Human frataxin, the Friedreich ataxia deficient protein, interacts with mitochondrial respiratory chain.

Friedreich ataxia (FRDA) is a rare, inherited neurodegenerative disease caused by an expanded GAA repeat in the first intron of the FXN gene, leading to transcriptional silencing and reduced expression of frataxin. Frataxin participates in the mitochondrial assembly of FeS clusters, redox cofactors of the respiratory complexes I, II and III. To date it is still unclear how frataxin deficiency culminates in the decrease of bioenergetics efficiency in FRDA patients' cells. We previously demonstrated that in healthy cells frataxin is closely attached to the mitochondrial cristae, which contain both the FeS cluster assembly machinery and the respiratory chain complexes, whereas in FRDA patients' cells with impaired respiration the residual frataxin is largely displaced in the matrix. To gain novel insights into the function of frataxin in the mitochondrial pathophysiology, and in the upstream metabolic defects leading to FRDA disease onset and progression, here we explored the potential interaction of frataxin with the FeS cluster-containing respiratory complexes I, II and III. Using healthy cells and different FRDA cellular models we found that frataxin interacts with these three respiratory complexes. Furthermore, by EPR spectroscopy, we observed that in mitochondria from FRDA patients' cells the decreased level of frataxin specifically affects the FeS cluster content of complex I. Remarkably, we also found that the frataxin-like protein Nqo15 from T. thermophilus complex I ameliorates the mitochondrial respiratory phenotype when expressed in FRDA patient's cells. Our data point to a structural and functional interaction of frataxin with complex I and open a perspective to explore therapeutic rationales for FRDA targeted to this respiratory complex.

Duodenal alpha-Synuclein Pathology and Enteric Gliosis in Advanced Parkinson's Disease.

BACKGROUND: The role of the gut-brain axis has been recently highlighted as a major contributor to Parkinson's disease (PD) physiopathology, with numerous studies investigating bidirectional transmission of pathological protein aggregates, such as α-synuclein (αSyn). However, the extent and the characteristics of pathology in the enteric nervous system have not been fully investigated. OBJECTIVE: We characterized αSyn alterations and glial responses in duodenum biopsies of patients with PD by employing topography-specific sampling and conformation-specific αSyn antibodies. METHODS: We examined 18 patients with advanced PD who underwent Duodopa percutaneous endoscopic gastrostomy and jejunal tube procedure, 4 untreated patients with early PD (disease duration <5 years), and 18 age- and -sex-matched healthy control subjects undergoing routine diagnostic endoscopy. A mean of four duodenal wall biopsies were sampled from each patient. Immunohistochemistry was performed for anti-aggregated αSyn (5G4) and glial fibrillary acidic protein antibodies. Morphometrical semiquantitative analysis was performed to characterize αSyn-5G4+ and glial fibrillary acidic protein-positive density and size. RESULTS: Immunoreactivity for aggregated α-Syn was identified in all patients with PD (early and advanced) compared with controls. αSyn-5G4+ colocalized with neuronal marker ß-III-tubulin. Evaluation of enteric glial cells demonstrated an increased size and density when compared with controls, suggesting reactive gliosis. CONCLUSIONS: We found evidence of synuclein pathology and gliosis in the duodenum of patients with PD, including early de novo cases. Future studies are required to evaluate how early in the disease process duodenal pathology occurs and its possible contribution to levodopa effect in chronic patients. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

PAK6-mediated phosphorylation of PPP2R2C regulates LRRK2-PP2A complex formation.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are a common cause of inherited and sporadic Parkinson's disease (PD) and previous work suggests that dephosphorylation of LRRK2 at a cluster of heterologous phosphosites is associated to disease. We have previously reported subunits of the PP1 and PP2A classes of phosphatases as well as the PAK6 kinase as regulators of LRRK2 dephosphorylation. We therefore hypothesized that PAK6 may have a functional link with LRRK2's phosphatases. To investigate this, we used PhosTag gel electrophoresis with purified proteins and found that PAK6 phosphorylates the PP2A regulatory subunit PPP2R2C at position S381. While S381 phosphorylation did not affect PP2A holoenzyme formation, a S381A phosphodead PPP2R2C showed impaired binding to LRRK2. Also, PAK6 kinase activity changed PPP2R2C subcellular localization in a S381 phosphorylation-dependent manner. Finally, PAK6-mediated dephosphorylation of LRRK2 was unaffected by phosphorylation of PPP2R2C at S381, suggesting that the previously reported mechanism whereby PAK6-mediated phosphorylation of 14-3-3 proteins promotes 14-3-3-LRRK2 complex dissociation and consequent exposure of LRRK2 phosphosites for dephosphorylation is dominant. Taken together, we conclude that PAK6-mediated phosphorylation of PPP2R2C influences the recruitment of PPP2R2C to the LRRK2 complex and PPP2R2C subcellular localization, pointing to an additional mechanism in the fine-tuning of LRRK2 phosphorylation.

Divergent Effects of G2019S and R1441C LRRK2 Mutations on LRRK2 and Rab10 Phosphorylations in Mouse Tissues.

Mutations in LRRK2 cause familial Parkinson's disease and common variants increase disease risk. LRRK2 kinase activity and cellular localization are tightly regulated by phosphorylation of key residues, primarily Ser1292 and Ser935, which impacts downstream phosphorylation of its substrates, among which Rab10. A comprehensive characterization of LRRK2 activity and phosphorylation in brain as a function of age and mutations is missing. Here, we monitored Ser935 and Ser1292 phosphorylation in midbrain, striatum, and cortex of 1, 6, and 12 months-old mice carrying G2019S and R1441C mutations or murine bacterial artificial chromosome (BAC)-Lrrk2-G2019S. We observed that G2019S and, at a greater extent, R1441C brains display decreased phospho-Ser935, while Ser1292 autophosphorylation increased in G2019S but not in R1441C brain, lung, and kidney compared to wild-type. Further, Rab10 phosphorylation, is elevated in R1441C carrying mice, indicating that the effect of LRRK2 mutations on substrate phosphorylation is not generalizable. In BAC-Lrrk2-G2019S striatum and midbrain, Rab10 phosphorylation, but not Ser1292 autophosphorylation, decreases at 12-months, pointing to autophosphorylation and substrate phosphorylation as uncoupled events. Taken together, our study provides novel evidence that LRRK2 phosphorylation in mouse brain is differentially impacted by mutations, brain area, and age, with important implications as diagnostic markers of disease progression and stratification.

An Agonist of the CXCR4 Receptor Strongly Promotes Regeneration of Degenerated Motor Axon Terminals.

The activation of the G-protein coupled receptor CXCR4 by its ligand CXCL12α is involved in a large variety of physiological and pathological processes, including the growth of B cells precursors and of motor axons, autoimmune diseases, stem cell migration, inflammation, and several neurodegenerative conditions. Recently, we demonstrated that CXCL12α potently stimulates the functional recovery of damaged neuromuscular junctions via interaction with CXCR4. This result prompted us to test the neuroregeneration activity of small molecules acting as CXCR4 agonists, endowed with better pharmacokinetics with respect to the natural ligand. We focused on NUCC-390, recently shown to activate CXCR4 in a cellular system. We designed a novel and convenient chemical synthesis of NUCC-390, which is reported here. NUCC-390 was tested for its capability to induce the regeneration of motor axon terminals completely degenerated by the presynaptic neurotoxin α-Latrotoxin. NUCC-390 was found to strongly promote the functional recovery of the neuromuscular junction, as assayed by electrophysiology and imaging. This action is CXCR4 dependent, as it is completely prevented by AMD3100, a well-characterized CXCR4 antagonist. These data make NUCC-390 a strong candidate to be tested in human therapy to promote nerve recovery of function after different forms of neurodegeneration.