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Human Molecular Genetics Pages 813-822  

Formation of polyglutamine inclusions in non-CNS tissue
Introduction
Results
   Detection of polyQ inclusions in non-CNS tissues
   Ultrastructure of skeletal muscle inclusions
   Shrinkage/atrophy of organs in R6/2 transgenic mice
   Appearance of nuclear inclusions in non-CNS tissues
   Investigation into the basis of the muscle atrophy in lines R6/1 and R6/2
   Muscle fibre type analysis
   Ultrastructural analysis
Discussion
   Inclusion formation
   Distribution of inclusions
   The relationship between neuronal inclusions and pathology
   The relationship between non-neuronal inclusions and pathology
   Non-CNS pathology and inclusions in HD
   Conclusion
Materials And Methods
   Mice
   Antibodies, immunocytochemistry, histology and ultrastructure
   Morphometry
Abbreviations
Acknowledgements
References

Formation of polyglutamine inclusions in non-CNS tissue

Formation of polyglutamine inclusions in non-CNS tissue

Kirupa Sathasivam+, Carl Hobbs1,+, Mark Turmaine2, Laura Mangiarini, Amarbirpal Mahal, Fabien Bertaux, Erich E. Wanker3, Pat Doherty1, Stephen W. Davies2 and Gillian P. Bates*

Medical and Molecular Genetics, GKT Medical and Dental School, King's College, 8th Floor, Guy's Tower, Guy's Hospital, London SE1 9RT, UK, 1Experimental Pathology, GKT Medical and Dental School, King's College, 4th Floor, Hodgkin Building, Guy's Hospital, London SE1 9RT, UK, 2Department of Anatomy and Developmental Biology, University College London, Gower Street, London WC1E 6BT, UK and 3Max Planck Institute for Molecular Genetics, Ihnestrasse 73, Berlin Dahlem, Germany

Received November 19, 1998; Revised and Accepted February 11, 1999

Huntington's disease (HD) is one of a class of inherited progressive neurodegenerative disorders that are caused by a CAG/polyglutamine repeat expansion. We have previously generated mice that are transgenic for exon 1 of the HD gene carrying highly expanded CAG repeats which develop a progressive movement disorder and weight loss with similarities to HD. Neuronal inclusions composed of the exon 1 protein and ubiquitin are present in specific brain regions prior to onset of the phenotype, which in turn occurs long before specific neurodegeneration can be detected. In this report we have extended the search for polyglutamine inclusions to non-neuronal tissues. Outside the central nervous system (CNS), inclusions were identified in a variety of post-mitotic cells. This is consistent with a concentration-dependent nucleation and aggregation model of inclusion formation and indicates that brain-specific factors are not necessary for this process. To possibly gain insights into the wasting that is observed in the human disease, we have conducted a detailed analysis of the timing and progression of inclusion formation in skeletal muscle and an investigation into the cause of the severe muscle atrophy that occurs in the mouse model. The formation of inclusions in non-CNS tissues will be particularly useful with respect to in vivo monitoring of pharmaceutical agents selected for their ability to prevent polyglutamine aggregation in vitro, without the requirement that the agent can cross the blood-brain barrier in the first instance.

INTRODUCTION

Huntington's disease (HD) is one of a family of inherited, late onset, neurodegenerative disorders that are caused by a polyglutamine (polyQ) expansion. The other members of this group include dentatorubral pallidoluysian atrophy (DRPLA), spinal and bulbar muscular atrophy (SBMA) and the spinocerebellar ataxias (SCA1-3, 6 and 7) (1). Comparison of the genetics and molecular biology of these diseases shows that they have a number of features in common, namely a dominant pattern of inheritance, anticipation and the broadly comparable sizes of the normal and expanded repeat ranges. The proteins containing these polyQ expansions are unrelated, in most cases of unknown function and have extensively overlapping expression patterns (1,2).

We have previously generated a mouse model of HD that is transgenic for exon 1 of the human HD gene, containing highly expanded CAG repeats, under the control of the HD promoter. Three lines [R6/1, (CAG)115; R6/2, (CAG)145; R6/5, (CAG)128-156] develop a progressive neurological phenotype, the symptoms of which include a movement disorder and weight loss with similarities to HD (3,4). On the basis of home cage behaviour, line R6/2 has an age of onset of ~2 months. However, a more recent detailed assessment of motor function has shown that deficits can be detected at between 5 and 6 weeks and that the age at which a phenotype is apparent is dependent upon the sensitivity of the test and the difficulty of a particular task (5,6). The disease progresses rapidly such that mice are rarely kept beyond 14 weeks of age.

Immunocytochemistry with antibodies to the truncated exon 1 protein and to ubiquitin identified neuronal intranuclear inclusions in brains from all symptomatic lines (7). At the ultrastructural level, inclusions can be observed in the absence of immunolabelling as pale staining granular and fibrillar structures devoid of a membrane. Only a single nuclear inclusion is detected per neuron. In line R6/2, inclusions are first identified in the cortex at 3.5 weeks and in the striatum at 4.5 weeks, prior to the onset of symptoms (7). In contrast, selective cell death is not observed in these structures until 14-17 weeks of age, suggesting that the symptoms are due to a neuronal dysfunction rather than as a result of neurodegeneration (M. Turmaine, A. Raza, A. Mahal, L. Mangiarini, G.P. Bates and S.W. Davies, submitted for publication). Coincident with the first appearance of inclusions, a decrease in brain weight is observed (7). It has not yet been determined whether this is the consequence of cell loss, cell shrinkage, loss of processes or a reduction in the intercellular volume. In the two lines showing a slower progression, R6/1 and R6/5, inclusions are also observed in neurites and occasionally in astrocytes (M. Turmaine, A. Raza, A. Mahal, L. Mangiarini, G.P. Bates and S.W. Davies, submitted for publication).

Insights into the structure of the polyQ inclusions have come from in vitro analysis of GST-huntingtin exon 1 fusion proteins (8). It was found that GST fusion proteins with (CAG)83 and (CAG)121 spontaneously formed highly insoluble aggregates with a fibrillar morphology. Removal of the GST tag from proteins with (CAG)51 also resulted in amyloid-like fibrils that showed green birefringence when stained with Congo red and examined under polarized light. Aggregation was not observed with proteins containing (CAG)20 or (CAG)30 repeats in the non-pathogenic range. This is consistent with the proposal by Perutz et al. (9) that polyQ stretches can form polar zippers via hydrogen bonding in a cross-[beta]-pleated amyloid-like structure. A detailed analysis of the concentration and polyQ length dependence of aggregation has shown that the ability to form these highly ordered structures closely correlates with the polyQ pathogenic threshold (10).

Neuronal inclusions are now clearly established as the pathological signature of polyQ disease and have been identified in post-mortem brains from HD (11-13), SCA1 (14), SCA3 (15), SCA7 (16), DRPLA (12,17) and SBMA patients (18). In all cases they are composed of the specific polyQ-containing protein and ubiquitin. Inclusions in affected neurons of SCA1 patients and an SCA1 transgenic mouse model stain positive for antibodies that detect the 20S proteasome and the molecular chaperone HDJ-2/HSDJ (19). Similarly, inclusions in HD patient brains and transgenic mice contain the 20S proteasome and several components of the 19S and 11S activation complexes (S.W. Davies, unpublished data).

The factors that govern the cell specificity of both inclusion formation and neurodegeneration for each of the polyQ diseases are not understood. The ectopic expression of a polyQ expansion within the hprt gene, introduced by gene targeting, has demonstrated that polyQ tracts are capable of aggregation in a protein context other than that already associated with disease (20). These mice developed a progressive neurological disorder associated with neuronal intranuclear inclusions, although neurodegeneration was absent. This clearly demonstrates that interactions specific to the polyQ diseases are not necessary for the formation of polyQ aggregation in neuronal populations.

In this report, we describe the distribution of polyQ inclusions in tissues outside the central nervous system (CNS) in the R6 transgenic mouse lines. We have conducted a detailed analysis of the timing and formation of inclusions in non-neuronal tissues in relation to organ shrinkage/atrophy. We have carried out a detailed investigation into the basis of the observed muscle atrophy in order to: (i) provide insights into the loss of muscle bulk observed in HD (21,22); and (ii) provide a parallel analysis of the formation and consequence of neuronal inclusions. The identification of inclusions in non-CNS tissues will be useful for monitoring in vivo trials of pharmaceutical agents designed to prevent polyQ aggregation.

RESULTS

Detection of polyQ inclusions in non-CNS tissues

In order to identify inclusions in non-CNS tissues, an R6/2 male mouse at 14 weeks of age was perfusion fixed and individual organs were paraffin wax embedded, sectioned and processed for immunohistochemistry using the CAG53b and anti-ubiquitin antibodies. Tissues and cell types in which inclusions were detected are summarized in Table 1 and examples are illustrated in Figure 1. In all cases, inclusions were only detected in cell nuclei; in most cases a single inclusion was present although some nuclei contained up to three inclusions (especially in cells in the liver, kidney and skeletal muscle). Inclusions were found in the muscle fibres of skeletal and cardiac muscle but were not present in the smooth muscle of the stomach wall. In the adrenal glands, inclusions were restricted to the medulla and the reticula layer of the cortex and they were not present in the glomerulosa and fasciculata layers. In the pancreas they were restricted to the islets of Langerhans, in the liver to the hepatocytes and in the kidney were identified at a low level in the tubular, interstitial and glomerular cells. They were found in the neuronal ganglion cells of the myenteric plexus of the stomach wall and Meissner's plexus of the duodenum. Inclusions were absent from skin, spleen, testis, coagulation gland, the mucosal cells of the stomach, duodenum, colon and buccal cavity and the white adipose tissue.


Figure 1. Intranuclear inclusions in non-CNS tissues. Sections were from an 8-week-old (a) and 14-week-old (b-f) R6/2 mouse immunoprobed with anti-ubiquitin antibodies (a) and CAG53b anti-huntingtin antibodies (b-f). Intranuclear inclusions are present in skeletal muscle (a), pancreas (b), liver (c), adrenal gland (d) and kidney (e) but not in spleen (f). Nuclei were counterstained with methyl green. Scale bar, 10 µm.

Table 1. Intranuclear inclusion-positive cell types
Tissue Inclusion-positive cell type
Skeletal muscle Muscle fibres
Heart Cardiac muscle
Liver Hepatocytes
Adrenal glands Medullary and reticula cells
Pancreas Islets of Langerhans
Kidney Tubular/interstitial/glomerular cells
Stomach wall Myenteric plexus
Duodenum Meissner's plexus


We had been specifically interested in investigating the presence of inclusions in tissues accessible by biopsy which could have an important application in monitoring clinical trials. Of the possible candidate tissues, skin, mucosal cells and skeletal muscle, inclusions were only identified in muscle.

Ultrastructure of skeletal muscle inclusions

An ultrastructural analysis of nuclear inclusions in skeletal muscle was performed. The inclusions appear as pale staining granular and fibrillar structures identical to those previously observed in neurons (Fig. 2). This electron microscopical analysis suggested that at least in some cases the appearance of more than one inclusion per nucleus at the light microscopy level could be accounted for by single inclusions in adjacent nuclei.


Figure 2. Ultrastructural analysis of nuclear inclusions in skeletal muscle from a 12-week-old R6/2 mouse. (a) Muscle fibre nucleus containing a nuclear inclusion (arrow) adjacent to the more darkly stained nucleolus. (b) Adjacent nuclei each containing a single inclusion. The morphology of the muscle fibres, including the mitochondria and motor end plate, are perfectly normal. Scale bar, 2 µm.


Shrinkage/atrophy of organs in R6/2 transgenic mice

A series of R6/2 male mice had previously been used to determine the timing of the progressive decrease in brain weight with respect to the loss in body weight (7). This comprised seven or eight mice at each time point ranging from 2 to 12 weeks. It was found that the decrease in brain weight occurred after 4 weeks, considerably earlier than the decrease in body weight, which commenced at ~7 weeks. This study has now been extended to determine the onset and extent of atrophy in other organs. To this end, the heart, liver, spleen, kidneys, thymus, testes and quadriceps and soleus muscles were dissected from the original series of male R6/2 mice and weighed (Fig. 3). The most dramatic atrophy occurred in the testes, which, like brain, began to decrease in weight after 4 weeks. By 8 weeks, the transgenic testes were 88% of the weight of the non-transgenic testes, after which point they plummeted in weight to 15% at 12 weeks. This testicular atrophy most likely underlies the loss of fertility that is encountered in the R6/2 males. Approximately 50% of R6/2 males are sterile, and of those that do breed, all are sterile by 8 weeks of age. The quadriceps and soleus muscles appear to develop normally until 6 weeks of age, after which time they atrophy to 42 and 47% of the weight of those dissected from the non-transgenic mice at 12 weeks. Similarly, weight loss begins in heart and kidney at 6 weeks and in liver at 8 weeks, decreasing to 55, 50 and 75% of the non-transgenic organs at 12 weeks, respectively.


Figure 3. Progressive organ atrophy in line R6/2. The quadriceps and soleus muscles (a), testes (b), liver (c), kidneys (d) and spleen (e) were dissected and weighed from a series of R6/2 male mice ranging in age from 2 to 12 weeks. Weight loss was detected in testes from 4 weeks, both muscles and kidney from 6 weeks and liver from 8 weeks. Transgenic tissue is represented by a solid line and non-transgenic tissue by a dotted line.


Appearance of nuclear inclusions in non-CNS tissues

An analysis of the onset and progression of inclusion formation was conducted in longitudinal sections taken from snap-frozen quadriceps muscle of R6/2 mice that were 2, 4, 6, 8, 12 and 14 weeks of age. In each case, three transgenic mice and a single non-transgenic littermate control were processed at each time point. Inclusions were not present at 4 weeks but could be seen by 6 weeks of age. Overall, a focal distribution of nuclei containing inclusions was observed, making quantification difficult. To gain an estimate of the frequency of inclusions, the number of inclusions present in ~300 nuclei in adjacent fields in 8 µm longitudinal sections was counted for each mouse. This suggested that the number of inclusions increased steadily from 6 (5 ± 1%) to 12 weeks (18 ± 3%). However, this analysis provides only an approximation and a more accurate figure will require extensive quantification over several muscle types.

To determine the appearance of inclusions in the other tissues, two transgenic male mice and one non-transgenic littermate were perfusion fixed and paraffin embedded sections were prepared at 4, 6, 8 and 10 weeks of age. In all cases, inclusions were not present at 4 weeks and were readily apparent by 8 weeks. At 6 weeks they were more prominent in pancreas, kidney and adrenals than in liver.

Investigation into the basis of the muscle atrophy in lines R6/1 and R6/2

In order to shed light on the loss of muscle bulk observed in the human disease, we set out to uncover the basis of the muscle atrophy that occurs in the R6 transgenic lines. Transverse sections from the quadriceps muscle of 14-week-old R6/2 and 15-month-old R6/1 mice were cut from snap-frozen tissue and stained using haematoxylin and eosin (H&E), haematoxylin-van Gieson (HVG) and the periodic acid-Schiff (PAS) assay. Acid phosphatase and NADH-tetrazolium reductase (NADH-TR) enzyme activities were measured and immunohistochemistry was performed with the anti-N-cam antibody (H28).

There was little evidence of myopathy as indicated by muscle fibre regeneration. If present, internal nuclei can be detected (particularly noticeable in the PAS stained sections) (Fig. 4). Whilst the frequency of internal nuclei was slightly elevated (4% in R6/1 and 1% in R6/2 as compared with 1 and 0.3% in their respective controls), this was not apparent to an extent that would be indicative of myopathy. Similarly, vesicular nuclei or fibre splitting could not be detected, which would also provide indications of myopathy. Regenerating fibres are uniformly immunoreactive for the anti-N-cam antibody and only 2% in R6/1 and 0.3% in R6/2 were evident as compared with 0.7 and 0.3% in their respective controls. The muscle atrophy is not caused by denervation. Examination of all sections revealed no evidence of a focal atrophy and the motor endplate distribution, as demonstrated by anti-N-cam immunohistochemistry, also appeared normal (Fig. 4). We could find no evidence of cellular infiltration in response to necrotic fibres on the basis of acid phosphatase activity or of the presence of granular or basophilic fibres on the H&E stained sections. The HVG stained sections showed no fibrosis, as is evident from the proliferation of the endomysial or perimysial connective tissue, or of the presence of myofibril central cores (Fig. 4). The PAS stained tissue showed no dramatic increase in the glycogen content of the transgene muscles or abnormalities in the basal membrane. In contrast, the transgene sections showed little staining, possibly reflecting the emaciation that had occurred in these mice. The PAS staining can distinguish type 1 and type 2 fibres on the basis of glycogen content and this indicated that there was probably no selective atrophy or specific fibre type grouping (Fig. 4), although this was pursued in more depth as outlined below.


Figure 4. Histological and immunocytochemical analysis of skeletal muscle from lines R6/1 and R6/2. Transverse frozen sections through quadriceps muscle from a 15-month-old non-transgenic littermate (a, e and i), a 15-month-old R6/1 transgenic (b, f and j), a 14-week-old non-transgenic littermate (c, g and k) and a 14-week-old R6/2 transgenic (d, h and l) were stained for PAS (a-d), immunoprobed with an anti-N-cam antibody (e-h) and stained for HVG (i-l). Nuclei were counterstained with haematoxylin. Scale bar, 40 µm

Muscle fibre type analysis

To further examine the possibility of a specific fibre type atrophy or fibre type grouping, NADH-TR activity was measured and immunohistochemistry was performed with antibodies to the specific myosin heavy chains present in the various muscle fibre types. Sections from the quadriceps muscle of a 14-week-old R6/2 mouse and 15-month-old R6/1 mouse were cut from snap-frozen tissue as before and the immunoreactivity to anti-type 2A myosin is shown in Figure 5. A morphometric analysis was also conducted. This analysis demonstrated that there was no fibre type grouping. The lesser fibre diameter was measured for the sections through the quadriceps muscle from the 14-week-old R6/2 mouse and from the extensor digitorum longus muscle of the R6/1 mouse. The normal distribution evident in these graphs (Fig. 5e and f) shows quite clearly that a comparatively uniform atrophy of all muscle fibres had occurred in the transgenics.

Ultrastructural analysis

Ultrastructural analysis of R6/2 quadriceps muscle at 14 weeks showed no evidence of myopathy or neuropathy. The only feature that distinguished it from non-transgenic muscle was the presence of intranuclear inclusions (Fig. 2). In contrast, the ultrastructure of skeletal muscle from the 15 month R6/1 mouse did show evidence of degeneration (Fig. 6). Non-specific ultrastructural changes included enlarged mitochondria with a well-preserved proliferation of cristae, less elaborate motor endplates with a reduction in the number of post-synaptic folds and endplate-associated fibrous structures.

   a - d
   e - f

Figure 5. Type 2A fibre typing and muscle fibre morphometry in R6/1 and R6/2 skeletal muscle. Frozen transverse sections from a 15-month-old non-transgenic littermate (a), a 15-month-old R6/1 transgenic (b), a 14-week-old non-transgenic littermate (c) and a 14-week-old R6/2 transgenic (d) were immunoprobed with an anti-myosin heavy chain type 2A antibody. This shows no evidence of type 2A fibre atrophy. Scale bar, 40 µm. The lesser fibre diameter was measured for the extensor digitorum longus muscle from an R6/1 mouse at 15 months (e) and the quadriceps muscle from an R6/2 mouse at 14 weeks (f). Dotted line, non-transgenic littermates; solid line, transgenics.


Figure 6. Ultrastructure of degenerative muscle in line R6/1. Non-specific ultrastructural changes which may consist of fibrillar structures (F), changes in motor end plate morphology (M) (a) reassociated muscle fibres (R) and enlarged mitochondria (b). Scale bar, 2 µm.


DISCUSSION

We have extended the search for polyQ inclusions to the non-CNS tissues of a transgenic mouse model of HD. Nuclear inclusions were found in post-mitotic cells of a number of tissues, including skeletal and cardiac muscle, liver, kidney, adrenal glands and pancreas. In most cases, the presence of inclusions correlates with a progressive decrease in size of the respective organ. An extensive histological analysis of skeletal muscle found no evidence of myopathy or neuropathy, but instead that the decrease in muscle bulk was caused by a uniform shrinkage across all muscle fibre types.

Inclusion formation

The molecular pathogenesis of polyQ disease is triggered by the gain of function that the polyQ expansion imparts to the protein in question. The gain of this new function must correlate precisely with the threshold at which the repeat expands into the pathogenic range and any proposed molecular mechanism of pathogenesis must account for the late onset of the disease. It has previously been shown by in vitro analysis that the likely gain of function is the ability of expanded polyQ repeats to form amyloid-like aggregates (8). More recently, a detailed study of the concentration and polyQ length dependence of the aggregation of polyQ tracts in the context of the HD protein has shown that aggregation occurs at a polyQ repeat length that shows a striking correlation with the pathogenic threshold observed in HD (10).

The nucleation and aggregation model for the formation of inclusions predicts that an initial slow nucleation step, possibly in equilibrium with the soluble form of the protein, is followed by a more rapid aggregation step (23). The predicted kinetics could account for the late onset of the disease both in humans and in the transgenic mice. Evidence from (i) immunohistochemistry on post-mortem brain material with antibodies spanning the huntingtin protein (11-13), (ii) cell culture models transfected with various portions of huntingtin (24-28) and (iii) in vitro aggregation experiments (8) indicates that it is an N-terminal fragment of huntingtin that is the precursor of aggregation and that is present in the inclusions. Therefore, the molecular pathogenesis of the human disease would require a cleavage/proteolysis step whereas, in the R6 mouse lines, the transgene only codes for the N-terminus, therefore by-passing this initial step in the disease pathogenesis. Therefore, in the mice the rate of formation of aggregates could be predicted to depend on polyQ length and the concentration of the transgene protein whereas in the human disease the cleavage/proteolytic event that generates the N-terminal precursor may provide an additional rate-limiting step. The precise nature of this fragment and the cleavage/proteolysis step involved in the disease are unknown.

Distribution of inclusions

The formation of inclusions in neurons is likely to occur as a result of the terminally differentiated nature of these cells allowing the critical concentration for aggregation to be reached. A mitotically active cell might be expected to dilute the concentration of the precursor molecule at each cell division. However, the identification of inclusions in non-proliferating astrocytes (S.W. Davies, unpublished data) indicates that they can form in mitotically active cells. Therefore, should inclusions be present in peripheral tissues, they could be predicted to occur similarly in post-mitotic cells and possibly mitotically active cells dependent upon the level of expression of the protein. An expression profile of huntingtin outside the CNS has not been studied in depth. In situ hybridization to human tissues showed expression of the HD gene to be uniformly low in the acinar, ductal and islet of Langerhans cells of the pancreas and hepatocytes, bile ducts and connective tissues of the liver. Developing sperm in testes expressed variable levels of HD mRNA, with immature spermatogonia expressing higher levels than maturing spermatids (29). It has not been possible to determine the level of the transgene protein by immunohistochemistry in the R6 lines as it cannot be distinguished from the mouse protein with available antibodies. However, if the level of transgene protein in non-CNS tissues is similarly low, a distribution of inclusions in post-mitotic cells, as identified in this report, is consistent with the reliance of inclusion formation upon the local concentration of the transgene protein.

In all cases, inclusions were identified in nuclei. In neurons, the normal distribution of huntingtin is reported to be extranuclear (30-32) and the mechanism by which the transgene protein enters the nucleus is unknown. The subcellular localization of huntingtin in peripheral tissues has been the subject of much less extensive scrutiny and a nuclear localization in some cell types cannot be ruled out. Indeed, a nuclear localization of huntingtin in cell cultures established from mouse embryonic fibroblasts, human skin fibroblasts and mouse neuroblastoma cells has previously been documented (33). Still, irrespective of the normal localization of huntingtin in these cell types, in all cases the transgene product can clearly enter and accumulate in the nucleus.

The relationship between neuronal inclusions and pathology

The phenotype of the R6 lines is caused by neurodysfunction and not neurodegeneration. In line R6/2, inclusions are present in the cortex and striatum (3-4 weeks) prior to the onset of symptoms (~8 weeks), which is long before specific neurodegeneration can be seen in these brain regions (14-17 weeks). Changes in the expression levels of specific neurotransmitter receptors can be detected from as early as 4 weeks (34), providing an insight into a possible mechanism of dysfunction. Shortly after the onset of symptoms, inclusions can be detected in the majority of, if not all, neurons. However, within the lifetime of the R6/2 mouse, cell death is the consequence of polyQ aggregation in only a small number of neurons residing in the cortex, striatum and cerebellum (S.W. Davies, unpublished data).

Immunohistochemical analysis of HD, DRPLA, SCA3 and SCA7 post-mortem brains has indicated that the distribution of inclusions overlaps with, but is also wider than, the classically described neuropathology (11,12,16). Therefore, in the lifetime of the patient, polyQ aggregation and the eventual presence of an inclusion, as detected by light microscopy, does not necessarily lead to the degeneration of the neuron in which it has formed. The pattern of neurodegeneration may arise from a selective susceptibility to polyQ aggregate toxicity.

The relationship between non-neuronal inclusions and pathology

Within the skeletal muscle of the R6/2 mice, there was a remarkable correlation between the appearance of inclusions at 6 weeks and the onset of muscle atrophy. We have conducted an extensive histological study and found no evidence of myopathy or neuropathy and at close to end stage the only consistent difference between the R6/2 muscle and that of the non-transgenic littermates was the size of the muscle fibres. It would appear that the muscle fibres have undergone a uniform shrinkage. We are not aware of other diseases in which a loss of muscle bulk is the result of this uniform atrophy that encompasses all muscle fibre types. Therefore, as in brain, degenerative changes are only observed after a protracted time course, in this case in the form of non-specific ultrastructural changes in a 15-month-old R6/1 mouse. These findings are in contrast to the muscle pathology observed in other myopathies that are caused by amyloid inclusions. Sporadic inclusion body myositis (s-IBM) is characterized by the presence of amyloid inclusions in the nucleus and cytoplasm of muscle fibres which have been found to contain [beta]-amyloid, two other epitopes of the amyloid precursor protein, hyperphosphorylated tau, [alpha]1-antichymotrypsin, apolipoprotein E, ubiquitin, presenilin-1 and cellular prion protein (35). There are three sequential and overlapping aspects of muscle fibre destruction in s-IBM: an attack on muscle fibres by cytotoxic T lymphocytes, vacuolar degeneration and a denervation atrophy of the muscle fibres. The hereditary inclusion body myopathies are similar to the s-IBMs except that they lack the lymphocytic inflammation (36). Recent transgenic models of these disorders have demonstrated a complex myopathy (37,38). Similarly, oculopharyngeal muscular dystrophy is caused by modest expansions of a GCG/alanine repeat expansion in the poly(A)-binding protein 2 gene, resulting in intranuclear inclusions within muscle fibres (39). This disease is also characterized morphologically by the presence of rimmed vacuoles (40). A uniform shrinkage of the muscle fibres has not been reported for any of these disesaes.

In addition to brain and muscle, we have found that the liver, kidney, heart and testis of the R6/2 mice also decrease in size. With the exception of testis, these organs contain inclusions in a subset of cell types, the appearance of which broadly correlates with the onset of weight loss. Is it possible that the presence of inclusions can lead to cell shrinkage? Comparison with other mouse models may argue against this as a general cell autonomous mechanism. No loss in brain weight was reported in mice in which the hprt gene had been targeted with a polyQ expansion, despite an extensive distribution of neuronal inclusions (20).

Recent reports of diabetes in R6/2 mice (41) are probably accounted for by the identification of inclusions in the pancreatic islets. It is likely that the presence of the polyQ inclusions directly leads to cellular dysfunction in these cells. In 72% of cases, type II diabetes is associated with the deposition of amylin between the endocrine cells and capillaries, usually penetrating into deep invaginations of the plasma membrane of the B cells. It is thought that the accumulation of amyloid in the islets is likely to impair islet function and may be a causal factor in the development of type II diabetes (42). Consistent with this hypothesis, mutations in the amylin gene are responsible for an early onset form of type II diabetes (43). The deposition of polyQ amyloid in pancreatic islets may similarly lead to an impairment of islet function. It is interesting that an increase in the frequency of diabetes in HD patients, as compared with the normal population, has been reported (44), although it is not known whether this is related to polyQ amyloid deposition. However, the diabetes may contribute to the loss of body weight observed in these mice.

Non-CNS pathology and inclusions in HD

There is little documentation of peripheral organ weight and/or histology in HD. One detailed study of liver morphology was conducted in response to the observation that HD patients commonly presented with a minor disturbance of liver function (45). This paper reported that in HD the liver tends to be smaller than normal and the histology suggested that hepatocytes in HD have a shortened survival time. In addition, a recent review (46) discussed an unpublished study of 122 HD cases in which organ weight was found to be significantly reduced for cases with increased severity of brain involvement (E.D. Bird, C. Hall and R.H. Myers, unpublished data). Weight loss was not uniform across all tissues and was most evident in the liver and heart and least evident in the kidney.

A recent study of the peripheral organs in SBMA identified non-neural inclusions in scrotal skin, dermis, kidney, heart and testis but not in spleen, liver and muscle (47). In this case, the cells containing inclusions were all mitotic cells, capable of mitosis in adulthood. Inclusions were nuclear and contained the N-terminus of the androgen receptor (AR). The authors suggest that this pattern could not be explained by expression levels alone and could be influenced by the presence of a protease that cleaves the N-terminal fragment from the full-length protein, specific proteins that interact with the mutant AR or different AR functional activities among the different tissues. It is not known whether inclusions form in HD peripheral organs as there are no ultrastructural reports or examples of immunocytochemistry using anti-N-terminal huntingtin antibodies to post-mortem HD tissues outside the CNS. As discussed above, in the R6 lines the probable initial step in the pathogenic pathway is missing, that of cleavage/proteolysis to generate the aggregation precursor. Therefore, any tissue variations in the distribution of factors involved in the generation of this fragment would be expected to impact on the cell specificity of inclusions. In addition, the R6 transgenic lines are clearly an extreme version of the disease, with polyQ expansions greater than that generally found in juvenile HD. Therefore, it may be necessary to look at juvenile cases to determine whether the distribution of non-neural inclusions seen in the mice might be mirrored in patients, and these studies will be undertaken when suitable tissues become available.

Conclusion

PolyQ inclusions have been identified in a number of post-mitotic non-CNS cells in a mouse model of HD. The most immediate application of these findings will be with respect to testing therapeutic interventions. A major effort will be invested in the identification of small molecules that can prevent or slow down the kinetics of aggregate formation. Nuclear inclusions in non-CNS tissues will be used to assess whether compounds, selected for their ability to prevent aggregation in vitro, can prevent or slow down inclusion formation in vivo without having to worry in the first instance whether they are capable of crossing the blood-brain barrier.

MATERIALS AND METHODS

Mice

Transgenic mouse lines R6/1 and R6/2 [Jackson codes B6CBA-TgN(HDexon1)61 and B6CBA-TgN(HDexon1)62] can be obtained from the Induced Mutant Resource (Jackson Laboratory, Bar Harbor, ME). Lines R6/1 and R6/2 were maintained by backcrossing to (C57BL/6×CBA)F1 mice. Genotyping and CAG repeat sizing was as previously described (4). The timing and progression of organ atrophy were assessed by weighing organs dissected from a series of male mice that had been perfusion fixed with 4% paraformaldehyde.

Antibodies, immunocytochemistry, histology and ultrastructure

The sources and working dilutions of antibodies were as follows: anti-ubiquitin (1:2000; Dako, Copenhagen, Denmark); CAG53b (1:5000; 7); anti-N-cam H28 (1:500; Boehringer Mannheim, Mannheim, Germany); anti-myosin heavy chain type 1 (A4.84), type 2 (N3.36) and type 2A (A474) skeletal muscle fibre antibodies (Simon Hughes, King's College, London, UK). Immunohistochemistry was performed as previously described (48) on 5 µm paraffin embedded peripheral tissue sections (49) from mice that had been perfusion fixed with paraformaldehyde/lysine/periodate fixative, with the exception of muscle for which 8 µm frozen sections were prepared (48). Ultrastructural analysis (48) and histological stains (49) were carried out as published.

Morphometry

The lesser muscle fibre diameter was measured by a semi-automated approach using the colour freelance program (Foster Findlay Associates, Newcastle upon Tyne, UK).

ABBREVIATIONS

AR, androgen receptor; CNS, central nervous system; DRPLA, dentatorubral pallidoluysian atrophy; HD, Huntington's disease; H&E, haematoxylin and eosin; HVG, haematoxylin-van Gieson; NADH-TR, NADH-tetrazolium reductase; PAS, periodic acid-Schiff base; polyQ, polyglutamine; s-IBM, sporadic inclusion body myositis; SBMA, spinal and bulbar muscular atrophy; SCA, spinocerebellar ataxia.

ACKNOWLEDGEMENTS

We wish to thank Anne Young, Stefan Buk, Rick H. Myers, Jean Bolt, Jean Louis Mandel, Adriano Aguzzi and Hans Lehrach for enlightening discussions, and Robin Poston for help with the muscle fibre morphometry. This work was supported by grants from the Wellcome Trust, European Union (BMH4 CT96 0244), Hereditary Disease Foundation (in the form of an award to G.P.B. from Harry Lieberman), the Huntington's Disease Society of America and the Special Trustees of Guy's Hospital.

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*To whom correspondence should be addressed. Tel: +44 171 955 4485; Fax: +44 171 955 4444; Email: gillian.bates@kcl.ac.uk
+These authors contributed equally to this work

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J. Carmichael, J. Chatellier, A. Woolfson, C. Milstein, A. R. Fersht, and D. C. Rubinsztein
Bacterial and yeast chaperones reduce both aggregate formation and cell death in mammalian cell models of Huntington's disease
PNAS, August 15, 2000; 97(17): 9701 - 9705.
[Abstract] [Full Text] [PDF]

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