leupeptins and Huntington-Disease

Huntington's disease (HD) is a deadly neurodegenerative disease with abnormal expansion of CAG repeats in the huntingtin gene. Mutant Huntingtin protein (mHTT) forms abnormal aggregates and intranuclear inclusions in specific neurons, resulting in cell death. Here, we tested the ability of a natural heat-shock protein 90 inhibitor, Gedunin, to degrade transfected mHTT in Neuro-2a cells and endogenous mHTT aggregates and intranuclear inclusions in both fibroblasts from HD patients and neurons derived from induced pluripotent stem cells from patients. Our data showed that Gedunin treatment degraded transfected mHTT in Neuro-2a cells, endogenous mHTT aggregates and intranuclear inclusions in fibroblasts from HD patients, and in neurons derived from induced pluripotent stem cells from patients in a dose- and time-dependent manner, and its activity depended on the proteasomal pathway rather than the autophagy route. These findings also showed that although Gedunin degraded abnormal mHTT aggregates and intranuclear inclusions in cells from HD patient, it did not affect normal cells, thus providing a new perspective for using Gedunin to treat HD.

Topics: Animals; Cell Culture Techniques; Fibroblasts; Heat-Shock Proteins; Humans; Huntingtin Protein; Huntington Disease; Induced Pluripotent Stem Cells; Intranuclear Inclusion Bodies; Leupeptins; Limonins; Mice; Mutant Proteins; Mutation; Neurons; Proteasome Endopeptidase Complex; Protein Aggregates; Transfection

Gastrodia elata Blume (Fam. Orchidaceae) is a traditional Chinese herbal medicine for treating headaches, dizziness, tetanus, epilepsy, and numbness of the limbs, which suggests that it has neuroprotective effect.. To validate the neuroprotection of Gastrodia elata in preventing neurodegenerations, such as Huntington's disease (HD).. MTT assay was used to validate the protection of Gastrodia elata. In pheochromocytoma (PC12) cell. Transient transfection of mutant huntingtin (Htt) in PC12 cell was used as an in vitro model of HD. Filter retardation assay was used to measure Htt-induced protein aggregations. Proteasome activity was monitored by transfection of pZsProSensor-1 and imaged by a confocal laser scanning microscope.. This protection of Gastrodia elata could be blocked by an A(2A)-R antagonist and a protein kinase A (PKA) inhibitor, indicating an A(2A)-R signaling event. Gastrodia elata could reverse mutant Htt-induced protein aggregations and proteasome de-activation through A(2A)-R signaling. In addition, activation of PKA tended to activate proteasome activity and reduce mutant Htt protein aggregations. The proteasome inhibitor, MG 132, blocked Gastrodia elata-mediated suppression of mutant Htt aggregations.. Gastrodia elata prevented mutant Htt aggregations and increased proteasomal activity by targeting the A(2A)-R through PKA-dependent pathway.

Topics: Adenosine A2 Receptor Agonists; Adenosine A2 Receptor Antagonists; Animals; Cyclic AMP-Dependent Protein Kinases; Cysteine Proteinase Inhibitors; Drugs, Chinese Herbal; Enzyme Inhibitors; Gastrodia; Huntingtin Protein; Huntington Disease; Leupeptins; Nerve Tissue Proteins; Neuroprotective Agents; Nuclear Proteins; PC12 Cells; Phytotherapy; Proteasome Endopeptidase Complex; Rats; Receptor, Adenosine A2A; Signal Transduction; Transfection; Ubiquitin; Ubiquitinated Proteins

Bcl-2-associated athanogene-1 (BAG1) is a multifunctional protein delivering chaperone-recognized unfolded substrates to the proteasome for degradation. It has been shown to be essential for proper CNS development in vivo, playing a crucial role in neuronal survival and differentiation. With regard to Huntington's disease, a sequestration of BAG1 into inclusion bodies and a neuroprotective effect in double transgenic mice have been reported. Here, we show that BAG1 reduces aggregation and accelerates degradation of mutant huntingtin (htt-mut). Moreover, it reduces nuclear levels of htt-mut. This effect can be overcome by over-expression of seven in absentia homolog 1, an E3 ligase negatively regulated by BAG1 and known to be involved in nuclear import of htt-mut. In vivo, BAG1 proved to be protective in a Drosophila melanogaster Huntington's disease model, preventing photoreceptor cell loss induced by htt-mut. In summary, we present BAG1 as a therapeutic tool modulating key steps in htt toxicity in vitro and ameliorating htt toxicity in vivo.

Topics: Animals; Animals, Genetically Modified; Cysteine Proteinase Inhibitors; Disease Models, Animal; DNA-Binding Proteins; Drosophila melanogaster; Gene Expression Regulation; Green Fluorescent Proteins; Humans; Huntingtin Protein; Huntington Disease; Leupeptins; Mice; Mutation; Nerve Tissue Proteins; Nuclear Proteins; Photoreceptor Cells; Proteins; Subcellular Fractions; Transcription Factors; Ubiquitin-Protein Ligases

In patients with Huntington's disease (HD), the proteolytic activity of the ubiquitin proteasome system (UPS) is reduced in the brain and other tissues. The pathological hallmark of HD is the intraneuronal nuclear protein aggregates of mutant huntingtin. We determined how to enhance UPS function and influence catalytic protein degradation and cell survival in HD. Proteasome activators involved in either the ubiquitinated or the non-ubiquitinated proteolysis were overexpressed in HD patients' skin fibroblasts or mutant huntingtin-expressing striatal neurons. Following compromise of the UPS, overexpression of the proteasome activator subunit PA28gamma, but not subunit S5a, recovered proteasome function in the HD cells. PA28gamma also improved cell viability in mutant huntingtin-expressing striatal neurons exposed to pathological stressors, such as the excitotoxin quinolinic acid and the reversible proteasome inhibitor MG132. These results demonstrate the specific functional enhancements of the UPS that can provide neuroprotection in HD cells.

Topics: Cell Survival; Cells, Cultured; Corpus Striatum; Fibroblasts; Humans; Huntingtin Protein; Huntington Disease; Leupeptins; Muscle Proteins; Nerve Tissue Proteins; Neurons; Nitro Compounds; Nuclear Proteins; Peptide Fragments; Propionates; Proteasome Endopeptidase Complex; Quinolinic Acid; Recombinant Fusion Proteins; RNA-Binding Proteins; Transduction, Genetic; Ubiquitin; Up-Regulation