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1.
Gene Ther ; 31(5-6): 345-351, 2024 05.
Artigo em Inglês | MEDLINE | ID: mdl-38467879

RESUMO

Adeno-associated viruses (AAV) are commonly used in the scientific field due to their diverse application range. However, AAV shedding, the release of virions from the host organism, can impact the safety of AAV-based approaches. An increasing number of authorities require the characterization of vector shedding in clinical trials. Recently, shedding of transduced laboratory animals has also gained attention regarding the necessary disposal measures of their waste products. However, no explicit international regulations for AAV-shedding waste exist. Generating insights into shedding dynamics becomes increasingly relevant to help authorities develop adequate regulations. To date, knowledge of AAV vector shedding in mice is very limited. Moreover, confirmation of functional shed AAV particles in mice is missing. Therefore, we examined feces, urine, and saliva of mice after CNS injection with AAV2/8. It revealed the presence of viral DNA fragments via qPCR for up to 4 days after injection. To examine AAV functionality we performed nested PCR and could not detect full-length viral genomes in any but two collected feces samples. Furthermore, a functional infection assay did not reveal evidence of intact AAV particles. Our findings are supposed to contribute murine shedding data as a foundation to help establish still lacking adequate biosafety regulations in the context of AAV shedding.


Assuntos
DNA Viral , Dependovirus , Vetores Genéticos , Eliminação de Partículas Virais , Animais , Dependovirus/genética , Camundongos , Vetores Genéticos/genética , Vetores Genéticos/administração & dosagem , DNA Viral/genética , Fezes/virologia , Camundongos Endogâmicos C57BL , Saliva/virologia , Humanos
2.
Eur J Neurosci ; 57(5): 739-761, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36656174

RESUMO

Increasing evidence suggests that astrocytes play an important role in the progression of Parkinson's disease (PD). Previous studies on our parkin knockout mouse demonstrated a higher accumulation of damaged mitochondria in astrocytes than in surrounding dopaminergic (DA) neurons, suggesting that Parkin plays a crucial role regarding their interaction during PD pathogenesis. In the current study, we examined primary mesencephalic astrocytes and neurons in a direct co-culture system and discovered that the parkin deletion causes an impaired differentiation of mesencephalic neurons. This effect required the parkin mutation in astrocytes as well as in neurons. In Valinomycin-treated parkin-deficient astrocytes, ubiquitination of Mitofusin 2 was abolished, whereas there was no significant degradation of the outer mitochondrial membrane protein Tom70. This result may explain the accumulation of damaged mitochondria in parkin-deficient astrocytes. We examined differential gene expression in the substantia nigra region of our parkin-KO mouse by RNA sequencing and identified an upregulation of the endoplasmic reticulum (ER) Ca2+ -binding protein reticulocalbin 1 (RCN1) expression, which was validated using qPCR. Immunostaining of the SN brain region revealed RCN1 expression mainly in astrocytes. Our subcellular fractionation of brain extract has shown that RCN1 is located in the ER and in mitochondria-associated membranes (MAM). Moreover, a loss of Parkin function reduced ATP-stimulated calcium-release in ER mesencephalic astrocytes that could be attenuated by siRNA-mediated RCN1 knockdown. Our results indicate that RCN1 plays an important role in ER-associated calcium dyshomeostasis caused by the loss of Parkin function in mesencephalic astrocytes, thereby highlighting the relevance of astrocyte function in PD pathomechanisms.


Assuntos
Cálcio , Retículo Endoplasmático , Doença de Parkinson , Ubiquitina-Proteína Ligases , Animais , Camundongos , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Neurônios Dopaminérgicos/metabolismo , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/patologia , Camundongos Knockout , Doença de Parkinson/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Regulação para Cima
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