The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system has been widely used for nuclear DNA editing to generate mutations or correct specific disease alleles. Despite its ...flexible application, it has not been determined if CRISPR/Cas9, originally identified as a bacterial defense system against virus, can be targeted to mitochondria for mtDNA editing. Here, we show that regular FLAG-Cas9 can localize to mitochondria to edit mitochondrial DNA with sgRNAs targeting specific loci of the mitochondrial genome. Expression of FLAG-Cas9 together with gRNA targeting Cox1 and Cox3 leads to cleavage of the specific mtDNA loci. In addition, we observed disruption of mitochondrial protein homeostasis following mtDNA truncation or cleavage by CRISPR/Cas9. To overcome nonspecific distribution of FLAG-Cas9, we also created a mitochondria-targeted Cas9 (mitoCas9). This new version of Cas9 localizes only to mitochondria; together with expression of gRNA targeting mtDNA, there is specific cleavage of mtDNA. MitoCas9-induced reduction of mtDNA and its transcription leads to mitochondrial membrane potential disruption and cell growth inhibition. This mitoCas9 could be applied to edit mtDNA together with gRNA expression vectors without affecting genomic DNA. In this brief study, we demonstrate that mtDNA editing is possible using CRISPR/Cas9. Moreover, our development of mitoCas9 with specific localization to the mitochondria should facilitate its application for mitochondrial genome editing.
Accompanied by increased life span, aging-associated diseases, such as metabolic diseases and cancers, have become serious health threats. Recent studies have documented that aging-associated ...diseases are caused by prolonged cellular stresses such as endoplasmic reticulum (ER) stress, mitochondrial stress, and oxidative stress. Thus, ameliorating cellular stresses could be an effective approach to treat aging-associated diseases and, more importantly, to prevent such diseases from happening. However, cellular stresses and their molecular responses within the cell are typically mediated by a variety of factors encompassing different signaling pathways. Therefore, a target-based drug discovery method currently being used widely (reverse pharmacology) may not be adequate to uncover novel drugs targeting cellular stresses and related diseases. The connectivity map (CMap) is an online pharmacogenomic database cataloging gene expression data from cultured cells treated individually with various chemicals, including a variety of phytochemicals. Moreover, by querying through CMap, researchers may screen registered chemicals in silico and obtain the likelihood of drugs showing a similar gene expression profile with desired and chemopreventive conditions. Thus, CMap is an effective genome-based tool to discover novel chemopreventive drugs.
The increasing lifespan in developed countries results in age-associated chronic diseases. Biological aging is a complex process associated with accumulated cellular damage by environmental or ...genetic factors with increasing age. Aging results in marked changes in brain structure and function. Age-related neurodegenerative diseases and disorders (NDDs) represent an ever-growing socioeconomic challenge and lead to an overall reduction in quality of life around the world. Alzheimer's disease (AD) and Parkinson's disease (PD) are most common degenerative neurological disorders of the central nervous system (CNS) in aging process. The low levels of acetylcholine and dopamine are major neuropathological feature of NDDs in addition to oxidative stress, intracellular calcium ion imbalance, mitochondrial dysfunction, ubiquitin-proteasome system impairment and endoplasmic reticulum stress. Current treatments minimally influence these diseases and are ineffective in curing the multifunctional pathological mechanisms. Synthetic neuroprotective agents sometimes have negative reactions as an adverse effect in humans. Recently, numerous ethnobotanical studies have reported that herbal medicines for the treatment or prevention of NDDs are significantly better than synthetic drug treatment. Medicinal herbs have traditionally been used around the world for centuries. Radix Glycyrrhizae (RG) is the dried roots and rhizomes of
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from the Leguminosae/Fabaceae family. It has been used for centuries in traditional medicine as a life enhancer, for the treatment of coughs and influenza, and for detoxification. Diverse chemical constituents from RG have reported including flavanones, chalcones, triterpenoid saponins, coumarines, and other glycosides. Among them, flavanone liquiritigenin (LG) and its precursor and isomer chalcone isoliquiritigenin (ILG) are the main bioactive constituents of RG. In the present review, we summarize evidence in the literature on the structure and phytochemical properties and pharmacological applications of LG and ILG in age-related diseases to establish new therapeutics to improve human health and lifespan.
Abstract Parkinson’s disease (PD) is characterized by selective degeneration of dopaminergic (DAergic) neurons in the substantia nigra pars compacta (SNpc). alpha-synuclein (alpha-syn) is known to ...regulate mitochondrial function and both PINK1 and Parkin have been shown to eliminate damaged mitochondria in PD. Mechanistic target of rapamycin (mTOR) is expressed in several distinct subcellular compartments and mediates the effects of nutrients, growth factors, and stress on cell growth. However, the contributions of these various regulators to DAergic cell death have been demonstrated mainly in culture with serum, which is known to dramatically influence endogenous growth rate and toxin susceptibility through nutrient and growth factor signaling. Therefore, we compared neurotoxicity induced by the mitochondrial inhibitor rotenone (ROT, 5 or 10 uM for 24 h) in SH-SY5Y cells cultured with 10% fetal bovine serum (FBS), 1% FBS, or 1% bovine serum albumin (BSA, serum-free). In addition, C57BL/6J mice were injected with 12 ug ROT into the right striatum, and brains examined by histology and Western blotting 2 weeks later for evidence of DAergic cell death and the underlying signaling mechanisms. ROT dose-dependently reduced SH-SY5Y cell viability in all serum groups without a significant effect of serum concentration. ROT injection also significantly reduced immunoreactivity for the DAergic cell marker tyrosine hydroxylase (TH) in both the mouse striatum and SNpc. Western blotting revealed that ROT inhibited TH and Parkin expression while increasing -syn and PINK1 expression in both SH-SY5Y cells and injected mice, consistent with disruption of mitochondrial function. Moreover, expression levels of the mTOR signaling pathway components mTORC, AMP-activated protein kinase (AMPK), ULK1, and ATG13 were altered in ROT-induced PD. Further, serum level influenced mTOR signaling in the absence of ROT and the changes in response to ROT. Signs of endoplasmic reticulum (ER) stress and altered expression of tethering proteins mediating mitochondria-associated ER contacts (MAMs) were also altered concomitant with ROT-induced neurodegeneration. Taken together, this study demonstrates that complex mechanism involving mitochondrial dysfunction, altered mTOR nutrient-sensing pathways, ER stress, and disrupted MAM protein dynamics are involved in DAergic neurodegeneration in response to ROT.
Mutations in parkin lead to early-onset autosomal recessive Parkinson’s disease (PD) and inactivation of parkin is thought to contribute to sporadic PD. Adult knockout of parkin in the ventral ...midbrain of mice leads to an age-dependent loss of dopamine neurons that is dependent on the accumulation of parkin interacting substrate (PARIS), zinc finger protein 746 (ZNF746), and its transcriptional repression of PGC-1α. Here we show that adult knockout of parkin in mouse ventral midbrain leads to decreases in mitochondrial size, number, and protein markers consistent with a defect in mitochondrial biogenesis. This decrease in mitochondrial mass is prevented by short hairpin RNA knockdown of PARIS. PARIS overexpression in mouse ventral midbrain leads to decreases in mitochondrial number and protein markers and PGC-1α–dependent deficits in mitochondrial respiration. Taken together, these results suggest that parkin loss impairs mitochondrial biogenesis, leading to declining function of the mitochondrial pool and cell death.
Dysfunctional parkin due to mutations or post-translational modifications contributes to dopaminergic neurodegeneration in Parkinson's disease (PD). Overexpression of parkin provides protection ...against cellular stresses and prevents dopamine cell loss in several PD animal models. Here we performed an unbiased high-throughput luciferase screening to identify chemicals that can increase parkin expression. Among promising parkin inducers, hydrocortisone possessed the most favorable profiles including parkin induction ability, cell protection ability, and physicochemical property of absorption, distribution, metabolism, and excretion (ADME) without inducing endoplasmic reticulum stress. We found that hydrocortisone-induced parkin expression was accountable for cell protection against oxidative stress. Hydrocortisone-activated parkin expression was mediated by CREB pathway since gRNA to CREB abolished hydrocortisone's ability to induce parkin. Finally, hydrocortisone treatment in mice increased brain parkin levels and prevented 6-hydroxy dopamine induced dopamine cell loss when assessed at 4 days after the toxin's injection. Our results showed that hydrocortisone could stimulate parkin expression via CREB pathway and the induced parkin expression was accountable for its neuroprotective effect. Since glucocorticoid is a physiological hormone, maintaining optimal levels of glucocorticoid might be a potential therapeutic or preventive strategy for Parkinson's disease.
Parkinson's disease (PD) is a well-known age-related neurodegenerative disorder associated with longer lifespans and rapidly aging populations. The pathophysiological mechanism is a complex progress ...involving cellular damage such as mitochondrial dysfunction and protein homeostasis. Age-mediated degenerative neurological disorders can reduce the quality of life and also impose economic burdens. Currently, the common treatment is replacement with levodopa to address low dopamine levels; however, this does not halt the progression of PD and is associated with adverse effects, including dyskinesis. In addition, elderly patients can react negatively to treatment with synthetic neuroprotection agents. Recently, natural compounds such as phytochemicals with fewer side effects have been reported as candidate treatments of age-related neurodegenerative diseases. This review focuses on mitochondrial dysfunction, oxidative stress, hormesis, proteostasis, the ubiquitin‒proteasome system, and autophagy (mitophagy) to explain the neuroprotective effects of using natural products as a therapeutic strategy. We also summarize the efforts to use natural extracts to develop novel pharmacological candidates for treatment of age-related PD.
Mutations in PTEN-induced putative kinase 1 (PINK1) and parkin cause autosomal-recessive Parkinson’s disease through a common pathway involving mitochondrial quality control. Parkin inactivation ...leads to accumulation of the parkin interacting substrate (PARIS, ZNF746) that plays an important role in dopamine cell loss through repression of proliferator-activated receptor gamma coactivator-1-alpha (PGC-1α) promoter activity. Here, we show that PARIS links PINK1 and parkin in a common pathway that regulates dopaminergic neuron survival. PINK1 interacts with and phosphorylates serines 322 and 613 of PARIS to control its ubiquitination and clearance by parkin. PINK1 phosphorylation of PARIS alleviates PARIS toxicity, as well as repression of PGC-1α promoter activity. Conditional knockdown of PINK1 in adult mouse brains leads to a progressive loss of dopaminergic neurons in the substantia nigra that is dependent on PARIS. Altogether, these results uncover a function of PINK1 to direct parkin-PARIS-regulated PGC-1α expression and dopaminergic neuronal survival.
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•PARIS is a PINK1 substrate•PINK1 phosphorylation of PARIS primes it for parkin ubiquitination and degradation•PINK1 controls PARIS-regulated PGC-1α levels and dopamine neuron survival•Conditional knockdown of PINK1 leads to PARIS-dependent loss of dopamine neurons
Lee et al. demonstrate that PARIS is a common substrate of PINK1 and parkin. PINK1 phosphorylation of PARIS primes it for ubiquitination and clearance by parkin. Thus, dysfunction of either PINK1 or parkin converges on PARIS accumulation, which leads to PGC-1α repression and dopamine neuron loss.
Senescent cells exhibit a reduced response to intrinsic and extrinsic stimuli. This diminished reaction may be explained by the disrupted transmission of nuclear signals. However, this hypothesis ...requires more evidence before it can be accepted as a mechanism of cellular senescence. A proteomic analysis of the cytoplasmic and nuclear fractions obtained from young and senescent cells revealed disruption of nucleocytoplasmic trafficking (NCT) as an essential feature of replicative senescence (RS) at the global level. Blocking NCT either chemically or genetically induced the acquisition of an RS-like senescence phenotype, named nuclear barrier-induced senescence (NBIS). A transcriptome analysis revealed that, among various types of cellular senescence, NBIS exhibited a gene expression pattern most similar to that of RS. Core proteomic and transcriptomic patterns common to both RS and NBIS included upregulation of the endocytosis-lysosome network and downregulation of NCT in senescent cells, patterns also observed in an aging yeast model. These results imply coordinated aging-dependent reduction in the transmission of extrinsic signals to the nucleus and in the nucleus-to-cytoplasm supply of proteins/RNAs. We further showed that the aging-associated decrease in Sp1 transcription factor expression was critical for the downregulation of NCT. Our results suggest that NBIS is a modality of cellular senescence that may represent the nature of physiological aging in eukaryotes.
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