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Human Molecular Genetics Advance Access originally published online on January 11, 2008
Human Molecular Genetics 2008 17(8):1137-1146; doi:10.1093/hmg/ddn003
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© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Huntingtin-associated protein-1 is a modifier of the age-at-onset of Huntington's disease

Silke Metzger1,{dagger}, Juan Rong2,{dagger}, Huu-Phuc Nguyen1, Austin Cape2, Juergen Tomiuk1, Anne S. Soehn1, Peter Propping3, Yun Freudenberg-Hua3, Jan Freudenberg4, Liang Tong5, Shi-Hua Li2, Xiao-Jiang Li2,* and Olaf Riess1

1 Department of Medical Genetics, University of Tuebingen, 72076 Tuebingen, Germany 2 Department of Human Genetics, Emory University, Atlanta, GA 30322, USA 3 Institute of Human Genetics, University of Bonn, 52111 Bonn, Germany 4 Department of Neurology, Institute of Human Genetics, University of California, San Francisco, CA 94143-2922, USA 5 Department of Biological Sciences, Columbia University, New York, NY 10027, USA

* To whom correspondence should be addressed at: Department of Human Genetics, Emory University School of Medicine, 615 Michael St. Room 347, Atlanta, GA 30322, USA. Tel: +1 4047273290; Fax: +1 4047273949; Email: xiaoli{at}genetics.emory.edu

Received November 20, 2007; Revised December 21, 2007; Accepted January 5, 2008


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
A polyglutamine repeat expansion of more than 36 units in a protein called huntingtin (htt) is the only known cause of Huntington's disease (HD). The expanded repeat length is inversely correlated with the age-at-onset (AAO), however, the onset age among HD patients with CAG repeats below 60 units varies considerably. In addition to environmental factors, genetic factors different from the expanded CAG repeat length can modify the AAO of HD. We hypothezised that htt interacting proteins might contribute to this variation in the AAO and investigated human htt-associated protein-1 (HAP1) using genetic and functional assays. We identified six polymorphisms in the HAP1 gene including one that substitutes methionine (M441) for threonine (T441) at amino acid 441. Analyzing 980 European HD patients, we found that patients homozygous for the M441 genotype show an 8-year delay in the AAO. Functional assays demonstrated that human M441-HAP1 interacts with mutant htt more tightly than does human T441-HAP1, reduces soluble htt degraded products and protects against htt-mediated toxicity. We thus provide genetic and functional evidence that the M441-HAP1 polymorphism modifies the AAO of HD.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
Huntington's disease (HD) is a progressive neurodegenerative disorder, which is associated with motor dysfunction, cognitive deficits and psychiatric disturbances that manifest commonly at the age of 35–50 years (1). The underlying genetic cause is an expanded CAG repeat of more than 36 units in the IT15 gene encoding huntingtin (htt) (2), which is inversely correlated with the age-at-onset (AAO) (3–6). Since the expanded CAG repeat determines only 42–73% of the variance in the AAO (7,8), other genetic and environmental factors are thought to modify the course of HD (9). Based on siblingship analyses, familial (genetic) factors might account for up to 19% of the AAO variance in addition to the expanded CAG repeat (10). Indeed, studies with genome approaches have supported the existence of additional genetic factors that modify the AAO of HD (11,12). Also, there is association of the AAO of HD with the polymorphisms of the IT15 gene itself (CCGn repeat, delta2642 polymorphism, CAG repeat length of the normal allele) (8,13–17), and of gene products potentially involved in the HD pathway such as the GluR6 kainate receptor locus (18), the NR2A and NR2B receptor genes (19) and the UCHL1 gene (20,21). However, most of these studies have been initiated in small patient samples and did not reach statistical power for final conclusion. Furthermore, none of the associated polymorphisms was analyzed for their functional implication.

We have recently analyzed a large number of htt interacting proteins and other candidates for their potential modification of the AAO (20,22). By continuously searching for htt interacting proteins as potential genetic modifiers, we identified several unknown polymorphisms in the gene encoding human htt-associated protein-1 (HAP1). HAP1 was the first protein identified to interact with htt. It binds more tightly to mutant htt than wild-type htt (23). HAP1 is enriched in brain and colocalizes with htt in the cytoplasm of the neurons. Its association with specific organelles, such as mitochondria, microtubules or synaptic vesicles, is almost identical to that for htt (24). Through the association with microtubule-dependent trafficking proteins such as dynactin p150 and kinesin light chain (KLC), HAP1 is involved in axonal trafficking (25,26). In addition, the association of HAP1 to endosomal organelles, the type 1 inositol 1,4,5-triphospate receptor (IP3R1) and the androgen receptor, as well as its involvement in BDNF transport support a role for HAP1 in intracellular trafficking and endocytosis (24,27–29). Based on these observations, HAP1 is suggested to play a role as a trafficking protein or as an adaptor between cargos and intracellular transporters. Similarly, htt also participates in intracellular transport processes. For example, HAP1 and htt are co-transported bidirectionally in axons (30). Thus, HAP1 is an excellent protein to study for its potential modifying influence at the AAO in HD.

In the present study, we identified several polymorphisms in HAP1 and investigated their association with the AAO of more than 900 European HD patients. We found that one polymorphic amino acid change at position 441 leads to an 8-years delayed onset of first symptoms. This polymorphic HAP1 (M441) has a stronger interaction with mutated htt, reduces degradation of htt and decreases neurodegeneration. Thus, the M441 polymorphism presents the first genetic modifier of AAO in HD, which has been identified by association studies and confirmed by functional assays.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
The M441 polymorphism is associated with the AAO in HD
In our cohort of 980 HD patients, the expanded HD allele accounts for ~53% of the variance in the AAO (R2 = 0.527) and shows a very strong influence on the disease onset (P < 0.0001; Supplementary Material, Table S2). Sequencing the human HAP1 gene in unaffected individuals revealed the presence of six polymorphisms. Subsequent studies confirmed the presence of these polymorphisms in 679–968 Middle European HD patients (Supplementary Material, Table S1). Initial analysis of these HAP1 polymorphisms in HD patients, regardless of their CAG repeats, did not demonstrate their significant effects on the AAO of HD. For example, neither variance (P = 0.200) nor covariance (P = 0.381) for the polymorphism M441 reached statistical significance when its copy number and the CAG repeat units were not considered (Supplementary Material, Table S2). We divided the analyzed HD patients into two groups of <60 and ≥60 CAG units. In this way, we revealed a significant effect of the M441 genotype on the AAO of HD (P = 0.015) (Table 1). HD patients with <60 CAG units and the homozygous M441 genotype develop their first symptoms at the average age of 52.5 years (SD 11.32), which is about 8 years later than that of HD patients with other genotypes (TT or TM) (Table 2). This finding suggests that the delaying effect of M441 on the AAO of HD only occurs in homozygous M441 individuals and is thus dependent on the copy number of M441. Although HD patients with ≥60 CAG repeats and the heterozygous M441 genotype also show about an 8-years delay in AAO (27.49 versus 19.17) when compared with those with other genotypes (Table 2), the limited number of patients in this group did not yield statistical significance for the effect of M441 on the AAO of HD patients with ≥60 CAGs. We did not find homozygous M441 patients with ≥60 CAGs so that the effect of M441 on the AAO of HD with ≥60 CAGs remains to be investigated. The correlation of CAG repeat size and the mean AAO in the different HAP1 genotypes is depicted in Supplementary Material, Figure S1. Overall, the M441 polymorphism constitutes 1.2% of the variance in AAO in the presence of expanded HD alleles smaller than 60 CAG repeats. This represents 2.5% of the variance that cannot be accounted for by the expanded CAG repeat in the HD gene. Estimation of haplotype frequencies for M441 and other identified HAP1 polymorphisms showed that M441 exists on one common haplotype, suggesting that M441 could be an evolutionary recent polymorphism that might be specific for the Middle European population (Fig. 1).


Figure 1
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  		Figure 1. Haplotype frequency distribution estimated from the genotypes of all coding SNPs that were detected in the European population. Common non-synonymous SNPs are highlighted, the remaining SNPs are either synonymous or rare. M441 exclusively exists on the major haplotype, suggesting that it could be an evolutionary recent polymorphism and confer a population-specific effect. Haplotype frequencies were estimated by the program snphap that implements an expectation maximization algorithm to calculate maximum likelihood frequencies (http://www-gene.cimr.cam.ac.uk/clayton/software/snphap. txt).

 


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  		Table 1. Effect of the human HAP1 T441M polymorphism (M441) on the age-at-onset of HD

 


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  		Table 2. Mean ages-at-onset of the different human HAP1 genotypes

 
The M441 polymorphism is associated with a stronger interaction with mutated htt
Human HAP1 (hHAP1) is primarily characterized by {alpha}-helices and contains several domains for different phosphorylations and protein–protein interactions (Fig. 2A). Previous studies localized the htt binding site to the amino acid 323–416 region in hHAP1 (31). In the polymorphic form of hHAP1 (M441), amino acid residue threonine (T441), which is conserved in hHAP1 and rodent HAP1, is replaced by methionine. The amino acid change is located at the end of an {alpha}-helix, whose function is not yet defined. As the HAP1 protein is an interactor of htt (23), we first analyzed the influence of changing T441 to M441 on the interaction of hHAP1 with htt. Htt immunoprecipitation shows that M441 binds mutated htt more tightly than T441 in transfected HEK293 cells (Fig. 2B and C). The precipitation of transfected htt was specific to the anti-htt antibody (Supplementary Material, Fig. S1A). In contrast, M441 does not alter the interaction with wild-type htt. Also, interactions of hHAP1 with other proteins such as IP3R1, Kalirin, KLC and p150, which have been reported previously (25–27,32,33), are not influenced by the M441 polymorphism (Supplementary Material, Table S3).


Figure 2
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  		Figure 2. Domain organization and sequence motifs in human HAP1 and its interaction with htt. (A) hHAP1 contains several potential phosphorylation sites [tyrosine kinase (green), casein kinase II (yellow), protein kinase c (black), cAMP- and cGMP-dependent protein kinase (white)]. It also consists of several domains including those suitable for interaction with the extracellular matrix [leucine zipper (red), glycosaminogycan attachment site (light blue), cell attachment sequence (dark blue), and N-myristoylation site (orange)]. T441 or M441 is located at the end of an {alpha}-helix. (B) HEK293 cells were transfected separately with T441, M441 and N208-120Q. The cell lysates were then mixed for immunoprecipitating N208-120Q in order to have the same input for each immunoprecipitation. The blots were probed with antibodies to hHAP1 (T441 or M441) and htt (upper panel). Densitometry analysis of the ratio of bound to input (lower panel) verified that mutant htt was co-precipitated with more M441 than T441. *P<0.05. (C) HEK293 cells were co-transfected with N208-120Q and T441 or M441. Immunoprecipitation of htt also shows that more M441 than T441 (arrow) was co-precipitated, indicating a stronger interaction between htt and M441. NT, non-transfected cells.

 
The M441 polymorphism is associated with large cytoplasmic htt aggregates
Previous studies have indicated that hHAP1 and htt have similar cellular distribution patterns (24). However, mutant htt is also localized in the nucleus and forms intranuclear aggregates, whereas hHAP1 is in the cytoplasm. Cytoplasmic aggregates formed by N-terminal htt fragments are another characteristic feature of HD (34). Similarly, overexpression of HAP1A leads to cytoplasmic puncta that resemble stigmoid bodies in neuronal cells in the brain (24,29,33). Coexpressing hHAP1 (T441 or M441) and htt in HEK293 cells showed a colocalization of hHAP1 with some cytoplasmic htt aggregates, which were formed by N-terminal mutant htt (1–208 amino acids) with 120Q (N208-120Q). This colocalization was also seen in cells transfected with N-terminal htt (208 amino acids) containing a moderate (44Q) polyQ tract (Fig. 3). The colocalization of hHAP1 with htt aggregates supports the interaction between hHAP1 and mutant htt, which was also demonstrated by immunoprecipitation. Interestingly, M441 colocalizes with more htt aggregates than does T441 (Fig. 3). We found that 30.2±1.8% and 39.4±4.2% (mean±SD) of htt aggregates contained T441 and M441 signals, respectively. Large htt aggregates did not show obvious HAP1 staining, suggesting that the extensive aggregation of htt can reduce its interaction with HAP1.


Figure 3
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  		Figure 3. M441 colocalizes with more htt aggregates in transfected cells. (A) Low magnification images (100x) showing that co-transfection of M441 with N208-120Q in HEK293 cells increased the number of htt aggregates when compared with cotransfection with T441. (B and C) High magnification images (400x) showing that htt aggregates (arrows) contains more M441 than T441 in HEK293 cells that expressed N208-120Q (B) or N208-44Q (C).

 
The M441 polymorphism influences htt degradation
Since degradation of htt to smaller fragments is a critical step leading to HD pathology, we further investigated whether the increased association of M441 with htt can influence htt degradation. We transfected HEK293 cells with N208-120Q htt that contained the HA epitope at its C-terminus. Immunostaining of transfected cells with antibodies to htt (EM48) and the C-terminal HA epitope shows that more EM48-positive aggregates than HA epitope-containing aggregates are present in transfected cells (Fig. 4A), indicating that proteolytic cleavage of transfected htt occurs in cells and leads to the formation of more htt aggregates that can only be labeled by EM48. Western blotting also shows smear and small htt fragments in transfected cells, which appear to be decreased in the presence of M441 (Fig. 4B). To confirm this, we isolated soluble and pellet fractions of transfected cells. More aggregated htt was seen in the pellet fraction in the presence of M441 (Fig. 4C), which is consistent with increased htt aggregates in M441 transfected cells (Fig. 3A) and suggests that M441 can stabilize htt aggregates via its increased association with mutant htt. In addition, M441 reduced the amount of soluble degraded htt products in the pellet fraction (Fig. 4C), which also suggests that M441 can stabilize htt aggregates or binds mutant htt more tightly. In turn, the increased association of M441 with mutant htt could reduce the interactions of htt with other proteins or its toxicity.


Figure 4
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  		Figure 4. M441 reduces the amount of degraded htt products in cells. (A) Staining of N208-120Q-HA transfected HEK293 cells showing that some htt aggregates (arrows) have lost the HA epitope, suggesting that they are formed by degraded products. (B) Co-transfection of N208-120Q-HA with M441 resulted in less degraded htt products (bracket) than cells co-transfected with N208-120Q-HA with T441. (C) Fractionation of transfected HEK293 cells showing more htt aggregates (upper panel) and less htt fragments (middle panel) in the pellet fractions (P) in the presence of M441. The bottom panel shows the staining of the same blots with the antibody to hHAP1 (T441 or M441). The pellet fraction from 18 000g centrifugation contains more aggregated htt than the soluble (S) fraction. T, total cell lysates. Two soluble and pellet fractions are shown from each transfection.

 
The M441 polymorphism is associated with reduced htt-mediated toxicity
To examine whether M441 can reduce htt toxicity, we co-expressed htt (N208-23Q or N208-120Q) with T441 or M441 in cultured rat brain cortical neurons (Fig. 5). Apoptotic neurons with nuclear DNA fragmentation were evident in cells expressing mutated htt with 120 CAG repeats. Co-expression of hHAP1 did lead to a reduction of apoptosis in the primary neurons examined. Additionally, we observed that M441 reduced the number of degenerated neurons to a greater extent than T441 (Fig. 5A and B), suggesting a protective effect of M441. Counting the percentage of apoptotic neurons also verified the greater protection of M441 against htt-induced neuronal degeneration (Fig. 5C). Expression of T441 or M441 alone did not result in cell degeneration (Supplementary Material, Fig. S1B and Fig. 5C).


Figure 5
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  		Figure 5. The protective effect of hHAP1 on htt-associated cytotoxicity. (A) Transfection of cultured rat brain cortical neurons with GFP and N208-23Q or N208-120Q. Note that mutant htt (N208-120Q) causes nuclear fragmentation (arrowhead) of cultured neurons. (B) Co-transfection of N208-120Q with T441 or M441 can protect against neuronal degeneration. Note that M441 also colocalizes with large htt aggregates (arrows). (C) Counting the percentage of degenerating transfected neurons showing that M441 has greater protection against htt toxicity than does T441. *P = 0.0186 (n = 6) when comparing N208-120Q plus M441 and N-208-120Q plus T441. **P<0.01 when compared with the N208-120Q group.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
Here we report for the first time the identification of a genetic modifier for HD, which has functional relevance for the disease pathogenesis. We demonstrate that the M441 polymorphism is associated with an 8-year-delay on the AAO of HD. The AAO and the course of HD are mainly determined by the number of expanded CAG repeats in the IT-15 gene, which results in an elongated polyglutamine stretch htt (7). Most patients have repeat sizes of less than 60 CAGs and often show symptoms in midlife. Large expansions of more than 60 CAGs are generally associated with a juvenile disease onset appearing in about 5–10% of all HD cases (35). The M441 polymorphism modifies the AAO in HD patients with repeat expansions of <60 CAGs and an adult onset of the disease. In adult HD patients, variability in AAO is much higher than in juvenile HD and is more likely influenced by additional genetic factors. In the HD patients of the current study, the M441 polymorphism represents 2.5% of the variance that cannot be accounted for by the expanded CAG repeat in the IT-15 gene.

Previous studies already suggested that HAP1 can protect against htt and polyQ toxicity (reviewed in 36). For instance, HAP1 suppresses androgen-induced apoptosis in spinal and bulbar muscular atrophy (SBMA), inhibits degradation of the epidermal growth factor receptor (EGFR) and protects cells expressing mutant htt (28,37,38). A more general neuroprotective role for HAP1 has been suggested based on its distribution in brain regions (39) and based on transcriptional studies in the R6/2 model of HD (40). Here we performed several functional assays supporting the role of HAP1 as a protective factor against htt-mediated neurodegeneration. In particular, we have shown the increased binding of M441 to mutant htt, which is also reflected by the increased colocalization of M441 with htt aggregates. Aggregation of N-terminal fragments of mutant htt displays one of the most noticeable pathologic features in HD. Htt aggregates could be pathogenic, non-toxic or even protective (34,41,42). Although the role of htt aggregates remains controversial, it is clear that generation of N-terminal mutant htt fragments by proteolytic cleavage is an initial step towards HD neuropathology (43–45). Thus, under the in vivo condition when mutant htt is expressed at the endogenous level and forms aggregates slowly in the brain, it is likely that M441 binds soluble mutant htt more tightly than T441, at least partially inhibiting its degradation or its interactions with other proteins and thereby reducing htt toxicity and delaying the AAO of HD. Alternatively, the interaction of HAP1 with mutant htt may promote the formation of aggregated htt and reduce the level of soluble mutant htt that could be more toxic than aggregated htt.

Neurodegeneration in HD mostly occurs in the medium spiny neurons of the striatum and the cortex and is the result of various effects such as excitotoxicity, oxidative stress or reduced neurotrophic expression (27,46–49). A toxic gain-of-function of mutant htt is thought to underlie HD pathogenesis, because heterozygous Hdh knockout mice have a normal phenotype, whereas transgenic mice overexpressing mutant htt show severe neurological symptoms (50,51). Expansion of the polyglutamine stretch in htt leads to altered interactions with various proteins, including HAP1, and therefore affects the function of htt and its interacting proteins. If these abnormal interactions contribute to neurodegeneration in HD, htt-interacting or -associated proteins could influence the pathogenic events (52) and can be candidates in the search for disease modification.

Several studies have searched for genetic modifiers for HD (11,22,53). Genome-wide scans localized potential linkage sites of genetic modifiers to human chromosomes 4p16, 6p21–23, 6q23–24 and 18q22, and different polymorphisms were identified to associate with the AAO of HD (11,12,16–18,20). However, a true association or modifying role should be verified by the functional studies of the relevance of genetic modifiers to htt toxicity. The M441 polymorphism presents the first functionally defined modifier in HD pathogenesis. The identification of the M441-HAP1 polymorphism will draw further attention towards the role of HAP1 in the pathogenesis of HD in several new directions. First, the protective effect of HAP1 can help the development of therapies for HD. Secondly, it would be interesting to explore the role of the M441-HAP1 polymorphism in vesicle trafficking (54) or synaptic transmission (55) in vivo and whether it influences the function of other HAP1 interactors. Finally, the strategy of investigating the M441-HAP1 polymorphism outlined in the present study may help analyze other modifying factors of AAO in HD.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
HD patients
Overall we examined 980 unrelated Middle European HD patients (20,22). The AAO of HD patients was estimated as the age at which motor or cognitive symptoms first occurred. The mean AAO of HD patients was 45.2 years (SD 13.4) and ranged from 5 to 85 years. CAG repeat lengths in the IT15 gene had been tested in all patients, and CAG numbers have been standardized in a reference laboratory. The number of the expanded CAG repeats ranged from 39 to 90 with a median repeat number of 44. All participating individuals gave informed consent according to the Declaration of Helsinki.

Sequencing of the human HAP1 gene
The entire coding region of the human HAP1 gene (GenBank accession no. isoform 2: NM_003949; isoform 1: NM_177977 [GenBank] ) was initially sequenced in a total of 60 unaffected European individuals. In this initial sample, the frequencies for the minor alleles of the six analyzed polymorphisms were: K4R (g.11A>G): 0.490; S58T (g.172T>A): 0.170; S357L (g.6304C>T): 0.216; g.6202C>T: 0.216; R437W (g.7215C>T): 0.410; T441M (isoform 2: g.7073C>T; isoform 1: g.7537C>T; dbSNP: rs4523977): 0.221 (56). Genotyping of the different human HAP1 polymorphisms was performed by different methods depending on the best approach for the respective polymorphism. PCR amplification according to standard conditions preceded each. Subsequently, the M441 polymorphism was detected by a Wave® DNA Fragment Analysis system (Transgenomic, Inc., San Jose, CA) following the manufacturer's recommendations. The specific column temperature was 58.5°C with a gradient of the B buffer of 58–68%. Indistinct peak pattern was collected and sequenced by CEQ 8000 Dye Terminator Cycle Sequencing (Beckman Coulter, Inc., Fullerton, CA, USA) with a specific primer. The polymorphisms K4R, S58T, S357L and g.6202C>T were determined by restriction analysis with specific enzymes. The respective PCR products were incubated with 2 U EarI (K4R), 1.5 U MboI (S58T), 1.5 U BsrI (S357L) and 3 U HhaI (g.6202C>T) according to the manufacturer's instructions (New England Biolabs, Inc., Beverly, MA, USA). The R437W polymorphism was detected by pyrosequencing with the PSQ 96MA system (Biotage AB, Uppsala, Sweden).

Reagents
The following antibodies were used: rabbit polyclonal antibodies against human HAP1 and htt (EM48) and mouse anti-htt (mEM48) (23,24,54), GAPDH (Abcam, Cambridge, UK), a Flag® tag (Sigma, St Louis, MO, USA) and KLC (26); mouse monoclonal antibodies against the HA epitope (Cell Signaling, Danvers, MA, USA), {gamma}-tubulin (Sigma, St Loui, MO, USA), V5 tag (Invitrogen, Carlsbad, CA, USA) and c-myc tag (Sigma, St Louis, MO, USA).

Structure analysis
Analysis of the protein structure of HAP1 was performed using The PredictProtein Server (http://cubic.bioc.columbia.edu/predictprotein/predictprotein.html).

Interaction studies
The wild-type isoform of human HAP1 (GenBank accession no. NM_177977 [GenBank] ) is designated as T441 and the polymorphic isoform (T441M) (dbSNP: rs4523977) as M441. To investigate the interaction between HAP1 and htt, cultured HEK293 cells were transfected with pcDNA-T441- or M441-HAP1 and pRK-N208-120Q or pRK-N208-44Q htt. Forty-eight hours after transfection, cells were fixed with 4% paraformaldehyde and subjected to immunofluorescent staining. The images were taken and analyzed with a Zeiss microscope (Axiovert 200 MOT) with a digital camera and the Openlab software (Improvision). Immunoprecipitation was performed as described previously (23,31). To determine interactions of T441 and M441 with type 1 inositol 1,4,5-triphospate receptor (IP3R1), Kalirin and KLC, cultured HEK293 cells were transiently transfected by using LipofectamineTM 2000 (Invitrogen, Carlsbad, CA, USA), as directed by the manufacturer, with pCMV Tag4A-T441 or -M441 and the respective interaction partners pcDNA-IP3R1 (gift from I. Bezprozvanny, Department of Physiology, University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75390, USA), peak10-Kalirin (generated by B. Eipper, Edge Biosystems, Gaithersburg, MD, USA) or pRK-KLC (26). Forty-eight hours after transfection cells were lysed with 300 µl RIPA lysis buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.1% SDS, 0.5% deoxycholic acid, 1% Triton X-100). A total of 1000µg protein was incubated with 40µl anti-Flag® agarose (Sigma, St Louis, MO, USA), washed with PBS, and resuspended in 15 µl of SDS–PAGE sample buffer. For immunoblotting, the samples were separated on 7.5–12% acrylamide gels, transferred to nitrocellulose membranes and detected with specific antibodies and the ECLTM western blotting detection reagents (GE Healthcare, Chalfont St Giles, UK).

Cell viability assay
For neuronal cell culture, cortical neurons were isolated from the rat brain cortex at embryonic day 18. Four days after culturing, neurons were co-transfected with htt (pRK-N208-23Q or N208-120Q) and hHAP1 (pCDNA-T441 or -M441) using lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) in serum-free medium. Five hours later, the medium was replaced with neurobasal/B27 medium. After 36 h, neurons were fixed in 4% paraformaldehyde. Confocal imaging was performed using the 63x oil immersion objective lens and a Zeiss LSM 510 confocal microscope system. The proportion of apoptotic neurons was determined by counting cells (about 40 neurons/each group, n = 6) with condensed nucleus or multiple apoptotic bodies.

Statistical analysis
Allele and genotype frequencies for a descriptive statistical analysis of the examined hHAP1 polymorphisms were investigated by JMP® Version 5.1 (SAS institute, Inc., Cory, NC, USA). Using the framework of generalized linear models in analysis of variance and covariance (GLM procedure of SAS® Version 9.1, SAS Institute, Inc., Cory, NC, USA, 2003), we tested the modifying role of the respective hHAP1 polymorphisms on the AAO of HD. First, we applied a model of analysis of variance with the polymorphisms and the expanded HD allele as independent variables and the AAO of HD as dependent variable. The goodness-of-fit was evaluated by the proportion of variation in the AAO explained by the coefficient of determination (R2). We obtained the best fit of our data by logarithmic transformation of the AAO and the CAG repeat number in HD. To determine the effect of the polymorphisms on AAO by an analysis of variance, the effect of the expanded HD allele (HD CAG) was calculated alone, as well as with different polymorphisms. When the respective factors were added to the effect of the expanded HD allele ({Delta}R2), a change of R2 indicated a relative improvement of the model. This method identified the percentage of the variance that was attributable to the candidate modifier loci when there was a significant P-value (P ≤ 0.05). Depending on the respective polymorphisms, some patients could not be genotyped and were therefore excluded from statistical analyses. To determine the least significant number of patients necessary for a significant effect of the respective polymorphisms, we performed a power analysis using JMP® Version 5.1 (SAS institute, Inc., Cory, NC, USA).

To verify the effect of M441 on the AAO of HD, we applied a model to analyze covariance with both the expanded HD allele and a grouped variable of <60 or ≥60 CAG units. The differential effects of CAG repeat length on the AAO in the three hHAP1 genotypes on amino acid position 441 were tested by combining individual linear regressions within genotype classes (NLIN procedure of SAS® Version 9.1, SAS Institute, Inc., Cory, NC, USA, 2003). Using likelihood-ratio statistics, the minimum parameter set was determined (slopes within genotype classes correspond to the effect of CAG repeat length; intercepts represent a basic susceptibility). {chi}2 test was used to check the data for homogeneity. The differences in the AAO of HD with different hHAP1 genotypes were determined by a two-tailed t-test (TTest procedure of SAS® Version 9.1, SAS Institute, Inc., Cory, NC, USA, 2003). The group variances were equal. To analyze the effect of T441 and M441 on htt toxicity, statistical significance (P < 0.05) was assessed using the Student's t-test whenever two groups were compared. When analyzing multiple groups, we employed ANOVA with Scheffé's post hoc test to determine statistical significance. Data are presented as mean±SEM. Calculations were performed with SigmaPlot 4.11 and Prism (version 4) software.


   SUPPLEMENTARY MATERIAL
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
FUNDING
 REFERENCES
 
Supplementary Material is available at HMG Online.


   FUNDING
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
REFERENCES
 
This study was supported by the GeNeMove Network for hereditary movement disorders (01GM0603); the German Human Genome Project (DHGP2); the German National Genome Research Network (NGFN); and the National Institutes of Health grants NS045016 (S.-H.L.), NS036232 and AG019206 (X.-J.L.).


   ACKNOWLEDGEMENTS
 
We thank M.E. MacDonald (Massachusetts General Hospital, Boston, MA, USA) and R.H. Myers (Boston School of Medicine, Boston, MA, USA) for reading the manuscript and advice. We are grateful to the patients for participating in the study, and in particular to the following neurologists and geneticists for providing DNA samples of HD patients: F. Laccone (Department of Medical Genetics, Vienna, Austria), S. Didonato and C. Gellera (National Institute of Neurology Carlo Besta, Milano, Italy), H.W. Lange (Rehazentrum, Düsseldorf, Germany), H. Weirich-Schwaiger (Medical University of Innsbruck, Innsbruck, Austria), B. Melegh (University of Pécs, Pécs, Hungary), J.T. Epplen (University of Bochum, Germany), J. Zaremba (Institute of Psychiatry and Neurology, Warsaw, Poland), A.N. Basak (Bogazici University Bebek Istanbul, Istanbul, Turkey), J. Zidovska (University and Teaching Hospital, Prague, Czech Rebublic), M. Pandolfo (Erasme Hospital, Brussels, Belgium), L. Kadasi (Slovak Academy of Sciences, Bratislava, Slovakia), M. Kvasnicova (Department of Clinical Genetics, Banska Bystrica, Slovakia), B.H.F. Weber (University of Regensburg, Regensburg, Germany), F. Kreuz (Klinikum für Psychiatrie, Chemnitz, Germany), M. Dose (BKH-Taufkirchen, Taufkirchen, Germany) and M. Stuhrmann (University of Hannover, Hannover, Germany). Additionally, we thank I. Bezprozvanny (Department of Physiology, University of Texas Southwestern Medical Center at Dallas, Dallas, USA) and B. Eipper (Edge Biosystems, Gaithersburg, USA) for the cDNA of the IP3R1 and Kalirin.

Conflict of Interest statement. None declared.


   FOOTNOTES
 
{dagger}The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors. Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 

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M. Swami, A. E. Hendricks, T. Gillis, T. Massood, J. Mysore, R. H. Myers, and V. C. Wheeler
Somatic expansion of the Huntington's disease CAG repeat in the brain is associated with an earlier age of disease onset
Hum. Mol. Genet., August 15, 2009; 18(16): 3039 - 3047.
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