Effects of photobiomodulation on colon cancer cell line HT29 according to mitochondria.

BACKGROUND/AIM: Although photobiomodulation therapy (PBMt) is available to alleviate post-operative side effects of malignant diseases, its application is still controversial due to some potential of cancer recurrence and occurrence of a secondary malignancy. We investigated effect of PBMt on mitochondrial function in HT29 colon cancer cells. METHODS: HT29 cell proliferation was determined with MTT assay after PBMt. Immunofluorescent staining was performed to determine mitochondrial biogenesis and reactive oxygen species (ROS). Mitochondrial membrane potential was measured with Mitotracker. Western blotting was executed to determine expression of fission, fusion, UCP2, and cyclin B1 and D1 proteins. In vivo study was performed by subcutaneously inoculating cancer cells into nude mice and immunohistochemistry was done to determine expression of FIS1, MFN2, UCP2, and p-AKT. RESULTS: The proliferation and migration of HT29 cells reached maximum with PBMt (670 nm, light emitting diode, LED) at 2.0 J/cm2 compared to control (P < 0.05) with more expression of cyclin B1 and cyclin D1 (P < 0.05). Immunofluorescent staining showed that ROS and mitochondrial membrane potential were enhanced after PBMt compared to control. ATP synthesis of mitochondria was also higher in the PBMt group than in the control (P < 0.05). Expression levels of fission and fusion proteins were significantly increased in the PBMt group than in the control (P < 0.05). Electron microscopy revealed that the percentage of mitochondria showing fission was not significantly different between the two groups. Oncometabolites including D-2-hydoxyglutamate in the supernatant of cell culture were higher in the PBMt group than in the control with increased UCP2 expression (P < 0.05). Both tumor size and weight of xenograft in nude mice model were bigger and heavier in the PBMt group than in the control (P < 0.05). Immunohistologically, mitochondrial biogenesis proteins UCP2 and p-AKT in xenograft of nude mice were expressed more in the PBMt group than in the control (P < 0.05). CONCLUSIONS: Treatment with PBM using red light LED may induce proliferation and progression of HT29 cancer cells by increasing mitochondrial activity and fission.

L-Theanine Prolongs the Lifespan by Activating Multiple Molecular Pathways in Ultraviolet C-Exposed Caenorhabditis elegans.

L-theanine, a unique non-protein amino acid, is an important bioactive component of green tea. Previous studies have shown that L-theanine has many potent health benefits, such as anti-anxiety effects, regulation of the immune response, relaxing neural tension, and reducing oxidative damage. However, little is known concerning whether L-theanine can improve the clearance of mitochondrial DNA (mtDNA) damage in organisms. Here, we reported that L-theanine treatment increased ATP production and improved mitochondrial morphology to extend the lifespan of UVC-exposed nematodes. Mechanistic investigations showed that L-theanine treatment enhanced the removal of mtDNA damage and extended lifespan by activating autophagy, mitophagy, mitochondrial dynamics, and mitochondrial unfolded protein response (UPRmt) in UVC-exposed nematodes. In addition, L-theanine treatment also upregulated the expression of genes related to mitochondrial energy metabolism in UVC-exposed nematodes. Our study provides a theoretical basis for the possibility that tea drinking may prevent mitochondrial-related diseases.

Naringenin, a flavanone constituent from Sea buckthorn pulp extract, prevents ultraviolet (UV)-B radiation-induced skin damage via alleviation of impaired mitochondrial dynamics mediated inflammation in human dermal fibroblasts and Balb/c mice models.

Ultraviolet-B (UV-B) irradiation has been reported to cause oxidative stress and inflammation-mediated skin photo-damage. Furthermore, mitochondrial dynamics have been implicated to play a critical role in these processes. For the first time, we describe in this study how UVB-induced aberrant mitochondrial dynamics and inflammation interact in primary human dermal fibroblasts (HDFs). Our findings demonstrated that UV-B irradiation induced -impairment in mitochondrial dynamics by increasing mitochondrial fragmentation in HDFs. Imbalanced mitochondrial dynamics lead to the activation of NFкB and pro-inflammatory cytokines. The current study further aimed to investigate the protective effect of Naringenin (a naturally occurring flavonoid isolated from Sea buckthorn fruit pulp) against UV-B-induced mitochondrial fragmentation and inflammation in HDFs and Balb/c mice. Although Naringenin has been shown to have anti-inflammatory and antioxidant potential, its effects and mechanisms of action on UVB-induced inflammation remained unclear. We observed that Naringenin restored the UV-B-induced imbalance in mitochondrial fission and fusion in HDFs. It also inhibited the phosphorylation of NFкB and reduced the generation of pro-inflammatory cytokines. Naringenin also alleviated UV-B-induced oxidative stress by scavenging the reactive oxygen species and up-regulating the cellular antioxidant enzymes (Catalase and Nrf2). Topical application of Naringenin to the dorsal skin of Balb/c mice exposed to UV-B radiation prevented mitochondrial fragmentation and progression of inflammatory responses. Naringenin treatment prevented neutrophil infiltration and epidermal thickening in mice's skin. These findings provide an understanding for further research into impaired mitochondrial dynamics as a therapeutic target for UV-B-induced inflammation. Our findings imply that Naringenin could be developed as a therapeutic remedy against UVB-induced inflammation.

Mitochondria-localized lncRNA HITT inhibits fusion by attenuating formation of mitofusin-2 homotypic or heterotypic complexes.

Long noncoding RNAs (lncRNAs) are emerging as essential players in multiple biological processes. Mitochondrial dynamics, comprising the continuous cycle of fission and fusion, are required for healthy mitochondria that function properly. Despite long-term recognition of its significance in cell-fate control, the mechanism underlying mitochondrial fusion is not completely understood, particularly regarding the involvement of lncRNAs. Here, we show that the lncRNA HITT (HIF-1α inhibitor at translation level) can specifically localize in mitochondria. Cells expressing higher levels of HITT contain fragmented mitochondria. Conversely, we show that HITT knockdown cells have more tubular mitochondria than is present in control cells. Mechanistically, we demonstrate HITT directly binds mitofusin-2 (MFN2), a core component that mediates mitochondrial outer membrane fusion, by the in vitro RNA pull-down and UV-cross-linking RNA-IP assays. In doing so, we found HITT disturbs MFN2 homotypic or heterotypic complex formation, attenuating mitochondrial fusion. Under stress conditions, such as ultraviolet radiation, we in addition show HITT stability increases as a consequence of MiR-205 downregulation, inhibiting MFN2-mediated fusion and leading to apoptosis. Overall, our data provide significant insights into the roles of organelle (mitochondria)-specific resident lncRNAs in regulating mitochondrial fusion and also reveal how such a mechanism controls cellular sensitivity to UV radiation-induced apoptosis.

Role of Mitochondria in Radiation Responses: Epigenetic, Metabolic, and Signaling Impacts.

Until recently, radiation effects have been considered to be mainly due to nuclear DNA damage and their management by repair mechanisms. However, molecular biology studies reveal that the outcomes of exposures to ionizing radiation (IR) highly depend on activation and regulation through other molecular components of organelles that determine cell survival and proliferation capacities. As typical epigenetic-regulated organelles and central power stations of cells, mitochondria play an important pivotal role in those responses. They direct cellular metabolism, energy supply and homeostasis as well as radiation-induced signaling, cell death, and immunological responses. This review is focused on how energy, dose and quality of IR affect mitochondria-dependent epigenetic and functional control at the cellular and tissue level. Low-dose radiation effects on mitochondria appear to be associated with epigenetic and non-targeted effects involved in genomic instability and adaptive responses, whereas high-dose radiation effects (>1 Gy) concern therapeutic effects of radiation and long-term outcomes involving mitochondria-mediated innate and adaptive immune responses. Both effects depend on radiation quality. For example, the increased efficacy of high linear energy transfer particle radiotherapy, e.g., C-ion radiotherapy, relies on the reduction of anastasis, enhanced mitochondria-mediated apoptosis and immunogenic (antitumor) responses.

Effect of photobiomodulation on mitochondrial dynamics in peripheral nervous system in streptozotocin-induced type 1 diabetes in rats.

There is no effective treatment to halt peripheral nervous system damage in diabetic peripheral neuropathy. Mitochondria have been at the center of discussions as important factors in the development of neuropathy in diabetes. Photobiomodulation has been gaining clinical acceptance as it shows beneficial effects on a variety of nervous system disorders. In this study, the effects of photobiomodulation (904 nm, 45 mW, 6.23 J/cm2, 0.13 cm2, 60 ns pulsed time) on mitochondrial dynamics were evaluated in an adult male rat experimental model of streptozotocin-induced type 1 diabetes. Results presented here indicate that photobiomodulation could have an important role in preventing or reversing mitochondrial dynamics dysfunction in the course of peripheral nervous system damage in diabetic peripheral neuropathy. Photobiomodulation showed its effects on modulating the protein expression of mitofusin 2 and dynamin-related protein 1 in the sciatic nerve and in the dorsal root ganglia neurons of streptozotocin-induced type 1 diabetes in rats.

Cdk5-mediated Drp1 phosphorylation drives mitochondrial defects and neuronal apoptosis in radiation-induced optic neuropathy.

Radiation-induced optic neuropathy (RION) is a devastating complication following external beam radiation therapy (EBRT) that leads to acute vision loss. To date, no efficient, available treatment for this complication, due partly to the lack of understanding regarding the developmental processes behind RION. Here, we report radiation caused changes in mitochondrial dynamics by regulating the mitochondrial fission proteins dynamin-related protein 1 (Drp1) and fission-1 (Fis1). Concurrent with an excessive production of reactive oxygen species (ROS), both neuronal injury and visual dysfunction resulted. Further, our findings delineate an important mechanism by which cyclin-dependent kinase 5 (Cdk5)-mediated phosphorylation of Drp1 (Ser616) regulates defects in mitochondrial dynamics associated with neuronal injury in the development of RION. Both the pharmacological inhibition of Cdk5 by roscovitine and the inhibition of Drp1 by mdivi-1 inhibited mitochondrial fission and the production of ROS associated with radiation-induced neuronal loss. Taken together, these findings may have clinical significance in preventing the development of RION.

Loss of dynamin-related protein 1 (Drp1) does not affect epidermal development or UVB-induced apoptosis but does accelerate UVB-induced carcinogenesis.

BACKGROUND: Mitochondrial morphology is controlled by fission and fusion. Dynamin-related protein 1 (Drp1, dynamin-1-like protein (Dnml1)) regulates mitochondrial fission, which is associated with cell division and apoptosis. We previously reported that DRP1 is indispensable for cell growth in cutaneous squamous cell carcinoma. However, little is known about Drp1 in normal epidermis/keratinocytes. OBJECTIVES: We investigated the function of Drp1 in normal epidermis/keratinocytes. METHODS: Epidermis-specific Drp1 knockout (EKO) mice were analyzed. RESULTS: Epidermal development in the EKO mice were indistinguishable from those in the wild-type (WT) mice. Ultrastructural analysis and immunohistochemistry revealed that the mitochondria of keratinocytes in the EKO mice were neither elongated nor constricted. Drp1 knockdown did not diminish the cell growth of normal human keratinocytes. Both in vivo and in vitro, UVB-induced apoptosis in the EKO epidermis and keratinocytes did not differ from that in the WT mice. In chronic UVB-irradiation, the loss of Drp1 sensitized the epidermis to the development of skin tumors. Clinically, DRP1 is expressed more highly in sun-exposed skin than in non-exposed skin in individuals under age 40, but not in those over age 60. CONCLUSION: EKO mice demonstrate that Drp1 is dispensable for the development and apoptosis of the epidermis. Drp1 plays critical roles in malignant tumors; thus, the molecular machinery of mitochondrial dynamics involving Drp1 could be a novel therapeutic target for malignant keratinocytic lesions. On the other hand, the anti-tumorigenic role of Drp1 in chronic UVB-induced carcinogenesis need to be further investigated.

Mitochondrial fission promotes radiation-induced increase in intracellular Ca2+ level leading to mitotic catastrophe in mouse breast cancer EMT6 cells.

Mitochondrial dynamics are crucial for cellular survival in response to various stresses. Previously, we reported that Drp1 promoted mitochondrial fission after x-irradiation and its inhibition resulted in reduced cellular radiosensitivity and mitotic catastrophe. However, the mechanisms of radiation-induced mitotic catastrophe related to mitochondrial fission remain unclear. In this study, we investigated the involvement of cellular ATP production, ROS generation, and Ca2+ levels in mitotic catastrophe in EMT6 cells. Knockdown of Drp1 and Fis1, which are mitochondrial fission regulators, resulted in elongated mitochondria and significantly attenuated cellular radiosensitivity. Reduced mitochondrial fission mainly decreased mitotic catastrophe rather than necrosis and apoptosis after irradiation. Cellular ATP contents in Drp1 and Fis1 knockdown cells were similar to those in control cells. N-acetylcysteine and 2-glucopyranoside ascorbic acid have no effect on mitotic catastrophe after irradiation. The cellular [Ca2+]i level increased after irradiation, which was completely suppressed by Drp1 and Fis1 inhibition. Furthermore, BAPTA-AM significantly reduced radiation-induced mitotic catastrophe, indicating that cellular Ca2+ is a key mediator of mitotic catastrophe induction after irradiation. These results suggest that mitochondrial fission is associated with radiation-induced mitotic catastrophe via cytosolic Ca2+ regulation.

Neuroprotective effect of anodal transcranial direct current stimulation on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity in mice through modulating mitochondrial dynamics.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the accumulation of protein inclusions and the loss of dopaminergic neurons. Abnormal mitochondrial homeostasis is thought to be important for the pathogenesis of PD. Transcranial direct current stimulation (tDCS), a noninvasive brain stimulation technique, constitutes a promising approach for promoting recovery of various neurological conditions. However, little is known about its mechanism of action. The present study elucidated the neuroprotective effects of tDCS on the mitochondrial quality control pathway in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse model. We used the MPTP-induced neurotoxicity in vivo model. Mice were stimulated for 5 consecutive days with MPTP treatment. After observation of behavioral alteration using the rotarod test, mice were sacrificed for the measurement of the PD- and mitochondrial quality control-related protein levels in the substantia nigra. tDCS improved the behavioral alterations and changes in tyrosine hydroxylase levels in MPTP-treated mice. Furthermore, tDCS attenuated mitochondrial damage, as indicated by diminished mitochondrial swelling and mitochondrial glutamate dehydrogenase activity in the MPTP-induced PD mouse model. MPTP significantly increased mitophagy and decreased mitochondrial biogenesis-related proteins. These changes were attenuated by tDCS. Furthermore, MPTP significantly increased fission-related protein dynamin-related protein 1 with no effect on fusion-related protein mitofusin-2, and tDCS attenuated these changes. Our findings demonstrated the neuroprotective effect of anodal tDCS on the MPTP-induced neurotoxic mouse model through suppressing excessive mitophagy and balancing mitochondrial dynamics. The neuroprotective effect of anodal tDCS with modulation of mitochondrial dynamics provides a new therapeutic strategy for the treatment of PD.

Photobiomodulation preconditioning prevents cognitive impairment in a neonatal rat model of hypoxia-ischemia.

Neonatal hypoxia-ischemia (HI) injury caused by oxygen deprivation is the most common cause of mortality and severe neurologic deficits in neonates. The present work evaluated the preventative effect of photobiomodulation (PBM) preconditioning, and its underlying mechanism of action on brain damage in an HI model in neonatal rats. According to the optimal time response of ATP levels in brain samples removed from normal rats, a PBM preconditioning (PBM-P) regimen (808 nm CW laser, 1 cm2 spot, 100 mW/cm2 , 12 J/cm2 ) was delivered to the scalp 6 hours before HI. PBM-P significantly attenuated cognitive impairment, volume shrinkage in the brain, neuron loss, dendritic and synaptic injury after HI. Further mechanistic investigation found that PBM-P could restore HI-induced mitochondrial dynamics and inhibit mitochondrial fragmentation, followed by a robust suppression of cytochrome c release, and prevention of neuronal apoptosis by inhibition of caspase activation. Our work suggests that PBM-P can attenuate HI-induced brain injury by maintaining mitochondrial dynamics and inhibiting the mitochondrial apoptotic pathway.

Photobiomodulation for Global Cerebral Ischemia: Targeting Mitochondrial Dynamics and Functions.

Hypothermia is currently the only approved therapy for global cerebral ischemia (GCI) after cardiac arrest; however, it unfortunately has multiple adverse effects. As a noninvasive procedure, photobiomodulation (PBM) therapy has emerged as a potential novel treatment for brain injury. PBM involves the use of low-level laser light therapy to influence cell behavior. In this study, we evaluated the therapeutic effects of PBM treatment with an 808-nm diode laser initiated 6 h after GCI. It was noted that PBM dose-dependently protected against GCI-induced neuronal death in the vulnerable hippocampal CA1 subregion. Functional assessments demonstrated that PBM markedly preserved both short-term (a week) and long-term (6 months) spatial learning and memory function following GCI. Further mechanistic studies revealed that PBM post-treatment (a) preserved healthy mitochondrial dynamics and suppressed substantial mitochondrial fragmentation of CA1 neurons, by reducing the detrimental Drp1 GTPase activity and its interactions with adaptor proteins Mff and Fis1 and by balancing mitochondrial targeting fission and fusion protein levels; (b) reduced mitochondrial oxidative damage and excessive mitophagy and restored mitochondrial overall health status and preserved mitochondrial function; and (c) suppressed mitochondria-dependent apoptosome formation/caspase-3/9 apoptosis-processing activities. Additionally, we validated, in an in vitro ischemia model, that cytochrome c oxidase served as a key PBM target for mitochondrial function preservation and neuroprotection. Our findings suggest that PBM serves as a promising therapeutic strategy for the functional recovery after GCI, with mechanisms involving PBM's preservation on mitochondrial dynamics and functions and the inhibition of delayed apoptotic neuronal death in GCI.

Effects of biphasic and monophasic electrical stimulation on mitochondrial dynamics, cell apoptosis, and cell proliferation.

Currently, electrical stimulation (ES) is used to induce changes in various tissues and cellular processes, but its effects on mitochondrial dynamics and mechanisms are unknown. The aim of this study was to compare the effects of monophasic and biphasic, anodal, and cathodal ES on apoptosis, proliferation, and mitochondrial dynamics in neuroblastoma SH-SY5Y cells. Cells were cultured and treated with ES. Alamar blue assay was performed to measure cell proliferation. The proteins expression of apoptotic-related proteins Bcl-2 associated X (Bax), B cell lymphoma 2 (Bcl-2), optic-atrophy-1 (OPA1), mitofusin2 (Mfn2), phosphorylated dynamin-related protein 1 at serine 616 (p-DRP1), and total dynamin-related protein 1 (Total-DRP1) were also determined. The results showed that monophasic anodal and biphasic anodal/cathodal (Bi Anod) ES for 1 hr at 125 pulses per minute (2.0 Hz) produced the most significant increase in cell proliferation. In addition, monophasic anodal and Bi Anod ES treated cells displayed a significant increase in the levels of anti-apoptotic protein Bcl-2, whereas the Bax levels were not changed. Moreover, the levels of Mfn2 were increased in the cells treated by Bi Anod, and OPA1 was increased by monophasic anodal and Bi Anod ES, indicating increased mitochondrial fusion in these ES-treated cells. However, the levels of mitochondrial fission indicated by DRP1 remained unchanged compared with non-stimulated cells. These findings were confirmed through visualization of mitochondria using Mitotracker Deep Red, demonstrating that monophasic anodal and Bi Anod ES could induce pro-survival effects in SH-SY5Y cells through increasing cell proliferation and mitochondrial fusion. Future research is needed to validate these findings for the clinical application of monophasic anodal and Bi Anod ES.

Mitochondrial dynamics (fission and fusion) and collagen production in a rat model of diabetic wound healing treated by photobiomodulation: comparison of 904 nm laser and 850 nm light-emitting diode (LED).

OBJECTIVE: Mitochondrial dysfunction has been associated with the development of diabetes mellitus which is characterized by disorders of collagen production and impaired wound healing. This study analyzed the effects of photobiomodulation (PBM) mediated by laser and light-emitting diode (LED) on the production and organization of collagen fibers in an excisional wound in an animal model of diabetes, and the correlation with inflammation and mitochondrial dynamics. METHODS: Twenty Wistar rats were randomized into 4 groups of 5 animals. Groups: (SHAM) a control non-diabetic wounded group with no treatment; (DC) a diabetic wounded group with no treatment; (DLASER) a diabetic wounded group irradiated by 904 nm pulsed laser (40 mW, 9500 Hz, 1 min, 2.4 J); (DLED) a diabetic wounded group irradiated by continuous wave LED 850 nm (48 mW, 22 s, 1.0 J). Diabetes was induced by injection with streptozotocin (70 mg/kg). PBM was carried out daily for 5 days followed by sacrifice and tissue removal. RESULTS: Collagen fibers in diabetic wounded skin were increased by DLASER but not by DLED. Both groups showed increased blood vessels by atomic force microscopy. Vascular endothelial growth factor (VEGF) was higher and cyclooxygenase (COX2) was lower in the DLED group. Mitochondrial fusion was higher and mitochondrial fusion was lower in DLED compared to DLASER. CONCLUSION: Differences observed between DLASER and DLED may be due to the pulsed laser and CW LED, and to the higher dose of laser. Regulation of mitochondrial homeostasis may be an important mechanism for PBM effects in diabetes.

Different mitochondrial fragmentation after irradiation with X-rays and carbon ions in HeLa cells and its influence on cellular apoptosis.

Although mitochondria are known to play an important role in radiation-induced cellular damage, the mechanisms by which ionizing radiation modulates mitochondrial dynamics are largely unknown. In this study, human cervical carcinoma cell line HeLa was used to demonstrate the different modes of mitochondrial network in response to different quality radiations such as low linear energy transfer (LET) X-rays and high-LET carbon ions. Mitochondria fragmented into punctate and clustered ones upon carbon ion irradiation in a dose- and LET-dependent manner, which was associated with apoptotic cell death. In contrast, low-dose X-ray irradiation promoted mitochondrial fusion while mitochondrial fission was detected until the radiation dose was more than 1 Gy. This fission was driven by ERK1/2-mediated phosphorylation of Drp1 on Serine 616. Inhibition of mitochondrial fragmentation suppressed the radiation-induced apoptosis and thus enhanced the resistance of cells to carbon ions and high-dose X-rays, but not for cells irradiated with X-rays at the low dose. Our results suggest that radiations of different qualities cause diverse changes of mitochondrial dynamics in cancer cells, which play an important role in determining the cell fate.

Fragmentation level determines mitochondrial damage response and subsequently the fate of cancer cells exposed to carbon ions.

OBJECTIVES: Although mitochondria are known to play an important role in radiation-induced cellular damage response, the mechanisms of how radiation elicits mitochondrial responses are largely unknown. MATERIALS AND METHODS: Human cervical cancer cell line HeLa and human breast cancer cell lines MCF-7 and MDA-MB-231 were irradiated with high LET carbon ions at low (0.5 Gy) and high (3 Gy) doses. Mitochondrial functions, dynamics, mitophagy, intrinsic apoptosis and total apoptosis, and survival fraction were investigated after irradiation. RESULTS: We found that carbon ions irradiation induced two different mitochondrial morphological changes and corresponding responses in cancer cells. Cells exposed to carbon ions of 0.5 Gy exhibited only modestly truncated mitochondria, and subsequently damaged mitochondria could be eliminated through mitophagy. In contrast, mitochondria within cells insulted by 3 Gy radiation split into punctate and clustered ones, which were associated with apoptotic cell death afterward. Inhibition of mitochondrial fission by Drp1 or FIS1 knockdown or with the Drp1 inhibitor mdivi-1 suppressed mitophagy and potentiated apoptosis after irradiation at 0.5 Gy. However, inhibiting fission led to mitophagy and increased cell survival when cells were irradiated with carbon ions at 3 Gy. CONCLUSION: We proposed a stress response model to provide a mechanistic explanation for the mitochondrial damage response to high-LET carbon ions.

Calmodulin-dependent protein kinase II (CaMKII) mediates radiation-induced mitochondrial fission by regulating the phosphorylation of dynamin-related protein 1 (Drp1) at serine 616.

Mitochondrial dynamics are suggested to be indispensable for the maintenance of cellular quality and function in response to various stresses. While ionizing radiation (IR) stimulates mitochondrial fission, which is mediated by the mitochondrial fission protein, dynamin-related protein 1 (Drp1), it remains unclear how IR promotes Drp1 activation and subsequent mitochondrial fission. Therefore, we conducted this study to investigate these concerns. First, we found that X-irradiation triggered Drp1 phosphorylation at serine 616 (S616) but not at serine 637 (S637). Reconstitution analysis revealed that introduction of wild-type (WT) Drp1 recovered radiation-induced mitochondrial fission, which was absent in Drp1-deficient cells. Compared with cells transfected with WT or S637A Drp1, the change in mitochondrial shape following irradiation was mitigated in S616A Drp1-transfected cells. Furthermore, inhibition of CaMKII significantly suppressed Drp1 S616 phosphorylation and mitochondrial fission induced by IR. These results suggest that Drp1 phosphorylation at S616, but not at S637, is prerequisite for radiation-induced mitochondrial fission and that CaMKII regulates Drp1 phosphorylation at S616 following irradiation.

Radiation induces progenitor cell death, microglia activation, and blood-brain barrier damage in the juvenile rat cerebellum.

Posterior fossa tumors are the most common childhood intracranial tumors, and radiotherapy is one of the most effective treatments. However, irradiation induces long-term adverse effects that can have significant negative impacts on the patient's quality of life. The purpose of this study was to characterize irradiation-induced cellular and molecular changes in the cerebellum. We found that irradiation-induced cell death occurred mainly in the external germinal layer (EGL) of the juvenile rat cerebellum. The number of proliferating cells in the EGL decreased, and 82.9% of them died within 24 h after irradiation. Furthermore, irradiation induced oxidative stress, microglia accumulation, and inflammation in the cerebellum. Interestingly, blood-brain barrier damage and blood flow reduction was considerably more pronounced in the cerebellum compared to other brain regions. The cerebellar volume decreased by 39% and the migration of proliferating cells to the internal granule layer decreased by 87.5% at 16 weeks after irradiation. In the light of recent studies demonstrating that the cerebellum is important not only for motor functions, but also for cognition, and since treatment of posterior fossa tumors in children typically results in debilitating cognitive deficits, this differential susceptibility of the cerebellum to irradiation should be taken into consideration for future protective strategies.

Quantification and Characterization of UVB-Induced Mitochondrial Fragmentation in Normal Primary Human Keratinocytes.

UV irradiation is a major environmental factor causing skin dryness, aging and cancer. UVB in particular triggers cumulative DNA damage, oxidative stress and mitochondrial dysfunction. The objective of our study was to provide both qualitative and quantitative analysis of how mitochondria respond to UVB irradiation in normal human epidermal keratinocytes (NHEK) of healthy donors, with the rationale that monitoring mitochondrial shape will give an indication of cell population fitness and enable the screening of bioactive agents with UVB-protective properties. Our results show that NHEK undergo dose-dependent mitochondrial fragmentation after exposure to UVB. In order to obtain a quantitative measure of this phenomenon, we implemented a novel tool for automated quantification of mitochondrial morphology in live cells based on confocal microscopy and computational calculations of mitochondrial shape descriptors. This method was used to substantiate the effects on mitochondrial morphology of UVB irradiation and of knocking-down the mitochondrial fission-mediating GTPase Dynamin-related protein 1 (DRP1). Our data further indicate that all the major mitochondrial dynamic proteins are expressed in NHEK but that their level changes were stronger after mitochondrial uncoupler treatment than following UVB irradiation or DRP1 knock-down. Our system and procedures might be of interest for the identification of cosmetic or dermatologic UVB-protective agents.

Far-infrared radiation protects viability in a cell model of Spinocerebellar Ataxia by preventing polyQ protein accumulation and improving mitochondrial function.

Far infrared radiation (FIR) is currently investigated as a potential therapeutic strategy in various diseases though the mechanism is unknown. Presently, we tested if FIR mediates beneficial effects in a cell model of the neurodegenerative disease spinocerebellar ataxia type 3 (SCA3). SCA3 is caused by a mutation leading to an abnormal polyglutamine expansion (PolyQ) in ataxin-3 protein. The consequent aggregation of mutant ataxin-3 results in disruption of vital cell functions. In this study, neuroblastoma cells (SK-N-SH) was transduced to express either non-pathogenic ataxin-3-26Q or pathogenic ataxin-3-78Q proteins. The cells expressing ataxin-3-78Q demonstrated decreased viability, and increased sensitivity to metabolic stress in the presence rotenone, an inhibitor of mitochondrial respiration. FIR exposure was found to protect against these effects. Moreover, FIR improved mitochondrial respiratory function, which was significantly compromised in ataxin-3-78Q and ataxin-3-26Q expressing cells. This was accompanied by decreased levels of mitochondrial fragmentation in FIR treated cells, as observed by fluorescence microscopy and protein expression analysis. Finally, the expression profile LC3-II, Beclin-1 and p62 suggested that FIR prevent the autophagy inhibiting effects observed in ataxin-3-78Q expressing cells. In summary, our results suggest that FIR have rescuing effects in cells expressing mutated pathogenic ataxin-3, through recovery of mitochondrial function and autophagy.