Metformin attenuates cuprizone-induced mitochondrial dysfunction and senescence-associated changes in primary neuronal cells.

SUMMARYα-synuclein (α-syn) aggregation and accumulation drive neurodegeneration in Parkinson’s disease (PD). The substantia nigra of patients with PD contains excess iron, yet the underlying mechanism accounting for this iron accumulation is unclear. Here, we show that misfolded α-syn activates microglia, which release interleukin 6 (IL-6). IL-6, via its trans-signaling pathway, induces changes in the neuronal iron transcriptome that promote ferrous iron uptake and decrease cellular iron export via a pathway we term the cellular iron sequestration response, or CISR. The brains of patients with PD exhibit molecular signatures of the IL-6-mediated CISR. Genetic deletion of IL-6, or treatment with the iron chelator deferiprone, reduces pathological α-syn toxicity in a mouse model of sporadic PD. These data suggest that IL-6-induced CISR leads to toxic neuronal iron accumulation, contributing to synuclein-induced neurodegeneration.

The number and morphology of mitochondria within a cell are precisely regulated by the mitochondrial fission and fusion machinery. The human protein, hFis1, participates in mitochondrial fission by recruiting the Drp1 into the mitochondria. Using short hairpin RNA, we reduced the expression levels of hFis1 in mammalian cells. Cells lacking hFis1 showed sustained elongation of mitochondria and underwent significant cellular morphological changes, including enlargement, flattening, and increased cellular granularity. In these cells, staining for acidic senescence-associated beta-galactosidase activity was elevated, and the rate of cell proliferation was greatly reduced, indicating that cells lacking hFis1 undergo senescence-associated phenotypic changes. Reintroduction of the hFis1 gene into hFis1-depleted cells restored mitochondrial fragmentation and suppressed senescence-associated beta-galactosidase activity. Moreover, depletion of both hFis1 and OPA1, a critical component of mitochondrial fusion, resulted in extensive mitochondrial fragmentation and markedly rescued cells from senescence-associated phenotypic changes. Intriguingly, sustained elongation of mitochondria was associated with decreased mitochondrial membrane potential, increased reactive oxygen species production, and DNA damage. The data indicate that sustained mitochondrial elongation induces senescence-associated phenotypic changes that can be neutralized by mitochondrial fragmentation. Thus, one of the key functions of mitochondrial fission might be prevention of the sustained extensive mitochondrial elongation that triggers cellular senescence.

A growing set of observations points to mitochondrial dysfunction, iron accumulation, oxidative damage and chronic inflammation as common pathognomonic signs of a number of neurodegenerative diseases that includes Alzheimer’s disease, Huntington disease, amyotrophic lateral sclerosis, Friedrich’s ataxia and Parkinson’s disease. Particularly relevant for neurodegenerative processes is the relationship between mitochondria and iron. The mitochondrion upholds the synthesis of iron–sulfur clusters and heme, the most abundant iron-containing prosthetic groups in a large variety of proteins, so a fraction of incoming iron must go through this organelle before reaching its final destination. In turn, the mitochondrial respiratory chain is the source of reactive oxygen species (ROS) derived from leaks in the electron transport chain. The co-existence of both iron and ROS in the secluded space of the mitochondrion makes this organelle particularly prone to hydroxyl radical-mediated damage. In addition, a connection between the loss of iron homeostasis and inflammation is starting to emerge; thus, inflammatory cytokines like TNF-alpha and IL-6 induce the synthesis of the divalent metal transporter 1 and promote iron accumulation in neurons and microglia. Here, we review the recent literature on mitochondrial iron homeostasis and the role of inflammation on mitochondria dysfunction and iron accumulation on the neurodegenerative process that lead to cell death in Parkinson’s disease. We also put forward the hypothesis that mitochondrial dysfunction, iron accumulation and inflammation are part of a synergistic self-feeding cycle that ends in apoptotic cell death, once the antioxidant cellular defense systems are finally overwhelmed.

Mitochondria are crucial organelles in the production of energy and in the control of signalling cascades. A machinery of pro-fusion and fission proteins regulates their morphology and subcellular localization. In muscle this results in an orderly pattern of intermyofibrillar and subsarcolemmal mitochondria. Muscular atrophy is a genetically controlled process involving the activation of the autophagy-lysosome and the ubiquitin-proteasome systems. Whether and how the mitochondria are involved in muscular atrophy is unknown. Here, we show that the mitochondria are removed through autophagy system and that changes in mitochondrial network occur in atrophying muscles. Expression of the fission machinery is per se sufficient to cause muscle wasting in adult animals, by triggering organelle dysfunction and AMPK activation. Conversely, inhibition of the mitochondrial fission inhibits muscle loss during fasting and after FoxO3 overexpression. Mitochondrial-dependent muscle atrophy requires AMPK activation as inhibition of AMPK restores muscle size in myofibres with altered mitochondria. Thus, disruption of the mitochondrial network is an essential amplificatory loop of the muscular atrophy programme.

Selective regional iron accumulation is a hallmark of several neurodegenerative diseases, including Alzheimer’s disease and Parkinson’s disease. The underlying mechanisms of neuronal iron dyshomeostasis have been studied, mainly in a gene-by-gene approach. However, recent high-content phenotypic screens using CRISPR/Cas9-based gene perturbations allow for the identification of new pathways that contribute to iron accumulation in neuronal cells. Herein, we perform a bioinformatic analysis of a CRISPR-based screening of lysosomal iron accumulation and the functional genomics of human neurons derived from induced pluripotent stem cells (iPSCs). Consistent with previous studies, we identified mitochondrial electron transport chain dysfunction as one of the main mechanisms triggering iron accumulation, although we substantially expanded the gene set causing this phenomenon, encompassing mitochondrial complexes I to IV, several associated assembly factors, and coenzyme Q biosynthetic enzymes. Similarly, the loss of numerous genes participating through the complete macroautophagic process elicit iron accumulation. As a novelty, we found that the impaired synthesis of glycophosphatidylinositol (GPI) and GPI-anchored protein trafficking also trigger iron accumulation in a cell-autonomous manner. Finally, the loss of critical components of the iron transporters trafficking machinery, including MON2 and PD-associated gene VPS35, also contribute to increased neuronal levels. Our analysis suggests that neuronal iron accumulation can arise from the dysfunction of an expanded, previously uncharacterized array of molecular pathways.

Inflammation and iron accumulation are present in a variety of neurodegenerative diseases that include Alzheimer's disease and Parkinson's disease. The study of the putative association between inflammation and iron accumulation in central nervous system cells is relevant to understand the contribution of these processes to the progression of neuronal death. In this study, we analyzed the effects of the inflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) and of lipopolysaccharide on total cell iron content and on the expression and abundance of the iron transporters divalent metal transporter 1 (DMT1) and Ferroportin 1 (FPN1) in neurons, astrocytes and microglia obtained from rat brain. Considering previous reports indicating that inflammatory stimuli induce the systemic synthesis of the master iron regulator hepcidin, we identified brain cells that produce hepcidin in response to inflammatory stimuli, as well as hepcidin-target cells. We found that inflammatory stimuli increased the expression of DMT1 in neurons, astrocytes, and microglia. Inflammatory stimuli also induced the expression of hepcidin in astrocytes and microglia, but not in neurons. Incubation with hepcidin decreased the expression of FPN1 in the three cell types. The net result of these changes was increased iron accumulation in neurons and microglia but not in astrocytes. The data presented here establish for the first time a causal association between inflammation and iron accumulation in brain cells, probably promoted by changes in DMT1 and FPN1 expression and mediated in part by hepcidin. This connection may potentially contribute to the progression of neurodegenerative diseases by enhancing iron-induced oxidative damage.

Pro-inflammatory cytokine-mediated ferroportin down-regulation contributes to the nigral iron accumulation in lipopolysaccharide-induced Parkinsonian models

The microtubule-associated protein tau has risk alleles for both Alzheimer's disease and Parkinson's disease and mutations that cause brain degenerative diseases termed tauopathies. Aggregated tau forms neurofibrillary tangles in these pathologies, but little is certain about the function of tau or its mode of involvement in pathogenesis. Neuronal iron accumulation has been observed pathologically in the cortex in Alzheimer's disease, the substantia nigra (SN) in Parkinson's disease and various brain regions in the tauopathies. Here we report that tau-knockout mice develop age-dependent brain atrophy, iron accumulation and SN neuronal loss, with concomitant cognitive deficits and parkinsonism. These changes are prevented by oral treatment with a moderate iron chelator, clioquinol. Amyloid precursor protein (APP) ferroxidase activity couples with surface ferroportin to export iron, but its activity is inhibited in Alzheimer's disease, thereby causing neuronal iron accumulation. In primary neuronal culture, we found loss of tau also causes iron retention, by decreasing surface trafficking of APP. Soluble tau levels fall in affected brain regions in Alzheimer's disease and tauopathies, and we found a similar decrease of soluble tau in the SN in both Parkinson's disease and the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model. These data suggest that the loss of soluble tau could contribute to toxic neuronal iron accumulation in Alzheimer's disease, Parkinson's disease and tauopathies, and that it can be rescued pharmacologically.

Mesenchymal Stem Cells Ameliorate Cuprizone-Induced Demyelination by Targeting Oxidative Stress and Mitochondrial Dysfunction.

Amyotrophic lateral sclerosis (ALS), characterized by degeneration of spinal motor neurons, consists of sporadic and familial forms. One cause of familial ALS is missense mutations in the superoxide dismutase 1 (SOD1) gene. Iron accumulation occurs in the CNS of both forms of ALS; however, its contribution to the pathogenesis of ALS is not known. We examined the role of iron in a transgenic mouse line overexpressing the human SOD1(G37R) mutant. We show that multiple mechanisms may underlie the iron accumulation in neurons and glia in SOD1(G37R) transgenic mice. These include dysregulation of proteins involved in iron influx and sensing of intracellular iron; iron accumulation in ventral motor neurons secondary to blockage of anterograde axonal transport; and increased mitochondrial iron load in neurons and glia. We also show that treatment of SOD1(G37R) mice with an iron chelator extends life span by 5 weeks, accompanied by increased survival of spinal motor neurons and improved locomotor function. These data suggest that iron chelator therapy might be useful for the treatment of ALS.

Brain degenerative disorders, which include some neurodevelopmental disorders and age-associated diseases, cause debilitating neurological deficits and are generally fatal. A large body of emerging evidence indicates that iron accumulation in neurons within specific regions of the brain plays an important role in the pathogenesis of many of these disorders. Iron homeostasis is a highly complex and incompletely understood process involving a large number of regulatory molecules. Our review provides a description of what is known about how iron is obtained by the body and brain and how defects in the homeostatic processes could contribute to the development of brain diseases, focusing on Alzheimer's disease and Parkinson's disease as well as four other disorders belonging to a class of inherited conditions referred to as neurodegeneration based on iron accumulation (NBIA) disorders. A description of potential therapeutic approaches being tested for each of these different disorders is provided.

Iron accumulation in the substantia nigra is a hallmark of Parkinson's disease (PD), but its cellular drivers remain unclear. Oligodendrocytes, the most iron-rich cells in the brain, have been implicated in PD pathology. Our previous studies showed that 6-hydroxydopamine (6-OHDA) promotes iron accumulation in neurons and astrocytes by increasing iron influx and decreasing efflux. However, its effects on oligodendrocyte iron metabolism remain unknown. In this study, we examined how 6-OHDA affects iron homeostasis and inflammatory gene expression in MO3.13 oligodendrocytes. Using MTT, calcein-AM fluorescence assays, RT-PCR, and Western blotting, we compared undifferentiated and differentiated cells. In undifferentiated oligodendrocytes, 6-OHDA increased transferrin receptor 1 (TfR1) and iron regulatory protein 1 (IRP1) while reducing ferroportin 1 (FPN1), resulting in enhanced iron uptake and reduced export. In contrast, differentiated cells showed decreased TfR1 and IRP1 and increased FPN1, promoting iron efflux. 6-OHDA also induced stage-specific inflammatory responses. In undifferentiated cells, IL-1β and TNF-α mRNA levels rose in a dose-dependent manner, whereas differentiated cells selectively upregulated IL-1β. These results suggest that undifferentiated oligodendrocytes undergo iron-related inflammation that may promote differentiation, while differentiated cells respond with a more restricted cytokine profile. This study is the first to demonstrate that 6-OHDA promotes iron accumulation in undifferentiated oligodendrocytes by disrupting the IRP1-mediated balance between TfR1 and FPN1. Moreover, 6-OHDA induces distinct inflammatory responses depending on the stage of oligodendrocyte differentiation. These findings highlight the dual role of oligodendrocytes as both iron reservoirs and modulators of the neuroinflammatory microenvironment, providing new insights into the cellular mechanisms underlying nigral iron accumulation in PD, and suggesting that oligodendrocytes play a critical regulatory role in PD pathogenesis.

Aging is accompanied by impaired mitochondrial function and accumulation of senescent cells. Mitochondrial dysfunction contributes to senescence by increasing the levels of reactive oxygen species and compromising energy metabolism. Senescent cells secrete a senescence‐associated secretory phenotype (SASP) and stimulate chronic low‐grade inflammation, ultimately inducing inflammaging. Mitochondrial dysfunction and cellular senescence are two closely related hallmarks of aging; however, the key driver genes that link mitochondrial dysfunction and cellular senescence remain unclear. Here, we aimed to elucidate a novel role of carnitine acetyltransferase (CRAT) in the development of mitochondrial dysfunction and cellular senescence in dermal fibroblasts. Transcriptomic analysis of skin tissues from young and aged participants showed significantly decreased CRAT expression in intrinsically aged skin. CRAT downregulation in human dermal fibroblasts recapitulated mitochondrial changes in senescent cells and induced SASP secretion. Specifically, CRAT knockdown caused mitochondrial dysfunction, as indicated by increased oxidative stress, disruption of mitochondrial morphology, and a metabolic shift from oxidative phosphorylation to glycolysis. Mitochondrial damage induced the release of mitochondrial DNA into the cytosol, which activated the cyclic GMP‐AMP synthase (cGAS)‐stimulator of interferon genes (STING) and NF‐ĸB pathways to induce SASPs. Consistently, fibroblast‐specific CRAT‐knockout mice showed increased skin aging phenotypes in vivo, including decreased cell proliferation, increased SASP expression, increased inflammation, and decreased collagen density. Our results suggest that CRAT deficiency contributes to aging by mediating mitochondrial dysfunction‐induced senescence.

Metformin accelerates myelin recovery and ameliorates behavioral deficits in the animal model of multiple sclerosis via adjustment of AMPK/Nrf2/mTOR signaling and maintenance of endogenous oligodendrogenesis during brain self-repairing period.

Muscle fiber type specific alterations of mitochondrial respiratory function and morphology in aged female mice