Effects of Class II-Selective Histone Deacetylase Inhibitor on Neuromuscular Function and Disease Progression in SOD1-ALS Mice.

Emerging evidence indicates that transcriptome alterations due to epigenetic deregulation concur to ALS pathogenesis. Accordingly, pan-histone deacetylase (HDAC) inhibitors delay ALS development in mice, but these compounds failed when tested in ALS patients. Possibly, lack of selectivity toward specific classes of HDACs weakens the therapeutic effects of pan-HDAC inhibitors. Here, we tested the effects of the HDAC Class II selective inhibitor MC1568 on disease evolution, motor neuron survival as well as skeletal muscle function in SOD1G93A mice. We report that HDACs did not undergo expression changes during disease evolution in isolated motor neurons of adult mice. Conversely, increase in specific Class II HDACs (-4, -5 and -6) occurs in skeletal muscle of mice with severe neuromuscular impairment. Importantly, treatment with MC1568 causes early improvement of motor performances that vanishes at later stages of disease. Notably, motor improvement is not paralleled by reduced motor neuron degeneration but by increased skeletal muscle electrical potentials, reduced activation of mir206/FGFBP1-dependent muscle reinnervation signaling, and increased muscle expression of myogenic genes.

Effect of Class II HDAC inhibition on glutamate transporter expression and survival in SOD1-ALS mice.

Transcriptional deregulation emerges as a key pathogenetic mechanism in ALS pathogenesis, and non-class-specific histone deacetylase (HDACs) inhibitors proved of therapeutic efficacy in preclinical models of ALS. When tested in patients, however, these drugs failed, probably because of a lack of selectivity toward pathogenetic HDACs. Here, we studied the effects of MC1568, an inhibitor of Class-II HDACs which have been reported to contribute to ALS pathogenesis. We focused on transcriptional regulation of glutamate transporter EAAT2, whose reduced expression may contribute to motor neuron degeneration in ALS. We report that MC1568 highly increased EAAT2 transcripts in primary cultures of mouse glia, but these increases did not correlate with increased glutamate uptake capacity. Accordingly, we found that MC1568 augmented protein expression of EAAT2 together with its sumoylation, a post-translational modification typically altering protein function and localization. When tested in SOD1G93A mice, however, MC1568 fully restored the reduced spinal cord expression of EAAT2 and glutamate uptake up to control levels. A prolonged treatment with MC1568 (from onset to end stage) was unable to prolong survival of mice. Data reveal a key role of Class-II HDACs in expression and function of glutamate transporter, further corroborating preclinical and clinical evidence that the sole restoration of glutamate uptake is not of therapeutic relevance to ALS therapy.

Post onset, oral rapamycin treatment delays development of mitochondrial encephalopathy only at supramaximal doses.

Mitochondrial encephalopathies are fatal, infantile neurodegenerative disorders caused by a deficit of mitochondrial functioning, for which there is urgent need to identify efficacious pharmacological treatments. Recent evidence shows that rapamycin administered both intraperitoneally or in the diet delays disease onset and enhances survival in the Ndufs4 null mouse model of mitochondrial encephalopathy. To delineate the clinical translatability of rapamycin in treatment of mitochondrial encephalopathy, we evaluated the drug's effects on disease evolution and mitochondrial parameters adopting treatment paradigms with fixed daily, oral doses starting at symptom onset in Ndufs4 knockout mice. Molecular mechanisms responsible for the pharmacodynamic effects of rapamycin were also evaluated. We found that rapamycin did not affect disease development at clinically-relevant doses (0.5 mg kg-1). Conversely, an oral dose previously adopted for intraperitoneal administration (8 mg kg-1) delayed development of neurological symptoms and increased median survival by 25%. Neurological improvement and lifespan were not further increased when the dose raised to 20 mg kg-1. Notably, rapamycin at 8 mg kg-1 did not affect the reduced expression of respiratory complex subunits, as well as mitochondrial number and mtDNA content. This treatment regimen however significantly ameliorated architecture of mitochondria cristae in motor cortex and cerebellum. However, reduction of mTOR activity by rapamycin was not consistently found within the brain of knockout mice. Overall, data show the ability of rapamycin to improve ultrastructure of dysfunctional mitochondria and corroborate its therapeutic potential in mitochondrial disorders. The non-clinical standard doses required, however, raise concerns about its rapid and safe clinical transferability.

Dysregulation of sphingosine 1 phosphate receptor-1 (S1P1) signaling and regulatory lymphocyte-dependent immunosuppression in a model of post-fingolimod MS rebound.

Fingolimod affords protection from MS by sequestering lymphocytes in secondary lymphoid organs via down regulation of their sphingosine 1 phosphate receptor (S1P1). Unexpectedly, accumulating evidence indicates that patients who discontinue fingolimod treatment may be at risk of rehearsal of magnetic resonance (MR) and clinical disease activity, sometimes featuring dramatic rebound. We therefore developed in vivo and in vitro models of post-fingolimod MS rebound to unravel its cellular and molecular mechanisms. The impact of fingolimod withdrawal on T regulatory lymphocytes was also investigated by means of cytofluorimetric analysis and antigen-specific lymphocyte proliferation assays. We show that mice with relapsing-remitting experimental autoimmune encephalomyelitis (EAE) undergo extremely severe, chronic disease rebound upon discontinuation of fingolimod. Remarkably, rebound is preceded by a burst of S1P1 overexpression in lymph node-entrapped lymphocytes that correlates with subsequent massive lymphocyte egress and widespread CNS immune infiltration. Also, consistent with the ability of S1P1 to counteract polarization and function of T regulatory lymphocytes their number and suppression of effector T cells is reduced by fingolimod suspension. Data disclose the first pathogenic mechanisms of post-fingolimod rebound that may be targeted for therapeutic intervention.

Pharmacological NAD-Boosting Strategies Improve Mitochondrial Homeostasis in Human Complex I-Mutant Fibroblasts.

Mitochondrial disorders are devastating genetic diseases for which efficacious therapies are still an unmet need. Recent studies report that increased availability of intracellular NAD obtained by inhibition of the NAD-consuming enzyme poly(ADP-ribose) polymerase (PARP)-1 or supplementation with the NAD-precursor nicotinamide riboside (NR) ameliorates energetic derangement and symptoms in mouse models of mitochondrial disorders. Whether these pharmacological approaches also improve bioenergetics of human cells harboring mitochondrial defects is unknown. It is also unclear whether the same signaling cascade is prompted by PARP-1 inhibitors and NR supplementation to improve mitochondrial homeostasis. Here, we show that human fibroblasts mutant for the NADH dehydrogenase (ubiquinone) Fe-S protein 1 (NDUFS1) subunit of respiratory complex I have similar ATP, NAD, and mitochondrial content compared with control cells, but show reduced mitochondrial membrane potential. Interestingly, mutant cells also show increased transcript levels of mitochondrial DNA but not nuclear DNA respiratory complex subunits, suggesting activation of a compensatory response. At variance with prior work in mice, however, NR supplementation, but not PARP-1 inhibition, increased intracellular NAD content in NDUFS1 mutant human fibroblasts. Conversely, PARP-1 inhibitors, but not NR supplementation, increased transcription of mitochondrial transcription factor A and mitochondrial DNA-encoded respiratory complexes constitutively induced in mutant cells. Still, both NR and PARP-1 inhibitors restored mitochondrial membrane potential and increased organelle content as well as oxidative activity of NDUFS1-deficient fibroblasts. Overall, data provide the first evidence that in human cells harboring a mitochondrial respiratory defect exposure to NR or PARP-1, inhibitors activate different signaling pathways that are not invariantly prompted by NAD increases, but equally able to improve energetic derangement.

CoQ10-containing eye drops prevent UVB-induced cornea cell damage and increase cornea wound healing by preserving mitochondrial function.

PURPOSE: We evaluated the potential protective effects of Coenzyme Q10 (CoQ10) on human corneal cells and rabbit eyes after ultraviolet B (UVB) exposure and a model of wound healing in rabbit eyes after corneal epithelium removal. METHODS: Human corneal epithelium cells (HCE) were exposed to a source of UVB radiation (312 nM) in the presence of different CoQ10 concentrations or vehicle. The mitochondrial function and cell survival were evaluated by means of 3-(4,5-dimethylthiazole-2-yl)2,5-diphenyl-tetrazolium (MTT) reduction and lactic dehydrogenase (LDH) release. Furthermore, quantitation of oxygen consumption and mitochondrial membrane potential were conducted. In vivo rabbit models were adopted to evaluate the effect of CoQ10 on UVB-induced conjunctival vessel hyperemia and corneal recovery after ethanol induced corneal lesion. RESULTS: In UVB-exposed HCE cells, CoQ10 addition led to an increased survival rate and mitochondrial function. Furthermore, oxygen consumption was maintained at control levels and adenosine triphosphate (ATP) decline was completely prevented in the CoQ10-treated cells. Interestingly, in an in vivo model, CoQ10 was able dose-dependently to reduce UVB-induced vessel hyperemia. Finally, in a model of corneal epithelium removal, 12 hours from surgery, animals treated with CoQ10 showed a reduction of damaged area in respect to vehicle controls, which lasted until 48 hours. CONCLUSIONS: We demonstrated that CoQ10 reduces corneal damages after UVB exposure in vivo and in vitro by preserving mitochondrial function. Also, for the first time to our knowledge we showed that the administration of CoQ10 after corneal epithelium removal promotes corneal wound healing.

PARP inhibition delays progression of mitochondrial encephalopathy in mice.

Mitochondrial disorders are deadly childhood diseases for which therapeutic remedies are an unmet need. Given that genetic suppression of the nuclear enzyme poly (adenine diphosphate-ribose) polymerase(PARP)-1 improves mitochondrial functioning, we investigated whether pharmacological inhibition of the enzyme affords protection in a mouse model of a mitochondrial disorder. We used mice lacking the Ndufs4 subunit of the respiratory complex I (Ndufs4 knockout [ KO] mice); these mice undergo progressive encephalopathy and die around postnatal day 50. Mice were treated daily with the potent PARP inhibitor N-(6-oxo-5,6-dihydrophenanthridin-2-yl)-(N,N-dimethylamino)acetamide hydrochloride (PJ34); neurological parameters, PARP activity, and mitochondrial homeostasis were evaluated. We found that mice receiving N-(6-oxo-5,6-dihydrophenanthridin-2-yl)-(N,N-dimethylamino)acetamide hydrochloride from postnatal day 30 to postnatal day 50 show reduced neurological impairment, and increased exploratory activity and motor skills compared with vehicle-treated animals. However, drug treatment did not delay or reduce death. We found no evidence of increased PARP activity within the brain of KO mice compared with heterozygous, healthy controls. Conversely, a 10-day treatment with the PARP inhibitor significantly reduced basal poly(ADP-ribosyl)ation in different organs of the KO mice, including brain, skeletal muscle, liver, pancreas, and spleen. In keeping with the epigenetic role of PARP-1, its inhibition correlated with increased expression of mitochondrial respiratory complex subunits and organelle number. Remarkably, pharmacological targeting of PARP reduced astrogliosis in olfactory bulb and motor cortex, but did not affect neuronal loss of KO mice. In light of the advanced clinical development of PARP inhibitors, these data emphasize their relevance to treatment of mitochondrial respiratory defects.

Succinate dehydrogenase subunit B mutations modify human neuroblastoma cell metabolism and proliferation.

Paragangliomas (PGLs) are rare neuroendocrine tumours. About 30-40 % of these tumours are mutated in one of the different susceptibility genes, including those encoding the different subunits of the succinate dehydrogenase, a complex involved both in the tricarboxylic acid cycle and in the oxygen transport chain. The aim of this work was to investigate whether SDHB mutations may account for alterations in cell metabolism and functions. Since human PGL cell lines are not available, we used the neuroblastoma cell line (SK-N-AS) stably transfected with the wild-type human SDHB or different SDHB-mutated constructs carrying some significant mutations found in our patients affected by PGLs. Similarly to succinate dehydrogenase (SDH)-mutated tumour cells, mutated SK-N-AS clones showed reduced SDH enzyme activity. All clones showed normal citrate synthase activity, reduced oxygen consumption and reduced carbonic anhydride production, thus demonstrating a decreased in mitochondrial metabolism. In two of the three mutated SK-N-AS, we also found an increase in HIF1α expression. Surprisingly and unexpectedly, in all the SDHB-mutated clones, we found a significant decrease in glucose uptake and in lactate culture medium concentration, suggesting also a decrease of cytosolic metabolism. Finally, we found that these energetic changes were associated to an increase in cell proliferation and migration. Overall, these data demonstrate that although SDHB mutations significantly downregulate both mitochondrial and cytoplasmic cellular metabolism, these mutations are associated to an upregulation of some cellular functions, such as growth rate and invasiveness.

Nicotinamide phosphoribosyltransferase (NAMPT) activity is essential for survival of resting lymphocytes.

NAD biosynthesis is emerging as a key regulator of immune cell functions. Accordingly, inhibitors of the NAD-synthesizing enzyme nicotinamide phosphoribosyltransferase (NAMPT) have anti-inflammatory effects, counteract hematological malignancies and are being tested in clinical trials. Still, their effect on different cell types still waits to be fully investigated. Here we show that the NAMPT inhibitor FK866 induces NAD depletion in various mouse organs but selectively causes dramatic atrophy of the spleen red pulp. Accordingly, in cultured mouse lymphocytes exposed to FK866, NAD contents drop to 50% of basal values within 2 days, a condition sufficient to prompt complete cell death. Cultures of human lymphocytes are more resistant to FK866 and sustain a 50% NAD reduction for 5 days before dying. Death of both cell types can be prevented by different NAD precursors, indicating critical NAD homeostasis in lymphocytes. Indeed, inhibition of the NAD-consuming enzyme poly(ADP-ribose) polimerase-1 suffices to prevent FK866-induced NAD depletion and death of both lymphocyte types. Poly(ADP-ribose) polymerase-1-null lymphocytes also undergo lower NAD depletion and reduced cell death when exposed to the drug. At variance with other cell types, neither apoptosis nor autophagy are exclusively responsible for lymphocyte death by FK866, consistent with a general impairment of lymphocyte homeostasis following NAD depletion. Data demonstrate a unique sensitivity of resting lymphocytes to NAD-depleting agents, providing new hints of relevance to lymphocyte biology and therapeutic interventions with NAMPT inhibitors.

Insight into molecular and functional properties of NMNAT3 reveals new hints of NAD homeostasis within human mitochondria.

Among the enzymes involved in NAD homeostasis, nicotinamide mononucleotide adenylyltransferases (NMNAT1-3) are central to intracellular NAD formation. Although NMNAT3 is postulated to be a mitochondrial enzyme contributing to NAD-dependent organelle functioning, information on endogenous proteins is lacking. We report that in human cells a single gene nmnat3 localized on chromosome 3 codes for two mRNA splice variants NMNATv1 and FKSG76, whereas the previously reported NMNAT3v2 transcript is not present. However, NMNAT3v1 and FKSG76 proteins are not detectable, consistent with the finding that an upstream ORF in their mRNAs negatively regulates translation. NMNAT3v1 transfection demonstrates that the protein is cytosolic and inactive, whereas FKSG76 is mitochondrial but operates NAD cleavage rather than synthesis. In keeping with the lack of NMNAT3, we show that extracellular NAD, but not its metabolic precursors, sustains mitochondrial NAD pool in an ATP-independent manner. Data of the present study modify the scenario of the origin of mitochondrial NAD by showing that, in human cells, NMNAT3 is absent in mitochondria, and, akin to plants and yeast, cytosolic NAD maintains the mitochondrial NAD pool.

Neurological basis of AMP-dependent thermoregulation and its relevance to central and peripheral hyperthermia.

Therapeutic hypothermia is of relevance to treatment of increased body temperature and brain injury, but drugs inducing selective, rapid, and safe cooling in humans are not available. Here, we show that injections of adenosine 5'-monophosphate (AMP), an endogenous nucleotide, promptly triggers hypothermia in mice by directly activating adenosine A1 receptors (A1R) within the preoptic area (POA) of the hypothalamus. Inhibition of constitutive degradation of brain extracellular AMP by targeting ecto 5'-nucleotidase, also suffices to prompt hypothermia in rodents. Accordingly, sensitivity of mice and rats to the hypothermic effect of AMP is inversely related to their hypothalamic 5'-nucleotidase activity. Single-cell electrophysiological recording indicates that AMP reduces spontaneous firing activity of temperature-insensitive neurons of the mouse POA, thereby retuning the hypothalamic thermoregulatory set point towards lower temperatures. Adenosine 5'-monophosphate also suppresses prostaglandin E2-induced fever in mice, having no effects on peripheral hyperthermia triggered by dioxymetamphetamine (ecstasy) overdose. Together, data disclose the role of AMP, 5'-nucleotidase, and A1R in hypothalamic thermoregulation, as well and their therapeutic relevance to treatment of febrile illness.

The NAD metabolome--a key determinant of cancer cell biology.

NAD is a vital molecule in all organisms. It is a key component of both energy and signal transduction--processes that undergo crucial changes in cancer cells. NAD(+)-dependent signalling pathways are many and varied, and they regulate fundamental events such as transcription, DNA repair, cell cycle progression, apoptosis and metabolism. Many of these processes have been linked to cancer development. Given that NAD(+)-dependent signalling reactions involve the degradation of the molecule, permanent nucleotide resynthesis through different biosynthetic pathways is crucial for incessant cancer cell proliferation. This necessity supports the targeting of NAD metabolism as a new therapeutic concept for cancer treatment.

Ischemic neuroprotection by TRPV1 receptor-induced hypothermia.

Although treatment of stroke patients with mild hypothermia is a promising therapeutic approach, chemicals inducing prompt and safe reduction of body temperature are an unmet need. We measured the effects of the transient receptor potential vanilloid-1 (TRPV1) agonist rinvanil on thermoregulation and ischemic brain injury in mice. Intraperitoneal or intracerebroventricular injection of rinvanil induces mild hypothermia that is prevented by the receptor antagonist capsazepine. Both intraischemic and postischemic treatments provide permanent neuroprotection in animals subjected to transient middle cerebral artery occlusion (MCAo), an effect lost in mice artificially kept normothermic. Data indicate that TRPV1 receptor agonists are promising candidates for hypothermic treatment of stroke.

Pharmacological effects of exogenous NAD on mitochondrial bioenergetics, DNA repair, and apoptosis.

During the last several years, evidence that various enzymes hydrolyze NAD into bioactive products prompted scientists to revisit or design strategies able to increase intracellular availability of the dinucleotide. However, plasma membrane permeability to NAD and the mitochondrial origin of the dinucleotide still wait to be clearly defined. Here, we report that intracellular NAD contents increased upon exposure of cell lines or primary cultures to exogenous NAD (eNAD). NAD precursors could not reproduce the effects of eNAD, and they were not found in the incubating medium containing eNAD, thereby suggesting direct cellular eNAD uptake. We found that in mitochondria of cells exposed to eNAD, NAD and NADH as well as oxygen consumption and ATP production were increased. Conversely, DNA repair, a well known NAD-dependent process, was unaltered upon eNAD exposure. We also report that eNAD conferred significant cytoprotection from apoptosis triggered by staurosporine, C2-ceramide, or N-methyl-N'-nitro-N-nitrosoguanidine. In particular, eNAD reduced staurosporine-induced loss of mitochondrial membrane potential and ensuing caspase activation. Of importance, pharmacological inhibition or silencing of the NAD-dependent enzyme SIRT1 abrogated the ability of eNAD to provide protection from staurosporine, having no effect on eNAD-dependent protection from C2-ceramide or N-methyl-N'-nitro-N-nitrosoguanidine. Taken together, our findings, on the one hand, strengthen the hypothesis that eNAD crosses the plasma membrane intact and, on the other hand, provide evidence that increased NAD contents significantly affects mitochondrial bioenergetics and sensitivity to apoptosis.

Poly(ADP-ribose) polymerase-1 is a nuclear epigenetic regulator of mitochondrial DNA repair and transcription.

Poly(ADP-ribose) polymerase-1 (PARP-1) is a NAD-consuming enzyme with an emerging key role in epigenetic regulation of gene transcription. Although PARP-1 expression is characteristically restricted to the nucleus, a few studies report the mitochondrial localization of the enzyme and its ability to regulate organelle functioning. Here, we show that, despite exclusive nuclear localization of PARP-1, mitochondrial homeostasis is compromised in cell lines exposed to PARP-1 pharmacological inhibitors or small interfering RNA. PARP-1 suppression reduces integrity of mitochondrial DNA (mtDNA), as well as expression of mitochondria-encoded respiratory complex subunits COX-1, COX-2, and ND-2. Accordingly, PARP-1 localizes at promoters of nuclear genes encoding both the mtDNA repair proteins UNG1, MYH1, and APE1 and the mtDNA transcription factors TFB1M and TFB2M. It is noteworthy that poly(ADP-ribosyl)ation is required for nuclear gene expression of these mitochondrial proteins. Consistent with these findings, PARP-1 suppression impairs mitochondrial ATP production. Our results indicate that PARP-1 plays a central role in mitochondrial homeostasis by epigenetically regulating nuclear genes involved in mtDNA repair and transcription. These data might have important implications in pharmacology of PARP-1 inhibitors as well as clinical oncology and aging.