Development of Generic Equivalence Margins for Early-Stage Implementation of Equivalence Testing for Potency Assays.

Relative potency assays for biological therapeutics require statistical evaluation to demonstrate similarity between the dose-response curves of a reference standard and the test samples. We developed an equivalence testing approach that can be utilized for the complete potency assay life cycle, from early development until commercialization. This approach was based on the use of generic equivalence margins to enable equivalence testing at the beginning of assay development, when the body of assay-specific data is still very limited. Generic equivalence margins for equivalence testing of four-parameter logistic curve fits were established for bioassays and binding assays spanning a variety of designs, formats, and read-outs. We also established that equivalence testing using ratios of the reference standard and test sample was superior to equivalence testing using absolute differences. Based on a large body of historical data, generic equivalence margins were determined for the curve upper asymptote, slope, and dynamic range. Furthermore, we developed a road map to guide the implementation of generic or assay-specific margins to ensure the appropriate data analysis approach is being applied during the assay life cycle.

Low-density lipoprotein receptor-related protein 1 is a novel modulator of radial glia stem cell proliferation, survival, and differentiation.

The LDL family of receptors and its member low-density lipoprotein receptor-related protein 1 (LRP1) have classically been associated with a modulation of lipoprotein metabolism. Current studies, however, indicate diverse functions for this receptor in various aspects of cellular activities, including cell proliferation, migration, differentiation, and survival. LRP1 is essential for normal neuronal function in the adult CNS, whereas the role of LRP1 in development remained unclear. Previously, we have observed an upregulation of LewisX (LeX) glycosylated LRP1 in the stem cells of the developing cortex and demonstrated its importance for oligodendrocyte differentiation. In the current study, we show that LeX-glycosylated LRP1 is also expressed in the stem cell compartment of the developing spinal cord and has broader functions in the developing CNS. We have investigated the basic properties of LRP1 conditional knockout on the neural stem/progenitor cells (NSPCs) from the cortex and the spinal cord, created by means of Cre-loxp-mediated recombination in vitro. The functional status of LRP1-deficient cells has been studied using proliferation, differentiation, and apoptosis assays. LRP1 deficient NSPCs from both CNS regions demonstrated altered differentiation profiles. Their differentiation capacity toward oligodendrocyte progenitor cells (OPCs), mature oligodendrocytes and neurons was reduced. In contrast, astrocyte differentiation was promoted. Moreover, LRP1 deletion had a negative effect on NSPCs proliferation and survival. Our observations suggest that LRP1 facilitates NSPCs differentiation via interaction with apolipoprotein E (ApoE). Upon ApoE4 stimulation wild type NSPCs generated more oligodendrocytes, but LRP1 knockout cells showed no response. The effect of ApoE seems to be independent of cholesterol uptake, but is rather mediated by downstream MAPK and Akt activation. GLIA 2016 GLIA 2016;64:1363-1380.

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.

Cellular prion protein participates in amyloid-ß transcytosis across the blood-brain barrier.

The blood-brain barrier (BBB) facilitates amyloid-ß (Aß) exchange between the blood and the brain. Here, we found that the cellular prion protein (PrP(c)), a putative receptor implicated in mediating Aß neurotoxicity in Alzheimer's disease (AD), participates in Aß transcytosis across the BBB. Using an in vitro BBB model, [(125)I]-Aß(1-40) transcytosis was reduced by genetic knockout of PrP(c) or after addition of a competing PrP(c)-specific antibody. Furthermore, we provide evidence that PrP(c) is expressed in endothelial cells and, that monomeric Aß(1-40) binds to PrP(c). These observations provide new mechanistic insights into the role of PrP(c) in AD.

LRP1 mediates bidirectional transcytosis of amyloid-ß across the blood-brain barrier.

According to the "amyloid hypothesis", the amyloid-ß (Aß) peptide is the toxic intermediate driving Alzheimer's disease (AD) pathogenesis. Recent evidence suggests that the low density lipoprotein receptor-related protein 1 (LRP1) transcytoses Aß out of the brain across the blood-brain barrier (BBB). To provide genetic evidence for LRP1-mediated transcytosis of Aß across the BBB we analyzed Aß transcytosis across primary mouse brain capillary endothelial cells (pMBCECs) derived from wild-type and LRP1 knock-in mice. Here, we show that pMBCECs in vitro express functionally active LRP1. Moreover, we demonstrate that LRP1 mediates transcytosis of [(125)I]-Aß(1-40) across pMBCECs in both directions, whereas no role for LRP1-mediated Aß degradation was detected. Analysis of [(125)I]-Aß(1-40) transport across pMBCECs generated from mice harboring a knock-in mutation in the NPxYxxL endocytosis/sorting domain of endogenous LRP1 revealed a reduced Aß clearance from brain-to-blood and blood-to-brain compared with wild-type derived pMBCECs. Therefore, for the first time, we present genetic evidence that LRP1 modulates the pathogenic actions of soluble Aß in the brain by clearing Aß across the BBB.

Inactivation of the proximal NPXY motif impairs early steps in LRP1 biosynthesis.

The proximal NPXY and distal NPXYXXL motifs in the intracellular domain of LRP1 play an important role in regulation of the function of the receptor. The impact of single and double inactivating knock-in mutations of these motifs on receptor maturation, cell surface expression, and ligand internalization was analyzed in mutant and control wild-type mice and MEFs. Single inactivation of the proximal NPXY or in combination with inactivation of the distal NPXYXXL motif are both shown to be associated with an impaired maturation and premature proteasomal degradation of full-length LRP1. Therefore, only a small mature LRP1 pool is able to reach the cell surface resulting indirectly in severe impairment of ligand internalization. Single inactivation of the NPXYXXL motif revealed normal maturation, but direct impairment of ligand internalization. In conclusion, the proximal NPXY motif proves to be essential for early steps in the LRP1 biosynthesis, whereas NPXYXXL appears rather relevant for internalization.

Mechanisms of C-reactive protein-induced blood-brain barrier disruption.

BACKGROUND AND PURPOSE: Increased mortality after stroke is associated with brain edema formation and high plasma levels of the acute phase reactant C-reactive protein (CRP). The aim of this study was to examine whether CRP directly affects blood-brain barrier stability and to analyze the underlying signaling pathways. METHODS: We used a cell coculture model of the blood-brain barrier and the guinea pig isolated whole brain preparation. RESULTS: We could show that CRP at clinically relevant concentrations (10 to 20 microg/mL) causes a disruption of the blood-brain barrier in both approaches. The results of our study further demonstrate CRP-induced activation of surface Fcgamma receptors CD16/32 followed by p38-mitogen-activated protein kinase-dependent reactive oxygen species formation by the NAD(P)H-oxidase. The resulting oxidative stress increased myosin light chain kinase activity leading to an activation of the contractile machinery. Blocking myosin light chain phosphorylation prevented the CRP-induced blood-brain barrier breakdown and the disruption of tight junctions. CONCLUSIONS: Our data identify a previously unrecognized mechanism linking CRP and brain edema formation and present a signaling pathway that offers new sites of therapeutic intervention.