Common and Rare Variants in TMEM175 Gene Concur to the Pathogenesis of Parkinson's Disease in Italian Patients.

Parkinson's disease (PD) represents the most common neurodegenerative movement disorder. We recently identified 16 novel genes associated with PD. In this study, we focused the attention on the common and rare variants identified in the lysosomal K+ channel TMEM175. The study includes a detailed clinical and genetic analysis of 400 cases and 300 controls. Molecular studies were performed on patient-derived fibroblasts. The functional properties of the mutant channels were assessed by patch-clamp technique and co-immunoprecipitation. We have found that TMEM175 was highly expressed in dopaminergic neurons of the substantia nigra pars compacta and in microglia of the cerebral cortex of the human brain. Four common variants were associated with PD, including two novel variants rs2290402 (c.-10C > T) and rs80114247 (c.T1022C, p.M341T), located in the Kozak consensus sequence and TM3II domain, respectively. We also disclosed 13 novel highly penetrant detrimental mutations in the TMEM175 gene associated with PD. At least nine of these mutations (p.R35C, p. R183X, p.A270T, p.P308L, p.S348L, p. L405V, p.R414W, p.P427fs, p.R481W) may be sufficient to cause the disease, and the presence of mutations of other genes correlated with an earlier disease onset. In vitro functional analysis of the ion channel encoded by the mutated TMEM175 gene revealed a loss of the K+ conductance and a reduced channel affinity for Akt. Moreover, we observed an impaired autophagic/lysosomal proteolytic flux and an increase expression of unfolded protein response markers in patient-derived fibroblasts. These data suggest that mutations in TMEM175 gene may contribute to the pathophysiology of PD.

NCOA4 links iron bioavailability to DNA metabolism.

Iron is essential for deoxyribonucleotides production and for enzymes containing an Fe-S cluster involved in DNA replication and repair. How iron bioavailability and DNA metabolism are coordinated remains poorly understood. NCOA4 protein mediates autophagic degradation of ferritin to maintain iron homeostasis and inhibits DNA replication origin activation via hindrance of the MCM2-7 DNA helicase. Here, we show that iron deficiency inhibits DNA replication, parallel to nuclear NCOA4 stabilization. In iron-depleted cells, NCOA4 knockdown leads to unscheduled DNA synthesis, with replication stress, genome instability, and cell death. In mice, NCOA4 genetic inactivation causes defective intestinal regeneration upon dextran sulfate sodium-mediated injury, with DNA damage, defective cell proliferation, and cell death; in intestinal organoids, this is fostered by iron depletion. In summary, we describe a NCOA4-dependent mechanism that coordinates iron bioavailability and DNA replication. This function prevents replication stress, maintains genome integrity, and sustains high rates of cell proliferation during tissue regeneration.

NCOA4-mediated ferritinophagy in macrophages is crucial to sustain erythropoiesis in mice.

Nuclear receptor coactivator 4 (NCOA4) promotes ferritin degradation and Ncoa4-ko mice in a C57BL/6 background show microcytosis and mild anemia, aggravated by iron deficiency. To understand tissue-specific contributions of NCOA4-mediated ferritinophagy we explored the effect of Ncoa4 genetic ablation in the iron-rich Sv129/J strain. Increased body iron content protects these mice from anemia and, in basal conditions, Sv129/J Ncoa4-ko mice show only microcytosis; nevertheless, when fed a low-iron diet they develop a more severe anemia compared to that of wild-type animals. Reciprocal bone marrow (BM) transplantation from wild-type donors into Ncoa4-ko and from Ncoa4-ko into wild-type mice revealed that microcytosis and susceptibility to iron deficiency anemia depend on BM-derived cells. Reconstitution of erythropoiesis with normalization of red blood count and hemoglobin concentration occurred at the same rate in transplanted animals independently of the genotype. Importantly, NCOA4 loss did not affect terminal erythropoiesis in iron deficiency, both in total and specific BM Ncoa4-ko animals compared to controls. On the contrary, upon a low iron diet, spleen from wild-type animals with Ncoa4-ko BM displayed marked iron retention compared to (wild-type BM) controls, indicating defective macrophage iron release in the former. Thus, erythropoietin administration failed to mobilize iron from stores in Ncoa4-ko animals. Furthermore, Ncoa4 inactivation in thalassemic mice did not worsen the hematologic phenotype. Overall our data reveal a major role for NCOA4-mediated ferritinophagy in macrophages to favor iron release for erythropoiesis, especially in iron deficiency.