Background Intracellular calcium [Ca2+]i is pivotal in driving aldosterone secretion. However, knowledge of the [Ca2+]i dynamics underlying aldosterone excess in primary aldosteronism (PA), the most common form of endocrine hypertension, is lacking due to the difficulty of obtaining aldosterone-producing adenoma (APA) cells ex vivo. Methods We used the Ca2+-sensitive Fura-2 dye and live confocal microscopy to investigate variations in [Ca2+]i under basal conditions and after angiotensin II (Ang II) stimulation in primary aldosterone-producing CD56+ cells, immunomagnetically isolated ex vivo from surgically treated PA patients. Results We found that the resting [Ca2+]i levels were consistently and markedly lower in APA cells (n = 14, 0.53; IQR: 0.33 - 0.79) compared with paired cells from the normal adjacent adrenal cortex (AAC) (n = 14, 0.80; IQR: 0.65 - 0.90, p = 0.021). While both cell types exhibited similar spontaneous oscillations, Ang II [1 nM] stimulation increased the number of cells exhibiting [Ca2+]i oscillations (p = 0.004) and induced a higher frequency of oscillations (p = 0.002) in APA than in AAC. Conclusion This study demonstrates prominent differences between APA and AAC cells in [Ca2+]i dynamics, with more marked oscillatory responses upon Ang II stimulation. These differences may underpin the increased aldosterone production, a hallmark of human PA.

Hereditary-Motor-Neuropathies (dHMNs) are clinically and genetically heterogeneous neurological disorders characterized by degeneration of peripheral motoneurons. We previously identified two sigma-1 receptor (Sigma-1R) variants (p.E138Q; p.E150K) in dHMN Italian patients that behave as "loss-of-function" mutations in neuroblastoma cell lines. Here, we characterize the functional effects of Sigma-1R mutation in primary fibroblasts from homozygous patients bearing the E150K mutation, and matched controls, by performing biochemical, gene expression, immunofluorescence and Ca2+ imaging analysis. Our results show that Sigma-1R expression and distribution is significantly altered in patient fibroblasts. Moreover, patient cells present a general derangement of cell homeostasis as revealed by impairment of global Ca2+ dynamics, disorganization of the ER-mitochondria tethers, enhancement of the autophago-lysosomal pathway and blunting of mitochondrial aerobic metabolism compared to controls. These findings highlight the crucial role of Sigma-1R in the maintenance of cell and protein homeostasis, inter-organelle communication and intracellular Ca2+ signalling, supporting the notion that Sigma-1R is protective for motor neuron activity and its down-regulation and/or loss-of-function, as in the case of the E150K mutation, might play the key role in the neuronal degeneration in dHMN patients.

Introduction: Primary aldosteronism (PA), the most common curable salt-dependent form of arterial hypertension, features renal K + loss and enhanced Na + reabsorption. Hypothesis: We assessed the hypothesis that alterations of electrolyte, water, and Tonicity-responsive Enhancer Binding Protein/NFAT5 mRNA content occur in the skin of PA patients and are corrected by surgical cure. Methods: We obtained skin biopsies in 80 subjects: 49 consecutive consenting PA patients, optimally treated with a mineralocorticoid receptor antagonist (MRA) before adrenalectomy; 6 patients with essential hypertension, and 25 normotensive controls. We measured Na + , K + and water content with atomic absorption spectrometry after ashing and NFAT5 mRNA with digital droplet PCR. The PA patients were retested after adrenalectomy. Results: We discovered that the skin biopsy specimen dry weight (DW) was higher at surgery than follow-up (p<0.001) and correlated directly with electrolyte and water content (all p<0.01), indicating the need for adjusting electrolyte and water data for DW. Surgical cure of PA markedly increased skin DW-adjusted K + (from 1.14±0.1 µg/mg to 2.81±0.27 µg/mg, p<0.001) and water content (from 2.92±1.4 mg/mg to 3.85±0.23 mg/mg, p<0.001), but left DW-adjusted skin Na + content unaffected. In the PA patients NFAT5 mRNA copy number was higher (p=0.03) than in the normotensive controls and decreased after surgery (p=0.03). Conclusions: In conclusion, surgical cure, but not optimal MRA treatment corrected the prominent skin cell K + depletion that occurred despite normal serum K + levels. The lack of overt skin Na + accumulation in PA patients during MRA treatment could be explained by enhanced skin Na + lymphatic drainage due to the documented activation of the skin NFAT5/TonEBP pathway.

BACKGROUND:Primary aldosteronism (PA), the most common curable salt-dependent form of arterial hypertension, features renal K+ loss and enhanced Na+ reabsorption. We investigated whether the electrolyte, water, and TonEBP (tonicity-responsive enhancer binding protein)/NFAT5 (nuclear factor of activated T cells 5) content is altered in the skin of patients with PA and corrected by surgical cure. METHODS:We obtained skin biopsies from 80 subjects: 49 consecutive patients with PA, optimally treated with a mineralocorticoid receptor antagonist; 6 essential hypertensives; and 25 normotensive controls. We measured Na+, K+, water content with atomic absorption spectroscopy after ashing, and NFAT5 mRNA with digital droplet polymerase chain reaction. The patients with PA were retested after adrenalectomy. RESULTS:We discovered a higher dry weight of the skin biopsy specimen at surgery than at follow-up (P<0.001) and a direct correlation with electrolyte and water content (all P<0.01), indicating the need for dry weight adjustment of electrolyte and water data. Surgical cure of PA markedly increased skin dry weight-adjusted K+ (from 1.14±0.1 to 2.81±0.27 µg/mg; P<0.001) and water content (from 2.92±1.4 to 3.85±0.23 mg/mg; P<0.001), but left dry weight-adjusted skin Na+ content unaffected. In patients with PA at baseline, NFAT5 mRNA was higher (P=0.031) than in normotensive controls and decreased after surgery (P=0.035). CONCLUSIONS:Despite mineralocorticoid receptor antagonist treatment ensuring normokalemia, the patients with PA had a skin cell K+ depletion that was corrected by adrenalectomy. The activated NFAT5/TonEBP pathway during mineralocorticoid receptor antagonist administration suggests enhanced skin Na+ lymphatic drainage and can explain the lack of overt skin Na+ accumulation in patients with PA. Its deactivation after surgical cure can account for the lack of skin Na+ decrease postadrenalectomy. REGISTRATION:URL: https://www.clinicaltrials.gov; Unique identifier: NCT06090617.

This study will test the hypothesis that primary aldosteronism (PA) involves alterations in Na+, K+, and water content in the skin that are corrected by adrenalectomy. In skin biopsies, we will measure the content of Na+, K+, water, by physical-chemical methods and the osmotic-stress-responsive transcription factor Tonicity-responsive Enhancer Binding Protein (TonEBP, NFAT5) mRNA copy number by droplet digital PCR, in sex-balanced cohorts of 18 -75-year-old consecutive consenting patients with unilateral and bilateral PA, primary (essential) hypertension, and normotension. Before surgery, the patients with unilateral PA will receive the mineralocorticoid receptor antagonist (MRA) canrenone at doses that correct hypokalemia and high blood pressure values. They will be reassessed in an identical way one month after surgical cure, while off MRA. PA patients not selected for adrenalectomy will similarly be assessed at diagnosis and follow-up while on stable MRA treatment. Since a pilot study showed a direct correlation of dry weight (DW) with skin electrolytes and water content and significant differences of biopsy DW between surgery and follow-up, meaningful comparison of the skin cations and water content and TonEBP mRNA copy number, between specimen obtained at different time points, will require DW- and total mRNA-adjustment, respectively. This study will provide novel information on the skin Na+, K+ and water content in PA, the paradigm of salt-dependent hypertension, and novel knowledge on the effect of surgical cure of hyperaldosteronism. The TonEBP-mediated regulation of Na+, K+ and water content in the skin will also be unveiled. Trial Registration number: NCT06090617. Date of Registration: 2023-10-19.

Transcription of mitochondrial DNA generates long polycistronic precursors whose nucleolytic cleavage yields the individual mtDNA-encoded transcripts. In most cases, this cleavage occurs at the 5′- and 3′-ends of tRNA sequences by the concerted action of RNAseP and RNaseZ/ELAC2 endonucleases, respectively. Variants in the ELAC2 gene have been predominantly linked to severe to mild cardiomyopathy that, in its milder forms, is accompanied by variably severe neurological presentations. Here, we report five patients from three unrelated families. Four of the patients presented mild to moderate cardiomyopathy and one died at 1 year of age, one patient had no evidence of cardiomyopathy. The patients had variable neurological presentations that included intellectual disability, ataxia, refractory epilepsy, neuropathy and deafness. All patients carried previously unreported missense and nonsense variants. Enzymatic analyses showed multiple OXPHOS deficiencies in biopsies from two patients, whereas immunoblot analyses revealed a decreased abundance of ELAC2 in fibroblasts from three patients. Northern blot analysis revealed an accumulation of unprocessed mt-tRNAVal-precursor consistent with the role of ELAC2 in transcript processing. Our study expands the genetic spectrum of ELAC2-linked disease and suggests that cardiomyopathy is not an invariably present clinical hallmark of this pathology.

The notion of mitochondria being involved in the decoding and shaping of intracellular Ca2+ signals has been circulating since the end of the 19th century. Despite that, the molecular identity of the channel that mediates Ca2+ ion transport into mitochondria remained elusive for several years. Only in the last decade, the genes and pathways responsible for the mitochondrial uptake of Ca2+ began to be cloned and characterized. The gene coding for the pore-forming unit of the mitochondrial channel was discovered exactly 10 years ago, and its product was called mitochondrial Ca2+ uniporter or MCU. Before that, only one of its regulators, the mitochondria Ca2+ uptake regulator 1, MICU1, has been described in 2010. However, in the following years, the scientific interest in mitochondrial Ca2+ signaling regulation and physiological role has increased. This shortly led to the identification of many of its components, to the description of their 3D structure, and the characterization of the uniporter contribution to tissue physiology and pathology. In this review, we will summarize the most relevant achievements in the history of mitochondrial Ca2+ studies, presenting a chronological overview of the most relevant and landmarking discoveries. Finally, we will explore the impact of mitochondrial Ca2+ signaling in the context of muscle physiology, highlighting the recent advances in understanding the role of the MCU complex in the control of muscle trophism and metabolism.

The cyclic AMP (cAMP) signalling cascade is necessary for cell homeostasis and plays important roles in many processes. This is particularly relevant during ageing and age-related diseases, where drastic changes, generally decreases, in cAMP levels have been associated with the progressive decline in overall cell function and, eventually, the loss of cellular integrity. The functional relevance of reduced cAMP is clearly supported by the finding that increases in cAMP levels can reverse some of the effects of ageing. Nevertheless, despite these observations, the molecular mechanisms underlying the dysregulation of cAMP signalling in ageing are not well understood. Compartmentalization is widely accepted as the modality through which cAMP achieves its functional specificity; therefore, it is important to understand whether and how this mechanism is affected during ageing and to define which is its contribution to this process. Several animal models demonstrate the importance of specific cAMP signalling components in ageing, however, how age-related changes in each of these elements affect the compartmentalization of the cAMP pathway is largely unknown. In this review, we explore the connection of single components of the cAMP signalling cascade to ageing and age-related diseases whilst elaborating the literature in the context of cAMP signalling compartmentalization.

Autophagy is a highly regulated degradative process crucial for maintaining cell homeostasis. This important catabolic mechanism can be nonspecific, but usually occurs with fine spatial selectivity (compartmentalization), engaging only specific subcellular sites. While the molecular machines driving autophagy are well understood, the involvement of localized signaling events in this process is not well defined. Among the pathways that regulate autophagy, the cyclic AMP (cAMP)/protein kinase A (PKA) cascade can be compartmentalized in distinct functional units called microdomains. However, while it is well established that, depending on the cell type, cAMP can inhibit or promote autophagy, the role of cAMP/PKA microdomains has not been tested. Here we show not only that the effects on autophagy of the same cAMP elevation differ in different cell types, but that they depend on a highly complex sub-compartmentalization of the signaling cascade. We show in addition that, in HT-29 cells, in which autophagy is modulated by cAMP rising treatments, PKA activity is strictly regulated in space and time by phosphatases, which largely prevent the phosphorylation of soluble substrates, while membrane-bound targets are less sensitive to the action of these enzymes. Interestingly, we also found that the subcellular distribution of PKA type-II regulatory PKA subunits hinders the effect of PKA on autophagy, while displacement of type-I regulatory PKA subunits has no effect. Our data demonstrate that local PKA activity can occur independently of local cAMP concentrations and provide strong evidence for a link between localized PKA signaling events and autophagy.

Protein kinase CK2, a pleiotropic and constitutively active kinase, is strictly involved in different diseases, especially in cancer. Many efforts have been carried out to develop specific CK2 inhibitors and recently, it has been evidenced that ferulic acid (FA) represents a promising, albeit cell impermeable, CK2 inhibitor. In the present study, the potential of a nanotechnological approach to cope with intracellular CK2 regulation was explored. Surface-Active Maghemite Nanoparticles (SAMNs), coupling magnetism with photoluminescence, a new feature of SAMNs here described for the first time, were chosen as dual imaging nanocarrier for FA. The self-assembled nanodevice (SAMN@FA) displayed a significant CK2 inhibitory activity in vitro. Moreover, effective cellular internalization of SAMN@FA in cancer cells was proved by direct visualization of the photoluminescent nanocarrier by confocal microscopy and was corroborated by phosphorylation levels of endogenous CK2 targets. The proposed trimodal nanodevice, representing the first example of cellular CK2 nano-inhibition, paves the way for novel active nanocarriers as appealing theranostic tool for future biomedical applications.

The combination of different nanomaterials has been investigated during the past few decades and represents an exciting challenge for the unexpected emerging properties of the resulting nano-hybrids. Spermidine (Spd), a biogenic polyamine, has emerged as a useful functional monomer for the development of carbon quantum dots (CQDs). Herein, an electrostatically stabilized ternary hybrid, constituted of iron oxide-DNA (the core) and spermidine carbon quantum dots (CQDSpds, the shell), was self-assembled and fully characterized. The as-obtained nano-hybrid was tested on HeLa cells to evaluate its biocompatibility as well as cellular uptake. Most importantly, besides being endowed by the magnetic features of the core, it displayed drastically enhanced fluorescence properties in comparison with parent CQDSpds and it is efficiently internalized by HeLa cells. This novel ternary nano-hybrid with multifaceted properties, ranging from fluorescence to superparamagnetism, represents an interesting option for cell tracking.

Casein kinase 2 (CK2) is a tetrameric protein kinase composed of 2 catalytic (α and α′) and 2 regulatory β subunits. Our study provides the first molecular and cellular characterization of the different CK2 subunits, highlighting their individual roles in skeletal muscle specification and differentiation. Analysis of C2C12 cell knockout for each CK2 subunit reveals that: 1) CK2β is mandatory for the expression of the muscle master regulator myogenic differentiation 1 in proliferating myoblasts, thus controlling both myogenic commitment and subsequent muscle‐specific gene expression and myotube formation; 2) CK2α is involved in the activation of the muscle‐specific gene program; and 3) CK2α′ activity regulates myoblast fusion by mediating plasma membrane translocation of fusogenic proteins essential for membrane coalescence, like myomixer. Accordingly, CK2α′ overexpression in C2C12 cells and in mouse regenerating muscle is sufficient to increase myofiber size and myonuclei content via enhanced satellite cell fusion. Consistent with these results, pharmacological inhibition of CK2 activity substantially blocks the expression of myogenic markers and muscle cell fusion both in vitro in C2C12 and primary myoblasts and in vivo in mouse regenerating muscle and zebrafish development. Overall, our work describes the specific and coordinated functions of CK2 subunits in orchestrating muscle differentiation and fusogenic activity, highlighting CK2 relevance in the physiopathology of skeletal muscle tissue.—Salizzato, V., Zanin, S., Borgo, C., Lidron, E., Salvi, M., Rizzuto, R., Pallafacchina, G., Donella‐Deana, A. Protein kinase CK2 subunits exert specific and coordinated functions in skeletal muscle differentiation and fusogenic activity. FASEB J. 33, 10648–10667 (2019). www.fasebj.org

Ca2+ ion is universally considered the most versatile second messenger responsible for decoding and regulating the majority of the signaling pathways within the cell. The study of intracellular Ca2+ concentration ([Ca2+]i) dynamics is consequently of primary importance for the interpretation of cellular biology. This chapter will present a relatively simple, largely diffused, and nevertheless robust method to measure variations of [Ca2+]i by the use of the Ca2+-sensitive chemical dye Fura-2. A general protocol for the assessment of [Ca2+]i in adherent cells, applicable to a variety of cell systems, will be first presented. Then, the implementation of Fura-2 to detect [Ca2+]i in two specific cell types, namely, human adrenocortical cells and primary skin fibroblasts, will be discussed in more particulars. Finally, the procedure to monitor Ca2+ influx through the plasma membrane using Fura-2 will be described.

In 2011, using exomes sequencing Choi et al. in about 34% of 22 aldosterone-producing adenoma (APA) discovered mutations in the selectivity filter of the KCNJ5 gene, which codes for the Kir3.4 K+ channel. This channel is highly expressed in the aldosterone-producing cells of the zona glomerulosa (ZG) and plays a key role in maintaining cell hyperpolarized (Fig. 1, Panels A and B). The Authors identified two recurrent functional variants (G151R and L168R) in this highly conserved region of the K+ channel, which allows the exit of K+ from the cells in a selective fashion, for example, with exclusion of other ions. Using cloning techniques and site-directed mutagenesis they could show that mutant-transfected cells in vitro exhibit perturbation in both size and charge of the selectivity filter, resulting in permeability to Na+, Na+ influx, and membrane depolarization. After this discovery other mutations in APA were found. Practically all of them result in increased cytosolic calcium with ensuing activation of the biosynthesis of aldosterone. These somatic mutations are herein described along with their functional consequences and the mechanisms whereby they lead to hyperaldosteronism.

Root growth is a fundamental process in plants and assures nutrient and water uptake required for efficient photosynthesis and metabolism. Postembryonic development of roots is controlled by the functionality of the meristem. Several hormones and signaling molecules regulate the size of the meristem, and among them, auxins play a major role. Protein kinase CK2, along with the chaperone protein HSP90, has been found to be involved in the regulation of auxin transport. Here, we show that p23-1, a cochaperone of HSP90, is phosphorylated by CK2 in Arabidopsis. We identified Ser201 as the major CK2 target site in p23-1 and demonstrated that phosphorylation of this site is necessary for normal root development. Moreover, we shed light on the nature of CK2 in Arabidopsis, showing that the three catalytic isoforms, CK2 αA, αB and αC, are proteins of approximately 40 kDa. Our results increase knowledge of the connection among HSP90, p23-1 and CK2 in Arabidopsis, suggesting the existence of a possible common root development mechanism controlled by these signaling molecules.

Context: The G protein-coupled estrogen receptor (GPER) mediates an aldosterone secretagogue effect of 17 beta-estradiol in human HAC15 adrenocortical cells after estrogen receptor beta blockade. Because GPER mediates mineralocorticoid receptor-independent aldosterone effects in other cell types, we hypothesized that aldosterone could modulate its own synthesis via GPER activation. Methods: HAC15 cells were exposed to aldosterone in the presence or absence of canrenone, a mineralocorticoid receptor antagonist, and/or of the selective GPER antagonist G36. Aldosterone synthase (CYP11B2) mRNA and protein levels changes were the study end points. Similar experiments were repeated in strips obtained ex vivo from aldosterone-producing adenoma (APA) and in GPER-silenced HAC15 cells. Results: Aldosterone markedly increased CYP11B2 mRNA and protein expression (vs untreated samples, P < 0.001) in both models by acting via GPER, because these effects were abolished by G36 (P < 0.01) and not by canrenone. GPER-silencing (P < 0.01) abolished the aldosterone-induced increase of CYP11B2, thus proving that aldosterone acts via GPER to augment the step-limiting mitochondrial enzyme (CYP11B2) of its synthesis. Angiotensin II potentiated the GPER-mediated effect of aldosterone on CYP11B2. Coimmunoprecipitation studies provided evidence for GPER-angiotensin type-1 receptor heterodimerization. Conclusion: We propose that this autocrine-paracrine mechanism could enhance aldosterone biosynthesis under conditions of immediate physiological need in which the renin-angiotensinaldosterone system is stimulated as, for example, hypovolemia. Moreover, as APA overexpresses GPER this mechanism could contribute to the aldosterone excess that occurs in primary aldosteronism in a seemingly autonomous fashion from angiotensin II.

In the last few decades, a large body of experimental evidence has highlighted the complex role for mitochondria in eukaryotic cells: they are not only the site of aerobic metabolism (thus providing most of the ATP supply for endergonic processes) but also a crucial checkpoint of cell death processes (both necrosis and apoptosis) and autophagy. For this purpose, mitochondria must receive and decode the wide variety of physiological and pathological stimuli impacting on the cell. The “old” notion that mitochondria possess a sophisticated machinery for accumulating and releasing Ca2+, the most common and versatile second messenger of eukaryotic cells, is thus no surprise. What may be surprising is that the identification of the molecules involved in mitochondrial Ca2+transport occurred only in the last decade for both the influx (the mitochondrial Ca2+uniporter, MCU) and the efflux (the sodium calcium exchanger, NCX) pathways. In this review, we will focus on the description of the amazing molecular complexity of the MCU complex, highlighting the numerous functional implications of the tissue-specific expression of the variants of the channel pore components (MCU/MCUb) and of the associated proteins (MICU 1, 2, and 3, EMRE, and MCUR1).

Distal hereditary motor neuropathies (dHMNs) are clinically and genetically heterogeneous neurological conditions characterized by degeneration of the lower motor neurons. So far, 18 dHMN genes have been identified, however, about 80% of dHMN cases remain without a molecular diagnosis. By a combination of autozygosity mapping, identity-by-descent segment detection and whole-exome sequencing approaches, we identified two novel homozygous mutations in the SIGMAR1 gene (p.E138Q and p.E150K) in two distinct Italian families affected by an autosomal recessive form of HMN. Functional analyses in several neuronal cell lines strongly support the pathogenicity of the mutations and provide insights into the underlying pathomechanisms involving the regulation of ER-mitochondria tethering, Ca2+ homeostasis and autophagy. Indeed, in vitro, both mutations reduce cell viability, the formation of abnormal protein aggregates preventing the correct targeting of sigma-1R protein to the mitochondria-associated ER membrane (MAM) and thus impinging on the global Ca2+ signalling. Our data definitively demonstrate the involvement of SIGMAR1 in motor neuron maintenance and survival by correlating, for the first time in the Caucasian population, mutations in this gene to distal motor dysfunction and highlight the chaperone activity of sigma-1R at the MAM as a critical aspect in dHMN pathology.

By derivatizing the purely competitive CK2 inhibitor N1-(4,5,6,7-tetrabromo-1H-benzimidazol-2-yl)-propane-1,3-diamine (K137) at its 3-amino position with a peptidic fragment composed of three or four glutamic or aspartic acid residues, a new family of bisubstrate inhibitors has been generated whose ability to simultaneously interact with both the ATP and the phosphoacceptor substrate-binding sites has been probed by running mixed competition kinetics and by mutational mapping of the kinase residues implicated in substrate recognition. The most effective bisubstrate inhibitor, K137-E4, interacts with three functional regions of the kinase: the hydrophobic pocket close to the ATP-binding site, the basic residues of the p+1 loop that recognizes the acidic determinant at position n+1 and the basic residues of α-helixC that recognize the acidic determinant at position n+3. Compared with the parent inhibitor (K137), K137-E4 is severalfold more potent (IC50 25 compared with 130 nM) and more selective, failing to inhibit any other kinase as drastically as CK2 out of 140 enzymes, whereas 35 kinases are inhibited more potently than CK2 by K137. K137-E4 is unable to penetrate the cell and to inhibit endogenous CK2, its pro-apoptotic efficacy being negligible compared with cell-permeant inhibitors; however, it readily inhibits ecto-CK2 on the outer cell surface, reducing the phosphorylation of several external phosphoproteins. Inhibition of ecto-CK2 by K137-E4 is accompanied by a slower migration of cancer cells as judged by wound healing assays. On the basis of the cellular responses to K137-E4, we conclude that ecto-CK2 is implicated in cell motility, whereas its contribution to the pro-survival role of CK2 is negligible.