scholarly journals Intravenous infusion of iPSC-derived neural precursor cells increases acid β-glucosidase function in the brain and lessens the neuronopathic phenotype in a mouse model of Gaucher disease

2019 ◽  
Vol 28 (20) ◽  
pp. 3406-3421 ◽  
Author(s):  
Yanyan Peng ◽  
Benjamin Liou ◽  
Venette Inskeep ◽  
Rachel Blackwood ◽  
Christopher N Mayhew ◽  
...  
Keyword(s):  
Mouse Model ◽  
Gaucher Disease ◽  
Fluorescent Protein ◽  
Therapeutic Potential ◽  
Wild Type Mouse ◽  
Induced Pluripotent Stem Cell ◽  
Precursor Cells ◽  
Mrna Levels ◽  
Green Fluorescent ◽  
The Brain

Abstract Gaucher disease (GD) is caused by GBA1 mutations leading to functional deficiency of acid-β-glucosidase (GCase). No effective treatment is available for neuronopathic GD (nGD). A subclass of neural stem and precursor cells (NPCs) expresses VLA4 (integrin α4β1, very late antigen-4) that facilitates NPC entry into the brain following intravenous (IV) infusion. Here, the therapeutic potential of IV VLA4+NPCs was assessed for nGD using wild-type mouse green fluorescent protein (GFP)-positive multipotent induced pluripotent stem cell (iPSC)-derived VLA4+NPCs. VLA4+NPCs successfully engrafted in the nGD (4L;C*) mouse brain. GFP-positive cells differentiated into neurons, astrocytes and oligodendrocytes in the brainstem, midbrain and thalamus of the transplanted mice and significantly improved sensorimotor function and prolonged life span compared to vehicle-treated 4L;C* mice. VLA4+NPC transplantation significantly decreased levels of CD68 and glial fibrillary acidic protein, as well as TNFα mRNA levels in the brain, indicating reduced neuroinflammation. Furthermore, decreased Fluoro-Jade C and NeuroSilver staining suggested inhibition of neurodegeneration. VLA4+NPC-engrafted 4L;C* midbrains showed 35% increased GCase activity, reduced substrate [glucosylceramide (GC, −34%) and glucosylsphingosine (GS, −11%)] levels and improved mitochondrial oxygen consumption rates in comparison to vehicle-4L;C* mice. VLA4+NPC engraftment in 4L;C* brain also led to enhanced expression of neurotrophic factors that have roles in neuronal survival and the promotion of neurogenesis. This study provides evidence that iPSC-derived NPC transplantation has efficacy in an nGD mouse model and provides proof of concept for autologous NPC therapy in nGD.

scholarly journals Habenular Kiss1 Neurons Modulate the Serotonergic System in the Brain of Zebrafish

Endocrinology ◽  
2012 ◽  
Vol 153 (5) ◽  
pp. 2398-2407 ◽  
Author(s):  
Satoshi Ogawa ◽  
Kai We Ng ◽  
Priveena Nair Ramadasan ◽  
Fatima Megala Nathan ◽  
Ishwar S. Parhar
Keyword(s):  
Fluorescent Protein ◽  
Serotonergic System ◽  
Raphe Nuclei ◽  
Transgenic Zebrafish ◽  
Mrna Levels ◽  
Interpeduncular Nucleus ◽  
Hypothalamic Nuclei ◽  
Green Fluorescent ◽  
Raphé Nuclei ◽  
The Brain

The Kiss1/KISS1 gene has recently been implicated as a potent hypothalamic regulator of reproductive functions, in particular, the onset of puberty in mammals. In zebrafish (Danio rerio), there are two kiss1 homologues (kiss1 and kiss2) expressed in the brain: Kiss2-expressing neurons in the hypothalamic nuclei are considered potent regulators of reproduction, whereas the role of Kiss1-expressing neurons in the habenula remains unknown. We first analyzed the expression of kiss1 mRNA in a transgenic zebrafish, in which the habenula-interpeduncular nucleus (IPN) pathway is labelled with green fluorescent protein, and our application of a biocytin neural tracer into the habenula showed the presence of neuronal projections of Kiss1 neurons to the ventral IPN. Therefore, we speculated that kiss1 neurons might regulate the serotonergic system in the raphe. However, laser microdissection followed by real-time PCR revealed the expression of Kiss1 receptor (kissr1) mRNA in the habenula and the ventral IPN but not in the dorsal IPN or the serotonergic neurons in the raphe nuclei. Dual-fluorescent in situ hybridization revealed the coexpression of kiss1 and kissr1 mRNA in the habenula. Administration of Kiss1 significantly decreased the level of kiss1 mRNA (0.3- to 0.5-fold, P < 0.001), but the level of c-fos mRNA was increased (∼3-fold, P < 0.05) in the ventral habenula, suggesting that there is autocrine regulation of the kiss1 gene. Kiss1 administration significantly increased the c-fos mRNA levels in the raphe nuclei (2.5-fold, P < 0.001) and genes involved in the regulation of serotonin levels (pet1 and slc6a4a; 3.3- and 2.2-fold, P < 0.01). These findings suggest that the autocrine-regulated habenular Kiss1 neurons indirectly regulate the serotonergic system in the raphe nuclei through the IPN in the zebrafish.

scholarly journals Systemic AAV6-synapsin-GFP administration results in lower liver biodistribution, compared to AAV1&2 and AAV9, with neuronal expression following ultrasound-mediated brain delivery

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Danielle Weber-Adrian ◽  
Rikke Hahn Kofoed ◽  
Joseph Silburt ◽  
Zeinab Noroozian ◽  
Kairavi Shah ◽  
...  
Keyword(s):  
Gene Delivery ◽  
Intravenous Injection ◽  
Viral Vectors ◽  
Focused Ultrasound ◽  
Fluorescent Protein ◽  
Gfp Expression ◽  
Peripheral Organs ◽  
Neuronal Expression ◽  
Green Fluorescent ◽  
The Brain

AbstractNon-surgical gene delivery to the brain can be achieved following intravenous injection of viral vectors coupled with transcranial MRI-guided focused ultrasound (MRIgFUS) to temporarily and locally permeabilize the blood–brain barrier. Vector and promoter selection can provide neuronal expression in the brain, while limiting biodistribution and expression in peripheral organs. To date, the biodistribution of adeno-associated viruses (AAVs) within peripheral organs had not been quantified following intravenous injection and MRIgFUS delivery to the brain. We evaluated the quantity of viral DNA from the serotypes AAV9, AAV6, and a mosaic AAV1&2, expressing green fluorescent protein (GFP) under the neuron-specific synapsin promoter (syn). AAVs were administered intravenously during MRIgFUS targeting to the striatum and hippocampus in mice. The syn promoter led to undetectable levels of GFP expression in peripheral organs. In the liver, the biodistribution of AAV9 and AAV1&2 was 12.9- and 4.4-fold higher, respectively, compared to AAV6. The percentage of GFP-positive neurons in the FUS-targeted areas of the brain was comparable for AAV6-syn-GFP and AAV1&2-syn-GFP. In summary, MRIgFUS-mediated gene delivery with AAV6-syn-GFP had lower off-target biodistribution in the liver compared to AAV9 and AAV1&2, while providing neuronal GFP expression in the striatum and hippocampus.

scholarly journals A new transgene mouse model using an extravesicular EGFP tag to elucidate the in vivo function of extracellular vesicles

2021 ◽  
Author(s):  
Mikkel Oernfeldt Noegaard ◽  
Lasse Bach Steffensen ◽  
Didde Hansen ◽  
Ernst-Martin Fuchtbauer ◽  
Morten Buch Engelund ◽  
...  
Keyword(s):  
Mouse Model ◽  
Extracellular Vesicles ◽  
Fluorescent Protein ◽  
Urine Samples ◽  
Egfp Expression ◽  
Reporter Mice ◽  
Plasma And Urine Samples ◽  
Co Precipitation ◽  
Green Fluorescent

The in vivo function of cell-derived extracellular vesicles (EVs) is challenging to establish since cell-specific EVs are difficult to isolate. We therefore created an EV reporter using CD9 to display enhanced green fluorescent protein (EGFP) on the EV surface. CD9-EGFP expression in cells did not affect EV size and concentration, but allowed for co-precipitation of EV markers TSG101 and ALIX from cell-conditioned medium by anti-GFP immunoprecipitation. We created a transgenic mouse where CD9-EGFP was inserted in the inverse orientation and double-floxed, ensuring Cre recombinase-dependent EV reporter expression. We crossed the EV reporter mice with mice expressing Cre ubiquitously (CMV-Cre), in cardiomyocytes (AMHC-Cre) and kidney epithelium (Pax8-Cre), respectively. The mice showed tissue-specific EGFP expression, and plasma and urine samples were used to immunoprecipitate EVs. CD9-EGFP EVs was detected in plasma samples from CMV-Cre/CD9-EGFP and AMHC-Cre/CD9-EGFP mice, but not in PAX8-Cre/CD9-EGFP mice. On the other hand, CD9-EGFP EVs were detected in urine samples from CMV-Cre/CD9-EGFP and PAX8-Cre/CD9-EGFP mice, but not AMHC-Cre/CD9-EGFP, indicating that plasma EVs are not filtered to the urine. In conclusion, our EV reporter mouse model enables Cre-dependent EV labeling, providing a new approach to study cell-specific EVs in vivo and gain new insight into their physiological and pathophysiological function.

scholarly journals Covalently tethering siRNA to hydrogels for localized, controlled release and gene silencing

2019 ◽  
Vol 5 (8) ◽  
pp. eaax0801 ◽  
Author(s):  
Minh Khanh Nguyen ◽  
Cong Truc Huynh ◽  
Alex Gilewski ◽  
Samantha E. Wilner ◽  
Keith E. Maier ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Therapeutic Potential ◽  
Sirna Delivery ◽  
Hydrolytic Degradation ◽  
Nucleic Acid Delivery ◽  
Covalent Bonds ◽  
Disulfide Linkages ◽  
Green Fluorescent ◽  
Interfering Rna ◽  
Transfection Agent

Small interfering RNA (siRNA) has found many applications in tissue regeneration and disease therapeutics. Effective and localized siRNA delivery remains challenging, reducing its therapeutic potential. Here, we report a strategy to control and prolong siRNA release by directly tethering transfection-capable siRNA to photocrosslinked dextran hydrogels. siRNA release is governed via the hydrolytic degradation of ester and/or disulfide linkages between the siRNA and hydrogels, which is independent of hydrogel degradation rate. The released siRNA is shown to be bioactive by inhibiting protein expression in green fluorescent protein–expressing HeLa cells without the need of a transfection agent. This strategy provides an excellent platform for controlling nucleic acid delivery through covalent bonds with a biomaterial and regulating cellular gene expression, which has promising potential in many biomedical applications.

Nuclear localization of angiotensinogen in astrocytes

2005 ◽  
Vol 288 (2) ◽  
pp. R539-R546 ◽  
Author(s):  
Mikhiela Sherrod ◽  
Xuebo Liu ◽  
Xiaoji Zhang ◽  
Curt D. Sigmund
Keyword(s):  
Fusion Protein ◽  
Nuclear Localization ◽  
Fluorescent Protein ◽  
Primary Cultures ◽  
Endocrine Function ◽  
Astrocytoma Cell ◽  
Protein Encoding ◽  
Localization Signal ◽  
Green Fluorescent ◽  
The Brain

In the brain, angiotensinogen (AGT) is primarily expressed in astrocytes; brain ANG II derived from locally produced AGT has been shown to influence blood pressure. To better understand the molecular basis of AGT expression in the brain, we identified a human astrocytoma cell line, CCF-STTG1, that expresses endogenous AGT mRNA and produces AGT protein. Studies examining CCF-STTG1 cell AGT after N- and O-glycosidase suggest that AGT may not be posttranslationally modified by glycosylation in these cells as it is in plasma. Small amounts of AGT (5% of HepG2) were detected in the culture medium, suggesting a low rate of AGT secretion. Immunocytochemical examination of AGT in CCF-STTG1 cells revealed mainly nuclear localization. Although this has not been previously reported, it is consistent with nuclear localization of other serpin family members. To examine this further, we generated a fusion protein consisting of green fluorescent protein (GFP) and human AGT and examined subcellular localization by confocal microscopy after confirming expression of the fusion protein by Western blot. In CCF-STTG1 cells, a control GFP construct lacking AGT was mainly localized in the cytoplasm, whereas the GFP-AGT fusion protein was primarily localized in the nucleus. To map the location of a potential nuclear localization signal, overlapping 500-bp fragments of human AGT cDNA were fused in frame downstream of GFP. Although four of the fusion proteins exhibited either perinuclear or cytoplasmic localization, one fusion protein encoding the COOH terminus of AGT was localized in the nucleus. Importantly, nuclear localization of human AGT was confirmed in primary cultures of glial cells isolated from transgenic mice expressing the human AGT under the control of its own endogenous promoter. Our results suggest that AGT may have a novel intracellular role in the brain apart from its predicted endocrine function.

scholarly journals Iba1+/NG2+ Macrophage-Like Cells Expressing a Variety of Neuroprotective Factors Ameliorate Ischemic Damage of the Brain

2009 ◽  
Vol 30 (3) ◽  
pp. 603-615 ◽  
Author(s):  
Anna Smirkin ◽  
Hiroaki Matsumoto ◽  
Hisaaki Takahashi ◽  
Akihiro Inoue ◽  
Masahiko Tagawa ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Fluorescent Protein ◽  
Artery Occlusion ◽  
Brain Lesions ◽  
Ischemic Lesion ◽  
Ischemic Brain ◽  
Green Fluorescent ◽  
High Level ◽  
Cerebral Artery Occlusion ◽  
The Brain

In a transient 90-min middle cerebral artery occlusion (MCAO) model of rats, a large ischemic lesion is formed where macrophage-like cells massively accumulate, many of which express a macrophage marker, Iba1, and an oligodendrocyte progenitor cell marker, NG2 chondroitin sulfate proteoglycan (NG2); therefore, the cells were termed BINCs (Brain Iba1+/NG2+Cells). A bone marrow transplantation experiment using green-fluorescent protein-transgenic rats showed that BINCs were derived from bone marrow. 5-Fluorouracil (5FU) injection at 2 days post reperfusion (2 dpr) markedly reduced the number of BINCs at 7 dpr, causing enlargement of necrotic volumes and frequent death of the rats. When isolated BINCs were transplanted into 5FU-aggravated ischemic lesion, the volume of the lesion was much reduced. Quantitative real-time RT-PCR showed that BINCs expressed mRNAs encoding bFGF, BMP2, BMP4, BMP7, GDNF, HGF, IGF-1, PDGF-A, and VEGF. In particular, BINCs expressed IGF-1 mRNA at a very high level. Immunohistochemical staining showed that IGF-1-expressing BINCs were found not only in rat but also human ischemic brain lesions. These results suggest that bone marrow-derived BINCs play a beneficial role in ischemic brain lesions, at least in part, through secretion of neuroprotective factors.

Relative Strengths and Regulation of Different Promoter-Associated Sequences for Various blaSHV Genes and Their Relationships to β-Lactam Resistance

2017 ◽  
Vol 27 (2) ◽  
pp. 91-101 ◽  
Author(s):  
Yao Zhai ◽  
Zhao Zhang ◽  
Zhanwei Wang ◽  
Yusheng Chen ◽  
Qi Wang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Green Fluorescent Protein ◽  
Mrna Expression ◽  
Inhibitory Concentration ◽  
Fluorescent Protein ◽  
Mrna Levels ◽  
Associated Sequences ◽  
Regulation Function ◽  
Green Fluorescent ◽  
Mrna Expression Levels

<b><i>Aims:</i></b> This work investigated the relative strengths of different <i>bla</i><sub>SHV</sub> promoter-associated sequences and their regulation function in <i>bla</i><sub>SHV</sub> expression and β-lactam resistance. <b><i>Methods:</i></b> Recombinant plasmids with the promoter-associated sequences (P-W, P-S, P-IS, and P-WPD), <i>tac</i> promoter, and combined fragments of promoter and <i>bla</i><sub>SHV</sub> were separately constructed and transformed into <i>Escherichia coli</i> DH5α. The relative strengths of the promoters indicated by the intensities of green fluorescent protein and the mRNA expression levels of <i>bla</i><sub>SHV</sub> were compared. The minimum inhibitory concentration and extended spectrum β-lactamase phenotypes were evaluated. <b><i>Results:</i></b> The relative strengths were ranked as P-<i>tac</i> > P-WPD > P-IS > P-S > P-W. The mRNA expression and β-lactam resistance levels of the different promoter-associated sequence groups were generally consistent with the strength rank, but the extent of <i>gfp</i> and <i>bla</i><sub>SHV</sub> mRNA levels varied significantly in each group. The β-lactam resistance levels were inconsistent with the strength rank in certain <i>bla</i><sub>SHV</sub> groups. In relation to the different promoter-associated sequences,<i> bla</i><sub>SHV-ESBLs</sub> displayed significantly different change modes of β-lactam resistance compared with <i>bla</i><sub>SHV-non-ESBLs</sub>. <b><i>Conclusion:</i></b> The mRNA expression and β-lactam resistance of the <i>bla</i><sub>SHV</sub> showed consistencies and inconsistencies with the strengths of the promoter-associated sequences. The mechanisms accounting for these discrepancies need further investigation.

scholarly journals Transgenic Mice Expressing Green Fluorescent Protein under the Control of the Corticotropin-Releasing Hormone Promoter

Endocrinology ◽  
2009 ◽  
Vol 150 (12) ◽  
pp. 5626-5632 ◽  
Author(s):  
Tamar Alon ◽  
Ligang Zhou ◽  
Cristian A. Pérez ◽  
Alastair S. Garfield ◽  
Jeffrey M. Friedman ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Artificial Chromosome ◽  
Brain Regions ◽  
Ingestive Behavior ◽  
Physiological Processes ◽  
Functional Relevance ◽  
Green Fluorescent ◽  
And Function ◽  
The Brain

Abstract CRH is widely expressed in the brain and is of broad functional relevance to a number of physiological processes, including stress response, parturition, immune response, and ingestive behavior. To delineate further the organization of the central CRH network, we generated mice expressing green fluorescent protein (GFP) under the control of the CRH promoter, using bacterial artificial chromosome technology. Here we validate CRH-GFP transgene expression within specific brain regions and confirm the distribution of central GFP-producing cells to faithfully recapitulate that of CRH-expressing cells. Furthermore, we confirm the functional integrity of a population of GFP-producing cells by demonstrating their apposite responsiveness to nutritional status. We anticipate that this transgenic model will lend itself as a highly tractable tool for the investigation of CRH expression and function in discrete brain regions.

Comparison of the therapeutic potential of adult and embryonic neural precursor cells in a rat model of Parkinson disease

2008 ◽  
Vol 108 (1) ◽  
pp. 149-159 ◽  
Author(s):  
Kenichiro Muraoka ◽  
Tetsuro Shingo ◽  
Takao Yasuhara ◽  
Masahiro Kameda ◽  
Wen Ji Yuen ◽  
...  
Keyword(s):  
Parkinson Disease ◽  
Rat Model ◽  
Fluorescent Protein ◽  
Therapeutic Potential ◽  
Precursor Cells ◽  
Neural Precursor Cells ◽  
Neural Precursor ◽  
Therapeutic Effects ◽  
Adenoviral Infection

Object The therapeutic effects of adult and embryonic neural precursor cells (NPCs) were evaluated and their therapeutic potential compared in a rat model of Parkinson disease. Methods Adult NPCs were obtained from the subventricular zone and embryonic NPCs were taken from the ganglionic eminence of 14-day-old embryos. Each NPC type was cultured with epidermal growth factor. The in vitro neuronal differentiation rate of adult NPCs was approximately equivalent to that of embryonic NPCs after two passages. Next, the NPCs were transfected with either green fluorescent protein or glial cell line–derived neurotrophic factor (GDNF) by adenoviral infection and transplanted into the striata in a rat model of Parkinson disease (PD) induced by unilateral intrastriatal injection of 6-hydroxydopamine. An amphetamine-induced rotation test was used to evaluate rat behavioral improvement, and immunohistochemical analysis was performed to compare grafted cell survival, differentiation, and host tissue changes. Results The rats with GDNF-transfected NPCs had significantly fewer amphetamine-induced rotations and less histological damage. Except for the proportion of surviving grafted cells, there were no significant differences between adult and embryonic NPCs. Conclusions Adult and embryonic NPCs have a comparable therapeutic potential in a rat model of PD.

Sign in / Sign up

Export Citation Format

Share Document