Fig 2: Expression analysis of TMEM175 in human and mouse tissues. a, b qPCR experiments showed that TMEM175 mRNA is highly expressed in the human brain (brown bar) and mouse midbrain and forebrain (blue and yellow bar) at P45. Results were normalized by GAPDH. Data are expressed as absolute values with SD calculated from three biological replicates. c Single-cell RNA sequencing analysis of human substantia nigra (SN) and cerebral cortex showed the highest expression of TMEM175 mRNA in dopaminergic neurons of SN and in microglia of the cerebral cortex. Abbreviations: SN, substantia nigra; ODC, oligodendrocytes; OPC, oligodendrocyte progenitor cell

Fig 3: MRI analysis of PD patient. Three-dimensional T1-weighted images showing widespread cortical atrophy in a PD patient carrying the TMEM175 p.P14L mutation (coronal slice, left; axial slice, right)

Fig 4: Mutations in TMEM175 alter channel activity. a Typical I-V relation obtained by applying a single voltage ramp (from - 100 to + 100 mV, 200 ms) on a representative HEK293 cell transfected with wt TMEM175-GFP-fused construct (red line), TMEM175-M341T polymorphic variant (gray line), and TMEM175 R35C, P308L, A270T, and L405V rare deleterious variants (black line). Note the increase in the current in cell overexpressing the wt plasmid. b Histogram representing the mean current density measured at + 30 mV in different experimental conditions, as indicated. Note the increase in current density in cells overexpressing the wt TMEM175 protein compared to non-transfected cells (n = 8 and n = 13 biological replicates, respectively). Current density measured in cells transfected with the mutant proteins TMEM175 R35C, P308L, A270T, and L405V (n = 5, n = 6, n = 6, n = 7 biological replicates, respectively) was similar to that measured in non-transfected cells, while the polymorphic variant TMEM175 M341T showed an increase of the K + current across the plasma membrane that was intermediate between that detected with the wt and mutant protein (n = 6 biological replicates). Data (mean ± SEM) were analyzed with Student’s t-test and plotted as histogram representation. (*) indicates a p value < 0.05. c On the top representative immunoblotting of anti-GFP in immunoprecipitated protein with anti-Akt in MN9D cells expressing GFP-conjugated TMEM175 wt and mutant proteins. In the left, total proteins blotted for input control. On the bottom, representative histogram of the fold of wt densitometric ratio between the anti-GFP and anti-Akt blots (n = 3 biological replicates). Note the loss of binding between Akt and the mutant TMEM175 alleles P308L, L405V, and M341T expressed in stable cell line. The mean ± SEM of three biological replicates were analyzed with Student’s t-test. Data were plotted as histogram representation. (**) indicates a p value < 0.01, (***) indicates a p value < 0.001

Fig 5: TMEM175 mutations affect the expression of markers involved in the autophagy-lysosomal pathway and unfolded protein response (UPR). qPCR experiments were performed in human dermal fibroblasts, under basal conditions (HBSS -) and under serum starvation (1 h) (HBSS +) to examine the expression of genes involved in the surveillance of autophagy-lysosomal and URP pathways. In the graph, dermal fibroblasts of healthy subjects (mean value of 5 healthy subject-derived cells: CNT1-CNT5) are indicated as red bars and dermal fibroblasts of PD patients (mean value of 6 patient-derived cells: PD3, PD5, PD14, PD16, PD17, and PD22 carrying the mutations p.S348L, p.P427fs, p.R35Ca, p.R35C.b, p.R414W, and p.R481W respectively (Table 4) are indicated as black bars. The mean ± SD of three biological replicates was analyzed with Student’s t-test. Data were plotted as histogram representation. (*) indicates a p value < 0.05, (**) indicates a p value < 0.01, (***) indicates a p value < 0.001