Faculty Bibliography

Implantation rate decreases and miscarriage rate increases with advancing maternal age. The oocyte must be the locus of reproductive aging because donation of oocytes from younger to older women abrogates the effects of aging on fecundity. Nuclear transfer experiments in a mouse model of reproductive aging show that the reproductive aging phenotype segregates with the nucleus rather than the cytoplasm. A number of factors within the nucleus have been hypothesized to mediate reproductive aging, including disruption of cohesions, reduced chiasma, aneuploidy, disrupted meiotic spindles, and DNA damage caused by chronic exposure to reactive oxygen species. We have proposed telomere attrition as a parsimonious way to explain these diverse effects of aging on oocyte function. Telomeres are repetitive sequences of DNA and associated proteins, which form a loop (t loop) at chromosome ends. Telomeres prevent the blunt end of DNA from triggering a DNA damage response. Previously, we showed that experimental telomere shortening phenocopies reproductive aging in mice. Telomere shortening causes reduced synapsis and chiasma, chromosome fusions, embryo arrest and fragmentation, and abnormal meiotic spindles. Telomere length of polar bodies predicts the fragmentation of human embryos. Telomerase, the reverse transcriptase capable of reconstituting shortened telomeres, is only minimally active in oocytes and preimplantation embryos. Intriguingly, during the first cell cycles following activation, telomeres robustly elongate via a DNA double-strand break mechanism called alternative lengthening of telomeres (ALTs). Alternative lengthening of telomere takes place even in telomerase-null mice. This mechanism of telomere elongation previously had been found only in cancer cells lacking telomerase activity. We propose that ALT elongates telomeres across generations but does so at the cost of extensive genomic instability in preimplantation embryos.

Defective autophagy contributes to Alzheimer disease (AD) pathogenesis although evidence is conflicting on whether multiple stages are impaired. Here, for the first time, we have comprehensively evaluated the entire autophagic process specifically in CA1 pyramidal neurons of hippocampus from early and late-stage AD subjects and nondemented controls. CA1 neurons aspirated by laser capture microdissection were analyzed using a custom-designed microarray comprising 578 neuropathology- and neuroscience-associated genes. Striking upregulation of autophagy-related genes, exceeding that of other gene ontology groups, reflected increases in autophagosome formation and lysosomal biogenesis beginning at early AD stages. Upregulated autophagosome formation was further indicated by elevated gene and protein expression levels for autophagosome components and increased LC3-positive puncta. Increased lysosomal biogenesis was evidenced by activation of MiTF/TFE family transcriptional regulators, particularly TFE3 (transcription factor binding to IGHM enhancer 3) and by elevated expression of their target genes and encoded proteins. Notably, TFEB (transcription factor EB) activation was associated more strongly with glia than neurons. These findings establish that autophagic sequestration is both competent and upregulated in AD. Autophagosome-lysosome fusion is not evidently altered. Despite this early disease response, however, autophagy flux is progressively impeded due to deficient substrate clearance, as reflected by autolysosomal accumulation of LC3-II and SQSTM1/p62 and expansion of autolysosomal size and total area. We propose that sustained induction of autophagy in the face of progressively declining lysosomal clearance of substrates explains the uncommonly robust autophagic pathology and neuritic dystrophy implicated in AD pathogenesis.

Reticulon-4 (RTN4) (also called Nogo-A/B) and the atlastin (ATL) GTPases have critical roles in the formation of tubules and sheets of the standard peripheral endoplasmic reticulum (ER) in mammalian cells. The interferon-inducible protein MxA (myxovirus resistance protein A) is a dynamin-family, atlastin-like GTPase with membrane-binding and tubulation activity. While MxA has been investigated extensively for its antiviral effects, less is known about its role in cellular physiology in the uninfected cell. Over the last 15 years MxA has been represented to localize to "subcompartments of the smooth ER." However these prior studies did not include ER structural proteins as markers. In contrast, using RTN4 as an ER marker, we discovered that MxA expressed in human cell lines associated with large tubuloreticular structures that were distinct from the standard RTN4/ATL3-based ER. Our methods included immunofluorescence studies, thin-section EM, and single and double-label immunoEM. MxA generated large variably sized tubuloreticular structures that were RTN-4 negative. In contrast to HAtagged ATL3 which colocalized with RTN4 and increased ER sheets at three-way junctions, HA-tagged MxA tubules were independent of RTN4 and MxA did not affect the standard RTN4-based ER. Thus, remarkably, one and the same cell contained two distinct tubuloreticular systems - one based on RTN4/ATL3 and the other on MxA. The MxA-reticulum is a novel organelle which is positive for EEA1, clathrin light chain, Rab5, Rab11 and GRP78/BiP, but not for LAMP2, Rab7 and syntaxin 17. The present studies advance the novel paradigm that different atlastin-like GTPases can generate distinct ER-like tubuloreticular systems within the same cell. The new data require reinterpretion of prior studies in the MxA field over the last decade

In vivo imaging modalities provide powerful tools for the noninvasive longitudinal characterization of preclinical cancer models. Medulloblastoma (MB) is the most common malignant brain tumor in children, and the subject of intense research, much of which involves mouse models. Manganese-enhanced magnetic resonance imaging (MEMRI) produces unparalleled images of the cerebellum, the site of most MBs [1,2]. For this reason, longitudinal MEMRI of preclinical medulloblastoma models enables analysis of the region of origin, monitoring of tumor progression, and treatment response evaluation. In this study, we present the initial MEMRI characterization of a novel mouse medulloblastoma model with an activating mutation in the Smo gene, which exhibit different growth characteristics than those observed in previous studies of Ptch1 knockout mice [1]. SmoM2 mice were engineered by crossing Atoh1-CreER [3] male mice with homozygous R26-floxedSTOP-SmoM2 females [4]. The SmoM2 mutation was induced by subcutaneous injection of low dose (1mug/g) Tamoxifen (TMX) at postnatal day P2. Biweekly imaging sessions using 7-Tesla MRI (Bruker) began at postnatal day P21. MnCl2 (50-60 mg/kg) was injected intraperitoneally 24 hours before imaging. Scan protocol: 1 min low-resolution pilot, 20 min 150mum resolution T1-weighted GE sequence (TE/TR = 4/30 ms; FA = 20degree; FOV = 19.2 mm x 19.2 mm x 12 mm; Matrix = 128 x 128 x 80). Images were analyzed in 3-space using Amira and Fiji. Morphological characterization was corroborated with histology as shown in Fig1. Longitudinal MEMRI results are summarized in Fig2. Based on our preliminary results, all SmoM2 mice had preneoplastic lesions, while approximately half developed into full tumor morphology (n=21). Of the mice with tumors, approximately 72% developed bilateral tumors and the remaining developed tumors in either the right or left hemisphere. Approximately 50% of animals with bilateral tumors exhibited regression in one lateral tumor and progression in the other, or progression in both tumors (n=8). General disease progression is as follows: at approximately postnatal week W3, small lesions are apparent in the majority of interlobule spaces including the mid vermis; at ~W7, regions of proliferative lesion thickening are apparent and smaller lesions regress; at ~W13 significant tumor encroachment into the forebrain as well as expansion of the third and fourth ventricles are apparent. Tumors were observed to originate in the posterior hemispheres, shift and compress the normal appearing cerebellum as they progress, and finally encroach into the forebrain. Estimated tumor volume doubling time is approximately 4.5 days at early timepoints (W11.5). Noticeable symptoms - including delayed tail-pull reflex, ataxia, and hydrocephalus - in SmoM2 mice were apparent as early as W10. In addition to qualitative understanding of tumor progression, we have manually segmented and quantified tumor volume at these key timepoints in an effort to produce a unified growth model. Current efforts in automated segmentation and hierarchical clustering-based classification of tumors will guide upcoming preclinical trials of anticancer therapeutics

Primordial germ cells (PGCs) in many species associate intimately with endodermal cells, but the significance of such interactions is largely unexplored. Here, we show that Caenorhabditis elegans PGCs form lobes that are removed and digested by endodermal cells, dramatically altering PGC size and mitochondrial content. We demonstrate that endodermal cells do not scavenge lobes PGCs shed, but rather, actively remove lobes from the cell body. CED-10 (Rac)-induced actin, DYN-1 (dynamin) and LST-4 (SNX9) transiently surround lobe necks and are required within endodermal cells for lobe scission, suggesting that scission occurs through a mechanism resembling vesicle endocytosis. These findings reveal an unexpected role for endoderm in altering the contents of embryonic PGCs, and define a form of developmentally programmed cell remodelling involving intercellular cannibalism. Active roles for engulfing cells have been proposed in several neuronal remodelling events, suggesting that intercellular cannibalism may be a more widespread method used to shape cells than previously thought.

The development of the mammalian cerebellum is orchestrated by both cell-autonomous programs and inductive environmental influences. Here, we describe the main processes of cerebellar ontogenesis, highlighting the neurogenic strategies used by developing progenitors, the genetic programs involved in cell fate specification, the progressive changes of structural organization, and some of the better-known abnormalities associated with developmental disorders of the cerebellum.

Background: Immunological complexity in atherosclerosis warrants targeted treatment of specific inflammatory cells that aggravate the disease. With the initiation of large phase III trials investigating immunomodulatory drugs for atherosclerosis, cardiovascular disease treatment enters a new era. Accordingly, numerous small molecules have been developed to modulate immune cell function, many of which are promising immunotherapy candidates. However, effective immunotherapies require precise effects only on pathological immune cells without causing side effects on the healthy tissues or other immune cells. Results: We here propose a radically different approach that implement and evaluate in vivo a combinatorial library of nanoparticles with distinct physiochemical properties and differential immune cell specificities. The library's nanoparticles are based on endogenous high-density lipoprotein (HDL), which can preferentially deliver therapeutic compounds to pathological immune cells in atherosclerotic plaques^1,2. Using the Apoe ^-/- mouse model of atherosclerosis, we quantitatively evaluated the library's immune cell specificity by combining nanomaterial characterization, in vitro assays, optical imaging, and immunological techniques (a). These distinct physiochemical properties among the library nanoparticles resulted in an approximate 6-fold difference in promoting cholesterol efflux from macrophages, 10-fold difference among blood half-lives, 3.35-fold difference in relative aorta-to-liver accumulation, and 3.84-fold difference in relative aortic-to-splenic macrophage accumulation. In a proof-of-concept study, we identified an ideal drug-delivery nanoparticle with a long blood half-life, low liver retention, and high specificity to atherosclerotic macrophages. We formulated into the nanoparticle (Rx-HDL) a liver receptor X agonist (GW3965), whose high liver toxicity failed its clinical translation. Compared to an undesirable nanoparticle with poor properties for drug delivery (Rx-PLGA-HDL), Rx-HDL minimally retained in the liver while still efficiently delivered GW3965 to atherosclerotic plaques, revealed by in vivo PET imaging and ex vivo flow cytometry. In a one-week intensive treatment regimen in atherosclerotic mice, Rx-HDL totally abolished GW3965's liver toxicity (b ). Finally, a 6-week long-term treatment with Rx-HDL produced significant therapeutic effects on atherosclerotic plaques (c). Conclusion: In this study, for the first time, we demonstrate a systemic in vivo immune cell screening of a nanoparticle library can produce effective immunotherapy for atherosclerosis. Screening the immune cell specificity of nanoparticles can be employed to develop tailored therapies for atherosclerosis and other inflammatory diseases. [IMAGE PRESENTED]

Small-molecule fluorophores are important tools for advanced imaging experiments. We previously reported a general method to improve small, cell-permeable fluorophores which resulted in the azetidine-containing 'Janelia Fluor' (JF) dyes. Here, we refine and extend the utility of these dyes by synthesizing photoactivatable derivatives that are compatible with live-cell labeling strategies. Once activated, these derived compounds retain the superior brightness and photostability of the JF dyes, enabling improved single-particle tracking and facile localization microscopy experiments.

Autophagy and endocytosis deliver unneeded cellular materials to lysosomes for degradation. Beyond processing cellular waste, lysosomes release metabolites and ions that serve signaling and nutrient sensing roles, linking the functions of the lysosome to various pathways for intracellular metabolism and nutrient homeostasis. Each of these lysosomal behaviors is influenced by the intraluminal pH of the lysosome, which is maintained in the low acidic range by a proton pump, the vacuolar ATPase (v-ATPase). New reports implicate altered v-ATPase activity and lysosomal pH dysregulation in cellular aging, longevity, and adult-onset neurodegenerative diseases, including forms of Parkinson Disease and Alzheimer Disease. Genetic defects of subunits composing the v-ATPase or v-ATPase-related proteins occur in an increasingly recognized group of familial neurodegenerative diseases. Here, we review the expanding roles of the v-ATPase complex as a platform regulating lysosomal proteolysis and cellular homeostasis. We discuss the unique vulnerability of neurons to persistent low level lysosomal dysfunction and review recent clinical and experimental studies that link dysfunction of the v-ATPase complex to neurodegenerative diseases across the age spectrum.