Human Molecular Genetics, 2003, Vol. 12, No. 13 1621-1629
DOI: 10.1093/hmg/ddg163
© 2003 Oxford University Press
Nucleocytoplasmic transport signals affect the age at onset of abnormalities in knock-in mice expressing polyglutamine within an ectopic protein context
1Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL 35294, USA, 2Department of Neurology, University of Michigan, Ann Arbor, MI 48109-0585, USA, 3Geriatrics Research, Education, and Clinical Center, Ann Arbor VAMC, Ann Arbor, MI 48109, USA and 4Department of Neurobiology, University of Alabama at Birmingham, Birmingham, AL 35294, USA
Received March 24, 2003; Accepted April 28, 2003
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
In order to better understand the role of nuclear localization of polyglutamine in the human CAG repeat disorders, gene targeting was used to add either nuclear localization (NLS) or nuclear export (NES) signals to versions of the mouse Hprt protein containing expanded polyglutamine (HprtQ150). The NLS increased levels of nuclear HprtQ150 protein in the mouse brain and hastened both the presentation of neuronal intranuclear inclusions (NIIs) and the onset of behavioral abnormalities. The NES reduced levels of nuclear HprtQ150 protein in mouse brain and delayed both the presentation of NIIs and the onset of behavioral abnormalities. Together these results indicate the nucleus is the primary site of toxicity in HprtQ150 mice. Furthermore, the signals did not alter the relative regional distribution of NIIs, suggesting that factors other than nuclear access dictate the regional specificity of NII formation in this mouse model.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Inheritance of expanded CAG/polyglutamine repeats causes at least nine distinct neurological diseases. These include Huntington's disease (HD), spinobulbar muscular atrophy (SBMA), dentatorubral pallidoluysian atrophy (DRPLA) and several spinocerebellar ataxias (SCAs) (1). These diseases share many characteristics including an inverse correlation between repeat length and age of onset (2) as well as the presence of nuclear or cytoplasmic protein aggregates containing polyglutamine flanked by portions of the disease protein (3,4). These similarities suggest a common molecular mechanism as do experiments in mice where long polyglutamines are expressed with little or no additional protein (5,6). Polyglutamine is also toxic when expressed within a context not related to the disease proteins. Mice expressing a polyglutamine stretch coded by 150 CAGs in the X-linked Hprt gene show late-onset progressive neurological abnormalities and develop neuronal intranuclear inclusions (NIIs) containing both Hprt protein and ubiquitin (7). This phenotype depended on repeat length and was not found in Hprt knock-out mice used as controls. Thus Hprt protein, which normally serves as an enzyme to catalyze a chemical reaction involved in purine metabolism, served as a carrier of toxic polyglutamine. Since wild-type Hprt protein is cytoplasmic and the version with a long glutamine repeat was found in the nucleus as NIIs approximately coincident with neurological abnormalities, it was hypothesized that nuclear localization of a portion of the HprtQ150 protein was an important step in the neuronal dysfunction of this mouse line (7). In humans, some of the polyglutamine disease proteins are nuclear, some cytoplasmic and others are found in both cellular compartments (8). For those restricted to the cytoplasm, however, aggregates containing a portion of the disease protein have been found in the nucleus. Several mouse and tissue culture studies show a positive correlation between accumulation of nuclear polyglutamine disease protein and toxicity (reviewed in 9). Other studies, however, implicate sites outside the nucleus including the cytoplasm (10), mitochondria (11) and synaptic vesicles (12). These contrasting conclusions may be due in part to differences in protein context, which may alter the primary subcellular site of toxicity.
To address the importance of nuclear accumulation of polyglutamine when expressed outside the normal context of the disease proteins, we created variants of the HprtQ150 protein containing nucleocytoplasmic transport signals. Our results show that the level of nuclear HprtQ150 protein correlates with the onset of behavioral and neuroanatomic abnormalities while extranuclear levels do not. Together these data support the view that the nucleus is the primary site of toxicity in HprtQ150 mice.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Choice of experimental system
Our previous work had shown that the addition of a stretch of
150 glutamines into the endogenous mouse Hprt protein caused late-onset progressive neurological abnormalities and a distinct regional pattern of NII formation in the mouse brain (7). To determine the role of nuclear localization in this toxicity, we created and compared six gene targeted variants of Hprt. The use of gene targeting facilitated the comparison of mice by controlling for genomic context, thus providing the highest likelihood of identical levels of Hprt expression for the variants. The use of Hprt also allowed the study of polyglutamine in a context not related to the disease proteins. Disease-specific protein motifs might directly affect subcellular localization or alter the toxic effects of such localization. Such disease-specific regions might be nuclear localization (NLS) or nuclear export signal (NES) sites, binding sites for proteins with such signals, binding sites for proteins restricted to one subcellular compartment, or binding sites for proteins that are toxic when delivered to specific subcellular sites. Hprt protein contains no homology to known NLSs and has no known binding partners other than itself. Although Hprt protein is small enough (26 kDa) that it might be able to freely diffuse through a nuclear pore (13,14), it is not normally found in the nucleus. Exclusion from the nucleus might be due to its larger tetrameric structure (15), or the presence of a functional NES.
Construction of variants
Constructs to gene target exon 3 of the endogenous mouse Hprt locus were used to make variants coding for full-length Hprt proteins with additions after amino acid 51. Two variants contained the insertion of a myc tag, an NLS and a repeat of either 150 or nine glutamines (alleles designated hprtNLSQ150 and hprtNLSQ9, respectively). Two variants contained the insertion of a myc tag, an NES and a repeat of either 150 or 11 glutamines (alleles designated hprtNESQ150 and hprtNESQ11, respectively). Two variants were also made with the insertion of a myc tag and a repeat of either 150 or 38 glutamines. These alleles were designated hprtNosignalQ150 and hprtNosignalQ38 where ‘Nosignal’ represents the absence of an added nucleocytoplasmic transport signal. Gene targeting using these constructs eliminated endogenous Hprt activity in ES cells allowing selection with 6-thioguanine. Resistant colonies were screened by PCR across the modified area and correct gene targeting was confirmed by Southern analysis (Fig. 1). Modification of the single X-linked Hprt locus of male ES cells resulted in loss of a band representing wild-type and gain of a band representing the modified allele. Southern blots were repeated with at least three different restriction enzymes to confirm proper targeting in each clone. Targeting was further confirmed by western analyses of ES cells showing the addition of a myc tag to each of the full length Hprt variant proteins (data not shown). Mouse lines were made by injection of ES cells into blastocysts as described previously (7). The analyses of mouse lines described below revealed no differences among the three short repeat control lines which were indistinguishable from wild-type mice for all behavioral and neuroanatomic assays performed. Thus, this work focuses on comparisons of mice expressing long polyglutamine versions of Hprt protein. We refer to these collectively as HprtQ150 variants.
|
Analysis of HprtQ150 protein from mouse brain
The efficacy of the added nucleocytoplasmic signals was determined by analysis of nuclear and extranuclear protein isolated from the brains of HprtQ150 variants at 6 and 8 months of age. Western analysis comparing nuclear protein revealed NLS HprtQ150 protein in greater abundance than the other two variants. A representative western blot is shown in Figure 2. A greater amount of nuclear HprtQ150 protein was found in all 16 comparisons between NLS and NES variants and all 15 comparisons between NLS and Nosignal variants (n=5–7 mice of each genotype). Thus, the NLS caused a noticeable and reproducible increase in the amount of nuclear HprtQ150 protein in mouse brain. Similar analysis for the NES HprtQ150 protein revealed its presence was usually lower in the nucleus compared to the other variants. The abundance of nuclear NES HprtQ150 protein was always less than nuclear NLS HprtQ150 protein (16 comparisons, n=5–7 mice of each genotype). In 11 comparisons of nuclear fractions six revealed less NES HprtQ150 protein than Nosignal protein, four showed no difference and one showed slightly more NES HprtQ150 protein than the Nosignal version (n=5–7 mice of each genotype). Taken together, these comparisons show the NES lessens the probability that a mouse will accumulate nuclear HprtQ150 protein by 6–8 months of age. The signal intensities of extranuclear NES and extranuclear Nosignal HprtQ150 protein did not differ noticeably by western blot analysis (Fig. 2). In contrast, the NLS version had noticeably reduced levels of extranuclear HprtQ150 protein when compared to the other variants (Fig. 2). This reduction was found in all 21 comparisons made between at least five mice of each genotype.
|
Analysis of NII distribution of HprtQ150 variants at different ages
The addition of 150 glutamines to the endogenous mouse Hprt protein caused a distinct regional pattern of ubiquitin immunoreactive NIIs (7). In this study, as in the previous study, we found NIIs but no evidence of neurodegeneration or neurite aggregates. Furthermore, both studies revealed only one NII was present per nucleus and the size of the inclusions increased with age. Figure 3 shows ubiquitin immunostaining of inclusions from 12-month-old variants. Mice expressing NLS HprtQ150 protein had larger inclusions than the other age-matched variants, perhaps as a result of NIIs being formed earlier in the NLS HprtQ150 mice (described below).
|
An experienced neuroanatomist (ST-G) blinded to genotype subjectively graded NII load in six brain regions on a scale of 0–3 (see Table 1). Thirty-three mice were analysed including three to five mice from each of the nine groups representing the three HprtQ150 variants at three different ages. Six-month-old mice showed few, and in most cases no, NIIs. At 8–9 months of age NLS HprtQ150 mice showed more ubiquitin positive inclusions than the other two variants. This trend continued for mice 10–12 months of age. By 8–9 months of age four of four Nosignal HprtQ150 mice had inclusions compared with only one of three NES HprtQ150 mice. These data show that the proportion of neurons with ubiquitinated NIIs was greater with increasing age and correlated with the relative amount of nuclear protein for each of the variants (NLS>Nosignal >NES). The same general trends were revealed using the anti c-myc antibody, but fewer inclusions were revealed when staining the same brains (data not shown). The lesser efficiency of the anti c-myc antibody in revealing NIIs might be due to differences in antibody affinity, the loss of c-myc epitope by proteolysis, or an alteration of the conformation or accessibility of the c-myc epitope after aggregation. The one exceptional NES HprtQ150 mouse (ID no. 9, Table 1) with numerous inclusions at 8 months of age suggests either environmental factors or genetic modifiers unlinked to Hprt influence NII load.
|
A striking aspect of the data shown in Table 1 is that each mouse had a similar regional distribution of NIIs. For example, within each mouse the parabrachial and parabigeminal nuclei had higher NII loads than the striatum and globus pallidus (see Table 1). The relative regional distribution of NIIs did not change with the addition of NLS or NES sequences. Since these signals alter the concentration of nuclear HprtQ150 protein and the age NIIs are observed, factors other than nuclear access seem to dictate the relative NII load of these different regions.
Effects of signals on behavioral abnormalities of HprtQ150 mice
Over 100 mice were assessed for abnormalities previously reported for the HprtQ150 line (7). These mice included between 15 and 17 mice for each of seven groups—three groups for the HprtQ150 variants, three groups for short repeat variants, and one group for wild-type mice. Each mouse was assessed once every 2 weeks starting at 7 weeks of age. Trials continued for one year or until death. The bi-weekly examination included a test for activity upon opening a cage, a tail suspension test, the accelerating rotarod and a determination of weight. Seizures occurring during the trials were noted.
As in previous studies, more HprtQ150 mice than short repeat controls remained inactive after removing the cage top. Short repeat controls did not differ from wild-type for any of the assessments made in this work and the results from these four groups are considered together as controls. Figure 4A shows that the median age of onset for inactivity in the NLS HprtQ150 variants was one month earlier than for the NES and Nosignal HprtQ150 variants (P<0.05, Kruskal–Wallis non-parametric ANOVA with Dunn's post test, K–W w/D). The NES had no discernable effect on the onset or frequency of the abnormal inactivity.
|
Figure 4B shows wild-type mice and short repeat variants were consistently able to escape a tail suspension by climbing onto the observer's hand. HprtQ150 variants lost that ability at different ages. NLS HprtQ150 mice exhibited the earliest loss as shown by the comparison of the three HprtQ150 variants between 4 and 6 months of age (P<0.05, K–W w/D). Later the NLS and Nosignal HprtQ150 variants completely lost the ability to escape while NES HprtQ150 mice showed resistance to the loss. Between 8 and 10 months of age NES HprtQ150 mice performed better on this test than both of the other HprtQ150 variants (P<0.05, K–W w/D). This test also revealed the NES caused a delay in onset of clasping behavior (Fig. 4C; P<0.05, Fisher Exact test, FE).
Seizures that occurred during the bi-weekly assays are displayed in Figure 4D. No seizures were observed during these trials for short repeat controls, but one wild type mouse had a single seizure. For the five trials of HprtQ150 variants 7–9 months of age 11 of 17 NLS, 12 of 16 Nosignal and four of 15 NES mice had seizures. Although the NLS had no effect, the NES made it less likely that a 7- to 9-month-old HprtQ150 variant would have a seizure (P<0.05, FE).
At 6 months of age the undisturbed nocturnal activity of the mice was assessed by an infrared beam activity monitor (described previously in 7). Two methods were used to assess the activity. The first, shown in Figure 4E, was to measure the proportion of 2 min periods where a mouse was actively roaming the cage. A mouse breaking three or more of the six beams crossing the bottom of the cage was considered roaming, since the length of the mouse body is long enough to break only two beams at one time. The short repeat and wild-type control animals were actively roaming during 50% of the 2 min periods during two 12 h dark cycles. The NLS and Nosignal HprtQ150 variants were less active, roaming only 25% of the periods. The NES HprtQ150 variants were roaming during 37% of the periods. These results are consistent with the NLS having no effect on this assay or having an effect earlier than 6 months of age. The NES reduces or delays the reduction in activity caused by the HprtQ150 allele (P<0.05, K–W w/D). A similar effect was noted for 6-month-old mice when nocturnal vertical activity was measured by total upper beam breaks (Fig. 4F, P<0.05, K–W w/D).
We previously reported that HprtQ150 mice have an earlier age of death than short repeat controls (7). Short repeat controls and wild-type mice survived beyond 24 months, an age by which all HprtQ150 variants had died. The median ages at death for the NLS, Nosignal and NES HprtQ150 variants were 10
, 11 and 12 months respectively (Fig. 5). A significant difference was found between NLS HprtQ150 mice and NES HprtQ150 mice (P<0.05, K–W w/D). This result suggests that the NLS hastened death, the NES delayed death or both. These three possible explanations are consistent with the hypothesis that nuclear localization of HprtQ150 protein is an important determinant of early death in these mice.
|
In a previous study we observed impairment in performance on the rotarod for the HprtQ150 line JO1 (7). Although the rotarod results collected for this study show the same decrease in performance when comparing long repeat mice to short repeat controls, we did not obtain statistically significant differences attributable to the presence of nucleocytoplasmic transport signals (data not shown). Furthermore, the late-onset weight gain observed in the JO1 line was not seen in the HprtQ150 variants studied here. This difference might be caused by the addition of a myc tag to the Hprt variants used in this work or an unlinked genetic modifier that was fixed in the JO1 colony and lost in the development of the colonies for this work.
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
To evaluate the role of nuclear localization in polyglutamine pathology we generated knock-in mouse lines expressing a pathological polyglutamine protein with signals that control its abundance in the nucleus. The effects of these signals are consistent with the modulation of a toxicity rather than the creation of a novel one, since their addition to the HprtQ150 protein did not result in mice with grossly different pathologies. For most of the abnormalities found in these mice either the NES delayed or the NLS hastened its onset when compared with the Nosignal HprtQ150 variant. These results support the conclusion that nuclear localization is a critical determinant of the onset of these abnormalities.
Is the nucleus the primary site of toxicity in HprtQ150 mice, or is the nuclear accumulation secondary to an insult that occurs elsewhere in the cell? Damage outside the nucleus may affect nuclear integrity and homeostasis. For example, interruption of mitochondrial function can reduce cellular energy levels to the point where nuclear functions are compromised, perhaps including the exclusion of HprtQ150 protein. Any such scenario, however, would be difficult to reconcile with our observation that the NLS accelerates toxicity while reducing HprtQ150 protein levels outside the nucleus. The nucleus seems to be the primary site of toxicity in the HprtQ150 variants.
The proportion of the total Nosignal HprtQ150 protein found in the nucleus is small when compared with that of the extranuclear compartment (compare lanes 4–8 in Fig. 2), yet altering these small amounts correlates with changes in the onset of symptoms. Our observations suggest that vulnerability of a compartment has more influence on toxicity than overall cellular concentration. Furthermore, these results highlight the possibility that small amounts of a polyglutamine containing protein in a compartment might cause problems. Thus, arguments based on the absence of a disease protein from the nucleus or other compartment should be considered in light of the sensitivity of detection provided in the experiment (16).
The results presented here complement other studies where nuclear levels of polyglutamine disease proteins were altered and corresponding changes were noted in the pathology (1,9). For example, the removal of a functional NLS from an expanded polyglutamine version of the ataxin-1 protein eliminated toxicity in transgenic mice (17). A transgenic mouse model for SBMA displayed abnormalities dependent upon nuclear localization of an expanded polyglutamine version of the androgen receptor (18). Furthermore, transgenic mice with nucleocytoplasmic transport signal sequences added to an HD exon 1 transgene with expanded CAG repeats showed alteration of the well-studied R6/2 phenotype in a manner consistent with a nuclear site of toxicity (C. Benn and G. Bates, personal communication). The results from these studies show that polyglutamine in other protein contexts also create toxicities enhanced by nuclear localization. The many limitations of mouse and tissue culture studies leave open the possibility that the molecular mechanism of toxicity in human polyglutamine disease may have extranuclear components dependent on disease-specific protein context.
The question of whether NIIs are required for HprtQ150 mice to have a neuronal toxicity was first presented by Ordway et al. (7) in the larger context of whether the aggregates seen in the human diseases protect against, are coincidental to, or necessary for toxicity (7,19). This theme has been expanded upon and addressed by overexpression of polyglutamine expanded disease transgenes in tissue culture and mice (17,20–23). The nucleocytoplasmic transport signals added to the HprtQ150 protein in this study altered the timing of presentation of NIIs, yet the positive correlation between the presence of NIIs and the age at onset of behavioral abnormalities remained.
In each of the human polyglutamine repeat diseases there are regionally specific neuropathological features, including differences in regional and cellular distribution of aggregates (1,2,9). A commonly proposed explanation for the regional specificity of NIIs involves cell specific differences in nuclear accessibility (8). In the HprtQ150 mice there are regional differences in the proportion of neurons containing NIIs (7). If nuclear accessibility of the HprtQ150 protein were a major determinant of regional NII load in this mouse model, the NLS and NES signals would be expected to alter the relative distribution of NIIs in the brain. The regional specificity remained unaltered by these signals, suggesting that factors other than nuclear access dictate cell specificity of NII formation in HprtQ150 mice. Such factors might be cell-specific promoters of aggregation such as chaperones (24–26) or a difference in each cell's ability to destroy a developing aggregate (21).
The role of nuclear function in the polyglutamine diseases has been implicated by alterations in transcript levels and the process of transcription (27). The presence of NIIs and abnormalities in nuclear morphology have also been described (3,4). Whether these are primary effects or secondary responses to cellular damage have yet to be determined. Our results support the view that the nucleus is an important site of toxicity in the polyglutamine diseases and support the development of therapies designed to prevent or reduce nuclear access.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Generation of Hprt variants
The sequence 5'TCGACCATAC GGGCCCAAGA AGAAGAGGA AGGTGGAATT CCTGAGGGAG CAAAAGTTGA TCAGCGAGGA AGATCTTGCT GTTGGAGCTC AC-(CAG)nGTGAG3' was inserted into the XhoI site of exon3 of an 11.5 kb murine Hprt gene targeting construct. This codes for an in-frame insertion of PYGPKKKRKVEFLREQKLISEEDLAVGAH(Q)nVSR, which includes the NLS of SV40 large T antigen, a c-myc tag and either nine or 145 glutamines between residue 51 and 52 of Hprt protein. At the same location we inserted the sequence 5'TCGACCACTT CGGCTCTGAA GCTGGCTGGC CTCGATATCT CGACCATACG GGCCCAAGAA GAAGAGGAAG GTGGAATTCC TGAGGGAGCA AAAGTTGATC AGCGAGGAAG ATCTTGCTGT TGGAGCTCAC (CAG)nGTGAG3'. This encodes PLALKLAGLDIEFLREQKLISEEDLAVGAH(Q)nVSR, which includes the NES of the Protein Kinase A inhibitory subunit, a c-myc epitope, and either 11 or 146 glutamines. Also at the same XhoI site we inserted 5'TCGACCAGAA TTCCTGAGGG AGCAAAAGTT GATCAGCGAG GAAGATCTTG CTGTTGGAGC TCAC(CAG)nGTG AG3' encoding PEFLREQKLISEEDLAVGAH(Q)nVSR which contains a c-myc epitope and either a 38 or 146 glutamine repeat. The insertions and their junctions with exon 3 were fully sequenced.
Gene targeting and selection for the loss of function of the mouse Hprt gene in R1 ES cells was carried out as previously described (7). 6-Thioguanine resistant colonies were screened for the modifications by PCR (described in 7) to verify the presence of the modification with a correctly sized CAG repeat. Southern analysis using BanII, BglII and PvuII digested DNA and the probe shown in Figure 1 was used to verify correct gene targeting. In addition to targeted fragments indicating the correct size, the loss of the wild-type length fragment indicates replacement-type recombination took place at the Hprt locus rather than insertion-type recombination. Western blots of proteins from targeted ES cells probed with the anti-myc tag antibody 9E10 or the anti-polyglutamine antibody 1C2 were used to confirm that the full-length Hprt protein contained the desired modification in targeted ES cells (data not shown). Properly targeted ES cells were injected into 3.5-day-old blastocysts and transferred to uteri of foster mothers to produce chimeras. These were bred to C57BL/6J mice and offspring carrying the hprt variants were bred to create the hemizygous and homozygous hprt variants used in this study.
Protein analyses
ES cells were grown on 100 mm plates to 70–90% confluency, rinsed twice then harvested by scraping in cold phosphate buffered saline (PBS). To verify proper gene targeting at the protein level cells were suspended in lysis buffer and centrifuged at 1300g for 5 min, the pellet was discarded, and 40–50 µg of protein of each sample was separated on a 12.5% SDS–polyacrylamide gel. The gel was electrotransferred to nitrocellulose membrane, blocked for 30 min with 5% powdered milk in tris buffered saline plus 0.05% tween20 (TBST), then treated overnight at 4°C with an anti-myc tag antibody (9E10 ascites) at 6 µg/ml or anti-polyglutamine antibody (1C2, Chemicon) at a 1 : 20 000 dilution in blocking buffer. The primary antibody was then detected by a horseradish peroxidase conjugated secondary antibody (Jackson Immuno Research Laboratories, West Grove, PA, USA) followed by enzymatic chemiluminesence (SuperSignal West Pico, Pierce) and autoradiography.
Brain protein was fractionated to a post-nuclear supernatant (PNS) fraction and a nuclear fraction by the modification of a previously described procedure (28). This procedure involves a hypotonic disruption of the cell membrane, and nuclear isolation by repeated centrifugation/wash steps including centrifugations through a sucrose cushion. Mice were sacrificed by cervical dislocation and the brain was quickly removed and rinsed in PBS to remove excess debris. The hemispheres were separated with a clean scalpel. One hemisphere was placed in 10% formalin to fix for immunohistochemical analyses. The other hemisphere was homogenized in a 5 ml dounce homogenizer with 4.5 ml of lysis buffer (10 mM Tris pH 7.5, 10 mM NaCl, 3 mM MgCl2 and 0.05% NP40) with protease inhibitors [Aprotinin (2 µg/ml), Leupeptins (2 µg/ml), Pepstatin A (1 µg/ml), phenylmethylsulfonyl fluoride (PMSF, 0.1 µg/ml)]. The homogenate was aliquoted into one tube of 0.5 ml (frozen as whole brain lysate) and four tubes of 1 ml each for fractionation as described previously (28). Nuclear protein preparations contained a small amount of precipitate, which was separated from soluble protein by centrifugation at 8000 rpm. Precipitate was resuspended with 25 µl of lysis buffer, quantitated, and solubilized with SDS sample buffer. To provide a complete representation of the protein in the nucleus, this solubilized precipitate was analysed on western blots alongside an equal amount of the nuclear supernatant fraction. Signal representing the HprtQ150 variants was never found in the supernatant of the nuclear fraction (data not shown). Each sample was western blotted as above with the 1C2 antibody (1 : 10 000 dilution, Chemicon). These blots were stripped and probed with 
-tubulin (B-5-1-2, sigma-aldrich) and histone (MAB052, Chemicon) specific antibodies to determine the purity of fractions and as a loading control.
Behavioral experiments
Animals were housed in cages of two or three animals in a room with a 12 h light cycle. Tests began at age 7±1 week. Tests were performed every 2 weeks as follows: mice in their home cage were placed in a laminar flow hood and the cage lid and feed rack were immediately removed. Mice rearing on their hind legs or moving all four feet to a new location within 15 sec after removal of lid were scored as ‘active’. Next each mouse was suspended for 1 min by holding their tails 0.5 cm from the base. Mice that brought either both front or both rear paws towards the abdomen and held them for at least 3 s were scored as ‘clasping’. Mice that climbed onto the observer's hand were scored as ‘escaping’. Mice were then rested for at least one minute and then placed on a rotarod treadmill (Ugo Basilie, Varese, Italy), a 3 cm diameter drum that increased its rotation from 4 to 40 rpm over ten 30 s intervals. Time spent on the rotarod was recorded up to a maximum of 300 s. The original HprtQ150 line JO1 (7) had seizures directly observable as tonic–clonic type convulsive spells and verified by electroencephalogram. During the behavioral trials described here some mice had the same type of convulsive spell and were noted as having seizures. Mice were also monitored at 6 months of age for their undisturbed activity. Up to 20 cages with one mouse each were placed into a monitor that transmitted infrared beams through standard cage bottoms (29 cm lengthx19 cm widthx13 cm height). Each beam break and the 2 min period in which it was broken was recorded and stored on computer. Six lower beams can be broken by a mouse walking along the cage bottom (2.5 cm above the cage floor spaced 4.5 cm apart) and two upper beams require rearing or climbing to break (7 cm above the cage floor, 1.8 cm from the cage wall). Because inactive mice can break a lower beam repeatedly by repeated movement of one body part (e.g. during scratching or grooming) we measured the number of 2 min periods in which three or more lower beams were broken, a measure termed ‘roaming’. Mice were placed in the apparatus for a 24 h adaptation period followed by a 48 h data collection period through at least two 12 h dark cycles.
Neuroanatomy
Mice were sacrificed by cervical dislocation, the brain was quickly excavated and rinsed in PBS to remove excess debris. Brains were halved with one hemisphere being used to prepare protein (described above) and the other fixed in 10% formalin. After 1 week of fixation, brains were cryoprotected in 20% sucrose. Each brain was coded to obscure the genotype and age of the mouse. Sections of 30–40 µM were cut in the horizontal plane and stained with either a rabbit polyclonal anti-myc tag antibody (1 : 200, Cell Signaling) or an anti-ubiquitin antibody (1 : 250, Dako). Diaminobenzidine staining was performed using an IgG Elite ABC kit (Vector Laboratories). Sections containing the region of interest were scanned for NIIs and graded on a scale of 0 to 3 (see Table 1 for regions and scale).
|
ACKNOWLEDGEMENTS |
|---|
This work is dedicated to the memory of Dr M.F. Perutz. We would like to thank Mr Andrew Crouse and Drs David Bedwell, Gail Johnson, Dick Jope, Mathieu Lesort, and especially Dr Kumar Pandya for experimental and intellectual advice, and Dr Tim Townes for the use of his microinjection equipment. This work was supported by grants from the Hereditary Disease Foundation Cure HD initiative and the NIH/NINDS grants R01 NS34492 (P.J.D.) and R01 NS38166 (R.L.A.).
|
FOOTNOTES |
|---|
* To whom correspondence should be addressed. Email: pdetloff{at}bmg.bhs.uab.edu
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
- Zoghbi, H.Y. and Orr, H.T. (2000) Glutamine repeats and neurodegeneration. A. Rev. Neurosci., 23, 217–247.[CrossRef][Web of Science][Medline]
-
Gusella, J.F., McNeil, S., Persichetti, F., Srinidhi, J., Novelletto, A., Bird, E., Faber, P., Vonsattel, J.P., Myers, R.H. and MacDonald, M.E. (1996) Huntington's disease. Cold Spring Harb. Symp. Quant. Biol., 61, 615–626.
[Abstract/Free Full Text] - Skinner, P.J., Koshy, B.T., Cummings, C.J., Klement, I.A., Helin, K., Servadio, A., Zoghbi, H.Y. and Orr, H.T. (1997) Ataxin-1 with an expanded glutamine tract alters nuclear matrix-associated structures. Nature, 389, 971–974.[CrossRef][Medline]
- Davies, S.W., Turmaine, M., Cozens, B.A., DiFiglia, M., Sharp, A.H., Ross, C.A., Scherzinger, E., Wanker, E.E., Mangiarini, L. and Bates, G.P. (1997) Formation of neuronal intranuclear inclusions underlies the neurological dysfunction in mice transgenic for the HD mutation. Cell, 90, 537–548.[CrossRef][Web of Science][Medline]
- Ikeda, H., Yamaguchi, M., Sugai, S., Aze, Y., Narumiya, S. and Kakizuka, A. (1996) Expanded polyglutamine in the Machado–Joseph disease protein induces cell death in vitro and in vivo. Nat. Genet., 13, 196–202.[CrossRef][Web of Science][Medline]
- Mangiarini, L., Sathasivam, K., Seller, M., Cozens, B., Harper, A., Hetherington, C., Lawton, M., Trottier, Y., Lehrach, H., Davies, S.W. et al. (1996) Exon 1 of the HD gene with an expanded CAG repeat is sufficient to cause a progressive neurological phenotype in transgenic mice. Cell, 87, 493–506.[CrossRef][Web of Science][Medline]
- Ordway, J.M., Tallaksen-Greene, S., Gutekunst, C.A., Bernstein, E.M., Cearley, J.A., Wiener, H.W., Dure, L.S.III., Lindsey, R., Hersch, S.M., Jope, R.S. et al. (1997) Ectopically expressed CAG repeats cause intranuclear inclusions and a progressive late onset neurological phenotype in the mouse. Cell, 91, 753–763.[CrossRef][Web of Science][Medline]
- Ross, C.A. (1997) Intranuclear neuronal inclusions: a common pathogenic mechanism for glutamine-repeat neurodegenerative diseases? Neuron, 19, 1147–1150.[CrossRef][Web of Science][Medline]
- Ross, C.A. (2002) Polyglutamine pathogenesis: emergence of unifying mechanisms for Huntington's disease and related disorders. Neuron, 35, 819–822.[CrossRef][Web of Science][Medline]
-
Hackam, A.S., Singaraja, R., Zhang, T., Gan, L. and Hayden, M.R. (1999) In vitro evidence for both the nucleus and cytoplasm as subcellular sites of pathogenesis in Huntington's disease. Hum. Mol. Genet., 8, 25–33.
[Abstract/Free Full Text] - Panov, A.V., Gutekunst, C.A., Leavitt, B.R., Hayden, M.R., Burke, J.R., Strittmatter, W.J. and Greenamyre, J.T. (2002) Early mitochondrial calcium defects in Huntington's disease are a direct effect of polyglutamines. Nat. Neurosci., 5, 731–736.[Web of Science][Medline]
- Li, H., Li, S.H., Johnston, H., Shelbourne, P.F. and Li, X.J. (2000) Amino-terminal fragments of mutant huntingtin show selective accumulation in striatal neurons and synaptic toxicity. Nat. Genet., 25, 385–389.[CrossRef][Web of Science][Medline]
- Gorlich, D. and Kutay, U. (1999) Transport between the cell nucleus and the cytoplasm. A. Rev. Cell Dev. Biol., 15, 607–660.[CrossRef][Web of Science][Medline]
- Gorlich, D. and Mattaj, I.W. (1996) Nucleocytoplasmic transport. Science, 271, 1513–1518.[Abstract]
- Caskey, C.T. and Kruh, G.D. (1979) The HPRT locus. Cell, 16, 1–9.[CrossRef][Web of Science][Medline]
- Huynh, D.P., Figueroa, K., Hoang, N. and Pulst, S.M. (2000) Nuclear localization or inclusion body formation of ataxin-2 are not necessary for SCA2 pathogenesis in mouse or human. Nat. Genet., 26, 44–50.[CrossRef][Web of Science][Medline]
- Klement, I.A., Skinner, P.J., Kaytor, M.D., Yi, H., Hersch, S.M., Clark, H.B., Zoghbi, H.Y. and Orr, H.T. (1998) Ataxin-1 nuclear localization and aggregation: role in polyglutamine-induced disease in SCA1 transgenic mice. Cell, 95, 41–53.[CrossRef][Web of Science][Medline]
- Katsuno, M., Adachi, H., Kume, A., Li, M., Nakagomi, Y., Niwa, H., Sang, C., Kobayashi, Y., Doyu, M. and Sobue, G. (2002) Testosterone reduction prevents phenotypic expression in a transgenic mouse model of spinal and bulbar muscular atrophy. Neuron, 35, 843–854.[CrossRef][Web of Science][Medline]
- Sisodia, S.S. (1998) Nuclear inclusions in glutamine repeat disorders: are they pernicious, coincidental, or beneficial? Cell, 95, 1–4.[CrossRef][Web of Science][Medline]
- Saudou, F., Finkbeiner, S., Devys, D. and Greenberg, M.E. (1998) Huntingtin acts in the nucleus to induce apoptosis but death does not correlate with the formation of intranuclear inclusions. Cell, 95, 55–66.[CrossRef][Web of Science][Medline]
- Yamamoto, A., Lucas, J.J. and Hen, R. (2000) Reversal of neuropathology and motor dysfunction in a conditional model of Huntington's disease. Cell, 101, 57–66.[CrossRef][Web of Science][Medline]
-
Yu, Z.X., Li, S.H., Nguyen, H.P. and Li, X.J. (2002) Huntingtin inclusions do not deplete polyglutamine-containing transcription factors in HD mice. Hum. Mol. Genet., 11, 905–914.
[Abstract/Free Full Text] -
Cooper, J.K., Schilling, G., Peters, M.F., Herring, W.J., Sharp, A.H., Kaminsky, Z., Masone, J., Khan, F.A., Delanoy, M., Borchelt, D.R. et al. (1998) Truncated N-terminal fragments of huntingtin with expanded glutamine repeats form nuclear and cytoplasmic aggregates in cell culture. Hum. Mol. Genet., 7, 783–790.
[Abstract/Free Full Text] -
Jana, N.R., Tanaka, M., Wang, G. and Nukina, N. (2000) Polyglutamine length-dependent interaction of Hsp40 and Hsp70 family chaperones with truncated N-terminal huntingtin: their role in suppression of aggregation and cellular toxicity. Hum. Mol. Genet., 9, 2009–2018.
[Abstract/Free Full Text] -
Krobitsch, S. and Lindquist, S. (2000) Aggregation of huntingtin in yeast varies with the length of the polyglutamine expansion and the expression of chaperone proteins. Proc. Natl Acad. Sci. USA, 97, 1589–1594.
[Abstract/Free Full Text] - Cummings, C.J., Mancini, M.A., Antalffy, B., DeFranco, D.B., Orr, H.T. and Zoghbi, H.Y. (1998) Chaperone suppression of aggregation and altered subcellular proteasome localization imply protein misfolding in SCA1. Nat. Genet., 19, 148–154.[CrossRef][Web of Science][Medline]
- Cha, J.H. (2000) Transcriptional dysregulation in Huntington's disease. Trends Neurosci., 23, 387–392.[CrossRef][Web of Science][Medline]
-
Greenwood, J.A. and Johnson, G.V. (1995) Localization and in situ phosphorylation state of nuclear tau. Exp. Cell Res., 220, 332–337.[CrossRef][Web of Science][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  C.-E. Wang, H. Zhou, J. R. McGuire, V. Cerullo, B. Lee, S.-H. Li, and X.-J. Li Suppression of neuropil aggregates and neurological symptoms by an intracellular antibody implicates the cytoplasmic toxicity of mutant huntingtin J. Cell Biol., October 20, 2008; 181(5): 803 - 816. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. L. Benn, T. Sun, G. Sadri-Vakili, K. N. McFarland, D. P. DiRocco, G. J. Yohrling, T. W. Clark, B. Bouzou, and J.-H. J. Cha Huntingtin Modulates Transcription, Occupies Gene Promoters In Vivo, and Binds Directly to DNA in a Polyglutamine-Dependent Manner J. Neurosci., October 15, 2008; 28(42): 10720 - 10733. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. M. Van Raamsdonk, Z. Murphy, E. J. Slow, B. R. Leavitt, and M. R. Hayden Selective degeneration and nuclear localization of mutant huntingtin in the YAC128 mouse model of Huntington disease Hum. Mol. Genet., December 15, 2005; 14(24): 3823 - 3835. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. L. Benn, C. Landles, H. Li, A. D. Strand, B. Woodman, K. Sathasivam, S.-H. Li, S. Ghazi-Noori, E. Hockly, S. M.N.N. Faruque, et al. Contribution of nuclear and extranuclear polyQ to neurological phenotypes in mouse models of Huntington's disease Hum. Mol. Genet., October 15, 2005; 14(20): 3065 - 3078. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Schilling, A. V. Savonenko, A. Klevytska, J. L. Morton, S. M. Tucker, M. Poirier, A. Gale, N. Chan, V. Gonzales, H. H. Slunt, et al. Nuclear-targeting of mutant huntingtin fragments produces Huntington's disease-like phenotypes in transgenic mice Hum. Mol. Genet., August 1, 2004; 13(15): 1599 - 1610. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||