1. |
- Genove, G., et al.
(författare)
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Photoreceptor degeneration, structural remodeling and glial activation : a morphological study on a genetic mouse model for pericyte deficiency
- 2014
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Ingår i: Neuroscience. - : Elsevier BV. - 0306-4522 .- 1873-7544. ; 279, s. 269-284
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Tidskriftsartikel (refereegranskat)abstract
- Interaction between pericytes and endothelial cells via platelet-derived growth factor B (PDGF-B) signaling is critical for the development of the retinal microvasculature. The PDGF-B retention motif controls the spatial distribution range of the growth factor in the vicinity of its producing endothelial cells allowing its recognition by PDGF receptor beta-(PDGFR-beta)-carrying pericytes; this promotes recruitment of pericytes to the vascular basement membrane. Impairment of the PDGF-B signaling mechanism causes development of vascular abnormalities, and in the retina this consequently leads to defects in the neurological circuitry. The vascular pathology in the pdgf-b(ret/ret) (PDGF-B retention motif knockout) mouse retina has been previously reported; our study investigates the progressive neuronal defects and changes in the retinal morphology of this pericyte-deficient mouse model. Immunohistochemical analysis revealed retinal injuries to occur as early as postnatal day (P) 10 with substantial damage progressing from P15 and onward. Vascular abnormalities were apparent from P10, however, prominent neuronal defects were mostly observed from P15, beginning with the compromised integrity of the laminated retinal structure characterized by the presence of rosettes and focally distorted regions. Photoreceptor degeneration was observed by loss of both rod and cone cells, including the disassembly and altered structure of their synaptic terminals. Significant shortening of cone outer segments was observed from P10 and later stages; however, decrease in cone density was only observed at P28. Disorganization and dendrite remodeling of rod bipolar cells also added to the diminished neural and synaptic integrity. Moreover, in response to retinal injuries, Muller and microglial cells were observed to be in the reactive phenotype from P15 and onward. Such a sequence of events indicates that the pdgf-b(ret/ret) mouse model displays a short time frame between P10 and P15, during which the retina shifts to a retinopathic phase by the development of prominently altered morphological features.
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2. |
- Ladds, Marcus J. G. W., et al.
(författare)
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A DHODH inhibitor increases p53 synthesis and enhances tumor cell killing by p53 degradation blockage
- 2018
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- The development of non-genotoxic therapies that activate wild-type p53 in tumors is of great interest since the discovery of p53 as a tumor suppressor. Here we report the identification of over 100 small-molecules activating p53 in cells. We elucidate the mechanism of action of a chiral tetrahydroindazole (HZ00), and through target deconvolution, we deduce that its active enantiomer (R)-HZ00, inhibits dihydroorotate dehydrogenase (DHODH). The chiral specificity of HZ05, a more potent analog, is revealed by the crystal structure of the (R)-HZ05/DHODH complex. Twelve other DHODH inhibitor chemotypes are detailed among the p53 activators, which identifies DHODH as a frequent target for structurally diverse compounds. We observe that HZ compounds accumulate cancer cells in S-phase, increase p53 synthesis, and synergize with an inhibitor of p53 degradation to reduce tumor growth in vivo. We, therefore, propose a strategy to promote cancer cell killing by p53 instead of its reversible cell cycle arresting effect.
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3. |
- Mollick, Tanzina, 1986-, et al.
(författare)
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Human neural progenitor cells decrease photoreceptor degeneration, normalize opsin distribution and support synapse structure in cultured porcine retina
- 2016
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Ingår i: Brain Research. - Amsterdam, Netherlands : Elsevier BV. - 0006-8993 .- 1872-6240. ; 1646, s. 522-534
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Tidskriftsartikel (refereegranskat)abstract
- Retinal neurodegenerative disorders like retinitis pigmentosa, age-related macular degeneration, diabetic retinopathy and retinal detachment decrease retinal functionality leading to visual impairment. The pathological events are characterized by photoreceptor degeneration, synaptic disassembly, remodeling of postsynaptic neurons and activation of glial cells. Despite intense research, no effective treatment has been found for these disorders. The current study explores the potential of human neural progenitor cell (hNPC) derived factors to slow the degenerative processes in adult porcine retinal explants. Retinas were cultured for 3 days with or without hNPCs as a feeder layer and investigated by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), immunohistochemical, western blot and quantitative real time-polymerase chain reaction (qRT-PCR) techniques. TUNEL showed that hNPCs had the capacity to limit photoreceptor cell death. Among cone photoreceptors, hNPC coculture resulted in better maintenance of cone outer segments and reduced opsin mislocalization. Additionally, maintained synaptic structural integrity and preservation of second order calbindin positive horizontal cells was also observed. However, Müller cell gliosis only seemed to be alleviated in terms of reduced Müller cell density. Our observations indicate that at 3 days of coculture, hNPC derived factors had the capacity to protect photoreceptors, maintain synaptic integrity and support horizontal cell survival. Human neural progenitor cell applied treatment modalities may be an effective strategy to help maintain retinal functionality in neurodegenerative pathologies. Whether hNPCs can independently hinder Müller cell gliosis by utilizing higher concentrations or by combination with other pharmacological agents still needs to be determined.
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4. |
- Mollick, Tanzina, 1986-
(författare)
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Photoreceptor degeneration, second order neuron remodeling and glia reactivity in an in vivo and in vitro model of retinal neurodegeneration
- 2016
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Photoreceptors have the ability to last during the entire lifespan of an individual. Being the first line of neurons in the visual transduction pathway, their health and maintenance is eminent for proper retinal function. However, photoreceptors are susceptible to neurodegenerative retinal dystrophies. A number of retinal pathologies such as retinitis pigmentosa, age-related amacular degeneration and diabetic retinopathy have been linked to photoreceptor death. Moreover, photoreceptor degeneration has been shown to affect downstream inner nuclear layer cells as well as induce reactive responses from Müller cells and microglia. Since current treatments are ineffective in preventing the degeneration of these neurons, intense research is still underway to discover novel treatment modalities. In this thesis, photoreceptor degeneration was assessed in an in vivo and in vitro model of neurodegeneration. Moreover, a possible mode of preserving these neurons by the use of human neural progenitor cells (hNPCs) was investigated. The in vivo pdgf-bret/ret (platelet derived growth factor-b retention motif knockout) mouse model, which shows severe vascular pathology as a result of detachment of pericytes from the vascular endothelium, was studied during the first postnatal month. In a short time span, i.e. between postnatal day (P)10 and P15, retinopathic features were observed. Photoreceptor degeneration related to cell death, cone outer segment (OS) shortening and synapse disassembly in the outer plexiform layer (OPL) was seen. The second order rod bipolar cells underwent remodeling and the Müller cells became gliotic with increased expression of GFAP (glial fibrillary acidic protein). Microglial cells were also observed to convert to their reactive amoeboid-like phenotype. These features seemed to become more severe in the older P28 mutants. In the in vitro porcine retinal explant model, photoreceptor death significantly increased by 3 days in vitro (div). This was associated with loss of cone OSs, opsin mislocalization and loss of synaptic integrity in the OPL. Horizontal cell death and remodeling was also observed together with a severe gliotic response from the Müller cells. Human neural progenitor cell cocultured explants for 3 div had the ability to preserve photoreceptor survival by means of OS conservation, better opsin trafficking and maintaining synaptic integrity. However, Müller cell gliosis was only mitigated by a decreased density of GFAP immunoreactive Müller cells. In conclusion, both the in vivo and in vitro model of neurodegeneration demonstrate the vulnerability of photoreceptors to various mechanisms of retinal injury. Interestingly, hNPC derived neurotrophic factors had neuroprotective qualities in 3 div porcine retinal explants.
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5. |
- Mollick, Tanzina
(författare)
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Retinal tumorigenesis and neurodegeneration : strategies to promote tumor cell death and support retinal cell survival to preserve vision
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Our vision is central to our quality of life enabling us to interact with our environment and lead productive lives. The ability to see begins in the retina, one of the paramount features of the eye, as it takes on the task of converting light signals to nerve impulses that are relayed to the visual centers of the brain. Proper functioning of the retina is essential for our visual experience. In this thesis, two different aspects of retinal dysfunction are highlighted together with potential therapeutic interventions. One of these is the childhood cancer retinoblastoma, which is characterized by uncontrolled cell proliferation after the loss of the RB1 gene. And, the other is retinal neurodegeneration which is characterized by the dysfunction and death of the retinal cells. We targeted the pyrimidine ribonucleotide synthesis pathway as a possible strategy to prevent retinoblastoma cell growth and to promote cell death. In the case of retinal neurodegeneration, we explored the neuroprotective effect of human neural progenitor cell (hNPC) derived neurotrophic factors to support retinal cell survival. The rationale to target pyrimidine ribonucleotide synthesis as a strategy to treat retinoblastoma cells was based on our findings in paper I. In this paper we discovered a novel dihydroorotate dehydrogenase (DHODH) inhibitor after screening a library of compounds for their ability to increase p53 transcription factor activity in two reporter cell lines, the ARN8 melanoma cell line and the T22 mouse fibroblasts. Amongst the hit compounds, we selected compound HZ00 because it was more active on the ARN8 cells than on the T22 fibroblasts and due to its favorable medicinal chemistry properties. Regarding the mechanism of action of HZ00 on the p53 pathway, we observed that it was able to induce p53 synthesis without affecting p53 mRNA levels. Moreover, HZ00 was able to kill cancer cells and also synergize with an inhibitor of p53 degradation both in vitro and in vivo. During our studies on the identification of the target for HZ00, we observed that short treatment times with HZ00 caused ARN8 cells to accumulate in S phase and long treatments led to an increase in the proportion of cells in SubG1. Moreover, the supplementation with an excess of uridine was able to prevent the cell death effect. Based on these and other facts, we eventually narrowed down the target for HZ00 to DHODH, and confirmed this finding with enzymatic assays using purified DHODH. A further search for more potent analogues of HZ00 led to the identification of HZ05. HZ05 was able to accumulate a number of different cancer cells in S phase, which was followed by increase in SubG1 levels. However, the U2OS cell line had a higher propensity to accumulate in S phase. Further analysis showed an increase in p53 expression in the S phase cells. When we pretreated the U2OS cells with HZ05 followed by nutlin-3a, cell death was observed. This indicated that DHODH inhibitors could sensitize cancer cells to p53 degradation inhibitors by accumulating them in S phase with high p53 levels. Retinoblastoma is typically a TP53 wild-type tumor characterized by inactivation of the RB1 gene. This deficiency leads to the loss of the cell cycle’s major G1-S checkpoint protein Rb. In paper I, we described that DHODH inhibitors can activate p53 and also cause cancer cells to accumulate in S phase, and that this is eventually followed by death. Since retinoblastoma cells already have a dysfunctional G1-S checkpoint, the use of DHODH inhibitors seemed like a rational approach to promote retinoblastoma cell death. In paper II, we investigated the potential of the DHODH inhibitor brequinar to reduce retinoblastoma cell growth and promote cell death, both as a single agent and in combination with a nucleoside transport inhibitor, dipyridamole. Similar to the effects seen in paper I, we saw that brequinar as a single agent was able to accumulate retinoblastoma cells in S phase and to an extent, also cause an increase in cell death. However, the response was slow and required a treatment time of 6 days. When we treated the retinoblastoma cells with a combination of brequinar and dipyridamole, the cells responded with an S phase accumulation as early as 24 hours after treatment, with most of them driven towards cell death with increasing time. This synergistic effect was also seen with other DHODH and nucleoside transport inhibitors. Moreover, the combination treatment was effective in the presence of uridine at physiologic plasma concentrations. Further investigation showed activation of caspases 3 and 7 as well as an increased expression of cleaved PARP-1, indicating the onset of apoptosis. Additionally, the treatment of a p53 mutant retinoblastoma cell line also responded to brequinar and the combination treatment, suggesting that targeting pyrimidine ribonucleotide synthesis could be an attractive strategy to eliminate both p53 wild-type and p53 mutant retinoblastoma cells. In paper III and IV, we investigated the neurodegenerative events in an in vivo and in vitro model of retinal neurodegeneration with an emphasis on photoreceptor degeneration, second order neuron remodeling and glia reactivity. Furthermore, in paper IV we assessed the neuroprotective potential of hNPC derived neurotrophic factors in porcine retinal explant cultures. In paper III, the in vivo pdgf-bret/ret mouse model showed severe vascular defects due to the detachment of pericytes from the vascular endothelium. Degenerative events were followed on postnatal day (P) 7, 10, 15 and 28. These events were quite evident at P15 and worsened by P28, and included photoreceptor cell death, shortening of the cone outer segments and synaptic disassembly in the outer plexiform layer (OPL). Rod bipolar cells underwent remodeling and the Müller cells showed increased expression of GFAP (glial fibrillary acidic protein). The microglia also changed to their reactive amoeboid-like phenotype. For the in vitro porcine retinal explant model in paper IV, photoreceptor death increased significantly by 3 days in vitro. This was associated to the loss of the cone outer segments, mislocalization of opsin and synaptic disassembly in the OPL. Furthermore, we observed a loss and remodeling of horizontal cells, as well as severe gliosis of the Müller cells. The hNPC cocultured explants were observed to maintain photoreceptor survival through preservation of the cone outer segments, better opsin trafficking and retaining synaptic integrity. However, Müller cell gliosis was only alleviated by a decreased density of GFAP immunoreactive Müller cells. Both the in vivo and in vitro model of neurodegeneration demonstrated the vulnerability of photoreceptors to different mechanisms of retinal injury. The hNPC derived neurotrophic factors had the potential to preserve photoreceptors in the porcine retinal explants, but were not able to completely eliminate Müller cell gliosis.
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