Entrevistas & Notas / Interviews & Notes by David Krizaj
Faith & Form (Vol. XLVI, No.2), 2013
In April 2012, members of the Forum for Architecture, Culture and Spirituality (acs.forum.org) ga... more In April 2012, members of the Forum for Architecture, Culture and Spirituality (acs.forum.org) gathered at the Mayan ruins of Chichén Itza in the Yucatan Peninsula to explore contemporary and timeless modes of the sacred. I (i.e., Michael J. Crosbie) asked our group members to keep journals of their experiences in Chichén and to document their reflections with sketches and photos. The following is a slice through their recorded words and images.
Papers by David Krizaj
Diabetes, 2015
Diabetic retinopathy (DR) is the leading cause of blindness in the working-age population in the ... more Diabetic retinopathy (DR) is the leading cause of blindness in the working-age population in the United States. The vision-threatening processes of neuroglial and vascular dysfunction in DR occur in concert, driven by hyperglycemia and propelled by a pathway of inflammation, ischemia, vasodegeneration, and breakdown of the blood retinal barrier. Currently, no therapies exist for normalizing the vasculature in DR. Here we show that a single intravitreal dose of adeno-associated virus serotype 2 encoding a more stable, soluble, and potent form of angiopoietin 1 (AAV2.COMP-Ang1) can ameliorate the structural and functional hallmarks of DR in Ins2Akita mice, with sustained effects observed through six months. In early DR, AAV2.COMP-Ang1 restored leukocyte-endothelial interaction, retinal oxygenation, vascular density, vascular marker expression, vessel permeability, retinal thickness, inner retinal cellularity, and retinal neurophysiological response to levels comparable to non-diabetic controls. In late DR, AAV2.COMP-Ang1 enhanced the therapeutic benefit of intravitreally-delivered endothelial colony-forming cells by promoting their integration into the vasculature and thereby stemming further visual decline. AAV2.COMP-Ang1 single-dose gene therapy can prevent neurovascular pathology, support vascular regeneration, and stabilize vision in DR.
Channels (Austin, Tex.), Jan 4, 2015
A perennial challenge in neuroscience research has been to elucidate the role of astrocytes, the ... more A perennial challenge in neuroscience research has been to elucidate the role of astrocytes, the most numerous cell type in the CNS, at all levels of brain function from development and cognition to trauma and death of the organism. The many types of astrocyte tend to have in common the regulation of water/ion transport, metabolic homeostasis and inflammatory signaling. Prominent expression of volume-sensitive ion channels and aquaporins ensures that even small activity-induced changes in extracellular ion concentrations result in astroglial swelling, which in turn impacts on the concentration/diffusion of ions and neurotransmitters across the extracellular space. Because astrocyte swelling, exacerbated by neuronal overexcitation, ischemia and/or inflammation can drive excitotoxic death of neurons in diabetes, seizures, stroke/ischemia and neurodegenerative/retinal diseases, it represents a prevalent source of surgical concern. 1 We recently identified the osmosensitive TRPV4 (transient receptor potential isoform 4) channel in retinal glia as a potential target for polyunsaturated fatty acids (PUFAs) commonly associated with brain swelling and inflammation, and elucidated its role in Ca 2C homeostasis, swelling and reactive gliosis. Both normal and pathological CNS activity generate free PUFAs, with the predominant elevation of arachidonic acid (AA), a cell diffusible, C20:4n6 longchain fatty acid product of phospholipase A2 (PLA2). Normally a constituent of membrane phospholipids, AA is released following Ca 2C -dependent activation of PLA2 and/or combined activation of phospholipase C and diacylglycerol lipase, reaching extracellular concentrations up to 0.5 mM. 3 AA is typically produced and released by astroglia but can be taken up into neurons where it affects a variety of intrinsic and synaptic signaling mechanisms. AA regulates cellular signaling as a standalone 2nd messenger and/or by acting through its thromboxane, leukotriene, prostaglandin and/or epoxyeicosatrienoic acid (EET) metabolites. The AA pathway was suggested to promote inflammation by exacerbating glial swelling, neuronal damage and CNS edema, 5,6 however, the relationship between PUFA signaling, swelling and Ca 2C homeostasis is not well understood.
Cell death & disease, 2013
Modulation of Ca(2+) within cells is tightly regulated through complex and dynamic interactions b... more Modulation of Ca(2+) within cells is tightly regulated through complex and dynamic interactions between the plasma membrane and internal compartments. In this study, we exploit in vivo imaging strategies based on genetically encoded Ca(2+) indicators to define changes in perikaryal Ca(2+) concentration of intact photoreceptors. We developed double-transgenic zebrafish larvae expressing GCaMP3 in all cones and tdTomato in long-wavelength cones to test the hypothesis that photoreceptor degeneration induced by mutations in the phosphodiesterase-6 (Pde6) gene is driven by excessive [Ca(2+)]i levels within the cell body. Arguing against Ca(2+) overload in Pde6 mutant photoreceptors, simultaneous analysis of cone photoreceptor morphology and Ca(2+) fluxes revealed that degeneration of pde6c(w59) mutant cones, which lack the cone-specific cGMP phosphodiesterase, is not associated with sustained increases in perikaryal [Ca(2+)]i. Analysis of [Ca(2+)]i in dissociated Pde6β(rd1)mouse rods sho...
PURPOSE OF THE STUDY:
Many blinding diseases of the inner retina are associated with degeneration... more PURPOSE OF THE STUDY:
Many blinding diseases of the inner retina are associated with degeneration and loss of retinal ganglion cells (RGCs). Recent evidence implicates several new signaling mechanisms as causal agents associated with RGC injury and remodeling of the optic nerve head. Ion channels such as Transient receptor potential vanilloid isoform 4 (TRPV4), pannexin-1 (Panx1) and P2X7 receptor are localized to RGCs and act as potential sensors and effectors of mechanical strain, ischemia and inflammatory responses. Under normal conditions, TRPV4 may function as an osmosensor and a polymodal molecular integrator of diverse mechanical and chemical stimuli, whereas P2X7R and Panx1 respond to stretch- and/or swelling-induced adenosine triphosphate release from neurons and glia. Ca(2+) influx, induced by stimulation of mechanosensitive ion channels in glaucoma, is proposed to influence dendritic and axonal remodeling that may lead to RGC death while (at least initially) sparing other classes of retinal neuron. The secondary phase of the retinal glaucoma response is associated with microglial activation and an inflammatory response involving Toll-like receptors (TLRs), cluster of differentiation 3 (CD3) immune recognition molecules associated with the T-cell antigen receptor, complement molecules and cell type-specific release of neuroactive cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β). The retinal response to mechanical stress thus involves a diversity of signaling pathways that sense and transduce mechanical strain and orchestrate both protective and destructive secondary responses.
CONCLUSIONS:
Mechanistic understanding of the interaction between pressure-dependent and independent pathways is only beginning to emerge. This review focuses on the molecular basis of mechanical strain transduction as a primary mechanism that can damage RGCs. The damage occurs through Ca(2+)-dependent cellular remodeling and is associated with parallel activation of secondary ischemic and inflammatory signaling pathways. Molecules that mediate these mechanosensory and immune responses represent plausible targets for protecting ganglion cells in glaucoma, optic neuritis and retinal ischemia.
... Degeneration . . . . . 437 SS Ni Dhubhghaill, MT Cahill, M. Campbell, L. Cassidy, MM Humphrie... more ... Degeneration . . . . . 437 SS Ni Dhubhghaill, MT Cahill, M. Campbell, L. Cassidy, MM Humphries, and P. Humphries 51 ... permeability. Slit-Robo signaling also plays an important role in embry-onic and tumor angiogenesis. In ...
Transient receptor potential vanilloid-3 (TRPV3) is a member of the TRPV subfamily of TRP ion cha... more Transient receptor potential vanilloid-3 (TRPV3) is a member of the TRPV subfamily of TRP ion channels. The physiological functions of TRPV3 are not fully understood, in part, due to a lack of selective agonists and antagonists that could both facilitate the elucidation of roles for TRPV3 in mammalian physiology as well as potentially serve as therapeutic agents to modulate conditions for which altered TRPV3 function has been implicated. In this study, the Microsource Spectrum Collection was screened for TRPV3 agonists and antagonists using alterations in calcium flux in TRPV3 overexpressing human embryonic kidney-293 (HEK-293) cells. The antispasmodic agent drofenine was identified as a new TRPV3 agonist. Drofenine exhibited similar potency to the known TRPV3 agonists 2-aminoethoxydiphenylboronate (2-APB) and carvacrol in HEK-293 cells, but greater selectivity for TRPV3 based on a lack of activation of TRPA1, V1, V2, V4, or M8. Multiple inhibitors were also identified, but all of the compounds were either inactive or not specific. Drofenine activated TRPV3 via interactions with the residue, H426, which is required for TRPV3 activation by 2-APB. Drofenine was a more potent agonist of TRPV3 and more cytotoxic than either carvacrol or 2-APB in human keratinocytes and its effect on TRPV3 in HaCaT cells was further demonstrated using the antagonist icilin. Due to the lack of specificity of existing TRPV3 modulators and the expression of multiple TRP channels in cells/tissue, drofenine may be a valuable probe for elucidating TRPV3 functions in complex biological systems. Identification of TRPV3 as a target for drofenine may also suggest a mechanism by which drofenine acts as a therapeutic agent. Abbreviations 2-APB, 2-aminoethoxydiphenylboronate; HEK-293, human embryonic kidney-293 cells; TRPA1, transient receptor potential ankyrin-1; TRPV1, transient receptor potential vanilloid-1; TRPV3, transient receptor potential vanilloid-3.
Activity-dependent shifts in ionic concentrations and water that accompany neuronal and glial act... more Activity-dependent shifts in ionic concentrations and water that accompany neuronal and glial activity can generate osmotic forces with biological consequences for brain physiology. Active regulation of osmotic gradients and cellular volume requires volume-sensitive ion channels. In the vertebrate retina, critical support to volume regulation is provided by Müller astroglia, but the identity of their osmosensor is unknown. Here, we identify TRPV4 channels as transducers of mouse Müller cell volume increases into physiological responses. Hypotonic stimuli induced sustained [Ca(2+)]i elevations that were inhibited by TRPV4 antagonists and absent in TRPV4(-/-) Müller cells. Glial TRPV4 signals were phospholipase A2- and cytochrome P450-dependent, characterized by slow-onset and Ca(2+) waves, and, in excess, were sufficient to induce reactive gliosis. In contrast, neurons responded to TRPV4 agonists and swelling with fast, inactivating Ca(2+) signals that were independent of phospholipase A2. Our results support a model whereby swelling and proinflammatory signals associated with arachidonic acid metabolites differentially gate TRPV4 in retinal neurons and glia, with potentially significant consequences for normal and pathological retinal function.
Stargardt type 3 (STGD3) disease is a juvenile macular dystrophy caused by mutations in the ELOVL... more Stargardt type 3 (STGD3) disease is a juvenile macular dystrophy caused by mutations in the ELOVL4 (Elongation of very long chain fatty acids 4) gene. Its protein product, ELOVL4, is an elongase required for the biosynthesis of very long-chain polyunsaturated fatty acids (VLC-PUFAs). It is unclear whether photoreceptor degeneration in STGD3 is caused by loss of VLC-PUFAs or by mutated ELOVL4 protein trafficking/aggregation. We therefore generated conditional knockout (cKO) mice with Elovl4 ablated in rods or cones and compared their phenotypes to transgenic (TG) animals that express the human STGD3-causing ELOVL4 STGD3 allele. Gas chromatography-mass spectrometry was used to assess C 30 -C 34 VLC-PUFA and N-retinylidene-N-retinylethanolamine content; electroretinography was used to measure phototransduction and outer retinal function; electron microscopy was used for retinal ultrastructure; and the optomotor tracking response was used to test scotopic and photopic visual performance. Elovl4 transcription and biosynthesis of C 30 -C 34 VLC-PUFAs in rod cKO and TG retinas were reduced up to 98%, whereas the content of docosahexaenoic acid was diminished in TG, but not rod cKO, retinas. Despite the near-total loss of the retinal VLC-PUFA content, rod and cone cKO animals exhibited no electrophysiological or behavioral deficits, whereas the typical rod-cone dystrophic pattern was observed in TG animals. Our data suggest that photoreceptor-specific VLC-PUFA depletion is not sufficient to induce the STGD3 phenotype, because depletion alone had little effect on photoreceptor survival, phototransduction, synaptic transmission, and visual behavior. P roper development and functioning of the mammalian nervous system depends on adequate levels of C 14 -C 22 longchain polyunsaturated fatty acids (LC-PUFAs), such as omega-6 (n6) (i.e., arachidonic acid, C 20 :4n6) and omega-3 (n3) [i.e., eicosapentaenoic acid (EPA; C 20 :5n3) and docosahexaenoic acid (DHA; C 22 :6n3)] species, and "very long-chain" (VLC) (C 24 -C 36 ) PUFAs. In contrast to abundant and ubiquitous expression of LC-PUFAs, VLC-PUFA biosynthesis is restricted to select types of cells (spermatocytes, fibroblasts, keratinocytes, and photoreceptors) where they may be necessary to stabilize highly curved cellular membranes (1-3). Both LC-and VLC-PUFAs are generated through an elongation process in the endoplasmic reticulum (ER) where resident Elongation of very long-chain fatty acids 2 (ELOVL2) and ELOVL4 elongases add two-carbon units to the acyl backbone (2, 4).
Transmission of visual signals at the first retinal synapse is associated with changes in calcium... more Transmission of visual signals at the first retinal synapse is associated with changes in calcium concentration in photoreceptors and bipolar cells. We investigated how loss of plasma membrane Ca 2+ ATPase isoform 2 (PMCA2), the calcium transporter isoform with the highest affinity for Ca 2+ /calmodulin, affects transmission of rod-and cone-mediated responses. PMCA2 expression in the neuroblast layer was observed soon after birth; in the adult, PMCA2 was expressed in inner segments and synaptic terminals of rod photoreceptors, in rod bipolar cells, and in most inner retinal neurons but was absent from cones. To determine the role of PMCA2 in retinal signaling, we compared morphology and light responses of retinas from control mice and deafwaddler dfw 2J mice, which lack functional PMCA2 protein. The cytoarchitecture of retinas from control and dfw 2J mice was indistinguishable at the light microscope level. Suction electrode recordings revealed no difference in the sensitivity or amplitude of outer segment light responses of control and dfw 2J rods. However, rod-mediated ERG b-wave responses in dfw 2J mice were ∼45% smaller and significantly slower than those of control mice. Furthermore, recordings from individual rod bipolar cells showed that the sensitivity of transmission at the rod output synapse was reduced by ∼50%. No changes in the amplitude or timing of cone-mediated ERG responses were observed. These results suggest that PMCA2-mediated Ca 2+ extrusion modulates the amplitude and timing of the high-sensitivity rod pathway to a much greater extent than that of the cone pathway.
Sustained increase in intraocular pressure represents a major risk factor for eye disease yet the... more Sustained increase in intraocular pressure represents a major risk factor for eye disease yet the cellular mechanisms of pressure transduction in the posterior eye are essentially unknown. Here we show that the mouse retina expresses mRNA and protein for the polymodal TRPV4 cation channel known to mediate osmo-and mechanotransduction. TRPV4 antibodies labeled perikarya, axons and dendrites of retinal ganglion cells (RGCs) and intensely immunostained the optic nerve head. Müller glial cells, but not retinal astrocytes or microglia, also expressed TRPV4 immunoreactivity. The selective TRPV4 agonists 4α-PDD and GSK1016790A elevated [Ca 2+ ] i in dissociated RGCs in a dose-dependent manner whereas the TRPV1 agonist capsaicin had no effect on [Ca 2+ ] RGC . Exposure to hypotonic stimulation evoked robust increases in [Ca 2+ ] RGC . RGC responses to TRPV4-selective agonists and hypotonic stimulation were absent in Ca 2+ -free saline and were antagonized by the nonselective TRP channel antagonists Ruthenium Red and gadolinium, but were unaffected by the TRPV1 antagonist capsazepine. TRPV4-selective agonists increased the spiking frequency recorded from intact retinas recorded with multielectrode arrays. Sustained exposure to TRPV4 agonists evoked dose-dependent apoptosis of RGCs. Our results demonstrate functional TRPV4 expression in RGCs and suggest that its activation mediates response to membrane stretch leading to elevated [Ca 2+ ] i and augmented excitability. Excessive Ca 2+ influx through TRPV4 predisposes RGCs to activation of Ca 2+ -dependent pro-apoptotic signaling pathways, indicating that TRPV4 is a component of the response mechanism to pathological elevations of intraocular pressure.
Papers (English) by David Krizaj
Frontiers of Architectural Research, May 15, 2017
Built environments can induce contemplative states, but direct evidence for their impact on the b... more Built environments can induce contemplative states, but direct evidence for their impact on the brain is lacking. This exploratory work investigated brain correlates of internal states elicited by architecture designed for contemplative experience. Functional MRI and self-reports of 12 architects were assessed to study their responses to photographs of ordinary and contemplative architectures. Images of contemplative buildings: (1) induced attentive, receptive, and absorbing experiences and diminished internal dialogue; (2) involved decreased engagement of prefrontal cortex; and (3) activated the occipital lobe, precentral gyrus, and inferior parietal lobule. They suggest that viewing buildings designed for contemplation may evoke experiential and brain signatures that consistently differ from those induced by buildings that serve everyday functions. The depth of such externally induced states was inversely correlated with the engagement of the Default Mode Network. Our study points toward a novel avenue for investigating how contemplation can be cultivated in the human brain/mind.
ANFA 2014 Conference - Presenters Abstracts, Sep 2014
Within the context of contemporary neuroscience and clinical research in meditation, this pilot s... more Within the context of contemporary neuroscience and clinical research in meditation, this pilot study uses fMRI scans to gauge and compare the neurophenomenological response that contemplative and ordinary buildings elicit from 12 subjects. The result indicates not only that there are clearly different reactions to the two types of built environments but also, and more significantly, that the phenomenological and neural correlates of the architecturally-induced contemplation share many similarities with internally-generated meditation while displaying important differences that have more in common with peak/flow/aesthetic psychosomatic states than with meditative conditions
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Entrevistas & Notas / Interviews & Notes by David Krizaj
Papers by David Krizaj
Many blinding diseases of the inner retina are associated with degeneration and loss of retinal ganglion cells (RGCs). Recent evidence implicates several new signaling mechanisms as causal agents associated with RGC injury and remodeling of the optic nerve head. Ion channels such as Transient receptor potential vanilloid isoform 4 (TRPV4), pannexin-1 (Panx1) and P2X7 receptor are localized to RGCs and act as potential sensors and effectors of mechanical strain, ischemia and inflammatory responses. Under normal conditions, TRPV4 may function as an osmosensor and a polymodal molecular integrator of diverse mechanical and chemical stimuli, whereas P2X7R and Panx1 respond to stretch- and/or swelling-induced adenosine triphosphate release from neurons and glia. Ca(2+) influx, induced by stimulation of mechanosensitive ion channels in glaucoma, is proposed to influence dendritic and axonal remodeling that may lead to RGC death while (at least initially) sparing other classes of retinal neuron. The secondary phase of the retinal glaucoma response is associated with microglial activation and an inflammatory response involving Toll-like receptors (TLRs), cluster of differentiation 3 (CD3) immune recognition molecules associated with the T-cell antigen receptor, complement molecules and cell type-specific release of neuroactive cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β). The retinal response to mechanical stress thus involves a diversity of signaling pathways that sense and transduce mechanical strain and orchestrate both protective and destructive secondary responses.
CONCLUSIONS:
Mechanistic understanding of the interaction between pressure-dependent and independent pathways is only beginning to emerge. This review focuses on the molecular basis of mechanical strain transduction as a primary mechanism that can damage RGCs. The damage occurs through Ca(2+)-dependent cellular remodeling and is associated with parallel activation of secondary ischemic and inflammatory signaling pathways. Molecules that mediate these mechanosensory and immune responses represent plausible targets for protecting ganglion cells in glaucoma, optic neuritis and retinal ischemia.
Papers (English) by David Krizaj
Many blinding diseases of the inner retina are associated with degeneration and loss of retinal ganglion cells (RGCs). Recent evidence implicates several new signaling mechanisms as causal agents associated with RGC injury and remodeling of the optic nerve head. Ion channels such as Transient receptor potential vanilloid isoform 4 (TRPV4), pannexin-1 (Panx1) and P2X7 receptor are localized to RGCs and act as potential sensors and effectors of mechanical strain, ischemia and inflammatory responses. Under normal conditions, TRPV4 may function as an osmosensor and a polymodal molecular integrator of diverse mechanical and chemical stimuli, whereas P2X7R and Panx1 respond to stretch- and/or swelling-induced adenosine triphosphate release from neurons and glia. Ca(2+) influx, induced by stimulation of mechanosensitive ion channels in glaucoma, is proposed to influence dendritic and axonal remodeling that may lead to RGC death while (at least initially) sparing other classes of retinal neuron. The secondary phase of the retinal glaucoma response is associated with microglial activation and an inflammatory response involving Toll-like receptors (TLRs), cluster of differentiation 3 (CD3) immune recognition molecules associated with the T-cell antigen receptor, complement molecules and cell type-specific release of neuroactive cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β). The retinal response to mechanical stress thus involves a diversity of signaling pathways that sense and transduce mechanical strain and orchestrate both protective and destructive secondary responses.
CONCLUSIONS:
Mechanistic understanding of the interaction between pressure-dependent and independent pathways is only beginning to emerge. This review focuses on the molecular basis of mechanical strain transduction as a primary mechanism that can damage RGCs. The damage occurs through Ca(2+)-dependent cellular remodeling and is associated with parallel activation of secondary ischemic and inflammatory signaling pathways. Molecules that mediate these mechanosensory and immune responses represent plausible targets for protecting ganglion cells in glaucoma, optic neuritis and retinal ischemia.