Biology subjects

Pham, T.

Publications and source records attributed to Pham, T..

64 records · Page 4Linked to original sources

bioRxiv2020

Conserved and Divergent Modulation of Calcification in Atherosclerosis and Aortic Valve Disease by Tissue Extracellular Vesicles

BackgroundFewer than 50% of patients develop calcification of both atherosclerotic plaques and aortic valves, implying differential pathogenesis. While circulating extracellular vesicles (EVs) act as biomarkers of cardiovascular diseases, tissue-entrapped EVs associate with early mineralization, but their contents, function, and contributions to disease remain unknown. ResultsGlobal proteomics of human carotid artery endarterectomies and calcified aortic valves from a total of 27 donors/patients revealed significant over-representation of proteins with vesicle-associated pathways/ontologies common to both diseases. We exploited enzymatic digestion, serial (ultra)centrifugation and OptiPrep density-gradient separation to isolate EV populations from diseased arteries and valves. Mass spectrometry found 22 EV marker proteins to be highly enriched in the four least-dense OptiPrep fractions while extracellular matrix proteins predominated in denser fractions, as confirmed by CD63 immunogold electron microscopy and nanoparticle tracking analysis. Proteomics and miRNA-sequencing of OptiPrep-enriched tissue EVs quantified 1,104 proteins and 123 miR cargoes linked to 5,182 target genes. Pathway networks of proteins and miR targets common to artery and valve tissue EVs revealed a shared regulation of Rho GTPase and MAPK intracellular signaling cascades. 179 proteins and 5 miRs were significantly altered between artery and valve EVs; multi-omics integration determined that EVs differentially modulated cellular contraction and p53-mediated transcriptional regulation in diseased vascular vs. valvular tissue. ConclusionsOur findings delineate a strategy to isolate, purify, and study protein and RNA cargoes from EVs entrapped in fibrocalcific tissues. Multi-omics and network approaches implicated tissue-resident EVs in human cardiovascular disease.

systems biology↗

bioRxiv2020

A taxonomy of transcriptomic cell types across the isocortex and hippocampal formation

The isocortex and hippocampal formation are two major structures in the mammalian brain that play critical roles in perception, cognition, emotion and learning. Both structures contain multiple regions, for many of which the cellular composition is still poorly understood. In this study, we used two complementary single-cell RNA-sequencing approaches, SMART-Seq and 10x, to profile [~]1.2 million cells covering all regions in the adult mouse isocortex and hippocampal formation, and derived a cell type taxonomy comprising 379 transcriptomic types. The completeness of coverage enabled us to define gene expression variations across the entire spatial landscape without significant gaps. We found that cell types are organized in a hierarchical manner and exhibit varying degrees of discrete or continuous relatedness with each other. Such molecular relationships correlate strongly with the spatial distribution patterns of the cell types, which can be region-specific, or shared across multiple regions, or part of one or more gradients along with other cell types. Glutamatergic neuron types have much greater diversity than GABAergic neuron types, both molecularly and spatially, and they define regional identities as well as inter-region relationships. For example, we found that glutamatergic cell types between the isocortex and hippocampal formation are highly distinct from each other yet possess shared molecular signatures and corresponding layer specificities, indicating their homologous relationships. Overall, our study establishes a molecular architecture of the mammalian isocortex and hippocampal formation for the first time, and begins to shed light on its underlying relationship with the development, evolution, connectivity and function of these two brain structures.

neuroscience↗

bioRxiv2020

A novel drug-combination screen in zebrafish identifies epigenetic small molecule candidates for Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a severe neuromuscular disorder and is one of the most common muscular dystrophies. There are currently few effective therapies to treat the disease, although many small-molecule approaches are being pursued. Specific histone deacetylase inhibitors (HDACi) can ameliorate DMD phenotypes in mouse and zebrafish animal models and have also shown promise for DMD in clinical trials. However, beyond these HDACi, other classes of epigenetic small molecules have not been broadly and systematically studied for their benefits for DMD. Here, we performed a novel chemical screen of a library of epigenetic compounds using the zebrafish dmd model. We identified candidate pools of epigenetic compounds that improve skeletal muscle structure in dmd zebrafish. We then identified a specific combination of two drugs, oxamflatin and salermide, that significantly rescued dmd zebrafish skeletal muscle degeneration. Furthermore, we validated the effects of oxamflatin and salermide in an independent laboratory. Our results provide novel, effective methods for performing a combination small-molecule screen in zebrafish. Our results also add to the growing evidence that epigenetic small molecules may be promising candidates for treating DMD.

pharmacology and toxicology↗

bioRxiv2019

Characterization of GRK5 as a novel regulator of rhabdomyosarcoma tumor cell growth and self-renewal

Rhabdomyosarcoma (RMS) is the most common soft-tissue pediatric sarcoma. Treatment options remain limited, presenting an urgent need for novel therapeutic targets. Using a high-throughput siRNA screen against the human kinome, we identified GRK5, a G-protein receptor kinase, as a novel regulator of RMS tumor cell growth and self-renewal. Through functional assays in vitro and in vivo, we show that GRK5 regulates cell cycling in a kinase-independent manner to promote RMS tumor cell growth. GRK5 interacts with NFAT to facilitate autoregulation of NFAT1 expression in a kinase independent manner, and loss of NFAT1 phenocopies GRK5 loss-of-function effects on cell cycle arrest. Self-renewal of RMS, required for recapitulation of tumor heterogeneity, is significantly reduced with loss of GRK5 due to increased cell death. Treatment of human RMS xenografts in mice with CCG-215022, a GRK5-selective inhibitor, reduces tumor growth of RMS. GRK5 represents a novel therapeutic target for the treatment of RMS. Statement of SignificanceGRK5 promotes growth and self-renewal of RMS, thereby representing a novel therapeutic target for improving survival outcomes of RMS patients. GRK5 regulates RMS tumor cell growth in a kinase-independent manner through direct interaction with NFAT1. This finding promises novel drug design, targeting non-kinase domains of GRK5.

cancer biology↗

bioRxiv2019

Cellular and synaptic adaptations of neural circuits processing skylight polarization in the fly

Specialized ommatidia harboring polarization-sensitive photoreceptors exist in the dorsal rim area (DRA) of virtually all insects. Although downstream elements have been described both anatomically and physiologically throughout the optic lobes and the central brain of different species, little is known about their cellular and synaptic adaptations and how these shape their functional role in polarization vision. We have previously shown that in the DRA of Drosophila melanogaster, two distinct types of modality-specific distal medulla cell types (Dm-DRA1 and Dm-DRA2) are post-synaptic to long visual fiber photoreceptors R7 and R8, respectively. Here we describe additional neuronal elements in the medulla neuropil that manifest modality-specific differences in the DRA region, including DRA-specific neuronal morphology, as well as differences in the structure of pre- or post-synaptic membranes. Furthermore, we show that certain cell types (medulla tangential cells and octopaminergic neuromodulatory cells) specifically avoid contacts with polarization-sensitive photoreceptors. Finally, while certain transmedullary cells are specifically absent from DRA medulla columns, other subtypes show specific wiring differences while still connecting the DRA to the lobula complex, as previously been described in larger insects. This hints towards a complex circuit architecture with more than one pathway connecting polarization-sensitive DRA photoreceptors with the central brain.

neuroscience↗

bioRxiv2019

Modality-specific circuits for skylight orientation in the fly visual system

In the fly optic lobe [~]800 highly stereotypical columnar microcircuits are arranged retinotopically to process visual information. Differences in cellular composition and synaptic connectivity within functionally specialized columns remains largely unknown. Here we describe the cellular and synaptic architecture in medulla columns located downstream of photoreceptors in the dorsal rim area (DRA), where linearly polarized skylight is detected for guiding orientation responses. We show that only in DRA medulla columns, both R7 and R8 photoreceptors target to the bona fide R7 target layer where they form connections with previously uncharacterized, modality-specific Dm neurons: Two morphologically distinct DRA-specific cell types (termed Dm-DRA1 and Dm-DRA2) stratify in separate sublayers and exclusively contact polarization-sensitive DRA inputs, while avoiding overlaps with color-sensitive Dm8 cells. Using the activity-dependent GRASP and trans-Tango techniques, we confirm that DRA R7 cells are synaptically connected to Dm-DRA1, whereas DRA R8 form synapses with Dm-DRA2. Finally, using live imaging of ingrowing pupal photoreceptor axons, we show that DRA R7 and R8 termini reach layer M6 sequentially, thus separating the establishment of different synaptic connectivity in time. We propose that a duplication of R7[->]Dm circuitry in DRA ommatidia serves as an ideal adaptation for detecting linearly polarized skylight using orthogonal e-vector analyzers.

neuroscience↗

bioRxiv2019

A novel mechanism for amplification of sensory responses by the amygdala-TRN projections.

Many forms of behavior require selective amplification of neuronal representations of relevant environmental signals. Following emotional learning, sensory stimuli drive enhanced responses in the sensory cortex. However, the brain circuits that underlie emotionally driven control of the sensory representations remain poorly understood. Here we identify a novel pathway between the basolateral amygdala (BLA), an emotional learning center in the mouse brain, and the inhibitory nucleus of the thalamus (TRN). We demonstrate that activation of this pathway amplifies sound-evoked activity in the central auditory pathway. Optogenetic activation of BLA suppressed spontaneous, but not tone-evoked activity in the auditory cortex (AC), effectively amplifying tone-evoked responses in AC. Anterograde and retrograde viral tracing identified robust BLA projections terminating at TRN. Optogenetic activation of amygdala-TRN pathway mimicked the effect of direct BLA activation, amplifying tone-evoked responses in the auditory thalamus and cortex. The results are explained by a computational model of the thalamocortical circuitry. In our model, activation of TRN by BLA suppresses spontaneous activity in thalamocortical cells, and as a result, thalamocortical neurons are primed to relay relevant sensory input. These results demonstrate a novel circuit mechanism for shining a neural spotlight on behaviorally relevant signals and provide a potential target for treatment of neuropsychological disorders, in which emotional control of sensory processing is disrupted.

neuroscience↗

bioRxiv2018

Electrical synapses regulate both subthreshold and population activity of principal cells in response to transient inputs within canonical feedforward circuits

As information about the world traverses the brain, the signals exchanged between neurons are passed and modulated by synapses, or specialized contacts between neurons. While neurotransmitter-based synapses tend to be either relay excitatory or inhibitory pulses of influence on the postsynaptic neuron, electrical synapses, composed of plaques of gap junction channels, are always-on transmitters that can either excite or inhibit a coupled neighbor. A growing body of evidence indicates that electrical synapses, similar to their chemical counterparts, are modified in strength during physiological neuronal activity. The synchronizing role of electrical synapses in neuronal oscillations has been well established, but their impact on transient signal processing in the brain is much less understood. Here we constructed computational models based on the canonical feedforward neuronal circuit, and included electrical synapses between inhibitory interneurons. We provided discrete closely-timed inputs to the circuits, and characterize the influence of electrical synapses on both the subthreshold summation and spike trains in the output neuron. Our simulations highlight the diverse and powerful roles that electrical synapses play even in simple circuits. Because these canonical circuits are represented widely throughout the brain, we expect that these are general principles for the influence of electrical synapses on transient signal processing across the brain.\n\nAuthor SummaryThe role that electrical synapses play in neural oscillations, network synchronization and rhythmicity is well established, but their role neuronal processing of transient inputs is much less understood. Here we used computational models of canonical feedforward circuits and networks to investigate how the strength of electrical synapses regulates the flow of transient signals passing through those circuits. We show that because the influence of electrical synapses on coupled neighbors can be either inhibitory or excitatory, their role in network information processing is heterogeneous.. Because of the widespread existence of electrical synapses between interneurons as well as a growing body of evidence for their plasticity, we expect such effects play a significant role in how the brain processes transient inputs.

neuroscience↗

bioRxiv2018

A centrosome asymmetry switch in fly neural stem cells

Centrosomes, the main microtubule organizing centers (MTOCs) of metazoan cells, contain an older mother and a younger daughter centriole. Stem cells either inherit the mother or daughter centriole-containing centrosome, providing a possible mechanism for biased delivery of cell fate determinants. However, the dynamics and mechanisms regulating centrosome asymmetry and biased centrosome segregation are unclear. Using 3D-Structured Illumination Microscopy (3D-SIM) and live cell imaging we show that in fly neural stem cells (neuroblasts) the mitotic kinase Polo and its centriolar protein substrate Centrobin (Cnb) dynamically relocalize from the mother to the daughter centriole during mitosis. This mechanism generates a centrosome, containing two molecularly distinct centrioles by telophase. Cnbs timely relocalization is regulated by Polo-mediated phosphorylation whereas Polos daughter centriole enrichment requires both Wdr62 and Cnb. Based on optogenetic protein mislocalization experiments we propose that the establishment of centriole asymmetry in mitosis primes biased interphase MTOC activity, necessary for correct spindle orientation.

cell biology↗

bioRxiv2017

Electrical synapses between inhibitory neurons shape the responses of principal neurons to transient inputs in the thalamus

As multimodal sensory information proceeds to the cortex, it is intercepted and processed by the nuclei of the thalamus. The main source of inhibition within thalamus is the reticular nucleus (TRN), which collects signals both from thalamocortical relay neurons and from thalamocortical feedback. Within the reticular nucleus, neurons are densely interconnected by connexin36-based gap junctions, known as electrical synapses. Electrical synapses have been shown to coordinate neuronal rhythms, including thalamocortical spindle rhythms, but their role in shaping or modulating transient activity is less understood. We constructed a four-cell model of thalamic relay and TRN neurons, and used it to investigate the impact of electrical synapses on closely timed inputs delivered to thalamic relay cells. We show that the electrical synapses of the TRN assist cortical discrimination of these inputs through effects of truncation, delay or inhibition of thalamic spike trains. We expect that these are principles whereby electrical synapses play similar roles in processing of transient activity in excitatory neurons across the brain.

neuroscience↗