Biology subjects

Sastre, D.

Publications and source records attributed to Sastre, D..

4 recordsLinked to original sources

bioRxiv2023

Robust nuclease-dead S. aureus dCas9-mediated alpha-synuclein knockdown in substantia nigra in a humanized mouse model of Parkinson's disease

Parkinsons disease (PD) is becoming increasingly prevalent due to an aging society, which places a substantial disease burden on patients and their families and an annual cost estimated at 52 billion dollars. However, no approved disease modulatory therapies that halt disease progression are available. Alpha-synuclein is a critical therapeutic target found in aggregated form in Lewy bodies which is the diagnostic hallmark of PD. Familial autosomal dominant forms of PD can present with causative exonic point mutations, copy number multiplications, and non-coding risk variants in the alpha-synuclein gene. The disease onset, severity, and progression depend on the gene expression levels of the alpha-synuclein. Here, we demonstrate that Streptococcus aureus dCas9 (sadCas9)-mediated CRISPR interference (CRISPRi) reduces alpha-synuclein mRNA and protein levels in a humanized mouse model. The mechanism of action is based on the principle that a complementary single guide RNA (sgRNA) recruits the sadCas9 protein to the promoter region of alpha-synuclein and modulates target gene transcription, leading to reduced gene expression. We show robust downregulation of alpha-synuclein in neurons after unilateral stereotactic injection into the substantia nigra of adult mice 1 and 6 months after surgery. This work shows proof of concept that viral-mediated sadCas9 CRISPR interference can be a promising therapeutic strategy to reduce alpha-synuclein in vivo. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=168 SRC="FIGDIR/small/556425v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@4d9247org.highwire.dtl.DTLVardef@beb419org.highwire.dtl.DTLVardef@21e0f9org.highwire.dtl.DTLVardef@391d5a_HPS_FORMAT_FIGEXP M_FIG C_FIG eTOC synopsisThis study focuses on the development of an AAV9 nuclease-dead S. aureus CRISPR/Cas9 expression system designed to target the human SNCA promoter. The objective is to achieve downregulation of -synuclein (a-syn) expression. After performing surgery and introducing the expression system, the levels of a-syn were measured at 1 month and 6 months post-surgery. The results indicate a significant downregulation of a-syn at both time points. Furthermore, it was observed that the initial immune response to the system attenuated over time, reaching control levels at the 6-month mark. These findings suggest the potential of this expression system for long-term downregulation of a-syn and provide insights into the immune response dynamics associated with its use.

neuroscience↗

bioRxiv2023

A generic binding pocket for small molecule IKs activators at the extracellular inter-subunit interface of KCNQ1 and KCNE1 channel complexes

The cardiac IKs ion channel comprises KCNQ1, calmodulin, and KCNE1 in a dodecameric complex which provides a repolarizing current reserve at higher heart rates and protects from arrhythmia syndromes that cause fainting and sudden death. Pharmacological activators of IKs are therefore of interest both scientifically and therapeutically for treatment of IKs loss-of-function disorders. One group of chemical activators are only active in the presence of the accessory KCNE1 subunit and here we investigate this phenomenon using molecular modeling techniques and mutagenesis scanning in mammalian cells. A generalized activator binding pocket is formed extracellularly by KCNE1, the domain-swapped S1 helices of one KCNQ1 subunit and the pore/turret region made up of two other KCNQ1 subunits. A few residues, including K41, A44 and Y46 in KCNE1, W323 in the KCNQ1 pore, and Y148 in the KCNQ1 S1 domain, appear critical for the binding of structurally diverse molecules, but in addition, molecular modeling studies suggest that induced fit by structurally different molecules underlies the generalized nature of the binding pocket. Activation of IKs is enhanced by stabilization of the KCNQ1-S1/KCNE1/pore complex, which ultimately slows deactivation of the current, and promotes outward current summation at higher pulse rates. Our results provide a mechanistic explanation of enhanced IKscurrents by these activator compounds and provide a map for future design of more potent therapeutically useful molecules.

pharmacology and toxicology↗

bioRxiv2023

Nuclease-dead S. aureus Cas9 downregulates alpha-synuclein and reduces mtDNA damage and oxidative stress levels in patient-derived stem cell model of Parkinson's disease

Parkinsons disease (PD) is one of the most common neurodegenerative diseases, but no disease modifying therapies have been successful in clinical translation presenting a major unmet medical need. A promising target is alpha-synuclein or its aggregated form, which accumulates in the brain of PD patients as Lewy bodies. While it is not entirely clear which alpha-synuclein protein species is disease relevant, mere overexpression of alpha-synuclein in hereditary forms leads to neurodegeneration. To specifically address gene regulation of alpha-synuclein, we developed a CRISPR interference (CRISPRi) system based on the nuclease dead S. aureus Cas9 (SadCas9) fused with the transcriptional repressor domain Krueppel-associated box to controllably repress alpha-synuclein expression at the transcriptional level. We screened single guide (sg)RNAs across the SNCA promoter and identified several sgRNAs that mediate downregulation of alpha-synuclein at varying levels. CRISPRi downregulation of alpha-synuclein in iPSC-derived neuronal cultures from a patient with an SNCA genomic triplication showed functional recovery by reduction of oxidative stress and mitochondrial DNA damage. Our results are proof-of-concept in vitro for precision medicine by targeting the SNCA gene promoter. The SNCA CRISPRi approach presents a new model to understand safe levels of alpha-synuclein downregulation and a novel therapeutic strategy for PD and related alpha-synucleinopathies.

neuroscience↗

bioRxiv2019

Focused screening reveals functional effects of microRNAs differentially expressed in colorectal cancer

BackgroundColorectal cancer (CRC) is still a leading cause of death worldwide. Recent studies have pointed to an important role of microRNAs carcinogenesis. In fact, several microRNAs have been described as aberrantly expressed in CRC tissues and in the serum of patients. More specifically, microRNAs with dual roles in both cancer and stem cell survival represent a potential source of novel molecular targets in CRC due to their described functions in normal and deregulated proliferation. However, the functional outcomes of microRNA aberrant expression still need to be explored at the cellular level. Here, we aimed to investigate the effects of microRNAs involved in the control of pluripotency of stem cells in the proliferation and cell death of a colorectal cancer cell line.\n\nMethodsWe performed transfection of 31 microRNA mimics in HCT116 CRC cells. Cell proliferation and cell death were measured after 4 days of treatment using fluorescence staining in a high content screening platform. Total number of live and dead cells were automatically counted and analyzed. To reveal mRNA targets, we used an oligonucleotide microarray. Functional classification of targets was done using DAVID tool. Gene expression of potential mRNA targets was performed by qPCR.\n\nResultsTwenty microRNAs altered the proliferation of HCT116 cells in comparison to control. Three microRNAs significantly repressed cell proliferation and induced cell death simultaneously (miR-22-3p, miR-24-3p, and miR-101-3p). Interestingly, all anti-proliferative microRNAs in our study had been previously described as poorly expressed in the CRC samples and were implicated in the disease. Microarray analysis of miR-101-3p targets revealed Wnt and cancer as pathways regulated by this microRNA. Specific repression of anti-apoptotic isoform of MCL-1, a member of the BCL-2 family, was also identified as a possible mechanism for miR-101-3p anti-proliferative/pro-apoptotic effect.\n\nConclusionsmicroRNAs described as upregulated in CRC tend to induce proliferation in vitro, whereas microRNAs described as poorly expressed in CRC halt proliferation and induce cell death in vitro. Selective inhibition of anti-apoptotic MCL-1 contributes to anti-tumoral activity of miR-101-3p.

cancer biology↗