This document is a thesis examining the effects of recombinant adeno-associated viral arginase 1 in transgenic mice with tau pathology. The introduction discusses Alzheimer's disease and tauopathies, L-arginine metabolism and associated pathways, and the mouse model used. The goal was to identify the effects of overexpressing arginase 1 in hippocampal neurons using viral vectors in this mouse model. Histological experiments included staining for various proteins and markers to analyze the effects.
Citalopram enhances action inhibition systems in Parkinson’s diseaseZheng Ye
1) The study investigated whether the selective serotonin reuptake inhibitor (SSRI) citalopram could reduce impulsivity and enhance response inhibition systems in patients with Parkinson's disease (PD).
2) The results showed that PD impaired both action restraint and cancellation compared to healthy controls. Citalopram improved behavioral performance on inhibition tasks in PD patients depending on disease severity.
3) Citalopram enhanced activation in the right inferior frontal gyrus, a brain region involved in response inhibition. The degree of enhancement correlated with reductions in impulsivity, supporting the role of serotonin in regulating inhibitory control.
This document reports on a study that tested the effects of isoxazole 9 (Isx-9), a small synthetic molecule, on adult hippocampal neurogenesis in rats. The study found that administering Isx-9 for 14 days potentiated cell proliferation and increased the number of immature neurons in the hippocampal dentate gyrus. Isx-9 treatment also completely reversed the reduction in cell proliferation and neuronal commitment observed in vehicle-treated animals that were subjected to repeated handling and injections. These findings demonstrate that Isx-9 has promising pro-neurogenic properties and could help mitigate stress-induced deficits in adult hippocampal neurogenesis.
This study investigated the mechanisms controlling the diurnal expression and trafficking of the astrocytic protein Fabp7. The key findings were:
1) Fabp7 mRNA and protein are enriched in the astrocytic perisynaptic compartment and exhibit a daily oscillation in their intracellular distribution in the hippocampus.
2) Northern blotting revealed a coordinated daily oscillation in the polyadenylation of Fabp7 mRNA throughout the mouse brain.
3) Fabp7 mRNA was found to coimmunoprecipitate with CPEB1, a protein that regulates subcellular trafficking and translation of synaptic plasticity mRNAs, in mouse astrocytes.
4) The 3'UTR of Fabp7
dkNET Webinar: A New Approach to the Study of Energy Balance and Obesity usin...dkNET
Abstract
We report a web-based tool for analysis of experiments using indirect calorimetry to measure physiological energy balance. CalR simplifies the process to import raw data files, generate plots, and determine the most appropriate statistical tests for interpretation. Analysis using the generalized linear model (which includes ANOVA and ANCOVA) allows for flexibility in interpreting diverse experimental designs, including those of obesity and thermogenesis. Users also may produce standardized output files for an experiment that can be shared and subsequently re-evaluated using CalR. This framework will provide the transparency necessary to enhance consistency, rigor, and reproducibility. The CalR analysis software will greatly increase the speed and efficiency with which metabolic experiments can be organized, analyzed per accepted norms, and reproduced and has become a standard tool for the field. CalR is accessible at https://CalRapp.org/
The top 4 key questions that our tool can answer:
1. Can I reproducibly and transparently analyze indirect calorimetry experiments in under 10 minutes?
2. How hard is it to use Analysis of Covariance (ANCOVA) to determine whether my groups of animals are significantly different?
3. Is there an automated, reproducible way to exclude “noisy” outlier data from our indirect calorimetry experiments?
4. What are the key factors in determining metabolic rate of mice?
Presenter: Alexander Banks, PhD, principal investigator and assistant professor at Harvard Medical School and the Beth Israel Deaconess Medical Center.
dkNET Webinar Information: https://dknet.org/about/webinar
The document reports on a study investigating the role of the p38 mitogen-activated protein kinase p38γ in Alzheimer's disease (AD) pathogenesis. The study found that depletion of p38γ exacerbated neuronal excitotoxicity, cognitive deficits, neuronal circuit abnormalities, and premature mortality in an AD mouse model overexpressing amyloid-β (Aβ). In contrast, increasing p38γ activity abolished these Aβ-induced deficits. Furthermore, mimicking site-specific tau phosphorylation by p38γ alleviated Aβ-induced neuronal death and excitotoxicity. The findings suggest p38γ phosphorylation of tau at specific sites inhibits Aβ toxicity in early AD, challenging the view that tau phosphorylation is purely pathogenic.
This study investigated how cocaine withdrawal affects anxiety and activity in the dorsal raphe (DR), a brain region involved in serotonin signaling and anxiety regulation. Mice were administered cocaine or saline in a binge-like pattern over 10 days. Anxiety-like behavior was assessed at different withdrawal timepoints using elevated plus maze and open field tests. Electrophysiological recordings from DR serotonin neurons found increased inhibitory currents during withdrawal, especially at 25 hours, indicating heightened GABA activity. Blocking 5-HT2C receptors in the DR prevented both the increased GABA activity and anxiety-like behavior during withdrawal. This suggests 5-HT2C receptors mediate anxiety produced by cocaine withdrawal, potentially by regulating GABA signaling in the DR serotonin system.
Caspase activation contributes to astrogliosis without inducing apoptosis in astrocytes. The study found that treating cultured neonatal rat astrocytes with dibutryl cAMP or beta-amyloid peptide, stimuli known to induce astrogliosis, led to increased caspase activity and expression of active caspase-3 without cell death. Inhibition of caspases attenuated the increased expression of glutamine synthetase and fibroblast growth factor-2, markers of astrogliosis. The results suggest caspases play a non-apoptotic role in regulating astrogliosis in astrocytes following brain injury.
Citalopram enhances action inhibition systems in Parkinson’s diseaseZheng Ye
1) The study investigated whether the selective serotonin reuptake inhibitor (SSRI) citalopram could reduce impulsivity and enhance response inhibition systems in patients with Parkinson's disease (PD).
2) The results showed that PD impaired both action restraint and cancellation compared to healthy controls. Citalopram improved behavioral performance on inhibition tasks in PD patients depending on disease severity.
3) Citalopram enhanced activation in the right inferior frontal gyrus, a brain region involved in response inhibition. The degree of enhancement correlated with reductions in impulsivity, supporting the role of serotonin in regulating inhibitory control.
This document reports on a study that tested the effects of isoxazole 9 (Isx-9), a small synthetic molecule, on adult hippocampal neurogenesis in rats. The study found that administering Isx-9 for 14 days potentiated cell proliferation and increased the number of immature neurons in the hippocampal dentate gyrus. Isx-9 treatment also completely reversed the reduction in cell proliferation and neuronal commitment observed in vehicle-treated animals that were subjected to repeated handling and injections. These findings demonstrate that Isx-9 has promising pro-neurogenic properties and could help mitigate stress-induced deficits in adult hippocampal neurogenesis.
This study investigated the mechanisms controlling the diurnal expression and trafficking of the astrocytic protein Fabp7. The key findings were:
1) Fabp7 mRNA and protein are enriched in the astrocytic perisynaptic compartment and exhibit a daily oscillation in their intracellular distribution in the hippocampus.
2) Northern blotting revealed a coordinated daily oscillation in the polyadenylation of Fabp7 mRNA throughout the mouse brain.
3) Fabp7 mRNA was found to coimmunoprecipitate with CPEB1, a protein that regulates subcellular trafficking and translation of synaptic plasticity mRNAs, in mouse astrocytes.
4) The 3'UTR of Fabp7
dkNET Webinar: A New Approach to the Study of Energy Balance and Obesity usin...dkNET
Abstract
We report a web-based tool for analysis of experiments using indirect calorimetry to measure physiological energy balance. CalR simplifies the process to import raw data files, generate plots, and determine the most appropriate statistical tests for interpretation. Analysis using the generalized linear model (which includes ANOVA and ANCOVA) allows for flexibility in interpreting diverse experimental designs, including those of obesity and thermogenesis. Users also may produce standardized output files for an experiment that can be shared and subsequently re-evaluated using CalR. This framework will provide the transparency necessary to enhance consistency, rigor, and reproducibility. The CalR analysis software will greatly increase the speed and efficiency with which metabolic experiments can be organized, analyzed per accepted norms, and reproduced and has become a standard tool for the field. CalR is accessible at https://CalRapp.org/
The top 4 key questions that our tool can answer:
1. Can I reproducibly and transparently analyze indirect calorimetry experiments in under 10 minutes?
2. How hard is it to use Analysis of Covariance (ANCOVA) to determine whether my groups of animals are significantly different?
3. Is there an automated, reproducible way to exclude “noisy” outlier data from our indirect calorimetry experiments?
4. What are the key factors in determining metabolic rate of mice?
Presenter: Alexander Banks, PhD, principal investigator and assistant professor at Harvard Medical School and the Beth Israel Deaconess Medical Center.
dkNET Webinar Information: https://dknet.org/about/webinar
The document reports on a study investigating the role of the p38 mitogen-activated protein kinase p38γ in Alzheimer's disease (AD) pathogenesis. The study found that depletion of p38γ exacerbated neuronal excitotoxicity, cognitive deficits, neuronal circuit abnormalities, and premature mortality in an AD mouse model overexpressing amyloid-β (Aβ). In contrast, increasing p38γ activity abolished these Aβ-induced deficits. Furthermore, mimicking site-specific tau phosphorylation by p38γ alleviated Aβ-induced neuronal death and excitotoxicity. The findings suggest p38γ phosphorylation of tau at specific sites inhibits Aβ toxicity in early AD, challenging the view that tau phosphorylation is purely pathogenic.
This study investigated how cocaine withdrawal affects anxiety and activity in the dorsal raphe (DR), a brain region involved in serotonin signaling and anxiety regulation. Mice were administered cocaine or saline in a binge-like pattern over 10 days. Anxiety-like behavior was assessed at different withdrawal timepoints using elevated plus maze and open field tests. Electrophysiological recordings from DR serotonin neurons found increased inhibitory currents during withdrawal, especially at 25 hours, indicating heightened GABA activity. Blocking 5-HT2C receptors in the DR prevented both the increased GABA activity and anxiety-like behavior during withdrawal. This suggests 5-HT2C receptors mediate anxiety produced by cocaine withdrawal, potentially by regulating GABA signaling in the DR serotonin system.
Caspase activation contributes to astrogliosis without inducing apoptosis in astrocytes. The study found that treating cultured neonatal rat astrocytes with dibutryl cAMP or beta-amyloid peptide, stimuli known to induce astrogliosis, led to increased caspase activity and expression of active caspase-3 without cell death. Inhibition of caspases attenuated the increased expression of glutamine synthetase and fibroblast growth factor-2, markers of astrogliosis. The results suggest caspases play a non-apoptotic role in regulating astrogliosis in astrocytes following brain injury.
Effect of kidney targated thymoquinone nanosuspension on LPS Shital Magar
This document provides background information and outlines the objectives and methodology for a dissertation studying the effect of kidney targeted thymoquinone nanosuspension on lipopolysaccharide (LPS) and cecal ligation and puncture (CLP)-induced acute kidney injury. The primary objectives are to prepare and characterize a thymoquinone nanosuspension formulation and evaluate its protective effects on LPS and CLP-induced acute kidney injury in rats. The experimental design involves administering various doses of the nanosuspension to rats in LPS and CLP models of acute kidney injury and analyzing outcomes through biochemical, histological, and molecular assays. The document discusses the hypothesized mechanisms of LPS and CLP-induced acute kidney injury
This document summarizes research on the physiological properties and potential health benefits and risks of exogenous melatonin supplementation. It discusses melatonin's role as an antioxidant and its potential anticancer, circadian rhythm regulating, and neuroprotective effects. However, it also notes that early morning melatonin administration may impair psychomotor performance and vigilance. More research is still needed to fully understand melatonin's long-term impacts and benefits, especially regarding cancer treatments and effects on memory.
This study examined the effects of chronic exposure to radiofrequency electromagnetic fields (RF-EMF) on energy balance functions in developing rats. Rats were exposed to 900 MHz RF-EMF at 1 V/m for 5 weeks starting in their juvenile period. Their feeding behavior, sleep patterns, and thermoregulation were recorded at air temperatures of 24°C and 31°C and compared to unexposed controls. The results showed that RF-EMF exposure increased rapid eye movement (REM) sleep episodes independently of temperature, and affected other sleep parameters depending on temperature. Exposure also induced peripheral vasoconstriction and slightly increased daytime food intake. Most effects were temperature-dependent. RF-EMF exposure appeared to
This study examined the effects of cerium oxide nanoparticles (CeO2 NPs) on disease progression in a transgenic mouse model of amyotrophic lateral sclerosis (ALS). The mice expressed a mutation in the SOD1 gene associated with familial ALS. Following disease onset, mice were given injections of either saline (control) or CeO2 NPs. Mice treated with CeO2 NPs showed improved motor performance and increased survival time, particularly female mice. The results suggest CeO2 NPs may be a potential treatment for slowing progression of ALS and other neurodegenerative diseases associated with oxidative stress.
This study investigated the mechanisms by which D-pinitol induces vasodilation in mouse mesenteric arteries. The results showed that D-pinitol caused concentration-dependent vasodilation that was endothelium-dependent and nitric oxide (NO)-mediated. D-pinitol increased phosphorylation of eNOS at its active site and decreased phosphorylation at its inactive site. It also increased NO production in mesenteric arteries through a mechanism involving calcium-calmodulin activation of eNOS. Administration of D-pinitol reduced systolic blood pressure in mice. These findings suggest D-pinitol causes vasodilation by activating eNOS and increasing NO bioavailability through calcium-calmodulin.
Effect of naringenin on 3 np induced huntington’s disease like symptoms by es...IJARIIT
The main aim of this study was to investigate the effect of Naringenin, a flavonoid on 3-Nitropropionic acid (3-NP)-
induced Huntington’s disease like symptoms by estimations of motor co-ordination and behavioral parameters. 3-NP is an
irreversible inhibitor of complex II in the mitochondria. 3-NP-induced neurodegeneration has been widely used as an animal
model of Huntington’s disease (HD). It replicates the pathology of HD by causing oxidative stress. Naringenin is a polyphenolic
compound, a bioflavonoid, known to have a neuroprotective effect in a rat model of Alzheimer’s disease. In the present study,
the neuroprotective effect of Naringenin on 3-NP induced oxidative stress in the rat was determined by behavioral parameters.
Rats were induced with 3-NP (15 mg/kg) intraperitoneally for 21 days and rats induced with 3-NP were treated with Naringenin
(25mg/kg and 75mg/kg) for 21 days. 3-NP caused a decline in motor function in the neurological score, locomotor activity, and
impaired rotarod activity. Naringenin treatment significantly improved grip strength indicating an improvement in motor
performance, alterations in % spontaneous alternations. These findings suggest the antioxidant potential of Naringenin
flavonoid against 3-Nitropionic acid induced neurotoxicity. However, more investigations are required to elucidate the cellular
mechanisms of Naringenin against 3-Nitropropionic acid induced Huntington’s disease like symptoms.
Improving Animal Modeling with 24/7 Home Cage Monitoring in Bioexclusion & Bi...InsideScientific
https://insidescientific.com/webinar/improving-animal-modeling-24/7-home-cage-monitoring-bioexclusion-biocontainment-mouse-housing-system-tecniplast
Recently, a surging response to the COVID-19 pandemic has led to an exponential increase in study support for biocontainment and bioexclusion research. Mouse models are being rapidly developed in both areas, and biosafe housing of these animal models is critical. Additionally, non-invasive home cage monitoring can improve the translational value of these research models.
Locomotor activity patterns can be monitored 24/7 as a diagnostic tool for biosecurity studies. Researchers, staff and animals alike will also benefit from a decreased need for animal handling, caging manipulations and animal monitoring.
This webinar will be most valuable for institutions where biocontainment and bioexclusion work is being considered or conducted, and for researchers who wish to better understand what can be achieved through continuous measurement of animal welfare based of use of non-invasive activity monitoring.
1) The document discusses Tay-Sachs disease (TSD), a neurodegenerative lysosomal storage disorder caused by mutations in the HEXA gene resulting in β-hexosaminidase A deficiency and GM2 ganglioside accumulation.
2) Accumulation of GM2 gangliosides is thought to trigger apoptosis, inflammation and synaptic dysfunction, though the specific mechanisms are unclear.
3) The author proposes a potential new therapeutic strategy for TSD involving silencing the GM2 activator protein to prevent GM2 transport to lysosomes and accumulation, combined with anti-inflammatory drugs. This approach aims to address current issues in understanding TSD pathogenesis and improving patient outcomes.
1) Mice lacking the inhibitory synapse cell adhesion molecule neuroligin 2 (NL2) were found to exhibit increased anxiety-like behavior.
2) While these NL2-deficient mice appeared to have a decrease in the density of inhibitory synaptic puncta, electron microscopy revealed no actual change in inhibitory synapse numbers.
3) This suggests that NL2 deletion impairs the function of inhibitory synapses without decreasing their numbers, and this decrease in inhibitory synaptic function correlates with increased anxiety in the mice.
Ecophysiological Impacts of Climate Change: Performance, Fitness and ExtinctionInsideScientific
Join Caroline Williams, PhD and Eric Riddell, PhD as they discuss their research involving climate change and the ecophysiological effects of changing global temperatures on organismal biology and survival.
Key Topics Include:
Energy use models using microclimate data can accurately predict whole winter energy use.
Colder soils in a warmer world: Snow modulates a trade-off between cold exposure and energy use for soil dwelling organisms.
How processes at the genetic level can influence ecological processes, such as geographic ranges.
Complex methods to make realistic predictions on the ecological impact of climate change.
This study investigated the optimal ratio of methionine to methionine plus cysteine (M:M+C) in the diets of growing pigs under normal conditions and during immune system stimulation (ISS). Thirty-six pigs were fed one of five diets varying in M:M+C ratio and their nitrogen balance was measured before and during two periods of ISS induced by injecting lipopolysaccharide. ISS reduced nitrogen balance and the optimal dietary M:M+C ratio increased during ISS, suggesting methionine is preferentially used during an immune response. The researchers concluded that ISS results in a higher optimal dietary M:M+C ratio in growing pigs.
Thermal Physiology: The Effects of Environmental Temperatures on Energy Expen...InsideScientific
Mice are generally an excellent model of human biology with nearly identical metabolic pathways. In contrast, the 3000-fold difference in body mass causes huge differences in thermal physiology and energy homeostasis. Humans generally live in a thermoneutral environment, while mice live and are typically studied below thermoneutrality. A mouse housed singly at 22 °C devotes 42% of its energy expenditure to maintaining its body temperature; the corresponding value in humans is approximately 0%. Understanding this different physiology is important, allowing one to avoid incorrect application of mouse observations to humans. It also boosts elucidation of physiology that is subtle or difficult to study in humans.
The goal is to understand thermal physiology and to use it to develop conditions under which mice better model humans. This is important for studying the effectiveness of drug treatments for metabolic diseases, like obesity and diabetes. Marc and Oksana discuss what thermoneutrality means in the mouse and the concept of the thermoneutral point. They also explore the effects of cold, hot, and near-thermoneutral environments on mouse energy expenditure, body temperature, and behavior.
This study investigated the effects of a single early life seizure induced by kainic acid (KA-ELS) on hippocampal synaptic plasticity and glutamate receptor function in rats. The key findings were:
1) Enhanced long-term potentiation (LTP) was observed 7 days, but not 2 days, after KA-ELS compared to controls, indicating inappropriate enhancement of synaptic connections in the acute period following seizure.
2) α-amino-3-hydroxy-5-methyl-isoxazole-propionic acid (AMPA) receptor desensitization was decreased 7 days after KA-ELS, despite no changes in AMPA receptor expression, phosphorylation, or membrane insertion.
This document summarizes the effects of environmental chemical exposures on epigenetics and disease. It finds that environmental pollutants like air pollution and chemicals like arsenic and aluminum can cause epigenetic changes including DNA methylation, histone modifications, and microRNA expression. These epigenetic alterations have been linked to various diseases. The document provides a table listing specific epigenetic changes found for different environmental chemicals and the diseases studied. It concludes that while many reports link environmental exposures to epigenetic changes, most have not been directly associated with disease outcomes, and more research is still needed.
IDN5706 is a semi-synthetic derivative of hyperforin that has neuroprotective effects. The document reports on experiments examining the effects of IDN5706 on field excitatory postsynaptic potentials (fEPSPs) in mouse hippocampal slices. The results suggest that IDN5706 activates TRPC3/6/7 channels, improving synaptic response and protecting against reductions in fEPSPs caused by amyloid beta oligomers. IDN5706 also improved spatial memory in mice, and this effect was blocked by a TRPC channel blocker. Computational modeling further supported the idea that IDN5706 activates TRPC channels.
Kouvaros S and Papatheodoropoulos C, (2016). Major dorsoventral differences i...Stylianos Kouvaros
1) The study examines differences in modulation of the local hippocampal CA1 network between the dorsal and ventral hippocampus by NMDA, mGlu5, adenosine A2A, and cannabinoid CB1 receptors.
2) Activation of NMDA receptors reduced excitatory transmission more in the ventral hippocampus than in the dorsal hippocampus by an adenosine A1 receptor-independent mechanism. In the dorsal hippocampus, NMDA receptors reduced inhibition and enhanced postsynaptic excitability.
3) Co-activation of mGlu5 and NMDA receptors strongly potentiated their effects in the dorsal hippocampus but had no potentiating effect in the ventral hippocampus. This potentiation in the dorsal hippocampus required adenos
Un modelo representa de forma simplificada las relaciones entre variables para controlar situaciones reales. Los modelos educativos dependen de la concepción de la educación, docentes, aprendizaje y estudiantes. El diseño curricular es el primer paso para planificar, organizar e implementar un currículum de acuerdo a un modelo educativo, definiendo qué, cómo, cuándo y cómo evaluar lo que se enseña.
The document provides examples of converting between binary, decimal, and hexadecimal number systems. It includes converting individual binary, decimal, and hexadecimal numbers as well as converting numbers that have fractional parts. The conversions are done by dividing the number by the base (2 for binary, 10 for decimal, 16 for hexadecimal) and representing the remainders as the digits of the number in that number system.
El documento describe el papel del juego en la educación inicial. Explica que el juego es una forma de aprendizaje y enseñanza, y motiva a los niños a aprender a través del disfrute. También destaca que los juegos y juguetes son herramientas educativas importantes que ayudan a los niños a desarrollar habilidades sociales y cognitivas. El documento concluye que el juego desde una edad temprana ayuda a los niños a resolver problemas, desarrollar la imaginación y establecer vínculos
El documento describe cinco técnicas comunes para la recolección de datos: entrevistas, encuestas, observación, diagramas de flujo y diccionarios de datos. Las entrevistas y encuestas se utilizan para obtener información y opiniones de las personas, mientras que la observación, los diagramas de flujo y los diccionarios de datos ayudan a comprender procesos y sistemas. Todas estas técnicas proporcionan información valiosa para el desarrollo de sistemas de información.
Effect of kidney targated thymoquinone nanosuspension on LPS Shital Magar
This document provides background information and outlines the objectives and methodology for a dissertation studying the effect of kidney targeted thymoquinone nanosuspension on lipopolysaccharide (LPS) and cecal ligation and puncture (CLP)-induced acute kidney injury. The primary objectives are to prepare and characterize a thymoquinone nanosuspension formulation and evaluate its protective effects on LPS and CLP-induced acute kidney injury in rats. The experimental design involves administering various doses of the nanosuspension to rats in LPS and CLP models of acute kidney injury and analyzing outcomes through biochemical, histological, and molecular assays. The document discusses the hypothesized mechanisms of LPS and CLP-induced acute kidney injury
This document summarizes research on the physiological properties and potential health benefits and risks of exogenous melatonin supplementation. It discusses melatonin's role as an antioxidant and its potential anticancer, circadian rhythm regulating, and neuroprotective effects. However, it also notes that early morning melatonin administration may impair psychomotor performance and vigilance. More research is still needed to fully understand melatonin's long-term impacts and benefits, especially regarding cancer treatments and effects on memory.
This study examined the effects of chronic exposure to radiofrequency electromagnetic fields (RF-EMF) on energy balance functions in developing rats. Rats were exposed to 900 MHz RF-EMF at 1 V/m for 5 weeks starting in their juvenile period. Their feeding behavior, sleep patterns, and thermoregulation were recorded at air temperatures of 24°C and 31°C and compared to unexposed controls. The results showed that RF-EMF exposure increased rapid eye movement (REM) sleep episodes independently of temperature, and affected other sleep parameters depending on temperature. Exposure also induced peripheral vasoconstriction and slightly increased daytime food intake. Most effects were temperature-dependent. RF-EMF exposure appeared to
This study examined the effects of cerium oxide nanoparticles (CeO2 NPs) on disease progression in a transgenic mouse model of amyotrophic lateral sclerosis (ALS). The mice expressed a mutation in the SOD1 gene associated with familial ALS. Following disease onset, mice were given injections of either saline (control) or CeO2 NPs. Mice treated with CeO2 NPs showed improved motor performance and increased survival time, particularly female mice. The results suggest CeO2 NPs may be a potential treatment for slowing progression of ALS and other neurodegenerative diseases associated with oxidative stress.
This study investigated the mechanisms by which D-pinitol induces vasodilation in mouse mesenteric arteries. The results showed that D-pinitol caused concentration-dependent vasodilation that was endothelium-dependent and nitric oxide (NO)-mediated. D-pinitol increased phosphorylation of eNOS at its active site and decreased phosphorylation at its inactive site. It also increased NO production in mesenteric arteries through a mechanism involving calcium-calmodulin activation of eNOS. Administration of D-pinitol reduced systolic blood pressure in mice. These findings suggest D-pinitol causes vasodilation by activating eNOS and increasing NO bioavailability through calcium-calmodulin.
Effect of naringenin on 3 np induced huntington’s disease like symptoms by es...IJARIIT
The main aim of this study was to investigate the effect of Naringenin, a flavonoid on 3-Nitropropionic acid (3-NP)-
induced Huntington’s disease like symptoms by estimations of motor co-ordination and behavioral parameters. 3-NP is an
irreversible inhibitor of complex II in the mitochondria. 3-NP-induced neurodegeneration has been widely used as an animal
model of Huntington’s disease (HD). It replicates the pathology of HD by causing oxidative stress. Naringenin is a polyphenolic
compound, a bioflavonoid, known to have a neuroprotective effect in a rat model of Alzheimer’s disease. In the present study,
the neuroprotective effect of Naringenin on 3-NP induced oxidative stress in the rat was determined by behavioral parameters.
Rats were induced with 3-NP (15 mg/kg) intraperitoneally for 21 days and rats induced with 3-NP were treated with Naringenin
(25mg/kg and 75mg/kg) for 21 days. 3-NP caused a decline in motor function in the neurological score, locomotor activity, and
impaired rotarod activity. Naringenin treatment significantly improved grip strength indicating an improvement in motor
performance, alterations in % spontaneous alternations. These findings suggest the antioxidant potential of Naringenin
flavonoid against 3-Nitropionic acid induced neurotoxicity. However, more investigations are required to elucidate the cellular
mechanisms of Naringenin against 3-Nitropropionic acid induced Huntington’s disease like symptoms.
Improving Animal Modeling with 24/7 Home Cage Monitoring in Bioexclusion & Bi...InsideScientific
https://insidescientific.com/webinar/improving-animal-modeling-24/7-home-cage-monitoring-bioexclusion-biocontainment-mouse-housing-system-tecniplast
Recently, a surging response to the COVID-19 pandemic has led to an exponential increase in study support for biocontainment and bioexclusion research. Mouse models are being rapidly developed in both areas, and biosafe housing of these animal models is critical. Additionally, non-invasive home cage monitoring can improve the translational value of these research models.
Locomotor activity patterns can be monitored 24/7 as a diagnostic tool for biosecurity studies. Researchers, staff and animals alike will also benefit from a decreased need for animal handling, caging manipulations and animal monitoring.
This webinar will be most valuable for institutions where biocontainment and bioexclusion work is being considered or conducted, and for researchers who wish to better understand what can be achieved through continuous measurement of animal welfare based of use of non-invasive activity monitoring.
1) The document discusses Tay-Sachs disease (TSD), a neurodegenerative lysosomal storage disorder caused by mutations in the HEXA gene resulting in β-hexosaminidase A deficiency and GM2 ganglioside accumulation.
2) Accumulation of GM2 gangliosides is thought to trigger apoptosis, inflammation and synaptic dysfunction, though the specific mechanisms are unclear.
3) The author proposes a potential new therapeutic strategy for TSD involving silencing the GM2 activator protein to prevent GM2 transport to lysosomes and accumulation, combined with anti-inflammatory drugs. This approach aims to address current issues in understanding TSD pathogenesis and improving patient outcomes.
1) Mice lacking the inhibitory synapse cell adhesion molecule neuroligin 2 (NL2) were found to exhibit increased anxiety-like behavior.
2) While these NL2-deficient mice appeared to have a decrease in the density of inhibitory synaptic puncta, electron microscopy revealed no actual change in inhibitory synapse numbers.
3) This suggests that NL2 deletion impairs the function of inhibitory synapses without decreasing their numbers, and this decrease in inhibitory synaptic function correlates with increased anxiety in the mice.
Ecophysiological Impacts of Climate Change: Performance, Fitness and ExtinctionInsideScientific
Join Caroline Williams, PhD and Eric Riddell, PhD as they discuss their research involving climate change and the ecophysiological effects of changing global temperatures on organismal biology and survival.
Key Topics Include:
Energy use models using microclimate data can accurately predict whole winter energy use.
Colder soils in a warmer world: Snow modulates a trade-off between cold exposure and energy use for soil dwelling organisms.
How processes at the genetic level can influence ecological processes, such as geographic ranges.
Complex methods to make realistic predictions on the ecological impact of climate change.
This study investigated the optimal ratio of methionine to methionine plus cysteine (M:M+C) in the diets of growing pigs under normal conditions and during immune system stimulation (ISS). Thirty-six pigs were fed one of five diets varying in M:M+C ratio and their nitrogen balance was measured before and during two periods of ISS induced by injecting lipopolysaccharide. ISS reduced nitrogen balance and the optimal dietary M:M+C ratio increased during ISS, suggesting methionine is preferentially used during an immune response. The researchers concluded that ISS results in a higher optimal dietary M:M+C ratio in growing pigs.
Thermal Physiology: The Effects of Environmental Temperatures on Energy Expen...InsideScientific
Mice are generally an excellent model of human biology with nearly identical metabolic pathways. In contrast, the 3000-fold difference in body mass causes huge differences in thermal physiology and energy homeostasis. Humans generally live in a thermoneutral environment, while mice live and are typically studied below thermoneutrality. A mouse housed singly at 22 °C devotes 42% of its energy expenditure to maintaining its body temperature; the corresponding value in humans is approximately 0%. Understanding this different physiology is important, allowing one to avoid incorrect application of mouse observations to humans. It also boosts elucidation of physiology that is subtle or difficult to study in humans.
The goal is to understand thermal physiology and to use it to develop conditions under which mice better model humans. This is important for studying the effectiveness of drug treatments for metabolic diseases, like obesity and diabetes. Marc and Oksana discuss what thermoneutrality means in the mouse and the concept of the thermoneutral point. They also explore the effects of cold, hot, and near-thermoneutral environments on mouse energy expenditure, body temperature, and behavior.
This study investigated the effects of a single early life seizure induced by kainic acid (KA-ELS) on hippocampal synaptic plasticity and glutamate receptor function in rats. The key findings were:
1) Enhanced long-term potentiation (LTP) was observed 7 days, but not 2 days, after KA-ELS compared to controls, indicating inappropriate enhancement of synaptic connections in the acute period following seizure.
2) α-amino-3-hydroxy-5-methyl-isoxazole-propionic acid (AMPA) receptor desensitization was decreased 7 days after KA-ELS, despite no changes in AMPA receptor expression, phosphorylation, or membrane insertion.
This document summarizes the effects of environmental chemical exposures on epigenetics and disease. It finds that environmental pollutants like air pollution and chemicals like arsenic and aluminum can cause epigenetic changes including DNA methylation, histone modifications, and microRNA expression. These epigenetic alterations have been linked to various diseases. The document provides a table listing specific epigenetic changes found for different environmental chemicals and the diseases studied. It concludes that while many reports link environmental exposures to epigenetic changes, most have not been directly associated with disease outcomes, and more research is still needed.
IDN5706 is a semi-synthetic derivative of hyperforin that has neuroprotective effects. The document reports on experiments examining the effects of IDN5706 on field excitatory postsynaptic potentials (fEPSPs) in mouse hippocampal slices. The results suggest that IDN5706 activates TRPC3/6/7 channels, improving synaptic response and protecting against reductions in fEPSPs caused by amyloid beta oligomers. IDN5706 also improved spatial memory in mice, and this effect was blocked by a TRPC channel blocker. Computational modeling further supported the idea that IDN5706 activates TRPC channels.
Kouvaros S and Papatheodoropoulos C, (2016). Major dorsoventral differences i...Stylianos Kouvaros
1) The study examines differences in modulation of the local hippocampal CA1 network between the dorsal and ventral hippocampus by NMDA, mGlu5, adenosine A2A, and cannabinoid CB1 receptors.
2) Activation of NMDA receptors reduced excitatory transmission more in the ventral hippocampus than in the dorsal hippocampus by an adenosine A1 receptor-independent mechanism. In the dorsal hippocampus, NMDA receptors reduced inhibition and enhanced postsynaptic excitability.
3) Co-activation of mGlu5 and NMDA receptors strongly potentiated their effects in the dorsal hippocampus but had no potentiating effect in the ventral hippocampus. This potentiation in the dorsal hippocampus required adenos
Un modelo representa de forma simplificada las relaciones entre variables para controlar situaciones reales. Los modelos educativos dependen de la concepción de la educación, docentes, aprendizaje y estudiantes. El diseño curricular es el primer paso para planificar, organizar e implementar un currículum de acuerdo a un modelo educativo, definiendo qué, cómo, cuándo y cómo evaluar lo que se enseña.
The document provides examples of converting between binary, decimal, and hexadecimal number systems. It includes converting individual binary, decimal, and hexadecimal numbers as well as converting numbers that have fractional parts. The conversions are done by dividing the number by the base (2 for binary, 10 for decimal, 16 for hexadecimal) and representing the remainders as the digits of the number in that number system.
El documento describe el papel del juego en la educación inicial. Explica que el juego es una forma de aprendizaje y enseñanza, y motiva a los niños a aprender a través del disfrute. También destaca que los juegos y juguetes son herramientas educativas importantes que ayudan a los niños a desarrollar habilidades sociales y cognitivas. El documento concluye que el juego desde una edad temprana ayuda a los niños a resolver problemas, desarrollar la imaginación y establecer vínculos
El documento describe cinco técnicas comunes para la recolección de datos: entrevistas, encuestas, observación, diagramas de flujo y diccionarios de datos. Las entrevistas y encuestas se utilizan para obtener información y opiniones de las personas, mientras que la observación, los diagramas de flujo y los diccionarios de datos ayudan a comprender procesos y sistemas. Todas estas técnicas proporcionan información valiosa para el desarrollo de sistemas de información.
Uso de comandos insert, update y delete en bases de datos de sql serverJulio Guevara
Este documento describe los pasos para manipular datos en una base de datos SQL Server mediante los comandos INSERT, UPDATE y DELETE. Primero se selecciona la base de datos, luego se agregan registros a las tablas "Usuarios" y "Autores" usando INSERT. Después, se actualizan campos en las tablas "Autores" y "Libros" con UPDATE. Finalmente, se eliminan registros de la tabla "Usuarios" con DELETE.
Este documento describe diferentes técnicas para la recolección de datos utilizadas por analistas, incluyendo entrevistas, encuestas, observación, diagramas de flujo y diccionarios de datos. Explica que estas herramientas ayudan a los analistas a desarrollar sistemas de información mediante la recopilación de información relevante. Además, proporciona detalles sobre cómo aplicar específicamente cada técnica y los tipos de información que cada una puede generar.
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Este documento describe los pasos para agregar índices y restricciones a las tablas de una base de datos de SQL Server. Primero, elimina los datos existentes de las tablas mediante comandos TRUNCATE. Luego, agrega un índice único agrupado, una restricción DEFAULT y una clave primaria a la tabla Usuarios. A continuación, agrega dos índices, uno agrupado y uno no agrupado, y una clave primaria a la tabla Autores. Finalmente, agrega dos índices, dos restricciones FOREIGN KEY y
El documento habla sobre la importancia de la privacidad y la seguridad en línea. Explica que los usuarios deben tomar medidas para proteger su información personal en Internet, como usar contraseñas seguras y actualizadas, y estar atentos al phishing. También enfatiza que las empresas deben implementar medidas de seguridad sólidas para proteger los datos de los clientes.
Asavari G. Joshi is seeking a career opportunity where she can utilize her skills and continuously upgrade her knowledge. She has an M.E. in Signal Processing from JSPM's Imperial College of Engineering & Research in Pune and a B.E. in Electronics and Telecommunication from Padmabhooshan Vasantraodada Patil Institute of Technology. Her technical skills include programming languages like C, C++, and MATLAB. She has experience with projects in image processing, embedded design, and wireless channel equalization. She also has publications in international journals and has participated in conferences. Asavari is achievement-oriented, a committed team player, and self-motivated.
Asavari G. Joshi is seeking a career opportunity where she can utilize her skills and continuously upgrade her knowledge. She has an M.E. in Signal Processing from JSPM's Imperial College of Engineering & Research in Pune and a B.E. in Electronics and Telecommunication from Padmabhooshan Vasantraodada Patil Institute of Technology. Her technical skills include programming languages like C, C++, and MATLAB. She has experience with projects in image processing, embedded design, and wireless channel equalization. She also has publications in international journals and has participated in conferences. Asavari is achievement-oriented, a committed team player, and self-motivated.
This document discusses the role of various factors in neurodegenerative diseases such as Amyotrophic lateral sclerosis (ALS), Alzheimer's disease, and Parkinson's disease. It notes that geographic clusters of ALS have been observed in areas such as Guam and Japan. It also discusses the metabolic cooperation between neurons and astrocytes in the brain, highlighting how disruptions to this interaction can lead to neurodegenerative disorders. The document reviews literature on various pathogenic mechanisms and potential environmental and genetic risk factors involved in these conditions.
This document summarizes a book titled "Role of Plants, environmental toxins and physical neurotoxicological factors in Amyotrophic lateral sclerosis, Alzheimer Disease and Other Neurodegenerative Diseases." It has 10 authors from various countries. The book aims to observe the effects of plant neurotoxins, physical factors, and geography on neurodegenerative diseases like ALS, PD, and AD. It reviews literature on geographic clusters of these diseases and genetic mutations. It also discusses the brain's high metabolic needs, interactions between neurons and astrocytes, and how impairments in these systems can lead to neurodegeneration. The book presents figures and intends to develop new hypotheses and therapies based on contributing pathogenic factors.
This document summarizes research on amyotrophic lateral sclerosis (ALS), including its pathogenesis, genetic factors, and potential new treatment strategies. It discusses how ALS results from damage to motor neurons in the brain and spinal cord from a combination of increased toxicity from substances like glutamate, calcium ions, and protein aggregates as well as weaknesses in the motor neuron system from genetic mutations and deficits in neurotrophic factors. While the cause is still unclear, various studies suggest roles for inflammation, immune responses, oxidative stress, cytoskeletal abnormalities, and dysregulation of autophagy and microRNAs. The document reviews several studies examining these pathways and their implications for understanding ALS and identifying new therapeutic targets. It also proposes experimental models to
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The document discusses DNA repair mechanisms in human lymphocytes. It describes how oxidized purines in DNA are repaired through the base excision repair pathway involving the OGG1 glycosylase. This repair requires activation by the transcription factor NF-YA, which upregulates OGG1 expression when DNA is at risk of oxidation. The repair of oxidized bases by OGG1 in human lymphocytes is slow but can be activated by phytohemagglutinin. Disruption of this global repair of oxidized purines may be due to downregulation of OGG1. A second document discusses how green tea acts as an antioxidant to protect against oxidative stress and influence DNA damage and repair through activation of the Nrf2 transcription factor and upreg
Insulin resistance and cancer the role of insulin and IGFsBladimir Viloria
This document discusses the role of insulin and insulin-like growth factors (ILFs) such as IGF1 and IGF2 in linking insulin resistance to cancer risk. It summarizes evidence that ILPs regulate cell proliferation, differentiation, and apoptosis through signaling pathways involved in carcinogenesis. While some epidemiological studies found associations between ILP levels and cancer risk, the data is still debated. The document reviews how ILP signaling through insulin and IGF receptors can activate pathways driving cell growth and inhibiting cell death, thus playing a role in cancer development and progression. It proposes ILPs may represent a link between the pathogenic processes of insulin resistance, inflammation, diabetes, and certain cancers.
1521-0103/346/2/318–327$25.00 http://dx.doi.org/10.1124/jpet.113.202994
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS J Pharmacol Exp Ther 346:318–327, August 2013
Copyright ª 2013 by The American Society for Pharmacology and Experimental Therapeutics
Selective and Potent Agonists and Antagonists for Investigating
the Role of Mouse Oxytocin Receptors
Marta Busnelli, Elisabetta Bulgheroni, Maurice Manning, Gunnar Kleinau, and Bice Chini
CNR, Institute of Neuroscience, Milan, Italy (M.B., E.B., B.C.); Department of Biotechnology and Translational Medicine,
Università degli Studi di Milano, Milan, Italy (M.B.); Department of Biochemistry and Cancer Biology, University of Toledo,
Toledo, Ohio (M.M.); and Institute of Experimental Pediatric Endocrinology, Charité-Universitätsmedizin Berlin, Berlin, Germany
(G.K.)
Received January 8, 2013; accepted May 29, 2013
ABSTRACT
The neuropeptides oxytocin (OT) and vasopressin (AVP) have
been shown to play a central role in social behaviors; as
a consequence, they have been recognized as potential drugs to
treat neurodevelopmental and psychiatric disorders character-
ized by impaired social interactions. However, despite the basic
and preclinical relevance of mouse strains carrying genetic
alterations in the OT/AVP systems to basic and preclinical
translational neuroscience, the pharmacological profile of mouse
OT/AVP receptor subtypes has not been fully characterized. To
fill in this gap, we have characterized a number of OT and AVP
agonists and antagonists at three murine OT/AVP receptors
expressed in the nervous system as follows: the oxytocin
(mOTR) and vasopressin V1a (mV1aR) and V1b (mV1bR)
subtypes. These three receptors were transiently expressed in
vitro for binding and intracellular signaling assays, and then
a homology model of the mOTR structure was constructed to
investigate how its molecular features compare with human
and rat OTR orthologs. Our data indicate that the selectivity
profile of the natural ligands, OT and AVP, is conserved in
humans, rats, and mice. Furthermore, we found that the
synthetic peptide [Thr4Gly7]OT (TGOT) is remarkably selective
for the mOTR and, like the endogenous OT ligand, activates Gq
and Gi and recruits b-arrestins. Finally, we report three
antagonists that exhibit remarkably high affinities and selectiv-
ities at mOTRs. These highly selective pharmacological tools
will contribute to the investigation of the specific physiologic
and pathologic roles of mOTR for the development of selective
OT-based therapeutics.
Introduction
Central oxytocin (OT) and vasopressin (AVP) effects are
mediated by three G-protein-coupled receptors (GPCRs) that
are evolutionarily highly conserved and closely related, with
overall homology varying from 40 to 85%: the vasopressin 1a
receptor (V1aR), the vasopressin 1b receptor (V1bR), and the
OT receptor (OTR) (Barberis et al., 1999; Birnbaumer, 2000;
Zingg and Laporte, 2003). OT and AVP are also structurally
very simila ...
1521-0103/346/2/318–327$25.00 http://dx.doi.org/10.1124/jpet.113.202994
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS J Pharmacol Exp Ther 346:318–327, August 2013
Copyright ª 2013 by The American Society for Pharmacology and Experimental Therapeutics
Selective and Potent Agonists and Antagonists for Investigating
the Role of Mouse Oxytocin Receptors
Marta Busnelli, Elisabetta Bulgheroni, Maurice Manning, Gunnar Kleinau, and Bice Chini
CNR, Institute of Neuroscience, Milan, Italy (M.B., E.B., B.C.); Department of Biotechnology and Translational Medicine,
Università degli Studi di Milano, Milan, Italy (M.B.); Department of Biochemistry and Cancer Biology, University of Toledo,
Toledo, Ohio (M.M.); and Institute of Experimental Pediatric Endocrinology, Charité-Universitätsmedizin Berlin, Berlin, Germany
(G.K.)
Received January 8, 2013; accepted May 29, 2013
ABSTRACT
The neuropeptides oxytocin (OT) and vasopressin (AVP) have
been shown to play a central role in social behaviors; as
a consequence, they have been recognized as potential drugs to
treat neurodevelopmental and psychiatric disorders character-
ized by impaired social interactions. However, despite the basic
and preclinical relevance of mouse strains carrying genetic
alterations in the OT/AVP systems to basic and preclinical
translational neuroscience, the pharmacological profile of mouse
OT/AVP receptor subtypes has not been fully characterized. To
fill in this gap, we have characterized a number of OT and AVP
agonists and antagonists at three murine OT/AVP receptors
expressed in the nervous system as follows: the oxytocin
(mOTR) and vasopressin V1a (mV1aR) and V1b (mV1bR)
subtypes. These three receptors were transiently expressed in
vitro for binding and intracellular signaling assays, and then
a homology model of the mOTR structure was constructed to
investigate how its molecular features compare with human
and rat OTR orthologs. Our data indicate that the selectivity
profile of the natural ligands, OT and AVP, is conserved in
humans, rats, and mice. Furthermore, we found that the
synthetic peptide [Thr4Gly7]OT (TGOT) is remarkably selective
for the mOTR and, like the endogenous OT ligand, activates Gq
and Gi and recruits b-arrestins. Finally, we report three
antagonists that exhibit remarkably high affinities and selectiv-
ities at mOTRs. These highly selective pharmacological tools
will contribute to the investigation of the specific physiologic
and pathologic roles of mOTR for the development of selective
OT-based therapeutics.
Introduction
Central oxytocin (OT) and vasopressin (AVP) effects are
mediated by three G-protein-coupled receptors (GPCRs) that
are evolutionarily highly conserved and closely related, with
overall homology varying from 40 to 85%: the vasopressin 1a
receptor (V1aR), the vasopressin 1b receptor (V1bR), and the
OT receptor (OTR) (Barberis et al., 1999; Birnbaumer, 2000;
Zingg and Laporte, 2003). OT and AVP are also structurally
very simila ...
The document examines the effects of the TACE inhibitor BMS-561392 on APP processing both in vitro and in vivo. In cell cultures expressing APP, BMS-561392 reduced secretion of sAPPα without increasing Aβ production. Conversely, a BACE inhibitor decreased sAPPβ and Aβ without affecting sAPPα. In vivo infusion of BMS-561392 into mouse brains decreased sAPPα levels but did not significantly change steady-state Aβ levels. The findings suggest that under normal conditions, BACE and TACE do not compete for APP as a substrate, though they share localization in the trans-Golgi network. Inhibition of TACE may therefore reduce TNFα levels in
1) ALS is a progressive neurodegenerative disease that affects motor neurons, leading to muscle paralysis. It was first described in 1874 but its pathogenesis remains unclear.
2) ALS may be caused by both increased toxicity from substances like RNA proteins and weakness of the motor neuron system. Genetic mutations and environmental toxins are also implicated in some cases.
3) The document proposes new experimental and drug delivery approaches to better understand disease mechanisms and improve treatment of ALS.
This document discusses perspectives in molecular imaging and immunotherapies for Alzheimer's disease (AD). It provides background on AD, reviewing current theories for its pathogenesis including the cholinergic, amyloid-beta, and tau hypotheses. Imaging technologies like positron emission tomography (PET) and magnetic resonance imaging (MRI) can help with early diagnosis by detecting biomarkers for different stages of AD in cerebrospinal fluid and brain tissue. Combining molecular imaging systems may improve accuracy. Current and future immunotherapies aim to treat or prevent AD by targeting its underlying molecular mechanisms.
1) Rapamycin treatment significantly increased survival and delayed disease progression in a mouse model of Leigh syndrome (mitochondrial disease) that was deficient in the Ndufs4 gene.
2) Rapamycin treated mice did not develop neurological lesions in the brain that were present in untreated mice, and had reduced neurological symptoms and inflammation.
3) While rapamycin did not improve mitochondrial function directly, it induced metabolic changes in the mice including increased amino acid catabolism and decreased glycolysis, which helped alleviate symptoms of the mitochondrial disease.
This document summarizes a project investigating whether mitochondrial DNA-encoded oxidative phosphorylation (OXPHOS) transcripts are dysregulated in the blood of patients with mild cognitive impairment (MCI) and Alzheimer's disease (AD) compared to controls. The student researcher designed primers, conducted quantitative real-time PCR on blood samples from controls and patients, and found several mtDNA-encoded OXPHOS transcripts were significantly more abundant in MCI and AD patients. This suggests peripheral changes in mitochondrial gene expression occur early in AD and could provide biomarkers for early diagnosis and monitoring disease progression and treatment responses.
The Role Of G Protein Coupled ReceptorssAngela Hays
- G protein-coupled receptors (GPCRs) are seven transmembrane receptors located on cell surfaces that play an important role in intracellular signaling pathways and crucial physiological processes.
- When a ligand binds to a GPCR, it activates a heterotrimeric G protein within the cell. This leads to the production of second messengers like DAG and IP3, which mediate different cellular functions such as muscle contraction.
- The IP3 receptor releases intracellular calcium stores when bound by IP3, increasing cytosolic calcium levels and activating calcium-dependent signaling pathways.
The document discusses a study examining the progression of experimental prion disease in mice with compromised DNA repair capacity. The study found that mice lacking genes responsible for repairing oxidative DNA damage developed prion disease at an accelerated clinical course compared to wildtype mice. This suggests oxidative DNA damage contributes to prion disease pathogenesis. The study analyzed levels of disease-associated prion protein and markers of neurodegeneration in brain tissue over time between the two mouse models.
This study investigated how increased expression of the cytokine interleukin-6 (IL-6) in astrocytes alters the effects of acute ethanol on synaptic function in the hippocampus. Transgenic mice with elevated astrocyte expression of IL-6 were used to model increased IL-6 production induced by ethanol exposure. Results showed that acute ethanol enhanced synaptic transmission and long-term potentiation in hippocampal slices from IL-6 transgenic mice, whereas ethanol typically reduces these measures in non-transgenic mice. Additionally, IL-6 transgenic mice showed higher brain activity during ethanol withdrawal compared to non-transgenic mice. These findings suggest that increased astrocyte IL-6 production induced by ethanol can lead to neuroadaptive changes that alter how
Caffeine Blocks HIV-1 Tat-Induced Amyloid Beta Production and Tau Phosphoryla...Mahmoud Lotfy Soliman
Caffeine may help reduce Alzheimer's disease pathology associated with HIV infection by blocking the effects of the HIV protein Tat. The study found that:
1) HIV Tat increased amyloid-beta and phosphorylated tau levels in neural cells, markers of Alzheimer's disease, but caffeine prevented these increases.
2) HIV Tat disrupted endolysosome function in neural cells, but caffeine blocked these effects of Tat and preserved normal endolysosome functioning.
3) Caffeine on its own decreased amyloid-beta levels and may help protect against Alzheimer's disease progression in people living with HIV through its effects on endolysosome function and Tat protein activity.
1) The document discusses the relationship between the immune system and brain development/function. Alterations in immune function can impact neurodevelopment and be associated with various neuropsychiatric disorders.
2) Studies show that drugs like fingolimod that modulate sphingosine-1-phosphate receptors and prevent lymphocyte egress from lymph nodes can significantly reduce relapse rates and disability progression in multiple sclerosis patients.
3) Autism disorders may involve abnormalities in certain brain areas and a complex symptomatology related to genetic and environmental factors that can disrupt normal brain growth and the immune situation. The immune status, specific time periods, microenvironment, and genetics may all provide insights into autism pathogenesis.
Chronic Neuroinflammation in Alzheimer’s Disease New Perspectives Animal Mode...Niloo Karunaweera
This review article discusses chronic neuroinflammation in Alzheimer's disease and potential new animal models and drug candidates. Specifically:
1) Chronic neuroinflammation is now considered a major factor in Alzheimer's pathogenesis, but current transgenic animal models do not fully replicate the degree of inflammation, neurodegeneration, and cognitive decline seen in humans.
2) A new potential animal model is the GFAP-IL6 mouse, which shows substantial neurodegeneration, motor/cognitive decline from 6 months, mimicking human Alzheimer's features better than existing models.
3) Three candidate drugs - curcumin, apigenin, and tenilsetam - are proposed that could be tested in this new animal model to
Chronic Neuroinflammation in Alzheimer’s Disease New Perspectives Animal Mode...
Thesis by Andrii Kovalenko
1. Kovalenko 1
Thesis by: Andrii Kovalenko
The effect of Recombinant Adeno Associated Viral Arginase 1 in Transgenic
TetO (MAPT P301L) Responsive Mice and the Impact on Tau
Examining Committee: Leslie Sandusky, PhD1, Maj-Linda Selenica, PhD1
Thesis chair: Daniel C. Lee, PhD1
4/29/2016
1
Byrd Alzheimer's Institute, University of South Florida, Tampa, FL 33613, USA; College of
Pharmacy, Department of Pharmaceutical Sciences, University of South Florida, Tampa, FL
33612, USA.
2. Kovalenko 2
Table of Contents
Abstract
1. Introduction ………………………………………………………………………….……… 4
1.1 Focus of the project
1.2 Introduction to Dementias and Alzheimer’s Disease
1.3 L-Arginine catabolism and associated metabolic pathways
1.4 Mouse Model: tetO(MAPT*P301L)Tg
1.5 Viral Construct
1.6 Hypothesis
2. Materials and Methods ……………………………………………………………….…….. 11
2.1 Animals
2.2 Stereotaxic Surgeries
2.3 Tissue collection
2.4 Immunohistochemistry
2.5 Nissl Staining
2.6 Analysis
3. Results ……………………………………………………………………………….….….. 14
3.1 Immunostaining for green fluorescent protein (GFP)
3.2 Immunostaining for arginase 1 (ARG1) and quantification as percent area stained
3.3 Immunostaining for hemagglutinin (HA) tag and its quantification
as a percent area stained
3.4 Immunostaining for total tau (H150) and its quantification as a percent area stained
3.5 Immunostaining for pshosphorylated tau (pSer199/202) and its quantification
as a percent area stained
3.6 Immunostaining for a marker of microglial activation, Iba-1 and its quantification
as a percent area stained
3.7 Immunostaining for a marker of neuronal integrity, NeuN and its quantification
as a percent area stained
3.8 Nissl staining and quantification of hippocampal volume
4. Discussion……………………………………………………………………………….….. 22
4.1 AAV9-tTA-GFP induces expression of green fluorescent protein in nTg and
tetO(MAPT*P301L)Tg mice
4.2 AAV9-tTA-ARG1 induces expression of arginase 1 (ARG1) and hemagglutinin tag
(HA) in nTg and tetO(MAPT*P301L)Tg mice
4.3 AAV9-tTA-ARG reduces levels of total tau in tetO(MAPT*P301L)Tg mice
4.4 AAV9-tTA-ARG1 reduces levels of phosphorylated tau (pSer199/202) in
tetO(MAPT*P301L)Tg mice
4.5 AAV9-tTA-ARG1 modifies microglia activation in nTg mice, quantified by
measuring the levels of Iba-1
4.6 AAV9-tTA-GFP and AAV9-tTA-ARG1 modify levels of NeuN in nTg mice but not
in tetO(MAPT*P301L)Tg mice
4.7 Neither of treatments modified the hippocampal volume of nTg and
tetO(MAPT*P301L)Tg mice assessed by quantification of Nissl bodies
5. Conclusion……………………………………………………………………………….…..24
6. References…………………………………………………………………………………... 25
3. Kovalenko 3
Acknowledgements:
I would like to express my deep gratitude to my mentors, Dr. Daniel Lee and his post-
doctoral fellow, Dr. Leslie Sandusky for their patient guidance, encouragement and useful
critiques of this research work. I’m also very grateful to Dr. Maj-Linda Selenica, who agreed to
supervise this project. I cannot express enough my appreciation for all the learning opportunities,
provided by my committee. I’m thankful to Jerry B. Hunt, a technician in the laboratory of Dr.
Daniel Lee, for generous sharing of his knowledge and expertise.
I am grateful to my beloved parents for their untiring love and support.
4. Kovalenko 4
Abstract:
Alzheimer’s disease (AD) is the most prevalent neurodegenerative disease in the USA.
More than 95% of affected individuals are age 65 or older. With 2.5 million diagnosed cases of
AD and a least as many undiagnosed ones, it is estimated to cost the United States more than 400
billions of dollars in the form of healthcare, long-term care and unpaid man-hours of caregivers.
These numbers are expected to triple by 2050 due to the aging of the population. Therefore, it is
essential to investigate the pathogenesis and create new therapeutic approaches to this
economically and socially devastating disease (Alzheimer's, 2014).
Arginine is a semi-essential amino acid catabolism of which is the center of synthesis of
nitric oxide (NO) and polyamines. Among known functions of arginine are effects on vascular
health and oxidative stress via NO, activation of microglial cells and astrocytes, regulation of
inflammation, modulation of glucose metabolism and influence on tissue repair and cellular
proliferation via polyamines (Yi et al., 2009). Evidently, many of these processes, as well as
disturbances and alterations in L-arginine metabolism influence progression of Alzheimer’s
disease (Yi et al., 2009). This project aids to the growing knowledge about L-arginine
metabolism and demonstrates that it is a viable target for novel therapeutic approaches to the
Alzheimer’s disease.
The goal of this project was the identification of the effects of overexpression of Arg1 in
hippocampal neurons using a bilateral intracranial hippocampal injection of adeno-associated
virus serotype 9 (rAAV9) carrying a tetracycline-controlled transactivator protein (tTA) inducing
tau pathology in the tetO(MAPT*P301L)Tg mice. This project is focused on the
immunohistochemical analysis of several hallmark proteins and markers in an animal model of
Alzheimer’s disease. Histological experiments included immunohistochemical stains for green
fluorescent protein (GFP), arginase 1 (ARG1), total tau (H150), phosphorylated tau
(pSer199/202), hexaribonuceotide binding protein (NeuN), ionized Calcium-binding adapter
molecule 1 (IBA1), hemagglutinin (HA) tag on viral arginase 1, and Nissl staining.
5. Kovalenko 5
1. Introduction:
1.1 Focus of the project.
The goal of this project was the identification of the effects of overexpression of Arg1 in
hippocampal neurons using a bilateral intracranial hippocampal injection of adeno-associated
virus serotype 9 (rAAV9) carrying a tetracycline-controlled transactivator protein (tTA) inducing
tau pathology in the tetO(MAPT*P301L)Tg mice. This project is focused on the
immunohistochemical analysis of several hallmark proteins and markers in an animal model of
Alzheimer’s disease. Histological experiments included immunohistochemical stains for green
fluorescent protein (GFP), arginase 1 (ARG1), total tau (H150), phosphorylated tau
(pSer199/202), hexaribonuceotide binding protein (NeuN), ionized Calcium-binding adapter
molecule 1 (IBA1), hemagglutinin (HA) tag on viral arginase 1, and Nissl staining.
1.2 Introduction to dementias and Alzheimer’s disease.
Dementia is a broad term, for diseases that are primarily characterized by decline in
memory, language, learning or other cognitive abilities that affect the individual’s capability to
perform every-day tasks (Alzheimer's, 2014). Tauopathies comprise of more than 20
neurodegenerative diseases (Berger et al., 2007) including Alzheimer’s disease (AD) that
accounts for 60-80% of progressive neurodegenerative disorders in elderly patients (Alzheimer's,
2014). About 5.3 million people are thought to be currently affected by AD in the US with half
of them being undiagnosed (Alzheimer's, 2015). By 2050, the number of affected people is
thought to triple due to the aging of the US population. In 2014 total annual payments for health
care services were estimated to be more than $226 billion. The contribution of family members’
care and volunteers was evaluated as more than $220 billion (Alzheimer's, 2015). It is clear that
Alzheimer’s disease is of great economic importance and extensive research on pathogenesis and
development of new approaches to treatment and prevention of AD is essential today to avoid
enormous monetary and human expenses in the future.
Neurodegeneration in AD is associated with aggregates of erroneously cleaved amyloid
precursor protein (APP), resulting in so-called β-amyloid plaques outside neurons, and
accumulation of hyper-phosphorylated tau, microtubule-stabilizing protein, forming
neurofibrillary tangles (NFTs) inside neurons (Nagy et al., 1998). Although advancements in the
field have been made, the pathogenesis of AD still appears unclear as new risk factors and gene
variants emerge (Mudher & Lovestone, 2002). In recent years, the role of immunity in the
development of AD has been widely investigated. Recent findings suggest that local immune-
mediated amino acid catabolism is a possible mechanism of aging-associated neurodegeneration
(Kan et al., 2015; Potenza, Nacci, & Mitolo-Chieppa, 2001; Yi et al., 2009).
1.3 L-Arginine metabolism and its implications in Immune Responses.
L-arginine is a semi-essential amino acid that plays a central role in numerous metabolic
activities. (Figure 1.) L-arginine is a major substrate of several competing metabolic pathways:
the nitric oxide synthase (NOS) pathway that leads to production of nitric oxide (NO) and
citruline, and the arginase (ARG) pathway, essential for synthesis of polyamines (PAs) (Kan et
al., 2015; Vural, Sirin, Yilmaz, Eren, & Delibas, 2009; Yi et al., 2009).
6. Kovalenko 6
Figure 1. From "L-arginine and Alzheimer's disease." (Yi et al., 2009).
NOS, Nitric oxide synthase exists in three known isoforms: neuronal (nNOS), inducible (iNOS)
and endothelial (eNOS). Arginase exists in two known isoforms: ARG1 and ARG2. ODC,
ornithine decarboxylase; MTA, methylthioadenosine; D-SAM, decarboxylase SAM.
Nitric Oxide (NO) is an important metabolic factor. It is a vasodilator, an important
neurotransmitter and has been suggested to play an important role in long-term potentiation
(LTP) and long-term depression (LTD) electrophysiology models of learning and cognition
(Vural et al., 2009). L-arginine and NO have been associated with atherosclerosis and platelet
formation (Yi et al., 2009) and some researchers proposed that AD is primarily a neurovascular
disease (de la Torre, 2002; Yi et al., 2009). L-arginine and NO may play a role in oxidative
stress, although the nature of this role remains a subject of controversy (Yi et al., 2009). Some
researchers have argued that NO can serve as a free radical and results in generation of reactive
oxygen species (ROS) such as peroxynitrite, which at high concentrations can be broken in more
ROS (NO2+, NO, OH), also able to induce significant oxidative stress (Corzo, Zas, Rodriguez,
Fernandez-Novoa, & Cacabelos, 2007; Malinski, 2007). Overproduction of NO is thought to
have the potential to induce cytotoxicity (Law, Gauthier, & Quirion, 2001). However, some
research suggests that NO also has a protective effect against reactive oxygen species as H2O2
and superoxide, and low levels of NO may cause cerebrovascular intracytoplasmic tissue
damage, disrupting mitochondrial metabolism and resulting in AD (de la Torre & Stefano, 2000).
Conversely, significantly higher total nitrite concentration and much lower arginase
concentration were found in patients with AD (Vural et al., 2009).
L-arginine has repeatedly been shown to play an important role in macrophage
cytotoxicity and inflammation (Hibbs, Taintor, & Vavrin, 1987). The polarized activation state
of macrophages (M1 or M2) is largely determined by the prevalent mode of arginine metabolism
in an evolving immune response (Mills, 2012; Rath, Muller, Kropf, Closs, & Munder, 2014).
Macrophages exhibiting classical (M1) activation state are characterized by expression of NOS
converting arginine into nitric oxide (NO) that can be metabolized further to form reactive
7. Kovalenko 7
nitrogen species (Rath et al., 2014). M1 macrophages have the main function of killing/fighting
(Mills, 2012). Macrophages exhibiting alternative (M2) activation show increased expression of
Arg that competes with NOS for L-arginine (Rath et al., 2014). These macrophages and PAs and
proline pathways downstream of the ARG pathway have the main function of healing/repairing
and were shown to play an important role in cellular proliferation (Mills, 2012; Rath et al.,
2014). With increasing age, more microglia appear to be classically activated (M1) than
alternatively activated (M2) (Lee et al., 2013). Recent results from untargeted metabolomic
analysis of human plasma indicate that subjects with mild cognitive impairment (MCI) had a
greater chance of conversion of MCI to AD when impairments of L-arginine and PA metabolic
pathways were also present (Graham et al., 2015). Altered prostaglandin biosynthesis in patients
with MCI who converted to AD may indicate an inflammatory response (Graham et al., 2015).
Polyamines (PAs), putrescine, spermidine, and spermine are downstream metabolites of
L-arginine through arginase and ornithine decarboxylase (ODC) (Liu, Gupta, Jing, & Zhang,
2008). These organic cations can be biosynthesized by mammalian cells or gut microbiota, or
ingested with food (Pegg, 2009). Primary and secondary amino groups of these organic aliphatic
molecules are fully protonated at physiological pH and give PAs their positive charge (Liu et al.,
2008). PAs have a wide variety of functions due to electrostatic attraction to negatively charged
molecules including acidic proteins, nucleic acids (DNA and RNA) and phospholipids (Park &
Igarashi, 2013). Among the most important functions of PAs are regulation of gene expression
on the levels of transcription (binding to DNA and affecting its structure), translation (binding to
mRNAs, ribosomes), and regulation of posttranslational modifications (Pegg, 2009). Because the
spectrum of PAs functions is so broad, virtually all metabolic reactions are affected directly or
indirectly by them (Pegg, 2009). This is the reason for strict regulation of PAs via biosynthesis,
efflux, catabolism and uptake, controlled by various feedback mechanisms that maintain the
intracellular concentration of PAs within a relatively narrow range (Park & Igarashi, 2013; Pegg,
2009).
PAs are essential for cellular proliferation (Minois, Carmona-Gutierrez, & Madeo, 2011).
Disruptions of PAs biosynthesis result in defective neurons, as in case with the Snyder-Robinson
syndrome, a developmental disease in which mutant gene encoding for spermine synthase
manifests, in mental retardation, skeletal defects, facial asymmetry etc. (Pegg, 2009). PAs have
been shown to play a central role in sustaining neuronal progenitor cells (NPC) proliferation in
the subgranular cell layer (SCL) of the dentate gyrus (DG) and sub-ventricular zone (SVZ), the
only two regions where neurogenesis is thought to happen in the adult brain (Malaterre et al.,
2004). Multiple earlier studies have shown aging-related alterations in ODC activity and levels
of polyamines in human’s and model animals’ CNS (Morrison, Becker, Ang, & Kish, 1995;
Morrison & Kish, 1995; Virgili, Necchi, Scherini, & Contestabile, 2001; Vivo et al., 2001).
These findings have led to a suggestion that ODC/PA metabolism alterations are part of the
normal process of aging and the aging-related decline in cognitive abilities and neurogenesis that
is seen across species (Liu et al., 2008). Aging has been shown to have an effect on ODC/PA
metabolic pathways in neuronal circuits associated with learning and memory (Liu et al., 2008).
Notably, different regions of the hippocampus, CA1, CA3, and DG, that play different roles in
memory processing have been shown to have different alterations of polyamine metabolic
pathways (Liu et al., 2008).
Disruption of the circadian clock, manifesting in disrupted sleep/wake cycles are the
major cause of institutionalization of patients with AD (Duncan et al., 2012). Alterations in
circadian neural oscillators within the hypothalamic suprachiasmatic nucleus (SCN) are likely to
8. Kovalenko 8
be responsible for these disruptions. Recent research provided evidence for interplay between
the circadian rhythms, feeding-dependent mechanisms and polyamine metabolism (Zwighaft et
al., 2015). Polyamines have been shown to play an important role in the regulation of circadian
rhythms in the suprachiasmatic nucleus, residing in the hypothalamus and to modulate the
interaction between PER2 and CRY1, crucial for circadian oscillations (Zwighaft et al., 2015).
Polyamine metabolism and related enzymes have been shown to exhibit diurnal rhythmicity and
an association between the age-related decline in polyamine levels and longer circadian periods
has been detected.(Zwighaft et al., 2015). Dietary PA supplementation restored the length of
circadian period back to normal (Zwighaft et al., 2015).
Calorie restriction is well known for increasing longevity, promoting healthy aging,
delaying brain senescence and delaying neurodegeneration (Delic et al., 2015; Fusco et al., 2012;
Wang et al., 2015). Cyclic adenosine monophosphate is an important secondary messenger that
was shown to delay aging effects and improve ageing-associated phenotypes in aged mice,
mimicking the effects of calorie restriction (Wang et al., 2015). Arginase 1 and its products, PAs
were shown to act downstream from cAMP and induce axonal growth (Cai et al., 2002). Direct
inhibition of PAs synthesis blocked the ability of cAMP to overcome inhibition by MAG that
prevents axonal regeneration in adult neurons (Cai et al., 2002). More recent research results
suggest that spermidine increasing longevity through autophagy (Eisenberg et al., 2009).
Autophagy is an essential mechanism of disposal of unwanted or damaged molecules and
organelles. Upregulation of autophagy was shown to result in an increase of life span (Minois et
al., 2011). Arginase 1 and PAs, therefore, have the potential to become the therapeutic agent that
is more specific than cAMP in mimicking beneficial effects of caloric restriction without its
setbacks (Cai et al., 2002). In mice, chronic supplementation of diet with spermidine has been
shown to promote longevity (de Cabo, Carmona-Gutierrez, Bernier, Hall, & Madeo, 2014).
Epigenetic changes leading to deacetylation of histones, resulting in reduction of ROS, necrosis
and increased autophagy likely account for beneficial effects of spermidine (Eisenberg et al.,
2009). Alternatively, the interaction between PAs and the circadian clocks described earlier may
be at least partially responsible for this effect (Zwighaft et al., 2015).
In the mouse model of tauopathy Tg4510 expressing the P301L mutant form of human
tau, caloric restriction didn’t have consistent effects on tau deposition and didn’t rescue increased
activation of astrocytes and microglia (Brownlow et al., 2014). Neither did it restore the
functions of mitochondria in the brain (Delic et al., 2015). Caloric restriction seems to improve
short-term memory in Tg4510 (Brownlow et al., 2014).
Inhibition of arginase, which converts L-arginine into L-ornithine, and ornithine
decarboxylase (ODC), which converts L-ornithine into putrescine, protects (CVN-AD) mice
from AD-like pathology (Kan et al., 2015). CVN-AD mice model is claimed to display the
cardinal characteristics of AD progression, including amyloid-beta plaques, phosphorylated tau
protein, significant death of neurons in hippocampus, spatial memory impairments and marked
inflammatory component (Hoos et al., 2013). The spatial correlation of Arg1 localization with
that of Aß, Iba-1 and CD11c have lead to the suggestion that CD11c+ microglia are the likely
source of arginase-1 production that was implicated in the development of AD-like pathology in
CVN-AD mice. Supposedly, an accumulation of arginine-1 resulted in depletion of tissue
arginine (Kan et al., 2015)that lead to amino acid depletion responses in susceptible cells, that
are known to result in cell death when arginine deprivation is sustained (Kuma & Mizushima,
2010). These results suggest that overexpression of neuronal cytosolic arginase (ARG1; ARG2 is
an isoform of ARG found in mitochondria) can be used to outcompete the increased
9. Kovalenko 9
consumption of arginine by activated microglia and increase the bioavailability of arginine for
neurons, preventing neuronal death and development of AD-like pathology. More
conventionally, microglia activation and its role in plaque maintenance and periplaque
neuropathology were thought to be a secondary process developing later during disease
progression (Wyss-Coray & Rogers, 2012). Most recent research demonstrates a region-specific
increase of phagocytic microglia in pre-plaque brains (Hong et al., 2016), suggesting that
synapse loss, a major correlate of cognitive decline in AD (Mucke & Selkoe, 2012), can be
mediated early through microglia and immune-related pathways (Hong et al., 2016).
1.4 Mouse Model: tetO(MAPT*P301L)Tg
The tau protein is encoded by a single gene on chromosome 17q21 in humans (Neve,
Harris, Kosik, Kurnit, & Donlon, 1986). P301L mutation on MAPT reduces binding of tau to
microtubules and increases the aggregation of the abnormal form of tau with phosphorylated
serine 202 (Fontaine et al., 2015). Bigenic mice rTg4510 expressing P301L mutant MAPT that
can be suppressed by doxycycline (Dox) have lead to several advances in the field as showing
that NFTs are not sufficient to cause neuronal death and cognitive decline (Santacruz et al.,
2005). TetO(MAPT*P301L)Tg mice used in this experiment allow Tet-on/Tet-off expression of
P301L human tau driven by virally expressed tetracycline-controlled transactivator protein (tTA)
and are useful in generating animals with AD-like pathology.
1.5 Virus
In E.coli, the Tet repressor protein (TetR) regulates the gene of the tetracycline resistance
operon, binding to the tet operator sequences (tetO). Tetracycline-controlled transactivator
protein (tTA) is a 37kDa protein that is a fusion of TetR and the Herpes simplex virus VP16
activation domain (Triezenberg, Kingsbury, & McKnight, 1988), an addition of which converts
TetR into Tet activator. Adeno-associated viral vectors are widely used in research, pre-clinical
and clinical gene delivery studies, as they can induce a long-term stable gene expression without
increased inflammation or cytotoxicity (Gray et al., 2011).
With the use of adeno-associated viral (AAV) vectors, packaging of compact promoters
becomes critical (Gray et al., 2011). The CBA promoter is a hybrid CMV enhancer/Chicken β-
actin (CBA) promoter provides cell-specific gene expression in the higher neurons of the central
nervous systems, but much lower in motor neurons (Gray et al., 2011).
Combination of the power of currently available transgenic tetracycline mouse model of
human tau (tetO(MAPT*P301L)Tg) with flexibility of viral transduction and a second transgene
(e.g. GFP, or ARG1-HA) provides a few significant advantages (Hunt et al., 2015):
1. Regional control over the expression of MAPT P301L and therapeutic protein by selection of
the serotype of AAV, cell specificity of the used promoter and the injection site.
2. Temporal control over the expression of MAPT P301L by choosing the time of tetO regulated
gene expression and addition or subtraction of doxycycline from rodent’s diet.
3. Ability to monitor the pathological accumulation of tau and/or it’s spreading from neurons in
which the pathology was activated by tTA carried by the rAAV construct.
4. Activation of pathology and expression of therapeutic protein happens in the same neurons,
infected by AAV.
10. Kovalenko 10
For this study, tTA2 cDNA was cloned downstream of the minimal CBA promoter
followed by an SV40 PolyA and TetO with minimal CMV promoter followed by either the gene
coding for arginine 1 fused with hemagglutinin or by the gene coding for the green fluorescent
protein (GFP) (Figure 2). (tTA and tetO promoter were generously provided by Ronald J.
Mandel from the University of Florida). AAV serotype 9 was used because it is known for
providing larger distribution areas than other serotypes (Carty et al., 2010).
Figure 2. Schematic representation of [A] the bicistronic rAAV9 construct and regulation of
TetO-driven genes: Arg1-HA, GFP and [B] MAPT*P301L transgene. TR2, Terminal repeat
(TR) of AAV type 2; CBA, hybrid Cytomegalovirus enhancer/chicken β-actin promoter; TetR,
Tet repressor protein; AV16, Herpes simplex virus activation domain; tTA, tetracycline-
controlled transactivator protein; SV40 polyA, Seminal virus 40 AATAAA hexanucleotide
11. Kovalenko 11
polyadenylation signal; TetO, tet operator sequence; pMin ΔCMV, minimal promoter
Cytomegalovirus delta. ARG1-HA, the gene encoding for arginine 1 with hemagglutinin tag on
it; GFP, green fluorescent protein; BGH Poly A, bovine growth hormone polyadenylation
sequence; MAPT*P301L, human tau gene bearing the Pro to Leu mutation at codon 301 of tau.
1.6 Hypothesis
Previous research has shown that adenoviral expression of ARG1 in the CNS of Tg4510
mice significantly reduces deposition of phosphorylated tau species and tangle pathology,
possibly through modulation of several kinases capable of phosphorylating human tau, decreased
inflammation and activation of autophagy (Hunt et al., 2015).
It was hypothesized that overexpression of AAV9-tTA-ARG1 impacts tau levels,
inflammation, and neuronal integrity in hippocampi of tetO(MAPT*P301L)Tg mice, compared
to AAV9-tTA-GFP and AAV9-tTA-Empty Capsid.
2. Materials and Methods
Protocols for histological experiments, tissue mounting, scanning and analyzing were obtained
from Dr. Daniel Lee and Dr. Leslie Sandusky.
2.1 Animals.
Twenty non-transgenic and 20 hemizygous tetO(MAPT*P301L)Tg mice were
generously provided by Dr. Chad Dickey from the Department of Molecular Medicine in the
Byrd Alzheimer’s Institute. The Institutional Animal Care and Use Committee of the University
of South Florida approved all protocols for experiments on animals. Mice were kept at standard
vivarium conditions with the twelve-hour light/dark cycle and given ad libitum access to food
and water.
2.2 Stereotaxic Surgeries.
After full anesthesia with isofluorane, the position of mice was fixed in a stereotaxic
frame. Both non-transgenic and tetO(MAPT*P301L)Tg mice received a bilateral intracranial
convection enhanced delivery (C.E.D.) of virus in the dentate gyrus (DG) area of the
hippocampus (HPC). Following coordinates from bregma were used: anterior/posterior (AP): -
2.7mm, medial/lateral (ML): ±2.7mm, dorsal/ventral (DV): -3.0mm. A 10uL Hamilton Syringe
was used to deliver a 2uL per site injection of a solution containing either AAV9-Empty Capsid
or AAV9-tTA-GFP (Green Fluorescent Protein) or AAV9-tTA-Arg1 (Arginase 1) at a constant
flow rate of 1.5uL/min.
12. Kovalenko 12
Group N Genotype Treatment
1 5 nTg AAV9-EC
2 6 nTg AAV9-tTA-GFP
3 7 nTg AAV9-tTA-ARG1
4 5 tetO(MAPT*P301L)Tg AAV9-Empty Capsid
5 8 tetO(MAPT*P301L)Tg AAV9-tTA-GFP
6 7 tetO(MAPT*P301L)Tg AAV9-tTA-ARG1
Table 1: Animals grouped by genotype and by treatment. Three groups of
tetO(MAPT*P301L)Tg mice received bilateral intracranial C.E.D. of either AAV9-tTA-Empty
Capsid, or AAV9-tTA-GFP (green fluorescent protein), or AAV9-tTA-ARG1 (arginase 1).
Treatments were repeated in three control groups of non-transgenic mice, resulting in six groups
total. N - animals per group.
2.3 Tissue collection.
Following a viral incubation of four months, mice received a lethal injection of SomnaSol
(024351, Henry Schein) and were transcardially perfused with 0.9% saline solution. Brains were
quickly harvested and separated into two halves. One half was dissected into 8 regions: anterior
cortex, prefrontal cortex, hippocampus, striatum cortex, thalamus, substantia nigra, cerebellum
and “rest of brain” to be used for future studies. This half will be used for analyses using
standard western blots techniques and are out of the scope of this project. Another half was fixed
in 4% Paraformaldehyde solution (pH 7.4) for 24 hours to be later analyzed by conventional
immunohistochemical techniques and staining for Nissl bodies. Following the fixation step,
tissue was stored at 4°C in Dulbecco’s Phosphate Buffered Saline with Sodium Azide
(DPBS+Azide, pH 7.4) for period of 9 months before being cryoprotected and sectioned.
Following recipe of DPBS+Azide solution was used: 137mM NaCl (S640-3, Fisher Scientific),
8mM Na2HPO4 (S374-1, Fisher Scientific), 1.47mM KH2PO4 (00746, Chem-Impex Intl., Inc.),
268nM KCl (P954-1, Sigma Aldrich), 1.2mM CaCl2•2H2O (C3306-500, Sigma Aldrich), 246nM
of MgCl2•6H2O (M2670-500, Sigma Aldrich), 108mM sodium azide (BP922I-500, Fisher
Scientific) in 18.2MΩ-cm pure water purified with PURELAB Ultra (ELGA).
2.4 Immunohistochemistry.
Hemispheres selected for immunohistochemical experiments were cryoprotected by
submerging into 30% sucrose solution for 3 days, and then horizontally sectioned using a sliding
microtome into 25um thick sections for free-floating immunohistochemistry. Every twelve’s
section was cut 50um thick to be used for the Nissl staining. Tissue was stored at 4°C in
DPBS+Azide (pH 7.4)
Standard free-floating immunohistochemistry procedures were used to immunostain
25um thick sections equally spaced at 300um apart. Six sections per mouse were immunostained
with each of the following antibodies: chicken anti-GFP (ab13970, Abcam), rabbit anti-tau H150
(sc-5587, Santa Cruz Biotechnology), chicken anti-ARG1 (A generous gift of Sidney Morris
from University of Pittsburg), rabbit anti-tau pSer199/202 (54963-025, AnaSpec), anti-HA
biotinylated (13636200, Roche), NeuN (ABN78, Millipore), Iba-1 (019-19741, Wako). Free-
floating sections were incubated in primary antibody overnight at room temperature, then
13. Kovalenko 13
incubated in matching secondary antibody (VectorLabs) for 2 hours (this step was skipped when
primary antibodies were biotinylated), and then incubated for 1 hour in avidin-biotin complex
reagent (PK4000, VectorLabs). Color development was done using 3,3’-Diaminobenzidine
(D5673-25G, Sigma) enhanced with nickelous ammonium sulfate (N48-500, Fisher Scientific).
Stained tissue was mounted on glass slides and dehydrated by sequential submerging in 25%,
50%, 75%, and 100% ethanol. Then tissue was cleared with Histo-Clear (HS-200, National
Diagnostics) and glass coverslipped with DPX (360294H, VWR) mounting media. Microscope
slides with stained tissue sections were scanned with a Zeiss Mirax-Scan 150 slide scanner.
Scans were analyzed using methods described in section 2.6 Analysis, and positive signal was
quantified as percent area stained.
2.5 Nissl Staining.
50um thick brain sections equally spaced at 275um apart were selected and mounted on glass
slides and air-dried. After rehydrating tissue by 10 quick dips in millipure H2O, sections were
stained with 0.05% cresyl violet solution for 7 minutes. Then tissue was destained by with 30
quick dips in acidic water (6 drops of 17.5M glacial acetic acid per 200mL of H2O) and rinsed
with millipure H2O. After dehydration by sequential submerging in 75%, 95% (111000190,
Pharmco-AAPER) and 100% ethanol (111000200, Pharmco-AAPER) for 1 minute each,
sections were cleared with Histo-Clear (HS-200, National Diagnostics) and glass coverslipped
with DPX mounting media (360294H, VWR). Slides were scanned with a Zeiss Mirax-Scan 150
slide scanner and hippocampal regions were analyzed using methods described in section 2.6
Analysis. Group differences in staining were analyzed using SPSS statistical software (Ver. 23,
IBM) using one-way ANOVA.
2.6 Analysis.
Microscope slides with stained tissue sections were scanned with a Zeiss Mirax-Scan
150 slide scanner. Analysis was performed with IAE+NearCYTE WSI Analysis software (Ver.
1.9.2.5, Created by Andrew Lesniak). Positive staining was quantified by HSV (hue, saturation,
value) segmentation as percent area. Methods of unbiased stereology were used to quantify the
hippocampal volumes with assistance from the Stereologer system (Tampa-St. Petersburg, FL).
The hippocampal volume (mm^3) was estimated using the Cavalieri-point counting method
(Mouton, 2011). All values obtained from a single mouse brain (6 sections per animal) were
averaged. One-way ANOVA followed by Fisher’s PLSD post hoc were performed using SPSS
statistics software (Ver. 23, IBM). Group differences with p<0.05 were considered to be
statistically significant. Graphs were generated using GraphPad Prism (Ver. 5.01, LaJolla). One
animal from group 3 (nTg mouse injected with AAV9-tTA-ARG1) was excluded from analysis
of immunostaining for ARG1 as an outlier.
14. Kovalenko 14
3. Results
3.1 Immunostaining for green fluorescent protein (GFP).
Figure 3. Analysis using a one-way ANOVA revealed a significant effect of group on
green fluorescent protein (GFP) [F(5, 32) = 1.545, p<0.001]. [B, A, D] Post hoc analyses using
Fisher PLSD revealed a significant difference in the amount of detected green fluorescent protein
(GFP) between non-transgenic (nTg) mice that received intracranial C.E.D. of AAV9-tTA-GFP
(M=0.951, SD=0.569) and nTg mice that received AAV9-Empty Capsid (M=0.008; SD<0.001),
as well as compared to nTg mice injected with AAV9-tTA-ARG1 (M=0.015, SD=0.009). [E, D,
F] Fisher PLSD post hoc analysis also revealed that in tetO(MAPT*P301L)Tg mice, injection
with AAV9-tTA-GFP lead to significantly higher expression of GFP (M=0.947, SD=0.66)
compared to injection with AAV9-Empty Capsid (M=0.011, SD=0.003) as well as compared to
injection with AAV9-tTA-ARG1 (M=0.019, SD=0.020).
[B, E] Importantly, there was no significant difference in GFP expression between
tetO(MAPT*P301L)Tg and nTg mice that received intracranial C.E.D. of AAV9-tTA-GFP.
*p<0.05.
nTg tetO(MAPT*P301L)Tg
0.0
0.5
1.0
1.5
2.0
AAV9-Empty Capsid
AAV9-tTA-GFP
AAV9-tTA-ARG1
GFP
* * * *
Genotype
%Area+SD
15. Kovalenko 15
3.2 Immunostaining for arginase 1 (ARG1) and quantification as percent area stained.
Figure 4. Analysis using a one-way ANOVA revealed a significant effect of group on
expression of ARG1 [F(5, 31) =4.564, p=0.003]. [D, A, B] Post hoc analyses using Fisher PLSD
revealed a significant difference in the amount of detected ARG1 between nTg mice that
received intracranial C.E.D. of AAV9-tTA-ARG1 (M=0.0.435, SD=0.327) and nTg mice that
received AAV9-Empty Capsid (M=0.003; SD<0.004), as well as compared to nTg mice injected
with AAV9-tTA-GFP (M=0.000, SD=0.000). [F, D, E] Fisher PLSD post hoc analysis revealed
that in tetO(MAPT*P301L)Tg mice, injection with AAV9-tTA-ARG1 lead to significantly
higher expression of GFP (M=0.228, SD=0.372) compared to injection with AAV9-Empty
Capsid (M=0.001, SD=0.001) as well as compared to injection with AAV9-tTA-GFP (M=0.005,
SD=0.016).
[D, F] No statistically significant difference was observed between tetO(MAPT*P301L)Tg and
nTg animals injected with AAV9-tTA-ARG1. However, a trend of reduction in ARG1
expression in tauopathy model was observed compared to wild type-mice (p=0.088). *p<0.05.
nTg tetO(MAPT*P301L)Tg
0.0
0.2
0.4
0.6
0.8
1.0
AAV9-Empty Capsid
AAV9-tTA-GFP
AAV9-tTA-ARG1
Arginase (ARG1)
*
*
*
*
Genotype
%Area+SD
16. Kovalenko 16
3.3 Immunostaining for hemagglutinin (HA) tag and its quantification as a percent area
stained.
Figure 5. Analysis using a one-way ANOVA revealed a significant effect of group on
expression of hemagglutinin [F(5, 32) =5.033, p=0.002]. [D, A, B] Post hoc analyses using
Fisher PLSD revealed a significant difference in the amount of detected HA tag between nTg
mice that received intracranial C.E.D. of AAV9-tTA-ARG1 (M=0.0.435, SD=0.327) and nTg
mice that received AAV9-Empty Capsid (M=0.003; SD<0.004), as well as compared to nTg
mice injected with AAV9-tTA-GFP (M=0.000, SD=0.000). [F, D, E] Fisher PLSD post hoc
analysis revealed that in tetO(MAPT*P301L)Tg mice, injection with AAV9-tTA-ARG1 lead to
significantly higher expression of GFP (M=0.228, SD=0.372) compared to injection with AAV9-
Empty Capsid (M=0.001, SD=0.001) as well as compared to injection with AAV9-tTA-GFP
(M=0.005, SD=0.016).
[D, F] No statistically significant difference in % area for HA was observed
tetO(MAPT*P301L)Tg and nTg animals injected with AAV9-tTA-ARG1. However, a trend of
reduction in HA expression in tetO(MAPT*P301L)Tg was observed compared to nTg mice
(p=0.068). *p<0.05.
nTg tetO(MAPT*P301L)Tg
0.0
0.1
0.2
0.3
0.4
0.5
AAV9-Empty Capsid
AAV9-tTA-GFP
AAV9-tTA-ARG1
HA biotin
*
*
*
*
Genotype
%Area+SD
17. Kovalenko 17
3.4 Immunostaining for total tau (H150) and its quantification as a percent area stained.
Figure 6. Analysis using a one-way ANOVA revealed a significant effect of group on the
expression of total tau (H150), [F(5, 32) =13.917, p=0.000]. [A, B, C] Post hoc Fisher PLSD
analysis revealed no significant difference between nTg animals injected with AAV9-Empty
Capsid (M=0.0052, SD=0.003), AAV9-tTA-GFP (M=0.031, SD=0.061) and AAV9-tTA-ARG1
(M=0.006, SD=0.005). [D, E] Levels of detected H150 were significantly higher in
tetO(MAPT*P301L)Tg mice injected with AAV9-tTA-GFP (M=0.636, SD=0.334) than in mice
of the same genotype injected with AAV9-Empty Capsid (M=0.033, SD=0.026). [E, F] Levels
of H150 were significantly lower in tetO(MAPT*P301L)Tg mice injected with AAV9-tTA-
ARG1 (M=0.186, SD=0.199) compared to tetO(MAPT*P301L)Tg mice that received AAV9-
tTA-GFP (M=0.636, SD=0.334). [D, F] Difference in H150 expression was between
tetO(MAPT*P301L)Tg mice injected with AAV9-Empty Capsid and AAV9-tTA-ARG1 found
to be statistically insignificant as analyzed by post hoc Fisher PLSD (p=0.159). *p<0.05.
nTg tetO(MAPT*P301L)Tg
0.0
0.2
0.4
0.6
0.8
1.0
AAV9-Empty Capsid
AAV9-tTA-GFP
AAV9-tTA-ARG1
Total tau (H150)
* *
*
Genotype
%Area+SD
18. Kovalenko 18
3.5 Immunostaining for pshosphorylated tau (pSer199/202) and its quantification as a percent
area stained.
Figure 7. Analysis using a one-way ANOVA revealed a significant effect of group on
expression of phosphorylated tau (pSer199/202), [F(5, 32) =11.931, p=0.000]. [A, B, C] Post hoc
Fisher PLSD analysis revealed no significant difference between nTg animals injected with
AAV9-Empty Capsid (M=0.005, SD=0.004), AAV9-tTA-GFP (M=0.005, SD=0.005) and
AAV9-tTA-ARG1 (M=0.015, SD=0.025). [D, E] Levels of detected pSer199/202 were
significantly higher in tetO(MAPT*P301L)Tg mice injected with AAV9-tTA-GFP (M=0.098,
SD=0.048) than in mice of the same genotype injected with AAV9-Empty Capsid (M=0.012,
SD=0.010). [E, F] Levels of pSer199/202 were significantly lower in tetO(MAPT*P301L)Tg
mice injected with AAV9-tTA-ARG1 (M=0.044, SD=0.029) compared to
tetO(MAPT*P301L)Tg mice that received AAV9-tTA-GFP (M=0.098, SD=0.048). [D, F]
Difference in pSer199/202 expression between tetO(MAPT*P301L)Tg mice injected with
AAV9-Empty Capsid (M=0.012, SD=0.010) and AAV9-tTA-ARG1 (M=0.044, SD=0.029)
found to be statistically insignificant, however a trend of an increase in phosphorylated tau was
observed as analyzed by post hoc Fisher PLSD (p=0.058). *p<0.05.
nTg tetO(MAPT*P301L)Tg
0.00
0.05
0.10
0.15
0.20
AAV9-Empty Capsid
AAV9-tTA-GFP
AAV9-tTA-ARG1
*
Phosphorylated tau (pSer199/202)
*
*
Genotype
%Area+SD
19. Kovalenko 19
3.6 Immunostaining for a marker of microglial activation, Iba-1 and its quantification as a
percent area stained.
Figure 8. There was no statistically significant difference between groups as determined
by one-way ANOVA [F(5,32)=1.48, p=0.224]. [A, C] The Fisher PLSD post hoc analysis
determined that levels of detected Iba-1 were significantly lower in the group of nTg mice
injected with AAV9-Empty Capsid (M=4.359, SD=1.029) compared to the group of nTg mice
injected with AAV9-tTA-ARG1 (M=5.590, SD=1.105). [D, F] There was no significant
difference detected in groups of tetO(MAPT*P301L)Tg mice injected with AAV9-Empty Capsid
(M=5.211, SD=0.619) and with AAV9-tTA-ARG1 (M=4.915, SD=0.872). *p<0.05.
nTg tetO(MAPT*P301L)Tg
0
2
4
6
8
AAV9-Empty Capsid
AAV9-tTA-GFP
AAV9-tTA-ARG1
*
Microglia Activation (Iba-1)
Genotype
%Area+SD
20. Kovalenko 20
3.7 Immunostaining for a marker of neuronal integrity, NeuN and its quantification as a
percent area stained.
Figure 9. Analysis using a one-way ANOVA didn’t reveal a statistically significant
between-group differences in neuronal integrity, assessed by quantification of NeuN as a
positive % area stained [F(5,32)=2.312, p=0.067]. [A, B, C] Further analysis, using post hoc
Fisher PLSD revealed significant decrease in positive % area stained for NeuN in nTg mice
injected with AAV9-Empty Capsid (M=5.604, SD=1.529), compared to AAV9-tTA-GFP
(M=3.499, SD=0.625) and to AAV9-tTA-ARG1 (M=4.274, SD=1.596). [A, D] Levels of
detected NeuN protein were significantly lower in tetO(MAPT*P301L)Tg mice that received an
injection with AAV9-Empty Capsid (M=3.466, SD=1.81), compared to nTg mice that received
AAV9-Empty Capsid injection (M=5.604, SD=1.529). [D, E, F] No statistically significant
difference was found between tetO(MAPT*P301L)Tg animals injected with AAV9-Empty
Capsid (M=3.466, SD=1.81), AAV9-tTA-GFP (M=4.217, SD=1.134), AAV9-tTA-ARG1
(M=3.218, SD=1.250). *p<0.05.
nTg tetO (MAPT P301L)Tg
0
2
4
6
8
AAV9-Empty Capsid
AAV9-tTA-GFP
AAV9-tTA-ARG1
NeuN
*
*
*
Genotype
%Area+SD
21. Kovalenko 21
3.8 Nissl staining and quantification of hippocampal volume.
nTg tetO(MAPT*P301L)Tg
0.0
0.1
0.2
0.3
0.4
AAV9-Empty Capsid
AAV9-tTA-GFP
AAV9-tTA-ARG1
Nissl
Genotype
Volume(mm^3)+SD
Figure 10. There was no statistically significant difference between groups as determined
by one-way ANOVA [F(5,32)=0.857, p=0.52]. Post hoc analyses using Fisher PLSD didn’t
reveal any significant difference in volume of Nissl bodies between groups. *p<0.05
22. Kovalenko 22
4. Discussion.
4.1 AAV9-tTA-GFP induces expression of green fluorescent protein in nTg and
tetO(MAPT*P301L)Tg mice.
Immunohistochemical stain of brain sections for the green fluorescent protein (GFP) and
quantification of neurons in which GFP was expressed, as a percent area stained was performed
(Figure 3). GFP expression was shown to be successfully induced by intracranial bilateral
injection with AAV9-tTA-GFP in both nTg and tetO(MAPT*P301L)Tg mice. [B, E] No
significant difference in levels of GFP was observed, suggesting that neuronal connectivity was
not affected in tetO(MAPT*P301L)Tg mouse model after it’s activation by tet transactivator
protein driving expression of both viral GFP and human Tau (MAPT*P301L).
4.2 AAV9-tTA-ARG1 induces expression of arginase 1 (ARG1) and hemagglutinin tag (HA)
in nTg and tetO(MAPT*P301L)Tg mice.
Immunohistochemical stain of brain sections for arginase 1 (ARG1) and hemagglutinin
tag (HA) fused with arginase 1 was performed (Figure 4, Figure 5). Neurons expressing ARG1
and HA were quantified as a percent area stained. Statistical analysis of groups of mice injected
with AAV9-tTA-ARG1 using a post hoc Fisher PLSD revealed no significant effect of genotype
on levels of expression of ARG1 and HA. However, a trend of reduction of levels of ARG1
expression was observed in tetO(MAPT*P301L)Tg compared to nTg mice (p=0.088) ([C, F] -
Figure 4). As gene for HA was fused with the gene for ARG1 on a viral vector, neurons,
expressing ARG1 were expected to coexpress HA. Therefore, a reduction in expression of ARG1
would correlate with the reduction in expression of HA. Indeed, this is exactly what has been
observed. A trend of reduction of levels of HA in tetO(MAPT*P301L)Tg mice injected with
AAV9-tTA-ARG1 compared to nTg mice that received the same treatment (p=0.068) ([C, F] –
Figure 5).
4.3 AAV9-tTA-ARG reduces levels of total tau in tetO(MAPT*P301L)Tg mice.
Immunohistochemical stain of brain sections for total tau (H150), and it quantitative
analysis for percent area stained were performed (Figure 6). [A-C] No H150 was detected in nTg
mice regardless of treatment, providing clear evidence for correct genotyping of nTg mice. [D,
E] A significant increase of levels of H150 in tetO(MAPT*P301L)Tg mice injected with AAV9-
tTA-GFP compared to genotypically identical mice injected with AAV9-Empty Capsid was
observed. These results provide indisputable evidence for successful Tet-on activation of tetO
regulated MAPT*P301L gene expression by viral tetracycline-controlled transactivator protein.
[E, F] A significant decrease in levels of H150 in tetO(MAPT*P301L)Tg mice injected with
AAV9-tTA-ARG1 compared to Tg mice injected with AAV9-tTA-GFP to levels, insignificantly
different from those, detected in Tg mice injected with AAV9-Empty Capsid, provides evidence
for the importance of arginine metabolism for the development of tau pathology.
23. Kovalenko 23
4.4 AAV9-tTA-ARG1 reduces levels of phosphorylated tau (pSer199/202) in
tetO(MAPT*P301L)Tg mice.
Immunohistochemical stain of brain sections for phosphorylated tau (pSer199/202), and
it quantitative analysis for percent area stained were performed (Figure 7). [A-C] No
pSer199/202 form of tau was detected in nTg mice regardless of treatment. [D, E] Tauopathy
was successfully induced by addition of tetracycline-controlled transactivator protein into the
system. [E, F] Arginase 1 (ARG1) had a significant therapeutic effect on the level of
pSer199/202, significantly reducing it. [D, F] In tetO(MAPT*P301L)Tg mice treated with
AAV9-tTA-ARG1, levels of pSer199/202 were not significantly higher from baseline levels,
established by AAV9-Empty Capsid (p=0.058) but a trend was observed, and an increase of
group's N-value could decrease standard deviation, making a difference statistically significant.
4.5 AAV9-tTA-ARG1 modifies microglia activation in nTg mice, quantified by measuring the
levels of Iba-1.
Immunohistochemical stain of brain sections for ionized calcium binding adapter protein 1 (Iba1)
aka allograft inflammatory factor 1 (AIF1) that is restrictedly expressed in activated
macrophage/microglia (Kanazawa, Ohsawa, Sasaki, Kohsaka, & Imai, 2002) was performed,
followed by quantitative analysis as the percent area stained (Figure 8). [A, C] nTg mice that
received an injection with AAV9-tTA-ARG1 showed significantly higher microglial activation
compared to nTg mice injected with AAV9-Empty Capsid. [D, F] This pattern was not present in
tetO(MAPT*P301L)Tg mice that received identical treatments.
4.6 AAV9-tTA-GFP and AAV9-tTA-ARG1 modify levels of NeuN in nTg mice but not in
tetO(MAPT*P301L)Tg mice.
Immunohistochemical stain of brain sections for NeuN protein that is known to be expressed
only in nervous tissue (Gusel'nikova & Korzhevskiy, 2015) was conducted and quantified as a
percent area stained to evaluate neuronal integrity in brains of nTg and tetO(MAPT*P301L)Tg
mice (Figure 9). [A, D] Significantly lower levels of NeuN were detected in
tetO(MAPT*P301L)Tg mice injected with AAV9-Empty Capsid, compared to nTg mice that
received an identical treatment. [A, B, C] nTg mice that received an injection with AAV9-Empty
Capsid showed significantly lower percent area stained for NeuN compared to nTg mice injected
with AAV9-tTA-GFP and AAV9-tTA-ARG1. The reduction in NeuN protein is commonly
associated with loss of neuronal integrity and neuronal damage; furthermore, it was shown that
NeuN nuclear protein disappears from dying pyramidal neurons of the hippocampus
(Gusel'nikova & Korzhevskiy, 2015). However, varying levels of phosphorylation of NeuN has
been observed to alter the ability of this protein to bind anti-NeuN antibodies (Gusel'nikova &
Korzhevskiy, 2015) and reduction of percent area stained can indicate changes in
phosphorylation patterns rather than the neuronal loss.
24. Kovalenko 24
4.7 Neither of treatments modified the hippocampal volume of nTg and
tetO(MAPT*P301L)Tg mice assessed by quantification of Nissl bodies.
Nissl staining was conducted and hippocampal volume was quantified in mm^3 to assess the
neuronal loss and atrophy (Figure 10). [A-F] The volume of neurons in the hippocampal regions
remained the same regardless of genotype or type of injected virus. No significant difference was
found across groups.
5. Conclusion.
The results of this project describe the effects of bicistronic virus carrying tetracycline-
controlled transactivator protein (tTA) on tet-responsive transgenic mouse model of tau
pathology. This approach provided important advantages over other available models of
tauopathies such as regional and temporal control over the expression of both therapeutic agent
and pathology. Furthermore, alteration of the type of utilized AAV serotype in future
experiments allows a possibility of fine-tuning of viral distribution. The results of this
experiment show that such dual activator-responder approach can be used to bring advances in
the field.
AAV9-tTA-ARG1 successfully induced expression of arginase 1 (ARG1) in both nTg
and tetO(MAPT*P301L)Tg mice. AAV9-tTA-ARG1 successfully decreased both total tau
(H150) and phosphorylated tau (pSer199/202) compared to AAV9-tTA-GFP. AAV9-tTA-ARG1
increased microglia activation in nTg mice but not in tetO(MAPT*P301L)Tg mice. The
hippocampal volume of nTg and tetO(MAPT*P301L)Tg mice was not significantly affected by
intracranial hippocampal injections of AAV9. Viral injections had an effect on the neuronal
integrity of nTg but not tetO(MAPT*P301L)Tg mice.
The results of this experiment show that Arginase 1 and arginine metabolism may serve as a
viable therapeutic target for treatment of Alzheimer’s disease and other tauopathies. Further
research with larger groups may be necessary to decrease the standard error of the sampling
distribution and allow detection of significant changes (ARG1, HA, NeuN, Iba-1). Particularly, a
trend of reduction in levels of ARG1 in tetO(MAPT*P301L)Tg mice that received injection of
AAV9-tTA-ARG1 was detected when compared to nTg mice that received the same treatment
(p=0.088). Even more significant trend was observed for levels of viral ARG1 assessed by
staining for HA tag fused to viral ARG1 (p=0.068). These trends of differences may become
statistically significant once larger groups are used. Future studies may include an additional
control group in which mice wouldn’t be injected to see how the injection itself affects
tetO(MAPT*P301L)Tg mice. Furthermore, double immunofluorescence experiments may be
performed to provide further evidence for co-expression of both therapeutic agent and mutant
MAPT in the same neurons.
Acknowledgment: This work was supported by Alzheimer’s Association (MNIRDG).
Thank you to Chad Dickey for tetO MAPT P301L mice.
25. Kovalenko 25
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