bims-midbra 2022-04-17 papers

bims-midbra

Biomed News

on Mitochondrial dynamics in brain cells

Issue of 2022‒04‒17
twelve papers selected by
Ana Paula Mendonça
University of Padova

High-Resolution Imaging of Mitochondria and Mitochondrial Nucleoids in Differentiated SH-SY5Y Cells.
The Role of Impaired Mitochondrial Dynamics in MFN2-Mediated Pathology.
CLOCK regulates Drp1 mRNA stability and mitochondrial homeostasis by interacting with PUF60.
Drosophila Primary Neuronal Cultures as a Useful Cellular Model to Study and Image Axonal Transport.
Vitamin K2 Modulates Mitochondrial Dysfunction Induced by 6-Hydroxydopamine in SH-SY5Y Cells via Mitochondrial Quality-Control Loop.
Induction of Mitochondrial Fragmentation and Mitophagy after Neonatal Hypoxia-Ischemia.
Defects of Nutrient Signaling and Autophagy in Neurodegeneration.
Cerebellar Pathology in an Inducible Mouse Model of Friedreich Ataxia.
Assessment of Mitochondrial Trafficking as a Surrogate for Fast Axonal Transport in Human Induced Pluripotent Stem Cell-Derived Spinal Motor Neurons.
Morphine-induced microglial immunosuppression via activation of insufficient mitophagy regulated by NLRX1.
PINK1-mediated Drp1S616 phosphorylation modulates synaptic development and plasticity via promoting mitochondrial fission.
Use of Microfluidics Chambers to Image Axonal transport in Adult Sensory Neurons.

Methods Mol Biol. 2022 ;2431 291-310

High-Resolution Imaging of Mitochondria and Mitochondrial Nucleoids in Differentiated SH-SY5Y Cells.

Emily Annuario, Kristal Ng, Alessio Vagnoni.

  Mitochondria are highly dynamic organelles which form intricate networks with complex dynamics. Mitochondrial transport and distribution are essential to ensure proper cell function, especially in cells with an extremely polarised morphology such as neurons. A layer of complexity is added when considering mitochondria have their own genome, packaged into nucleoids. Major mitochondrial morphological transitions, for example mitochondrial division, often occur in conjunction with mitochondrial DNA (mtDNA) replication and changes in the dynamic behaviour of the nucleoids. However, the relationship between mtDNA dynamics and mitochondrial motility in the processes of neurons has been largely overlooked. In this chapter, we describe a method for live imaging of mitochondria and nucleoids in differentiated SH-SY5Y cells by instant structured illumination microscopy (iSIM). We also include a detailed protocol for the differentiation of SH-SY5Y cells into cells with a pronounced neuronal-like morphology and show examples of coordinated mitochondrial and nucleoid motility in the long processes of these cells.

Keywords:  Axonal transport; Instant structured illumination microscopy (iSIM); Mitochondria; Mitochondrial DNA; Mitochondrial fission; Neuronal differentiation; Nucleoids; SH-SY5Y cells; Superresolution

DOI:  https://doi.org/10.1007/978-1-0716-1990-2_15
Front Cell Dev Biol. 2022 ;10 858286

The Role of Impaired Mitochondrial Dynamics in MFN2-Mediated Pathology.

Mashiat Zaman, Timothy E Shutt.

  The Mitofusin 2 protein (MFN2), encoded by the MFN2 gene, was first described for its role in mediating mitochondrial fusion. However, MFN2 is now recognized to play additional roles in mitochondrial autophagy (mitophagy), mitochondrial motility, lipid transfer, and as a tether to other organelles including the endoplasmic reticulum (ER) and lipid droplets. The tethering role of MFN2 is an important mediator of mitochondrial-ER contact sites (MERCs), which themselves have many important functions that regulate mitochondria, including calcium homeostasis and lipid metabolism. Exemplifying the importance of MFN2, pathogenic variants in MFN2 are established to cause the peripheral neuropathy Charcot-Marie-Tooth Disease Subtype 2A (CMT2A). However, the mechanistic basis for disease is not clear. Moreover, additional pathogenic phenotypes such as lipomatosis, distal myopathy, optic atrophy, and hearing loss, can also sometimes be present in patients with CMT2A. Given these variable patient phenotypes, and the many cellular roles played by MFN2, the mechanistic underpinnings of the cellular impairments by which MFN2 dysfunction leads to disease are likely to be complex. Here, we will review what is known about the various functions of MFN2 that are impaired by pathogenic variants causing CMT2A, with a specific emphasis on the ties between MFN2 variants and MERCs.

Keywords:  CMT2A; MFN2; lipid homeostasis; mitochondria; mitochondrial dynamics; mitochondrial endoplasmic reticulum contact sites; mitophagy; mtDNA

DOI:  https://doi.org/10.3389/fcell.2022.858286
Cell Rep. 2022 Apr 12. pii: S2211-1247(22)00387-4. [Epub ahead of print]39(2): 110635

CLOCK regulates Drp1 mRNA stability and mitochondrial homeostasis by interacting with PUF60.

Lirong Xu, Jiaxin Lin, Yutong Liu, Bingxuan Hua, Qianyun Cheng, Changpo Lin, Zuoqin Yan, Yaping Wang, Ning Sun, Ruizhe Qian, Chao Lu.

  Circadian genes such as Clock, Bmal1, Cryptochrome1/2, and Period1/2/3 constitute the precise circadian system. ClockΔ19 is a commonly used mouse model harboring a circadian clock gene mutation, which lacks the EXON-19-encoded 51 amino acids. Previous reports have shown that ClockΔ19 mice have severe metabolic abnormalities. Here, we report that the mitochondria of ClockΔ19 mice exhibit excessive fission and dysfunction. We also demonstrate that CLOCK binds to the RNA-binding protein PUF60 through its EXON 19. Further, we find that PUF60 directly maintains mitochondrial homeostasis through regulating Drp1 mRNA stability, while the association with CLOCK can competitively inhibit this function. In ClockΔ19 mice, CLOCKΔ19 releases PUF60, leading to enhanced Drp1 mRNA stability and persistent mitochondrial fission. Our results reveal a direct post-transcriptional role of CLOCK in regulating mitochondrial homeostasis via Drp1 mRNA stability and that the loss of EXON 19 of CLOCK in ClockΔ19 mice leads to severe mitochondrial homeostasis disorders.

Keywords:  CP: Metabolism; CP: Molecular biology; Clock; Drp1; PUF60; mRNA stability; mitochondrial fission

DOI:  https://doi.org/10.1016/j.celrep.2022.110635
Methods Mol Biol. 2022 ;2431 429-449

Drosophila Primary Neuronal Cultures as a Useful Cellular Model to Study and Image Axonal Transport.

André Voelzmann, Natalia Sanchez-Soriano.

  The use of primary neuronal cultures generated from Drosophila tissue provides a powerful model for studies of transport mechanisms. Cultured fly neurons provide similarly detailed subcellular resolution and applicability of pharmacology or fluorescent dyes as mammalian primary neurons. As an experimental advantage for the mechanistic dissection of transport, fly primary neurons can be combined with the fast and highly efficient combinatorial genetics of Drosophila, and genetic tools for the manipulation of virtually every fly gene are readily available. This strategy can be performed in parallel to in vivo transport studies to address relevance of any findings. Here we will describe the generation of primary neuronal cultures from Drosophila embryos and larvae, the use of external fluorescent dyes and genetic tools to label cargo, and the key strategies for live imaging and subsequent analysis.

Keywords:  Axonal transport; Drosophila primary neurons; Dynein; Kinesin; Live imaging; Lysotracker; Mitochondria; Mitotracker; Motors; Organelles

DOI:  https://doi.org/10.1007/978-1-0716-1990-2_23
Nutrients. 2022 Apr 04. pii: 1504. [Epub ahead of print]14(7):

Vitamin K2 Modulates Mitochondrial Dysfunction Induced by 6-Hydroxydopamine in SH-SY5Y Cells via Mitochondrial Quality-Control Loop.

Hengfang Tang, Zhiming Zheng, Han Wang, Li Wang, Genhai Zhao, Peng Wang.

  Vitamin K2, a natural fat-soluble vitamin, is a potent neuroprotective molecule, owing to its antioxidant effect, but its mechanism has not been fully elucidated. Therefore, we stimulated SH-SY5Y cells with 6-hydroxydopamine (6-OHDA) in a proper dose-dependent manner, followed by a treatment of vitamin K2. In the presence of 6-OHDA, cell viability was reduced, the mitochondrial membrane potential was decreased, and the accumulation of reactive oxygen species (ROS) was increased. Moreover, the treatment of 6-OHDA promoted mitochondria-mediated apoptosis and abnormal mitochondrial fission and fusion. However, vitamin K2 significantly suppressed 6-OHDA-induced changes. Vitamin K2 played a significant part in apoptosis by upregulating and downregulating Bcl-2 and Bax protein expressions, respectively, which inhibited mitochondrial depolarization, and ROS accumulation to maintain mitochondrial structure and functional stabilities. Additionally, vitamin K2 significantly inhibited the 6-OHDA-induced downregulation of the MFN1/2 level and upregulation of the DRP1 level, respectively, and this enabled cells to maintain the dynamic balance of mitochondrial fusion and fission. Furthermore, vitamin K2 treatments downregulated the expression level of p62 and upregulated the expression level of LC3A in 6-OHDA-treated cells via the PINK1/Parkin signaling pathway, thereby promoting mitophagy. Moreover, it induced mitochondrial biogenesis in 6-OHDA damaged cells by promoting the expression of PGC-1α, NRF1, and TFAM. These indicated that vitamin K2 can release mitochondrial damage, and that this effect is related to the participation of vitamin K2 in the regulation of the mitochondrial quality-control loop, through the maintenance of the mitochondrial quality-control system, and repair mitochondrial dysfunction, thereby alleviating neuronal cell death mediated by mitochondrial damage.

Keywords:  PINK1/Parkin signaling pathway; ROS; apoptosis; mitochondrial biogenesis; mitochondrial dysfunction; mitochondrial quality-control loop; mitophagy; vitamin K2

DOI:  https://doi.org/10.3390/nu14071504
Cells. 2022 Apr 01. pii: 1193. [Epub ahead of print]11(7):

Induction of Mitochondrial Fragmentation and Mitophagy after Neonatal Hypoxia-Ischemia.

Syam Nair, Anna-Lena Leverin, Eridan Rocha-Ferreira, Kristina S Sobotka, Claire Thornton, Carina Mallard, Henrik Hagberg.

  Hypoxia-ischemia (HI) leads to immature brain injury mediated by mitochondrial stress. If damaged mitochondria cannot be repaired, mitochondrial permeabilization ensues, leading to cell death. Non-optimal turnover of mitochondria is critical as it affects short and long term structural and functional recovery and brain development. Therefore, disposal of deficient mitochondria via mitophagy and their replacement through biogenesis is needed. We utilized mt-Keima reporter mice to quantify mitochondrial morphology (fission, fusion) and mitophagy and their mechanisms in primary neurons after Oxygen Glucose Deprivation (OGD) and in brain sections after neonatal HI. Molecular mechanisms of PARK2-dependent and -independent pathways of mitophagy were investigated in vivo by PCR and Western blotting. Mitochondrial morphology and mitophagy were investigated using live cell microscopy. In primary neurons, we found a primary fission wave immediately after OGD with a significant increase in mitophagy followed by a secondary phase of fission at 24 h following recovery. Following HI, mitophagy was upregulated immediately after HI followed by a second wave at 7 days. Western blotting suggests that both PINK1/Parkin-dependent and -independent mechanisms, including NIX and FUNDC1, were upregulated immediately after HI, whereas a PINK1/Parkin mechanism predominated 7 days after HI. We hypothesize that excessive mitophagy in the early phase is a pathologic response which may contribute to secondary energy depletion, whereas secondary mitophagy may be involved in post-HI regeneration and repair.

Keywords:  metabolism; mitochondria; mitochondrial fission; mitophagy; neonatal brain injury; neonatal hypoxia–ischemia; reactive oxygen species

DOI:  https://doi.org/10.3390/cells11071193
Front Cell Dev Biol. 2022 ;10 836196

Defects of Nutrient Signaling and Autophagy in Neurodegeneration.

Jon Ondaro, Haizea Hernandez-Eguiazu, Maddi Garciandia-Arcelus, Raúl Loera-Valencia, Laura Rodriguez-Gómez, Andrés Jiménez-Zúñiga, Julen Goikolea, Patricia Rodriguez-Rodriguez, Javier Ruiz-Martinez, Fermín Moreno, Adolfo Lopez de Munain, Ian James Holt, Francisco Javier Gil-Bea, Gorka Gereñu.

  Neurons are post-mitotic cells that allocate huge amounts of energy to the synthesis of new organelles and molecules, neurotransmission and to the maintenance of redox homeostasis. In neurons, autophagy is not only crucial to ensure organelle renewal but it is also essential to balance nutritional needs through the mobilization of internal energy stores. A delicate crosstalk between the pathways that sense nutritional status of the cell and the autophagic processes to recycle organelles and macronutrients is fundamental to guarantee the proper functioning of the neuron in times of energy scarcity. This review provides a detailed overview of the pathways and processes involved in the balance of cellular energy mediated by autophagy, which when defective, precipitate the neurodegenerative cascade of Parkinson's disease, frontotemporal dementia, amyotrophic lateral sclerosis or Alzheimer's disease.

Keywords:  AMPK; autophagy; glucose metabolism; lipid metabolism; mTORC1 (mechanistic target of rapamycin complex 1); mitochondrial metabolism; neurodegenerative diseases; nutrient sensing pathways

DOI:  https://doi.org/10.3389/fcell.2022.836196
Front Neurosci. 2022 ;16 819569

Cerebellar Pathology in an Inducible Mouse Model of Friedreich Ataxia.

Elizabeth Mercado-Ayón, Nathan Warren, Sarah Halawani, Layne N Rodden, Lucie Ngaba, Yi Na Dong, Joshua C Chang, Carlos Fonck, Fulvio Mavilio, David R Lynch, Hong Lin.

  Friedreich ataxia (FRDA) is an autosomal recessive neurodegenerative disorder caused by deficiency of the mitochondrial protein frataxin. Lack of frataxin causes neuronal loss in various areas of the CNS and PNS. In particular, cerebellar neuropathology in FRDA patients includes loss of large principal neurons and synaptic terminals in the dentate nucleus (DN), and previous studies have demonstrated early synaptic deficits in the Knockin-Knockout mouse model of FRDA. However, the exact correlation of frataxin deficiency with cerebellar neuropathology remains unclear. Here we report that doxycycline-induced frataxin knockdown in a mouse model of FRDA (FRDAkd) leads to synaptic cerebellar degeneration that can be partially reversed by AAV8-mediated frataxin restoration. Loss of cerebellar Purkinje neurons and large DN principal neurons are observed in the FRDAkd mouse cerebellum. Levels of the climbing fiber-specific glutamatergic synaptic marker VGLUT2 decline starting at 4 weeks after dox induction, whereas levels of the parallel fiber-specific synaptic marker VGLUT1 are reduced by 18-weeks. These findings suggest initial selective degeneration of climbing fiber synapses followed by loss of parallel fiber synapses. The GABAergic synaptic marker GAD65 progressively declined during dox induction in FRDAkd mice, while GAD67 levels remained unaltered, suggesting specific roles for frataxin in maintaining cerebellar synaptic integrity and function during adulthood. Expression of frataxin following AAV8-mediated gene transfer partially restored VGLUT1/2 levels. Taken together, our findings show that frataxin knockdown leads to cerebellar degeneration in the FRDAkd mouse model, suggesting that frataxin helps maintain cerebellar structure and function.

Keywords:  cerebellum; frataxin; gene therapy; glutamatergic and GABAergic synapses; mitochondria dynamics

DOI:  https://doi.org/10.3389/fnins.2022.819569
Methods Mol Biol. 2022 ;2431 311-322

Assessment of Mitochondrial Trafficking as a Surrogate for Fast Axonal Transport in Human Induced Pluripotent Stem Cell-Derived Spinal Motor Neurons.

Arpan R Mehta, Siddharthan Chandran, Bhuvaneish T Selvaraj.

  Axonal transport is essential for the development, function, and survival of the nervous system. In an energy-demanding process, motor proteins act in concert with microtubules to deliver cargoes, such as organelles, from one end of the axon to the other. Perturbations in axonal transport are a prominent phenotype of many neurodegenerative diseases, including amyotrophic lateral sclerosis. Here, we describe a simple method to fluorescently label mitochondrial cargo, a surrogate for fast axonal transport, in human induced pluripotent stem cell-derived motor neurons. This method enables the sparse labeling of axons to track directionality of movement and can be adapted to assess not only the cell autonomous effects of a genetic mutation on axonal transport but also the cell non-autonomous effects, through the use of conditioned medium and/or co-culture systems.

Keywords:  Axonal transport; Human induced pluripotent stem cell; Mitochondria; Motor neuron

DOI:  https://doi.org/10.1007/978-1-0716-1990-2_16
J Neuroinflammation. 2022 Apr 12. 19(1): 87

Morphine-induced microglial immunosuppression via activation of insufficient mitophagy regulated by NLRX1.

Jialing Peng, Jingrui Pan, Hongxuan Wang, Jingjing Mo, Lihuan Lan, Ying Peng.

  BACKGROUND: Chronic morphine exposure induces immunosuppression in the peripheral and central nervous system, resulting in susceptibility of patients to invading pathogens. Mitophagy is a crucial regulator of inflammation, and dysregulated mitophagy may cause immunosuppression, but whether mitophagy is linked with morphine-induced immunosuppression in the brain remains unknown. NLRX1 is the only mitochondrially localized NOD family receptor protein which serves as a critical regulator in immunity and mitophagy activation, but it remains an enigma how NLRX1 functions in the crosstalk between microglial inflammatory defense and mitophagy in the presence of morphine.METHODS: Primary microglia and astrocytes, BV2 and MA cell lines were utilized. Mice were stimulated with repeated morphine treatment to mimic chronic morphine exposure, and activation of mitophagy, lysosomal functions, and inflammation were assayed in specific brain regions and immune organs with or without NLRX1-silencing.
RESULTS: Morphine induced microglial mitophagy in a LC3 (microtubule-associated proteins light chain 3)-dependent manner, which was mediated by NLRX1. Contrastingly, morphine impaired lysosomal functions, including generation, acidification and mitophagosome-lysosome fusion, thus leading to insufficient mitophagy activation in microglia. NLRX1-silencing inhibited mitophagy activity and rescued lysosomal functions including generation and acidification in microglia. The NLRX1-mediated incomplete mitophagy in microglial cells contributed to immunosuppression and vulnerability towards pathogenic challenge after morphine treatment. In vivo, NLRX1-mediated microglial mitophagy activation by morphine was mainly located in the murine brain cortex, striatum, and cerebellum, where NLRX1 functioned as a negative immune regulator and facilitated septic shock. Collectively, microglial immune responses to septic shock were amenable to NLRX1 silencing in the brain with morphine treatment.
CONCLUSION: Morphine activated insufficient mitophagy in microglia which was regulated by NLRX1, ultimately leading to host immunosuppression and susceptible conditions in the brain.

Keywords:  Immunosuppression; Microglia; Mitophagy; Morphine; NLRX1

DOI:  https://doi.org/10.1186/s12974-022-02453-7
Signal Transduct Target Ther. 2022 Apr 15. 7(1): 103

PINK1-mediated Drp1S616 phosphorylation modulates synaptic development and plasticity via promoting mitochondrial fission.

Qingtao Gao, Runyi Tian, Hailong Han, Jesse Slone, Caifang Wang, Xiao Ke, Tongmei Zhang, Xiangyu Li, Yuhong He, Panlin Liao, Fang Wang, Ye Chen, Shiqing Fu, Kexuan Zhang, Fangfang Zeng, Yingxuan Yang, Zhuo Li, Jieqiong Tan, Jiada Li, Youming Lu, Taosheng Huang, Zhonghua Hu, Zhuohua Zhang.

Dynamic change of mitochondrial morphology and distribution along neuronal branches are essential for neural circuitry formation and synaptic efficacy. However, the underlying mechanism remains elusive. We show here that Pink1 knockout (KO) mice display defective dendritic spine maturation, reduced axonal synaptic vesicles, abnormal synaptic connection, and attenuated long-term synaptic potentiation (LTP). Drp1 activation via S616 phosphorylation rescues deficits of spine maturation in Pink1 KO neurons. Notably, mice harboring a knockin (KI) phosphor-null Drp1S616A recapitulate spine immaturity and synaptic abnormality identified in Pink1 KO mice. Chemical LTP (cLTP) induces Drp1S616 phosphorylation in a PINK1-dependent manner. Moreover, phosphor-mimetic Drp1S616D restores reduced dendritic spine localization of mitochondria in Pink1 KO neurons. Together, this study provides the first in vivo evidence of functional regulation of Drp1 by phosphorylation and suggests that PINK1-Drp1S616 phosphorylation coupling is essential for convergence between mitochondrial dynamics and neural circuitry formation and refinement.

DOI: https://doi.org/10.1038/s41392-022-00933-z
Methods Mol Biol. 2022 ;2431 271-288

Use of Microfluidics Chambers to Image Axonal transport in Adult Sensory Neurons.

Maria Fransiska Emily, Lokesh Agrawal, Paolo Barzaghi, Miki Otsuki, Marco Terenzio.

  Transport of cargoes along axons is crucial for ensuring effective neuronal function and survival. Lysosomes, which are membrane-bound organelles responsible for the degradation of macromolecules, are among the many cargoes being transported. Compartmentalized systems that allow for the separation of the somatic compartment from the axonal network, are widely used in the field of neurobiology and in the study of axonal transport in particular. Among the various solutions available, microfluidics chambers that take advantage of fluidic separation between different compartments, have seen widespread adoption. Said chambers are made of polydimethylsiloxane (PDMS), a transparent, gas permeable compound, which is compatible with fluorescence microscopy, and have significantly positively impacted cellular neuroscience, drastically increasing our understanding of axonal peripheral signaling. Here we describe a two-layered microfluidics chamber, engineered to allow for the culture of adult sensory neurons. This device was designed to promote the proper placement of adult sensory neurons in the somatic chamber in proximity of the microgrooves. We detail the production of the master mold, how to fabricate and assemble the device and how to disaggregate and load the cells in it. In addition, we provide details on how to conduct and analyze an axonal transport experiment using a custom made script in MATLAB designed by our laboratory.

Keywords:  Adult Sensory Neurons; Axons; Intracellular Transport; Lysosomes; Microfluidics Chambers

DOI:  https://doi.org/10.1007/978-1-0716-1990-2_14