Biology subjects

Cremonesi, A.

Publications and source records attributed to Cremonesi, A..

3 recordsLinked to original sources

bioRxiv2024

Base editing of Ptbp1 in neurons alleviates symptoms in a mouse model for Parkinson's disease

Parkinsons disease (PD) is a multifactorial disease caused by irreversible progressive loss of dopaminergic neurons (DANs). Recent studies have reported successful conversion of astrocytes into DANs by repressing polypyrimidine tract binding protein 1 (PTBP1), which led to the rescue of motor symptoms in a chemically-induced mouse model of PD. However, several follow-up studies have questioned the validity of this astrocyte to DAN conversion model. In this study, we devised an adenine base editing strategy to downregulate PTBP1 in astrocytes and neurons in a chemically-induced PD mouse model. While PTBP1 downregulation in astrocytes had no effect, we observed that PTBP1 downregulation in neurons of the substantia nigra pars compacta and striatum resulted in the expression of the DAN marker tyrosine hydroxylase (TH) in non-dividing neurons, which was associated with an increase in striatal dopamine concentrations and a rescue of forelimb akinesia and spontaneous rotations. Phenotypic analysis using multiplexed iterative immunofluorescence imaging further revealed that most of the TH-positive cells in the striatum co-expressed the dopaminergic marker DAT and the pan-neuronal marker NEUN, with the majority of these triple-positive cells being classified as mature GABAergic neurons. Additional research is needed to fully elucidate the molecular mechanisms underlying the expression of the observed markers and understand how the formation of these cells contributes to the rescue of spontaneous motor behaviors. Nevertheless, our findings support a model where neuronal, but not astrocytic, downregulation of PTBP1 can mitigate symptoms in PD mice.

neuroscience↗

bioRxiv2024

Treatment of a genetic liver disease in mice through transient prime editor expression

Prime editing is a versatile genome editing technology that does not rely on DNA double-strand break formation and homology-directed repair (HDR). This makes it a promising tool for correcting pathogenic mutations in tissues consisting predominantly of postmitotic cells, such as the liver. While recent studies have already demonstrated proof-of-concept for in vivo prime editing, the use of viral delivery vectors resulted in prolonged prime editor (PE) expression, posing challenges for clinical application. Here, we developed an in vivo prime editing approach where we delivered the pegRNA using self-complementary adeno-associated viral (scAAV) vectors and the prime editor using nucleoside-modified mRNA encapsulated in lipid nanoparticles (LNPs). This methodology led to transient expression of the PE for 48h and 26% editing at the Dnmt1 locus using AAV doses of 2.5x1013 vector genomes (vg)/kg and a single dose of 3mg/kg mRNA-LNP. When targeting the pathogenic mutation in the Pahenu2 mouse model of phenylketonuria (PKU), we achieved 4.3% gene correction using an AAV dose of 2.5x1013 vg/kg and three doses of 2 mg/kg mRNA-LNP. Editing was specific to the liver and the intended locus, and was sufficient to reduce blood L-phenylalanine (Phe) levels from over 1500 {micro}mol/l to below the therapeutic threshold of 600 {micro}mol/l. Our study demonstrates the feasibility of in vivo gene correction in the liver with transient PE expression, bringing prime editing closer to clinical application.

bioengineering↗

bioRxiv2023

GLUD1 dictates muscle stem cell differentiation by controlling mitochondrial glutamate levels

Muscle stem cells (MuSCs) enable muscle growth and regeneration after exercise or injury. Upon activation MuSCs metabolically rewire to meet the changing demands of proliferation. Here we describe that primary changes in metabolism itself can dictate MuSC fate decisions to control differentiation and fusion. We found that glutamine anaplerosis into the TCA cycle decreases during MuSC differentiation and coincides with decreased expression of the mitochondrial glutamate deaminase GLUD1. Genetic deletion of Glud1 in proliferating MuSCs resulted in precocious differentiation and imbalanced fusion combined with loss of self-renewal in vitro and in vivo. Mechanistically, deleting Glud1 caused mitochondrial glutamate accumulation in proliferating MuSCs and inhibited the malate-aspartate shuttle (MAS). Restoring MAS activity by supplementation of alanine normalized differentiation. In conclusion, high GLUD1 activity in proliferating MuSCs prevents deleterious mitochondrial glutamate accumulation and inactivation of the MAS. It thereby acts as a compartment specific metabolic brake on MuSC differentiation. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=194 SRC="FIGDIR/small/560525v1_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@e3585dorg.highwire.dtl.DTLVardef@1be4a54org.highwire.dtl.DTLVardef@1f077org.highwire.dtl.DTLVardef@15220f1_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIGlutamine is the major TCA cycle substrate in MuSCs with decreasing contribution upon differentiation. C_LIO_LILoss of Glud1 impairs MuSC self-renewal capacity and causes imbalanced fusion in vitro and in vivo. C_LIO_LIGlud1 deletion leads to mitochondrial glutamate trapping and malate-aspartate shuttle (MAS) dysfunction. C_LIO_LIRestoration of MAS activity in Glud1 deficient MuSCs reverses precocious differentiation and imbalanced fusion. C_LI

cell biology↗