Rita La Rovere

The BH4 Domain of Anti-apoptotic Bcl-XL, but Not That of the Related Bcl-2, Limits the Voltage-dependent Anion Channel 1 (VDAC1)-mediated Transfer of Pro-apoptotic Ca2+ Signals to Mitochondria

Background: VDAC1 mediates the transfer of pro-apoptotic Ca2+ signals into mitochondria. Results: The BH4 domain of Bcl-XL, but not that of Bcl-2, targets VDAC1 and suppresses its pro-apoptotic Ca2+-flux properties. N-terminal VDAC1 peptide alleviates this effect of BH4-Bcl-XL. Conclusion: Bcl-XL via its BH4 domain inhibits VDAC1 activity. Significance: Bcl-2 and Bcl-XL differ in their BH4 domain biology by regulating ER and mitochondrial Ca2+-transport systems, respectively. Excessive Ca2+ fluxes from the endoplasmic reticulum to the mitochondria result in apoptotic cell death. Bcl-2 and Bcl-XL proteins exert part of their anti-apoptotic function by directly targeting Ca2+-transport systems, like the endoplasmic reticulum-localized inositol 1,4,5-trisphosphate receptors (IP3Rs) and the voltage-dependent anion channel 1 (VDAC1) at the outer mitochondrial membranes. We previously demonstrated that the Bcl-2 homology 4 (BH4) domain of Bcl-2 protects against Ca2+-dependent apoptosis by binding and inhibiting IP3Rs, although the BH4 domain of Bcl-XL was protective independently of binding IP3Rs. Here, we report that in contrast to the BH4 domain of Bcl-2, the BH4 domain of Bcl-XL binds and inhibits VDAC1. In intact cells, delivery of the BH4-Bcl-XL peptide via electroporation limits agonist-induced mitochondrial Ca2+ uptake and protects against staurosporine-induced apoptosis, in line with the results obtained with VDAC1−/− cells. Moreover, the delivery of the N-terminal domain of VDAC1 as a synthetic peptide (VDAC1-NP) abolishes the ability of BH4-Bcl-XL to suppress mitochondrial Ca2+ uptake and to protect against apoptosis. Importantly, VDAC1-NP did not affect the ability of BH4-Bcl-2 to suppress agonist-induced Ca2+ release in the cytosol or to prevent apoptosis, as done instead by an IP3R-derived peptide. In conclusion, our data indicate that the BH4 domain of Bcl-XL, but not that of Bcl-2, selectively targets VDAC1 and inhibits apoptosis by decreasing VDAC1-mediated Ca2+ uptake into the mitochondria.

(R)‐α‐Lipoyl‐Glycyl‐L‐Prolyl‐L‐Glutamyl Dimethyl Ester Codrug as a Multifunctional Agent with Potential Neuroprotective Activities

The (R)‐α‐lipoyl‐glycyl‐L‐prolyl‐L‐glutamyl dimethyl ester codrug (LA‐GPE, 1) was synthesized as a new multifunctional drug candidate with antioxidant and neuroprotective properties for the treatment of neurodegenerative diseases. Physicochemical properties, chemical and enzymatic stabilities were evaluated, along with the capacity of LA‐GPE to penetrate the blood–brain barrier (BBB) according to an in vitro parallel artificial membrane permeability assay for the BBB. We also investigated the potential effectiveness of LA‐GPE against the cytotoxicity induced by 6‐hydroxydopamine (6‐OHDA) and H2O2 on the human neuroblastoma cell line SH‐SY5Y by using the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) reduction assay. Our results show that codrug 1 is stable at both pH 1.3 and 7.4, exhibits good lipophilicity (log P=1.51) and a pH‐dependent permeability profile. Furthermore, LA‐GPE was demonstrated to be significantly neuroprotective and to act as an antioxidant against H2O2‐ and 6‐OHDA‐induced neurotoxicity in SH‐SY5Y cells.

A glutathione derivative with chelating and in vitro neuroprotective activities: synthesis, physicochemical properties, and biological evaluation

Metal‐ion dysregulation and oxidative stress have been linked to the progressive neurological decline associated with neurodegenerative disorders such as Alzheimer’s and Parkinson’s diseases. Herein we report the synthesis and chelating, antioxidant, and in vitro neuroprotective activities of a novel derivative of glutathione, GS(HQ)H, endowed with an 8‐hydroxyquinoline group as a metal‐chelating moiety. In vitro results showed that GS(HQ)H may be stable enough to be absorbed unmodified and arrive intact to the blood–brain barrier, that it may be able to remove CuII and ZnII from the Aβ peptide without causing any copper or zinc depletion in vivo, and that it protects SHSY‐5Y human neuroblastoma cells against H2O2‐ and 6‐OHDA‐induced damage. Together, these findings suggest that GS(HQ)H could be a potential neuroprotective agent for the treatment of neurodegenerative diseases in which a lack of metal homeostasis has been reported as a key factor.

Endoplasmic Reticulum–Mitochondrial Ca2+ Fluxes Underlying Cancer Cell Survival

Calcium ions (Ca2+) are crucial, ubiquitous, intracellular second messengers required for functional mitochondrial metabolism during uncontrolled proliferation of cancer cells. The mitochondria and the endoplasmic reticulum (ER) are connected via “mitochondria-associated ER membranes” (MAMs) where ER–mitochondria Ca2+ transfer occurs, impacting the mitochondrial biology related to several aspects of cellular survival, autophagy, metabolism, cell death sensitivity, and metastasis, all cancer hallmarks. Cancer cells appear addicted to these constitutive ER–mitochondrial Ca2+ fluxes for their survival, since they drive the tricarboxylic acid cycle and the production of mitochondrial substrates needed for nucleoside synthesis and proper cell cycle progression. In addition to this, the mitochondrial Ca2+ uniporter and mitochondrial Ca2+ have been linked to hypoxia-inducible factor 1α signaling, enabling metastasis and invasion processes, but they can also contribute to cellular senescence induced by oncogenes and replication. Finally, proper ER–mitochondrial Ca2+ transfer seems to be a key event in the cell death response of cancer cells exposed to chemotherapeutics. In this review, we discuss the emerging role of ER–mitochondrial Ca2+ fluxes underlying these cancer-related features.

Myogenic potential of canine craniofacial satellite cells

The skeletal fibers have different embryological origin; the extraocular and jaw-closer muscles develop from prechordal mesoderm while the limb and trunk muscles from somites. These different origins characterize also the adult muscle stem cells, known as satellite cells (SCs) and responsible for the fiber growth and regeneration. The physiological properties of presomitic SCs and their epigenetics are poorly studied despite their peculiar characteristics to preserve muscle integrity during chronic muscle degeneration. Here, we isolated SCs from canine somitic [somite-derived muscle (SDM): vastus lateralis, rectus abdominis, gluteus superficialis, biceps femoris, psoas] and presomitic [pre-somite-derived muscle (PSDM): lateral rectus, temporalis, and retractor bulbi] muscles as myogenic progenitor cells from young and old animals. In addition, SDM and PSDM-SCs were obtained also from golden retrievers affected by muscular dystrophy (GRMD). We characterized the lifespan, the myogenic potential and functions, and oxidative stress of both somitic and presomitic SCs with the aim to reveal differences with aging and between healthy and dystrophic animals. The different proliferation rate was consistent with higher telomerase activity in PSDM-SCs compared to SDM-SCs, although restricted at early passages. SDM-SCs express early (Pax7, MyoD) and late (myosin heavy chain, myogenin) myogenic markers differently from PSDM-SCs resulting in a more efficient and faster cell differentiation. Taken together, our results showed that PSDM-SCs elicit a stronger stem cell phenotype compared to SDM ones. Finally, myomiR expression profile reveals a unique epigenetic signature in GRMD SCs and miR-206, highly expressed in dystrophic SCs, seems to play a critical role in muscle degeneration. Thus, miR-206 could represent a potential target for novel therapeutic approaches.

HA14-1 potentiates apoptosis in B-cell cancer cells sensitive to a peptide disrupting IP3 receptor / Bcl-2 complexes

Anti-apoptotic B-cell lymphoma 2 (Bcl-2) is commonly upregulated in hematological cancers, including B-cell chronic lymphocytic leukemia (B-CLL) and diffuse large B-cell lymphoma (DLBCL), thereby protecting neoplastic cells from oncogenic-stress-induced apoptosis. Bcl-2 executes its anti-apoptotic function at two different sites in the cell. At the mitochondria, Bcl-2 via its hydrophobic cleft interacts with pro-apoptotic Bcl-2 family members to inhibit apoptosis. At the endoplasmic reticulum (ER), Bcl-2 via its Bcl-2 homology (BH)4 domain, prevents excessive Ca(2+) signals by interacting with the inositol 1,4,5-trisphosphate receptor (IP3R), an intracellular Ca(2+)-release channel. A peptide tool (BIRD-2) that targets the BH4 domain of Bcl-2 reverses Bcl-2s inhibitory action on IP3Rs and can trigger pro-apoptotic Ca(2+)signals in B-cell cancer cells. Here, we explored whether HA14-1, a Bcl-2 inhibitor that also inhibits sarco/endoplasmic reticulum Ca(2+)-ATPases (SERCA), could potentiate BIRD-2-induced cell death. We measured apoptosis in Annexin V/7-AAD stained cells using flow cytometry and intracellular Ca(2+) signals in Fura2-AM-loaded cells using an automated fluorescent plate reader. HA14-1 potentiated BIRD-2-induced Ca(2+) release from the ER and apoptosis in both BIRD-2-sensitive DLBCL cell lines (SU-DHL-4) and in primary B-CLL cells. BIRD-2-resistant DLBCL cells (OCI-LY-1) were already very sensitive to HA14-1. Yet, although BIRD-2 moderately increased Ca(2+) levels in HA14-1-treated cells, apoptosis was not potentiated by BIRD-2 in these cells. These results further underpin the relevance of IP3R-mediated Ca(2+) signaling as a therapeutic target in the treatment of Bcl-2-dependent B-cell malignancies and the advantage of combination regimens with HA14-1 to enhance BIRD-2-induced cell death.

Myomir dysregulation and reactive oxygen species in aged human satellite cells

Satellite cells that reside on the myofibre surface are crucial for the muscle homeostasis and regeneration. Aging goes along with a less effective regeneration of skeletal muscle tissue mainly due to the decreased myogenic capability of satellite cells. This phenomenon impedes proper maintenance and contributes to the age-associated decline in muscle mass, known as sarcopenia. The myogenic potential impairment does not depend on a reduced myogenic cell number, but mainly on their difficulty to complete a differentiation program. The unbalanced production of reactive oxygen species in elderly people could be responsible for skeletal muscle impairments. microRNAs are conserved post-transcriptional regulators implicated in numerous biological processes including adult myogenesis. Here, we measure the ROS level and analyze myomiR (miR-1, miR-133b and miR-206) expression in human myogenic precursors obtained from Vastus lateralis of elderly and young subjects to provide the molecular signature responsible for the differentiation impairment of elderly activated satellite cells.

Effect of milrinone analogues on intracellular calcium increase in single living H9C2 cardiac cells

The synthesis of milrinone analogues where the 4-pyridyl moiety was replaced by an ester or amide group is reported. Only amide derivatives are able to support intracellular calcium influx following chemical depolarization with 60 mM KCl in a percentage varying from 20 to 45% of differentiated H9C2 cardiomyocytes. Those cells were differentiated after chronic exposure to 10 nM retinoic acid which induces the expression of voltage-gated calcium channels. Analogues of milrinone containing an ester function did not show significant activity.

Extracellular GTP is a Potent Water- Transport Regulator via Aquaporin 5 Plasma-Membrane Insertion in M1-CCD Epithelial Cortical Collecting Duct Cells

Background/Aims: Extracellular GTP is able to modulate some specific functions in neuron, glia and muscle cell models as it has been demonstrated over the last two decades. In fact, extracellular GTP binds its specific plasma membrane binding sites and induces signal transduction via [Ca2+]i increase. We demonstrate, for the first time, that extracellular GTP is able to modulate cell swelling in M1-CCD cortical collecting duct epithelial cells via upregulation of aquaporin 5 (AQP5) expression. Methods: We used videoimaging, immunocitochemistry, flow cytometry, confocal techniques, Western blotting and RT-PCR for protein and gene expression analysis, respectively. Results: We demonstrate that AQP5 mRNA is up-regulated 7 h after the GTP exposure in the cell culture medium, and its protein level is increased after 12-24 h. We show that AQP5 is targeted to the plasma membrane of M1-CCD cells, where it facilitates cell swelling, and that the GTP-dependent AQP5 up-regulation occurs via [Ca2+]i increase. Indeed, GTP induces both oscillating and transient [Ca2+]i increase, and specifically the oscillating kinetic appears to be responsible for blocking cell cycle in the S-phase while the [Ca2+]i influx, with whatever kinetic, seems to be responsible for inducing AQP5 expression. Conclusion: The role of GTP as a regulator of AQP5-mediated water transport in renal cells is of great importance in the physiology of renal epithelia, due to its possible physiopathological implications. GTP-dependent AQP5 expression could act as osmosensor. In addition, the data presented here suggest that GTP might play the same role in other tissues where rapid water transport is required for cell volume regulation and maintenance of the homeostasis.

A double point mutation at residues Ile14 and Val15 of Bcl‐2 uncovers a role for the BH4 domain in both protein stability and function

B‐cell lymphoma 2 (Bcl‐2) protein is the archetype apoptosis suppressor protein. The N‐terminal Bcl‐2‐homology 4 (BH4) domain of Bcl‐2 is required for the antiapoptotic function of this protein at the mitochondria and endoplasmic reticulum (ER). The involvement of the BH4 domain in Bcl‐2′s antiapoptotic functions has been proposed based on Gly‐based substitutions of the Ile14/Val15 amino acids, two hydrophobic residues located in the center of Bcl‐2′s BH4 domain. Following this strategy, we recently showed that a BH4‐domain‐derived peptide in which Ile14 and Val15 have been replaced by Gly residues, was unable to dampen proapoptotic Ca2+‐release events from the ER. Here, we investigated the impact of these mutations on the overall structure, stability, and function of full‐length Bcl‐2 as a regulator of Ca2+ signaling and cell death. Our results indicate that full‐length Bcl‐2 Ile14Gly/Val15Gly, in contrast to wild‐type Bcl‐2, (a) displayed severely reduced structural stability and a shortened protein half‐life; (b) failed to interact with Bcl‐2‐associated X protein (BAX), to inhibit the inositol 1,4,5‐trisphosphate receptor (IP3R) and to protect against Ca2+‐mediated apoptosis. We conclude that the hydrophobic face of Bcl‐2′s BH4 domain (Ile14, Val15) is an important structural regulatory element by affecting protein stability and turnover, thereby likely reducing Bcl‐2′s ability to modulate the function of its targets, like IP3R and BAX. Therefore, Bcl‐2 structure/function studies require pre‐emptive and reliable determination of protein stability upon introduction of point mutations at the level of the BH4 domain.