Biology subjects

Gonzalez-Aseguinolaza, G.

Publications and source records attributed to Gonzalez-Aseguinolaza, G..

5 recordsLinked to original sources

bioRxiv2025

Locus Coeruleus alpha-Synuclein Overexpression Induces Prodromal Parkinsonian Features in Mice

The locus coeruleus is one of the first brain regions to develop -synuclein pathology during the prodromal phase of Parkinsons Disease, contributing significantly to non-motor symptoms such as cognitive decline, mood alterations or sleep disturbances. However, the precise role of the locus coeruleus in the early stages of the disease remains unclear. To address this, we developed and characterized a novel mouse model based on the PRSx8-driven overexpression of human -synuclein in noradrenergic neurons of the locus coeruleus using an adeno-associated viral vector. Animals were assessed at 1 and 3 months post-injection using an integrated battery of histological, behavioural, neurochemical, and electrophysiological analyses. We observed robust accumulation of phosphorylated -synuclein in the locus coeruleus, along with widespread propagation to projection areas, including the hippocampus, prefrontal cortex, and dorsal raphe. Despite the absence of neuronal loss in the locus coeruleus, we identified reduced noradrenergic axonal integrity and marked decreases in both noradrenaline and serotonin levels, highlighting a disruption of neurochemical balance at an early stage. The electrophysiological data revealed transient alterations in the excitability and intrinsic properties of locus coeruleus neurons in a sex-dependent manner, suggesting that -synuclein pathology induces early functional disruption prior to overt neurodegeneration. Behavioural outcomes demonstrated selective cognitive deficits and mild anxiety-like behaviours, while other non-motor functions remained preserved. Importantly, although several pathological and functional alterations were evident in the initial phases, behavioural impairments were also maintained at later time points, indicating that locus coeruleus-driven pathology exerts long-lasting consequences. Interestingly, female mice exhibited colon shortening, providing evidence of a sex-specific pathological process at the level of the gut-brain axis. We believe that this model accurately replicates key prodromal features of Parkinsons disease, demonstrating that -synuclein-induced pathology in the locus coeruleus leads to functional circuit impairment and cognitive decline. Our findings emphasize the locus coeruleus as a critical site of early vulnerability in Parkinsons Disease and underscore the importance of sex as a biological variable influencing disease progression and therapeutic response.

neuroscience↗

bioRxiv2023

Protective Role of RIPK1 Scaffolding against HDV-Induced Hepatocyte cell death and the Significance of Cytokines in Mice

Hepatitis delta virus (HDV) infection represents the most severe form of human viral hepatitis; however, the mechanisms underlying its pathology remain incompletely understood. We recently developed an HDV mouse model by injecting adeno-associated viral vectors (AAV) containing replication-competent HBV and HDV genomes. This model replicates many features of human infection, including liver injury. Notably, the extent of liver damage can be diminished with anti-TNF- treatment. In the present study, we found that TNF- is mainly produced by macrophages. Downstream of the TNF- receptor (TNFR), the receptor-interacting serine/threonine-protein kinase 1 (RIPK1) serves as a cell fate regulator, playing roles in both cell survival and death pathways. In this study, we explored the function of RIPK1 and other host factors in HDV-induced cell death. We determined that the scaffolding function of RIPK1, and not its kinase activity, offers partial protection against HDV-induced apoptosis. A reduction in RIPK1 expression in hepatocytes through Cas9-mediated gene editing significantly intensifies HDV-induced damage. Contrary to our expectations, the protective effect of RIPK1 was not linked to TNF- or macrophage activation, as their absence did not alter the extent of damage. Intriguingly, in the absence of RIPK1, macrophages confer a protective role. However, in animals unresponsive to type-I IFNs, RIPK1 downregulation did not exacerbate the damage, suggesting RIPK1s role in shielding hepatocytes from type-I IFN-induced cell death. Interestingly, while the damage extent is similar between IFNAR KO and WT mice in terms of transaminase elevation, their cell death mechanisms differ. In conclusion, our findings reveal that HDV-induced type-I IFN production is central to inducing hepatocyte death, and RIPK1s scaffolding function offers protective benefits. Thus, type-I IFN together with TNF-, contribute to HDV-induced liver damage. These insights may guide the development of novel therapeutic strategies to mitigate HDV-induced liver damage and halt disease progression. Author summaryHepatitis D is the most aggressive form of viral hepatitis. Our manuscript underscores the complexity of HDV-induced liver damage, where both viral and host factors play significant roles. Previously, we demonstrated that pharmacological inhibition of TNF- reduced HDV-induced liver damage. This result was corroborated in the present study using TNF--deficient mice. Moreover, we reported that the expression of the HDV antigen might have a cytotoxic effect, and HDV replication induces a strong activation of the innate immune system, accompanied by a substantial production of IFN-{beta}. In this study, we discovered that RIPK1, a molecule described as a cell fate modulator acting downstream of TNF-, plays a protective role during HDV replication. Contrary to our expectations, neither TNF- nor macrophages, the primary producers of TNF-, contributed to this protective effect. Instead, it seems type I IFN was involved. Interestingly, the role of type I IFN in HBV-induced liver damage has recently been proposed. Furthermore, our data reveals that several mechanisms of hepatocyte death are at play simultaneously during HDV replication, with apoptosis being one of them. Additional studies are needed to identify other mechanisms involved. Finally, these findings suggest that therapies targeting TNF- and type-I IFN, or those increasing RIPK1 levels, might be effective in preventing or treating HDV-induced liver damage.

pathology↗

bioRxiv2023

Harnessing the power of whole human liver ex situ normothermic perfusion for preclinical AAV vector evaluation

Developing clinically predictive model systems for evaluating gene transfer and gene editing technologies has become increasingly important in the era of personalized medicine. Liver-directed gene therapies present a unique challenge due to the complexity of the human liver. In this work, we describe the application of whole human liver explants in an ex situ normothermic perfusion system to evaluate a set of fourteen natural and bioengineered adeno-associated viral (AAV) vectors directly in human liver, in the presence and absence of neutralizing human sera. Under non-neutralizing conditions, the recently developed AAV variants, AAV-SYD12 and AAV-LK03, emerged as the most functional variants in terms of cellular uptake and transgene expression. However, when assessed in the presence of human plasma containing anti-AAV neutralizing antibodies (NAbs), vectors of human origin, specifically those derived from AAV2/AAV3b, were extensively neutralized, whereas AAV8-derived variants performed efficiently. This study establishes the use of normothermic liver perfusion as an invaluable preclinical model for evaluating liver-targeted gene therapies and providing guidance for making essential decisions that promote the most effective translational programs.

molecular biology↗

bioRxiv2022

Assessment of pre-clinical liver models based on their ability to predict the liver-tropism of AAV vectors

The liver is a prime target for in vivo gene therapies using recombinant adeno-associated viral vectors (rAAV). Multiple clinical trials have been undertaken for this target in the past 15 years, however we are still to see market approval of the first liver-targeted AAV-based gene therapy. Inefficient expression of the therapeutic transgene, vector-induced liver toxicity and capsid, and/or transgene-mediated immune responses reported at high vector doses are the main challenges to date. One of the contributing factors to the insufficient clinical outcomes, despite highly encouraging preclinical data, is the lack of robust, biologically- and clinically-predictive preclinical models. To this end, this study reports findings of a functional evaluation of six AAV vectors in twelve preclinical models of the human liver, with the aim to uncover which model is the most relevant for the selection of AAV capsid variant for safe and efficient transgene delivery to primary human hepatocytes. The results, generated by studies in models ranging from immortalized cells, iPSC-derived and primary hepatocytes, and primary human hepatic organoids to in vivo models, increased our understanding of the strengths and weaknesses of each system. This should allow the development of novel gene therapies targeting the human liver.

bioengineering↗

bioRxiv2020

In vitro and In vivo Genetic Disease Modelling via NHEJ precise deletions using CRISPR/Cas9

Development of advanced gene and cell therapies for the treatment of genetic diseases requires confident animal and cellular models to test their efficacy and is crucial in the cases where no primary samples from patients are available. CRISPR/Cas9 technology, has become one of the most spread endonuclease tools for editing the genome at will. Moreover, it is known that the use of two guides tends to produce the precise deletion between the guides via NHEJ. Different distances between guides were tested (from 8 to 500 base pairs). We found that distances between the two cutting sites and orientation of Cas9 protein-DNA interaction are important for the efficiency within the optimal range (30-60 bp), we could obtain new genetically reproducible models for two rare disease, a Pyruvate Kinase Deficiency model, using human primary cells, and a (for in vivo primary hyperoxaluria therapeutic mouse model. We demonstrate that the use of a 2-guideRNAs at the optimal distance and orientation is a powerful strategy for disease modelling in those diseases where the availability of primary cells is limited.

bioengineering↗