Biology subjects

Tessari, I.

Publications and source records attributed to Tessari, I..

4 recordsLinked to original sources

bioRxiv2024

PAK6 promotes neuronal autophagy by regulating TFEB nuclear translocation.

Autophagy is a highly conserved homeostatic process essential for the bulk degradation of cytoplasmic components and aggregated proteins. Multiple evidence indicates that impairment of (macro)autophagy leads to neurodegeneration, such as Parkinson disease (PD). Our previous work showed that p21 activated kinase 6 (PAK6) interacts with the PD-associated leucine-rich repeat kinase (LRRK2) to promote neurite outgrowth in the mouse striatum; still the function of PAK6 in the brain is largely unknown. Here, we found that downregulation of neuronal but not glial mbt, the D. melanogaster homolog of PAK6, impairs autophagy-lysosomal function. PAK6 overexpression in cells and in C. elegans increases transcription factor EB (TFEB) nuclear translocation in a kinase activity-dependent manner. Mechanistically, PAK6 forms a complex with TFEB to regulate its nuclear localization in a manner dependent on phosphorylation of and binding to 14-3-3 proteins and phosphorylation of TFEB at S467. In line with its ability to promote neuronal autophagy, mbt downregulation exacerbates alpha-synuclein toxicity in Drosophila dopaminergic neurons. Moreover, PAK6 overexpression in the substantia nigra of mutant LRRK2 mice reduces the burden of phosphorylated alpha-synuclein in dopaminergic neurons. Altogether, our study uncovers a novel role of PAK6 as a positive regulator of autophagy via TFEB and suggests that modulating its activity may represent a way to selectively turn on autophagy in neurons, with implications for the treatment of neurodegenerative disorders.

neuroscience↗

bioRxiv2024

PAK6 rescues pathogenic LRRK2-mediated ciliogenesis and centrosomal cohesion defects in a mutation-specific manner

P21 activated kinase 6 (PAK6) is a serine-threonine kinase with physiological expression enriched in the brain and overexpressed in a number of human tumors. While the role of PAK6 in cancer cells has been extensively investigated, the physiological function of the kinase in the context of brain cells is poorly understood. Our previous work uncovered a link between PAK6 and the Parkinsons disease (PD)-associated kinase LRRK2, with PAK6 controlling LRRK2 activity and subcellular localization via phosphorylation of 14-3-3 proteins. Here, to gain more insights into PAK6 physiological function, we performed protein-protein interaction arrays and identified a subgroup of PAK6 binders related to ciliogenesis. We confirmed that endogenous PAK6 localizes at both the centrosome and the cilium, and positively regulates ciliogenesis not only in tumor cells but also in neurons and astrocytes. Strikingly, PAK6 rescues ciliogenesis and centrosomal cohesion defects associated with the G2019S but not the R1441C LRRK2 PD mutation. Since PAK6 binds LRRK2 via its GTPase/Roc-COR domain and the R1441C mutation is located in the Roc domain, we used microscale thermophoresis and AlphaFold2-based computational analysis to demonstrate that PD mutations in LRRK2 affecting the Roc-COR structure substantially decrease PAK6 affinity, providing a rationale for the differential protective effect of PAK6 toward the distinct forms of mutant LRRK2. Altogether, our study discloses a novel role of PAK6 in ciliogenesis and points to PAK6 as the first LRRK2 modifier with PD mutation-specificity.

neuroscience↗

bioRxiv2024

Identification and characterization of nanobodies acting as molecular chaperones for glucocerebrosidase through a novel allosteric mechanism

The enzyme glucocerebrosidase (GCase) catalyses the hydrolysis of glucosylceramide to glucose and ceramide within lysosomes. Homozygous or compound heterozygous mutations in the GCase-sencoding GBA1 gene cause the lysosomal storage disorder Gaucher disease, while heterozygous mutations are the most frequent genetic risk factor for Parkinsons disease. These mutations commonly affect GCase stability, trafficking or activity. Here, we report the development and characterization of nanobodies (Nbs) targeting and acting as chaperones for GCase. We identified several Nb families that bind with nanomolar affinity to GCase. Based on biochemical characterization, we grouped the Nbs in two classes: Nbs that improve the activity of the enzyme and Nbs that increase GCase stability in vitro. A selection of the most promising Nbs was shown to improve GCase function in cell models and positively impact the activity of the N370S mutant GCase. These results lay the foundation for the development of new therapeutic routes.

biochemistry↗

bioRxiv2020

PKA-mediated phosphorylation of SPG11/spatacsin regulates binding with a subset of 14-3-3 proteins

Mutations in SPG11, encoding spatacsin, constitute the major cause of autosomal recessive Hereditary Spastic Paraplegia (HSP) with thinning of the corpus callosum. Previous studies showed that spatacsin orchestrates cellular traffic events through the formation of a coat-like complex and its loss of function results in lysosomal and axonal transport impairments. However, the upstream mechanisms that regulate spatacsin trafficking are unknown. Here, using proteomics and CRISPR/Cas9-mediated tagging of endogenous spatacsin, we identified a subset of 14-3-3 proteins as physiological interactors of spatacsin. The interaction is modulated by Protein Kinase A (PKA)-dependent phosphorylation of spatacsin at Ser1955, which initiates spatacsin trafficking from the plasma membrane to the intracellular space. Our study provides novel insight in understanding spatacsin physio-pathological roles with mechanistic dissection of its associated pathways.

cell biology↗