Investigating the Glial Contribution to Persistent Neuropathic Pain

Persistent neuropathic pain is the coordinated activation and sensitization of glial and neuronal elements both peripherally and centrally. Here, we have investigated the role of glial fibrillary acidic protein (GFAP)-positive astrocytes in the central nervous system (CNS) and non-myelinating Schwann cells in the peripheral nervous system (PNS) and their individual contributions to persistent neuropathic pain. We used three-dimensional reconstruction of ultrastructural data to establish the morphological relationship between astrocyte processes and incoming C and A-delta fiber synapses with second-order pain neurons. We found that not only do astrocytes contact 100% of the C and A-delta fiber glomerular synapses, but they also provide a high degree of ensheathment of each glomerulus. This encapsulation of the glomerular synapses puts astrocytes in a position to potentially modulate neuronal activity and synapse structure. Next, we used two glial-specific transgenic mouse models to interfere vesicular gliotransmitter release. However, neither blocking IP3-dependent Ca2+ release or SNARE-dependent vesicle release had any effect on nociception. We then used two glial-specific transgenic mouse models that interfere with the NFkB-COX2 inflammatory pathway and observed a robust yet temporary alleviation of pain behavior from one to five weeks post-nerve injury. This finding indicates that the NFkB-COX2 signaling pathway in GFAP-positive glia is critical to the maintenance of a specific phase of persistent neuropathic pain. GFAP-positive glia include peripheral non-myelinating Schwann cells, which ensheath unmyelinated nociceptive neurons, as well as central astrocytes, which ensheath neuronal synapses throughout the CNS. We used the tet-Off transgenic mouse expression system in a novel manner to tease apart the peripheral vs central roles of GFAP-positive cells. The administration of oxytetracycline, a blood-brain barrier impermeable analog of doxycycline, was capable of turning off transgene expression in the PNS without affecting transgene expression in the CNS. The blockade of peripheral transgene expression reversed the alleviation of pain behavior post-nerve injury in mice with suppressed NFkB activity. Thus, the suppression of NFkB in astrocytes is insufficient to relieve mechanical sensitization following nerve injury. The implicates non-myelinating Schwann cells in an important role in the maintenance of a specific phase of persistent neuropathic pain from one to five weeks post-injury.Doctor of Philosoph

Brain-Targeted Drug Delivery

Brain diseases currently affect one in six people worldwide; they include a wide range of neurological diseases, from Alzheimer’s and Parkinson’s diseases to epilepsy, brain injuries, brain cancer, neuroinfections, and strokes. The treatment of these diseases is complex and limited due to the presence of the blood–brain barrier (BBB), which covers the entirety of the brain. The BBB not only has the function of protecting the brain from harmful substances; it is also a metabolic barrier and a transport regulator of nutrients/serum factors/neurotoxins. Knowing these characteristics when it comes to the treatment of brain diseases makes it easier to understand the lack of efficacy of therapeutic drugs, resulting from the innate resistance of the BBB to permeation. To overcome this limitation, drug delivery systems based on nanotechnology/microtechnology have been developed. Brain-targeted drug delivery enables targeted therapy with a higher therapeutic efficacy and fewer side effects because it targets moieties present in the drug delivery systems

Part I: Super-resolution Microscopy Method Development Part II: Investigations of Transcription Regulation by Chromosomal Organization in Bacteria

Part I: SMLM provides not only high-resolution images of molecular assemblies beyond the diffraction limit but also enables quantitative analysis of the dynamics and compositions. However, challenges in imaging and analysis due to cell geometry, resolution limit, and fluorophore properties impede the full potential of SMLM. To address these challenges, I first developed a single- molecule tracking methodology that minimizes the confinement of diffusing molecules to obtain accurate diffusion coefficients and transition rates. Next, I developed a methodology to improve three-dimensional (3D)-SMLM imaging by directly taking into account the variability of 3D point-spread-functions, which produces superior resolution compared to existing methodologies. Finally, I developed a method to correct for blinking-artifacts. Blinking-artifacts are caused by repeated localizations of the same fluorophores, which distort images and produce false nanoclusters. I derived a method to find the ”ground- truth” of the underlying pairwise distribution without any additional calibration. This ground truth enables me to identify the true underlying spatial distribution of molecules in the SMLM image, solving a problem that has long persisted in the field. Part II: It is well established that chromosomal organization dramatically influences transcription, but the underlying mechanisms remain elusive. We hypothesize that supercoiling constrained by the chromosomal topology has an effect on transcription rate and hence coordinates expression within the same topological domain. To examine this hypothesis, I developed a theoretical model to account directly for the buildup of supercoiling due to transcription in a DNA-loop. To investigate how the topology of the chromosome influences transcription further, I then developed the first in vivo assays to manipulate the formation of a “large” chromosomal DNA topological domain in E. coli cells to examine transcription activity of multiple genes enclosed in the domain. My experiments showed that domain formation decreases expression levels of genes both inside and outside the domain — demonstrating a ”long-range” cis-regulatory mechanism due to the “architecture” of the chromosome within bacteria. Finally, using quantitative SMLM, we investigated how ”large-scale” chromosome organization affects the spatial organization of RNA-polymerase (RNAP). We discovered RNAP clusters engaged in active ribosomal RNA synthesis; whose organization is “driven” by the chromosomal organization

Multiscale Simulations of Biological Membranes : The Challenge To Understand Biological Phenomena in a Living Substance

Biological membranes are tricky to investigate. They are complex in terms of molecular composition and structure, functional over a wide range of time scales, and characterized by nonequilibrium conditions. Because of all of these features, simulations are a great technique to study biomembrane behavior. A significant part of the functional processes in biological membranes takes place at the molecular level; thus computer simulations are the method of choice to explore how their properties emerge from specific molecular features and how the interplay among the numerous molecules gives rise to function over spatial and time scales larger than the molecular ones. In this review, we focus on this broad theme. We discuss the current state-of-the-art of biomembrane simulations that, until now, have largely focused on a rather narrow picture of the complexity of the membranes. Given this, we also discuss the challenges that we should unravel in the foreseeable future. Numerous features such as the actin-cytoskeleton network, the glycocalyx network, and nonequilibrium transport under ATP-driven conditions have so far received very little attention; however, the potential of simulations to solve them would be exceptionally high. A major milestone for this research would be that one day we could say that computer simulations genuinely research biological membranes, not just lipid bilayers.Peer reviewe

Psr1p interacts with SUN/sad1p and EB1/mal3p to establish the bipolar spindle

Regular Abstracts - Sunday Poster Presentations: no. 382During mitosis, interpolar microtubules from two spindle pole bodies (SPBs) interdigitate to create an antiparallel microtubule array for accommodating numerous regulatory proteins. Among these proteins, the kinesin-5 cut7p/Eg5 is the key player responsible for sliding apart antiparallel microtubules and thus helps in establishing the bipolar spindle. At the onset of mitosis, two SPBs are adjacent to one another with most microtubules running nearly parallel toward the nuclear envelope, creating an unfavorable microtubule configuration for the kinesin-5 kinesins. Therefore, how the cell organizes the antiparallel microtubule array in the first place at mitotic onset remains enigmatic. Here, we show that a novel protein psrp1p localizes to the SPB and plays a key role in organizing the antiparallel microtubule array. The absence of psr1+ leads to a transient monopolar spindle and massive chromosome loss. Further functional characterization demonstrates that psr1p is recruited to the SPB through interaction with the conserved SUN protein sad1p and that psr1p physically interacts with the conserved microtubule plus tip protein mal3p/EB1. These results suggest a model that psr1p serves as a linking protein between sad1p/SUN and mal3p/EB1 to allow microtubule plus ends to be coupled to the SPBs for organization of an antiparallel microtubule array. Thus, we conclude that psr1p is involved in organizing the antiparallel microtubule array in the first place at mitosis onset by interaction with SUN/sad1p and EB1/mal3p, thereby establishing the bipolar spindle.postprin

Removal of antagonistic spindle forces can rescue metaphase spindle length and reduce chromosome segregation defects

Regular Abstracts - Tuesday Poster Presentations: no. 1925Metaphase describes a phase of mitosis where chromosomes are attached and oriented on the bipolar spindle for subsequent segregation at anaphase. In diverse cell types, the metaphase spindle is maintained at a relatively constant length. Metaphase spindle length is proposed to be regulated by a balance of pushing and pulling forces generated by distinct sets of spindle microtubules and their interactions with motors and microtubule-associated proteins (MAPs). Spindle length appears important for chromosome segregation fidelity, as cells with shorter or longer than normal metaphase spindles, generated through deletion or inhibition of individual mitotic motors or MAPs, showed chromosome segregation defects. To test the force balance model of spindle length control and its effect on chromosome segregation, we applied fast microfluidic temperature-control with live-cell imaging to monitor the effect of switching off different combinations of antagonistic forces in the fission yeast metaphase spindle. We show that spindle midzone proteins kinesin-5 cut7p and microtubule bundler ase1p contribute to outward pushing forces, and spindle kinetochore proteins kinesin-8 klp5/6p and dam1p contribute to inward pulling forces. Removing these proteins individually led to aberrant metaphase spindle length and chromosome segregation defects. Removing these proteins in antagonistic combination rescued the defective spindle length and, in some combinations, also partially rescued chromosome segregation defects. Our results stress the importance of proper chromosome-to-microtubule attachment over spindle length regulation for proper chromosome segregation.postprin

Pathogenic potential of anti-ganglioside antibodies in a murine model of axonal Guillain-Barré syndrome

Guillian-Barré Syndrome (GBS) is the world’s leading cause of neuromuscular paralysis occurring in serologically and pathogenically distinct forms. GBS is believed to have an autoimmune basis, where antibodies raised during antecedent infections (eg Campylobacter jejuni) cross-react with self antigens, exemplifying the process of molecular mimicry. These self-antigens are gangliosides, which are glycolipid structures enriched in peripheral nerve in specific membrane compartments termed lipid rafts. To date, successful murine models of anti-GD1a and anti-Gq1b ganglioside mediated neuropathy exist. Clinical evidence supports the involvement of anti-GM1 antibodies in nerve injury, however generation of anti-GM1 antibody mediated neuropathy models remain an enigma, and to date, the only successful model is based in Japanese rabbits. This thesis aims to address the controversies surrounding anti-GM1 antibody mediated neuropathy by utilising a panel of anti-GM1 antibodies of differing specificity, and explores how the stereometric interactions of GM1 with lipid raft species underpin the pathogenic potential of these antibodies

Dichotomic role of NAADP/two-pore channel 2/Ca2+ signaling in regulating neural differentiation of mouse embryonic stem cells

Poster Presentation - Stem Cells and Pluripotency: abstract no. 1866The mobilization of intracellular Ca2+stores is involved in diverse cellular functions, including cell proliferation and differentiation. At least three endogenous Ca2+mobilizing messengers have been identified, including inositol trisphosphate (IP3), cyclic adenosine diphosphoribose (cADPR), and nicotinic adenine acid dinucleotide phosphate (NAADP). Similar to IP3, NAADP can mobilize calcium release in a wide variety of cell types and species, from plants to animals. Moreover, it has been previously shown that NAADP but not IP3-mediated Ca2+increases can potently induce neuronal differentiation in PC12 cells. Recently, two pore channels (TPCs) have been identified as a novel family of NAADP-gated calcium release channels in endolysosome. Therefore, it is of great interest to examine the role of TPC2 in the neural differentiation of mouse ES cells. We found that the expression of TPC2 is markedly decreased during the initial ES cell entry into neural progenitors, and the levels of TPC2 gradually rebound during the late stages of neurogenesis. Correspondingly, perturbing the NAADP signaling by TPC2 knockdown accelerates mouse ES cell differentiation into neural progenitors but inhibits these neural progenitors from committing to the final neural lineage. Interestingly, TPC2 knockdown has no effect on the differentiation of astrocytes and oligodendrocytes of mouse ES cells. Overexpression of TPC2, on the other hand, inhibits mouse ES cell from entering the neural lineage. Taken together, our data indicate that the NAADP/TPC2-mediated Ca2+signaling pathway plays a temporal and dichotomic role in modulating the neural lineage entry of ES cells; in that NAADP signaling antagonizes ES cell entry to early neural progenitors, but promotes late neural differentiation.postprin

Senescence

The book "Senescence" is aimed to describe all the phenomena related to aging and senescence of all forms of life on Earth, i.e. plants, animals and the human beings. The book contains 36 carefully reviewed chapters written by different authors, aiming to describe the aging and senescent changes of living creatures, i.e. plants and animals