Research Article: Amyloid‐beta 1‐40 is associated with alterations in NG2+ pericyte population ex vivo and in vitro

Date Published: February 17, 2018

Publisher: John Wiley and Sons Inc.

Author(s): Nina Schultz, Kristoffer Brännström, Elin Byman, Simon Moussaud, Henrietta M. Nielsen, Anders Olofsson, Malin Wennström.

http://doi.org/10.1111/acel.12728

Abstract

The population of brain pericytes, a cell type important for vessel stability and blood brain barrier function, has recently been shown altered in patients with Alzheimer’s disease (AD). The underlying reason for this alteration is not fully understood, but progressive accumulation of the AD characteristic peptide amyloid‐beta (Aβ) has been suggested as a potential culprit. In the current study, we show reduced number of hippocampal NG2+ pericytes and an association between NG2+ pericyte numbers and Aβ1‐40 levels in AD patients. We further demonstrate, using in vitro studies, an aggregation‐dependent impact of Aβ1‐40 on human NG2+ pericytes. Fibril‐EP Aβ1‐40 exposure reduced pericyte viability and proliferation and increased caspase 3/7 activity. Monomer Aβ1‐40 had quite the opposite effect: increased pericyte viability and proliferation and reduced caspase 3/7 activity. Oligomer‐EP Aβ1‐40 had no impact on either of the cellular events. Our findings add to the growing number of studies suggesting a significant impact on pericytes in the brains of AD patients and suggest different aggregation forms of Aβ1‐40 as potential key regulators of the brain pericyte population size.

Partial Text

One of the most well‐established hallmarks of Alzheimer’s disease (AD) is the progressive accumulation of the amyloid‐beta (Aβ) peptide, forming Aβ plaques in the brain. Brain areas containing such Aβ plaques display neuroinflammation, featured by activated glial cells, and substantial loss of neurons. Not only neurons and glial cells are affected in AD, but also the vascular network appears to be altered and reduced vessel density, dysfunctional blood–brain barrier (BBB) and microbleeds are commonly seen in AD patients (Iadecola, 2004; Kalaria, 2002; Zlokovic, 2005). A recent study conducted by Sengillo and colleagues has further showed a marked loss of pericytes in AD patients compared to age‐matched nondemented controls (Sengillo et al., 2013). These cells are very dynamic and versatile. In their mature state, they play a key role in vessel stabilization and vessel permeability, but in response to changes in their environment, pericytes become activated and take part in events such as vascular remodelling, inflammation (Bergers & Song, 2005; Paul et al., 2012) and clearance of neurotoxic substances (including Aβ) (Zlokovic, 2008). Hence, loss of pericytes leads not only to deleterious events, such as impaired BBB or vessel leakage (Quaegebeur, Segura & Carmeliet, 2010), but could also underlie the accumulation of Aβ in the AD brain.

Studies analysing brain pericytes by the use of immunohistochemical staining are upon recently very few and foremost performed on rodents. The reason for this shortage of studies are difficulties in staining pericytes in human brain tissue. Many of the pericyte‐specific markers are sensitive for overfixation and antigen retrieval methods used in stainings of paraffin sections. Our analysis of number of pericyte cell bodies in the ML of hippocampus was performed by the use of immunohistochemical stainings against the pericyte marker chondroitin proteoglycan NG2 and the pericyte/endothelial marker laminin α5. Both markers are expressed by pericytes on arterioles and capillaries (Stapor et al., 2014), but laminin α5 is additionally expressed by pericytes on postcapillary venules (Yousif et al., 2013). Importantly, pericytes are dynamic and versatile and the expression of markers, which includes also nestin, desmin, CD13, PDGFR‐ß and RGS5, fluctuates depending on activation, migration and vessel‐stabilization pericyte state. For example, NG2 is associated with both mature and active pericytes (migration, vascular remodelling and inflammation; Stapor et al., 2014), whereas pericytes expressing laminin are foremost found on quiescent and mature vessels (Hallmann et al., 2005). None of the markers mentioned above are found on all pericytes, and hence, immunohistochemistry based on one marker only enables analysis of a subset of the pericyte population. Our study showed significantly decreased number of NG2+ pericytes as well as decreased number of NG2+ pericytes/vessel length in AD patients compared to nondemented controls, but the analysis of laminin+ pericytes showed no significant differences in pericyte variables between the groups. This difference highlights the heterogeneity of the pericyte population (i.e. marker expression and localization) and proposes an impact of AD pathology on foremost activated pericytes. The reduction in NG2+ pericytes could be due to a direct impact on the pericyte population either by degeneration or formation of new NG2+ pericytes, but in view of our laminin result we cannot exclude the possibility that the pericyte population size remains the same and the difference is due to a downregulation of NG2 expression. Nevertheless, the results found in our study are in line with the previous study by Sengillo and colleagues, demonstrating a pericyte loss in AD patients (Sengillo et al., 2013). In that study, the number of mural cells (an umbrella term for smooth muscle cells and pericytes) was analysed by measuring the amount of PDGFR‐β+ (a marker for migrating immature mural cells (Song, Ewald, Stallcup, Werb & Bergers, 2005)) and CD13+ (marker for activated pericytes (Svensson, Ozen, Genove, Paul & Bengzon, 2015)) cells associated with vessels/capillaries in hippocampus. They found a 60% reduction in hippocampal mural cells and a 33% reduction in hippocampal pericytes, which is in the order of magnitude with our own study (approximately 26% reduction in NG2+ pericytes). In view of these findings, we conclude that different subsets of pericytes involved in migration, activation and remodelling (NG2+, PDGFR‐β+ and CD13+ pericytes) are affected by AD pathology, whereas mature and quiescent pericytes (laminin+) are less affected.

To conclude, our results confirm the previous study demonstrating pericyte alterations in AD patients and highlight Aβ1‐40 as a potential regulator of brain pericyte population. Moreover, our in vitro studies show that the regulatory role of Aβ1‐40 is dependent on aggregation form, where the monomeric appears to have a rescuing and mitogenic impact, oligomers are quiescent and fibrils induce toxicity. This is particularly interesting given the common notion that the oligomer form of the kin peptide, Aβ1‐42, is considered to be the culprit behind neuronal loss in AD. Hence, our results point out the differences between Aβ species and their aggregation forms in terms of toxicity on pericytes, a finding important to take into consideration when targeting the aggregation process of Aβ as a treatment for AD.

NS carried out the experiments, analysed data and drafted the manuscript; EB participated in scientific discussions and tissue preparation; KB and AO were involved in preparing and evaluating Aβ peptides; SM and HMN performed APOE genotyping; NBB supplied the brain tissue, neuropathological analyses, discussions regarding tissue pretreatment, interpreting and matching clinical data and advising on the approach of the study; MW designed the study and edited the final version of the manuscript. All authors read and approved the final manuscript.

The authors declare that they have no competing interests.

 

Source:

http://doi.org/10.1111/acel.12728

 

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