Faculty Bibliography

Attachment to an abusive caregiver has wide phylogenetic representation, suggesting that animal models are useful in understanding the neural basis underlying this phenomenon and subsequent behavioral outcomes. We previously developed a rat model, in which we use classical conditioning to parallel learning processes evoked during secure attachment (odor-stroke, with stroke mimicking tactile stimulation from the caregiver) or attachment despite adversity (odor-shock, with shock mimicking maltreatment). Here we extend this model to mice. We conditioned infant mice (postnatal day (PN) 7-9 or 13-14) with presentations of peppermint odor and either stroking or shock. We used (14) C 2-deoxyglucose (2-DG) to assess olfactory bulb and amygdala metabolic changes following learning. PN7-9 mice learned to prefer an odor following either odor-stroke or shock conditioning, whereas odor-shock conditioning at PN13-14 resulted in aversion/fear learning. 2-DG data indicated enhanced bulbar activity in PN7-9 preference learning, whereas significant amygdala activity was present following aversion learning at PN13-14. Overall, the mouse results parallel behavioral and neural results in the rat model of attachment, and provide the foundation for the use of transgenic and knockout models to assess the impact of both genetic (biological vulnerabilities) and environmental factors (abusive) on attachment-related behaviors and behavioral development.

The accumulation of Tau into aggregates is associated with key pathological events in frontotemporal lobe degeneration (FTD-Tau) and Alzheimer disease (AD). Recent data have shown that misfolded Tau can be internalized by cells in vitro (Frost, B., Jacks, R. L., and Diamond, M. I. (2009) J. Biol. Chem. 284, 12845-12852) and propagate pathology in vivo (Clavaguera, F., Bolmont, T., Crowther, R. A., Abramowski, D., Frank, S., Probst, A., Fraser, G., Stalder, A. K., Beibel, M., Staufenbiel, M., Jucker, M., Goedert, M., and Tolnay, M. (2009) Nat. Cell Biol. 11, 909-913; Lasagna-Reeves, C. A., Castillo-Carranza, D. L., Sengupta, U., Guerrero-Munoz, M. J., Kiritoshi, T., Neugebauer, V., Jackson, G. R., and Kayed, R. (2012) Sci. Rep. 2, 700). Here we show that recombinant Tau misfolds into low molecular weight (LMW) aggregates prior to assembly into fibrils, and both extracellular LMW Tau aggregates and short fibrils, but not monomers, long fibrils, nor long filaments purified from brain extract are taken up by neurons. Remarkably, misfolded Tau can be internalized at the somatodendritic compartment, or the axon terminals and it can be transported anterogradely, retrogradely, and can enhance tauopathy in vivo. The internalized Tau aggregates co-localize with dextran, a bulk-endocytosis marker, and with the endolysosomal compartments. Our findings demonstrate that exogenous Tau can be taken up by cells, uptake depends on both the conformation and size of the Tau aggregates and once inside cells, Tau can be transported. These data provide support for observations that tauopathy can spread trans-synaptically in vivo, via cell-to-cell transfer.

Most age-associated neurodegenerative diseases involve the aggregation of specific proteins within the nervous system. In Alzheimer disease, the insidious pathogenic process begins many years before the symptoms emerge, and the lesions that characterize the disease-senile plaques and neurofibrillary tangles-ramify systematically through the brain. We review evidence that the -amyloid and tau proteins, which aggregate to form senile plaques and neurofibrillary tangles, respectively, are induced to misfold and self-assemble by a process of templated conformational change that amplifies a toxic species. Recent data also indicate that the spread of these lesions from one site to another is mediated by the cellular uptake, transport, and release of endogenous seeds formed by the cognate proteins. This simple pathogenic principle suggests that the formation, trafficking, and metabolism of pathogenic protein seeds are promising therapeutic targets for Alzheimer disease and other neurodegenerative disorders.

Regulation of mRNA translation is a major modulator of gene expression, allowing cells to fine tune protein levels during growth and differentiation and in response to physiological signals and environmental changes. Mass-spectrometry and RNA-sequencing methods now enable global profiling of the translatome, but these still involve significant analytical and economical limitations. We developed a novel system-wide proteomic approach for direct monitoring of translation, termed PUromycin-associated Nascent CHain Proteomics (PUNCH-P), which is based on the recovery of ribosome-nascent chain complexes from cells or tissues followed by incorporation of biotinylated puromycin into newly-synthesized proteins. Biotinylated proteins are then purified by streptavidin and analyzed by mass-spectrometry. Here we present an overview of PUNCH-P, describe other methodologies for global translatome profiling (pSILAC, BONCAT, TRAP/Ribo-tag, Ribo-seq) and provide conceptual comparisons between these methods. We also show how PUNCH-P data can be combined with mRNA measurements to determine relative translation efficiency for specific mRNAs.

We characterize an inhibitory circuit motif in the Drosophila olfactory system, parallel inhibition, which differs from feedforward or feedback inhibition. Excitatory and GABAergic inhibitory projection neurons (ePNs and iPNs) each receive input from antennal lobe glomeruli and send parallel output to the lateral horn, a higher center implicated in regulating innate olfactory behavior. Ca(2+) imaging of specific lateral horn neurons as an olfactory readout revealed that iPNs selectively suppressed food-related odor responses, but spared signal transmission from pheromone channels. Coapplying food odorant did not affect pheromone signal transmission, suggesting that the differential effects likely result from connection specificity of iPNs, rather than a generalized inhibitory tone. Ca(2+) responses in the ePN axon terminals show no detectable suppression by iPNs, arguing against presynaptic inhibition as a primary mechanism. The parallel inhibition motif may provide specificity in inhibition to funnel specific olfactory information, such as food and pheromone, into distinct downstream circuits.

BACKGROUND: We previously demonstrated that the cell adhesion protein contactin2 (Cntn2) is enriched in Purkinje cells of the cardiac conduction system (CCS). The objective of this study was generation of a mouse embryonic stem cell (mESC) reporter line that allows identification of Purkinje-like cardiomyocytes in vitro. METHODS AND RESULTS: mESC were generated from transgenic mice carrying a BAC Cntn2-eGFP reporter gene and were subsequently transduced with lentivirus coding for a MHCalpha-mCherry cardiomyocyte reporter gene. Immunostaining analysis confirmed that mESC expressed markers of pluripotency (Oct3/4; Klf4) and spontaneously differentiated into cells of all three germ layers in the absence of LIF (alpha-smooth muscle actin, beta-tubulin, alpha-fetoprotein). Spontaneous or serum-free directed cardiac differentiation resulted in generation of spontaneously beating cardiomyocytes, of which single positive MHCalpha-mCherry cells comprised 57.76% +/- 3.7% and MHCalpha-mCherry/Cntn2-eGFP cells comprised 1.9% +/- 0.9% of the total population as determined by fluorescence-activated cell sorting (FACS) (n = 5 differentiation cultures). Quantitative real-time PCR analysis of FACS isolated double-positive cells verified cardiomyocyte-specific transcript expression (Mlc2v: 33-fold, P <.01; Nkx2.5: 178-fold, P <.001) compared to MHCalpha-mCherry/Cntn2-eGFP-negative noncardiomyocytes. Moreover, double-positive cells expressed significantly elevated levels of CCS-specific transcripts compared to mCherry single-positive cardiomyocytes (Cntn2: 31-fold, P <.001; Cx40: 8-fold, P <.01). Action potential recordings of double-positive cells demonstrated distinct plateau phase and elongated action potential duration (APD50 = 79.9 +/- 10.4 ms, APD90 = 170.2 +/- 17.5 ms, n = 11) compared with eGFP-negative cardiomyocytes (APD50 = 53.4 +/- 9.4 ms, APD90 = 120.6 +/- 17.3 ms, n = 15), consistent with their assignment as Purkinje-like derivatives. CONCLUSIONS: We established an mESC reporter line that allows for the identification and enrichment of ventricular CCS derivatives. This model should be useful for downstream studies of CCS development and pathology, including the role of Purkinje cells as arrhythmogenic triggers. Cntn2 may also be a useful marker of CCS-like cells derived from human embryonic stem and/or induced pluripotent stem cells.