Fig 2: Schematic diagram modeling the role of the Sp1–p11–TASK1 triad in neurodegeneration. a Physiological expression of the transcription factor Sp1 maintains baseline levels of p11, which regulates IME by controlling TASK1 trafficking from the endoplasmic reticulum (ER) to the plasma membrane. p11–TASK1 complex dissociation is required to overcome ER retention and normal recycling of TASK1 to the surface14. Under healthy conditions, when neuronal IME is fine-tuned, moderated Ca2+ influx, through GluRs and/or VSCCs, leads to physiological responses, which are controlled by mechanisms of intracellular Ca2+ buffering and extrusion. b In neurological conditions, over-expression and/or de novo expression of nNOS could be the switch on (star) to initiate a positive feedback loop that contributes to the degeneration and death of sick neurons. Usually, NO production in the nervous system is directed by Ca2+ influx driven by NMDA receptors. The calmodulin (CaM)–Ca2+ interaction, which is now enabled to activate nNOS, allows for the coupling of pathological NO synthesis to Ca2+ entry. NO reacts rapidly with the superoxide anion (O2-) to form the highly toxic product peroxynitrite (ONOO-) and/or promotes S-nitrosylation, triggering protein misfolding and aggregation, thus leading to neurotoxicity8 (omitted for simplicity). In addition, a persistent pathological concentration of NO, involving the sGC/PKG-RhoA/ROCK pathway (omitted for simplicity), induces Sp1 and stimulates the human p11 promoter depending on, at least, a Sp1-binding site (GC-box). Subsequent p11 upregulation sequesters TASK1 to the ER, thus disrupting the normal recycling of this subunit to the surface. This TASK1 uptake/replenishment unbalance enhances IME, which, in turn, increases the opening probability of VSCCs and, GluRs in the presence of glutamate, thereby exacerbating Ca2+ entry into the cell. Ca2+ overload might contribute to neurodegeneration and, finally, to neuron death. Whether NO of glial origin6 and/or other unidentified factors promote Sp1 expression/binding and/or p11 expression are not discarded and merit for further investigation

Fig 3: PRIP prevents PI(4,5)P2 recognition by OCRL1 but not by RhoA. (a–d) PI(4,5)P2 sedimentation assay was performed in a combination of EGFP-PRIP1, GST-PRIP2, Halo-OCRL1, and Halo-RhoA (a,c). Supernatant 1 (Sup1) contained Halo-OCRL1 (a) or Halo-OCRL1 and Halo-RhoA (c), as confirmed by immunoblotting using indicated antibodies. The fractions of Sup1 in (a or c) precipitated with liposomes [PC, 100% PC; 5% PIP2, PI(4,5)P2:PC = 5:95 (molar ratio)] in the presence (+) or absence (−) of recombinant PRIP1 or PRIP2 (b) or with liposomes (5% PIP2) in the presence of indicated doses of recombinant PRIP1 (d) by centrifugation, respectively. Western blotting of the obtained pellet fraction (Ppt2, b or d) and supernatant (Sup2, b) was performed using indicated antibodies (left side of each panel). Similar data were obtained from three independent experiments, and a set of representative images is shown. The image obtained in a high sensitivity mode (high, d) is shown. Each of the original blots is shown in Supplementary Fig. S8 for (d–f) or Supplementary Fig. S9 for (b).

Fig 4: Relative (A) RhoA, (B) ROCK1, and (C) LIMK2 mRNA expression in irradiated MC3T3-E1 cells in response to different doses of X-ray irradiation. *P<0.05 vs. 0 Gy. ROCK1, Rho associated kinase 1; LIMK2, LIM domain kinase 2.

Fig 5: The immunoblots of (A) active RhoA, (B) total RhoA, (C) normalized Rho GTP, (D) P-LIMK2, (E) LIMK2 and (F) normalized P-LIMK were analyzed using densitometry. The results are presented in bar graphs as mean ± S.D. *P<0.05. P-LIMK2, phosphorylated LIM domain kinase 2; LIMK2, LIM domain kinase 2.