Boxun Lu

Extracellular Calcium Controls Background Current and Neuronal Excitability via an UNC79-UNC80-NALCN Cation Channel Complex

In contrast to its extensively studied intracellular roles, the molecular mechanisms by which extracellular Ca(2+) regulates the basal excitability of neurons are unclear. One mechanism is believed to be through Ca(2+)s interaction with the negative charges on the cell membrane (the charge screening effect). Here we show that, in cultured hippocampal neurons, lowering [Ca(2+)](e) activates a NALCN channel-dependent Na(+)-leak current (I(L-Na)). The coupling between [Ca(2+)](e) and NALCN requires a Ca(2+)-sensing G protein-coupled receptor, an activation of G-proteins, an UNC80 protein that bridges NALCN to a large novel protein UNC79 in the same complex, and the last amino acid of NALCNs intracellular tail. In neurons from nalcn and unc79 knockout mice, I(L-Na) is insensitive to changes in [Ca(2+)](e), and reducing [Ca(2+)](e) fails to elicit the excitatory effects seen in the wild-type. Therefore, extracellular Ca(2+) influences neuronal excitability through the UNC79-UNC80-NALCN complex in a G protein-dependent fashion.

Peptide neurotransmitters activate a cation channel complex of NALCN and UNC-80

Several neurotransmitters act through G-protein-coupled receptors to evoke a ‘slow’ excitation of neurons. These include peptides, such as substance P and neurotensin, as well as acetylcholine and noradrenaline. Unlike the fast (approximately millisecond) ionotropic actions of small-molecule neurotransmitters, the slow excitation is not well understood at the molecular level, but can be mainly attributed to suppressing K+ currents and/or activating a non-selective cation channel. The molecular identity of this cation channel has yet to be determined; similarly, how the channel is activated and its relative contribution to neuronal excitability induced by the neuropeptides are unknown. Here we show that, in the mouse hippocampal and ventral tegmental area neurons, substance P and neurotensin activate a channel complex containing NALCN and a large previously unknown protein UNC-80. The activation by substance P through TACR1 (a G-protein-coupled receptor for substance P) occurs by means of a unique mechanism: it does not require G-protein activation but is dependent on Src family kinases. These findings identify NALCN as the cation channel activated by substance P receptor, and suggest that UNC-80 and Src family kinases, rather than a G protein, are involved in the coupling from receptor to channel.

Identification of NUB1 as a suppressor of mutant Huntingtin toxicity via enhanced protein clearance

Huntingtons disease is caused by expanded CAG repeats in HTT, conferring toxic gain of function on mutant HTT (mHTT) protein. Reducing mHTT amounts is postulated as a strategy for therapeutic intervention. We conducted genome-wide RNA interference screens for genes modifying mHTT abundance and identified 13 hits. We tested 10 in vivo in a Drosophila melanogaster Huntingtons disease model, and 6 exhibited activity consistent with the in vitro screening results. Among these, negative regulator of ubiquitin-like protein 1 (NUB1) overexpression lowered mHTT in neuronal models and rescued mHTT-induced death. NUB1 reduces mHTT amounts by enhancing polyubiquitination and proteasomal degradation of mHTT protein. The process requires CUL3 and the ubiquitin-like protein NEDD8 necessary for CUL3 activation. As a potential approach to modulating NUB1 for treatment, interferon-β lowered mHTT and rescued neuronal toxicity through induction of NUB1. Thus, we have identified genes modifying endogenous mHTT using high-throughput screening and demonstrate NUB1 as an exemplar entry point for therapeutic intervention of Huntingtons disease.

A novel human embryonic stem cell-derived Huntington's disease neuronal model exhibits mutant huntingtin (mHTT) aggregates and soluble mHTT-dependent neurodegeneration

Most neurodegenerative diseases are linked to aberrant accumulation of aggregation‐prone proteins. Among them, Huntingtons disease (HD) is caused by an expanded polyglutamine repeat stretch in the N terminus of the mutant huntingtin protein (mHTT), which gets cleaved and aggregates in the brain. Recently established human induced pluripotent stem cell‐derived HD neurons exhibit some disease‐relevant phenotypes and provide tools for HD research. However, they have limitations such as genetic heterogeneity and an absence of mHTT aggregates and lack a robust neurodegeneration phenotype. In addition, the relationship between the phenotype and mHTT levels has not been elucidated. Herein, we present a human embryonic stem cell (hESC)‐derived HD neuronal model expressing HT‐Texon1 fragments, which addresses the deficiencies enumerated above. The wild‐type and HD lines are derived from an isogenic background and exhibit insoluble mHTT aggregates and neurodegeneration. We also demonstrate a quantitative relationship between neurodegeneration and soluble monomeric (but not oligomeric or aggregated) mHTT levels. Reduction of ~10% of mHTT is sufficient to prevent toxicity, whereas ~90% reduction of wild‐type HTT is safe and well‐tolerated in these cells. A known HD toxicity modifier (Rhes) showed expected rescue of neurodegeneration. Therefore, the hESC‐derived neuronal models complement existing induced pluripotent stem cell‐derived neuronal models and provide valuable tools for HD research.—Lu, B., Palacino, J. A novel human embryonic stem cell‐derived Huntingtons disease neuronal model exhibits mutant huntingtin (mHTT) aggregates and soluble mHTT‐dependent neurodegeneration. FASEB J. 27, 1820–1829 (2013). www.fasebj.org

A striatal-enriched intronic GPCR modulates huntingtin levels and toxicity

Huntingtons disease (HD) represents an important model for neurodegenerative disorders and proteinopathies. It is mainly caused by cytotoxicity of the mutant huntingtin protein (Htt) with an expanded polyQ stretch. While Htt is ubiquitously expressed, HD is characterized by selective neurodegeneration of the striatum. Here we report a striatal-enriched orphan G protein-coupled receptor(GPCR) Gpr52 as a stabilizer of Htt in vitro and in vivo. Gpr52 modulates Htt via cAMP-dependent but PKA independent mechanisms. Gpr52 is located within an intron of Rabgap1l, which exhibits epistatic effects on Gpr52-mediated modulation of Htt levels by inhibiting its substrate Rab39B, which co-localizes with Htt and translocates Htt to the endoplasmic reticulum. Finally, reducing Gpr52 suppresses HD phenotypes in both patient iPS-derived neurons and in vivo Drosophila HD models. Thus, our discovery reveals modulation of Htt levels by a striatal-enriched GPCR via its GPCR function, providing insights into the selective neurodegeneration and potential treatment strategies. DOI: http://dx.doi.org/10.7554/eLife.05449.001

TR-FRET Assays of Huntingtin Protein Fragments Reveal Temperature and PolyQ Length-Dependent Conformational Changes

Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) technology is a widely used immunoassay that enables high-throughput quantitative measurements of proteins of interest. One of the well established examples is the TR-FRET assay for mutant huntingtin protein (HTT), which is the major cause of the neurodegenerative Huntingtons disease (HD). To measure the mutant HTT protein, the published assays utilize a polyQ antibody, MW1, paired with HTT N-terminal antibodies. MW1 has much higher apparent affinity to mutant HTT with expanded polyQ stretch than to wild-type HTT with shorter polyQ, and thus the assays detect mutant HTT preferentially. Here we report a reversible temperature dependent change of TR-FRET signals for HTT N-terminal fragments: the signals become higher when the temperature is lowered from room temperature to 4°C. Interestingly, the temperature sensitivity of the TR-FRET signals is much higher for the Q25 (wild-type) than for the Q72 (mutant) protein. We further revealed that it is likely due to a temperature and polyQ length-dependent structural or spatial change of HTT, which is potentially useful for understanding polyQ structure and toxicity.

A toxic mutant huntingtin species is resistant to selective autophagy

Protein misfolding is a common theme in neurodegenerative disorders including Huntingtons disease (HD). The HD-causing mutant huntingtin protein (mHTT) has an expanded polyglutamine (polyQ) stretch that may adopt multiple conformations, and the most toxic of these is the one recognized by antibody 3B5H10. Here we show that the 3B5H10-recognized mHTT species has a slower degradation rate due to its resistance to selective autophagy in human cells and brains, revealing mechanisms of its higher toxicity.

TR-FRET Assays for Endogenous Huntingtin Protein Level in Mouse Cells

High-throughput measurement of huntingtin (Htt) levels is useful for Huntingtons disease research. For example, identification of genetic or chemical modifiers that reduce Htt levels by high-throughput screening provides promising strategy for HD drug discovery. In the human cells, high-throughput measurement of Htt levels has been established based on the Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) technology, using the 2B7/MW1 antibody pair. Unfortunately, application of this assay in the mouse cells has been problematic due to discrepancies between TR-FRET signals and Western-blots, possibly caused by non-specific antibody binding. Here we report TR-FRET assays that are able to detect endogenous Htt levels of the mouse striatal cell line (STHdh).

A high-throughput-compatible assay to measure the degradation of endogenous Huntingtin proteins

Aim:The accumulation of disease-causing proteins is a common hallmark of many neurodegenerative disorders. Measuring the degradation of such proteins using high-throughput-compatible assays is highly desired for the identification of genetic and chemical modulators of degradation. For example, Huntingtons disease (HD) is an incurable hereditary neurodegenerative disorder caused by the cytotoxicity of mutant huntingtin protein (mHTT). The high-throughput measurement of mHTT degradation is important in HD drug discovery and research. Existing methods for such purposes have limitations due to their dependence on protein tags or pan protein synthesis inhibitors. Here, we report a high-throughput-compatible pulse-chase method (CH-chase) for the measurement of endogenous tag-free huntingtin protein (HTT) degradation based on Click chemistry and Homogeneous Time Resolved Fluorescence (HTRF) technologies.Methods:The pulsed-labeled proteins were conjugated with biotin using the click reaction strain-promoted alkyne-azide cycloaddition (SPAAC), and the chase signals were calculated by measuring the reduction percentage of the HTT HTRF signals after pull-down with streptavidin beads.Results:We validated that the signals were within the linear detection range and were HTT-specific. We successfully measured the degradation of endogenous HTT in a high-throughput-compatible format using 96-well plates. The predicted changes of HTT degradation by known modifiers were observed, which confirmed that the assay is suitable for the identification of HTT degradation modifiers.Conclusion:We have established the first high-throughput-compatible assay capable of measuring endogenous, tag-free HTT degradation, providing a valuable tool for HD research and drug discovery. The method could be applied to other proteins and can facilitate research on other neurodegenerative disorders and proteinopathies.

Suppression of MAPK11 or HIPK3 reduces mutant Huntingtin levels in Huntington's disease models.

Most neurodegenerative disorders are associated with accumulation of disease-relevant proteins. Among them, Huntington disease (HD) is of particular interest because of its monogenetic nature. HD is mainly caused by cytotoxicity of the defective protein encoded by the mutant Huntingtin gene (HTT). Thus, lowering mutant HTT protein (mHTT) levels would be a promising treatment strategy for HD. Here we report two kinases HIPK3 and MAPK11 as positive modulators of mHTT levels both in cells and in vivo. Both kinases regulate mHTT via their kinase activities, suggesting that inhibiting these kinases may have therapeutic values. Interestingly, their effects on HTT levels are mHTT-dependent, providing a feedback mechanism in which mHTT enhances its own level thus contributing to mHTT accumulation and disease progression. Importantly, knockout of MAPK11 significantly rescues disease-relevant behavioral phenotypes in a knockin HD mouse model. Collectively, our data reveal new therapeutic entry points for HD and target-discovery approaches for similar diseases.