Parenchymal border macrophages regulate the flow dynamics of the cerebrospinal fluid.

Macrophages are important players in the maintenance of tissue homeostasis1. Perivascular and leptomeningeal macrophages reside near the central nervous system (CNS) parenchyma2, and their role in CNS physiology has not been sufficiently well studied. Given their continuous interaction with the cerebrospinal fluid (CSF) and strategic positioning, we refer to these cells collectively as parenchymal border macrophages (PBMs). Here we demonstrate that PBMs regulate CSF flow dynamics. We identify a subpopulation of PBMs that express high levels of CD163 and LYVE1 (scavenger receptor proteins), closely associated with the brain arterial tree, and show that LYVE1+ PBMs regulate arterial motion that drives CSF flow. Pharmacological or genetic depletion of PBMs led to accumulation of extracellular matrix proteins, obstructing CSF access to perivascular spaces and impairing CNS perfusion and clearance. Ageing-associated alterations in PBMs and impairment of CSF dynamics were restored after intracisternal injection of macrophage colony-stimulating factor. Single-nucleus RNA sequencing data obtained from patients with Alzheimer's disease (AD) and from non-AD individuals point to changes in phagocytosis, endocytosis and interferon-γ signalling on PBMs, pathways that are corroborated in a mouse model of AD. Collectively, our results identify PBMs as new cellular regulators of CSF flow dynamics, which could be targeted pharmacologically to alleviate brain clearance deficits associated with ageing and AD.

Meprin ß knockout reduces brain Aß levels and rescues learning and memory impairments in the APP/lon mouse model for Alzheimer's disease.

ß-Site amyloid precursor protein (APP) cleaving enzyme-1 (BACE1) is the major described ß-secretase to generate Aß peptides in Alzheimer's disease (AD). However, all therapeutic attempts to block BACE1 activity and to improve AD symptoms have so far failed. A potential candidate for alternative Aß peptides generation is the metalloproteinase meprin ß, which cleaves APP predominantly at alanine in p2 and in this study we can detect an increased meprin ß expression in AD brain. Here, we report the generation of the transgenic APP/lon mouse model of AD lacking the functional Mep1b gene (APP/lon × Mep1b-/-). We examined levels of canonical and truncated Aß species using urea-SDS-PAGE, ELISA and immunohistochemistry in brains of APP/lon mouse × Mep1b-/-. Additionally, we investigated the cognitive abilities of these mice during the Morris water maze task. Aß1-40 and 1-42 levels are reduced in APP/lon mice when meprin ß is absent. Immunohistochemical staining of mouse brain sections revealed that N-terminally truncated Aß2-x peptide deposition is decreased in APP/lon × Mep1b-/- mice. Importantly, loss of meprin ß improved cognitive abilities and rescued learning behavior impairments in APP/lon mice. These observations indicate an important role of meprin ß within the amyloidogenic pathway and Aß production in vivo.

The metalloprotease ADAMTS4 generates N-truncated Aß4-x species and marks oligodendrocytes as a source of amyloidogenic peptides in Alzheimer's disease.

Brain accumulation and aggregation of amyloid-ß (Aß) peptides is a critical step in the pathogenesis of Alzheimer's disease (AD). Full-length Aß peptides (mainly Aß1-40 and Aß1-42) are produced through sequential proteolytic cleavage of the amyloid precursor protein (APP) by ß- and γ-secretases. However, studies of autopsy brain samples from AD patients have demonstrated that a large fraction of insoluble Aß peptides are truncated at the N-terminus, with Aß4-x peptides being particularly abundant. Aß4-x peptides are highly aggregation prone, but their origin and any proteases involved in their generation are unknown. We have identified a recognition site for the secreted metalloprotease ADAMTS4 (a disintegrin and metalloproteinase with thrombospondin motifs 4) in the Aß peptide sequence, which facilitates Aß4-x peptide generation. Inducible overexpression of ADAMTS4 in HEK293 cells resulted in the secretion of Aß4-40 but unchanged levels of Aß1-x peptides. In the 5xFAD mouse model of amyloidosis, Aß4-x peptides were present not only in amyloid plaque cores and vessel walls, but also in white matter structures co-localized with axonal APP. In the ADAMTS4-/- knockout background, Aß4-40 levels were reduced confirming a pivotal role of ADAMTS4 in vivo. Surprisingly, in the adult murine brain, ADAMTS4 was exclusively expressed in oligodendrocytes. Cultured oligodendrocytes secreted a variety of Aß species, but Aß4-40 peptides were absent in cultures derived from ADAMTS4-/- mice indicating that the enzyme was essential for Aß4-x production in this cell type. These findings establish an enzymatic mechanism for the generation of Aß4-x peptides. They further identify oligodendrocytes as a source of these highly amyloidogenic Aß peptides.

The concerted amyloid-beta clearance of LRP1 and ABCB1/P-gp across the blood-brain barrier is linked by PICALM.

The accumulation of neurotoxic amyloid-beta (Aß) in the brain is a characteristic hallmark of Alzheimer's disease (AD). The blood-brain barrier (BBB) provides a large surface area and has been shown to be an important mediator for removal of brain Aß. Both, the ABC transporter P-glycoprotein (ABCB1/P-gp) and the receptor low-density lipoprotein receptor-related protein 1 (LRP1) have been implicated to play crucial roles in Aß efflux from brain. Here, with immunoprecipitation experiments, co-immunostainings and dual inhibition of ABCB1/P-gp and LRP1, we show that both proteins are functionally linked, mediating a concerted transcytosis of Aß through endothelial cells. Late-onset AD risk factor Phosphatidylinositol binding clathrin assembly protein (PICALM) is associated with both ABCB1/P-gp and LRP1 representing a functional link and guiding both proteins through the brain endothelium. Together, our results give more mechanistic insight on Aß transport across the BBB and show that the functional interplay of different clearance proteins is needed for the rapid removal of Aß from the brain.

The LepR-mediated leptin transport across brain barriers controls food reward.

OBJECTIVE: Leptin is a key hormone in the control of appetite and body weight. Predominantly produced by white adipose tissue, it acts on the brain to inhibit homeostatic feeding and food reward. Leptin has free access to circumventricular organs, such as the median eminence, but entry into other brain centers is restricted by the blood-brain and blood-CSF barriers. So far, it is unknown for which of its central effects leptin has to penetrate brain barriers. In addition, the mechanisms mediating the transport across barriers are unclear although high expression in brain barriers suggests an important role of the leptin receptor (LepR). METHODS: We selectively deleted LepR in brain endothelial and epithelial cells of mice (LepRbeKO). The expression of LepR in fenestrated vessels of the periphery and the median eminence as well as in tanycytes was not affected. RESULTS: Perfusion studies showed that leptin uptake by the brain depended on LepR in brain barriers. When being fed with a rewarding high-fat diet LepRbeKO mice gained more body weight than controls. The aggravated obesity of LepRbeKO mice was due to hyperphagia and a higher sensitivity to food reward. CONCLUSIONS: The LepR-mediated transport of leptin across brain barriers in endothelial cells lining microvessels and in epithelial cells of the choroid plexus controls food reward but is apparently not involved in homeostatic control of feeding.

Low density lipoprotein receptor-related protein 1 mediated endocytosis of ß1-integrin influences cell adhesion and cell migration.

The low density lipoprotein receptor-related protein 1 (LRP1) has been shown to interact with ß1-integrin and regulate its surface expression. LRP1 knock-out cells exhibit altered cytoskeleton organization and decreased cell migration. Here we demonstrate coupled endocytosis of LRP1 and ß1-integrin and the involvement of the intracellular NPxY2 motif of LRP1 in this process. Mouse embryonic fibroblasts harboring a knock in replacement of the NPxY2 motif of LRP1 by a multiple alanine cassette (AAxA) showed elevated surface expression of ß1-integrin and decreased ß1-integrin internalization rates. As a consequence, cell spreading was altered and adhesion rates were increased in our cell model. Cells formed more focal adhesion complexes, whereby in vitro cell migration rates were decreased. Similar results could be observed in a corresponding mouse model, the C57Bl6 LRP1 NPxYxxL knock in mice, therefore, the biochemistry of cellular adhesion was altered in primary cortical neurons. In vivo cell migration experiments demonstrated a disturbance of neuroblast cell migration along the rostral migratory stream. In summary, our results indicate that LRP1 interacts with ß1-integrin mediating integrin internalization and thus correlates with downstream signaling of ß1-integrin such as focal adhesion dynamics. Consequently, the disturbance of this interaction resulted in a dysfunction in in vivo and in vitro cell adhesion and cell migration.

Endothelial LRP1 transports amyloid-ß(1-42) across the blood-brain barrier.

According to the neurovascular hypothesis, impairment of low-density lipoprotein receptor-related protein-1 (LRP1) in brain capillaries of the blood-brain barrier (BBB) contributes to neurotoxic amyloid-ß (Aß) brain accumulation and drives Alzheimer's disease (AD) pathology. However, due to conflicting reports on the involvement of LRP1 in Aß transport and the expression of LRP1 in brain endothelium, the role of LRP1 at the BBB is uncertain. As global Lrp1 deletion in mice is lethal, appropriate models to study the function of LRP1 are lacking. Moreover, the relevance of systemic Aß clearance to AD pathology remains unclear, as no BBB-specific knockout models have been available. Here, we developed transgenic mouse strains that allow for tamoxifen-inducible deletion of Lrp1 specifically within brain endothelial cells (Slco1c1-CreER(T2) Lrp1(fl/fl) mice) and used these mice to accurately evaluate LRP1-mediated Aß BBB clearance in vivo. Selective deletion of Lrp1 in the brain endothelium of C57BL/6 mice strongly reduced brain efflux of injected [125I] Aß(1-42). Additionally, in the 5xFAD mouse model of AD, brain endothelial-specific Lrp1 deletion reduced plasma Aß levels and elevated soluble brain Aß, leading to aggravated spatial learning and memory deficits, thus emphasizing the importance of systemic Aß elimination via the BBB. Together, our results suggest that receptor-mediated Aß BBB clearance may be a potential target for treatment and prevention of Aß brain accumulation in AD.