Human Molecular Genetics, 2002, Vol. 11, No. 6 633-640
© 2002 Oxford University Press
Early phenotypes that presage late-onset neurodegenerative disease allow testing of modifiers in Hdh CAG knock-in mice
Molecular Neurogenetics Unit and 1Molecular Neuropathology, Massachusetts General Hospital, Charlestown, MA 02129, USA, 2Department of Neurology, Emory University School of Medicine, Atlanta, GA 30322, USA, 3Department of Neurosurgery, Brigham and Women’s Hospital, Boston, MA 02115, USA and 4Department of Pathology, Johns Hopkins University, Baltimore, MD 21205-2196, USA
Received October 18, 2001; Revised and Accepted January 14, 2002.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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In Huntington’s disease (HD), CAG repeats extend a glutamine tract in huntingtin to initiate the dominant loss of striatal neurons and chorea. Neuropathological changes include the formation of insoluble mutant N-terminal fragment, as nuclear/neuropil inclusions and filter-trap amyloid, which may either participate in the disease process or be a degradative by-product. In young Hdh knock-in mice, CAGs that expand the glutamine tract in mouse huntingtin to childhood-onset HD lengths lead to nuclear accumulation of full-length mutant huntingtin and later accumulation of insoluble fragment. Here we report late-onset neurodegeneration and gait deficits in older HdhQ111 knock-in mice, demonstrating that the nuclear phenotypes comprise early stages in a disease process that conforms to genetic and pathologic criteria determined in HD patients. Furthermore, using the early nuclear-accumulation phenotypes as surrogate markers, we show in genetic experiments that the disease process, initiated by full-length mutant protein, is hastened by co-expression of mutant fragment; therefore, accrual of insoluble-product in already compromised neurons may exacerbate pathogenesis. In contrast, timing of early disease events was not altered by normal huntingtin or by mutant caspase-1, two proteins shown to reduce inclusions and glutamine toxicity in other HD models. Thus, potential HD therapies in man might be directed at different levels: preventing the disease-initiating mechanism or slowing the subsequent progression of pathogenesis.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Expanded CAG repeats in the Huntington’s disease (HD) gene extend an N-terminal glutamine tract in a novel
350 kDa protein, huntingtin, causing dominantly inherited loss of vulnerable neurons in the striatum and choreiform movements (1,2). Genotype–phenotype studies in HD patients have revealed critical features of the disease mechanism: dominance of mutant huntingtin over the normal protein, progressivity with polyglutamine length and, in homozygotes, little discernible effect of the second mutant allele (3). Fulfillment of these HD genetic criteria has implicated an aggregation-promoting property of polyglutamine, typically measured in the context of short mutant fragment, in the disease process in man (4,5).
It has been proposed, from a variety of over-expression systems, that this toxic polyglutamine property could act from a short mutant fragment (6), perhaps liberated from full-length mutant protein in HD patients by caspase cleavage (7). However, findings in Hdh CAG knock-in mice, in which the glutamine tract of mouse huntingtin is extended to 92 or 111 residues, suggest that the 350 kDa mutant protein, is likely to be the site of initial toxicity (8,9). The results of immunoblot and immunostaining experiments support a mechanism, which conforms fully to the HD genetic criteria that leads to the accumulation of full-length mutant huntingtin in the nucleus of striatal neurons, with subsequent accrual of mutant fragment (as intranuclear inclusions and filter trap aggregate) and neuronal atrophy (8,9). Moreover, in Hdh CAG knock-in mice with 80 and 150 glutamines, the nuclear phenotypes are followed by mutant fragment neuropil deposits in the striatum and by terminal projection fields (10), or by reactive gliosis and low penetrant mild gait deficits (11), respectively; changes that suggest long-term neuropathological consequences that parallel disease in man.
Thus, the evidence indicates that the same polyglutamine property may act from the context of the full-length mutant protein to initiate a progressive disease cascade, and then, acting from subsequent mutant fragment, may propel the formation of aggregate that could, but need not contribute to the disease process (12). Furthermore, attempts to inhibit polyglutamine aggregation, measured with short over-expressed fragment, have now yielded factors that slow aggregation and/or toxicity (13–18), providing potential modifiers of the disease process in HD patients.
Hdh knock-in mice, with an implied series of events the timing of which is due primarily to CAG length rather than other factors, may provide optimal models with which to elucidate the role of mutant fragment and polyglutamine aggregation in a disease process that mirrors that in HD patients. Here we show that early striatal phenotypes accurately predict overt disease, providing surrogate markers for tests to determine whether co-expressed mutant fragment and two proteins that modify glutamine aggregation and toxicity in other HD models, normal huntingtin (19,20) and mutant caspase-1 (13), alter the timing of the disease cascade.
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RESULTS |
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Neurodegeneration and gait deficits in HdhQ111 knock-in mice
Hdh CAG knock-in mice with different CAG expansions exhibit similar neuropathologic phenotypes, although at different ages (8,10,11), with the longest repeats producing neurologic deficits (11). Therefore, to more precisely delineate stages in a progressive disease process, we assessed the timing of these changes in a single Hdh knock-in line, HdhQ111 mice. The striatal phenotypes are summarized in Figure 1A. Normal cytoplasmic EM48 stain detected at 5 weeks invariably yielded to diffuse nuclear-reactivity by 6 weeks (eight of eight brains). EM48 puncta were detected by 5 months (three of three brains), and nuclear inclusions were evident by 12 months of age (two of two brains), although at 10 months the latter phenotype was not fully penetrant (two of five brains). Later, at 17 months, the nuclear phenotypes were augmented by EM48 reactive deposits in striatal neuropil and projection fields (substantia nigra and globus pallidus; five of five brains), confirming axonal changes (10). By 24 months of age, reported neurodegenerative changes, toluidine blue-stained neurons and reactive gliosis (10,11,21), were observed in
50% of the HdhQ111 striata (five of ten brains). Consistent with incomplete penetrance, the proportion of toluidine blue-reactive striatal neurons was low (up to 3.5%), although in each case these mutant striata exhibited intense glial fibrillary acidic protein (GFAP) stain, indicating a glial cell response to neuronal injury. Notably, TUNEL staining of adjacent sections did not reveal pyknotic nuclei characteristic of apoptosis (data not shown), suggesting a degenerative process that is instead subtle and progressive.
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When neurologic function was assessed, using accelerating rotarod and ‘paw-clasping’ tests (8,11), HdhQ111 mice and their wild-type littermates were indistinguishable at 15 months of age (data not shown). At 24 months, they also did not differ in paw-clasp behavior (data not shown). However, at this age, tunnel walks to assess motor function (8,10,11) revealed subtle gait deficits. As summarized in Figure 1B, cohorts of heterozygous and homozygous HdhQ111 mice exhibited significantly shortened stride and imprecise hind–fore paw placement, compared to their wild-type littermates. Thus, mutant huntingtin with 111 glutamines initiates a disease process which, starting with nuclear accumulation phenotypes in striatal neurons, leads to a degenerative response and motor deficits by 24 months of age. The timing of the earliest phenotypes can be readily judged with few mice, but mouse-to-mouse variation of the late-onset changes indicates a role for additional factors and the need to look at older mice to reveal fully penetrant phenotypes.
A disease cascade due to a mechanism that fulfills HD genetic criteria
The temporal series of changes in HdhQ111 mice implied a similar disease process in the other lines of Hdh knock-in mice, whose timing may be CAG-size dependent. A comparison of the HdhQ111 data with those reported for four other Hdh CAG knock-in lines (8,10,11) (Fig. 2) confirms a striatal-specific disease process that is hastened by additional glutamines and dominant over the normal protein (in heterozygotes). Interestingly, onset of the disease process is slightly accelerated in homozygotes. Therefore, fulfillment of the critical HD genetic criteria suggests that this 2 year series of pathologic changes, which includes DNA damage (22,23), is likely to occur in presymptomatic, at-risk HD individuals and is also likely to forecast eventual striatal degeneration and neurological deficit.
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Accumulated mutant huntingtin fragment accelerates early disease events
The genotype–phenotype data are consistent with a pathogenic process that is initiated by full-length mutant huntingtin, as nuclear accumulation of the
350 kDa protein precedes other changes in striatal neurons (8). However, consistent with a previous proposal (10), these data do not preclude a role for accumulating mutant fragment in the ongoing disease process. Therefore, we tested the effect of added mutant N-terminal product by assessing early nuclear phenotypes in HdhQ111 mice bearing the N171–82Q transgene, expressing huntingtin N-terminus (residues 1–171) with 82 glutamines (24). Expression of the N171–82Q fragment, driven by the prion gene promotor, is relatively low in the striatum compared to other brain areas (24), permitting an assessment of HdhQ111 nuclear phenotypes that dominate in the striatum. As shown in Figure 3, early nuclear EM48 staining and later SDS-insoluble aggregate, filter-trapped from striatal extracts, increase noticeably when transgene and HdhQ111 allele are combined, compared to either lesion alone. Immunoblot analyses of soluble striatal proteins do not reveal increased levels (or novel) N-terminal fragment with co-expression of the N171–82Q transgene (data not shown). Moreover, quantification of the EM48-staining data supports a synergistic effect of the single N171–82Q transgene that is dependent on HdhQ111 dosage, worsening dramatically in homozygotes. These genotype–phenotype results demonstrate that mutant huntingtin fragment accelerates early pathological changes in HdhQ111 knock-in striatum.
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Wild-type huntingtin and dominant-negative caspase-1 do not affect early disease events
Consequently, we tested whether two gene products, normal huntingtin (19,20) and mutant caspase-1 (13), shown to reduce inclusions and glutamine toxicity in HD cellular and transgenic mouse models, alter the disease process. The impact of normal huntingtin on early nuclear changes was assessed in progeny of Hdh CAG knock-in and Hdhex4/5 knock-out parents (25), heterozygous and hemizygous littermates expressing a single dose of mutant huntingtin in the presence or absence of normal protein, respectively. In Figure 4 (left panel), nuclear EM48 stain is nearly identical in HdhQ111/Hdhex4/5 and HdhQ111/Hdh+ striata, and a closely similar pattern of nuclear inclusions is detected in HdhQ92/Hdhex4/5 and HdhQ92/Hdh+ striata. Therefore, although the nuclear phenotypes are slightly hastened with two copies of mutant huntingtin, there is no impact of the normal protein.
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Similarly, neuronal overexpression of dominant-negative caspase-1, tested in HdhQ111 mice carrying the ICEC285G transgene (26), also failed to alter the timing of diffuse nuclear EM48 stain and later inclusions, in HdhQ111 striatum. Figure 4 (right panel) shows very similar EM48 staining patterns in striata of HdhQ111 littermates with or without the transgene. Thus, early disease events are not obviously affected by dominant-negative caspase-1.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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We have demonstrated a glutamine length-dependent pathogenic cascade in accurate genetic models of HD. This 2 year series of pathologic changes, likely to occur in presymptomatic, at-risk HD individuals, forecasts eventual striatal degeneration and neurological deficit. The temporal order of the changes, which is not accessible in presymptomatic HD patients or easily reconstructable from the accrued damage of either end-stage HD post-mortem brain (27–29), or transgenic mouse brain (21,30), suggests a gradual neuronal decline. As the earliest nuclear accumulation phenotype involves the
350 kDa mutant protein (8), the genotype–phenotype data also support full-length mutant huntingtin, rather than N-terminal fragment, as the site of the earliest disease-initiating glutamine toxicity property. Delineation of stages in the pathologic process reveals limited variability in the earliest changes, but striking mouse-to-mouse variation for late-onset phenotypes, such as neurodegeneration and motor impairment. This suggests a role for modifying factors (genetic, environmental and stochastic) that may act at each successive stage, with the timing of late-onset phenotypes reflecting the accumulated impact of many different modifiers. This finding also argues that studies of late-onset phenotypes may require either large cohorts of mice, or smaller numbers of very old mice to ensure full penetrance. In contrast, the predictable timing of early pathologic changes, which accurately foreshadow later disease phenotypes, facilitates studies of disease modifiers by providing surrogate markers that can be evaluated at a few weeks of age using small groups of animals.
Overexpression of a mutant N171–82Q huntingtin fragment accelerated early disease events in Hdh knock-in striatum. This finding implies that in Hdh knock-in and HD brain, the accumulation of mutant fragment, which must be generated from full-length mutant huntingtin, and which forms inclusions, may hasten the disease process. Thus, therapeutic intervention aimed at reducing the burden of insoluble mutant huntingtin fragment may attenuate early stages of the disease mechanism. Notably, the death of the N171–82Q transgenic mice by 5–6 months (24) precludes an assessment of whether mutant fragment exacerbates subsequent neurodegeneration or neurologic deficit in Hdh knock-in striatum.
Recent evidence from cell culture and transgenic HD models has uncovered an intriguing neuroprotective function of wild-type huntingtin (19,20,31). However, wild-type huntingtin did not affect the timing of early disease correlates in Hdh knock-in mice. Therefore, in typical HD heterozygotes, the disease process is unaffected by the normal protein, although in homozygotes it is mildly hastened by a second dose of mutant protein. Thus, huntingtin’s reported protective activity may involve downstream events later in the disease process in sensitized neurons, acting, for example, via upregulation of neuroprotectant BDNF (32).
Early disease stages in Hdh knock-in mice were also unaffected by the presence of dominant-negative caspase-1 (ICEC285G). This finding, which contrasts with reduced inclusion burden in ICEC285G/HD exon 1 fragment transgenic striatum (13), argues that caspase-1 is unlikely to be involved in initiating the nuclear phenotype in Hdh knock-in mice. Nevertheless, consistent with the ICEC285G transgene conferring protection to an array of neuronal insults (33–36), inhibition of caspase-1-mediated cell death pathways may well afford a temporary reprieve later in the disease process. Thus, while modulating formation of inclusions and glutamine toxicity in sensitized neurons, the protective effects promoted by either normal huntingtin, or caspase-1 inhibition, appear unable to slow the ongoing disease-initiating process in Hdh knock-in striatum.
In summary, genotype–phenotype data in Hdh knock-in mice reveal that early nuclear accumulation of mutant huntingtin correlates with later striatal degeneration and motor deficits. The fulfillment of requisite HD genetic criteria suggests a similar chronic disease cascade in man, in asymptomatic at risk individuals, as well as symptomatic HD patients. Moreover, our findings argue strongly that, in addition to neuroprotection, the pathologic process may need to be attacked at the level of the initiating mechanism to achieve effective therapeutic strategies for this devastating disease.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Mice, genotyping and gait analysis
Mice were: HdhQ111 and HdhQ92 knock-in lines (129Sv/evxCD1) (8), Hdhex4/5 knock-out mice (129Sv/EvxCD1 background) (25), N171–82Q transgenic mice, expressing huntingtin amino acids 1–171 (with 82 glutamines) from the Prp promotor (C3H/HEJxC57BL/6J background) (24), and ICEC285G transgenic mice expressing dominant-negative caspase-1 (neuronal specific enolase promoter NSE-M17Z) (26). Genotyping was performed with specific Southern blot and PCR assays (8,22,24–26). Gait through a tunnel apparatus, with hind- and forepaws differentially painted with poster paint, was as reported (11,37). All tests and analyses were carried out on coded mice by investigators blinded to their genotypic status. t-tests were performed using the SigmaStat package (Jandel Scientific).
Immunostaining, histochemistry and filter-trap assay
Immunostaining with antibody EM48 (28) was on 7 µm coronal sections of periodate-lysine-paraformaldehyde (PLP)-perfused brain as described previously (8). All EM48 staining experiments were carried out under identical conditions. The intensity of diffuse EM48 stain, number of stained nuclei, and number of nuclei with EM48 reactive inclusions were quantified. Images (one dorsal and one ventral striatal field of EM48-stained sections from two or more brains for each genotype) were captured in Adobe Photoshop with a digital camera at 20x magnification (Olympus BX50 microscope). For mean nuclear stain intensity in arbitrary units (MNI), the ‘histogram’ function of Adobe Photoshop was used to convert the intensity of signal in all stained nuclei observed in a standard 2 inch-square region, to arbitrary units. Data from each sample were pooled, and data from multiple mice of each genotype to be compared were pooled. The ‘background’ neuropil signal, the mean value for 10 fields within the 2 inch square, was subtracted from the nuclear signal. The MNI was calculated from the pooled dataset, with the standard error (SE) (SigmaStat package). For the mean number of stained nuclei (MN), the number of diffusely stained nuclei was counted in eight 2 inch-square regions (four dorsal and four ventral). The data from each sample and the data from multiple mice of each genotype to be compared were pooled and used to calculate the mean number of stained nuclei and the SE (SigmaStat package). To quantify the total amount of nuclear EM48 stain we calculated the staining index (SI), which is the product of the MN and MNI. To quantify inclusion formation, the number of stained nuclei with an inclusion was counted in 10 fields throughout the striatum, viewed directly at 40x magnification. Data from multiple fields and from mice of each genotype were pooled and used to calculate the mean number of nuclei with an inclusion (MI) and SE (SigmaStat package). The MNI, MN and MI were compared for each genotype, or combination of genotypes, using a t-test (SigmaStat package).
Toluidine blue staining was carried out on 1.5 µm semi-thin sections on 10 HdhQ111 striata at 24 months of age, and on eight wild-type age-matched controls, as described (21). Dysmorphic toluidine-reactive neurons were quantified, using stained sections from two wild-type and six HdhQ111 striata. Ten frames (290 x 210 µm2) were picked at random from each animal, representing five frames in two sections at least 7.5 µm apart. Non-reactive ‘healthy’ neurons, with visible nucleoli, and darkly toluidine blue reactive neurons were counted. The proportion of toluidine-reactive neurons to non-stained neurons in wild-type striatum was 5/665 (0.8%) and, in four of six HdhQ111 striata, ranged from 3/342 (0.9%) to 10/283 (3.5%). Staining with anti-GFAP (DAKO Corporation, Carpinteria, CA) was on 50 µm free-floating coronal sections of brains perfused with 3% paraformaldehyde, 0.15% gluteraldehyde, 0.1 M phosphate buffer (pH 7.2) as described (28). TUNEL staining was performed on 50 µm sections using the procedure of Namura et al. (38) to detect incorporated biotinylated dUTP with the avidin biotin detection system (Vectastain ABC Kit, Vector Laboratories).
Filter-trap immunoblot assay to detect N-terminal aggregate was performed on SDS-insoluble striatal slice extracts, using equal loading of protein, as described (8).
Estimated timing for data drawn from the literature
Data depicted in Figure 2 were drawn from previous reports. The data for HdhQ92 and HdhQ111 mice are from the current work and from Wheeler et al. (8,22). Neuropil aggregates for Hdh4/Q80 mice are estimated as halfway between 11 months, when undetectable, and 27 months, when clearly detectable (10). Gait deficits were reported in some HdhCAG150 mice between 10 and 16 months of age (11), and are represented here at 13 months. Kennedy and Shelbourne (23) demonstrated very low levels of DNA instability at 9 months, increasing by 15 months, and onset is estimated here at 12 months.
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ACKNOWLEDGEMENTS |
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The authors thank Dr X.J.Li for EM48 antibody, and Ms C.Chakrabarti for technical assistance. This work was supported by NINDS grants NS16367 (HD Center Without Walls) (M.E.M., J.F.G., J.-P.V.), NS32765 (M.E.M.), NS35255 (C.-A.G., S.H.) and NS38144 and NS34172 (D.R.B.), an Anonymous Donor, the Hereditary Disease Foundation and the Huntington’s Disease Society of America (Coalition for the Cure). V.C.W. received a postdoctoral fellowship from the Hereditary Disease Foundation.
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +1 617 726 5089; Fax: +1 617 726 5735; Email: macdonam@helix.mgh.harvard.edu Present address:Steven Hersch, Department of Neurology, Massachusetts General Hospital, Boston MA 02114, USA
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