Angiotensin II (Ang II) has been shown to accelerate atherogenesis, and the cellular Ang II type ... more Angiotensin II (Ang II) has been shown to accelerate atherogenesis, and the cellular Ang II type 1 (AT(1))-receptor mediates most of Ang II-induced pro-atherogenic effects. In this study we have examined the effect of macrophage oxidative stress on cellular AT(1)-receptor expression. Mouse peritoneal macrophages (MPM) from apolipoprotein-E deficient (E(0)) mice at increasing ages (1 6 months) demonstrated an age-dependent increase in cellular lipid-peroxides (PD) content. In parallel, the AT(1)-receptor mRNA and protein levels both increased by up to 3.7-fold and 1.7-fold, respectively, in MPM from 6-month old mice compared with 1-month old mice. Vitamin E supplementation to E(0) mice significantly decreased the MPM PD content and macrophage AT(1)-receptor mRNA expression compared with placebo-treated mice. The role of oxidative stress in the cellular expression of AT(1)-receptors was further demonstrated by manipulation of macrophage glutathione content. Buthionine-sulfoximine, a glutathione synthesis inhibitor, increased MPM PD content and AT(1)-receptor mRNA expression, whereas L-2-oxothiazolidine-4-carboxylic acid, that contributes to glutathione synthesis, reduced macrophage PD and AT(1)-receptor mRNA expression. Incubation of MPM with oxidised low-density lipoproteins (LDL) led to a significant, dose-dependent and time-dependent increase in macrophage AT(1)-receptor mRNA and protein expression, compared with control cells. In contrast, native LDL or acetylated LDL did not significantly affect macrophage AT(1)-receptor mRNA expression. In conclusion, our findings suggest that oxidative stress in macrophages induces AT(1)-receptor expression. This phenomenon can stimulate the interaction of Ang II with macrophages and hence accelerate macrophage foam cell formation and early atherogenesis.
The widespread use of rodents in neuroscience has prompted the development of optimized viral var... more The widespread use of rodents in neuroscience has prompted the development of optimized viral variants for transduction of brain cells, in vivo. However, many of the viruses developed are less efficient in other model organisms, with birds being among the most resistant to infection by current viral tools. Resultantly, the use of genetically-encoded tools and methods in avian species is scarce; markedly holding the field back. We sought to bridge this gap by developing custom viruses towards the transduction of brain cells of the Japanese quail. We first develop a unique protocol for culturing primary neurons and glia from quail embryos, followed by characterization of cultures via immunostaining, single cell mRNA sequencing and patch clamp electrophysiology. We then leverage the cultures for the rapid screening of various viruses, only to find that all yielded very poor to no infection of cells in vitro. However, few infected neurons were obtained by AAV1 and AAV2. Scrutiny of the sequence of the AAV receptor found in quail led us to rationally design novel AAV variants, with one variant (AAV1-T593K; AAV1*) showing improved transduction efficiency in vitro and in vivo. Together, we present a novel culturing method, unique transcriptome profiles of quail’s brain cells and a custom-tailored AAV1 for transduction of quail neurons in vitro and in vivo.
Protein aggregation is a hallmark of many neurodegenerative diseases1,2. In order to cope with mi... more Protein aggregation is a hallmark of many neurodegenerative diseases1,2. In order to cope with misfolding and aggregation, cells have evolved an elaborate network of molecular chaperones, composed of different families3. But while chaperoning mechanisms for different families are well established, functional and regulatory diversification within chaperone families is still largely a mystery4,5. Here we decided to explore chaperone functional diversity, through the lens of pathological aggregation. We revealed that different naturally-occurring isoforms of DNAJ chaperones showed differential effects on different types of aggregates. We performed a chaperone screen for modulators of two neurodegeneration-related aggregating proteins, the Huntington’s disease-related HTT-polyQ, and the ALS-related mutant FUS (mutFUS). The screen identified known modulators of HTT-polyQ aggregation6,7, confirming the validity of our approach. Surprisingly, modulators of mutFUS aggregation were completel...
The N-methyl-D-aspartate receptors (NMDARs; GluNRS) are glutamate receptors, commonly located at ... more The N-methyl-D-aspartate receptors (NMDARs; GluNRS) are glutamate receptors, commonly located at excitatory synapses. Mutations affecting receptor function often lead to devastating neurodevelopmental disorders. We have identified two toddlers with different heterozygous missense mutations of the same, and highly conserved, glycine residue located in the ligand-binding-domain of GRIN2B: G689C and G689S. Structure simulations suggest severely impaired glutamate binding, which we confirm by functional analysis. Both variants show three orders of magnitude reductions in glutamate EC50, with G689S exhibiting the largest reductions observed for GRIN2B (~2000-fold). Moreover, variants multimerize with, and upregulate, GluN2Bwt-subunits, thus engendering a strong dominant-negative effect on mixed channels. In neurons, overexpression of the variants instigates suppression of synaptic GluNRs. Lastly, while exploring spermine potentiation as a potential treatment, we discovered that the varia...
Multimodal imaging of optically-cleared brains, ex vivo, by Magnetic Resonance Imaging (MRI) and ... more Multimodal imaging of optically-cleared brains, ex vivo, by Magnetic Resonance Imaging (MRI) and light microscopy (LM) presents unique opportunities for studying the brain at various scales and resolutions. However, CLARITY -cleared brains lack MRI contrast, implicating lipids as the major source of MRI contrast. We explored the ex vivo MRI compatibility of uDISCO, ECi and Scale -cleared brains. Surprisingly, uDISCO and ECi -cleared brains retain MRI contrast, whereas Scale-cleared brains show a severe loss of MRI contrast, as CLARITY. Determination of lipid-content in cleared samples shows that CLARITY, uDISCO and ECi are strongly delipidating, whereas Scale preserves most lipids. We conclude that MRI contrast can be associated with tissue expansion (and hyperhydration) rather than with lipid-content. Thus, we present two clearing methods compatible with ex vivo MRI.
Acetylcholinesterase (AChE) expression was found to be induced in the mammalian CNS, including th... more Acetylcholinesterase (AChE) expression was found to be induced in the mammalian CNS, including the retina, by different types of stress leading to cellular apoptosis. Here, we tested possible involvement of AChE in hyperglycemia-induced apoptosis in a retinal cell line. Y79 retinoblastoma cells were incubated in starvation media (1% FBS and 1 mg/ml glucose) for 16-24 h, and then exposed to hyperglycemic environment by raising extracellular glucose concentrations to a final level of 3.5 mg/ml or 6 mg/ml. Similar levels of mannitol were used as control for hyperosmolarity. Cells were harvested at different time intervals for analysis of apoptosis and AChE protein expression. Apoptosis was detected by the cleavage of Poly ADP-ribose polymerase (PARP) using western blot, and by Terminal deoxynucleotidyl-transferase-mediated dUTP nick-end-labeling (TUNEL) assay. AChE protein expression and activity was detected by western blot and by the Karnovsky and Roots method, respectively. Mission(...
Angiotensin II (Ang II) has been shown to accelerate atherogenesis, and the cellular Ang II type ... more Angiotensin II (Ang II) has been shown to accelerate atherogenesis, and the cellular Ang II type 1 (AT(1))-receptor mediates most of Ang II-induced pro-atherogenic effects. In this study we have examined the effect of macrophage oxidative stress on cellular AT(1)-receptor expression. Mouse peritoneal macrophages (MPM) from apolipoprotein-E deficient (E(0)) mice at increasing ages (1 6 months) demonstrated an age-dependent increase in cellular lipid-peroxides (PD) content. In parallel, the AT(1)-receptor mRNA and protein levels both increased by up to 3.7-fold and 1.7-fold, respectively, in MPM from 6-month old mice compared with 1-month old mice. Vitamin E supplementation to E(0) mice significantly decreased the MPM PD content and macrophage AT(1)-receptor mRNA expression compared with placebo-treated mice. The role of oxidative stress in the cellular expression of AT(1)-receptors was further demonstrated by manipulation of macrophage glutathione content. Buthionine-sulfoximine, a glutathione synthesis inhibitor, increased MPM PD content and AT(1)-receptor mRNA expression, whereas L-2-oxothiazolidine-4-carboxylic acid, that contributes to glutathione synthesis, reduced macrophage PD and AT(1)-receptor mRNA expression. Incubation of MPM with oxidised low-density lipoproteins (LDL) led to a significant, dose-dependent and time-dependent increase in macrophage AT(1)-receptor mRNA and protein expression, compared with control cells. In contrast, native LDL or acetylated LDL did not significantly affect macrophage AT(1)-receptor mRNA expression. In conclusion, our findings suggest that oxidative stress in macrophages induces AT(1)-receptor expression. This phenomenon can stimulate the interaction of Ang II with macrophages and hence accelerate macrophage foam cell formation and early atherogenesis.
The widespread use of rodents in neuroscience has prompted the development of optimized viral var... more The widespread use of rodents in neuroscience has prompted the development of optimized viral variants for transduction of brain cells, in vivo. However, many of the viruses developed are less efficient in other model organisms, with birds being among the most resistant to infection by current viral tools. Resultantly, the use of genetically-encoded tools and methods in avian species is scarce; markedly holding the field back. We sought to bridge this gap by developing custom viruses towards the transduction of brain cells of the Japanese quail. We first develop a unique protocol for culturing primary neurons and glia from quail embryos, followed by characterization of cultures via immunostaining, single cell mRNA sequencing and patch clamp electrophysiology. We then leverage the cultures for the rapid screening of various viruses, only to find that all yielded very poor to no infection of cells in vitro. However, few infected neurons were obtained by AAV1 and AAV2. Scrutiny of the sequence of the AAV receptor found in quail led us to rationally design novel AAV variants, with one variant (AAV1-T593K; AAV1*) showing improved transduction efficiency in vitro and in vivo. Together, we present a novel culturing method, unique transcriptome profiles of quail’s brain cells and a custom-tailored AAV1 for transduction of quail neurons in vitro and in vivo.
Protein aggregation is a hallmark of many neurodegenerative diseases1,2. In order to cope with mi... more Protein aggregation is a hallmark of many neurodegenerative diseases1,2. In order to cope with misfolding and aggregation, cells have evolved an elaborate network of molecular chaperones, composed of different families3. But while chaperoning mechanisms for different families are well established, functional and regulatory diversification within chaperone families is still largely a mystery4,5. Here we decided to explore chaperone functional diversity, through the lens of pathological aggregation. We revealed that different naturally-occurring isoforms of DNAJ chaperones showed differential effects on different types of aggregates. We performed a chaperone screen for modulators of two neurodegeneration-related aggregating proteins, the Huntington’s disease-related HTT-polyQ, and the ALS-related mutant FUS (mutFUS). The screen identified known modulators of HTT-polyQ aggregation6,7, confirming the validity of our approach. Surprisingly, modulators of mutFUS aggregation were completel...
The N-methyl-D-aspartate receptors (NMDARs; GluNRS) are glutamate receptors, commonly located at ... more The N-methyl-D-aspartate receptors (NMDARs; GluNRS) are glutamate receptors, commonly located at excitatory synapses. Mutations affecting receptor function often lead to devastating neurodevelopmental disorders. We have identified two toddlers with different heterozygous missense mutations of the same, and highly conserved, glycine residue located in the ligand-binding-domain of GRIN2B: G689C and G689S. Structure simulations suggest severely impaired glutamate binding, which we confirm by functional analysis. Both variants show three orders of magnitude reductions in glutamate EC50, with G689S exhibiting the largest reductions observed for GRIN2B (~2000-fold). Moreover, variants multimerize with, and upregulate, GluN2Bwt-subunits, thus engendering a strong dominant-negative effect on mixed channels. In neurons, overexpression of the variants instigates suppression of synaptic GluNRs. Lastly, while exploring spermine potentiation as a potential treatment, we discovered that the varia...
Multimodal imaging of optically-cleared brains, ex vivo, by Magnetic Resonance Imaging (MRI) and ... more Multimodal imaging of optically-cleared brains, ex vivo, by Magnetic Resonance Imaging (MRI) and light microscopy (LM) presents unique opportunities for studying the brain at various scales and resolutions. However, CLARITY -cleared brains lack MRI contrast, implicating lipids as the major source of MRI contrast. We explored the ex vivo MRI compatibility of uDISCO, ECi and Scale -cleared brains. Surprisingly, uDISCO and ECi -cleared brains retain MRI contrast, whereas Scale-cleared brains show a severe loss of MRI contrast, as CLARITY. Determination of lipid-content in cleared samples shows that CLARITY, uDISCO and ECi are strongly delipidating, whereas Scale preserves most lipids. We conclude that MRI contrast can be associated with tissue expansion (and hyperhydration) rather than with lipid-content. Thus, we present two clearing methods compatible with ex vivo MRI.
Acetylcholinesterase (AChE) expression was found to be induced in the mammalian CNS, including th... more Acetylcholinesterase (AChE) expression was found to be induced in the mammalian CNS, including the retina, by different types of stress leading to cellular apoptosis. Here, we tested possible involvement of AChE in hyperglycemia-induced apoptosis in a retinal cell line. Y79 retinoblastoma cells were incubated in starvation media (1% FBS and 1 mg/ml glucose) for 16-24 h, and then exposed to hyperglycemic environment by raising extracellular glucose concentrations to a final level of 3.5 mg/ml or 6 mg/ml. Similar levels of mannitol were used as control for hyperosmolarity. Cells were harvested at different time intervals for analysis of apoptosis and AChE protein expression. Apoptosis was detected by the cleavage of Poly ADP-ribose polymerase (PARP) using western blot, and by Terminal deoxynucleotidyl-transferase-mediated dUTP nick-end-labeling (TUNEL) assay. AChE protein expression and activity was detected by western blot and by the Karnovsky and Roots method, respectively. Mission(...
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Papers by Ronit Heinrich