Biology subjects

Martinez-Pizarro, A.

Publications and source records attributed to Martinez-Pizarro, A..

3 recordsLinked to original sources

bioRxiv2024

OLIGONUCLEOTIDES TARGETING THE SPLICE ACCEPTOR SITE DOWNSTREAM OF A MICROEXON AS AN INNOVATIVE THERAPY FOR AUTISM

BackgroundMicroexons are highly conserved and mostly neuronal-specific 3-27 nucleotide exons, enriched in genes linked to autism spectrum disorders (ASD). We have previously shown decreased inclusion of a neuronal specific 24 bp microexon (exon 4) of the translational regulator CPEB4 in brains of idiopathic ASD cases and that this leads to CPEB4 aggregation and subsequent under-expression of multiple high confidence ASD-risk genes. Furthermore, enhanced skipping of the CPEB4 microexon is also a novel etiological mechanism in schizophrenia (SCZ). MethodsIn this work we designed and tested in neuroblastoma cells a series of splice switching antisense oligonucleotides (SSO) targeting the CPEB4 genomic region surrounding the microexon. ResultsSSOs targeting candidate intronic regions near the microexon resulted in a decrease in microexon inclusion by blocking hnRNPC/PTPB1 binding, thus mimicking the isoform imbalance observed in ASD. However, based on the kinetic coupling model correlating transcriptional elongation with splicing regulation, we identified SSOs targeting downstream 3 splice site of exon 5 that favoured microexon inclusion in a dose-dependent manner and resulted in increased protein levels of FOXP1 and AUTS2, two high-confidence ASD risk genes that are known targets of CPEB4 and whose protein levels are reduced in ASD. ConclusionsThese results deepen our understanding of the complex splicing regulation of microexons and open new applications of SSOs to treat diseases such as ASD and SCZ that are characterized by altered microexon inclusion.

neuroscience↗

bioRxiv2023

PAH DEFICIENT PATHOLOGY IN HUMANIZED c.1066-11G>A PHENYLKETONURIA MICE

We have generated using CRISPR/Cas9 technology a partially humanized mouse model of the neurometabolic disease phenylketonuria (PKU), carrying the highly prevalent PAH variant c.1066-11G>A. This variant creates an alternative 3 splice site, leading to the inclusion of 9 nucleotides coding for 3 extra amino acids between Q355 and Y356 of the protein. Homozygous Pah c.1066-11A mice, with a partially humanized intron 10 sequence with the variant, accurately recapitulate the splicing defect and present almost undetectable hepatic PAH activity. They exhibit fur hypopigmentation, lower brain and body weight and reduced survival. Blood and brain phenylalanine levels are elevated, along with decreased tyrosine, tryptophan and monoamine neurotransmitter levels. They present behavioral deficits, mainly hypoactivity and diminished social interaction, locomotor deficiencies and an abnormal hind-limb clasping reflex. Changes in the morphology of glial cells, increased GFAP and Iba1 staining signals and decreased myelinization are observed. Hepatic tissue exhibits nearly absent PAH protein, reduced levels of chaperones DNAJC12 and HSP70 and increased autophagy markers LAMP1 and LC3BII, suggesting possible coaggregation of mutant PAH with chaperones and subsequent autophagy processing. This PKU mouse model with a prevalent human variant represents a useful tool for pathophysiology research and for novel therapies development.

pathology↗

bioRxiv2023

Regulating PCCA gene expression by modulation of pseudoexon splicing patterns to rescue enzyme activity in propionic acidemia

Pseudoexons are nonfunctional intronic sequences that can be activated by deep intronic sequence variation. Activation increases pseudoexon inclusion in mRNA and interferes with normal gene expression. The PCCA c.1285-1416A>G variation activates a pseudoexon and causes the severe metabolic disorder, propionic acidemia, by deficiency of the propionyl-CoA carboxylase enzyme encoded by PCCA and PCCB. We characterized this pathogenic pseudoexon activation event in detail and identified hnRNP A1 to be important for normal repression. The PCCA c.1285-1416A>G variation disrupts an hnRNP A1-binding splicing silencer and simultaneously creates a splicing enhancer. We demonstrate that blocking this region of regulation with splice-switching antisense oligonucleotides restores normal splicing and rescues enzyme activity in patient fibroblasts and in a cellular model created by CRISPR gene editing. The PCCA pseudoexon can be exploited as a gene-regulatory switch, as healthy tissues show relatively high levels of inclusion. By blocking inclusion of the non-activated wild type pseudoexon, we increase both PCCA and PCCB protein levels, which increases the activity of the heterododecameric enzyme. Surprisingly, we can increase enzyme activity from residual levels not only in patient fibroblasts harboring PCCA missense variants, but also those harboring PCCB missense variants. This could be a potential treatment strategy for propionic acidemia.

molecular biology↗