Characterisation of angiogenin uptake and signalling in astrocytes

Amyotrophic Lateral Sclerosis (ALS) is a progressive, fatal neurodegenerative disease of the motor system affecting both upper and lower motoneurons. The majority of ALS cases occur sporadically, however roughly 10 % show a hereditary component. Mutations in the hypoxiainducible factor angiogenin segregate with ALS pedigrees and the protein is expressed in motoneurons. Angiogenin shows potent neuroprotective properties in vitro and in vivo and application of recombinant human angiogenin to mixed spinal cord cultures revealed non-neuronal uptake of the protein. As the disease affects both motoneurons and non-neuronal neighbouring cells like astrocytes and microglia, a paracrine mechanism of neuroprotection has been suggested. This study was performed to gain insights into the mechanism of angiogenininduced neuroprotection via surrounding glial cells focusing on astrocytes. The vesicular internalisation of recombinant human angiogenin by primary astrocytes was investigated in culture using pharmacological inhibitors, cell transfection and immunocytochernistry. This revealed that angiogenin endocytosis by astrocytes is clathrin-dependent and involves dynamin for vesicle scission. Vesicle budding and trafficking do not require a functional microtubule network. A fraction of the internalised angiogenin is targeted for lysosomal degradation, while the majority remains in uncharacterised sorting endosomes. The receptor for angiogenin on astrocytes was identified to be the heparansulfate proteoglycan syndecan 4 by colocalisation studies and protein knock down. To further elucidate the role of angiogenin in paracrine neuroprotection, modification of the astrocyte secretome was investigated in response to angiogenin treatment by quantitative mass spectrometry. Metabolic protein labelling by Stable Isotope Labelling with Amino acids in Culture (SILAC) was optimised for primary astrocytes and proteins were identified by Fourier transform tandem mass spectrometry (nano-LC-FT-MSIMS). This screen identified over 1,500 proteins in the supernatant of primary astrocytes, many of which are known to exhibit extracellular location like components of the Extracellular Matrix (ECM), cytokines, growth factors and growth factor binding proteins. However, proteins with established intracellular function like splicing factors and ribosomal proteins were detected, too. Quantification using the MaxQuant software showed significant regulation of over 100 proteins in response to angiogenin treatment. The most strongly regulated proteins are involved in modifying the ECM or contribute to immune responses and might be responsible for the neuroprotective effects of angiogenin. These data also suggest the involvement of surrounding immune and endothelial cells in the biological activity of angiogenin. Additionally, local transfer of factors involved in protein translation from astrocytes to surrounding cells may be affected by angiogenin. In conclusion, this study has shed new light on the role of angiogenin in the complex cellular interplay in the Central Nervous System (CNS) focusing on astrocytes and how they interact with neighbouring cells. Further studies will be necessary to elucidate the functional signalling outcome of angiogenin focusing on its neuroprotective activities, in particular regarding ALS pathology with the aim of finding new therapies for this fatal disease

Motoneurons secrete angiogenin to induce RNA cleavage in astroglia.

Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disorder affecting motoneurons. Mutations in angiogenin, encoding a member of the pancreatic RNase A superfamily, segregate with ALS. We previously demonstrated that angiogenin administration shows promise as a neuroprotective therapeutic in studies using transgenic ALS mice and primary motoneuron cultures. Its mechanism of action and target cells in the spinal cord, however, are largely unknown. Using mixed motoneuron cultures, motoneuron-like NSC34 cells, and primary astroglia cultures as model systems, we here demonstrate that angiogenin is a neuronally secreted factor that is endocytosed by astroglia and mediates neuroprotection in paracrine. We show that wild-type angiogenin acts unidirectionally to induce RNA cleavage in astroglia, while the ALS-associated K40I mutant is also secreted and endocytosed, but fails to induce RNA cleavage. Angiogenin uptake into astroglia requires heparan sulfate proteoglycans, and engages clathrin-mediated endocytosis. We show that this uptake mechanism exists for mouse and human angiogenin, and delivers a functional RNase output. Moreover, we identify syndecan 4 as the angiogenin receptor mediating the selective uptake of angiogenin into astroglia. Our data provide new insights into the paracrine activities of angiogenin in the nervous system, and further highlight the critical role of non-neuronal cells in the pathogenesis of ALS

Successful Expansion but Not Complete Restriction of Tropism of Adeno-Associated Virus by In Vivo Biopanning of Random Virus Display Peptide Libraries

Targeting viral vectors to certain tissues in vivo has been a major challenge in gene therapy. Cell type-directed vector capsids can be selected from random peptide libraries displayed on viral capsids in vitro but so far this system could not easily be translated to in vivo applications. Using a novel, PCR-based amplification protocol for peptide libraries displayed on adeno-associated virus (AAV), we selected vectors for optimized transduction of primary tumor cells in vitro. However, these vectors were not suitable for transduction of the same target cells under in vivo conditions. We therefore performed selections of AAV peptide libraries in vivo in living animals after intravenous administration using tumor and lung tissue as prototype targets. Analysis of peptide sequences of AAV clones after several rounds of selection yielded distinct sequence motifs for both tissues. The selected clones indeed conferred gene expression in the target tissue while gene expression was undetectable in animals injected with control vectors. However, all of the vectors selected for tumor transduction also transduced heart tissue and the vectors selected for lung transduction also transduced a number of other tissues, particularly and invariably the heart. This suggests that modification of the heparin binding motif by target-binding peptide insertion is necessary but not sufficient to achieve tissue-specific transgene expression. While the approach presented here does not yield vectors whose expression is confined to one target tissue, it is a useful tool for in vivo tissue transduction when expression in tissues other than the primary target is uncritical

Management of anaphylaxis due to COVID-19 vaccines in the elderly

Older adults, especially men and/or those with diabetes, hypertension, and/or obesity, are prone to severe COVID-19. In some countries, older adults, particularly those residing in nursing homes, have been prioritized to receive COVID-19 vaccines due to high risk of death. In very rare instances, the COVID-19 vaccines can induce anaphylaxis, and the management of anaphylaxis in older people should be considered carefully. An ARIA-EAACI-EuGMS (Allergic Rhinitis and its Impact on Asthma, European Academy of Allergy and Clinical Immunology, and European Geriatric Medicine Society) Working Group has proposed some recommendations for older adults receiving the COVID-19 vaccines. Anaphylaxis to COVID-19 vaccines is extremely rare (from 1 per 100,000 to 5 per million injections). Symptoms are similar in younger and older adults but they tend to be more severe in the older patients. Adrenaline is the mainstay treatment and should be readily available. A flowchart is proposed to manage anaphylaxis in the older patients.Peer reviewe

Yeast Pathway Kit: A Method for Metabolic Pathway Assembly with Automatically Simulated Executable Documentation

We have developed the Yeast Pathway Kit (YPK) for rational and random metabolic pathway assembly in Saccharomyces cerevisiae using reusable and redistributable genetic elements. Genetic elements are cloned in a suicide vector in a rapid process that omits PCR product purification. Single-gene expression cassettes are assembled in vivo using genetic elements that are both promoters and terminators (TP). Cassettes sharing genetic elements are assembled by recombination into multigene pathways. A wide selection of prefabricated TP elements makes assembly both rapid and inexpensive. An innovative software tool automatically produces detailed self-contained executable documentation in the form of pydna code in the narrative Jupyter notebook format to facilitate planning and sharing YPK projects. A D-xylose catabolic pathway was created using YPK with four or eight genes that resulted in one of the highest growth rates reported on D-xylose (0.18 h(-1)) for recombinant S. cerevisiae without adaptation. The two-step assembly of single-gene expression cassettes into multigene pathways may improve the yield of correct pathways at the cost of adding overall complexity, which is offset by the supplied software tool.- This work was supported by the Fundacao para a Ciencia e Tecnologia Portugal (FCT) through Project MycoFat PTDC/AAC-AMB/120940/2010. F.A. was supported by FCT fellowship SFRH/BD/80934/2011. CBMA was supported by the strategic programme UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569) funded by national funds through the FCT I.P. and by the ERDF through the COMPETE2020 - Programa Operational Competitividade e Internacionalizacao (POCI). The authors wish to thank to Dr. Paula Goncalves for the pGXF1 vector, Dr. Daniel Schlieper for the pCAPs vector, Dr. Yukio Nagano for the pSU0 vector, Dr. Peter Kotter, University of Frankfurt, Germany, for the S. cereivise CEN.PK strains, and Dr. Nina Q Meinander for the p4** vectors.info:eu-repo/semantics/publishedVersio

Targeting of AAV capsid mutants selected on murine lung tissue <i>in vivo.</i>

<p>AAV luciferase vectors displaying selected or control capsids (wild-type or random insert VRRPRFW) were injected intravenously into female FVB mice. Tissue was harvested after 8 or 28 d, respectively, and processed as indicated. A: Evaluation of lung homing. Lung tissue was harvested 8 days after vector injection and the amount of AAV genomes was determined by quantitative PCR. Data represent mean values from n = 3 mice per group, analyzed in triplicates ± SD. B: <i>In vivo</i> lung gene transfer by selected AAV after intravenous injection. Lung tissue was harvested 28 days after vector injection, and luciferase activity was determined as relative light units (RLU) per mg protein. Bars indicate the median value, n = 5 mice per group (** = p<0.001 targeted vectors vs. wild-type and random insert control). C: <i>In vivo</i> transduction of various tissues in mice by AAV library mutants selected for lung transduction. Tissues were harvested and luciferase activity was determined as in 5B. The dotted line indicates the threshold beyond which luciferase expression data could be reliably delineated from background signal. Data represent mean values ± SEM, n = 5 mice per group. * p<0.05; ** p<0.01 targeted vectors vs. wild-type AAV-2. # p<0.05; ## p<0.01 targeted vectors vs. random insert control.</p

Peptides enriched in lung tissue during <i>in vivo</i> selection for lung-homing AAV after four rounds of selection

a<p>single letter code; shared amino acid patterns are highlighted in red letters</p>b<p>charge pattern of amino acid side chains: +, positively charged; − negatively charged; x, uncharged polar; y, nonpolar.</p

Gene delivery by AAV capsid mutants selected for breast cancer transduction <i>in vivo.</i>

<p>AAV luciferase vectors displaying selected peptides or controls (wild-type or VRRPRFW) were injected intravenously into female PymT tumor-bearing mice. After 8 d, representative tissues were harvested and luciferase activities were determined in individual tissues as relative light units (RLU) per mg protein. A: <i>In vivo</i> transduction of tumor tissue in PymT transgenic FVB mice by selected AAV mutants. Bars indicate the median, n = 5 mice per group. * p<0.05 targeted vectors vs. wild-type. # p<0.05 targeted vectors vs. random insert control. B: <i>In vivo</i> transduction of various non-cancerous tissues in PymT transgenic FVB mice by tumor-selected AAV mutants. The dotted line indicates the threshold beyond which luciferase expression data could be reliably delineated from background signal. Data represent mean values ± SEM, n = 5 mice per group. * p<0.05; ** p<0.01 targeted vectors vs. wild-type AAV-2. # p<0.05; ## p<0.01 targeted vectors vs. random insert control.</p

Kinetics of circulating AAV peptide library particles is similar to wild-type AAV.

<p>A random X₇ peptide library or wild-type AAV-2 viruses were injected intravenously at 1×10¹⁰ vg per mouse. Blood samples were collected after indicated time points and the amount of circulating viral particles in the serum was determined by real-time PCR. Data represent mean values from n = 3 mice per group, analyzed in triplicates ± SD.</p

Pathways used for selection of targeted viral capsids by screening random AAV display peptide libraries.

<p>For all selection pathways, genomic DNA containing <i>cap</i> gene fragments from internalized library viruses was extracted from the target cells or tissue. Library inserts were amplified by nested PCR and cloned back into the AAV library backbone plasmid pMT-202-6. The resulting pre-selected plasmid library was used to produce a secondary AAV library by transfection into 293T cells and subsequent superinfection with Ad5. Pre-selected AAV libraries were re-subjected to selection on the target cells <i>in vitro</i> or the target tissue <i>in vivo</i>. Preceding the amplification step, the library selection was done according to one of the following three pathways: Pathway A, <i>in vitro</i> selection: A random AAV display peptide library was incubated on primary breast cancer dissociation cultures derived from female tumor-bearing PymT mice. Non-internalized AAV library particles were removed by extensive washing followed by trypsin digestion prior to DNA extraction and AAV insert amplification. Pathway B, <i>in vivo</i>/<i>ex vivo</i> selection: A random AAV display peptide library was injected intravenously into female tumor-bearing PymT mice. After 24 hours, primary tumor cells of the injected mouse were prepared as in pathway A and grown <i>ex vivo</i> for 96 hours prior to DNA extraction and AAV insert amplification. Pathway C, <i>in vivo</i> selection: A random AAV display peptide library was injected as in pathway B in tumor-bearing mice (for selection of tumor-homing AAV) or wild-type mice (for selection of lung homing AAV), respectively. After 48 hours, the target tissue (tumor or lung, respectively) was removed and lysed, and DNA was extracted for AAV insert amplification.</p