The Mitochondrial m.3243A>G Mutation on the Dish, Lessons from In Vitro Models.

The m.3243A>G mutation in the tRNA Leu(UUR) gene (MT-TL1) is one of the most common pathogenic point mutations in human mtDNA. Patient symptoms vary widely and the severity of the disease ranges from asymptomatic to lethal. The reason for the high heterogeneity of m.3243A>G-associated disease is still unknown, and the treatment options are limited, with only supportive interventions available. Furthermore, the heteroplasmic nature of the m.3243A>G mutation and lack of specific animal models of mtDNA mutations have challenged the study of m.3243A>G, and, besides patient data, only cell models have been available for studies. The most commonly used cell models are patient derived, such as fibroblasts and induced pluripotent stem cell (iPSC)-derived models, and cybrid models where the mutant DNA is transferred to an acceptor cell. Studies on cell models have revealed cell-type-specific effects of the m.3243A>G mutation and that the tolerance for this mutation varies between cell types and between patients. In this review, we summarize the literature on the effects of m.3243A>G in cell models.

Neuron-astrocyte transmitophagy is altered in Alzheimer's disease.

Under physiological conditions in vivo astrocytes internalize and degrade neuronal mitochondria in a process called transmitophagy. Mitophagy is widely reported to be impaired in neurodegeneration but it is unknown whether and how transmitophagy is altered in Alzheimer's disease (AD). Here we report that the internalization of neuronal mitochondria is significantly increased in astrocytes isolated from AD mouse brains. We also demonstrate that the degradation of neuronal mitochondria by astrocytes is increased in AD mice at the age of 6 months onwards. Furthermore, we demonstrate for the first time a similar phenomenon between human neurons and AD astrocytes, and in murine hippocampi in vivo. The results suggest the involvement of S100a4 in impaired mitochondrial transfer between neurons and AD astrocytes together with significant increases in the mitophagy regulator and reactive oxygen species in aged AD astrocytes. These findings demonstrate altered neuron-supporting functions of AD astrocytes and provide a starting point for studying the molecular mechanisms of transmitophagy in AD.

Functional Characterization of Mechanosensitive Piezo1 Channels in Trigeminal and Somatic Nerves in a Neuron-on-Chip Model.

Mechanosensitive ion channels, Piezo1 and 2, are activated by pressure and involved in diverse physiological functions, including senses of touch and pain, proprioception and many more. Understanding their function is important for elucidating the mechanosensitive mechanisms of a range of human diseases. Recently, Piezo channels were suggested to be contributors to migraine pain generation. Migraine is typically characterized by allodynia and mechanical hyperalgesia associated with the activation and sensitization of trigeminal ganglion (TG) nerve fibers. Notably, migraine specific medicines are ineffective for other types of pain, suggesting a distinct underlying mechanism. To address, in a straightforward manner, the specificity of the mechanosensitivity of trigeminal vs. somatic nerves, we compared the activity of Piezo1 channels in mouse TG neurons vs. dorsal root ganglia (DRG) neurons. We assessed the functional expression of Piezo1 receptors using a conventional live calcium imaging setup equipped with a multibarrel application system and utilizing a microfluidic chip-based setup. Surprisingly, the TG neurons, despite higher expression of the Piezo1 gene, were less responsive to Piezo1 agonist Yoda1 than the DRG neurons. This difference was more prominent in the chip-based setup, suggesting that certain limitations of the conventional approach, such as turbulence, can be overcome by utilizing microfluidic devices with laminar solution flow.

NF-E2-related factor 2 activation boosts antioxidant defenses and ameliorates inflammatory and amyloid properties in human Presenilin-1 mutated Alzheimer's disease astrocytes.

Alzheimer's disease (AD) is a common dementia affecting a vast number of individuals and significantly impairing quality of life. Despite extensive research in animal models and numerous promising treatment trials, there is still no curative treatment for AD. Astrocytes, the most common cell type of the central nervous system, have been shown to play a role in the major AD pathologies, including accumulation of amyloid plaques, neuroinflammation, and oxidative stress. Here, we show that inflammatory stimulation leads to metabolic activation of human astrocytes and reduces amyloid secretion. On the other hand, the activation of oxidative metabolism leads to increased reactive oxygen species production especially in AD astrocytes. While healthy astrocytes increase glutathione (GSH) release to protect the cells, Presenilin-1-mutated AD patient astrocytes do not. Thus, chronic inflammation is likely to induce oxidative damage in AD astrocytes. Activation of NRF2, the major regulator of cellular antioxidant defenses, encoded by the NFE2L2 gene, poses several beneficial effects on AD astrocytes. We report here that the activation of NRF2 pathway reduces amyloid secretion, normalizes cytokine release, and increases GSH secretion in AD astrocytes. NRF2 induction also activates the metabolism of astrocytes and increases the utilization of glycolysis. Taken together, targeting NRF2 in astrocytes could be a potent therapeutic strategy in AD.

Astrocyte alterations in neurodegenerative pathologies and their modeling in human induced pluripotent stem cell platforms.

Astrocytes are the most abundant cell type in the brain. They were long considered only as passive support for neuronal cells. However, recent data have revealed many active roles for these cells both in maintenance of the normal physiological homeostasis in the brain as well as in neurodegeneration and disease. Moreover, human astrocytes have been found to be much more complex than their rodent counterparts, and to date, astrocytes are known to actively participate in a multitude of processes such as neurotransmitter uptake and recycling, gliotransmitter release, neuroenergetics, inflammation, modulation of synaptic activity, ionic balance, maintenance of the blood-brain barrier, and many other crucial functions of the brain. This review focuses on the role of astrocytes in human neurodegenerative disease and the potential of the novel stem cell-based platforms in modeling astrocytic functions in health and in disease.

Stem cells, mitochondria and aging.

Decline in metabolism and regenerative potential of tissues are common characteristics of aging. Regeneration is maintained by somatic stem cells (SSCs), which require tightly controlled energy metabolism and genomic integrity for their homeostasis. Recent data indicate that mitochondrial dysfunction may compromise this homeostasis, and thereby contribute to tissue degeneration and aging. Progeroid Mutator mouse, accumulating random mtDNA point mutations in their SSCs, showed disturbed SSC homeostasis, emphasizing the importance of mtDNA integrity for stem cells. The mechanism involved changes in cellular redox-environment, including subtle increase in reactive oxygen species (H2O2and superoxide anion), which did not cause oxidative damage, but disrupted SSC function. Mitochondrial metabolism appears therefore to be an important regulator of SSC fate determination, and defects in it in SSCs may underlie premature aging. Here we review the current knowledge of mitochondrial contribution to SSC dysfunction and aging. This article is part of a Special Issue entitled: Mitochondrial Dysfunction in Aging.

Tissue- and cell-type-specific manifestations of heteroplasmic mtDNA 3243A>G mutation in human induced pluripotent stem cell-derived disease model.

Mitochondrial DNA (mtDNA) mutations manifest with vast clinical heterogeneity. The molecular basis of this variability is mostly unknown because the lack of model systems has hampered mechanistic studies. We generated induced pluripotent stem cells from patients carrying the most common human disease mutation in mtDNA, m.3243A>G, underlying mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome. During reprogramming, heteroplasmic mtDNA showed bimodal segregation toward homoplasmy, with concomitant changes in mtDNA organization, mimicking mtDNA bottleneck during epiblast specification. Induced pluripotent stem cell-derived neurons and various tissues derived from teratomas manifested cell-type specific respiratory chain (RC) deficiency patterns. Similar to MELAS patient tissues, complex I defect predominated. Upon neuronal differentiation, complex I specifically was sequestered in perinuclear PTEN-induced putative kinase 1 (PINK1) and Parkin-positive autophagosomes, suggesting active degradation through mitophagy. Other RC enzymes showed normal mitochondrial network distribution. Our data show that cellular context actively modifies RC deficiency manifestation in MELAS and that autophagy is a significant component of neuronal MELAS pathogenesis.

Exome sequencing identifies mitochondrial alanyl-tRNA synthetase mutations in infantile mitochondrial cardiomyopathy.

Infantile cardiomyopathies are devastating fatal disorders of the neonatal period or the first year of life. Mitochondrial dysfunction is a common cause of this group of diseases, but the underlying gene defects have been characterized in only a minority of cases, because tissue specificity of the manifestation hampers functional cloning and the heterogeneity of causative factors hinders collection of informative family materials. We sequenced the exome of a patient who died at the age of 10 months of hypertrophic mitochondrial cardiomyopathy with combined cardiac respiratory chain complex I and IV deficiency. Rigorous data analysis allowed us to identify a homozygous missense mutation in AARS2, which we showed to encode the mitochondrial alanyl-tRNA synthetase (mtAlaRS). Two siblings from another family, both of whom died perinatally of hypertrophic cardiomyopathy, had the same mutation, compound heterozygous with another missense mutation. Protein structure modeling of mtAlaRS suggested that one of the mutations affected a unique tRNA recognition site in the editing domain, leading to incorrect tRNA aminoacylation, whereas the second mutation severely disturbed the catalytic function, preventing tRNA aminoacylation. We show here that mutations in AARS2 cause perinatal or infantile cardiomyopathy with near-total combined mitochondrial respiratory chain deficiency in the heart. Our results indicate that exome sequencing is a powerful tool for identifying mutations in single patients and allows recognition of the genetic background in single-gene disorders of variable clinical manifestation and tissue-specific disease. Furthermore, we show that mitochondrial disorders extend to prenatal life and are an important cause of early infantile cardiac failure.

The gene disrupted in Marinesco-Sjögren syndrome encodes SIL1, an HSPA5 cochaperone.

We identified the gene underlying Marinesco-Sjögren syndrome, which is characterized by cerebellar ataxia, progressive myopathy and cataracts. We identified four disease-associated, predicted loss-of-function mutations in SIL1, which encodes a nucleotide exchange factor for the heat-shock protein 70 (HSP70) chaperone HSPA5. These data, together with the similar spatial and temporal patterns of tissue expression of Sil1 and Hspa5, suggest that disturbed SIL1-HSPA5 interaction and protein folding is the primary pathology in Marinesco-Sjögren syndrome.

The Roles of Cystatin B in the Brain and Pathophysiological Mechanisms of Progressive Myoclonic Epilepsy Type 1.

Progressive myoclonic epilepsy type 1 (EPM1) is an autosomal recessive disorder, also known as Unverricht-Lundborg disease (ULD). EPM1 patients suffer from photo-sensitive seizures, stimulus-sensitive myoclonus, nocturnal myoclonic seizures, ataxia and dysarthria. In addition, cerebral ataxia and impaired GABAergic inhibition are typically present. EPM1 is caused by mutations in the Cystatin B gene (CSTB). The CSTB protein functions as an intracellular thiol protease inhibitor and inhibits Cathepsin function. It also plays a crucial role in brain development and regulates various functions in neurons beyond maintaining cellular proteostasis. These include controlling cell proliferation and differentiation, synaptic functions and protection against oxidative stress, likely through regulation of mitochondrial function. Depending on the differentiation stage and status of neurons, the protein localizes either to the cytoplasm, nucleus, lysosomes or mitochondria. Further, CSTB can also be secreted to the extracellular matrix for interneuron rearrangement and migration. In this review, we will review the various functions of CSTB in the brain and discuss the putative pathophysiological mechanism underlying EPM1.

Refractory congestive heart failure following delayed pericardectomy in a 12-year-old child with Mulibrey nanism due to a novel mutation in TRIM37.

UNLABELLED: Mulibrey nanism (MUL) is a rare autosomal recessive disorder with severe primordial growth retardation and multiorgan involvement, caused by mutations in TRIM37. Early clinical detection is important since more than 50 % of the patients develop congestive heart failure. We report a 12-year-old patient who presented in infancy with severe growth retardation, dysmorphic features, and cleft palate. Clinical diagnosis of MUL was established at the age of 5 years. Postmortem, molecular diagnostic confirmed MUL as a novel 1-bp deletion (c.1233delA) in exon 14 of the TRIM37 coding region. Cardiac examination at the age of 6 years revealed constrictive pericarditis with significant elevation of atrial filling pressures, consecutive hepatomegaly, and protein loosing enteropathy. Since the parents refused pericardectomy, surgery was delayed until the age of 12 years, when congestive heart failure deteriorated. Despite pericardectomy, the boy died from persistent right heart failure. CONCLUSION: Our report underlines the necessity of early clinical diagnosis of Mulibrey nanism. Careful cardiologic examination is required to detect constrictive pericarditis, which is a major factor of mortality in these patients. Pericardectomy should be performed early, to avoid sequelae of persisting congestive heart failure.

Generation of a human induced pluripotent stem cell line (UEFi004-A) from a patient with progressive myoclonic epilepsy type 1 (EPM1).

Progressive myoclonic epilepsy type 1 (EPM1) is an autosomal recessive disorder caused by mutations in the cystatin B gene (CSTB). Affected individual's manifest stimulus-sensitive and action myoclonus and tonic-clonic epileptic seizures. In this study, we have generated iPSCs from an EPM1 patient's skin fibroblasts with Sendai virus mediated transgene delivery. The iPSCs retained the patient specific promoter region expansion mutation, expressed pluripotency markers, differentiated into all three germ layers, and presented a normal karyotype. The line can in future be used to develop an in-vitro model for EPM1 and may help in understanding disease mechanisms at cellular and molecular level.

Modulating Mitochondrial DNA Heteroplasmy with Mitochondrially Targeted Endonucleases.

Mitochondria, mainly known as energy factories of eukaryotic cells, also exert several additional signaling and metabolic functions and are today recognized as major cellular biosynthetic and signaling hubs. Mitochondria possess their own genome (mitochondrial DNA-mtDNA), that encodes proteins essential for oxidative phosphorylation, and mutations in it are an important contributor to human disease. The mtDNA mutations often exist in heteroplasmic conditions, with both healthy and mutant versions of the mtDNA residing in patients' cells and the level of mutant mtDNA may vary between different tissues and organs and affect the clinical outcome of the disease. Thus, shifting the ratio between healthy and mutant mtDNA in patients' cells provides an intriguing therapeutic option for mtDNA diseases. In this review we describe current strategies for modulating mitochondrial heteroplasmy levels with engineered endonucleases including mitochondrially targeted TALENs and Zinc finger nucleases (ZFNs) and discuss their therapeutic potential. These gene therapy tools could in the future provide therapeutic help both for patients with mitochondrial disease as well as in preventing the transfer of pathogenic mtDNA mutations from a mother to her offspring.

Varied Responses to a High m.3243A>G Mutation Load and Respiratory Chain Dysfunction in Patient-Derived Cardiomyocytes.

The m.3243A>G mutation in mitochondrial tRNA-Leu(UUR) is one of the most common pathogenic mitochondrial DNA mutations in humans. The clinical manifestations are highly heterogenous and the causes for the drastic clinical variability are unknown. Approximately one third of patients suffer from cardiac disease, which often increases mortality. Why only some patients develop cardiomyopathy is unknown. Here, we studied the molecular effects of a high m.3243A>G mutation load on cardiomyocyte functionality, using cells derived from induced pluripotent stem cells (iPSC-CM) of two different m.3243A>G patients, only one of them suffering from severe cardiomyopathy. While high mutation load impaired mitochondrial respiration in both patients' iPSC-CMs, the downstream consequences varied. mtDNA mutant cells from a patient with no clinical heart disease showed increased glucose metabolism and retained cellular ATP levels, whereas cells from the cardiac disease patient showed reduced ATP levels. In this patient, the mutations also affected intracellular calcium signaling, while this was not true in the other patient's cells. Our results reflect the clinical variability in mitochondrial disease patients and show that iPSC-CMs retain tissue specific features seen in patients.

The role of the meningeal lymphatic system in local meningeal inflammation and trigeminal nociception.

A system of lymphatic vessels has been recently characterized in the meninges, with a postulated role in 'cleaning' the brain via cerebral fluid drainage. As meninges are the origin site of migraine pain, we hypothesized that malfunctioning of the lymphatic system should affect the local trigeminal nociception. To test this hypothesis, we studied nociceptive and inflammatory mechanisms in the hemiskull preparations (containing the meninges) of K14-VEGFR3-Ig (K14) mice lacking the meningeal lymphatic system. We recorded the spiking activity of meningeal afferents and estimated the local mast cells population, calcitonin gene-related peptide (CGRP) and cytokine levels as well as the dural trigeminal innervation in freshly-isolated hemiskull preparations from K14-VEGFR3-Ig (K14) or wild type C57BL/6 mice (WT). Spiking activity data have been confirmed in an acquired model of meningeal lymphatic dysfunction (AAV-mVEGFR3(1-4)Ig induced lymphatic ablation). We found that levels of the pro-inflammatory cytokine IL12-p70 and CGRP, implicated in migraine, were reduced in the meninges of K14 mice, while the levels of the mast cell activator MCP-1 were increased. The other migraine-related pro-inflammatory cytokines (basal and stimulated), did not differ between the two genotypes. The patterns of trigeminal innervation in meninges remained unchanged and we did not observe alterations in basal or ATP-induced nociceptive firing in the meningeal afferents associated with meningeal lymphatic dysfunction. In summary, the lack of meningeal lymphatic system is associated with a new balance between pro- and anti-migraine mediators but does not directly trigger meningeal nociceptive state.

Generation of a human induced pluripotent stem cell line (UEFi003-A) carrying heterozygous A673T variant in amyloid precursor protein associated with a reduced risk of Alzheimer's disease.

A673T mutation in the amyloid precursor protein (APP) is a rare variant associated with a reduced risk of late-onset Alzheimer's disease (AD) and age-related cognitive decline. The A673T mutation decreases beta-amyloid (Aß) production and aggregation in neuronal cultures in vitro. Here we have identified a Finnish non-diseased male individual carrying a heterozygous A673T mutation, obtained a skin biopsy sample from him, and generated an iPSC line using commercially available integration-free Sendai virus-based kit. The established iPSC line retained the mutation, expressed pluripotency markers, had a normal karyotype, and differentiated into all three germ layers in vitro.

Author Correction: Defects in mtDNA replication challenge nuclear genome stability through nucleotide depletion and provide a unifying mechanism for mouse progerias.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Metabolic alterations in Parkinson's disease astrocytes.

In Parkinson`s disease (PD), the loss of dopaminergic (DA) neurons in the substantia nigra pars compacta is associated with Lewy bodies arising from the accumulation of alpha-synuclein protein which leads ultimately to movement impairment. While PD has been considered a disease of the DA neurons, a glial contribution, in particular that of astrocytes, in PD pathogenesis is starting to be uncovered. Here, we report findings from astrocytes derived from induced pluripotent stem cells of LRRK2 G2019S mutant patients, with one patient also carrying a GBA N370S mutation, as well as healthy individuals. The PD patient astrocytes manifest the hallmarks of the disease pathology including increased expression of alpha-synuclein. This has detrimental consequences, resulting in altered metabolism, disturbed Ca2+ homeostasis and increased release of cytokines upon inflammatory stimulation. Furthermore, PD astroglial cells manifest increased levels of polyamines and polyamine precursors while lysophosphatidylethanolamine levels are decreased, both of these changes have been reported also in PD brain. Collectively, these data reveal an important role for astrocytes in PD pathology and highlight the potential of iPSC-derived cells in disease modeling and drug discovery.

Defects in mtDNA replication challenge nuclear genome stability through nucleotide depletion and provide a unifying mechanism for mouse progerias.

Mitochondrial DNA (mtDNA) mutagenesis and nuclear DNA repair defects are considered cellular mechanisms of ageing. mtDNA mutator mice with increased mtDNA mutagenesis show signs of premature ageing. However, why patients with mitochondrial diseases, or mice with other forms of mitochondrial dysfunction, do not age prematurely remains unknown. Here, we show that cells from mutator mice display challenged nuclear genome maintenance similar to that observed in progeric cells with defects in nuclear DNA repair. Cells from mutator mice show slow nuclear DNA replication fork progression, cell cycle stalling and chronic DNA replication stress, leading to double-strand DNA breaks in proliferating progenitor or stem cells. The underlying mechanism involves increased mtDNA replication frequency, sequestering of nucleotides to mitochondria, depletion of total cellular nucleotide pools, decreased deoxynucleoside 5'-triphosphate (dNTP) availability for nuclear genome replication and compromised nuclear genome maintenance. Our data indicate that defects in mtDNA replication can challenge nuclear genome stability. We suggest that defects in nuclear genome maintenance, particularly in the stem cell compartment, represent a unified mechanism for mouse progerias. Therefore, through their destabilizing effects on the nuclear genome, mtDNA mutations are indirect contributors to organismal ageing, suggesting that the direct role of mtDNA mutations in driving ageing-like symptoms might need to be revisited.