Tetracycline-controlled Transactivator Research Articles

Abstract 1442: Establishing a papillary renal cell carcinoma mouse model with activating Met mutation

Abstract Renal cell carcinoma (RCC) is a heterogeneous group of cancers of the nephron. Papillary renal cell carcinoma (PRCC) is the second most common type of RCC. c-Met activating mutations have been associated with Type 1 PRCC which are made up of small cells containing basophilic cytoplasm covering papillae and tubular structures. In patients with PRCC, c-MetH1112Y missense mutations are associated with both somatic and germline mutations. Previous functional studies indicate codon H1112 plays an important structural role in maintaining c-Met in the inactive conformation and alterations at this codon, including H1112Y, likely disrupt this interaction facilitating constitutive activation with increased c-MET activity. A c-MetH1112Y (c-MetHY) transgene with tetO promoter sequence was micro-injected into the mouse oocyte. Transgenic mice carrying this transgene have the ability to express this activated Met protein in cells expressing rtTA (reverse tetracycline-controlled transactivator) and when doxycycline is present. Mutant mice harboring this transgene along with additional trp53 mutations showed glomeruloid like Type 1 PRCC. Our in vivo studies confirmed the expression of c-MetHY gene within the kidney and Lrp2 and Ki67 staining indicated that PRCC arises from PT (proximal tubule). PRCC canonical markers CK7, AMACR and MET expression confirmed the Type1 PRCC in our mouse model. Additionally Pan-CK, E-Cad positive expression in mutant mice further confirmed PRCC arises from epithelial cells. We have generated and described type 1 PRCC mouse model with activating-MetHY mutation. This mouse model will help illustrate the oncogenic mechanism of Met activation and identify the critical events during progression of PRCC. Citation Format: Preet Lal, Li Ding, Feng Chen. Establishing a papillary renal cell carcinoma mouse model with activating Met mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1442.

A Review of Genetic and Gene Therapy for Parkinson's Disease.

Parkinson's disease (PD) is a syndrome with deterioration of neurons, with its onset starting in the '20s, known as the young beginning of Parkinson's to the late inception of the ailment in the 60s. The majority of the environmental risk associated with PD is age. The pathophysiology of PD is related to the accretion of synuclein alpha (SNCA) protein leading to toxicity. This toxicity further leads to a depletion in dopamine levels, creating both motor and non-motor symptoms. PD is the combination of genetic and environmental risk factors. Linkage and association studies provided data on autosomal dominant and recessive genes linked to PD. Current treatment regimes involve using levodopa, catechol-O-methyl transferase inhibitors, anticholinergics, and monoamine oxidase B (MAO-B) inhibitors. Genetic treatment is done by identifying possible targets. Gene therapy includes silencing, replacing, or correcting the flawed gene with a good gene. This therapy has the advantage of eliminating significant PD symptoms with fewer to no adverse effects than conventional treatment. These targets are organized into disease-modifying or non-disease modifying. The distinction between these two is that disease-modifying treatment stops the degeneration of neurons, while non-disease modifying treatment involves dopaminergic enzyme expression. In non-modifying targets, aromatic L-amino acid decarboxylase (AADC) therapy is used but not as a standalone, so the presentation of AADC, tyrosine hydroxylase (TH), and GTP cyclohydrolase 1 (GCH) is done together as a tricistronic system. With these developments, a drug named prosavin is under clinical phase 1 trial. Disease-modifying targets involve glial cell-derived neurotrophic factor (GDNF). Direct GDNF delivery reduces PD symptoms. This GDNF infusion technique works with a tetracycline-controlled transactivator. Gene therapy introduction into the treatment of PD would be beneficial as there would be lesser adverse effects seen as linked with conventional treatment involving levodopa, MAO-B inhibitors, and anticholinergics, among a few. This article discusses the genetic basis and genetic model of therapy for PD.

Activity of putative orexin neurons during cataplexy

It is unclear why orexin-deficient animals, but not wild-type mice, show cataplexy. The current hypothesis predicts simultaneous excitation of cataplexy-inhibiting orexin neurons and cataplexy-inducing amygdala neurons. To test this hypothesis, we measured the activity of putative orexin neurons in orexin-knockout mice during cataplexy episodes using fiber photometry. We created two animal models of orexin-knockout mice with a GCaMP6 fluorescent indicator expressed in putative orexin neurons. We first prepared orexin-knockout mice crossed with transgenic mice carrying a tetracycline-controlled transactivator transgene under the control of the orexin promoter. TetO-GCaMP6 was then introduced into mice via an adeno-associated virus injection or natural crossing. The resulting two models showed restricted expression of GCaMP6 in the hypothalamus, where orexin neurons should be located, and showed excitation to an intruder stress that was similar to that observed in orexin-intact mice in our previous study. The activity of these putative orexin neurons increased immediately before the onset of cataplexy-like behavior but decreased (approximately − 20% of the baseline) during the cataplexy-like episode. We propose that the activity of orexin neurons during cataplexy is moderately inhibited by an unknown mechanism. The absence of cataplexy in wild-type mice may be explained by basal or residual activity-induced orexin release, and emotional stimulus-induced counter activation of orexin neurons may not be necessary. This study will serve as a basis for better treatment of cataplexy in narcolepsy patients.

A novel genotyping method to determine copy number in a mouse line commonly used for inducible transgene expression in brain and spinal cord

Background: The NEFH-tTA mouse has the human neurofilament heavy polypeptide promoter directing tetracycline-controlled transactivator protein (tTa) expression to the brain and spinal cord, allowing tissue-specific and doxycycline-suppressible expression of a target gene. Current genotyping protocols can only differentiate between wild-type and transgenic animals. Being able to differentiate between hemizygous and homozygous animals would be beneficial in experiment planning and reducing animal numbers. Methods: We have identified the insertion site of the NEFH-tTA transgene via targeted locus amplification and next-generation sequencing. This was then used to design a multiplex PCR assay to distinguish between hemizygous and homozygous mice. Results: The NEFH-tTA transgene is located on chromosome 12. Our genotyping method can identify hemizygous and homozygous mice. Conclusions: The NEFH-tTA transgenic mouse line is a useful tool for studying a wide range of diseases including frontotemporal dementia and motor neuron disease, as well as other neurodevelopmental, neuromuscular or neurodegenerative disorders. We have designed and utilised a novel genotyping assay to distinguish between hemizygous and homozygous mice, involving a simple PCR assay. This is easily adaptable to a laboratory-specific protocol or machine, and will allow refinement of breeding strategies and a reduction in the number of animals that cannot be used in experiments.

A TDP-43 proteinopathy mouse model of motor neuron disease.

Motor neuron disease (MND) is characterized by both upper and lower motor neuron death. The average lifespan, after diagnosis, is ∼5 years, primarily due to respiratory failure and pneumonia with no effective treatment for the disease. Over 95% of all cases are pathologically characterized by cytoplasmic accumulation of hyperphosphorylated and ubiquitinated transactive response DNA binding protein 43 (TDP-43). While in frontotemporal dementia (FTD), ∼50% have TDP-43 pathology. The comorbidly seen in individuals with both FTD and MND has been shown to have an accelerated disease progression. We have developed an inducible aggressive mouse model that displays some of the behavioral and pathological features associated with MND and FTD-MND. A transgenic mouse containing three familial mutations in human TDP-43 was developed with a tetracycline response element (tetO-3X-TDP-43)(Cyagen). Double transgenic mice were generated by driving tetO-3XTDP-43 expression with the neurofilament heavy promoter expressing tetracycline-controlled transactivator protein ( NEFH-tTA) throughout the spinal cord and brain. Expression of the transgene was turned off (+DOX) until mice are weaned (P21). Mice were evaluated for motor an anxiety-like behavior. Pathological evaluation included biochemical solubility fractionation, mitochondrial isolation, immunohistochemistry, and primary neuronal culture. An age-dependent decrease in motor function leading to paralysis was observed at 45-60 days. This is associated with an increase in inflammation in mice expressing pathological TDP-43. Biochemical fragmentation of TDP-43 is seen in the spinal cord and brain. This aggressive model has the versatility to be used to drive expression in muscle, the hippocampus, and astrocytes to possibly help in our understanding of neurodegeneration in inclusion body myopathy, limbic predominant age-related TDP-43 encephalopathy (LATE), and other TDP-43 proteinopathies. It is important to develop animal models that recapitulate human disease in order to develop viable targets for therapeutic intervention.

The Tetracycline-Controlled Transactivator (Tet-On/Off) System in β-Cells Reduces Insulin Expression and Secretion in Mice.

Controllable genetic manipulation is an indispensable tool in research, greatly advancing our understanding of cell biology and physiology. However in β-cells, transgene silencing, low inducibility, ectopic expression, and off-targets effects are persistent challenges. In this study, we investigated whether an inducible Tetracycline (Tet)-Off system with β-cell–specific mouse insulin promoter (MIP)-itTA–driven expression of tetracycline operon (TetO)-CreJaw/J could circumvent previous issues of specificity and efficacy. Following assessment of tissue-specific gene recombination, β-cell architecture, in vitro and in vivo glucose-stimulated insulin secretion, and whole-body glucose homeostasis, we discovered that expression of any tetracycline-controlled transactivator (e.g., improved itTA, reverse rtTA, or tTA) in β-cells significantly reduced Insulin gene expression and decreased insulin content. This translated into lower pancreatic insulin levels and reduced insulin secretion in mice carrying any tTA transgene, independent of Cre recombinase expression or doxycycline exposure. Our study echoes ongoing challenges faced by fundamental researchers working with β-cells and highlights the need for consistent and comprehensive controls when using the tetracycline-controlled transactivator systems (Tet-On or Tet-Off) for genome editing.

Involvement of A5/A7 noradrenergic neurons and B2 serotonergic neurons in nociceptive processing: a fiber photometry study.

In the central nervous system, the A6 noradrenaline (NA) and the B3 serotonin (5-HT) cell groups are well-recognized players in the descending antinociceptive system, while other NA/5-HT cell groups are not well characterized. A5/A7 NA and B2 5-HT cells project to the spinal horn and form descending pathways. We recorded G-CaMP6 green fluorescence signal intensities in the A5/A7 NA and the B2 5-HT cell groups of awake mice in response to acute tail pinch stimuli, acute heat stimuli, and in the context of a non-noxious control test, using fiber photometry with a calcium imaging system. We first introduced G-CaMP6 in the A5/A7 NA or B2 5-HT neuronal soma, using transgenic mice carrying the tetracycline-controlled transactivator transgene under the control of either a dopamine β-hydroxylase or a tryptophan hydroxylase-2 promoters and by the site-specific injection of adeno-associated virus (AAV-TetO(3G)-G-CaMP6). After confirming the specific expression patterns of G-CaMP6, we recorded G-CaMP6 green fluorescence signals in these sites in awake mice in response to acute nociceptive stimuli. G-CaMP6 fluorescence intensity in the A5, A7, and B2 cell groups was rapidly increased in response to acute nociceptive stimuli and soon after, it returned to baseline fluorescence intensity. This was not observed in the non-noxious control test. The results indicate that acute nociceptive stimuli rapidly increase the activities of A5/A7 NA or B2 5-HT neurons but the non-noxious stimuli do not. The present study suggests that A5/A7 NA or B2 5-HT neurons play important roles in nociceptive processing in the central nervous system. We suggest that A5/A7/B2 neurons may be new therapeutic targets. All performed procedures were approved by the Institutional Animal Use Committee of Kagoshima University (MD17105) on February 22, 2018.

A modified Tet‐ON system minimizing leaky expression for cell‐type specific gene induction in medaka fish

The Tet-ON system is an important molecular tool for temporally and spatially-controlled inducible gene expression. Here, we developed a Tet-ON system to induce transgene expression specifically in the rod photoreceptors of medaka fish. Our modified reverse tetracycline-controlled transcriptional transactivator (rtTAm) with 5 amino acid substitutions dramatically improved the leakiness of the transgene in medaka fish. We generated a transgenic line carrying a self-reporting vector with the rtTAm gene driven by the Xenopus rhodopsin promoter and a tetracycline response element (TRE) followed by the green fluorescent protein (GFP) gene. We demonstrated that GFP fluorescence was restricted to the rod photoreceptors in the presence of doxycycline in larval fish (9days post-fertilization). The GFP fluorescence intensity was enhanced with longer durations of doxycycline treatment up to 72h and in a dose-dependent manner (5-45μg/ml). These findings demonstrate that the Tet-ON system using rtTAm allows for spatiotemporal control of transgene expression, at least in the rod photoreceptors, in medaka fish.

Cardiomyocyte Krüppel-Like Factor 5 Promotes De Novo Ceramide Biosynthesis and Contributes to Eccentric Remodeling in Ischemic Cardiomyopathy.

We previously showed that cardiomyocyte Krϋppel-like factor (KLF) 5 regulates cardiac fatty acid oxidation. As heart failure has been associated with altered fatty acid oxidation, we investigated the role of cardiomyocyte KLF5 in lipid metabolism and pathophysiology of ischemic heart failure. Using real-time polymerase chain reaction and Western blot, we investigated the KLF5 expression changes in a myocardial infarction (MI) mouse model and heart tissue from patients with ischemic heart failure. Using 2D echocardiography, we evaluated the effect of KLF5 inhibition after MI using pharmacological KLF5 inhibitor ML264 and mice with cardiomyocyte-specific KLF5 deletion (αMHC [α-myosin heavy chain]-KLF5-/-). We identified the involvement of KLF5 in regulating lipid metabolism and ceramide accumulation after MI using liquid chromatography-tandem mass spectrometry, and Western blot and real-time polymerase chain reaction analysis of ceramide metabolism-related genes. We lastly evaluated the effect of cardiomyocyte-specific KLF5 overexpression (αMHC-rtTA [reverse tetracycline-controlled transactivator]-KLF5) on cardiac function and ceramide metabolism, and rescued the phenotype using myriocin to inhibit ceramide biosynthesis. KLF5 mRNA and protein levels were higher in human ischemic heart failure samples and in rodent models at 24 hours, 2 weeks, and 4 weeks post-permanent left coronary artery ligation. αMHC-KLF5-/- mice and mice treated with ML264 had higher ejection fraction and lower ventricular volume and heart weight after MI. Lipidomic analysis showed that αMHC-KLF5-/- mice with MI had lower myocardial ceramide levels compared with littermate control mice with MI, although basal ceramide content of αMHC-KLF5-/- mice was not different in control mice. KLF5 ablation suppressed the expression of SPTLC1 and SPTLC2 (serine palmitoyltransferase [SPT] long-chain base subunit ()1 2, respectively), which regulate de novo ceramide biosynthesis. We confirmed our previous findings that myocardial SPTLC1 and SPTLC2 levels are increased in heart failure patients. Consistently, αMHC-rtTA-KLF5 mice showed increased SPTLC1 and SPTLC2 expression, higher myocardial ceramide levels, and systolic dysfunction beginning 2 weeks after KLF5 induction. Treatment of αMHC-rtTA-KLF5 mice with myriocin that inhibits SPT, suppressed myocardial ceramide levels and alleviated systolic dysfunction. KLF5 is induced during the development of ischemic heart failure in humans and mice and stimulates ceramide biosynthesis. Genetic or pharmacological inhibition of KLF5 in mice with MI prevents ceramide accumulation, alleviates eccentric remodeling, and increases ejection fraction. Thus, KLF5 emerges as a novel therapeutic target for the treatment of ischemic heart failure.

Improvement on the genetic engineering of an invasive agricultural pest insect, the cherry vinegar fly, Drosophila suzukii

BackgroundThe invasive fly Drosophila suzukii has become an established fruit pest in Europe, the USA, and South America with no effective and safe pest management. Genetic engineering enables the development of transgene-based novel genetic control strategies against insect pests and disease vectors. This, however, requires the establishment of reliable germline transformation techniques. Previous studies have shown that D. suzukii is amenable to transgenesis using the transposon-based vectors piggyBac and Minos, site-specific recombination (lox/Cre), and CRISPR/Cas9 genome editing.ResultsWe experienced differences in the usability of piggyBac-based germline transformation in different strains of D. suzukii: we obtained no transgenic lines in a US strain, a single rare transgenic line in an Italian strain, but observed a reliable transformation rate of 2.5 to 11% in a strain from the French Alps. This difference in efficiency was confirmed by comparative examination of these three strains. In addition, we used an attP landing site line to successfully established φC31-integrase-mediated plasmid integration at a rate of 10% and generated landing site lines with two attP sequences to effectively perform φC31-Recombinase Mediated Cassette Exchange (φC31-RMCE) with 11% efficiency. Moreover, we isolated and used the endogenous regulatory regions of Ds nanos to express φC31 integrase maternally to generate self-docking lines for φC31-RMCE. Besides, we isolated the promoter/enhancer of Ds serendipity α to drive the heterologous tetracycline-controlled transactivator (tTA) during early embryonic development and generated a testes-specific tTA driver line using the endogenous beta-2-tubulin (β2t) promoter/enhancer.ConclusionOur results provide evidence that the D. suzukii strain AM derived from the French Alps is more suitable for piggyBac germline transformation than other strains. We demonstrated the feasibility of using φC31-RMCE in the cherry vinegar fly and generated a set of lines that can be used for highly efficient integration of larger constructs. The φC31-based integration will facilitate modification and stabilization of previously generated transgenic lines that carry at least one attP site in the transgene construction. An early embryo-specific and a spermatogenesis-specific driver line were generated for future use of the binary expression system tet-off to engineer tissue- and stage-specific effector gene expression for genetic pest control strategies.

A novel genotyping method to determine copy number in a mouse line commonly used for inducible transgene expression in brain and spinal cord

Background: The NEFH-tTA mouse has the human neurofilament heavy polypeptide promoter directing tetracycline-controlled transactivator protein (tTa) expression to the brain and spinal cord, allowing tissue-specific and doxycycline-suppressible expression of a target gene. Current genotyping protocols can only differentiate between wild-type and transgenic animals. Being able to differentiate between hemizygous and homozygous animals would be beneficial in experiment planning and reducing animal numbers. Methods: We have identified the insertion site of the NEFH-tTA transgene via targeted locus amplification and next-generation sequencing. This was then used to design a multiplex PCR assay to distinguish between hemizygous and homozygous mice. Results: The NEFH-tTA transgene is located on chromosome 12. Our genotyping method can identify hemizygous and homozygous mice. Conclusions: The NEFH-tTA transgenic mouse line is a useful tool for studying a wide range of diseases including frontotemporal dementia and motor neuron disease, as well as other neurodevelopmental, neuromuscular or neurodegenerative disorders. We have designed and utilised a novel genotyping assay to distinguish between hemizygous and homozygous mice, involving a simple PCR assay. This is easily adaptable to a laboratory-specific protocol or machine, and will allow refinement of breeding strategies and a reduction in the number of animals that cannot be used in experiments.

Involvement of supralemniscal nucleus (B9) 5-HT neuronal system in nociceptive processing: a fiber photometry study

Nociception is important perception that has harmful influence on daily life of humans. As to main pain management system, some descending pathways are called descending antinociceptive systems (DAS). As main pathways of DAS, it is well known that dorsal raphe (B6/B7) - rostral ventromedial medulla (B3) - spinal dorsal horn includes serotonergic system. However, possible role of supralemniscal (B9) serotonin (5-HT) cell group in pain management is still open question. In this study, we measured activities of B9 5-HT neuronal cell bodies and B9 5-HT neuron-derived axons located in the locus coeruleus (LC) and ventral tegmental area (VTA), which are also main players of pain management, using fiber photometry system. We introduced the G-CaMP6 in B9 5-HT neurons using transgenic mice carrying a tetracycline-controlled transactivator transgene (tTA) under the control of a tryptophan hydroxylase-2 (TPH2) promoter and site-specific injection of adeno associated virus (AAV-TetO(3G)-G-CaMP6). After confirmation of specific expression of G-CaMP6 in the target population, G-CaMP6 fluorescence intensity in B9 group and LC/VTA groups was measured in awake mice exposed to acute tail pinch and heat stimuli. G-CaMP6 fluorescence intensity rapidly increased by both stimuli in all groups, but not significantly reacted by nonnociceptive control stimuli. The present results clearly indicate that acute nociceptive stimuli cause a rapid increase in the activities of B9-LC/B9-VTA 5-HTergic pathways, suggesting that B9 5-HT neurons play important roles in nociceptive processing.

Efficient tissue-type specific expression of target genes in a tetracycline-controlled manner from the ubiquitously active Eef1a1 locus

Using an efficient gene targeting approach, we developed a novel mouse line that expresses the tetracycline-controlled transactivator (tTA) from the constitutively active Eef1a1 locus in a Cre recombinase-inducible manner. The temporally and spatially controlled expression of the EF1-LSL-tTA knockin and activation of tTA-driven responder transgenes was tested using four transgenic lines that express Cre under tissue-specific promoters of the pancreas, mammary gland and other secretory tissues, as well as an interferon-inducible promoter. In all models, the endogenous Eef1a1 promoter facilitated a cell-type-specific activation of target genes at high levels without exogenous enhancer elements. The applicability of the EF1-LSL-tTA strain for biological experiments was tested in two studies related to mammary gland development and tumorigenesis. First, we validated the crucial role of active STAT5 as a survival factor for functionally differentiated epithelial cells by expressing a hyperactive STAT5 mutant in the mammary gland during postlactational remodeling. In a second experiment, we assessed the ability of the EF1-tTA to initiate tumor formation through upregulation of mutant KRAS. The collective results show that the EF1-LSL-tTA knockin line is a versatile genetic tool that can be applied to constitutively express transgenes in specific cell types to examine their biological functions at defined developmental stages.

Synergistic toxicity in an in vivo model of neurodegeneration through the co-expression of human TDP-43M337V and tauT175D protein

Although it has been suggested that the co-expression of multiple pathological proteins associated with neurodegeneration may act synergistically to induce more widespread neuropathology, experimental evidence of this is sparse. We have previously shown that the expression of Thr175Asp-tau (tauT175D) using somatic gene transfer with a stereotaxically-injected recombinant adeno-associated virus (rAAV9) vector induces tau pathology in rat hippocampus. In this study, we have examined whether the co-expression of human tauT175D with mutant human TDP-43 (TDP-43M337V) will act synergistically. Transgenic female Sprague-Dawley rats that inducibly express mutant human TDP-43M337V using the choline acetyltransferase (ChAT) tetracycline response element (TRE) driver with activity modulating tetracycline-controlled transactivator (tTA) were utilized in these studies. Adult rats were injected with GFP-tagged tau protein constructs in a rAAV9 vector through bilateral stereotaxic injection into the hippocampus. Injected tau constructs were: wild-type GFP-tagged 2N4R human tau (tauWT; n = 8), GFP-tagged tauT175D 2N4R human tau (tauT175D, pseudophosphorylated, toxic variant, n = 8), and GFP (control, n = 8). Six months post-injection, mutant TDP-43M337V expression was induced for 30 days. Behaviour testing identified motor deficits within 3 weeks after TDP-43 expression irrespective of tau expression, though social behaviour and sensorimotor gating remained unchanged. Increased tau pathology was observed in the hippocampus of both tauWT and tauT175D expressing rats and tauT175D pathology was increased in the presence of cholinergic neuronal expression of human TDP-43M337V. These data indicate that co-expression of pathological TDP-43 and tau protein exacerbate the pathology associated with either individual protein.

Hysteretic Genetic Circuit for Detection of Proteasomal Degradation in Mammalian Cells.

Synthetic hysteretic mammalian gene circuits generating sustained cellular responses to transient perturbations provide important tools to investigate complex cellular behaviors and reprogram cells for a variety of applications, ranging from protein production to cell fate decisions. The design rules of synthetic gene circuits with controlled hysteretic behaviors, however, remain uncharacterized. To identify the criteria for achieving predictable control of hysteresis, we built a genetic circuit for detection of proteasomal degradation (Hys-Deg). The Hys-Deg circuit is based on a tetracycline-controlled transactivator (tTA) variant engineered to interface with the ubiquitin proteasome system (UPS). The tTA variant activates its own expression, generating a positive feedback loop that is triggered by expression of another tTA gene that is constitutively regulated. Guided by predictive modeling, we characterized the hysteretic response of the Hys-Deg circuit. We demonstrated that control of the hysteretic response is achieved by modulating the ratio of expression of constitutive to inducible tTA. We also showed that the system can be finely tuned through dosage of the inducer tetracycline to calibrate the circuit for detection of the desired levels of UPS activation. This study establishes the design rules for building a hysteretic genetic circuit with an autoregulatory feedback loop and provides a synthetic memory module that could be easily integrated into regulatory gene networks to study and engineer complex cellular behaviors.

Pathogenic tau does not drive activation of the unfolded protein response

The unfolded protein response (UPR) is commonly associated with a range of neurodegenerative diseases, and targeting UPR components has been suggested as a therapeutic strategy. The UPR surveys protein folding within the endoplasmic reticulum. However, many of the misfolded proteins that accumulate in neurodegeneration are localized so that they do not directly cause endoplasmic reticulum triggers that activate this pathway. Here, using a transgenic mouse model and primary cell cultures along with quantitative PCR, immunoblotting, and immunohistochemistry, we tested whether the UPR is induced in in vivo and in vitro murine models of tauopathy that are based on expression of mutant tauP301L. We found no evidence for the UPR in the rTg4510 mouse model, in which mutant tau is transgenically expressed under the control of tetracycline-controlled transactivator protein. This observation was supported by results from acute experiments in which neuronal cultures expressed mutant tau and accumulated misfolded cytoplasmic tau aggregates but exhibited no UPR activation. These results suggest that the UPR is not induced as a response to tau misfolding and aggregation despite clear evidence for progressive cellular dysfunction and degeneration. We propose that caution is needed when evaluating the implied significance of the UPR as a critical determinant across major neurodegenerative diseases.

Acute nociceptive stimuli rapidly induce the activity of serotonin and noradrenalin neurons in the brain stem of awake mice

Nociception is an important type of perception that has major influence on daily human life. There are some descending pathways related to pain management and modulation, which are collectively known as the descending antinociceptive system (DAS). Noradrenalin (NA) in the locus coeruleus (LC) and serotonin (5-HT) in the rostral ventromedial medulla (RVM) are components of the DAS. Most 5-HT neurons in the dorsal raphe (DR) have ascending projections rather than descending projections, and they project to the thalamus that modulates nociception. Both the DAS and the DR are believed to be involved in pain-emotion symptoms. In this study, we utilized a fiber photometry system to specifically examine the activity of LC NA neurons and RVM/DR 5-HT neurons using mice carrying tetracycline-controlled transactivator transgene (tTA) under the control of either a dopamine β-hydroxylase promoter or a tryptophan hydroxylase-2 promoter and site-specific infection of an adeno-associated virus carrying a TetO G-CaMP6gene. After confirmation of specific expression of G-CaMP6 in the target populations, changes in green fluorescent signal intensity were recorded in awake mice upon exposure to acute nociceptive stimulation consisting of a pinch and application of heat (55°C) to the tail. Both stimuli resulted in rapid and transient (<15s) increases in the activity of LC NA neurons and RVM/DR 5-HT neurons while the control stimuli did not induce any changes. The present results clearly indicate that acute nociceptive stimuli increase the activity of LC NA neurons and RVM/DR 5HT neurons and suggest a possible therapeutic target for pain treatment.

Human sFLT1 Leads to Severe Changes in Placental Differentiation and Vascularization in a Transgenic hsFLT1/rtTA FGR Mouse Model.

The anti-angiogenic soluble fms-like tyrosine kinase 1 (sFLT1) is one of the candidates in the progression of preeclampsia, often associated with fetal growth restriction (FGR). Therapeutic agents against preeclampsia with/without FGR, as well as adequate transgenic sFLT1 mouse models for testing such agents, are still missing. Much is known about sFLT1–mediated endothelial dysfunction in several tissues; however, the influence of sFLT1 on placental and fetal development is currently unknown. We hypothesize that sFLT1 is involved in the progression of FGR by influencing placental differentiation and vascularization and is a prime candidate for interventional strategies. Therefore, we generated transgenic inducible human sFLT1/reverse tetracycline-controlled transactivator (hsFLT1/rtTA) mice, in which hsFLT1 is ubiquitously overexpressed during pregnancy in dams and according to the genetics in hsFLT1/rtTA homozygous and heterozygous fetuses. Induction of hsFLT1 led to elevated hsFLT1 levels in the serum of dams and on mRNA level in all placentas and hetero-/homozygous fetuses, resulting in FGR in all fetuses at term. The strongest effects in respect to FGR were observed in the hsFLT1/rtTA homozygous fetuses, which exhibited the highest hsFLT1 levels. Only fetal hsFLT1 expression led to impaired placental morphology characterized by reduced placental efficiency, enlarged maternal sinusoids, reduced fetal capillaries, and impaired labyrinthine differentiation, associated with increased apoptosis. Besides impaired placental vascularization, the expression of several transporter systems, such as glucose transporter 1 and 3 (Glut-1; Glut-3); amino acid transporters, solute carrier family 38, member one and two (Slc38a1; Slc38a2); and most severely the fatty acid translocase Cd36 and fatty acid binding protein 3 (Fabp3) was reduced upon hsFLT1 expression, associated with an accumulation of phospholipids in the maternal serum. Moreover, the Vegf pathway showed alterations, resulting in reduced Vegf, Vegfb, and Plgf protein levels and increased Bad and Caspase 9 mRNA levels. We suggest that hsFLT1 exerts an inhibitory influence on placental vascularization by reducing Vegf signaling, which leads to apoptosis in fetal vessels, impairing placental differentiation, and the nutrient exchange function of the labyrinth. These effects were more pronounced when both the dam and the fetus expressed hsFLT1 and ultimately result in FGR and resemble the preeclamptic phenotype in humans.

Brain-wide distribution of reporter expression in five transgenic tetracycline-transactivator mouse lines

The spatial pattern of transgene expression in tetracycline-controlled mouse models is governed primarily by the driver line used to introduce the tetracycline-controlled transactivator (tTA). Detailed maps showing where each tTA driver activates expression are therefore essential for designing and using tet-regulated models, particularly in brain research where cell type and regional specificity determine the circuits affected by conditional gene expression. We have compiled a comprehensive online repository of serial microscopic images showing brain-wide reporter expression for five commonly used tTA driver lines. We have spatially registered all images to a common three-dimensional mouse brain anatomical reference atlas for direct comparison of spatial distribution across lines. The high-resolution images and associated metadata are shared via the web page of the EU Human Brain Project. Images can be inspected using an interactive viewing tool that includes an optional overlay feature providing anatomical delineations and reference atlas coordinates. Interactive viewing is supplemented by semi-quantitative analyses of expression levels within anatomical subregions for each tTA driver line.

Promoter cross-talk affects the inducible expression of intronic shRNAs from the tetracycline response element.

RNA interference (RNAi), defined as double-stranded, RNA-mediated gene silencing, is a useful tool for functional genomic studies. Along with increasing information about genomic sequences due to the innovative development of genome-sequencing technologies, functional genomic technologies are needed to annotate the genome and determine the processes by which each gene is regulated. Lentiviral vectors have been used to efficiently deliver reagents, such as small interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs), into cells and tissues for functional genomic analyses. We developed a lentiviral vector that efficiently expresses intronic shRNA from the tetracycline regulatory element (TRE) promoter in a doxycycline-dependent manner. We developed a lentiviral vector system that contains reverse tetracycline-controlled transactivator 3 (rtTA3) and the TRE promoter, which are necessary for the doxycycline-inducible expression of shRNAs that are expressed as intronic miR-30a precursors. We then measured the cross-talk between the cytomegalovirus (CMV) and TRE promoters in the vector. We found that nearby promoters influence each other and that the TRE promoter should be located far from other promoters, such as the CMV promoter, in a vector. The orientation of a promoter with respect to other promoters also influences its transcriptional activity. A head-to-head orientation of the CMV and TRE promoters maintains the lowest level of transcription from TRE in the absence of doxycycline, compared to the tail-to-tail and head-to-tail orientations. Based on these findings, we were able to construct a lentiviral vector that faithfully expresses intronic miR-30a shRNA precursors in a doxycycline-inducible manner.