A cell-based screen for drugs to treat Huntington's disease
Introduction
Huntington's disease (HD) is caused by an expansion of a CAG repeat in the huntingtin gene, resulting in an expansion of a polyglutamine (poly Q) region near the N-terminus of the huntingtin protein (Htt) (The Huntington's Disease Collaborative Research Group, 1993). The mechanism(s) by which this expanded poly Q causes disease remains elusive, although many explanations have been proposed and are under active investigation Koshy and Zoghbi, 1997, Tobin and Signer, 2000. There is currently no treatment to prevent or palliate the progress of HD. Identification of clinically effective drugs that would decrease symptoms or increase survival would represent a major advance.
HD is characterized by progressive deficits in motor control and by affective and cognitive deterioration. Progression of the disease is accompanied by neuronal death in the striatum and, to a lesser extent, the cerebral cortex. The mode of neuronal death in HD continues to be debated, although considerable evidence suggests that apoptosis plays an important role (Hickey and Chesselet, 2003). Intraneuronal aggregates, both intra- and extranuclear, are prominent features in late stage HD, although whether these aggregates are causal, protective, or incidental remains hotly debated Bence et al., 2001, Davies et al., 1997, Hackam et al., 1998, Hughes and Olson, 2001, Kim et al., 1999, Perutz and Windle, 2001, Saudou et al., 1998, Waelter et al., 2001, Wang et al., 1999, Wellington et al., 2000. A variety of model systems have demonstrated that the exon 1 portion of Htt, which contains the expanded poly Q region, is sufficient to cause pathology Davies et al., 1997, Jana et al., 2001, Mangiarini et al., 1996, Martindale et al., 1998, Schilling et al., 1999, Wyttenbach et al., 2000.
In coordination with an NIH initiative to identify drugs of potential use in treating neurodegenerative diseases, we have developed and implemented a cell-based screen to test chemical compounds for their ability to protect against expanded-repeat Htt-mediated cell death. Since this assay measures cell death (monitored by the release of LDH into the culture medium), it is not dependent on specific hypotheses about the mechanism(s) by which neurons are killed in HD. It does, however, presuppose that neuronal death is an important aspect of HD, and that this neuronal death is cell-autonomous, that is, that it is not dependent on interneuronal connectivity in the intact brain. It also assumes that the cell death occurring rapidly in cultured cells overexpressing expanded repeat Htt is in some way a valid model for the neuronal death that occurs over tens of years in patients. These assumptions are relatively conservative compared with the hypothesis-driven assumptions based on the possible roles of aggregation, protein–protein binding, insolubility, and transcriptional alteration in HD pathogenesis. This assay therefore represents a relatively hypothesis-independent strategy to screen drugs for clinical use.
Because of their similarities to neurons, PC12 cells have provided a useful model for studying neuronal cell biology Chai et al., 1999, Furlong et al., 2000, Greene and Tischler, 1976, Li et al., 1999, Schubert et al., 1977, Song et al., 2002, Wyttenbach et al., 2001. In addition, PC12 cells are readily transfected, selected, and cloned. Both before and after terminal differentiation with NGF, PC12 cells exhibit many characteristics of mature neurons (Greene and Tischler, 1976), including the ability to undergo growth factor withdrawal-induced apoptotic cell death (Mills et al., 1997). As part of our efforts to understand the mechanisms of pathogenesis of HD, we have developed a cell culture model of HD using PC12 cells stably incorporating a plasmid that inducibly expresses an expanded poly Q (103 glutamines) form of exon 1 of Htt, fused to the marker EGFP (Kazantsev et al., 1999). For the sake of brevity, we will refer in this paper to the product of this synthetic truncated and fused construct as HttQ103. Transcription of the Htt transgene is driven by an ecdysone-regulated promoter so that the expression of HttQ103 can be turned on or off by the addition or removal of the nonsteroidal ecdysone analog tebufenozide (Suhr et al., 1998). These cells exhibit extensive (50%) and rapid (48 h) cell death after induction of HttQ103. Using these engineered cells, we have developed a medium-throughput 96-well plate-based assay to screen drugs for their ability to prevent the HttQ103-induced cell death, with the potential to screen 320 compounds/week.
In 2001, the NINDS created a drug library of 1040 compounds, selected in association with MicroSource Discovery Systems (Gaylordsville, CT), consisting mainly of FDA-approved drugs (Abbott, 2002) that were likely candidates for the treatment of neurodegenerative disorders. This library was distributed blindly to 27 laboratories for testing with various assays of potential relevance to neurological diseases. We applied these drugs to our PC12 cell-based assay system, and categorized them into seven groups depending on their effects. Only after the entire library was screened was the key provided to identify the compounds. Our screen revealed 18 compounds that completely protected against cell death at 48 h, and 51 that protected partially. Our assay was validated internally (by reproducibility, consistency, and similarities within pharmacological categories) as well as externally (successful identification from the blinded library of known protective compounds). At least two functional groups, the caspase inhibitors and the cannabinoids, show positive effects in this assay. This cell model therefore provides a unique and important advance in the search for drugs to treat HD as well as a new tool in the effort to understand the pathogenic mechanisms that underlie the progression of the disease.
Section snippets
Plasmids
The plasmids used for transfecting mammalian cells contained a synthetic DNA insert encoding exon 1 of human HttQ103 fused to EGFP. These inserts were gifts of Alex Kazantsev and David Housman (MIT), and encode the entire exon 1, including the proline-rich segment, and containing 103 mixed CAG/CAA repeats, fused to a C-terminal EGFP tag in pcDNA3.1/myc-HIS (Invitrogen). This insert, previously called 104Q/EGFP (Kazantsev et al., 1999), was subcloned into pBWN, a gift from Steve Suhr and Fred
Results
Inducing expression of HttQ103 in our PC12/HttQ103 cells by adding tebufenozide to the culture media caused a progressive accumulation of the HttQ103 peptide (green staining, Figs. 1B, E, H, K). Nontransfected PC12 cells showed no response to tebufenozide (data not shown). Shortly after induction, the HttQ103 was distributed diffusely throughout the cytoplasm (Fig. 1E), with a gradual coalescence into brightly fluorescent aggregates (Figs. 1H, K), which typically appeared in a juxtanuclear
Discussion
Development of simple systems to model disease processes is an essential step in the search for disease treatments and cures. This strategy is particularly important for human neurodegenerative diseases, which appear late in life, develop slowly, involve unknown pathogenic mechanisms, and are specific to humans. HD is one such disease that has a special advantage—the initial causal agent is known to be a particular genetic mutation that expands a region of repeated CAG triplets in the
Acknowledgements
We thank Fred Gage and Steve Suhr for the pBWN expression vector and the ecdysone analog tebufenozide, David Housman and Alex Kazantsev for the huntingtin-EGFP inserts, Kym Faul and Alec Dooley for GC-MS analysis of cannabinoid recoveries in cell culture samples, Jeff Gornbein for statistical consultation, and Nicole Makarenco for assistance in revising the manuscript. This work was supported by grants from NINDS (NS39591) to AJT and from the CURE-HD Initiative of the Hereditary Disease
References (53)
- et al.
Formation of neuronal intranuclear inclusions underlies the neurological dysfunction in mice transgenic for the HD mutation
Cell
(1997) - et al.
Huntingtin is a cytoplasmic protein associated with vesicles in human and rat brain neurons
Neuron
(1995) - et al.
Physiochemical properties, solubility, and protein binding of delta 9-tetrahydrocanninol
J. Pharm. Sci.
(1974) - et al.
Apoptosis in Huntington's disease
Prog. Neuro-Psychopharmacol. Biol. Psychiatry
(2003) - et al.
Exon 1 of the HD gene with an expanded CAG repeat is sufficient to cause a progressive neurological phenotype in transgenic mice
Cell
(1996) - et al.
Instability of the CAG repeat in immortalized fibroblast cell cultures from Huntington's disease transgenic mice
Brain Res.
(1999) - et al.
Differentiation to an NGF-dependent state and apoptosis following NGF removal both occur asynchronously in cultures of PC12 cells
Exp. Cell Res.
(1997) - et al.
Noradrenaline release-inhibiting receptors on PC12 cells devoid of α2- and CB1 receptors: similarities to presynaptic imidazoline and edg receptors
Neurochem. Int.
(2002) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays
J. Immunol. Methods
(1983)- et al.
Huntingtin acts in the nucleus to induce apoptosis but death does not correlate with the formation of intranuclear inclusions
Cell
(1998)
Expression of full-length polyglutamine-expanded Huntingtin disrupts growth factor receptor signaling in rat pheochromocytoma (PC12) cells
J. Biol. Chem.
Aggresomes formed by α-synuclein and synphilin-1 are cytoprotective
J. Biol. Chem.
Huntington's disease: the challenge for cell biologists
Trends Cell Biol.
Inhibiting caspase cleavage of huntingtin reduces toxicity and aggregate formation in neuronal and nonneuronal cells
J. Biol. Chem.
A simple statistical parameter for use in evaluation and validation of high throughput screening assays
J. Biomol. Screening
Neurologists strike gold in drug screen effort
Nature
Impairment of the ubiquitin–proteasome system by protein aggregation
Science
Analysis of the role of heat shock protein (Hsp) molecular chaperones in polyglutamine disease
J. Neurosci.
Truncated N-terminal fragments of huntingtin with expanded glutamine repeats form nuclear and cytoplasmic aggregates in cell culture
Hum. Mol. Genet.
Alpha-synuclein overexpression promotes aggregation of mutant huntingtin
Biochem. J.
Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor
Proc. Natl. Acad. Sci. U. S. A.
The influence of huntingtin protein size on nuclear localization and cellular toxicity
J. Cell Biol.
Antisense downregulation of mutant huntington in a cell model
J. Gene Med.
Therapeutic opportunities in polyglutamine disease
Nat. Med.
Inducible PC12 cell model of Huntington's disease shows toxicity and decreased histone acetylation
NeuroReport
Altered proteasomal function due to the expression of polyglutamine-expanded truncated N-terminal huntingtin induces apoptosis by caspase activation through mitochondrial cytochrome c release
Hum. Mol. Genet.
Cited by (116)
The Functional Compounds of Cannabis sativa
2023, Sustainable Food Science - A Comprehensive Approach: Volumes 1-4Inconsistencies in histone acetylation patterns among different HD model systems and HD post-mortem brains
2020, Neurobiology of DiseaseThermoregulatory disorders in Huntington disease
2018, Handbook of Clinical NeurologyCitation Excerpt :The original goal of this study was to identify objective and reliable biomarkers of disease onset and progression in transgenic mouse models as readouts for preclinical trials. As cannabinoids had emerged among potential candidate compounds that showed promise in large in vitro screens (Aiken et al., 2004) and as this class of drugs is known to produce hypothermia in mammals (Rawls and Benamar, 2011), we included body temperature in the set of metabolic parameters that were investigated. Body temperature was monitored telemetrically via subcutaneously implanted transponders placed in the interscapular space.