Andrea Wellington - Academia.edu (original) (raw)

Papers by Andrea Wellington

Research paper thumbnail of Early diabetes impairs ON sustained ganglion cell light responses and adaptation without cell death or dopamine insensitivity

Experimental Eye Research, Nov 1, 2020

Retinal signaling under dark-adapted conditions is perturbed during early diabetes. Additionally,... more Retinal signaling under dark-adapted conditions is perturbed during early diabetes. Additionally, dopamine, the main neuromodulator of retinal light adaptation, is diminished in diabetic retinas. However, it is not known if this dopamine deficiency changes how the retina responds to increased light or dopamine. Here we determine whether light adaptation is impaired in the diabetic retina, and investigate potential mechanism(s) of impairment. Diabetes was induced in C57BL/6J male mice via 3 intraperitoneal injections of streptozotocin (75mg/kg) and confirmed by blood glucose levels more than 200 mg/dL. After 6 weeks, whole-cell recordings of light-evoked and spontaneous inhibitory postsynaptic currents (IPSCs) or excitatory postsynaptic currents (EPSCs) were made from rod bipolar cells and ON sustained ganglion cells, respectively. Light responses were recorded before and after D1 receptor (D1R) activation (SKF-38393, 20 μM) or light adaptation (background of 950 photons•μm −2 •s −1). Retinal whole mounts were stained for either tyrosine hydroxylase and activated caspase-3 or GAD65/67, GlyT1 and RBPMS and imaged. D1R activation and light adaptation both decreased inhibition, but the disinhibition was not different between control and diabetic rod bipolar cells. However, diabetic ganglion cell light-evoked EPSCs were increased in the dark and showed reduced light adaptation. No differences were found in light adaptation of spontaneous EPSC parameters, suggesting upstream changes. No changes in cell density were found for dopaminergic, glycinergic or GABAergic amacrine cells, or ganglion cells. Thus, in early diabetes, ON sustained ganglion cells receive excessive excitation under darkand light-adapted conditions. Our results show that this is not attributable to loss in number or dopamine sensitivity of inhibitory amacrine cells or loss of dopaminergic amacrine cells.

Research paper thumbnail of Effects of imaging conditions on mitochondrial transport and length in larval motor axons of Drosophila

Comparative Biochemistry and Physiology A-molecular & Integrative Physiology, Oct 1, 2008

The distribution of mitochondria is sensitive to physiological stresses and changes in metabolic ... more The distribution of mitochondria is sensitive to physiological stresses and changes in metabolic demands. Consequently, it is important to carefully define the conditions facilitating live imaging of mitochondrial transport in dissected animal preparations. In this study, we examined Schneider's and the haemolymph-like solutions HL3 and HL6 for their suitability to image mitochondrial transport in motor axons of dissected Drosophila melanogaster larvae. Overall, mitochondrial transport kinetics in larval motor axons appeared similar among all three solutions. Unexpectedly, HL3 solution selectively increased the length of mitochondria in the context of the net-direction of transport. We also found that mitochondrial transport is sensitive to the extracellular Ca 2+ but not glutamate concentration. High concentrations of extracellular glutamate affected only the ratio between motile and stationary mitochondria. Our study offers a valuable overview of mitochondrial transport kinetics in larval motor axons of Drosophila under various conditions, guiding future studies genetically dissecting mechanisms of mitochondrial transport.

Research paper thumbnail of Impaired light adaptation in early diabetes is not accompanied by major cell death or dopamine receptor insensitivity

Investigative Ophthalmology & Visual Science, Jun 10, 2020

Research paper thumbnail of Impaired Light Adaptation of ON-Sustained Ganglion Cells in Early Diabetes Is Attributable to Diminished Response to Dopamine D4 Receptor Activation

Investigative Ophthalmology & Visual Science, Jan 25, 2022

Purpose Retinal neuronal signaling is disrupted early in diabetes, before the onset of the vascul... more Purpose Retinal neuronal signaling is disrupted early in diabetes, before the onset of the vascular pathologies associated with diabetic retinopathy. There is also growing evidence that retinal dopamine, a neuromodulator that mediates light adaptation, is reduced in early diabetes. Previously, we have shown that after 6 weeks of diabetes, light adaptation is impaired in ON-sustained (ON-s) ganglion cells in the mouse retina. The purpose of this study was to determine whether changes in the response to dopamine receptor activation contribute to this dysfunction. Methods Single-cell retinal patch-clamp recordings from the mouse retina were used to determine how activating dopamine type D4 receptors (D4Rs) changes the light-evoked and spontaneous excitatory inputs to ON-s ganglion cells, in both control and 6-week diabetic (STZ-injected) animals. Fluorescence in situ hybridization was also used to assess whether D4R expression was affected by diabetes. Results D4R activation decreased light-evoked and spontaneous inputs to ON-s ganglion cells in control and diabetic retinas. However, D4R activation caused a smaller reduction in light-evoked excitatory inputs to ON-s ganglion cells in diabetic retinas compared to controls. This impaired D4R signaling is not attributable to a decline in D4R expression, as there was no change in D4R mRNA density in the diabetic retinas. Conclusions These results suggest that the cellular response to dopamine signaling is disrupted in early diabetes and may be amenable to chronic dopamine supplementation therapy.

Research paper thumbnail of Drosophila Miro Is Required for Both Anterograde and Retrograde Axonal Mitochondrial Transport

The Journal of Neuroscience, Apr 29, 2009

Microtubule-based transport of mitochondria into dendrites and axons is vital for sustaining neur... more Microtubule-based transport of mitochondria into dendrites and axons is vital for sustaining neuronal function. Transport along microtubule tracks proceeds in a series of plus and minus end-directed movements that are facilitated by kinesin and dynein motors. How the opposing movements are controlled to achieve effective transport over large distances remains unclear. Previous studies showed that the conserved mitochondrial GTPase Miro is required for mitochondrial transport into axons and dendrites and serves as a Ca 2ϩ sensor that controls mitochondrial mobility. To directly examine Miro's significance for kinesin-and/or dynein-mediated mitochondrial motility, we live-imaged movements of GFP-tagged mitochondria in larval Drosophila motor axons upon genetic manipulations of Miro. Loss of Drosophila Miro (dMiro) reduced the effectiveness of both anterograde and retrograde mitochondrial transport by selectively impairing kinesin-or dynein-mediated movements, depending on the direction of net transport. Net anterogradely transported mitochondria exhibited reduced kinesin-but normal dynein-mediated movements. Net retrogradely transported mitochondria exhibited much shorter dynein-mediated movements, whereas kinesin-mediated movements were minimally affected. In both cases, the duration of short stationary phases increased proportionally. Overexpression (OE) of dMiro also impaired the effectiveness of mitochondrial transport. Finally, loss and OE of dMiro altered the length of mitochondria in axons through a mechanistically separate pathway. We suggest that dMiro promotes effective antero-and retrograde mitochondrial transport by extending the processivity of kinesin and dynein motors according to a mitochondrion's programmed direction of transport.

Research paper thumbnail of Spire contains actin binding domains and is related to ascidian posterior end mark-5

Development, 1999

Spire is a maternal effect locus that affects both the dorsal-ventral and anterior-posterior axes... more Spire is a maternal effect locus that affects both the dorsal-ventral and anterior-posterior axes of the Drosophila egg and embryo. It is required for localization of determinants within the developing oocyte to the posterior pole and to the dorsal anterior corner. During mid-oogenesis, spire mutants display premature microtubule-dependent cytoplasmic streaming, a phenotype that can be mimicked by pharmacological disruption of the actin cytoskeleton with cytochalasin D. Spire has been cloned by transposon tagging and is related to posterior end mark-5, a gene from sea squirts that encodes a posteriorly localized mRNA. Spire mRNA is not, however, localized to the posterior pole. SPIRE also contains two domains with similarity to the actin monomer-binding WH2 domain, and we demonstrate that SPIRE binds to actin in the interaction trap system and in vitro. In addition, SPIRE interacts with the rho family GTPases RHOA, RAC1 and CDC42 in the interaction trap system. Thus, our evidence su...

Research paper thumbnail of Drosophila Miro Is Required for Both Anterograde and Retrograde Axonal Mitochondrial Transport

Journal of Neuroscience, 2009

Microtubule-based transport of mitochondria into dendrites and axons is vital for sustaining neur... more Microtubule-based transport of mitochondria into dendrites and axons is vital for sustaining neuronal function. Transport along microtubule tracks proceeds in a series of plus and minus end-directed movements that are facilitated by kinesin and dynein motors. How the opposing movements are controlled to achieve effective transport over large distances remains unclear. Previous studies showed that the conserved mitochondrial GTPase Miro is required for mitochondrial transport into axons and dendrites and serves as a Ca 2ϩ sensor that controls mitochondrial mobility. To directly examine Miro's significance for kinesin-and/or dynein-mediated mitochondrial motility, we live-imaged movements of GFP-tagged mitochondria in larval Drosophila motor axons upon genetic manipulations of Miro. Loss of Drosophila Miro (dMiro) reduced the effectiveness of both anterograde and retrograde mitochondrial transport by selectively impairing kinesin-or dynein-mediated movements, depending on the direction of net transport. Net anterogradely transported mitochondria exhibited reduced kinesin-but normal dynein-mediated movements. Net retrogradely transported mitochondria exhibited much shorter dynein-mediated movements, whereas kinesin-mediated movements were minimally affected. In both cases, the duration of short stationary phases increased proportionally. Overexpression (OE) of dMiro also impaired the effectiveness of mitochondrial transport. Finally, loss and OE of dMiro altered the length of mitochondria in axons through a mechanistically separate pathway. We suggest that dMiro promotes effective antero-and retrograde mitochondrial transport by extending the processivity of kinesin and dynein motors according to a mitochondrion's programmed direction of transport.

Research paper thumbnail of Impaired light adaptation in early diabetes is not accompanied by major cell death or dopamine receptor insensitivity

Investigative Ophthalmology & Visual Science, Jun 10, 2020

Research paper thumbnail of Impaired light adaptation of ON-sustained ganglion cells in early diabetes is attributable to diminished dopamine D4 receptor sensitivity

Purpose: It has been known for some time that normal retinal signaling is disrupted early on in d... more Purpose: It has been known for some time that normal retinal signaling is disrupted early on in diabetes, before the onset of the vascular pathologies associated with diabetic retinopathy. There is growing evidence that levels of retinal dopamine, a neuromodulator that mediates light adaptation, may also be reduced in early diabetes. Previously, we have shown that after six weeks of diabetes in a mouse model, light adaptation is impaired at the level of ON-sustained (ON-s) ganglion cells. The purpose of this study was to determine whether changes in dopamine receptor sensitivity contribute to this dysfunction. Here we used single cell retinal patch-clamp recordings from the mouse retina to determine how activating dopamine type D4 receptors (D4Rs) changes the light-evoked and spontaneous excitatory inputs to ON-s ganglion cells, in both control and diabetic animals. We also used in-situ fluorescent hybridization to assess whether D4R expression was impacted by diabetes. We found that D4R activation had a smaller impact on light-evoked excitatory inputs to ON-s ganglion cells in diabetic retinas compared to controls. This impaired D4R signaling is not attributable to a decline in D4R expression, as we found increased D4R mRNA density in the outer plexiform layer in diabetic retinas. This suggests that the cellular machinery of dopaminergic signaling is itself disrupted in early diabetes and may be amenable to chronic dopamine supplementation therapy.

Research paper thumbnail of Impaired Light Adaptation of ON-Sustained Ganglion Cells in Early Diabetes Is Attributable to Diminished Response to Dopamine D4 Receptor Activation

Investigative Opthalmology & Visual Science

Research paper thumbnail of Early diabetes impairs ON sustained ganglion cell light responses and adaptation without cell death or dopamine insensitivity

Experimental eye research, 2020

Retinal signaling under dark-adapted conditions is perturbed during early diabetes. Additionally,... more Retinal signaling under dark-adapted conditions is perturbed during early diabetes. Additionally, dopamine, the main neuromodulator of retinal light adaptation, is diminished in diabetic retinas. However, it is not known if this dopamine deficiency changes how the retina responds to increased light or dopamine. Here we determine whether light adaptation is impaired in the diabetic retina, and investigate potential mechanism(s) of impairment. Diabetes was induced in C57BL/6J male mice via 3 intraperitoneal injections of streptozotocin (75 mg/kg) and confirmed by blood glucose levels more than 200 mg/dL. After 6 weeks, whole-cell recordings of light-evoked and spontaneous inhibitory postsynaptic currents (IPSCs) or excitatory postsynaptic currents (EPSCs) were made from rod bipolar cells and ON sustained ganglion cells, respectively. Light responses were recorded before and after D1 receptor (D1R) activation (SKF-38393, 20 μM) or light adaptation (background of 950 photons·μm-2 ·s-1)....

Research paper thumbnail of Wellington, A. et al. Spire contains actin binding domains and is related to ascidian posterior end mark-5. Development 126, 5267-5274

Development

Spire is a maternal effect locus that affects both the dorsal-ventral and anterior-posterior axes... more Spire is a maternal effect locus that affects both the dorsal-ventral and anterior-posterior axes of the Drosophila egg and embryo. It is required for localization of determinants within the developing oocyte to the posterior pole and to the dorsal anterior corner. During mid-oogenesis, spire mutants display premature microtubule-dependent cytoplasmic streaming, a phenotype that can be mimicked by pharmacological disruption of the actin cytoskeleton with cytochalasin D. Spire has been cloned by transposon tagging and is related to posterior end mark-5, a gene from sea squirts that encodes a posteriorly localized mRNA. Spire mRNA is not, however, localized to the posterior pole. SPIRE also contains two domains with similarity to the actin monomer-binding WH2 domain, and we demonstrate that SPIRE binds to actin in the interaction trap system and in vitro. In addition, SPIRE interacts with the rho family GTPases RHOA, RAC1 and CDC42 in the interaction trap system. Thus, our evidence su...

Research paper thumbnail of Drosophila Miro Is Required for Both Anterograde and Retrograde Axonal Mitochondrial Transport

Journal of Neuroscience, 2009

Microtubule-based transport of mitochondria into dendrites and axons is vital for sustaining neur... more Microtubule-based transport of mitochondria into dendrites and axons is vital for sustaining neuronal function. Transport along microtubules tracks proceeds in a series of plus-and minus-end directed movements that are facilitated by kinesin and dynein motors. How the opposing movements are controlled to achieve effective transport over large distances remains unclear. Previous studies showed that the conserved mitochondrial GTPase Miro is required for mitochondrial transport into axons and dendrites and serves as a Ca 2+ sensor that controls mitochondrial mobility. To directly examine Miro's significance for kinesin-and/or dynein-mediated mitochondrial motility, we live imaged movements of GFP-tagged mitochondria in larval Drosophila motor axons upon genetic manipulations of Miro. Loss of Drosophila Miro (dMiro) reduced the effectiveness of both anterograde and retrograde mitochondrial transport by selectively impairing kinesin-or dyneinmediated movements, depending on the direction of net transport. Net anterogradely transported mitochondria exhibited reduced kinesin-but normal dynein-mediated movements. Net retrogradely transported mitochondria exhibited much shorter dynein-mediated movements while kinesinmediated movements were minimally affected. In both cases, the duration of short stationary phases increased proportionally. Overexpression (OE) of dMiro also impaired the effectiveness of mitochondrial transport. Finally, loss and OE of dMiro altered the length of mitochondria in axons through a mechanistically separate pathway. We suggest that dMiro promotes effective antero-and retrograde mitochondrial transport by extending the processivity of kinesin and dynein motors according to a mitochondrion's programmed direction of transport.

Research paper thumbnail of Miro's N-Terminal GTPase Domain Is Required for Transport of Mitochondria into Axons and Dendrites

The Journal of neuroscience : the official journal of the Society for Neuroscience, Jan 8, 2015

Mitochondria are dynamically transported in and out of neuronal processes to maintain neuronal ex... more Mitochondria are dynamically transported in and out of neuronal processes to maintain neuronal excitability and synaptic function. In higher eukaryotes, the mitochondrial GTPase Miro binds Milton/TRAK adaptor proteins linking microtubule motors to mitochondria. Here we show that Drosophila Miro (dMiro), which has previously been shown to be required for kinesin-driven axonal transport, is also critically required for the dynein-driven distribution of mitochondria into dendrites. In addition, we used the loss-of-function mutations dMiroT25N and dMiroT460N to determine the significance of dMiro's N-terminal and C-terminal GTPase domains, respectively. Expression of dMiroT25N in the absence of endogenous dMiro caused premature lethality and arrested development at a pupal stage. dMiroT25N accumulated mitochondria in the soma of larval motor and sensory neurons, and prevented their kinesin-dependent and dynein-dependent distribution into axons and dendrites, respectively. dMiroT25N ...

Research paper thumbnail of The GTPase dMiro Is Required for Axonal Transport of Mitochondria to Drosophila Synapses

Neuron, 2005

Consistently, loss of mitochondria 4 Undergraduate Biology Research Program from photoreceptor te... more Consistently, loss of mitochondria 4 Undergraduate Biology Research Program from photoreceptor terminals is associated with blind-University of Arizona ness and a failure of synaptic transmission in the Dro-Tucson, Arizona 85721 sophila mutant milton (Stowers et al., 2002). In cultured 5 Department of Physiology hippocampal neurons, the number of dendritic mito-University of Toronto chondria correlates with the number and plasticity of Toronto, Ontario M5S 1A8 dendritic spines and synapses (Li et al., 2004). In addi-Canada tion, mitochondrial transport responds specifically to 6 Program in Neuroscience growth cone activity and nerve growth factor signaling University of Pennsylvania School of Medicine (Chada and Hollenbeck, 2004). Together, these studies Philadelphia, Pennsylvania 19104 underscore the functional significance of controlling the subcellular targeting of mitochondria. The molecular mechanisms that control the subcellu-Summary lar distribution of mitochondria involve long-distance transport along microtubules (MTs), which provide po-We have identified EMS-induced mutations in Drolar tracks for plus end-directed kinesin and minus endsophila Miro (dMiro), an atypical mitochondrial directed dynein motor proteins. Mitochondria, like vesi-GTPase that is orthologous to human Miro (hMiro). cles, display bidirectional motion where the cargo Mutant dmiro animals exhibit defects in locomotion stops, starts, and often changes direction (Welte, 2004; and die prematurely. Mitochondria in dmiro mutant Vale, 2003; Hollenbeck, 1996). Assuming that mitomuscles and neurons are abnormally distributed. Inchondria are simultaneously attached to two opposing stead of being transported into axons and dendrites, motors (De Vos et al., 2003), then net movement in one mitochondria accumulate in parallel rows in neuronal direction may be determined by the motor with the somata. Mutant neuromuscular junctions (NMJs) lack

Research paper thumbnail of Effects of imaging conditions on mitochondrial transport and length in larval motor axons of Drosophila

Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 2008

The distribution of mitochondria is sensitive to physiological stresses and changes in metabolic ... more The distribution of mitochondria is sensitive to physiological stresses and changes in metabolic demands. Consequently, it is important to carefully define the conditions facilitating live imaging of mitochondrial transport in dissected animal preparations. In this study, we examined Schneider's and the haemolymph-like solutions HL3 and HL6 for their suitability to image mitochondrial transport in motor axons of dissected Drosophila melanogaster larvae. Overall, mitochondrial transport kinetics in larval motor axons appeared similar among all three solutions. Unexpectedly, HL3 solution selectively increased the length of mitochondria in the context of the net-direction of transport. We also found that mitochondrial transport is sensitive to the extracellular Ca(2+) but not glutamate concentration. High concentrations of extracellular glutamate affected only the ratio between motile and stationary mitochondria. Our study offers a valuable overview of mitochondrial transport kinetics in larval motor axons of Drosophila under various conditions, guiding future studies genetically dissecting mechanisms of mitochondrial transport.

Research paper thumbnail of Drosophila Miro Is Required for Both Anterograde and Retrograde Axonal Mitochondrial Transport

Journal of Neuroscience, 2009

Microtubule-based transport of mitochondria into dendrites and axons is vital for sustaining neur... more Microtubule-based transport of mitochondria into dendrites and axons is vital for sustaining neuronal function. Transport along microtubule tracks proceeds in a series of plus and minus end-directed movements that are facilitated by kinesin and dynein motors. How the opposing movements are controlled to achieve effective transport over large distances remains unclear. Previous studies showed that the conserved mitochondrial GTPase Miro is required for mitochondrial transport into axons and dendrites and serves as a Ca(2+) sensor that controls mitochondrial mobility. To directly examine Miro's significance for kinesin- and/or dynein-mediated mitochondrial motility, we live-imaged movements of GFP-tagged mitochondria in larval Drosophila motor axons upon genetic manipulations of Miro. Loss of Drosophila Miro (dMiro) reduced the effectiveness of both anterograde and retrograde mitochondrial transport by selectively impairing kinesin- or dynein-mediated movements, depending on the direction of net transport. Net anterogradely transported mitochondria exhibited reduced kinesin- but normal dynein-mediated movements. Net retrogradely transported mitochondria exhibited much shorter dynein-mediated movements, whereas kinesin-mediated movements were minimally affected. In both cases, the duration of short stationary phases increased proportionally. Overexpression (OE) of dMiro also impaired the effectiveness of mitochondrial transport. Finally, loss and OE of dMiro altered the length of mitochondria in axons through a mechanistically separate pathway. We suggest that dMiro promotes effective antero- and retrograde mitochondrial transport by extending the processivity of kinesin and dynein motors according to a mitochondrion's programmed direction of transport.

Research paper thumbnail of Early diabetes impairs ON sustained ganglion cell light responses and adaptation without cell death or dopamine insensitivity

Experimental Eye Research, Nov 1, 2020

Retinal signaling under dark-adapted conditions is perturbed during early diabetes. Additionally,... more Retinal signaling under dark-adapted conditions is perturbed during early diabetes. Additionally, dopamine, the main neuromodulator of retinal light adaptation, is diminished in diabetic retinas. However, it is not known if this dopamine deficiency changes how the retina responds to increased light or dopamine. Here we determine whether light adaptation is impaired in the diabetic retina, and investigate potential mechanism(s) of impairment. Diabetes was induced in C57BL/6J male mice via 3 intraperitoneal injections of streptozotocin (75mg/kg) and confirmed by blood glucose levels more than 200 mg/dL. After 6 weeks, whole-cell recordings of light-evoked and spontaneous inhibitory postsynaptic currents (IPSCs) or excitatory postsynaptic currents (EPSCs) were made from rod bipolar cells and ON sustained ganglion cells, respectively. Light responses were recorded before and after D1 receptor (D1R) activation (SKF-38393, 20 μM) or light adaptation (background of 950 photons•μm −2 •s −1). Retinal whole mounts were stained for either tyrosine hydroxylase and activated caspase-3 or GAD65/67, GlyT1 and RBPMS and imaged. D1R activation and light adaptation both decreased inhibition, but the disinhibition was not different between control and diabetic rod bipolar cells. However, diabetic ganglion cell light-evoked EPSCs were increased in the dark and showed reduced light adaptation. No differences were found in light adaptation of spontaneous EPSC parameters, suggesting upstream changes. No changes in cell density were found for dopaminergic, glycinergic or GABAergic amacrine cells, or ganglion cells. Thus, in early diabetes, ON sustained ganglion cells receive excessive excitation under darkand light-adapted conditions. Our results show that this is not attributable to loss in number or dopamine sensitivity of inhibitory amacrine cells or loss of dopaminergic amacrine cells.

Research paper thumbnail of Effects of imaging conditions on mitochondrial transport and length in larval motor axons of Drosophila

Comparative Biochemistry and Physiology A-molecular & Integrative Physiology, Oct 1, 2008

The distribution of mitochondria is sensitive to physiological stresses and changes in metabolic ... more The distribution of mitochondria is sensitive to physiological stresses and changes in metabolic demands. Consequently, it is important to carefully define the conditions facilitating live imaging of mitochondrial transport in dissected animal preparations. In this study, we examined Schneider's and the haemolymph-like solutions HL3 and HL6 for their suitability to image mitochondrial transport in motor axons of dissected Drosophila melanogaster larvae. Overall, mitochondrial transport kinetics in larval motor axons appeared similar among all three solutions. Unexpectedly, HL3 solution selectively increased the length of mitochondria in the context of the net-direction of transport. We also found that mitochondrial transport is sensitive to the extracellular Ca 2+ but not glutamate concentration. High concentrations of extracellular glutamate affected only the ratio between motile and stationary mitochondria. Our study offers a valuable overview of mitochondrial transport kinetics in larval motor axons of Drosophila under various conditions, guiding future studies genetically dissecting mechanisms of mitochondrial transport.

Research paper thumbnail of Impaired light adaptation in early diabetes is not accompanied by major cell death or dopamine receptor insensitivity

Investigative Ophthalmology & Visual Science, Jun 10, 2020

Research paper thumbnail of Impaired Light Adaptation of ON-Sustained Ganglion Cells in Early Diabetes Is Attributable to Diminished Response to Dopamine D4 Receptor Activation

Investigative Ophthalmology & Visual Science, Jan 25, 2022

Purpose Retinal neuronal signaling is disrupted early in diabetes, before the onset of the vascul... more Purpose Retinal neuronal signaling is disrupted early in diabetes, before the onset of the vascular pathologies associated with diabetic retinopathy. There is also growing evidence that retinal dopamine, a neuromodulator that mediates light adaptation, is reduced in early diabetes. Previously, we have shown that after 6 weeks of diabetes, light adaptation is impaired in ON-sustained (ON-s) ganglion cells in the mouse retina. The purpose of this study was to determine whether changes in the response to dopamine receptor activation contribute to this dysfunction. Methods Single-cell retinal patch-clamp recordings from the mouse retina were used to determine how activating dopamine type D4 receptors (D4Rs) changes the light-evoked and spontaneous excitatory inputs to ON-s ganglion cells, in both control and 6-week diabetic (STZ-injected) animals. Fluorescence in situ hybridization was also used to assess whether D4R expression was affected by diabetes. Results D4R activation decreased light-evoked and spontaneous inputs to ON-s ganglion cells in control and diabetic retinas. However, D4R activation caused a smaller reduction in light-evoked excitatory inputs to ON-s ganglion cells in diabetic retinas compared to controls. This impaired D4R signaling is not attributable to a decline in D4R expression, as there was no change in D4R mRNA density in the diabetic retinas. Conclusions These results suggest that the cellular response to dopamine signaling is disrupted in early diabetes and may be amenable to chronic dopamine supplementation therapy.

Research paper thumbnail of Drosophila Miro Is Required for Both Anterograde and Retrograde Axonal Mitochondrial Transport

The Journal of Neuroscience, Apr 29, 2009

Microtubule-based transport of mitochondria into dendrites and axons is vital for sustaining neur... more Microtubule-based transport of mitochondria into dendrites and axons is vital for sustaining neuronal function. Transport along microtubule tracks proceeds in a series of plus and minus end-directed movements that are facilitated by kinesin and dynein motors. How the opposing movements are controlled to achieve effective transport over large distances remains unclear. Previous studies showed that the conserved mitochondrial GTPase Miro is required for mitochondrial transport into axons and dendrites and serves as a Ca 2ϩ sensor that controls mitochondrial mobility. To directly examine Miro's significance for kinesin-and/or dynein-mediated mitochondrial motility, we live-imaged movements of GFP-tagged mitochondria in larval Drosophila motor axons upon genetic manipulations of Miro. Loss of Drosophila Miro (dMiro) reduced the effectiveness of both anterograde and retrograde mitochondrial transport by selectively impairing kinesin-or dynein-mediated movements, depending on the direction of net transport. Net anterogradely transported mitochondria exhibited reduced kinesin-but normal dynein-mediated movements. Net retrogradely transported mitochondria exhibited much shorter dynein-mediated movements, whereas kinesin-mediated movements were minimally affected. In both cases, the duration of short stationary phases increased proportionally. Overexpression (OE) of dMiro also impaired the effectiveness of mitochondrial transport. Finally, loss and OE of dMiro altered the length of mitochondria in axons through a mechanistically separate pathway. We suggest that dMiro promotes effective antero-and retrograde mitochondrial transport by extending the processivity of kinesin and dynein motors according to a mitochondrion's programmed direction of transport.

Research paper thumbnail of Spire contains actin binding domains and is related to ascidian posterior end mark-5

Development, 1999

Spire is a maternal effect locus that affects both the dorsal-ventral and anterior-posterior axes... more Spire is a maternal effect locus that affects both the dorsal-ventral and anterior-posterior axes of the Drosophila egg and embryo. It is required for localization of determinants within the developing oocyte to the posterior pole and to the dorsal anterior corner. During mid-oogenesis, spire mutants display premature microtubule-dependent cytoplasmic streaming, a phenotype that can be mimicked by pharmacological disruption of the actin cytoskeleton with cytochalasin D. Spire has been cloned by transposon tagging and is related to posterior end mark-5, a gene from sea squirts that encodes a posteriorly localized mRNA. Spire mRNA is not, however, localized to the posterior pole. SPIRE also contains two domains with similarity to the actin monomer-binding WH2 domain, and we demonstrate that SPIRE binds to actin in the interaction trap system and in vitro. In addition, SPIRE interacts with the rho family GTPases RHOA, RAC1 and CDC42 in the interaction trap system. Thus, our evidence su...

Research paper thumbnail of Drosophila Miro Is Required for Both Anterograde and Retrograde Axonal Mitochondrial Transport

Journal of Neuroscience, 2009

Microtubule-based transport of mitochondria into dendrites and axons is vital for sustaining neur... more Microtubule-based transport of mitochondria into dendrites and axons is vital for sustaining neuronal function. Transport along microtubule tracks proceeds in a series of plus and minus end-directed movements that are facilitated by kinesin and dynein motors. How the opposing movements are controlled to achieve effective transport over large distances remains unclear. Previous studies showed that the conserved mitochondrial GTPase Miro is required for mitochondrial transport into axons and dendrites and serves as a Ca 2ϩ sensor that controls mitochondrial mobility. To directly examine Miro's significance for kinesin-and/or dynein-mediated mitochondrial motility, we live-imaged movements of GFP-tagged mitochondria in larval Drosophila motor axons upon genetic manipulations of Miro. Loss of Drosophila Miro (dMiro) reduced the effectiveness of both anterograde and retrograde mitochondrial transport by selectively impairing kinesin-or dynein-mediated movements, depending on the direction of net transport. Net anterogradely transported mitochondria exhibited reduced kinesin-but normal dynein-mediated movements. Net retrogradely transported mitochondria exhibited much shorter dynein-mediated movements, whereas kinesin-mediated movements were minimally affected. In both cases, the duration of short stationary phases increased proportionally. Overexpression (OE) of dMiro also impaired the effectiveness of mitochondrial transport. Finally, loss and OE of dMiro altered the length of mitochondria in axons through a mechanistically separate pathway. We suggest that dMiro promotes effective antero-and retrograde mitochondrial transport by extending the processivity of kinesin and dynein motors according to a mitochondrion's programmed direction of transport.

Research paper thumbnail of Impaired light adaptation in early diabetes is not accompanied by major cell death or dopamine receptor insensitivity

Investigative Ophthalmology & Visual Science, Jun 10, 2020

Research paper thumbnail of Impaired light adaptation of ON-sustained ganglion cells in early diabetes is attributable to diminished dopamine D4 receptor sensitivity

Purpose: It has been known for some time that normal retinal signaling is disrupted early on in d... more Purpose: It has been known for some time that normal retinal signaling is disrupted early on in diabetes, before the onset of the vascular pathologies associated with diabetic retinopathy. There is growing evidence that levels of retinal dopamine, a neuromodulator that mediates light adaptation, may also be reduced in early diabetes. Previously, we have shown that after six weeks of diabetes in a mouse model, light adaptation is impaired at the level of ON-sustained (ON-s) ganglion cells. The purpose of this study was to determine whether changes in dopamine receptor sensitivity contribute to this dysfunction. Here we used single cell retinal patch-clamp recordings from the mouse retina to determine how activating dopamine type D4 receptors (D4Rs) changes the light-evoked and spontaneous excitatory inputs to ON-s ganglion cells, in both control and diabetic animals. We also used in-situ fluorescent hybridization to assess whether D4R expression was impacted by diabetes. We found that D4R activation had a smaller impact on light-evoked excitatory inputs to ON-s ganglion cells in diabetic retinas compared to controls. This impaired D4R signaling is not attributable to a decline in D4R expression, as we found increased D4R mRNA density in the outer plexiform layer in diabetic retinas. This suggests that the cellular machinery of dopaminergic signaling is itself disrupted in early diabetes and may be amenable to chronic dopamine supplementation therapy.

Research paper thumbnail of Impaired Light Adaptation of ON-Sustained Ganglion Cells in Early Diabetes Is Attributable to Diminished Response to Dopamine D4 Receptor Activation

Investigative Opthalmology & Visual Science

Research paper thumbnail of Early diabetes impairs ON sustained ganglion cell light responses and adaptation without cell death or dopamine insensitivity

Experimental eye research, 2020

Retinal signaling under dark-adapted conditions is perturbed during early diabetes. Additionally,... more Retinal signaling under dark-adapted conditions is perturbed during early diabetes. Additionally, dopamine, the main neuromodulator of retinal light adaptation, is diminished in diabetic retinas. However, it is not known if this dopamine deficiency changes how the retina responds to increased light or dopamine. Here we determine whether light adaptation is impaired in the diabetic retina, and investigate potential mechanism(s) of impairment. Diabetes was induced in C57BL/6J male mice via 3 intraperitoneal injections of streptozotocin (75 mg/kg) and confirmed by blood glucose levels more than 200 mg/dL. After 6 weeks, whole-cell recordings of light-evoked and spontaneous inhibitory postsynaptic currents (IPSCs) or excitatory postsynaptic currents (EPSCs) were made from rod bipolar cells and ON sustained ganglion cells, respectively. Light responses were recorded before and after D1 receptor (D1R) activation (SKF-38393, 20 μM) or light adaptation (background of 950 photons·μm-2 ·s-1)....

Research paper thumbnail of Wellington, A. et al. Spire contains actin binding domains and is related to ascidian posterior end mark-5. Development 126, 5267-5274

Development

Spire is a maternal effect locus that affects both the dorsal-ventral and anterior-posterior axes... more Spire is a maternal effect locus that affects both the dorsal-ventral and anterior-posterior axes of the Drosophila egg and embryo. It is required for localization of determinants within the developing oocyte to the posterior pole and to the dorsal anterior corner. During mid-oogenesis, spire mutants display premature microtubule-dependent cytoplasmic streaming, a phenotype that can be mimicked by pharmacological disruption of the actin cytoskeleton with cytochalasin D. Spire has been cloned by transposon tagging and is related to posterior end mark-5, a gene from sea squirts that encodes a posteriorly localized mRNA. Spire mRNA is not, however, localized to the posterior pole. SPIRE also contains two domains with similarity to the actin monomer-binding WH2 domain, and we demonstrate that SPIRE binds to actin in the interaction trap system and in vitro. In addition, SPIRE interacts with the rho family GTPases RHOA, RAC1 and CDC42 in the interaction trap system. Thus, our evidence su...

Research paper thumbnail of Drosophila Miro Is Required for Both Anterograde and Retrograde Axonal Mitochondrial Transport

Journal of Neuroscience, 2009

Microtubule-based transport of mitochondria into dendrites and axons is vital for sustaining neur... more Microtubule-based transport of mitochondria into dendrites and axons is vital for sustaining neuronal function. Transport along microtubules tracks proceeds in a series of plus-and minus-end directed movements that are facilitated by kinesin and dynein motors. How the opposing movements are controlled to achieve effective transport over large distances remains unclear. Previous studies showed that the conserved mitochondrial GTPase Miro is required for mitochondrial transport into axons and dendrites and serves as a Ca 2+ sensor that controls mitochondrial mobility. To directly examine Miro's significance for kinesin-and/or dynein-mediated mitochondrial motility, we live imaged movements of GFP-tagged mitochondria in larval Drosophila motor axons upon genetic manipulations of Miro. Loss of Drosophila Miro (dMiro) reduced the effectiveness of both anterograde and retrograde mitochondrial transport by selectively impairing kinesin-or dyneinmediated movements, depending on the direction of net transport. Net anterogradely transported mitochondria exhibited reduced kinesin-but normal dynein-mediated movements. Net retrogradely transported mitochondria exhibited much shorter dynein-mediated movements while kinesinmediated movements were minimally affected. In both cases, the duration of short stationary phases increased proportionally. Overexpression (OE) of dMiro also impaired the effectiveness of mitochondrial transport. Finally, loss and OE of dMiro altered the length of mitochondria in axons through a mechanistically separate pathway. We suggest that dMiro promotes effective antero-and retrograde mitochondrial transport by extending the processivity of kinesin and dynein motors according to a mitochondrion's programmed direction of transport.

Research paper thumbnail of Miro's N-Terminal GTPase Domain Is Required for Transport of Mitochondria into Axons and Dendrites

The Journal of neuroscience : the official journal of the Society for Neuroscience, Jan 8, 2015

Mitochondria are dynamically transported in and out of neuronal processes to maintain neuronal ex... more Mitochondria are dynamically transported in and out of neuronal processes to maintain neuronal excitability and synaptic function. In higher eukaryotes, the mitochondrial GTPase Miro binds Milton/TRAK adaptor proteins linking microtubule motors to mitochondria. Here we show that Drosophila Miro (dMiro), which has previously been shown to be required for kinesin-driven axonal transport, is also critically required for the dynein-driven distribution of mitochondria into dendrites. In addition, we used the loss-of-function mutations dMiroT25N and dMiroT460N to determine the significance of dMiro's N-terminal and C-terminal GTPase domains, respectively. Expression of dMiroT25N in the absence of endogenous dMiro caused premature lethality and arrested development at a pupal stage. dMiroT25N accumulated mitochondria in the soma of larval motor and sensory neurons, and prevented their kinesin-dependent and dynein-dependent distribution into axons and dendrites, respectively. dMiroT25N ...

Research paper thumbnail of The GTPase dMiro Is Required for Axonal Transport of Mitochondria to Drosophila Synapses

Neuron, 2005

Consistently, loss of mitochondria 4 Undergraduate Biology Research Program from photoreceptor te... more Consistently, loss of mitochondria 4 Undergraduate Biology Research Program from photoreceptor terminals is associated with blind-University of Arizona ness and a failure of synaptic transmission in the Dro-Tucson, Arizona 85721 sophila mutant milton (Stowers et al., 2002). In cultured 5 Department of Physiology hippocampal neurons, the number of dendritic mito-University of Toronto chondria correlates with the number and plasticity of Toronto, Ontario M5S 1A8 dendritic spines and synapses (Li et al., 2004). In addi-Canada tion, mitochondrial transport responds specifically to 6 Program in Neuroscience growth cone activity and nerve growth factor signaling University of Pennsylvania School of Medicine (Chada and Hollenbeck, 2004). Together, these studies Philadelphia, Pennsylvania 19104 underscore the functional significance of controlling the subcellular targeting of mitochondria. The molecular mechanisms that control the subcellu-Summary lar distribution of mitochondria involve long-distance transport along microtubules (MTs), which provide po-We have identified EMS-induced mutations in Drolar tracks for plus end-directed kinesin and minus endsophila Miro (dMiro), an atypical mitochondrial directed dynein motor proteins. Mitochondria, like vesi-GTPase that is orthologous to human Miro (hMiro). cles, display bidirectional motion where the cargo Mutant dmiro animals exhibit defects in locomotion stops, starts, and often changes direction (Welte, 2004; and die prematurely. Mitochondria in dmiro mutant Vale, 2003; Hollenbeck, 1996). Assuming that mitomuscles and neurons are abnormally distributed. Inchondria are simultaneously attached to two opposing stead of being transported into axons and dendrites, motors (De Vos et al., 2003), then net movement in one mitochondria accumulate in parallel rows in neuronal direction may be determined by the motor with the somata. Mutant neuromuscular junctions (NMJs) lack

Research paper thumbnail of Effects of imaging conditions on mitochondrial transport and length in larval motor axons of Drosophila

Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 2008

The distribution of mitochondria is sensitive to physiological stresses and changes in metabolic ... more The distribution of mitochondria is sensitive to physiological stresses and changes in metabolic demands. Consequently, it is important to carefully define the conditions facilitating live imaging of mitochondrial transport in dissected animal preparations. In this study, we examined Schneider's and the haemolymph-like solutions HL3 and HL6 for their suitability to image mitochondrial transport in motor axons of dissected Drosophila melanogaster larvae. Overall, mitochondrial transport kinetics in larval motor axons appeared similar among all three solutions. Unexpectedly, HL3 solution selectively increased the length of mitochondria in the context of the net-direction of transport. We also found that mitochondrial transport is sensitive to the extracellular Ca(2+) but not glutamate concentration. High concentrations of extracellular glutamate affected only the ratio between motile and stationary mitochondria. Our study offers a valuable overview of mitochondrial transport kinetics in larval motor axons of Drosophila under various conditions, guiding future studies genetically dissecting mechanisms of mitochondrial transport.

Research paper thumbnail of Drosophila Miro Is Required for Both Anterograde and Retrograde Axonal Mitochondrial Transport

Journal of Neuroscience, 2009

Microtubule-based transport of mitochondria into dendrites and axons is vital for sustaining neur... more Microtubule-based transport of mitochondria into dendrites and axons is vital for sustaining neuronal function. Transport along microtubule tracks proceeds in a series of plus and minus end-directed movements that are facilitated by kinesin and dynein motors. How the opposing movements are controlled to achieve effective transport over large distances remains unclear. Previous studies showed that the conserved mitochondrial GTPase Miro is required for mitochondrial transport into axons and dendrites and serves as a Ca(2+) sensor that controls mitochondrial mobility. To directly examine Miro's significance for kinesin- and/or dynein-mediated mitochondrial motility, we live-imaged movements of GFP-tagged mitochondria in larval Drosophila motor axons upon genetic manipulations of Miro. Loss of Drosophila Miro (dMiro) reduced the effectiveness of both anterograde and retrograde mitochondrial transport by selectively impairing kinesin- or dynein-mediated movements, depending on the direction of net transport. Net anterogradely transported mitochondria exhibited reduced kinesin- but normal dynein-mediated movements. Net retrogradely transported mitochondria exhibited much shorter dynein-mediated movements, whereas kinesin-mediated movements were minimally affected. In both cases, the duration of short stationary phases increased proportionally. Overexpression (OE) of dMiro also impaired the effectiveness of mitochondrial transport. Finally, loss and OE of dMiro altered the length of mitochondria in axons through a mechanistically separate pathway. We suggest that dMiro promotes effective antero- and retrograde mitochondrial transport by extending the processivity of kinesin and dynein motors according to a mitochondrion's programmed direction of transport.