moving mice and monitor apoE levels over the course of several hours from a single mouse. Our technique enables assessment of brain apoE dynamics under physiological and pathophysiological conditions and in response to therapeutic interventions designed to affect apoE levels and lipidation within the brain.

Amyloidosis is a heterogeneous group of protein misfolding diseases characterized by deposition of amyloid proteins. The kidney is frequently affected, especially by immunoglobulin light chain (AL) and serum amyloid A (SAA) amyloidosis as the most common subgroups. Current diagnosis relies on histopathological examination, Congo red staining, or electron microscopy. Subtyping is done by immunohistochemistry; however, commercially available antibodies lack specificity. The purpose of this study was to identify and map amyloid proteins in formalin-fixed paraffin-embedded tissue sections using matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis in an integrated workflow. Renal amyloidosis and non-amyloidosis biopsies were processed for histological and MS analysis. Mass spectra corresponding to the congophilic areas were directly linked to the histological and MS images for correlation studies. Peptides for SAA and AL were detected by MALDI IMS associated to Congo red-positive areas. Sequence determination of amyloid peptides by LC-MS/MS analysis provided protein distribution and identification. Serum amyloid P component, apolipoprotein E, and vitronectin proteins were identified in both AA and AL amyloidosis, showing a strong correlation with Congo red-positive regions. Our findings highlight the utility of MALDI IMS as a new method to type amyloidosis in histopathological routine material and characterize amyloid-associated proteins that may provide insights into the pathogenetic process of amyloid formation. Graphical abstract Image correlation between hematoxylin and eosin, histochemistry (Congo red), and MALDI IMS of tissue sections from a patient affected with AA-amyloidosis. Hematoxylin and eosin staining ( HE ) shows glomerular structures ( black arrows ). Amyloid deposits appear in red in the Congo red-stained section in bright light ( CR ), and show an apple-green birefringence under polarized light ( GB , yellow arrows ). MALDI images for m/z = 1456.72, m/z = 1811.89 and m/z = 1156.6 correlate with Congo-red positive areas

Complications of atherosclerosis are the leading cause of morbidity and mortality worldwide. Various genetically modified mouse models are used to investigate disease trajectory with classical histology, currently the preferred methodology to elucidate plaque composition. Here, we show the strength of light-sheet fluorescence microscopy combined with deep learning image analysis for characterising and quantifying plaque burden and composition in whole aorta specimens. 3D imaging is a non-destructive method that requires minimal ex vivo handling and can be up-scaled to large sample sizes. Combined with deep learning, atherosclerotic plaque in mice can be identified without any ex vivo staining due to the autofluorescent nature of the tissue. The aorta and its branches can subsequently be segmented to determine how anatomical position affects plaque composition and progression. Here, we find the highest plaque accumulation in the aortic arch and brachiocephalic artery. Simultaneously, aortas can be stained for markers of interest (for example the pan immune cell marker CD45) and quantified. In ApoE−/− mice we observe that levels of CD45 reach a plateau after which increases in plaque volume no longer correlate to immune cell infiltration. All underlying code is made publicly available to ease adaption of the method.

PICALM rs3851179 and CLU rs11136000 have been recently associated with Alzheimer’s disease (AD). Investigating the effects of these genetic variants on the resting-state functional connectivity (rsFC) of the hippocampus may provide new insight into AD pathogenesis. We investigated the main effects and interactions of these two genetic variants on hippocampal rsFC in 283 healthy young adults. The hippocampus showed positive rsFC with the default mode network and negative rsFC with the fronto-parietal network. Risk PICALM G-allele carriers showed weaker negative rsFC compared with AA carriers, whereas risk CLU-CC carriers exhibited stronger positive and negative rsFC than T-allele carriers. There existed complex interactions between PICALM and CLU on the negative rsFC of the hippocampus. Moreover, we found an allele-dependent effect of CLU on hippocampal connectivity when an additive genetic model was applied to CLU. Most of these effects remained significant even after controlling for individual ApoE status. Our results suggest that PICALM and CLU risk genotypes exert differential impacts on the hippocampal rsFC in healthy young subjects. The complex interactions between PICALM and CLU should be considered when investigating the impact of these two genetic variants on the brain.