Eukaryotic cells respond to low-oxygen concentrations by upregulating hypoxic nuclear genes (hypoxic signaling). Although it has been shown previously that the mitochondrial respiratory chain is required for hypoxic signaling, its underlying role in this process has been unclear. Here, we find that yeast and rat liver mitochondria produce nitric oxide (NO) at dissolved oxygen concentrations below 20 microM. This NO production is nitrite (NO2-) dependent, requires an electron donor, and is carried out by cytochrome c oxidase in a pH-dependent fashion. Mitochondrial NO production in yeast is influenced by the YHb flavohemoglobin NO oxidoreductase, stimulates expression of the hypoxic nuclear gene CYC7, and is accompanied by an increase in protein tyrosine nitration. These findings demonstrate an alternative role for the mitochondrial respiratory chain under hypoxic or anoxic conditions and suggest that mitochondrially produced NO is involved in hypoxic signaling, possibly via a pathway that involves protein tyrosine nitration.
Mouse models for Huntington’s Disease and HD patients demonstrate motor and behavioral dysfunctions, such as progressive loss of coordination and memory, and share similar transcriptional profiles and striatal neuron atrophy. Clear differences between the mouse and human diseases include almost complete striatal degeneration and rarity of intranuclear inclusions in HD, and the fact that mice expressing full length mutant huntingtin do not demonstrate a shortened lifespan characteristic of HD. While no clinical interventions tested in mouse models to date have delayed disease progression, the mouse models provide an invaluable tool for both investigating the underlying pathogenic processes and developing new effective therapies. Inherent differences between humans and mice must be considered in the search for efficacious treatments for HD, but the striking similarities between human HD and mouse models support the view that these models are a biologically relevant system to support the identification and testing of potential clinical therapies.
Protein:protein interactions are among the most difficult to treat molecular mechanisms of disease pathology. Cystine-dense peptides have the potential to disrupt such interactions, and are used in drug-like roles by every clade of life, but their study has been hampered by a reputation for being difficult to produce, owing to their complex disulfide connectivity. Here we describe a platform for identifying target-binding cystine-dense peptides using mammalian surface display, capable of interrogating high quality and diverse scaffold libraries with verifiable folding and stability. We demonstrate the platform’s capabilities by identifying a cystine-dense peptide capable of inhibiting the YAP:TEAD interaction at the heart of the oncogenic Hippo pathway, and possessing the potency and stability necessary for consideration as a drug development candidate. This platform provides the opportunity to screen cystine-dense peptides with drug-like qualities against targets that are implicated for the treatment of diseases, but are poorly suited for conventional approaches.
The strains published by Menalled et al. (2003) and Lin et al. (2001) are known by alternative titles in the literature, either CAG140 or HdhQ140 for the Menalled et al. (2003) strain and either CAG150 or HdhQ150 for Lin et al. (2001). In preparing the manuscript, the strains were listed in the text and Table 1 as HdhQ140 and HdhQ150, respectively, but were referred to as CAG140 and CAG150, respectively, in Figure 1, a nomenclature error which was missed prior to publication. For the sake of consistency with the text, Figure 1 has been revised to name the strains first described by Menalled et al. (2003) and Lin et al. (2001) as HdhQ140 and HdhQ150, respectively. The corrected figure is included here and in the article available online.
The ability to quantitatively evaluate the impact of a potential therapeutic intervention for Huntington disease (HD) in animal models for the disease is a critical step in the pathway to development of an effective therapy for this devastating neurodegenerative disorder. We report here an approach that combines a cell-based assay's quantitative accuracy and direct relationship to molecular processes with the ability to directly monitor effects in HD model mouse neurons. To accomplish this goal, we have developed an accurate quantitative reporter assay for a transcript known to be down-regulated as an early consequence of mutant huntingtin expression. This system uses mouse strains carrying a GFP reporter for the expression of the dopamine receptor D2, expressed in the medium spiny neurons of the basal ganglion. This receptor consistently demonstrates reduced expression in patients and murine models, and the FACS-based assay gives a highly accurate and quantitative readout of this pathology in mouse neurons expressing mutant huntingtin. For four genetic models and one viral model, a highly reproducible time course of loss of reporter expression is observed. This quantitative measure of HD pathology can be used to measure the effects of HD therapeutics in small cohorts with high confidence. We further demonstrate that the introduction of an shRNA against the huntingtin transgene by virus can improve this pathological status in medium spiny neurons transduced with the construct. We believe this system can be of great utility in the validation of effective therapeutic interventions for HD.neurodegeneration | polyglutamine | viral vectors | gene expression | neuroprotection I n the search for effective therapeutic interventions for neurodegenerative diseases such as ALS, Parkinson disease, Alzheimer's disease, and Huntington disease (HD), cell and animal models for all of these conditions have been developed. Whereas cell models allow higher throughput and lower cost, any therapeutic intervention initially evaluated in a cell-culture model must subsequently be assessed in an appropriate animal model system before clinical trials in humans can be considered. However, the inherent variance in quantitation of the behavioral or pathological readouts in animals has practical consequences, requiring large cohorts to detect all but the most overt benefits, which limits experimental throughput. We sought to develop and implement an approach to this problem that combines the quantitative accuracy and direct relationship to molecular processes of a cell-based assay with the medical relevance of evaluating endogenous neurons in the animal brain.In the present study, we have focused on HD. The uniform genetic etiology of HD, caused by a poly(CAG) expansion within exon 1 of huntingtin (HTT) (1), has provided an excellent starting point for the derivation of animal models for the disease. A number of similar animal models have been developed and characterized by behavioral and/or morphometric assessments of pathology (reviewed in...
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