Apolipoprotein E2 Expression Alters Endosomal Pathways in a Mouse Model With Increased Brain Exosome Levels During Aging.

The polymorphic APOE gene is the greatest genetic determinant of sporadic Alzheimer's disease risk: the APOE4 allele increases risk, while the APOE2 allele is neuroprotective compared with the risk-neutral APOE3 allele. The neuronal endosomal system is inherently vulnerable during aging, and APOE4 exacerbates this vulnerability by driving an enlargement of early endosomes and reducing exosome release in the brain of humans and mice. We hypothesized that the protective effects of APOE2 are, in part, mediated through the endosomal pathway. Messenger RNA analyses showed that APOE2 leads to an enrichment of endosomal pathways in the brain when compared with both APOE3 and APOE4. Moreover, we show age-dependent alterations in the recruitment of key endosomal regulatory proteins to vesicle compartments when comparing APOE2 to APOE3. In contrast to the early endosome enlargement previously shown in Alzheimer's disease and APOE4 models, we detected similar morphology and abundance of early endosomes and retromer-associated vesicles within cortical neurons of aged APOE2 targeted-replacement mice compared with APOE3. Additionally, we observed increased brain extracellular levels of endosome-derived exosomes in APOE2 compared with APOE3 mice during aging, consistent with enhanced endosomal cargo clearance by exosomes to the extracellular space. Our findings thus demonstrate that APOE2 enhances an endosomal clearance pathway, which has been shown to be impaired by APOE4 and which may be protective due to APOE2 expression during brain aging.

Antibody targeting of E3 ubiquitin ligases for receptor degradation.

Most current therapies that target plasma membrane receptors function by antagonizing ligand binding or enzymatic activities. However, typical mammalian proteins comprise multiple domains that execute discrete but coordinated activities. Thus, inhibition of one domain often incompletely suppresses the function of a protein. Indeed, targeted protein degradation technologies, including proteolysis-targeting chimeras1 (PROTACs), have highlighted clinically important advantages of target degradation over inhibition2. However, the generation of heterobifunctional compounds binding to two targets with high affinity is complex, particularly when oral bioavailability is required3. Here we describe the development of proteolysis-targeting antibodies (PROTABs) that tether cell-surface E3 ubiquitin ligases to transmembrane proteins, resulting in target degradation both in vitro and in vivo. Focusing on zinc- and ring finger 3 (ZNRF3), a Wnt-responsive ligase, we show that this approach can enable colorectal cancer-specific degradation. Notably, by examining a matrix of additional cell-surface E3 ubiquitin ligases and transmembrane receptors, we demonstrate that this technology is amendable for 'on-demand' degradation. Furthermore, we offer insights on the ground rules governing target degradation by engineering optimized antibody formats. In summary, this work describes a strategy for the rapid development of potent, bioavailable and tissue-selective degraders of cell-surface proteins.

Profiling Basal Forebrain Cholinergic Neurons Reveals a Molecular Basis for Vulnerability Within the Ts65Dn Model of Down Syndrome and Alzheimer's Disease.

Basal forebrain cholinergic neuron (BFCN) degeneration is a hallmark of Down syndrome (DS) and Alzheimer's disease (AD). Current therapeutics have been unsuccessful in slowing disease progression, likely due to complex pathological interactions and dysregulated pathways that are poorly understood. The Ts65Dn trisomic mouse model recapitulates both cognitive and morphological deficits of DS and AD, including BFCN degeneration. We utilized Ts65Dn mice to understand mechanisms underlying BFCN degeneration to identify novel targets for therapeutic intervention. We performed high-throughput, single population RNA sequencing (RNA-seq) to interrogate transcriptomic changes within medial septal nucleus (MSN) BFCNs, using laser capture microdissection to individually isolate ~500 choline acetyltransferase-immunopositive neurons in Ts65Dn and normal disomic (2N) mice at 6 months of age (MO). Ts65Dn mice had unique MSN BFCN transcriptomic profiles at ~6 MO clearly differentiating them from 2N mice. Leveraging Ingenuity Pathway Analysis and KEGG analysis, we linked differentially expressed gene (DEG) changes within MSN BFCNs to several canonical pathways and aberrant physiological functions. The dysregulated transcriptomic profile of trisomic BFCNs provides key information underscoring selective vulnerability within the septohippocampal circuit. We propose both expected and novel therapeutic targets for DS and AD, including specific DEGs within cholinergic, glutamatergic, GABAergic, and neurotrophin pathways, as well as select targets for repairing oxidative phosphorylation status in neurons. We demonstrate and validate this interrogative quantitative bioinformatic analysis of a key dysregulated neuronal population linking single population transcript changes to an established pathological hallmark associated with cognitive decline for therapeutic development in human DS and AD.

Mitovesicles are a novel population of extracellular vesicles of mitochondrial origin altered in Down syndrome.

Mitochondrial dysfunction is an established hallmark of aging and neurodegenerative disorders such as Down syndrome (DS) and Alzheimer's disease (AD). Using a high-resolution density gradient separation of extracellular vesicles (EVs) isolated from murine and human DS and diploid control brains, we identify and characterize a previously unknown population of double-membraned EVs containing multiple mitochondrial proteins distinct from previously described EV subtypes, including microvesicles and exosomes. We term these newly identified mitochondria-derived EVs "mitovesicles." We demonstrate that brain-derived mitovesicles contain a specific subset of mitochondrial constituents and that their levels and cargo are altered during pathophysiological processes where mitochondrial dysfunction occurs, including in DS. The development of a method for the selective isolation of mitovesicles paves the way for the characterization in vivo of biological processes connecting EV biology and mitochondria dynamics and for innovative therapeutic and diagnostic strategies.