线粒体质量控制调控骨质疏松的研究机制进展

摘 要 线粒体是细胞内的信号中枢，在细胞生存、代谢和死亡过程中发挥关键作用。细胞需要维持线粒体的健康状态，并快速修复或清除受损的线粒体，以避免细胞死亡和组织损伤。线粒体质量控制（MQC）机制用于监测和维持线粒体的数量和质量，其中包括线粒体融合和裂变、线粒体生物发生和自噬等过程。骨质疏松症（OP）是一种常见的与衰老相关的疾病，其特征是骨量减少和易碎性骨折。研究发现，线粒体功能障碍在骨质疏松的发展中起着重要作用，线粒体异常功能和数量减少会导致骨细胞代谢紊乱，进而影响骨组织的结构和功能。因此，严格控制线粒体的质量和数量对于预防线粒体损伤对骨质疏松的病理影响至关重要。本综述旨在概述MQC所涉及的分子机制，总结目前MQC在骨质疏松进展中的复杂作用及潜在的治疗策略。     

Apoptosis and mitochondrial damage in INS-1 cells treated with alloxan

To evaluate the participation of mitochondrial damage, oxygen radicals and cell death in diabetes mellitus, we designed a way to investigate INS-1 cells, rat pancreatic beta-cell line, to die by treatment with alloxan which generate reactive oxygen species (ROS). Incubation of INS-1 cells with alloxan for 24 h resulted in a decrease in viability of cells as well as inhibition of glucose-stimulated insulin release; this could be prevented by antioxidants, vitamin E and butylated hydroxyanisol (BHA). The formation of a DNA ladder and the distribution of phosphatidylserine at the external surface of plasma membrane were observed as indicators of apoptosis in the cells treated with alloxan at concentrations below 0.5 mM. The formation of DNA ladder was prevented by vitamin E, BHA and catalase, suggesting that the ROS is involved in the process of apoptosis in INS-1 cells treated with alloxan. Lower levels of intracellular ATP, collapse of mitochondrial membrane potential and release of cytochrome c from mitochondria were also observed in INS-1 cells treated with alloxan, suggesting that alloxan caused the damage of mitochondria in cells and was related to the process of apoptosis. In contrast, rat liver RLC-18 cells treated with alloxan were not observed in the decrease of viability. It follows from the present study that mitochondrial damages by ROS generated from alloxan is linked to apoptosis in INS-1 cells.     

Tributyltin induces mitochondrial fission through Mfn1 degradation in human induced pluripotent stem cells

Organotin compounds, such as tributyltin (TBT), are well-known endocrine disruptors. TBT is also known to cause various forms of cytotoxicity, including neurotoxicity and immunotoxicity. However, TBT toxicity has not been identified in normal stem cells. In the present study, we examined the effects of TBT on cell growth in human induced pluripotent stem cells (iPSCs). We found that exposure to nanomolar concentrations of TBT decreased intracellular ATP levels and inhibited cell viability in iPSCs. Because TBT suppressed energy production, which is a critical function of the mitochondria, we further assessed the effects of TBT on mitochondrial dynamics. Staining with MitoTracker revealed that nanomolar concentrations of TBT induced mitochondrial fragmentation. TBT also reduced the expression of mitochondrial fusion protein mitofusin 1 (Mfn1), and this effect was abolished by knockdown of the E3 ubiquitin ligase membrane-associated RING-CH 5 (MARCH5), suggesting that nanomolar concentrations of TBT could induce mitochondrial dysfunction via MARCH5-mediated Mfn1 degradation in iPSCs. Thus, mitochondrial function in normal stem cells could be used to assess cytotoxicity associated with metal exposure.     

Mitochondrial division inhibitor (mdivi-1) inhibits proliferation and epithelial-mesenchymal transition via the NF-κB pathway in thyroid cancer cells

Excessively fragmented mitochondria have been reported in thyroid cancer (TC). Mitochondrial division inhibitor (mdivi-1), a putative inhibitor of dynamin-related protein 1 (Drp1), prevents mitochondrial fission and thereby restricts cell proliferation across several types of primary cancer. However, the role of mdivi-1 on TC has not been sufficiently studied. This research is intended to explore the therapeutic effect of mdivi-1 in TC cells. Results demonstrated that highly invasive TC cells displayed excessive mitochondrial fission with more fragmented mitochondria. Treatment with mdivi-1 inhibited mitochondrial fission in 8505C cells as indicated by transmission electron microscope (TEM). It also impaired the proliferation and increased apoptosis in 8505C and K1 cells as shown by plate cloning assay, cell viability assay, and apoptosis assay. Mdivi-1 treatment also attenuated migratory and invasive abilities in 8505C and K1 cells as shown by the transwell assay and the wound healing assay. And we noticed the same inhibition of mdivi-1 in cell migration and cell viability after the knockdown of Drp1 in 8505C cells. This demonstrated that mdivi-1 exerted an anti-tumor effect independently of Drp1 in 8505C cells. Moreover, mdivi-1 treatment reversed epithelial-mesenchymal transition (EMT) by inhibiting the NF-κB pathway in 8505C cells. The present findings demonstrate that mdivi-1 has a therapeutic role in thyroid carcinoma.     

Mitochondrial transplantation as a promising therapy for mitochondrial diseases

Mitochondrial diseases are a group of inherited or acquired metabolic disorders caused by mitochondrial dysfunction which may affect almost all the organs in the body and present at any age. However, no satisfactory therapeutic strategies have been available for mitochondrial diseases so far. Mitochondrial transplantation is a burgeoning approach for treatment of mitochondrial diseases by recovery of dysfunctional mitochondria in defective cells using isolated functional mitochondria. Many models of mitochondrial transplantation in cells, animals, and patients have proved effective via various routes of mitochondrial delivery. This review presents different techniques used in mitochondrial isolation and delivery, mechanisms of mitochondrial internalization and consequences of mitochondrial transplantation, along with challenges for clinical application. Despite some unknowns and challenges, mitochondrial transplantation would provide an innovative approach for mitochondrial medicine.     

Autofluorescence microscopy: a non-destructive tool to monitor mitochondrial toxicity

Visualization of NADH by fluorescence microscopy makes it possible to distinguish mitochondria inside living cells, allowing structure analysis of these organelles in a non-invasive way. Mitochondrial morphology is determined by the occurrence of mitochondrial fission and fusion. During normal cell function mitochondria appear as elongated tubular structures. However, cellular malfunction induces mitochondria to fragment into punctiform, vesicular structures. This change in morphology is associated with the generation of reactive oxygen species (ROS) and early apoptosis. The aim of this study is to demonstrate that autofluorescence imaging of mitochondria in living eukaryotic cells provides structural and morphological information that can be used to assess mitochondrial health. We firstly established the illumination conditions that do not affect mitochondrial structure and calculated the maximum safe light dose to which the cells can be exposed. Subsequently, sequential recording of mitochondrial fluorescence was performed and changes in mitochondrial morphology were monitored in a continuous non-destructive way. This approach was then used to assess mitochondrial toxicity induced by potential toxicants exposed to mammalian cells. Both mouse and human cells were used to evaluate mitochondrial toxicity of different compounds with different toxicities. This technique constitutes a novel and promising approach to explore chemical induced toxicity because of its reliability to monitor mitochondrial morphology changes and corresponding toxicity in a non-invasive way.     

The influence of chronic fluorosis on mitochondrial dynamics morphology and distribution in cortical neurons of the rat brain

The present study was designed to evaluate the effects of chronic fluorosis on the dynamics (including fusion and fission proteins), fragmentation, and distribution of mitochondria in the cortical neurons of the rat brain in an attempt to elucidate molecular mechanisms underlying the brain damage associated with excess accumulation of fluoride. Sixty Sprague–Dawley rats were divided randomly into three groups of 20 each, that is, the untreated control group (drinking water naturally containing <0.5 mg fluoride/l, NaF), the low-fluoride group (whose drinking water was supplemented with 10 mg fluoride/l) and the high-fluoride group (50 mg fluoride/l). After 6 months of exposure, the expression of mitofusin-1 (Mfn1), fission-1 (Fis1), and dynamin-related protein-1 (Drp1) at both the protein and mRNA levels were detected by Western blotting, immunohistochemistry, and real-time PCR, respectively. Moreover, mitochondrial morphology and distribution in neurons were observed by transmission electron or fluorescence microscopy. In the cortices of the brains of rats with chronic fluorosis, the level of Mfn1 protein was clearly reduced, whereas the levels of Fis1 and Drp1 were elevated. The alternations of expression of the mRNAs encoding all three of these proteins were almost the same as the corresponding changes at the protein levels. The mitochondria were fragmented and the redistributed away from the axons of the cortical neurons. These findings indicate that chronic fluorosis induces abnormal mitochondrial dynamics, which might in turn result in a high level of oxidative stress.     

Non-invasive imaging of ferucarbotran labeled INS-1E cells and rodent islets in vitro and in transplanted diabetic rats

Transplantation of pancreatic islets is a promising strategy for restoring insulin secretion in diabetes mellitus. To monitor transplanted islets, a method to evaluate the distribution in a non-invasive manner in vivo is needed. INS-1E, a stable differentiated insulin secreting cell line, and rodent islets were used to monitor cell transplantation by MRI. For labeling INS-1E cells in vitro, increasing concentrations of Resovist in culture medium were tested. For MR imaging in a clinical 3T scanner, we placed a layer of labeled INS-1E cells between two layers of 4% gelatin. Viability assay was performed. Cell function was evaluated by static incubation assay to assess insulin secretion. For in vivo imaging, iron labeled rodent islets were transplanted into the liver of streptozotocin induced diabetic rats and visualized by MRI. Blood sugar values were controlled and liver tissue was removed for histological analysis. SPIO labeled INS-1E cells did not show altered viability or reduced glucose stimulated insulin secretion in vitro. Double staining of labeled and unlabeled INS-1E cells showed no difference in the staining pattern. Labeling of rodent islets with SPIOs does not reduce their secretory activity or alter their viability. We visualized SPIO-labeled INS-1E cells and rat islets in vitro using a clinical 3T scanner. Diabetic rats transplanted with SPIO-labeled islets became normoglycemic. MR imaging successfully verified the distribution of labeled transplanted cells in vivo. Labeling INS-1E cells and rat islets with SPIOs does not alter their viability, while enabling MR imaging of labeled cells in vitro and within the living organism.     

Mitochondrial quality control mechanisms as molecular targets in cardiac ischemia–reperfusion injury

Mitochondrial damage is a critical contributor to cardiac ischemia/reperfusion (I/R) injury. Mitochondrial quality control (MQC) mechanisms, a series of adaptive responses that preserve mitochondrial structure and function, ensure cardiomyocyte survival and cardiac function after I/R injury. MQC includes mitochondrial fission, mitochondrial fusion, mitophagy and mitochondria-dependent cell death. The interplay among these responses is linked to pathological changes such as redox imbalance, calcium overload, energy metabolism disorder, signal transduction arrest, the mitochondrial unfolded protein response and endoplasmic reticulum stress. Excessive mitochondrial fission is an early marker of mitochondrial damage and cardiomyocyte death. Reduced mitochondrial fusion has been observed in stressed cardiomyocytes and correlates with mitochondrial dysfunction and cardiac depression. Mitophagy allows autophagosomes to selectively degrade poorly structured mitochondria, thus maintaining mitochondrial network fitness. Nevertheless, abnormal mitophagy is maladaptive and has been linked to cell death. Although mitochondria serve as the fuel source of the heart by continuously producing adenosine triphosphate, they also stimulate cardiomyocyte death by inducing apoptosis or necroptosis in the reperfused myocardium. Therefore, defects in MQC may determine the fate of cardiomyocytes. In this review, we summarize the regulatory mechanisms and pathological effects of MQC in myocardial I/R injury, highlighting potential targets for the clinical management of reperfusion.     

Subacute and sublethal ingestion of microcystin-LR impairs lung mitochondrial function by an oligomycin-like effect

Microcystin-LR (MC-LR) is a potent cyanotoxin that can reach several organs. However subacute exposure to sublethal doses of MC-LR has not yet well been studied. Herein, we evaluated the outcomes of subacute and sublethal MC-LR exposure on lungs. Male BALB/c mice were exposed to MC-LR by gavage (30 µg/kg) for 20 consecutive days, whereas CTRL mice received filtered water. Respiratory mechanics was not altered in MC-LR group, but histopathology disclosed increased collagen deposition, immunological cell infiltration, and higher percentage of collapsed alveoli. Mitochondrial function was extensively affected in MC-LR animals. Additionally, a direct in vitro titration of MC-LR revealed impaired mitochondrial function. In conclusion, MC-LR presented an intense deleterious effect on lung mitochondrial function and histology. Furthermore, MC-LR seems to exert an oligomycin-like effect in lung mitochondria. This study opens new perspectives for the understanding of the putative pulmonary initial mechanisms of damage resulting from oral MC-LR intoxication.     

Mitochondrial function in carbon tetrachloride-induced cirrhosis in the rat. Qualitative and quantitative defects

Mitochondrial function is impaired in patients and experimental animals with liver cirrhosis. The relationship between mitochondrial impairment and severity of cirrhosis is unknown, however. We therefore characterized the severity of cirrhosis in rats with phenobarbital/CCl4-induced cirrhosis by the aminopyrine breath test, a microsomal function test reflecting hepatocellular mass. Mitochondrial function was evaluated by measuring oxygen consumption, enzyme activities and ATP production in mitochondria isolated from cirrhotic (N = 8) and control livers (N = 4). Oxygen consumption and mitochondrial enzyme activities calculated per liver were significantly reduced in the presence of cirrhosis. This decrease corresponded to the loss of hepatocytes calculated from the reduction in aminopyrine breath test. The effect of atractylate, oligomycin and dinitrophenol on state 3 respiration was equal between the two groups. The respiratory control ratio was significantly reduced in mitochondria from cirrhotic livers with beta-hydroxybutyrate (4.01 +/- 0.94 vs 5.45 +/- 0.40), but not with succinate as substrate. The rate of ATP production was significantly decreased in mitochondria from cirrhotic rats for both substrates. In contrast, the static head (state 4) phosphate potential was fully developed after 10 min and was equal between the two groups. We conclude that cirrhosis of the liver leads to a loss of hepatocytes which is paralleled by reduced oxygen uptake and reduced mitochondrial enzyme activities.     

Attenuation of Aβ toxicity by promotion of mitochondrial fusion in neuroblastoma cells by liquiritigenin

Mitochondrial dynamics control mitochondrial morphology and function, and aberrations in these are associated with various neurodegenerative diseases including Alzheimer’s disease and Parkinson’s disease. To identify novel regulators of mitochondrial dynamics, we screened a phytochemical library and identified liquiritigenin as a potent inducer of mitochondrial fusion. Treatment with liquiritigenin induced an elongated mitochondrial morphology in SK-N-MC cells. In addition, liquiritigenin rescued mitochondrial fragmentation induced by knockout of mitochondrial fusion mediators such as Mfn1, Mfn2, and Opa1. Furthermore, we found that treatment with liquiritigenin notably inhibited mitochondrial fragmentation and cytotoxicity induced by Aβ in SK-N-MC cells.     

The antiepileptic drug topiramate preserves metabolism-secretion coupling in insulin secreting cells chronically exposed to the fatty acid oleate

Topiramate (Topamax), primarily prescribed against epilepsy, was reported to reduce body weight and to ameliorate glycemic control in obese patients with diabetes. In rodent models of obesity and diabetes, topiramate treatment counteracts hyperglycemia and increases insulin levels upon glucose tolerance test. These observations suggest that topiramate might exert direct action on insulin secreting cells, in particular regarding obesity associated β-cell dysfunction. In this study, INS-1E β-cells were exposed for 3 days to the fatty acid oleate (0.4 mM) and concomitantly treated with therapeutic concentrations of topiramate before measurements of insulin secretion and metabolic parameters. In healthy cells, topiramate had no acute or chronic effects on insulin release. Exposure of INS-1E cells to oleate for 3 days increased insulin release at basal 2.5 mM glucose and blunted the response to stimulatory glucose concentration (15 mM). Such lipotoxic effects were associated with impaired mitochondrial function, as evidenced by partial loss of resting mitochondrial membrane potential and reduced hyperpolarization in response to glucose. Oil-red-O staining and triglyceride measurements revealed lipid accumulation in oleate treated cells. Topiramate treatment counteracted oleate-induced lipid load and partially protected against mitochondrial membrane dysfunction. In particular, topiramate restored glucose stimulated insulin secretion, essentially by maintaining low insulin release at basal glucose. Topiramate increased expression of the nutrient sensor PPARα and of the mitochondrial fatty acid carrier CPT-1, correlating with enhancement of β-oxidation rate. The data demonstrate that a drug originally used as mood stabilizer exerts a direct action on β-cells, protecting against lipid-induced dysfunction.     

配伍对芒果苷在INS-1细胞药代动力学的影响及其在细胞内的分布

目的考察配伍黄柏对知母中芒果苷(mangiferin,MGF)在INS-1细胞药代动力学特征的影响,及芒果苷在正常和氧化损伤状态的INS-1细胞内的分布变化。方法以芒果苷单体、知母和知母-黄柏药对形式等剂量给药INS-1细胞,运用LC-MS/MS测定INS-1细胞中MGF的含量;设置正常组和模型组(140μmol·L^(-1 )H ₂O ₂处理INS-1细胞1 h),给药100μmol·L^(-1 ) MGF,分离线粒体、细胞核及胞质并检测其中的MGF。结果与单体给药相比,知母和药对给药后MGF在细胞内的浓度升高,药对组AUC_(0-t)明显高于单体组和知母组(P<0.01);INS-1细胞单次给药MGF 100μmol·L^(-1 )后,正常组MGF主要分布于细胞核,模型组MGF主要分布于胞质;与正常组相比,MGF在模型组的线粒体和胞质中C _max和AUC_(0-t)升高(P<0.01),细胞核中C _max和AUC_(0-t)明显降低(P<0.05)。结论配伍黄柏对知母有效成分芒果苷进入INS-1细胞具有促进作用;氧化损伤处理会改变MGF在INS-1细胞内的分布。     

Dehydroascorbate protection against dioxin-induced toxicity in the β-cell line INS-1E

Oxidative stress has been proposed as a mechanism of the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The aim of this research was to evaluate the protective effects of increased intracellular ascorbate levels against TCDD acute toxicity in the insulin-secreting β-cell line INS-1E. Ascorbate is considered a potent antioxidant, but its therapeutic efficacy is greatly limited by its slow achievement of high intracellular levels. This might be circumvented by administration of dehydroascorbate (DHA), which is transported at a much higher rate and undergoes rapid intracellular reduction to ascorbate. Indeed, 30 min incubation of INS-1E cells with various concentrations of DHA caused a remarkable, dose-related increase of the intracellular ascorbate levels. INS-1E cells preincubated with 0.5 and 1.0 mM DHA showed a greater viability than control cells after 1 h exposition to cytotoxic TCDD concentrations. In our experimental conditions, TCDD surprisingly failed to increase ROS production in INS-1E cells, but induced a dose-related mitochondrial depolarisation which was significantly improved by DHA preincubation. Furthermore, DHA preincubation completely prevented the low dose TCDD-induced inhibition of glucose-stimulated insulin secretion. Thus, our results suggest that DHA preincubation protects INS-1E cells against TCDD acute toxicity by partially preserving mitochondrial function.     

Superoxide anions mediate veratridine-induced cytochrome c release and caspase activity in bovine chromaffin cells

1. Mitochondrial mechanisms involved in veratridine-induced chromaffin cell death have been explored. 2. Exposure to veratridine (30 micro M, 1 h) produces cytochrome c release to the cytoplasm that seems to be mediated by superoxide anions and that is blocked by cyclosporin A (10 micro M), MnTBAP (10 nM), catalase (100 IU ml(-1)) and vitamin E (50 micro M). 3. Following veratridine treatment, there is an increase in caspase-like activity, blocked by vitamin E (50 micro M) and the mitochondrial permeability transition pore blocker cyclosporin A (10 micro M). 4. Superoxide anions open the mitochondrial permeability transition pore in isolated mitochondria, an effect that is blocked by vitamin E (50 micro M) and cyclosporin A (10 micro M), but not by the Ca2+ uniporter blocker ruthenium red (5 micro M). 5. These results strongly suggest that under the stress situation caused by veratridine, superoxide anions become important regulators of mitochondrial function in chromaffin cells. 6. Exposure of isolated bovine chromaffin mitochondria to Ca2+ results in mitochondrial swelling. This effect was prevented by ruthenium red (5 micro M) and cyclosporin A (10 micro M), while it was not modified by vitamin E (50 micro M). 7. Veratridine (30 micro M, 1 h) markedly decreased total glutathione and GSH content in bovine chromaffin cells. 8. In conclusion, superoxide anions seem to mediate veratridine-induced cytochrome c release, decrease in total glutathione, caspase activation and cell death in bovine chromaffin cells.     

Second generation antipsychotic-induced mitochondrial alterations: Implications for increased risk of metabolic syndrome in patients with schizophrenia

Metabolic syndrome (MetS) is seen more frequently in persons with schizophrenia than in the general population, and these metabolic abnormalities are further aggravated by second generation antipsychotic (SGA) drugs. Although the underlying mechanisms responsible for the increased prevalence of MetS among patients under SGA treatment are not well understood, alterations in mitochondria function have been implicated. We performed a comprehensive evaluation of the role of mitochondrial dysfunction in the pathophysiology of drug-induced MetS in schizophrenia. We found a downregulation in genes encoding subunits of the electron transport chain complexes (ETC), enzyme activity, and mitochondrial dynamics in peripheral blood cells from patients at high-risk for MetS. Additionally, we evaluated several markers of energy metabolism in lymphoblastoid cell lines from patients with schizophrenia and controls following exposure to antipsychotics. We found that the high-risk drugs clozapine and olanzapine induced a general down-regulation of genes involved in the ETC, as well as decreased activities of the corresponding enzymes, ATP levels and a significant decrease in all the functional parameters of mitochondrial oxygen consumption in cells from patients and controls. We also observed that the medium-risk SGA quetiapine decreased oxygen consumption and respiratory control ratio in controls and patients. Additionally, clozapine and olanzapine induced a downregulation of Drp1 and Mfn2 both in terms of mRNA and protein levels. Together, these data suggest that an intrinsic defect in multiple components of oxidative metabolism may contribute to the increased prevalence of MetS in patients under treatment with SGAs known to cause risk for MetS.     

棕榈酸对小鼠胰岛和INS-1E细胞作用的研究

目的:探讨高脂对INS-1E细胞和小鼠胰岛的慢性作用.方法:雌性NMRI小鼠6～10周龄,苯巴比妥腹腔麻醉,常规开腹,取小鼠胰腺组织,应用胶原酶技术消化胰腺分离胰岛.传代培养的INS-1E细胞和分离的小鼠胰岛分别在含与不含棕榈酸的RPMI1640中培养72 h,然后在含3.3、16.7 mmol/L葡萄糖的Krebs-Ringer缓冲液中培养60min,留取上清液行胰岛素测定.INS-1E细胞在含不同浓度的棕榈酸的RPMI1640中培养72 h,提取其总RNA,合成相应的cDNA,再行RT-PCR检测胰十二指肠同源异形盒1(Pdx1),胰岛素1、胰岛素2和葡萄糖转运子2的基因表达.结果:高脂培养后INS-1E细胞和小鼠胰岛的基础胰岛素分泌增加,糖刺激的胰岛素分泌减少,INS-1E细胞的胰岛素1,胰岛素2和葡萄糖转运子2的mRNA水平下降.结论:高脂显示了对胰岛B细胞的慢性毒性作用.     

Recent advances in mitochondrial diseases: From molecular insights to therapeutic perspectives

Mitochondria are double-membraned cytoplasmic organelles that are responsible for the production of energy in eukaryotic cells. The process is completed through oxidative phosphorylation (OXPHOS) by the respiratory chain (RC) in mitochondria. Thousands of mitochondria may be present in each cell, depending on the function of that cell. Primary mitochondria disorder (PMD) is a clinically heterogeneous disease associated with germline mutations in mitochondrial DNA (mtDNA) and/or nuclear DNA (nDNA) genes, and impairs mitochondrial structure and function. Mitochondrial dysfunction can be detected in early childhood and may be severe, progressive and often multi-systemic, involving a wide range of organs. Understanding epigenetic factors and pathways mutations can help pave the way for developing an effective cure. However, the lack of information about the disease (including age of onset, symptoms, clinical phenotype, morbidity and mortality), the limits of current preclinical models and the wide range of phenotypic presentations hamper the development of effective medicines. Although new therapeutic approaches have been introduced with encouraging preclinical and clinical outcomes, there is no definitive cure for PMD. This review highlights recent advances, particularly in children, in terms of etiology, pathophysiology, clinical diagnosis, molecular pathways and epigenetic alterations. Current therapeutic approaches, future advances and proposed new therapeutic plans will also be discussed.