Epigenetic Treatment of Persistent Viral Infections.

Preclinical Research Approximately 2,500 years ago, Hippocrates used the word herpes as a medical term to describe lesions that appeared to creep or crawl on the skin, advocating heat as a possible treatment. During the last 50 years, pharmaceutical research has made great strides, and therapeutic options have expanded to include small molecule antiviral agents, protease inhibitors, preventive vaccines for a handful of the papillomaviruses, and even cures for hepatitis C virus infections. However, effective treatments for persistent and recurrent viral infections, particularly the highly prevalent herpesviruses, continue to represent a significant unmet medical need, affecting the majority of the world's population. Exploring the population diversity of the human microbiome and the effects its compositional variances have on the immune system, health, and disease are the subjects of intense investigational research and study. Among the collection of viruses, bacteria, fungi, and single-cell eukaryotes that comprise the human microbiome, the virome has been grossly understudied relative to the influence it exerts on human pathophysiology, much as mitochondria have until recently failed to receive the attention they deserve, given their critical biomedical importance. Fortunately, cellular epigenetic machinery offers a wealth of druggable targets for therapeutic intervention in numerous disease indications, including those outlined above. With advances in synthetic biology, engineering our body's commensal microorganisms to seek out and destroy pathogenic species is clearly on the horizon. This is especially the case given recent breakthroughs in genetic manipulation with tools such as the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) gene-editing platforms. Tying these concepts together with our previous work on the microbiome and neurodegenerative and neuropsychiatric diseases, we suggest that, because mammalian cells respond to a viral infection by triggering a cascade of antiviral innate immune responses governed substantially by the cell's mitochondria, small molecule carnitinoids represent a new class of therapeutics with potential widespread utility against many infectious insults. Drug Dev Res 78 : 24-36, 2017. © 2016 Wiley Periodicals, Inc.

Epigenetic Treatment of Neurodegenerative Disorders: Alzheimer and Parkinson Diseases.

Preclinical Research In this review, we discuss epigenetic-driven methods for treating neurodegenerative disorders associated with mitochondrial dysfunction, focusing on carnitinoid antioxidant-histone deacetylase inhibitors that show an ability to reinvigorate synaptic plasticity and protect against neuromotor decline in vivo. Aging remains a major risk factor in patients who progress to dementia, a clinical syndrome typified by decreased mental capacity, including impairments in memory, language skills, and executive function. Energy metabolism and mitochondrial dysfunction are viewed as determinants in the aging process that may afford therapeutic targets for a host of disease conditions, the brain being primary in such thinking. Mitochondrial dysfunction is a core feature in the pathophysiology of both Alzheimer and Parkinson diseases and rare mitochondrial diseases. The potential of new therapies in this area extends to glaucoma and other ophthalmic disorders, migraine, Creutzfeldt-Jakob disease, post-traumatic stress disorder, systemic exertion intolerance disease, and chemotherapy-induced cognitive impairment. An emerging and hopefully more promising approach to addressing these hard-to-treat diseases leverages their sensitivity to activation of master regulators of antioxidant and cytoprotective genes, antioxidant response elements, and mitophagy. Drug Dev Res 77 : 109-123, 2016. © 2016 Wiley Periodicals, Inc.

Epigenetic Treatment of Neuropsychiatric Disorders: Autism and Schizophrenia.

Neuropsychiatric disorders are a heterogeneous group of conditions that often share underlying mitochondrial dysfunction and biological pathways implicated in their pathogenesis, progression, and treatment. To date, these disorders have proven notoriously resistant to molecular-targeted therapies, and clinical options are relegated to interventional types, which do not address the core symptoms of the disease. In this review, we discuss emerging epigenetic-driven approaches using novel acylcarnitine esters (carnitinoids) that act on master regulators of antioxidant and cytoprotective genes and mitophagic pathways. These carnitinoids are actively transported, mitochondria-localizing, biomimetic coenzyme A surrogates of short-chain fatty acids, which inhibit histone deacetylase and may reinvigorate synaptic plasticity and protect against neuronal damage. We outline these neuroprotective effects in the context of treatment of neuropsychiatric disorders such as autism spectrum disorder and schizophrenia.

Effects of transgenic sterilization constructs and their repressor compounds on hatch, developmental rate and early survival of electroporated channel catfish embryos and fry.

Channel catfish (Ictalurus punctatus) embryos were electroporated with sterilization constructs targeting primordial germ cell proteins or with buffer. Some embryos then were treated with repressor compounds, cadmium chloride, copper sulfate, sodium chloride or doxycycline, to prevent expression of the transgene constructs. Promoters included channel catfish nanos and vasa, salmon transferrin (TF), modified yeast Saccharomyces cerevisiae copper transport protein (MCTR) and zebrafish racemase (RM). Knock-down systems were the Tet-off (nanos and vasa constructs), MCTR, RM and TF systems. Knock-down genes included shRNAi targeting 5' nanos (N1), 3' nanos (N2) or dead end (DND), or double-stranded nanos RNA (dsRNA) for overexpression of nanos mRNA. These constructs previously were demonstrated to knock down nanos, vasa and dead end, with the repressors having variable success. Exogenous DNA affected percentage hatch (% hatch), as all 14 constructs, except for the TF dsRNA, TF N1 (T), RM DND (C), vasa DND (C), vasa N1 (C) and vasa N2 (C), had lower % hatch than the control electroporated with buffer. The MCTR and RM DND (T) constructs resulted in delayed hatch, and the vasa and nanos constructs had minimal effects on time of hatch (P < 0.05). Cadmium chloride appeared to counteract the slow development caused by the TF constructs in two TF treatments (P < 0.05). The 4 ppt sodium chloride treatment for the RM system decreased % hatch (P < 0.05) and slowed development. In the case of nanos constructs, doxycycline greatly delayed hatch (P < 0.05). Adverse effects of the transgenes and repressors continued for several treatments for the first 6 days after hatch, but only in a few treatments during the next 10 days. Repressors and gene expression impacted the yield of putative transgenic channel catfish fry, and need to be considered and accounted for in the hatchery phase of producing transgenically sterilized catfish fry and their fertile counterparts. This fry output should be considered to ensure that sufficient numbers of transgenic fish are produced for future applications and for defining repressor systems that are the most successful.

Mitochondrially-imported RNA in drug discovery.

The import of nuclear transcribed RNAs into mitochondria is an emerging area that presents a tremendous opportunity to develop human metabolic therapeutics. However, our knowledge base is quite limited. Much remains to be discovered regarding specific RNA localization and mechanisms of import. To identify novel RNAs imported into mitochondria, all RNAs within the mitochondria were characterized using next generation sequencing technology. Several nuclear transcribed RNAs were found within mitochondrial RNA (mtRNA) samples, including nuclear ribosomal RNAs, gamma satellite RNA and VL30 retroelement RNA. The presence of these RNAs within mitochondria coupled with RNA sequencing data from other laboratories investigating mtRNA processing, lead us to hypothesize that nuclease treatment of mitoplasts is insufficient for removing contaminating cytoplasmic RNAs. In contrast to traditional methodology, mitochondrial import was evaluated by qRT-PCR after stepwise removal of the outer mitochondrial membrane and subsequent lysis of mitochondria. This allowed identification of RNAs lost from the mitochondria with the same kinetics as mitochondrial DNA-transcribed RNAs. This approach provided an improved evaluation of nuclear RNA enrichment within mitochondrial membranes to characterize nuclease protection and mitochondrial import and identify false-positive detection errors. qRT-PCR results confirmed the presence of VL30 retroelement RNA within mitochondria and question the hypothesis that the RNA component of RNase P is imported. These results illustrate a reliable approach for evaluating the presence of RNAs within mitochondria and open new avenues of investigation relating to mtRNA biology and in targeting mitochondrial based therapeutics.

Antioxidant-mediated reversal of oxidative damage in mouse modeling of complex I inhibition.

Mitochondrial dysfunction is a key component of various aging-related pathologies of the brain that result in dementia. As such, it provides an important avenue in development of therapeutic interventions for a host of neurological disorders. A requirement for functional mitochondrial respiratory chain complex I (CI), to accomplish the normal physiological processes regulating memory, seems intuitive. In the present study, a synthetic lipoylcarnitine antioxidant (PMX-500FI; 100 mg/kg/day po) was administered to female ICR mice (3-4-month old) that were subsequently treated with the mitochondrial CI inhibitor, rotenone (400 mg/kg/day). After 1 week, rotenone-induced impairment of neuronal function was evaluated in the hippocampus, a brain region that is involved in regulating memory formation. Electrophysiological recordings in live brain slices showed that long-term potentiation (LTP) was reduced by rotenone exposure (P < 0.05) while pretreatment with PMX-500FI maintained LTP similar to control levels (P > 0.05). Potentiation during theta burst stimulation (TBS) was similar among treatment groups (P > 0.05); however, neurotransmitter release, which increased in control mice after TBS, was lower in rotenone treated mice (P < 0.05), and was accompanied by reduced basal synaptic transmission (P < 0.05), increased proapoptotic signaling and decreased extracellular signal-regulated kinase1/2 (ERK1/2) phosphorylation (P < 0.05). For each of these determinations, pretreatment with PMX-500FI alleviated the harmful effects of rotenone. These results illustrate that treatment with antioxidant PMX-500FI is protective against rotenone-induced impairment of neuronal bioenergetics in the mouse hippocampus, in regard to both excitatory synaptic physiology and proapoptotic signaling. The protective effect of PMX-500FI against rotenone-induced disruption of cellular bioenergetics may have important therapeutic implications for treating aging-related dementia and other diseases related to mitochondrial dysfunction and/or oxidative damage.

Bioprotective carnitinoids: lipoic acid, butyrate, and mitochondria-targeting to treat radiation injury: mitochondrial drugs come of age.

Preclinical Research Given nuclear-power-plant incidents such as the 2011 Japanese Fukushima-Daiichi disaster, an urgent need for effective medicines to protect against and treat the harmful biological effects of radiation is evident. To address such a challenge, we describe potential strategies herein including mitochondrial and epigenetic-driven methods using lipoic and butyric acid ester conjugates of carnitine. The antioxidant and other therapeutically beneficial properties of this class of agents may protect against ionizing radiation and resultant mitochondrial dysfunction. Recent studies of the compounds described herein reveal the potential-although further research and development is required to prove the effectiveness of this approach-to provide field-ready radiation-protective drugs.

Animal models of human mitochondrial DNA mutations.

BACKGROUND: Mutations in mitochondrial DNA (mtDNA) cause a variety of pathologic states in human patients. Development of animal models harboring mtDNA mutations is crucial to elucidating pathways of disease and as models for preclinical assessment of therapeutic interventions. SCOPE OF REVIEW: This review covers the knowledge gained through animal models of mtDNA mutations and the strategies used to produce them. Animals derived from spontaneous mtDNA mutations, somatic cell nuclear transfer (SCNT), nuclear translocation of mitochondrial genes followed by mitochondrial protein targeting (allotopic expression), mutations in mitochondrial DNA polymerase gamma, direct microinjection of exogenous mitochondria, and cytoplasmic hybrid (cybrid) embryonic stem cells (ES cells) containing exogenous mitochondria (transmitochondrial cells) are considered. MAJOR CONCLUSIONS: A wide range of strategies have been developed and utilized in attempts to mimic human mtDNA mutation in animal models. Use of these animals in research studies has shed light on mechanisms of pathogenesis in mitochondrial disorders, yet methods for engineering specific mtDNA sequences are still in development. GENERAL SIGNIFICANCE: Research animals containing mtDNA mutations are important for studies of the mechanisms of mitochondrial disease and are useful for the development of clinical therapies. This article is part of a Special Issue entitled Biochemistry of Mitochondria.

A New Approach to Treating Neurodegenerative Otologic Disorders.

Hearing loss, the most common neurological disorder and the fourth leading cause of years lived with disability, can have profound effects on quality of life. The impact of this "invisible disability," with significant consequences, economic and personal, is most substantial in low- and middle-income countries, where >80% of affected people live. Given the importance of hearing for communication, enjoyment, and safety, with up to 500 million affected globally at a cost of nearly $800 billion/year, research on new approaches toward prevention and treatment is attracting increased attention. The consequences of noise pollution are largely preventable, but irreversible hearing loss can result from aging, disease, or drug side effects. Once damage occurs, treatment relies on hearing aids and cochlear implants. Preventing, delaying, or reducing some degree of hearing loss may be possible by avoiding excessive noise and addressing major contributory factors such as cardiovascular risk. However, given the magnitude of the problem, these interventions alone are unlikely to be sufficient. Recent advances in understanding principal mechanisms that govern hearing function, together with new drug discovery paradigms designed to identify efficacious therapies, bode well for pharmaceutical intervention. This review surveys various causes of loss of auditory function and discusses potential neurological underpinnings, including mitochondrial dysfunction. Mitochondria mitigate cell protection, survival, and function and may succumb to cumulative degradation of energy production and performance; the end result is cell death. Energy-demanding neurons and vestibulocochlear hair cells are vulnerable to mitochondrial dysfunction, and hearing impairment and deafness are characteristic of neurodegenerative mitochondrial disease phenotypes. Beyond acting as cellular powerhouses, mitochondria regulate immune responses to infections, and studies of this phenomenon have aided in identifying nuclear factor kappa B and nuclear factor erythroid 2-related factor 2/antioxidant response element signaling as targets for discovery of otologic drugs, respectively, suppressing or upregulating these pathways. Treatment with free radical scavenging antioxidants is one therapeutic approach, with lipoic acid and corresponding carnitine esters exhibiting improved biodistribution and other features showing promise. These compounds are also histone deacetylase (HDAC) inhibitors, adding epigenetic modulation to the mechanistic milieu through which they act. These data suggest that new drugs targeting mitochondrial dysfunction and modulating epigenetic pathways via HDAC inhibition or other mechanisms hold great promise.

Epigenetic Treatment of Neurodegenerative Ophthalmic Disorders: An Eye Toward the Future.

Eye disease is one of the primary medical conditions that requires attention and therapeutic intervention in ageing populations worldwide. Further, the global burden of diabetes and obesity, along with heart disease, all lead to secondary manifestations of ophthalmic distress. Therefore, there is increased interest in developing innovative new approaches that target various mechanisms and sequelae driving conditions that result in adverse vision. The research challenge is even greater given that the terrain of eye diseases is difficult to landscape into a single therapeutic theme. This report addresses the burden of eye disease due to mitochondrial dysfunction, including antioxidant, autophagic, epigenetic, mitophagic, and other cellular processes that modulate the biomedical end result. In this light, we single out lipoic acid as a potent known natural activator of these pathways, along with alternative and potentially more effective conjugates, which together harness the necessary potency, specificity, and biodistribution parameters required for improved therapeutic outcomes.

Mouse models of mitochondrial complex I dysfunction.

Diseases of the mitochondria generally affect cells with high-energy demand, and appear to most profoundly affect excitatory cells that have localized high energy requirements, such as neurons and cardiac and skeletal muscle cells. Complex I of the mammalian mitochondrial respiratory chain is a very large, 45 subunit enzyme, and functional deficiency of complex I is the most frequently observed cause of oxidative phosphorylation (OXPHOS) disorders. Impairment of complex I results in decreased cellular energy production and is responsible for a variety of human encephalopathies, myopathies and cardiomyopathies. Complex I deficiency may be caused by mutations in any of the seven mitochondrial or 38 nuclear genes that encode complex I subunits or by mutations in various other nuclear genes that affect complex I assembly or function. Mouse models that faithfully mimic human complex I disorders are needed to better understand the role of complex I in health and disease and for evaluation of potential therapies for mitochondrial diseases. In this review we discuss existing mouse models of mitochondrial complex I dysfunction, focusing on those with similarities to human mitochondrial disorders. We also discuss some of the noteworthy murine genetic models in which complex I genes are not disrupted, but complex I dysfunction is observed, along with some of the more popular chemical compounds that inhibit complex I function and are useful for modeling complex I deficiency in mice. This article is part of a Directed Issue entitled: Bioenergetic dysfunction, adaptation and therapy.

Protection by an antioxidant of rotenone-induced neuromotor decline, reactive oxygen species generation and cellular stress in mouse brain.

Exposure to environmental toxins, including rotenone, results in central nervous system and systemic toxicity. Rotenone is a widely used pesticide and a mitochondrial complex I (CI) inhibitor. This study reports effectiveness of a synthetic lipoylcarnitine antioxidant compound, lipoylcarnitine methyl ester iodide (PMX-500F), for treatment of chronic rotenone induced neurological deficits in mice. Mice (C57BL/6NTac; two months of age) received oral administration of rotenone (30 mg/kg/day) or vehicle, preceded by intraperitoneal injection of PMX-500F (19 mg/kg) or vehicle for four weeks. In the Rota-rod test, rotenone treatment had no effect (P>0.05); however, PMX-500F treatment improved locomotor coordination and endurance (latency to fall time; P<0.05). For neuromuscular strength (wire hang test), rotenone treated mice showed reduced latency to fall compared to control mice (P<0.05). PMX-500F treatment improved the outcome in both control and rotenone exposed mice (P<0.05). Rotenone administration increased ROS generation in the forebrain and midbrain regions, but not in the cerebellum (P<0.05). Co-treatment with PMX-500F normalized the ROS in forebrain and midbrain regions to that of the control concentrations. In rotenone administered mice, activated stress-activated protein kinase/c-Jun NH2-terminal kinase (pSAPK/JNK) was higher in forebrain and midbrain lysates than in control mice (P<0.05) and treatment with PMX-500F reduced pSAPK/JNK to control levels. The pSAPK/JNK levels in the cerebellum were similar in all four groups (P>0.05). Total SAPK/JNK was not altered by either rotenone or PMX-500F treatment (P>0.05). These results illustrate that an antioxidant, here PMX-500F, provides protection against rotenone induced decline in neuromotor function, reactive oxygen species (ROS) generation and cellular stress.

D-galactose effectiveness in modeling aging and therapeutic antioxidant treatment in mice.

Accumulating evidence suggests that mitochondrial dysfunction and oxidative stress play major roles in aging. Chronic administration of D-galactose has been reported to cause deterioration of cognitive and motor skills that are similar to symptoms of aging and, therefore, is regarded as a model of accelerated aging. Because enhancing endogenous antioxidants is now widely regarded as an attractive therapy for conditions associated with mitochondrial oxidative stress, in the present study the effects of α-lipoic acid, L-carnitine, and PMX-500F on D-galactose treated mice were tested. Female mice were injected with (100 mg/kg) D-(+)-galactose for 6 weeks and some groups were treated with a daily dose of α-lipoic acid (5 mg/kg), L-carnitine (3.9 mg/kg), PMX-500F (11.9 mg/kg), or the vehicle (0.1 M Tris, pH 7.4). Control mice were treated with physiological saline. An accelerating Rota-Rod, open field test, and Y-maze test were performed, and serum lactate concentrations were analyzed. These analyses did not identify impairment in motor coordination, open-field activity, or spatial memory (p > 0.05). Similarly, serum lactate concentrations in D-galactose-treated mice were not elevated when compared to controls (p > 0.05). Treatment with the antioxidant compounds at the given concentrations did not result in any changes in the behavioral parameters tested. In conclusion, results of this study illustrate that chronic, short-term D-galactose treatment may not represent a suitable model for inducing readily detectable age-related neurobehavioral symptoms in mice.