Intranuclear inclusions and neuritic aggregates in transgenic mice expressing a mutant N-terminal fragment of huntingtin
Intranuclear inclusions and neuritic aggregates in transgenic mice expressing a mutant N-terminal fragment of huntingtin
Gabriele Schilling1, Mark W. Becher2,+, Alan H. Sharp1, Hyder A. Jinnah3, Kui Duan1, Joyce A. Kotzuk2,+, Hilda H. Slunt2, Tamara Ratovitski2, Jillian K. Cooper1, Nancy A. Jenkins4, Neal G. Copeland4, Donald L. Price2,3,5, Christopher A. Ross1,5,6 and David R. Borchelt2,*
1Department of Psychiatry, 2Department of Pathology, Division of Neuropathology, 3Department of Neurology, 5Department of Neuroscience and 6Program in Cellular and Molecular Medicine, Johns Hopkins University, Baltimore, MD 21205-2196, USA and 4Mammalian Genetics Laboratory, NCI Cancer Center, Frederick, MD, USA
Received October 7, 1998;Revised and Accepted December 9, 1998
Huntington’s disease (HD) is an inherited, neurodegenerative disorder caused by the expansion of a glutamine repeat in the N-terminus of the huntingtin protein. To gain insight into the pathogenesis of HD, we generated transgenic mice that express a cDNA encoding an N-terminal fragment (171 amino acids) of huntingtin with 82, 44 or 18 glutamines. Mice expressing relatively low steady-state levels of N171 huntingtin with 82 glutamine repeats (N171-82Q) develop behavioral abnormalities, including loss of coordination, tremors, hypokinesis and abnormal gait, before dying prematurely. In mice exhibiting these abnormalities, diffuse nuclear labeling, intranuclear inclusions and neuritic aggregates, all immunoreactive with an antibody to the N-terminus (amino acids 1-17) of huntingtin (AP194), were found in multiple populations of neurons. None of these behavioral or pathological phenotypes were seen in mice expressing N171-18Q. These findings are consistent with the idea that N-terminal fragments of huntingtin with a repeat expansion are toxic to neurons, and that N-terminal fragments are prone to form both intranuclear inclusions and neuritic aggregates.
Huntington’s disease (HD) is an autosomal dominant inherited, progressive, neurodegenerative disorder (1-6). The onset of HD is usually in mid-life and progresses to death over 15-20 years. The disorder is characterized by motor, cognitive and psychiatric symptoms. The motor changes involve loss of coordination of voluntary movements and involuntary movements, including chorea and dystonia. Pathological abnormalities seem to be restricted to the central nervous system (CNS), with preferential vulnerability in the caudate, putamen and deep layers of the cerebral cortex. In the striatum, loss of medium spiny neurons is most prevalent.
HD is caused by the expansion of a CAG repeat coding for polyglutamine in the N-terminal region of huntingtin (7). Normal individuals possess repeat lengths of 6-35 glutamines, whereas disease is associated with the inheritance of alleles with >36 repeats (8). Longer expansions are associated with an earlier age of onset (9,10). Glutamine expansions in the coding regions of polypeptides are the cause of a number of neurodegenerative diseases including HD, spinal bulbar muscular atrophy (SBMA), dentato-rubral and pallido-luysian atrophy (DRPLA), and several forms of spinal cerebellar ataxia (SCA). These diseases share a number of common features such as repeat length instability, anticipation and widespread expression of mutant polypeptides with selective loss of neurons (11,12).
In most of the polyglutamine diseases, intranuclear inclusions have been seen in subsets of CNS neurons (13-15). Intranuclear inclusions were first detected in a transgenic model of HD that was established by expressing the first exon of huntingtin (first 63 amino acids) with 115-156 glutamine repeats (16,17). Intranuclear inclusions in HD patient post-mortem brain react with N-terminal huntingtin antibodies but not antibodies to C-terminal regions of the protein (13,18). Moreover, a 40 kDa polypeptide that contains the polyglutamine tract is enriched in nuclear fractions from homogenates of HD brain (13). Together, these data suggest that intranuclear inclusions may be comprised of N-terminal fragments of huntingtin (19).
In HD, a pathologic accumulation of huntingtin in neurites has also been reported (13). These structures were found in both cortex and striatum of HD cases (13). However, these structures were not reported in the exon 1 transgenic model of HD (16,17), transgenic mice expressing a full-length human huntingtin with 89 glutamine repeats (20) or in animals that express a mutant hypoxanthine phosphoribosyltransferase polypeptide with 146 glutamine repeats (21).
In this study, we generated transgenic mice that express an N-terminally truncated huntingtin cDNA that contains 82 glutamines and encompasses the first 171 amino acids of huntingtin (N171-82Q). The expression of the transgene was directed by a mouse prion protein promoter vector, which drives the expression of foreign genes in virtually every neuron of the CNS (22). Our analysis of these animals demonstrates that mutant N-terminal fragments of huntingtin elicit behavioral and pathological abnormalities that recapitulate features of HD, including both nuclear inclusions and neuritic aggregates. Collectively, our findings provide support for the view that proteolytic processing of mutant huntingtin plays a role the generation of some of the pathological abnormalities associated with HD.
Multiple founders were identified for each construct (N171-82Q, N171-44Q and N171-18Q; Fig. 1), and subsets with the highest number of integrated copies were selected for breeding to produce stable lines of mice.
Figure1. The construct HD-N171 with normal or expanded glutamine repeats in the mouse prion protein promoter. The N-terminal piece (171 amino acids) of human huntingtin cDNA (including 18Q, 44Q and 82Q) flanked N-terminally with a SalI linker was cloned into the XhoI site of the mouse prion protein promoter. The stop codon is provided by the vector, adding an additional 13 amino acids (see Materials and Methods).
Northern blot analysis of offspring from these lines of mice demonstrated similar levels of transgene-derived mRNA in several lines of mice, including line 8 of N171-18Q mice, line 9 of N171-44Q mice and lines 6, 77, 81 and 100 of N171-82Q mice (Fig. 2). The non-transgenic mouse brain did not yield a band at the position that is specific for the transgene mRNA, showing the specificity of the probe for human huntingtin mRNA sequences. Under the hybridization stringency conditions used, mRNA for endogenous mouse huntingtin was only faintly visible at the top of the blot. In situ hybridization (data not shown) confirmed high levels of expression throughout the brain, except for cerebellar Purkinje cells, as previously seen with the prion promoter vector used here (22).
Figure2. Northern blots of brain mRNA from transgenic F3 offspring shows high levels of transgene mRNA. An overnight exposure of 5 µg of total RNA shows very high levels of expression in lines N171-82Q (lines 6, 77, 81 and 100), line 9 of N171-44Q and line 8 of N171-18Q. Several-fold lower expression was observed in lines 42 and 55 of N171-82Q and line 35 of N171-18Q. Non-transgenic mice show no hybridization at the size of the transgene product. Endogenous mouse huntingtin mRNA runs towards the top of the blot (above 28S) and is very faint compared with the expression of the transgene.
Immunoblots from brain homogenates immunostained with the polyclonal, anti-peptide antibody AP194 (23) (the epitope is the first 17 amino acids of huntingtin, which is 100% conserved between human and mouse) revealed that the steady-state levels of both normal and mutant HD-N171 polypeptides were lower than the level of endogenous full-length huntingtin protein (Fig. 3). In all immunoblots, transgene products were only visualized by the use of chemiluminescence technology and only after extended exposure to film. The transgene product could not be detected in extracts of brain from line 42 and 55 mice that express N171-82Q. In our best estimation, the levels of N171 polypeptides were comparable in the expanded lines 6, 77, 81 and 100 (44 kDa protein), the control transgenic line 8 (28 kDa protein) and the N171-44Q line 9 mice (35 kDa protein). The similarity in mRNA levels among these lines of mice supports this conclusion (Fig. 2).
Interestingly, the immunoblots of brain homogenates detected a second smaller transgene product in extracts of brain from N171-18Q and N171-44Q mice. These smaller products migrated at a position consistent with a protein that is ~10 kDa smaller than the presumptive full-length N171 fragments (28 kDa for N171-18Q, 35 kDa for N171-44Q and 44 kDa for N171-82Q). Because the AP194 antibody used to detect these proteins recognizes the N-terminal 17 amino acids of huntingtin, these smaller fragments must be C-terminally truncated. Moreover, because the polyglutamine tract begins very close to the N-terminus (at amino acid 18), these smaller polypeptides must contain the polyglutamine tracts. In the N171-82Q mutant lines, the C-terminally truncated product was observed much less consistently with AP194 antibodies. However, an N171-82Q truncation product could be detected with antibody (1C2), which recognizes long, polyglutamine tracts (Fig. 4), confirming that the truncation products contain the polyglutamine tract and that the N171-82Q protein is subject to cleavage. These fragments were detected in homogenates prepared in the presence of a cocktail of protease inhibitors and thus are likely to be products of in vivo cleavage.
Figure3. Immunoblots of transgenic mouse brain homogenates, probed with anti-huntingtin antibody, demonstrate low levels of transgene protein and bands of lower molecular mass suggestive of cleavage. Immunostaining was done with 1 µg/ml of AP194 (a rabbit anti-peptide antibody against the first 17 amino acids of huntingtin, which is 100% conserved between mouse and human). N171 fragments containing 18Q had an apparent molecular mass of 28 kDa; 44Q, 35 kDa; and 82Q, 44 kDa. Expression levels were comparable in lines N171-82Q (lines 6, 77, 81 and 100) and were ~5- to 10-fold lower than endogenous mouse huntingtin. These lines show a progressive phenotype including weight loss and abnormal gait, ending with premature death. Transgene products were not detectable in line N171-82Q (lines 42 and 55), and neither line exhibited a phenotype up to 2 years of age. The immunoreactive protein in lines carrying N171-82Q co-migrated with polypeptide produced in HEK-293 cells that had been transfected with an N171-82Q construct. In all the transgenic lines expressing the HD-N171 constructs, there appeared to be a cleavage product that included the polyglutamine tract (in some cases only visible with longer exposure).
Figure4. Detection of the N171-82Q cleavage band with 1C2. Immunoblots of whole brain homogenates from two HD-N171-82Q (line 81) mice and one non-transgenic animal were probed with 1C2 antibody (at a dilution of 1:1000). The signal of the N171-82Q transgene product (44 kDa) can be detected in both animals. In addition, a cleavage product, ~10 kDa smaller, was readily visible in both of the brains (marked with the asterisk). The 1C2 antibody does not detect a signal at 44 kDa (or at the size of the cleavage product) in the homogenate of the non-transgenic mouse. This blot confirms that the HD-N171-82Q fragment is C-terminally truncated and that the cleavage product includes the polyglutamine stretch.
At birth and for the first 1 or 2 months of life, the animals harboring the N171-82Q transgene appeared normal. Young animals (except in line 77) bred well. The first sign of phenotypic abnormality was a failure to gain weight. The weights of male F3 offspring were measured from several different lines of mice throughout their lifespan, beginning at 4 weeks of age (Fig. 5). Initially, transgenic and non-transgenic animals had similar weights but, beginning at 2 months of age, the N171-82Q mice (lines 6, 81 and 100 of N171-82Q mice) failed to gain weight. Moreover, loss of weight was observed in the last 4-6 weeks before death. The weight of mice from the control transgenic line 8 and non-transgenics increased steadily over this period.
Figure5. Weight loss in N171-82Q mice. Weight measurements of male F3 offspring demonstrate that N171-82Q mice do not gain weight as rapidly as N171-18Q and non-transgenic animals. Each point includes the weights of 4-6 transgenic mice (averaged) and non-transgenic mice. All mice start out with the same weight, but the weight of mice expressing N171-82Q increases more slowly than that of N171-18Q and non-transgenic mice. Prior to death, the weights of N171-82Q mice decrease.
Each of the lines of mutant mice expressing N171-82Q (lines 6, 77, 81 and 100) showed a shortened lifespan (Fig. 6). Line 77 was the most affected, showing a lifespan of only 2.5 months (possibly due to slightly higher levels of N171-82Q expression in this line). Mice in line 77 did not breed well, and eventually the line died out. Within each line of mice, the severity of the phenotype progressed at a similar rate over a period of several weeks and the mice died within a relatively narrow interval. Mice from lines 81 and 100 had lifespans of 5-6 months, whereas mice from line 6 died at 8-11 months of age. Handling-induced seizures were not observed in any of these lines. At autopsy, the animals showed no gross abnormalities in visceral organs and showed signs of food consumption. Blood glucose levels in endstage N171-82Q mice (n = 4) were similar to those in non-transgenic littermates (n = 4) (data not shown). Mice expressing N171-18Q (line 8) and N171-44Q (line 9) had lifespans that were comparable with those of non-transgenic littermates. At present, the cause of death in N171-82Q mice is unclear.
Figure6. Reduced lifespan of HD N171-82Q transgenic mice. Mice with N171-18Q had a normal lifespan. Line 77 of N171-82Q showed the shortest lifespan and died at ~2.5 months of age; this line could not be maintained (possibly due to slightly higher expression of the transgene protein). N171-82Q (line 81 and 100) lived to ~5-6 months, whereas animals from line 6 died at ~8-11 months of age. The cause of death remains unclear; there have been no seizures observed in these lines, and the animals appear to take food.
All three transgenic lines we established with N171-82Q (line 6, 81 and 100) showed an identical behavioral and pathologial phenotype. The N171-82Q animals exhibited progressive behavioral symptoms including tremors, loss of weight, uncoordination, hypokinesis, abnormal gait and frequent hindlimb clasping. In the last 4 weeks of life (a period we termed endstage), the N171-82Q mice were much smaller and less responsive to stimuli than non-transgenic littermates [compare Fig. 7A (non-transgenic) and B (N171-82Q transgenic)]. Endstage mice exhibited poor grooming and frequent clasping of the hindlimbs when suspended by the tail [compare Fig. 7C (non-transgenic) and D (N171-82Q transgenic)]. None of these motor impairments and behavioral phenotypes were observed in the control transgenic N171-18Q (line 8) mice up to 2 years of age. Moreover, these phenotypes have not been observed in mice expressing N171-44Q (line 9) up to 2 years of age.
To quantify the motor impairment, we assessed the performance of N171-82Q mice from lines 81 and 100 in a variable speed rotating rod task, measuring the length of time and rotational speed before animals fall (a short distance) from the rod. Initially, we compared 4-month-old mice from line N171-82Q-100 with age-matched non-transgenic littermates (Fig. 8A). Although the mutant mice improved their performance over the 4 day period, the motor skills of these animals were impaired overall when compared with their littermates. To analyze whether the motor impairment showed progression, we tested line N171-82Q from line 81 mice at 3 and 5 months of age, and compared these animals with age-matched transgenic N171-18Q mice from line 8 and non-transgenic mice. At 3 months of age, all animals tested showed similar levels of performance on the first day of the trial. However, over the next 3 days, mice from expanded line 81 failed to improve in their performance (Fig. 8B). At 5 months of age, the mutant line 81 mice were impaired from the first trial and stayed on the rod for less time than did 3-month-old animals (Fig. 8C). The performance of N171-18Q transgenic mice from line 8 mice was indistinguishable from that of non-transgenic mice at any age (Fig. 8B and C).
Neuropathological analyses of brains from N171-82Q mice from lines 6, 77, 81 and 100 indicated that, though slightly smaller, they were grossly normal, with no sign of abnormal development. Immunolabeling for glial fibrillary acidic protein (GFAP) also suggested that the brains of N171-82Q transgenic mice did not develop a severe astrocytic reaction. Light microscopic examination using hematoxylin/eosin and silver stains did not indicate severe loss of neurons in the cortex, hippocampus, striatum, cerebellum and brainstem. Whether more subtle losses of neurons occurred, as recently described in mice expressing full-length mutant huntingtin (20), will require further analysis, including stereological cell counting.
Immunocytochemical studies on frozen/free-floating sections with antibodies to the N-terminal 17 amino acids of huntingtin (AP194) and ubiquitin revealed diffuse nuclear staining and numerous nuclear inclusions in multiple neuronal populations in endstage N171-82Q mice, including the pyriform cortex (Fig. 9A), the caudate (Fig. 9C), the outer cortex (Fig. 9D), all areas of the hippocampus including CA1 (Fig. 9E) and the dentate gyrus (Fig. 9F), the amygdala (Fig. 9G) and the granule cell layer of the cerebellum (Fig. 9H). Inclusions were infrequent in the thalamus (Fig. 9I), globus pallidus (Fig. 9L) and brainstem reticular formation (Table 1). Nuclear inclusions were more readily distinguishable in non-counterstained sections. Inclusions and diffuse nuclear label were not detected in any region of the brain in the N171-18Q transgenic mice. An example of these data is provided by analyses of immunostaining in the pyriform cortex (Fig. 9B).
Figure7. Representative photographs of a normal mouse compared with an endstage mouse from line HD-N171-82Q line 81 at 5.5 months. As compared with non-transgenic mice (A), mice from line 81 were smaller and hypokinetic at this age (B). N171-82Q mice also exhibit a hunched posture, tremors, abnormal gait and poor grooming. When suspended by the tail, the non-transgenic mice show normal escape reflexes (hindlimbs spread) (C), whereas N171-82Q mice show frequent clasping of their hindlimbs (D).
Counterstaining of sections from the cortex and caudate of lines 6 and 81 N171-82Q mice clearly demonstrated the nuclear localization of the N7171-82Q inclusions (Fig. 10A-D). Moreover, the nuclear inclusions were recognized by antibodies to ubiquitin (Fig. 10F).
In addition to nuclear inclusions, immunostaining with huntingtin antibodies demonstrated neuritic pathology in several regions including the cerebral cortex (less frequent; data not shown), medial amygdala (Fig. 9J) and the subthalamic nucleus (Fig. 9K). The presence of neuritic and neuropil aggregates of huntingtin has been reported recently in HD post-mortem brain (13), suggesting an accumulation of mutant huntingtin in axons and dendrites. Staining sensitive for [beta]-sheet (congo red, thioflavin) did not detect inclusions in endstage animals (data not shown); however, these nuclear structures are much smaller than the [beta]-sheet structures found in Alzheimer’s disease where these stains are used routinely to disclose [beta] conformations.
Measures of oxidative stress in control and two lines of mutant (N171-82Q) transgenic mice
Carbonyl (nmol/mg)
Lipid peroxidation (nmol/mg)
Aconitase (U/mg protein)
Mn-SOD (U/mg protein)
N171-18Q-8
5.64 ± 0.36
1.53 ± 0.13
3.63 ± 0.08
7.10 ± 0.83
N171-82Q-81
6.60 ± 0.80
1.88 ± 0.14
4.15 ± 0.83
6.84 ± 0.25
N171-18Q-8
6.44 ± 0.25
1.88 ± 0.15
2.89 ± 0.12
5.90 ± 0.74
N171-82Q-100
6.01 ± 0.22
2.10 ± 0.23
3.22 ± 0.53
4.83 ± 0.67
n = 6 for all groups. Values are means ± SD.
Because of the large number of prior reports indicating oxidative stress as a mediator of neuronal damage in HD (24,25), a survey of oxidative stress markers was conducted. Comparison of forebrains from endstage mice of two lines of N171-82Q mice of control transgenic line 8 showed that there was no evidence for increased carbonyl proteins or lipid peroxidation products, and no loss of aconitase activity in transgenics (Table 2). The mutants also had normal levels of copper-zinc and manganese superoxide dismutase. Although these data do not eliminate the possibility that focal oxidative damage may play a role in the disease, these findings are not consistent with the idea that widespread severe oxidative stress contributes to the behavioral and pathological phenotype of the N171-82Q transgenic mice.
Figure8. Rotarod testing of two different lines of HD-N171-82Q mice demonstrates motor impairment and a progressive phenotype. (A) Four-month-old mice from line 100 of N171-82Q mice were compared with non-transgenic mice of the same age. Mice were measured in four trials/day on four consecutive days. Non-transgenic mice showed steady improvement in performance over the 4 day period, whereas N171-82Q mice improved only slightly. (B and C) The presence of motor impairment was confirmed in a second line of N171-82Q mice (line 81), and the progressiveness of the phenotype was measured by including two different timepoints of the disease. Compared with N171-18Q (line 8) and non-transgenic mice of the same age, line 81 of N171-82Q mice start out at the same level of performance but then fail to improve over the 4 days of trials. At 5 months of age, the N171-82Q mice show a poorer level of performance than the 3-month-old animals from the initial trial onward. The N171-18Q (line 8) and non-transgenics are indistinguishable at these timepoints. In all trials, n = 6 mice ± SD. Error bars are not shown when smaller than the point.
Figure9. Intranuclear neuronal inclusions and neuritic pathology in endstage N171-82Q mice. Frozen/free-floating sections from two lines of N171-82Q mice were immunostained with AP194 (at a dilution of 1:5000). Intranuclear inclusions were frequent in N171-82Q mice and absent in N171-18Q animals. For example, compare the pyriform cortex of an N171-82Q-6 animal (A) with that of a control transgenic animal N171-18Q (line 8) (B). Inclusions were abundant in striatum (C), cortex (D), hippocampus including CA1 (E) and dentate gyrus (F), amygdala (G) and granule cells of cerebellum (H). Inclusions were infrequent in the dorsal thalamus (I) and globus pallidus (L). Neuritic pathology was seen in several areas of the brain including the medial amygdala (J) and subthalamic nucleus (K). The sections were not counterstained, except (L). Original magnification for (A-D) was 252×; for the remaining panels it was 160×. Photomicrographs (A) and (D-K) were from N711-82Q line 6 mouse brain (8 months); (L) was from the same line (age 11 months); (B) is from N171-18Q (11 months); and (C) is from line 81 of N171-82Q transgenic mice (6.5 months).
Figure10. Intranuclear inclusions in caudate, cortex and amygdala in two different lines of N171-82Q endstage mice. (A-E) Immunocytochemistry of frozen/free-floating sections (30 µm), stained with AP194 (at a dilution of 1:5000) or (F) ubiquitin antibody (DAKO, dilution 1:10 000). Numerous inclusions (arrows) can be observed in N171-82Q (line 6, 11 months) in (A) caudate, (C) cortex and (E) amygdala. Similarly, in transgenic line 81 of N171-82Q mice (6.5 months), a high density of inclusions was detected in (B) caudate and (D) cortex. Intranuclear inclusions were reactive with ubiquitin in several areas including (F) amygdala of N171-82Q (line 6, 11 months) transgenic mice. Cells which had intranuclear inclusions usually had increased diffuse nuclear labeling for AP194 and ubiquitin. These pictures demonstrate that we observed very similiar neuropathology in two lines of N171-82Q endstage animals. Sections were lightly counterstained. Original magnification for all panels is 252×.
Our study records a number of new findings relevant to the pathogenesis of HD. First, we demonstrate that N-terminal fragments of huntingtin encompassing the first 171 amino acids and containing 82 glutamine repeats are sufficient to cause a number of neurological abnormalities and the formation of intranuclear inclusions, neuritic aggregates and diffuse nuclear localization of huntingtin. A recent description of transgenic mice expressing full-length mutant huntingtin (20) demonstrated that the mutant full-length protein, when overexpressed, can elicit behavioral abnormalities and selective degeneration of striatal neurons (20). Interestingly, in these mice, intranuclear inclusions were found in only 1% of neurons, and neuritic aggregates were not reported. Small N-terminal fragments of human huntingtin were also not reported. These findings suggest that full-length huntingtin is toxic but may be less prone to form intranuclear and neuritic inclusions in mice, perhaps because mice are deficient in the activities required to process human huntingtin proteolytically. In contrast, our N171-82Q fragments clearly readily aggregate to form both nuclear and neuritic inclusions. Because these latter structures are prevalent in the brains of HD cases (13), it seems likely that proteolytic truncation of mutant huntingtin is playing a role in the evolution of HD brain pathology.
Second, we frequently observed small structures in the neuropil that stained with antibodies to the N-terminus of huntingtin in our N171-82Q mice (Fig. 9J and K). Similar aggregations of mutant huntingtin in neurites have been observed in the cortex and striatum of patient brain (13). In the initial descriptions of transgenic mice expressing mutant exon 1 of huntingtin (16,17), these neuritic structures were not reported. Whether our N-171 fragment possesses information that makes these molecules more prone to produce neuritic pathology has yet to be determined. Nevertheless, to our knowledge, our animals are the first to reproduce the neuritic pathology (visible by light microscopy) that is becoming increasingly recognized as an important feature of HD pathology.
Finally, we noted that the density of intranuclear inclusions in different brain regions of our N171-82Q mice was decidedly non-uniform. Inclusions were relatively rare in neurons of the brainstem, thalamus and globus pallidus. In situ hybridization studies (data not shown) confirmed the high level expression of transgene mRNA in these cells, and thus the lack of inclusions is not due to a lack of expression, suggesting that the N171 constructs possess some information that imparts specificity. Elucidating the basis for neuronal specificity of inclusions, perhaps by varying the length of future huntingtin constructs, may provide important information on the mechanisms by which these inclusions form and the mechanisms for the selective injury of subsets of neurons in HD.
The steady-state levels of N171-18Q, N171-44Q and N171-82Q polypeptides appeared to be lower than that of endogenous huntingtin. Although it is possible that the aggregation of N171-82Q could lead to deceptively low levels of protein on immunoblots, the amount of N171-82Q protein per unit of mRNA appeared to be similar to that of N171-18Q protein. Thus, we do not believe that levels of N171-82Q transgene product reflect a loss of protein signal due to aggregation (26,27). Whether all N171 constructs extract from brain less efficiently than full-length protein is unknown and difficult to assess. On face value, our data suggest that the steady-state levels of N171-82Q polypeptides are not in excess of endogenous huntingtin and are likely to be significantly lower.
We observed that our 171 amino acid N-terminal fragments of huntingtin appeared to be subject to C-terminal truncation. From our estimations of relative molecular mass for the truncated product of N171-18Q, we predict that the C-terminus of the cleaved molecule falls near the C-terminus of exon 1. Because our homogenization buffers contained a cocktail of inhibitors to all classes of proteases, and because the relative ratio of cleaved and uncleaved polypeptides in N171-18Q (line 8) mice did not vary significantly among samples taken from different homogenizations, we assume that the observed cleavage occurred in vivo. The C-terminally truncated N171 fragment was detected more readily in line N171-18Q-8 and N171-44Q-9 mice than in N171-82Q mice. Whether the C-terminally truncated N171-82Q fragment is more difficult to detect because it is more prone to aggregate into SDS-insoluble structures or because N171-82Q is cleaved less efficiently is unclear. Thus, whether the cleavage we have observed here plays a role in the pathogenesis of HD is uncertain at present.
Apart from the abnormal localization of huntingtin that was detected in the brains of our endstage mice, the brains of these animals appeared to develop normally. No peripheral organs were grossly abnormal and there was no obvious cellular pathology in the heart, which is the only other organ that expresses foreign proteins from the prion protein vector at levels comparable with brain (22). Moreover, there was no obvious cell loss or change in cell structure in brain regions exhibiting high densities of intranuclear inclusions. Immunostaining for GFAP did not detect reactive gliosis. Thus, the behavioral phenotypes present in the N171-82Q mice appear to be the result of cell dysfunction rather than gross cell death. Whether more subtle losses of neurons have occurred in some brain regions is uncertain at present and will require stereological cell counting studies.
In summary, we have created transgenic mice expressing an N-terminal fragment of huntingtin with 82 glutamines. Our findings indicate that N171-82Q huntingtin fragments are sufficient to cause behavioral abnormalities, and to elicit the formation of intranuclear inclusions and neuritic aggregates in a subset of brain regions, pathology that is prominent in HD brain. Diffuse nuclear localization of expanded huntingtin may also contribute to pathology, as has been reported recently (28,29). Because the pathology of our animals closely mimics the abnormalities present in HD cases, we conclude that proteolytic processing of mutant huntingtin could play a role in the evolution of HD brain pathology.
The HD-N171-18Q, HD-N171-44Q and HD-N171-82Q (18 glutamine repeats, normal; 44 glutamine repeats, adult onset HD; and 82 glutamines, juvenile onset HD) constructs were obtained by PCR from genomic DNA of human lymphoblastoid cells using the primer sense 5[prime]-GGCCCGAGGCCTCCGGGACGTC-3[prime] and antisense 5[prime]-GGCTGAGGCAGCAGCGGCTGTGC-3[prime] with hot-start PCR. The products were purified and subcloned into the pCRII vector (Invitrogen, Carlsbad, CA). The fragments were digested with AlwNI and NcoI and ligated into a pBluescript plasmid containing a portion of the 5[prime] end of the huntingtin cDNA. The resulting plasmids were sequenced entirely to verify fragment orientation and nucleotide sequence veracity. A SalI linker (CGGTCGACCG; Stratagene, La Jolla, CA) was ligated to the 5[prime] end of the insert after digestion with EcoRI and filling in with DNA polymerase. The resulting plasmid contained the 5[prime] end of the huntingtin cDNA including none of the 5[prime]-untranslated sequence upstream of the start codon and extending to an XhoI site that occurs at base pair 823. This fragment was cloned into the XhoI site of the MoPrP.Xho vector as a SalI-XhoI fragment. Because these plasmids were built as intermediates in the assembly of longer huntingtin constructs, no stop codons were engineered into the cDNA sequences. Instead, the stop codon is provided by sequences in the 3[prime]-untranslated portion of the vector, adding 13 amino acids to the C-terminus of each of the three N171 constructs (LEPSCLFLRLLVV). Plasmids were digested with NotI, and the PrP vector sequences were separated on a low melting agarose gel (FMC Bioproducts, Rockland, ME). The construct containing huntingtin and the prion promoter was purified with [beta]-agarase (FMC Bioproducts) and microinjected into the male pronucleus of fertilized oocytes.
Transgenic animals were identified by PCR of genomic DNA extracted from mouse tail as previously described (21). A three-way PCR was used in genotyping. Two primers were complementary to the prion protein genomic DNA sequence: PrP-sense 5[prime]-CCTCTTGTGACTATGTGGACTGATGTCGG-3[prime], PrP-antisense 5[prime]-GTGGATACCCCCTCCCCCAGCCTAGACC-3[prime]. The amplified product of this reaction is 700 bp in length. The antisense primer is also complementary to the 3[prime]-untranslated portion of the PrP vector and, in combination with a third sense primer to the HD sequence (HD-591-5[prime]: 5[prime]-GAACTTTCAGCTACCAAGAAAGACCGTGT-3[prime]), generated a transgene-specific product that is 250 bp in length. The number of integrated transgene copies was established by Southern blot from mouse genomic DNA, after digestion with EcoRI. A fragment of N-terminal huntingtin cDNA (AlwNI-HindIII: HD nucleotide sequence 511-788) was radiolabeled and used to probe the Southern blots. Transgenic founder animals with >10 copies were bred to generate stable transgenic lines. Transgenic animals were mated continuously to hybrid (C3H/HEJ×C57 BL/6J F1) (Jackson Laboratory, Bar Harbor, ME) mice, and the lines were maintained on the hybrid background.
Total mRNA was extracted from mouse hemibrain by extraction in Trizol (Gibco BRL, Gaithersburg, MD), and 5 µg was fractionated by formaldehyde gel electrophoresis as described (30). The agarose gel was transferred onto Genescreen membrane (NEN, Wilmington, DE) and probed with a random primed labeled [32P]cDNA fragment of the HD cDNA (described above). The distribution of transgene mRNA expression was assessed by performing in situ hybridization as previously described (22), using a riboprobe.
Mouse brain was homogenized with 10 vol ice-cold 50 mM Tris-HCl (pH 7.4) and a cocktail of protease inhibitors (complete; Boehringer Mannheim, Indianapolis, IN). Homogenates of N171-82Q-transfected HEK-293 cells were loaded as a positive control. Proteins were separated by electrophoresis in 3-12% polyacrylamide gels, and transferred onto nitrocellulose membrane (Schleicher & Schuell, Keene, NH). Blots were blocked in 5% non-fat dry milk-phosphate-buffered saline (PBS) and probed with a polyclonal, affinity-purified antibody AP194 (peptide antibody, amino acids 1-17) (23) at a concentration of 1 µg/ml [in 3% bovine serum albumin (BSA)-PBS], overnight. Immunoblots probed with 1C2 antibody (31) were incubated at a dilution of 1:1000, overnight. After washing in 5% milk-PBS, the secondary antibody (goat anti-rabbit, goat anti-mouse; Boehringer Mannheim) was applied and subsequently detected by chemiluminescence (NEN).
Animals were anaesthetized in methoxyfluorane (Metofane; Mallinckrodt Veterinary, Nundelein, IL) and perfused with PLP [2% paraformaldehyde/75 mM d/l-lysine (Sigma, St Louis, MO) 10 mM sodium m-periodate (Sigma) in PBS pH 7.4] through the left cardiac ventricle. The brains were removed and post-fixed in PLP overnight, before transfer into PBS. The brains were dissected sagitally, one half was used for paraffin sections, the other for frozen sections (soaked overnight in 30% sucrose-PBS at 4°C, and frozen on crushed dry ice).
Inclusions were detected by immunostaining of free-floating frozen sections (30 µm) with a polyclonal N-terminal peptide (amino acids 1-17) antibody AP194 [dilutions for frozen sections: 1:5000; polyclonal anti-ubiquitin antibody (Dako, Carpentaria, CA): 1:10 000]. Paraffin-embedded tissue sections (8 µm) were immunostained with antibodies to huntingtin and ubiquitin at dilutions of 1:500 (32,33).
In the first trial, six 4-month-old naive mice from line N171-82Q-100 were compared with non-transgenic mice from the same age group. The second trial tested six naive 3-month-old animals from line 81 of N171-82Q mice and six additional 5-month old naive animals; age-matched N171-18Q-8 and non-transgenic mice were tested simultaneously. The mice were tested on a Rotarod device (Rotamex 4/8, Columbus Instruments International, Columbus, OH). The speed of the rod was set to increase from 4 to 40 r.p.m. over a 10 min period. The interval for the mice to fall from the rod was measured in four trials per day over a 4 day period. At least 10 min recovery time was allowed between trials (34). The data for each group of animals were averaged and plotted.
Aconitase enzyme activity was measured by modifying a standard two-step spectrophotometric assay (35) for use in a SpectraMax microplate reader (Molecular Device, Sunnyvale, CA). Total SOD enzyme activity was measured with the microplate reader by modifying a standard indirect inhibition assay with xanthine oxidase as a source of superoxide anions and nitroblue terazolium as chromogen. The activities of SOD 1 and SOD 2 were distinguished by including 5 mM potassium cyanide in the reaction to block the activity of SOD 2. Carbonyl proteins were measured by derivatization of carbonyl groups with dinitrophenylhydrazine, followed by separation of reagent from product by HPLC with a Zorbax GF-450 size exclusion column. Malondialdehyde, 4-hydroxynonenal and related aldehydes were measured by derivatization with N-methyl-2-phenylindole in acetonitrile (R&D Systems, Minneapolis, MN), and compared with a standard curve constructed with 4-hydroxynonenal.
Preliminary analysis of plastic sections, in collaboration with Dr Steven Hersch at Emory University, indicates that there is neuronal degeneration in the striatum of the 82Q mice.
This work was supported by the Hereditary Disease Foundation, the Huntington’s Disease Society of America ‘Coalition for the Cure’ and the NINDS (NS16375), the DeVelbiss fund and a bequest from the estate of Sar and Brita Levitan.
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