The blood-brain barrier (BBB) poses a major challenge for developing effective antibody therapies for neurological diseases. Using transcriptomic and proteomic profiling, we searched for proteins in mouse brain endothelial cells (BECs) that could potentially be exploited to transport antibodies across the BBB. Due to their limited protein abundance, neither antibodies against literature-identified targets nor BBB-enriched proteins identified by microarray facilitated significant antibody brain uptake. Using proteomic analysis of isolated mouse BECs, we identified multiple highly expressed proteins, including basigin, Glut1, and CD98hc. Antibodies to each of these targets were significantly enriched in the brain after administration in vivo. In particular, antibodies against CD98hc showed robust accumulation in brain after systemic dosing, and a significant pharmacodynamic response as measured by brain Aβ reduction. The discovery of CD98hc as a robust receptor-mediated transcytosis pathway for antibody delivery to the brain expands the current approaches available for enhancing brain uptake of therapeutic antibodies.

Increased iron levels and iron-mediated oxidative stress play an important role in the pathogenesis of many neurodegenerative diseases. The finding that mutations in the ferritin light polypeptide (FTL) gene cause a neurodegenerative disease known as neuroferritinopathy or hereditary ferritinopathy (HF) provided a direct connection between abnormal brain iron storage and neurodegeneration. HF is characterized by a severe movement disorder and by the presence of nuclear and cytoplasmic ferritin inclusion bodies in glia and neurons throughout the CNS and in tissues of multiple organ systems. Here we report that the expression in transgenic mice of a human FTL cDNA carrying a thymidine and cytidine insertion at position 498 (FTL498 -499InsTC) leads to the formation of nuclear and cytoplasmic ferritin inclusion bodies. As in HF, ferritin inclusions are seen in glia and neurons throughout the CNS as well as in cells of other organ systems. Our studies show histological, immunohistochemical, and biochemical similarities between ferritin inclusion bodies found in transgenic mice and in individuals with HF. Expression of the transgene in mice leads to a significant decrease in motor performance and a shorter life span, formation of ferritin inclusion bodies, misregulation of iron metabolism, accumulation of ubiquitinated proteins, and incorporation of elements of the proteasome into inclusions. This new transgenic mouse represents a relevant model of HF in which to study the pathways that lead to neurodegeneration in HF, to evaluate the role of iron mismanagement in neurodegenerative disorders, and to evaluate potential therapies for HF and related neurodegenerative diseases.

The presence or absence of core fucose in the Fc region N-linked glycans of antibodies affects their binding affinity toward FcγRIIIa as well as their antibody-dependent cell-mediated cytotoxicity (ADCC) activity. However, the quantitative nature of this structure-function relationship remains unclear. In this study, the in vitro biological activity of an afucosylated anti-CD20 antibody was fully characterized. Further, the effect of fucose reduction on Fc effector functions was quantitatively evaluated using the afucosylated antibody, its "regular" fucosylated counterpart and a series of mixtures containing varying proportions of "regular" and afucosylated materials. Compared with the "regular" fucosylated antibody, the afucosylated antibody demonstrated similar binding interactions with the target antigen (CD20), C1q and FcγRIa, moderate increases in binding to FcγRIIa and IIb, and substantially increased binding to FcγRIIIa. The afucosylated antibodies also showed comparable complement-dependent cytotoxicity activity but markedly increased ADCC activity. Based on EC 50 values derived from dose-response curves, our results indicate that the amount of afucosylated glycan in antibody samples correlate with both FcγRIIIa binding activity and ADCC activity in a linear fashion. Furthermore, the extent of ADCC enhancement due to fucose depletion was not affected by the FcγRIIIa genotype of the effector cells.

The membranous labyrinth of the inner ear establishes a precise geometrical topology so that it may subserve the functions of hearing and balance. How this geometry arises from a simple ectodermal placode is under active investigation. The placode invaginates to form the otic cup, which deepens before pinching off to form the otic vesicle. By the vesicle stage many genes expressed in the developing ear have assumed broad, asymmetrical expression domains. We have been exploring the possibility that these domains may reflect developmental compartments that are instrumental in specifying the location and identity of different parts of the ear. The boundaries between compartments are proposed to be the site of inductive interactions required for this specification. Our work has shown that sensory organs and the endolymphatic duct each arise near the boundaries of broader gene expression domains, lending support to this idea. A further prediction of the model, that the compartment boundaries will also represent lineage-restriction compartments, is supported in part by fate mapping the otic cup. Our data suggest that two lineagerestriction boundaries intersect at the dorsal pole of the otocyst, a convergence that may be critical for the specification of endolymphatic duct outgrowth. We speculate that the patterning information necessary to establish these two orthogonal boundaries may emanate, in part, from the hindbrain. The compartment boundary model of ear development now needs to be tested through a variety of experimental perturbations, such as the removal of boundaries, the generation of ectopic boundaries, and͞or changes in compartment identity.