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Human Molecular Genetics, 2003, Vol. 12, No. 7 711-725
DOI: 10.1093/hmg/ddg084
© 2003 Oxford University Press

PQBP-1 transgenic mice show a late-onset motor neuron disease-like phenotype

Tomohiro Okuda1,2, Hiroshi Hattori2, Sousuke Takeuchi3, Jun Shimizu3, Hiroko Ueda1, Jorma J. Palvimo4, Ichiro Kanazawa3,5, Hitoshi Kawano6, Masaya Nakagawa2 and Hitoshi Okazawa1,3,7,*

1Department of Molecular Therapeutics, Tokyo Metropolitan Institute for Neuroscience, 2-6, Musashi-dai, Fuchu, Tokyo 183-8526, Japan, 2Toyama Chemical Co., Ltd, 2-4-1, Shimo-Okui, Toyama 930-8508, Japan, 3Department of Neurology, Graduate School of Medicine, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-8655, Japan, 4Biomedicum Helsinki, Institute of Biomedicine, University of Helsinki, FIN-00014 Helsinki, Finland, 5National Center for Neurology and Psychiatry, 4-1-1, Ogawa-Higashi, Kodaira, Tokyo 187-8502, Japan, 6Department of Developmental Morphology, Tokyo Metropolitan Institute for Neuroscience, 2-6, Musashi-dai, Fuchu, Tokyo 183-8526, Japan and 7PRESTO, Japan Science and Technology Corporation (JST), 4-1-8, Honmachi, Kawagoe, Saitama 332-0012, Japan

Received September 19, 2002; Accepted February 1, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
A body of experimental evidence indicates that transcription and/or mRNA processing factors interacting with the polyglutamine disease gene products play crucial roles in the pathology. PQBP-1 is one of these factors and it has been shown to interact with the spinocerebellar ataxia type-1 (SCA1) disease gene product, ataxin-1. Our previous data suggested that relatively high expression of PQBP-1 in the cerebellum might explain the selective neuronal degeneration of SCA1. To further test whether PQBP-1 expression level regulates neuronal death, we generated transgenic mice of human PQBP-1 driven by a regulatory element for ubiquitous gene expression. The mice showed a late-onset and gradually progressive motor neuron disease-like phenotype, which might be related to neurogenic muscular atrophy observed in SCA1 patients. Ataxia could not be discriminated from predominant progressive weakness. Pathological examinations of the transgenic mice revealed loss of Purkinje and granular cells in the cerebellum as well as that of spinal motor neurons, corresponding to the pathology of human SCA1. These findings show that excessive action of PQBP-1 causes neuronal dysfunction and support PQBP-1 being involved in the pathology of SCA1.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
More than nine types of hereditary neurodegenerative diseases are caused by expansion of a polyglutamnine tract in the disease proteins including androgen receptor, huntingtin, ataxin-1, -2, -3, -6, -7, dentatorubral pallidoluysian atrophy (DRPLA) protein/atrophin-1 and TATA-binding protein (TBP) (for a review, see 1). Accumulation of the polyglutamine-containing proteins into neuronal intranuclear inclusion body (NII) is reported in Kennedy's disease (KD), Huntington's disease (HD), DRPLA, and spinocerebellar ataxia (SCA)-1, -3 and -7 (for review, see 2,3). Because NII sequesters normal nuclear proteins, it is considered to be a critical pathological process leading to neuronal dysfunction and/or death (4–7). Notably, sequestration of cAMP-responsive element-binding protein (CREB)-binding protein (CBP) into aggregates by disease proteins has been recently shown to play a critical role in the pathology of KD, HD and DRPLA (4–7). It has also been speculated that soluble disease proteins interact with transcription- or mRNA processing-related proteins in the nucleus and disturb essential nuclear functions before or during aggregation (8–10). Although polyglutamine diseases share these pathological processes, they obviously affect different sets of neurons and lead them to death in human pathology. Furthermore, instead of the neuron type-specific pathology, polyglutamine disease proteins are expressed ubiquitously throughout the brain (1). The selective neuronal death remains as an intriguing question for understanding the mechanism of polyglutamine diseases.

Several hypotheses have been proposed to explain the selective neuronal death. The first one is that transcriptional co-factors functioning in restricted areas of the brain bind to the disease proteins and mediate selective neuronal degeneration. Interaction of polyglutamine disease proteins with TATA-binding protein, PQBP-1, N-CoR, RED, ARA24, CBP, p53, TAFII130, ETO/MTG8, p160/GRIP1, Sp1 and C-terminal binding protein has been reported (4–8,11–18). Other nuclear proteins including SRC-1, TAFII30 and glucocorticoid receptor are sequestered into nuclear aggregates of cellular models (19–21). They might be able to explain selective neuronal death if they function in a cell-specific manner. In support of this hypothesis, a recent report on the conditional depletion of CREB showed a Huntington's disease phenotype with progressive neuronal degeneration in striatum and hippocampus (22). This observation, however, presented another question of why other neurons such as Purkinje cells were not affected although CREB expression was suppressed also in the cerebellum of the transgenic mice. Importantly, polyglutamine proteins can also disturb the function of transcriptional co-factors by inhibiting the histone acetyl-transferase (HAT) activity of CBP (23,24). HAT disturbance is implicated in the pathology, because suppression of the counteracting enzymes (HDACs) prevents neuronal death in the cell culture and Drosophila models (23,24). Although these transcription-related factors may mediate the selective neuronal death, we need further investigations to prove this hypothesis.

The second idea for selective neuronal death is that the aggregating form of disease protein is produced in restricted areas of the brain. This hypothesis is closely related to the toxic peptide theory that polyglutamine-containing peptides cleaved from the full-length mutant proteins are more harmful and preferentially deposit in the nucleus and/or in the axon terminal (25–29). Li's group reported that the N-terminal fragment of huntingtin is selectively accumulated in the striatal neurons, and proposed that the region-specific cleavage leads to the selective neuronal death (30). However, this view was challenged by a report showing that mutant huntingtin is more resistant to proteolysis and N-terminal peptides arise from normal huntingtin in diseased brains (31). The cleavage by caspases is known to occur with androgen receptor, ataxin-3 and atrophin-1/DRPLA gene product (32–35). N-terminal cleavage of ataxin-7 by unknown proteases was also reported (20). Although the similar region-specific processing of disease proteins may occur in these polyglutamine diseases, further analyses are necessary to confirm that the region-specific cleavage is a common rule for selective neuronal death.

The third hypothesis suggests that certain neuron type-specific effectors exist downstream of the disease protein-binding molecules and induce selective neuronal death. In fact, this story could be considered as a modified version of the first hypothesis. Huntingtin-interacting protein, Hip-1 (36,37), has been implicated in multiple functions including vesicle-trafficking, endocytosis and cortical actin cytoskeleton formation (for review, see 38). Hip-1 is expressed ubiquitously in different brain regions and thus difficult to induce selective neuronal death by itself. Recently a Hip-1-binding protein, Hippi, was shown to activate caspase-8 and participate in apoptosis (39,40). Although the expression of Hippi is not perfectly selective, it is highly expressed in cortico-striatal and striato-pallidal projection neurons (40), supporting the third hypothesis. LANP, a cerebellar nuclear protein that forms a complex with ataxin-1 and possibly mediates the pathology (41) might be classified into this group, considering its structural characteristics analogous to those of signal transduction molecules.

We have recently isolated a binding protein to the polyglutamine tract, PQBP-1, that is expressed predominantly in the brain regions susceptible to SCA-1 (12). We have shown that SCA-1 disease protein, ataxin-1, interacts with PQBP-1 and that the PQBP-1–ataxin-1 complex disturbs RNA polymerase II (8), matching with the first hypothesis for selective neuronal death. To test our previous result that the cerebellum-predominant expression profile of PQBP-1 is linked to the selective neuronal death, we generated transgenic mice overexpressing PQBP-1 by a ubiquitously active enhancer and promoter. The phenotype mimicked a late-onset form of motor neuron disease rather than cerebellar ataxia. Pathological examination of the transgenic mice revealed that the excessive and ubiquitous expression of PQBP-1 leads to remarkable neuronal loss in the spinal anterior horn, one of the vulnerable regions in SCA1 (42), as well as to reduction of cerebellar neurons. These findings suggest that PQBP-1 might be involved in selective neuronal degeneration of SCA1.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Generation of PQBP-1 transgenic mice
To generate transgenic mouse overexpressing PQBP-1, human PQBP-1 full-length cDNA was inserted downstream of cytomegalovirus enhancer and chicken ß-actin promoter that force ubiquitous gene expression (43). Genomic DNA was extracted from the tail, and genotyping was performed with Southern blot or PCR–Southern blot. In the latter analysis, PCR products amplified with primers specific to the integrated construct sequences were blotted with the PQBP-1 cDNA probe. The filters, washed either in high- or low-stringent conditions, indicated two transgene-positive mice (Fig. 1A). These founder mice (F0-14 and F0-32) grew normally and we could not point out any obvious deterioration of these mice in their youth. This phenotype was plausible because PQBP-1 alone, in the absence of ataxin-1 coexpression, does not remarkably suppress transcription or induce cell death (8). Therefore, we decided to observe their phenotype in aging.




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  		Figure 1. Impaired motor activity of the PQBP-1 transgenic mouse. (A) Genotype was checked by PCR–Southern blots in high- and low-stringent conditions (upper and lower panels, respectively). Arrows indicate the band derived from the integrated transgenes. Mouse line numbers are indicated above the panel. pCAGGS-PQBP1 plasmid and non-transgenic C57BL/6 mouse genomic DNA were used for PCR as positive and negative controls, respectively (P and N). In the low-stringent condition, PCR was performed with 35 cycles of the sequence, 1 min at 95°C, 2 min at 50°C and 1 min at 72°C, and the blot filter was washed in 0.2xSSC at 50°C for 30 min. In the high-stringent condition, PCR was performed with 35 cycles of the sequence, 1 min at 95°C, 2 min at 55°C, and 1 min at 72°C, and the filter was washed in 0.1xSSC at 60°C for 30 min. (B) When suspended by the tail, the transgenic mouse (F0-32) could not keep normal posture spreading the hind legs symmetrically like the control. (C) Impaired locomotive activity of the transgenic mouse. Arrowheads indicated inversion of the hind limb due to weakness. (D) Motor activity of the F0-32 and F2 transgenic mice (n=3) was estimated by four different tests. Age-matched control mice to each group (n=4) and young mice at the age of 4 months (n=4) were also tested. Examination was performed four times in each mouse, and the data are shown as mean±SD. The values of Rota-rod test declined in F0 and F2 mice statistically (P<0.01, Bonferroni's t-test).

 
F0-32 mouse was pregnant twice, whereas only two transgenic mice were generated among 14 F1 mice. The number of transgenic mice was clearly lower than expected. This might be due to mosaicism of the transgene in the founder mouse or due to an increased rate of intrauterine lethality by PQBP-1 transgene.

PQBP-1 mice show a late-onset motor neuron disease-like phenotype
Around 22 months, F0-32 showed an insidious onset of weakness in bilateral hind limbs. The mouse had a limp and walked only slowly. The symptom was progressive. It did not respond normally when hung by its tail (Fig. 1B). The weakness looked more remarkable in the right leg. The hind limbs sometimes inverted due to the hypotonus of leg muscles (Fig. 1C). Results of the spontaneous motor activity and the other motor function tests at the age of 26 months declined accordingly (Fig. 1D, F0). Neither involuntary movement nor epileptic seizure was observed during the course. It was difficult to discriminate cerebellar ataxia because the weakness of hind limbs masked it.

We could evaluate only one F1 mouse with similar examinations for motor activity from 12 to 14 months, but did not find any obvious decline in motor activity with these tests (data not shown). We could not examine another F1 mouse because it died before the age of onset in the founder mouse due to unknown reason. We therefore performed similar examinations with F2 mice at the age of 22 months. Some of the F2 mice (3/22) clearly showed abnormal responses when hung by the tails, whereas such responses were not observed in the age-matched control mice (n>40). The decline of motor activity in this group was confirmed statistically by Rota-rod examination (P<0.01, Bonferroni's t-test; Fig. 1D). These F2 mice were used for pathological examinations at 22 months. The rest of the F2 mice began to show similar limping of hind limbs at 24 months. Mean values of the motor functions in the other F2 mice were decreased to 80–90% of those in the control mice (data not shown). F0-14 died due to viral hepatitis at 2 months (F0-14 and F0-32 were generated in different sets of injections).

PQBP-1 expression in transgenic mice
F0, F1 and F2 PQBP-1 transgenic mice were dissected at the age of 26, 24 and 22 months, respectively. Macroscopically, all brain structures were well preserved in these mice. No malformation or atrophy was found. Although F0-32 was bloated, neither ascites nor abdominal tumor was found. Before histological analyses, we examined expression level of the PQBP-1 protein in brain tissues by immunohistochemistry with anti-PQBP-1 antibody. The stain intensity was clearly higher in neurons of the transgenic mice, including granule cells of the cerebellum, Purkinje cells and pontine neurons of the facial and trigeminal nerve nuclei (Fig. 2A–D). This finding, shared by F0 and F1 mice, indicated overexpression of PQBP-1 at the cellular level in the transgenic mice. We also performed western blot analyses with tissue samples of F2 mice. Although western blot mixes up various types of cells in the tissue, we observed increases of 10–80% in the signal of the ~37 kDa PQBP-1 band, corresponding to the full-length PQBP-1 protein (12), after correction with GAPDH (Fig. 2E). The increase was remarkable in the spinal cord (~80%) and moderate in the cerebellum (~20%), but hardly seen in the liver (Fig. 2E). The increase of PQBP-1 in skeleteal muscle was relatively small (~10%). In the cerebellum, the increase of the PQBP-1 protein detected by western blot looked small compared with the impressive stain of a part of granule cells in the cerebellum. The difference is probably due to the dilution effect by non-stained neurons, glial cells and axons composing the white matter in western blot. The pattern and the intensity of upper ladder bands corresponding to ubiquitinated PQBP-1 (12) varied among tissues. This might suggest distinct protein degradation metabolisms among tissues leading to different expression levels of the full-length PQBP-1 protein. Since the increase of the PQBP-1 protein level was relatively small, we examined the increase of mRNA in brain tissues of F2 mice with RT–PCR. As shown in Figure 2F, PQBP-1 mRNA was increased remarkably both in the cerebellum and the spinal cord (Fig. 2F). This finding confirmed transgenic expression of PQBP-1 mRNA. In addition, the result suggested that the PQBP-1 protein level is strictly regulated in a region-specific and cell-specific manner.



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  		Figure 2. PQBP-1 overexpression in neurons of transgenic mice. Paraffin sections of the cerebellum (A, B) and the pontine tegmentum (C, D) were immunostained with anti-PQBP-1 antibody. Granule and Purkinje cells in the cerebellum of the PQBP-1 transgenic mouse (B, D) showed higher signals when compared to those of the age-matched control mouse (A, C). G, granule cell layer; P, Purkinje cell layer; M, molecular cell layer. Granule cells were stained in the nuclei, while Purkinje cells were stained predominantly in the cytoplasm (B versus A). High stain signals were also seen in pontine neurons of the transgenic mouse (D versus C). Bar, 50 µm. (E) PQBP-1 protein expression in the transgenic mouse. Various tissues from a F2 transgenic mouse at the age of 22 months with no obvious symptom and from an age-matched control mouse were blotted with anti-PQBP-1 antibody. The relative signal intensity of the full-length PQBP-1 protein band (arrowhead) after correction with GAPDH, was increased to ~120% in the cerebellum and to ~180% in the spinal cord. The intensities of bands were calculated by Multi-bioimager (Amersham). (F) RT–PCR showing the PQBP-1 mRNA levels in the control (C) and F2 transgenic (Tg) mice. Both mouse and human cDNAs were amplified, and they matched to the length of positive control cDNA (PC) amplified from a plasmid. The lower panel shows GAPDH expression in equivalent samples. (G) Pol II expression in the cerebellum and spinal cord of the same transgenic mouse. We observed a faint band by longer exposures to X-ray films. Pol II protein seemed to be decreased in F2 transgenic mice.

 
Since PQBP-1 binds to RNA polymerase II (Pol II) and the interaction with mutant ataxin-1 decreases the active/hyperphosphorylated form (Pol IIO) in vitro, we tested whether expressions of the Pol II and Pol IIO proteins are affected by PQBP-1 trangene. We similarly performed western blot analyses of the cerebellar and spinal cord tissues of F2 mice with anti-Pol II N-terminal antibody. The signal of the total Pol II protein was decreased by the transgene in the cerebellum, while the band was very weak in the spinal cord (Fig. 2G). On the other hand, anti-phosphrylated C-terminal antibodies (H5 and H14) did not show a clear band in both tissues (data not shown). These results suggested that the level of Pol II activation is relatively low in the central nervous system and especially that the amount of the total Pol II protein is very low in the spinal cord. If we assume that Pol II repression is involved in neurodegeneration of the polyglutamine diseases, the low expression and activation levels of Pol II might correspond to the tissue specific vulnerability.

Excessive PQBP-1 affects spinal anterior horn neurons
We performed histological analyses with Kluver–Barrera (KB) and hematoxylin–eosin (HE) stains throughout the central nervous system. The most significant change was observed in the anterior horn of the spinal cord. Neurons in the anterior horn of the lumber spinal cord were clearly decreased (Fig. 3B versus A). In particular, large neurons judged as motor neurons were substantially reduced. There were some shrunken and dark neurons, although this type of cells also existed in the control (for example, see Figs 3A and 5A). Corresponding to the anterior horn lesion, anterior spinal roots showed severe reduction of myelinated fibers (Fig. 3E versus C). The change was not found in the posterior roots, indicating the selectivity of pathology. At the same level of the lumbar spinal cord, we found reactive astrogliosis (Fig. 3F versus D) further to support neuronal loss. The pathological dominance in the right anterior horn corresponded well to the symptomatic dominance in the right hind limb. Clinically, it is well known that similar focal dominance occurs in amyotrophic lateral sclerosis (ALS), which sometimes affects one limb at first then progresses to the other ones. In contrast, other parts of the spinal gray matter including the posterior and lateral horns were well preserved (data not shown).




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  		Figure 3. Loss of motor neurons in the anterior horn of the spinal cord. Paraffin sections of the spinal cord and roots at the lumbar level stained by KB (A–C, E). Large motor neurons in the anterior horn were decreased remarkably in the F0-32 PQBP-1 transgenic mouse (B) compared with those in the age-matched controls (A). Immunohistochemistry with anti-GFAP antibody showed reactive astrogliosis in the anterior horn (arrow heads, F) that was not found in the control (D). Nerve fiber density was obviously decreased in the anterior spinal root (AR) of the transgenic mouse, while the posterior spinal root (PR) was preserved (E versus C). Neurons in the posterior and lateral horns of the spinal cord were well preserved in the transgenic mouse (data not shown). Bar, 50 µm. (G–L) Anterior horn neurons were counted at the lumbar or thoracic level of the spinal cord in F0 (G, H), F1 (I, J) and F2 (K, L) mice. In F0 and F1, we could examine only one mouse, thus mean values from four independent sections are indicated. In F2, the data from three mice are shown as mean±SD. White bar, age-matched control mice; black bar, PQBP-1 transgenic mice. The numbers of anterior horn neurons at lumbar and thoracic levels were decreased in F0 and F1 mice, and the decrease was statistically confirmed in F2 mice (P<0.01).

 



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  		Figure 5. Reduction of Purkinje and granular cells in the cerebellum. Paraffin sections of cerebellum stained with KB (A–D). (A) Purkinje cells were decreased in the PQBP-1 transgenic mice compared to those in the age-matched controls (B versus A). It is noteworthy that dark neurons were found in the Purkinje cell layer of the control (A). The density of granule cells was lower in the PQBP-1 transgenic mouse than that in age-matched controls (D versus C). Bar, 50 µm. Purkinje (E, G, I) and granule cells (F, H, J) in F0 (E, F), F1 (G, H) and F2 (I, J) mice were counted in four independent areas. White bar, age-matched control mouse; black bar, PQBP-1 transgenic mice.

 
We quantified the number of neurons in the anterior horn of F0, F1 and F2 mice using multiple slices of the lumbar or thoracic spinal cord stained by KB and HE. Four independent quantifications of each level were summarized (Fig. 3G–L). Statistical analyses indicated that spinal motor neurons were decreased in F2 mice at the thoracic and lumbar levels (P<0.01, Bonferroni's t-test). Although F2 mice showed relatively mild symptoms, the results clearly showed that motor neurons were also affected in F2 generation. The difference of neuronal reduction among generations may reflect different timings of pathological examination. The quantitative analyses revealed that the number of the anterior horn neurons was also decreased at the thoracic level, although reduction of neurons and reactive astrogliosis was milder (data not shown).

Secondary changes of peripheral nerves and muscles
To exclude that the motor symptoms were due to primary disturbances of peripheral nerves or skeletal muscles, we examined sciatic and posterior peroneal nerves as well as femoral flexor and extensor muscles of the F0-32 transgenic mouse (Fig. 4). Both sciatic and posterior peroneal nerves showed substantial loss of myelinated fibers in all fascicles (Fig. 4A versus B). These nerves contained darkly stained axons and numerous myelin destructions after axonal retraction (myelin ovoid; Fig. 4A), indicating severe Wallerian degeneration. HE stain of femoral muscles showed angular fibers and small group atrophy (Fig. 4C), supporting neurogenic changes of muscles. Consistently, we also found target fibers (data not shown). These neuromuscular pathologies are similar to those of ALS, and clearly indicate that the chronic and active lesion of spinal motor neurons induced these secondary changes of peripheral nerves and muscles.



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  		Figure 4. Secondary changes in peripheral nerves and muscles. (A) Toluidine blue stain of the sciatic nerve of the PQBP-1 transgenic mouse. Myelinated fibers were remarkably decreased, and many myelin sheaths were relaxed or destroyed after retraction of axons by Wallerian degeneration (arrows). Some axoplasms were darkly stained, supporting axonal degeneration. (B) Myelinated fibers were distributed at a high density in the sciatic nerve of age-matched control mice. (C) Hematoxylin-eosin stain of the femoral flexor muscle of F0-32 showed many foci of small group atrophy (black arrows) and pyknotic nuclear clamp (gray arrows). Diameters of muscle fibers were not constant. (D) Femoral flexor muscle of an age-matched control mouse. Bar, 100 µm.

 
Excessive PQBP-1 affects cerebellum mildly
The cerebellum of F0-32 also showed mild reduction of Purkinje cells and granule cells (Fig. 5A–D). To confirm the impression, we calculated the number of Purkinje cells per length of the cell layer and the density of granular cells per area. Since the interval of Purkinje cells varies among cerebellar folia, we chose the first folium and counted the number of Purkinje cells per mm. At the same time, we counted the number of granule cells in the first folium per 0.01 mm2. These quantitative analyses proved reduction of Purkinje cells and granular cells in the cerebellum of F0-32 (Fig. 5E and F). Also in F1 mice, the number of Purkinje cells was clearly reduced, while granule cells were not significantly decreased (Fig. 5G and H). Purkinje and granule cells were not decreased statistically in F2 mice (Fig. 5I and J). This might be due to the younger age at which F2 mice were dissected.

Neurons do not show typical inclusions in PQBP-1 transgenic mice
We examined whether nuclear inclusion body was formed in neurons of the F0-32, F1 and F2 mice. With KB and HE stains, we could not find any nuclear or cytoplasmic inclusions in spinal, cerebellar, pontine or cerebral neurons. We further looked for inclusion bodies by immunohistochemistry with anti-ubiquitin polyclonal antibody. Some large motor neurons in the anterior horn of the lumbar spinal cord and a few Purkinje cells showed strong stains in the nuclear matrix but sparing the nucleolus, while similar ubiquitin stain was not observed in the spinal cord of control mice (Fig. 6). Meanwhile, we could not find definite nuclear inclusions reactive to anti-ubiquitin antibody both in affected and unaffected regions of the brain.



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  		Figure 6. Diffuse ubiquitin stain in the nuclei of spinal motor neurons. Paraffin sections of the lumbar spinal cord of F0-32 mouse were stained with anti-ubiquitin antibody. Some motor neurons in the lumbar spinal cord of the transgenic mouse showed extremely high signals in the nuclei (B), while those in age-matched controls did not show the nuclear dominant pattern (A). Bar, 50 µm.

 
To detect apoptosis, we also performed immunohistochemical analyses using anti-single-strand DNA antibody. However, we could find no obvious difference between the stain of transgenic mouse brain tissues and that of controls either in the anterior horn or in the cerebellum (data not shown), suggesting that apoptosis is not involved in neuronal death of the PQBP-1 transgenic mice.

AR is not likely to be involved in the dominance of spinal motor neuron lesions
In this study, we show that anterior horn neurons are most remarkably affected in PQBP-1 transgenic mice. Although anterior horn of the spinal cord is one of the most vulnerable regions in SCA1 (42), certain additional factors might be necessary to induce pathological dominance of the spinal cord to the cerebellum. PQBP-1 is a binding protein to the polyglutamine tract (12). If up-regulated, PQBP-1 protein might have a chance to interact with a normal form of certain other polyglutamine disease protein, functioning in spinal motor neurons, and the interaction might lead to the pathology observed in the transgenic mice. Androgen receptor (AR), that is expressed in spinal motor neurons and involved in the pathology of KD, is an obvious candidate molecule.

To test whether PQBP-1 can interfere with AR-dependent transcriptional regulation, we conducted reporter gene (chloramphenicol acetyltransferase, CAT) transfection assays. We used two different reporter plasmids, pCMV-ARE2-CAT and ptkARE2-CAT, for measurements of transcriptional repression via androgen response elements (ARE) and of transcriptional activation via AREs, respectively. We examined the effect of PQBP-1 on transcription in the presence or in the absence of rat or human AR (pSG5rAR or pSG5hAR, respectively). We found that PQBP-1 represses the basal transcription levels of ptkARE2-CAT reporter and pCMV-ARE2-CAT. The transcriptional repression by PQBP-1 independent of coexpressed transcription factors is consistent with our previous observation (8). However, co-transfection of AR did not enhance the transcriptional repression by PQBP-1 (data not shown). On the contrary, transcriptional repression tended to be attenuated by co-expression (data not shown). These results suggest that AR is not likely to be involved in the transcriptional repression and in the pathology of anterior horn neurons induced by PQBP-1.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
PQBP-1 interacts with the polyglutamine tract of multiple proteins including Brn-2 and ataxin-1 (8,12). Its affinity increases according to the length of the polyglutamine repeat. PQBP-1 binds to C-terminal domain of Pol II via its WW domain (8), and to U5 15 kDa via its unique domain (43,44). Because phosphorylation of the second serine in a repeat sequence of Pol II-CTD enhances interaction of PQBP-1 to Pol II (8), PQBP-1 is considered to be a bridging molecule between transcription and splicing that functions during RNA elongation. Our previous results indicated that mutant ataxin-1 interacts with the PQBP-1/Pol II complex, disturbs the transcription-mRNA processing link, and induces cell death by down-regulation of the active form of Pol II (Pol IIO) (8). This study shows that PQBP-1 overexpression leads to late-onset neuronal reduction in the central nervous system in vivo, agreeing with our previous results that PQBP-1 mediates toxicity of the polyglutamine disease protein in cultured cells (8) and increases vulnerability of primary cerebellar neurons to low potassium stress (45).

Recently, DNA microarray analysis of mRNA expression profile in the brain of mutant huntingtin transgenic mice, R6/2 revealed that Pol II mRNA expression is remarkably increased whereas total amount of the Pol II protein, active Pol IIO plus inactive Pol IIA, is clearly reduced (46). The reduction of the Pol II protein in the brain progresses with aging despite the compensatory up-regulation of mRNA expression. The group also showed sequestration of Pol II into the huntingtin aggregates by using an antibody that reacts with both Pol IIA and Pol IIO. Interestingly, perturbation of the Pol II proteins was also described in non-polyglutamine neurodegeneration (47). Mutant SMN, the causative gene for spinal muscular atrophy (SMA), induces reduction of Pol IIO in the nuclear matrix as well as sequestration of Pol IIA into the enlarged nuclear body (47). These data are consistent with our previous result that mutant ataxin-1 and PQBP-1 cooperatively reduces Pol IIO in the nuclear matrix (8), suggesting collectively that Pol II dysfunction is involved in neurodegeneration. Therefore, it is possible that excessive action of PQBP-1, a connector molecule between transcription and mRNA processing, leads to neuronal reduction in transgenic mice.

Our previous findings indicated that PQBP-1 induces cell death with a polyglutamine disease protein, ataxin-1 (8). However, it is not clear whether PQBP-1 is a modifier enhancing the toxicity of mutant ataxin-1 or an effector directly transferring the pathogenic effect of mutant ataxin-1. This study shows that PQBP-1 by itself has a weak toxicity inducing neuronal death. Since mutant polyglutamine proteins are not co-expressed in the transgenic mouse, the present study suggests that PQBP-1 could be considered as a candidate molecule to induce cell death downstream of the polyglutamine protein(s). Because nuclear aggregates are not found in our transgenic mice and because motor neurons of the transgenic mice show abnormal diffuse nuclear ubiquitination, this study might suggest that PQBP-1 triggers a cell death cascade bypassing aggregate formation. However, these ideas await further investigations including knock out (KO) mice or conditional KO mice. We planned to generate KO mice, but it was not successful, probably because PQBP-1 is expressed from the earliest stage of embryo (our unpublished observation) and plays an essential role in cells. Although PQBP-1 binds to normal ataxin-1 weakly in vitro (8), acceleration of the PQBP-1 toxicity by normal ataxin-1 does not seem to occur in our transgenic mice. Assuming that the interaction with normal ataxin-1 causes neuronal loss, pathological changes in the cerebellum should have been more severe than the spinal lesions. This consideration is also consistent with our previous observation that co-expression of normal ataxin-1 and PQBP-1 did not trigger cell death (8).

An intriguing conclusion of this study is that ubiquitous overexpression of the PQBP-1 gene still affects specific regions in the central nervous system. We could not find any obvious reduction of neurons in the brain regions other than the spinal anterior horn and the cerebellum. The distribution of pathological changes in nervous system of the PQBP-1 transgenic mice corresponded well with that of human SCA patients. Selective neuronal loss is not surprising in the transgenic mouse model regulated by a tissue specific enhancer. However, it is surprising that selective lesions are sometimes observed in mouse models driven by a ubiquitously active promoter. A recent report on conditional double knock-out of the Creb and Crem genes using Cre-loxP system also showed selective neuronal degeneration in striatum and hippocampus, although the Cre gene expression was regulated by the calcium/calmodulin-dependent kinase II-alpha gene promoter that forces ubiquitous expression in the brain (22). Our case also belongs to this category. Total expression of PQBP-1, the sum of endogenous and transgenic expressions, did not simply correspond to the extent of pathological changes. Instead, neuronal loss correlated with the increase of the full-length PQBP-1 protein by transgene (Fig. 1E). The latter report and our present study indicate that ubiquitous up- or down-regulation of specific molecules still induce selective neuronal degeneration mimicking polyglutamine diseases. Collectively, in the case of SCA1, PQBP-1 might be involved in the selective neuronal death at two steps. The first one is its cerebellum-dominant expression profile (8). The second one is the region-specific sensitivity to PQBP-1 in neuronal death, which we observed in this study. The latter might be relevant to the basal expression level of Pol II (Fig. 1F).

Although spinal motor neurons are sometimes reduced in SCA1 patients (42), the anterior horn is less severely affected than the cerebellum in usual. The discrepancy between human pathology and that of the PQBP-1 transgenic mice might be due to interaction of PQBP-1 with a normal form of polyglutamine disease protein other than ataxin-1 functioning predominantly in the mouse motor neurons. We tested this possibility by using normal AR, the KD-causative gene product that functions in spinal motor neurons. However, our data did not support normal AR being involved in the transcriptional repression by PQBP-1, i.e. the anterior horn pathology of the PQBP-1 transgenic mice. Another explanation for the pathology of transgenic mice could be difference of the PQBP-1 protein expression level (the sum of endogenous and transgene-induced expressions), because PQBP-1 itself weakly suppresses basal transcription (8).

Western blot analyses revealed that expression of the PQBP-1 protein was increased more remarkably in the spinal cord than in the cerebellum (Fig. 2E), although anti-PQBP-1 antibody stained anterior horn cells of the non-transgenic mice only very weakly (data not shown) and could not show the difference from the transgenic mice. The discrepancy between immunohistochemistry and western blot analysis might be due to de-calcification of vertebral bones for preparing spinal tissue samples. The treatment performed specifically for the spinal cord might have reduced the reactivity for anti-PQBP-1 antibody and blurred the difference. However, we found that the PQBP-1 stain in spinal motor neurons was weak even where the tissue was prepared without de-calcification (data not shown). Alternatively, the weak staining might suggest certain specific conditions in the nuclei of spinal motor neurons that mask the antigenisity of PQBP-1. For example, certain regulatory protein(s) might strictly conceal epitopes and/or functions of PQBP-1 in spinal motor neurons. Together with the results of RT–PCR showing equivalent increase of PQBP-1 mRNA in the spinal cord and in the cerebellum (Fig. 2F), the different changes of the PQBP-1 protein level suggest distinct protein metabolisms including protein degradation among different brain regions. This might be relevant to the selective neuronal reduction of the PQBP-1 transgenic mice and to the discrepancy between the human and transgenic mice pathologies. In addition, distinct expression levels of the total Pol II protein in the cerebellum and in the spinal cord (Fig. 2G) might lead to different vulnerabilities to PQBP-1 overexpression.

Taken together, this study shows that overexpression of PQBP-1 induces slowly progressive neuronal death homologous to human pathology. In the next step, we plan to clarify pathological mechanisms underlying the neuronal death observed in our PQBP-1 transgenic mice. For this purpose, we have started to analyze mRNA and protein expression profiles in the spinal and cerebellar neurons. We further need to analyze connections between Pol II dysfuction and neuronal death using multiple types of neuron. As discussed previously (48), highly complex cellular systems might underlie the cell specificity of polyglutamine diseases. However, we hope that these approaches lead to better understanding of selective neuronal death in neurodegenerative disorders.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Generation of PQBP-1 transgenic mice
A PQBP-1 full-length cDNA fragment was subcloned between XbaI and XhoI sites of pCAGGS (49). The 3.1 kb SalI and HindIII fragment cleaved out from the plasmid was injected into fertilized eggs of the C57BL/6 mice. For genotyping, genomic DNA was prepared from tail biopsy and used for Southern blot (50) or PCR-Southern blot analyses. In the latter, PCR was performed with the primers forward, TCCCCTTCTCCATCTCCA and reverse, CGCAACGGGCAGCGGCAT (corresponding to a promoter sequence and a human PQBP-1 cDNA sequence, respectively), then the blot was probed with a full-length PQBP-1 cDNA (12). The concrete PCR and Southern blot conditions are indicated in the figure legends.

Immunohistochemistry
Mice were transcardinally perfused with cold 4% parafolmaldehyde (PFA), and the brains, spinal cords and muscles were post-fixed in 4% PFA. Paraffin sections, 5–10 µm were de-paraffinized in xylene and rehydrated through an ethanol dilution series. Endogenous peroxidase was inactivated with 1% hydrogen peroxide in PBS for 30 min at room temperature. For ubiquitin staining, paraffin sections were treated with 99% formic acid for 5 min before peroxidase inactivation. Anti-PQBP-1 carboxyl-terminal polyclonal rabbit serum (1:2000 dilution), anti-ubiquitin polyclonal rabbit antibody (DAKO, 1:1000), and anti-GFAP rabbit polyclonal antibody (Chemicon, 1:500) were used as primary antibody. HRP-conjugated anti-rabbit antibody (DAKO, 1:400) was used as a secondary antibody, then visualized with VECTASTAIN ABC system (Vector Laboratories) and diaminobenzidine (DAB, Sigma). Anti-single-strand DNA antibody for detection of apoptosis was purchased from DAKO, and used according to the commercial protocol. Non-transgenic sibling mice were used as the age-matched control.

RT–PCR
Total RNA preparation and RT–PCR analysis were performed according to the method described previously (50). Primers for mouse PQBP-1, forward,: ATGCCGCTTCCTGTTGCG and reverse, TGGAAGAGGGGCCCAGCT, were used for RT–PCR. The resultant cDNA was 716 bp. The reverse primer sequence is completely conserved in human and mouse PQBP-1, while 16 of 18 nucleotides in the forward sequence are identical. We performed all the steps of RT–PCR using RNA LA PCR kit (AMV Ver.1.1, TaKaRa). Reverse transcription was performed at 42°C for 30 min. The thermal cycle condition was 94°C for 1 min, 50°C for 1 min, and 72°C for 2 min. Twenty-five cycles within the linear amplification range were used for the analysis. Primers for GAPDH were purchased from Clontech.

Quantitative analyses of the histology of transgenic mice
Paraffin sections of transgenic and age-matched control mice were stained with HE and KB according to the standard procedures. Multiple pictures were captured in the region of interest by Axiovision system (Zeiss), and the TIFF images were reconstructed with Adobe Photoshop (Adobe). Neurons on the combined image were directly counted by marking in more than four independent areas. Axonal diameters and densities were calculated with NIH image. Statistical analyses were basically performed with InStat 2.0 (Macintosh). Multiple pair-wise comparisons were performed using analysis of variance and Bonferroni t-test among groups of data.

Assessment of motor functions
Spontaneous activity and rearing scores correspond to horizontal and vertical movements measured by digital counters with the infrared sensor system (Neuroscience) for 30 min. For Rotarod examination, mice were placed on a rod rotating at 10 rpm, and the duration of remaining stay on the rod was measured. The Rotarod apparatus (SHINANO Industry, SN-445) has a rotor 3 cm in diameter, whose tracks are separated every 9.5 cm. In the wheel running test, the rolling number of the cage by a mouse placed in a narrow circular cage (Muromachi) was measured for 30 min. All the data were shown as mean±SD. Non-transgenic sibling mice were used as the age-matched control.

CAT assay
Ten micrograms of reporter and effector plasmids were transfected into 1x106 Hela cells with CellPhect transfection kit (Amersham Pharmacia Biotech) according to the commercial protocol with a minor modification. Briefly, to increase the transfection efficiency, Hela cells were treated 12 h after transfection with 15% glycerol in 1xBBS (pH 6.95) for 1 min. Thirty-six hours after transfection, cells were harvested with 0.25 M Tris–Cl (pH 7.5) and CAT assays were performed as described previously (51). Testosterone (Sigma) was added to the culture medium (final concentration 10 nM) after the glycerol shock. Construction of pCMV-ARE2-CAT, ptkARE2-CAT, pSG5rAR and pSG5hAR was described previously (52).


   ACKNOWLEDGEMENT
 
We thank Dr J.-i. Miyazaki (Osaka University) for pCAGGS plasmid, and appreciate critical discussions with Drs K. Tagawa and M. Hoshino in our department. This work was supported by a grant to H.O. from Japan Science Technology Corporation (JST), Japanese Ministry of Education, Culture, Sports, Science and Technology (12670596, 14370213) and by Toyama Chemical Co. Ltd.


   FOOTNOTES
 
* To whom correspondence should be addressed. Tel: +81 423253881; Fax: +81 423218678; Email: okazawa-tky{at}umin.ac.jp or okazawa{at}tmin.ac.jp Back


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