The Journal of Neuroscience, November 15, 2001, 21(22):8772–8781 Proteasomal-Dependent Aggregate Reversal and Absence of Cell Death in a Conditional Mouse Model of Huntington’s Disease Ester Martı´n-Aparicio,1 Ai Yamamoto,2 Fe´ lix Herna´ ndez,1 Rene´ Hen,2 Jesu´ s Avila,1 and Jose´ J. Lucas1 1Centro de Biologı´a Molecular “Severo Ochoa,” Consejo Superior de Investigaciones Cientı´ficas–Universidad Auto´ noma de Madrid, 28049 Madrid, Spain, and 2Center for Neurobiology and Behavior, Columbia University, New York, New York 10032 Neuronal intranuclear inclusions are a histopathological hall- aggregate formation, and5doftransgene suppression led to mark of Huntington’s disease. Nevertheless, the precise mech- aggregate disappearance. In mice, full reversal of aggregates anism by which they are formed and their relevance to neuronal and intranuclear mutant huntingtin was more rapid than re- cell death and/or dysfunction remains unclear. We recently ported previously and preceded the motor recovery by several generated a conditional mouse model of Huntington’s disease weeks. Furthermore, the proteasome inhibitor lactacystin inhib- (HD94) in which silencing expression of mutated huntingtin led ited the aggregate clearance observed in culture, thus indicat- to the disappearance of intranuclear aggregates and ameliora- ing that aggregate formation is a balance between the rate of tion of the behavioral phenotype. Here, we analyze primary huntingtin synthesis and its degradation by the proteasome. striatal neuronal cultures from HD94 mice to explore the dy- Finally, neither expression of the mutant huntingtin nor aggre- namics of aggregate formation and reversal, the possible gates compromised the viability of HD94 cultures. This corre- mechanisms involved, and the correlation between aggregates lated with the lack of cell death in symptomatic HD94 mice, and neuronal death. In parallel, we examine symptomatic adult thus demonstrating that neuronal dysfunction, and not cell loss, HD94 mice in similar studies and explored the relationship triggered by mutant huntingtin underlies symptomatology. between aggregate clearance and behavioral reversal. We re- Key words: Huntington’s disease; aggregates; conditional port that, in culture, aggregate formation and reversal were transgenic mouse model; fast reversal; proteasome; absence of rapid processes, such that2doftransgene expression led to cell death Huntington’s disease (HD) is a progressive, autosomal dominant, respective proteins. These triplet-repeat disorders share an inter- neurodegenerative disorder (Wexler et al., 1987). Patients suffer esting commonality: the presence of intraneuronal aggregates from motor dysfunction, cognitive decline, and psychological dis- (Ross, 1997; Nakamura et al., 2001). turbances over 10–15 years until death (Ambrose et al., 1994). The relevance of aggregate formation to the etiology of HD is The neuropathology is extremely restricted, with atrophy occur- unclear. Aggregates have been implicated as the trigger for neu- ring in the striatum and to a lesser extent in the cortex. rodegeneration, because both aggregates and pathogenicity are The mutation leading to HD is an expansion of CAG repeats caused by the same polyQ length threshold (Scherzinger et al., near the 5Ј end of the IT15 gene (The Huntington’s Disease 1997). Furthermore, in a transgenic mouse model, these aggre- Collaborative Research Group, 1993; Rubinsztein et al., 1994; gates precede the onset of symptomatology (Davies et al., 1997). Gusella and MacDonald, 1996). Although unaffected individuals Inclusions are also a feature in other non-polyQ disorders, such as Ͼ have 35 or fewer repeats, repeat lengths of 40 invariably lead to Alzheimer’s disease and Parkinson’s disease, thus suggesting that HD. The repeat sequence is translated into a polyglutamine protein aggregation might cause neurodegeneration through a stretch (polyQ) near the N terminus of the IT15 gene product general mechanism (Price et al., 1998; Tobin and Signer, 2000). huntingtin (htt). Eight other autosomal dominant neurological Moreover, proteins prone to aggregate, such as mutated hunting- diseases are also caused by a polyQ expansion mutation in their tin and cystic fibrosis proteins, have been reported to inhibit the ubiquitin proteasome system (UPS) (Bence et al., 2001). This Received July 2, 2001; revised Aug. 23, 2001; accepted Sept. 5, 2001. This work was supported by grants from Asociacio´n Espan˜ola de Corea de inhibition in turn altered the half-life of proteins involved in Huntington (ACHE), by the United States–Spain Commission for Scientific and apoptosis and cell survival (Jana et al., 2001). Nevertheless, Technological Cooperation, by the Hereditary Disease Foundation (HDF), and by whereas some studies using transfected cells support aggregate- an institutional grant from Fundacio´n Ramo´n Areces. E.M.-A. and A.Y. are recip- ients of predoctoral fellowships from ACHE and Fundacio´n Ferrer and from HDF, induced toxicity (Waelter et al., 2001), others dissociate inclusion respectively. We thank Drs. Javier Dı´az-Nido, Francisco Wandosell, Mar Pe´rez, and formation and cell survival (Kim et al., 1999) and even suggest Jose´ Gonza´lez-Castan˜o for helpful discussion and comments; Drs. Eric Kandel that intranuclear inclusions may reflect a protective mechanism and Mark Mayford for the use of the CaMKII ␣-tTA mice; Drs. Erich E. Wanker and Xiao-Jiang Li for kindly providing CAG53b and EM48 antibodies, respectively; against soluble htt-induced toxicity (Saudou et al., 1998). Drs. Victoria Arango and Mark Underwood for stereology; and Dr. Gine´s Morata Using a conditional animal model of HD that reversibly ex- for microscope and photography facility. We are also grateful to Carlos Sa´nchez, Raquel Cuadros, Helena Garuti, and Elena Langa for technical assistance. pressed a mutated htt fragment selectively in the forebrain Correspondence should be addressed to Jose´ J. Lucas, Centro de Biologı´a (HD94) (Yamamoto et al., 2000), we found that abolishing trans- Molecular “Severo Ochoa,” Consejo Superior de Investigaciones Cientı´ficas–Uni- gene expression in symptomatic mice leads to amelioration of the versidad Auto´noma de Madrid, Facultad Ciencias, Universidad Auto´noma de Ma- drid, Cantoblanco, 28049 Madrid, Spain. E-mail: [email protected]. behavioral phenotype and disappearance of inclusions. This dem- Copyright © 2001 Society for Neuroscience 0270-6474/01/218772-10$15.00/0 onstrated that inclusions are dynamic structures and that a con- Martı´n-Aparicio et al. • HD Aggregate Clearance Is Proteasome Dependent J. Neurosci., November 15, 2001, 21(22):8772–8781 8773 tinuous influx of the protein is required for disease progression. ␤-Galactosidase activity assays In this initial characterization, however, the minimum time the LacZ staining. Cultures were fixed for 20 min in 4% PFA in 5 mM cell required to clear the aggregates versus the time the organism MgCl2–PBS. Slides were incubated for 1 hr at 30°C in an X-gal solution: required to recover its motor behavior was not established, thus 1 mg/ml X-gal (Boehringer Mannheim, Indianapolis, IN), 5 mM potas- sium ferrocyanide, 5 mM potassium ferricyanide, and 2 mM MgCl2 in hindering the search for possible mechanisms that underlie the PBS. After staining, cultures were coverslipped with Fluoromount. recovery process. In light of this, we have established primary O-nitrophenyl ␤-D-calactopyranoside colorimetric assay. Cultures were neuronal cultures from HD94 mice. With this system, we explore washed twice with 5 mM MgCl2 in PBS. Cells were lysed in 250 mM Tris the dynamics of aggregate formation and reversal, the mechanism buffer, pH 8.0, with 0.1% Triton X-100. Enzymatic assay was performed responsible for reversal, and the correlation between aggregate as described previously (Sambrook et al., 1989). formation and cell death. As we gained more insight from the culture system, we then returned to the animal model and reex- Survival assays amined the issues sought in vitro. Propidium iodide–calcein staining. Twenty-four hours after plating, cul- tures were shifted to media without serum, B27, or both. After 24, 48, or 72 hr, cell viability was assessed by propidium iodide–calcein staining MATERIALS AND METHODS (Mattson et al., 1995). Calcein-AM is taken up and cleaved by esterases present within living cells and leads to a yellowish-green fluorescence, Animals whereas propidium iodide (PI) is taken up by only dead cells and become HD94 mice were generated as described previously (Yamamoto et al., orange-red fluorescent. In brief, neurons were incubated for 30 min with 2000). Mice were bred at the Centro de Biolog´ıa Molecular “Severo 2 ␮M PI (Sigma) and 1 ␮M calcein-AM (Molecular Probes, Eugene, OR). Ochoa” (Madrid, Spain) and Center for Neurobiology and Behavior After several brief rinses with each medium, cells were visualized by (Columbia, NY). Four to five mice were housed per cage, with food and fluorescence microscopy using a Zeiss (Oberkochen, Germany) Axiovert water available ad libitum. Mice were maintained in a temperature- 135 microscope. Four fields (selected at random) were analyzed per well controlled environment on a 12 hr light/dark cycle, with light onset at (ϳ300 cells per field) in two independent experiments. Cell death was 7:00 A.M. expressed as percentage of PI-positive cells from the total number of cells. Primary culture Hoechst 33342 nuclear staining. Neurons were fixed in 4% PFA in 5 mM Primary cultures of neurons and glia were prepared according to modi- MgCl2-PBS and pretreated with 0.2% Triton X-100 in PBS. After that, fications of established procedures (Huettner and Baughman, 1986; cultures were incubated with Hoechst dye 33342 (2.5 ␮g/ml; Biologics) Dubinsky, 1989). Pups were killed at postnatal day 1, and tail tissue and visualized under a fluorescence microscope. The percentage of samples were taken for ulterior genotyping by PCR. Striatal tissue was condensed or fragmented nuclei was counted for each genotype and then dissected and dissociated individually from each pup with the culture condition at 20ϫ magnification. Data are reported as the mean Ϯ Papain Dissociation System (Worthington, Freehold, NJ). The rest of SEM (n ϭ 3).