Retinal dysfunction, photoreceptor protein dysregulation and neuronal remodelling in the R6/1 mouse model of Huntington's disease

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Abstract

Huntington's disease (HD) is a progressive neurological disease characterised by motor dysfunction, cognitive impairment and personality changes. Previous work in HD patients and animal models of the disease has also highlighted retinal involvement. This study characterised the changes in retinal structure and function early within the progression of disease using the R6/1 mouse model of HD. The retinal phenotype was observed to occur at the same time in the disease process as other neurological deficits such as motor dysfunction (by 13 weeks of age). There was a specific functional deficit in cone response to the electroretinogram and using immunocytochemical techniques, this dysfunction was found to be likely due to a progressive and complete loss of cone opsin and transducin protein expression by 20 weeks of age. In addition, there was an increase in Müller cell gliosis and the presence of ectopic rod photoreceptor terminals. This retinal remodelling is also observed in downstream neurons, namely the rod and cone bipolar cells. While R6/1 mice exhibit significant retinal pathology simultaneously with other more classical HD alterations, this doesn't lead to extensive cell loss. These findings suggest that in HD, cone photoreceptors are initially targeted, possibly via dysregulation of protein expression or trafficking and that this process is subsequently accompanied by increased retinal stress and neuronal remodelling also involving the rod pathway. As retinal structure and connectivity are well characterised, the retina may provide a useful model tissue in which to characterise the mechanisms important in the development of neuronal pathology in HD.

Highlights

► Retinal phenotype in mouse model of Huntington's disease. ► Retinal change appeared by 13 weeks of age, co-incident with motor dysfunction. ► A functional cone deficit likely arose from loss of cone opsin and transducin protein. ► Ectopic rod photoreceptors and remodelled rod and cone bipolar cells were observed. ► There was an increase in Müller cell gliosis, yet limited cell loss.

Introduction

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder characterised by progressive development of motor, cognitive and psychiatric deficits which generally affects people from midlife and leads to a reduction in life expectancy (Reddy et al., 1999, Zoghbi and Orr, 2000). The cause of HD is an expansion in the CAG repeat region (encoding polyglutamine) within exon 1 of the Huntingtin gene (HTT) (Huntington's-Disease-Collaborative-Research-Group, 1993, Zoghbi and Orr, 2000). Individuals with 36 CAG repeats or more develop clinical symptoms of HD. Even though the huntingtin protein (Htt) is widely expressed in the brain, the disease usually leads to a selective loss of neurons, particularly within the striatum and cerebral cortex and to a lesser extent the hippocampus and sub-thalamus (Tobin and Signer, 2000). Despite ongoing advances, the normal function of Htt and the mechanisms leading to the progressive neurodegeneration are yet to be fully understood.

In addition to the classical neurological symptoms of HD, there is evidence that other tissues may also be directly affected. Studies have shown possible retinal involvement, with retinal increment thresholds higher in patients suffering HD compared with age matched controls, or patients with other neurological abnormalities (Paulus et al., 1993). However, while these retinal neurons show a functional alteration, post-mortem investigation of a single HD patient has shown no evidence for gross retinal pathology (Petrasch-Parwez et al., 2005). Further support for potential retinal changes in HD comes from studies investigating other polyglutamine neurological diseases, such as Spinocerebellar ataxia 7 (SCA7), in which patients develop extensive retinal degeneration (Michalik et al., 2004).

Retinal dysfunction and degeneration are consistently observed in rodent and Drosophila models of HD (Helmlinger et al., 2002, Jackson et al., 1998, Petrasch-Parwez et al., 2004). The first evidence of a retinal pathology associated with the expression of mutant huntingtin protein was reported in Drosophila, where photoreceptor degeneration was progressive and dependent on polyglutamine repeat length (Jackson et al., 1998). The HD mouse models, R6/1 and R6/2 have also been investigated with respect to retinal pathology (Helmlinger et al., 2002, Petrasch-Parwez et al., 2004). The R6/1 model has been reported to exhibit reductions in rod and cone function in addition to alterations in downstream neuronal function at a late stage of disease (32 weeks of age). These animals also exhibited disruption in the outer retina and photoreceptor degeneration at this age. Similar changes were observed in the retina of the R6/2 model although at earlier ages (approx. 10 weeks) (Helmlinger et al., 2002), in line with the general observation that R6/2 mice exhibit an accelerated form of disease.

The mechanism(s) leading to neuronal loss in HD is not fully understood, however, transcriptional dysregulation, excitotoxicity, energy dysfunction and formation of protein aggregates have all been implicated (Crook and Housman, 2011, Roze et al., 2008). Within the retina, reports have highlighted a possible role for a Ca2+-dependent increase in synaptic transmission in subsequent photoreceptor degeneration (Romero et al., 2008). Furthermore, gene expression profiling has revealed significant decreases in the expression of genes involved in the phototransduction and photoreceptor differentiation pathways in the R6/2 mouse (Abou-Sleymane et al., 2006). Another study has also detailed alterations in the expression of selective connexins, which are critical to the formation of retinal gap junctions (Petrasch-Parwez et al., 2004).

Most studies investigating retinal involvement in HD have characterised late stages in the disease process. Investigation of the early alterations in retinal pathology may provide insight into the disease mechanism within the retina and may help explain the apparent lack of any overt retinal degeneration in HD patients (Petrasch-Parwez et al., 2005). Thus, the aim of this study was to characterise changes in retinal structure and function early within the progression of disease using the R6/1 mouse model of HD. We show that at the same time when motor dysfunction becomes evident, there is significant retinal dysfunction characterised by a progressive and complete loss of cone opsin and transducin expression. In addition to this dysregulation of protein expression, there is an increase in retinal stress, cell death and remodelling of rod photoreceptors, as well as downstream neurons.

Section snippets

Animals

The R6/1 transgenic mouse line and age matched wild type (WT) control littermates of ages 7, 13 and 20 weeks were obtained from the Howard Florey Institute, University of Melbourne. The R6/1 hemizygote males were originally obtained from the Jackson Laboratory (Bar Harbor, ME, USA) (Mangiarini et al., 1996) and bred with CBB6 (CBAxC57/B6) F1 females to establish the R6/1 colony at the Howard Florey Institute (Nithianantharajah et al., 2008). Animals were maintained on a 12 hour light/dark cycle

Retinal human transgene expression and motor dysfunction in R6/1 mice

In order to validate the use of this model in the current study, the expression of the human transgene and motor dysfunction were assessed in R6/1 mice. Fig. 1A shows the expression of the mouse endogenous Htt gene (159 bp) in all retinal and brain samples. As expected, only the R6/1 animals show amplification of the human transgene (103 bp) in both retinal and brain samples. No amplified products were found in the respective negative controls. Fig. 1B shows motor function in WT and R6/1 mice at

Discussion

In addition to the tissues classically involved in HD (e.g. the striatum), previous work has shown the retina to be functionally altered in HD patients (Paulus et al., 1993) and exhibit late-stage degeneration in animal models. This study characterised early retinal alterations within the progression of disease in the R6/1 mouse model of HD. Retinal changes occurred at similar points in disease progression to other HD-related effects, such as motor dysfunction. Cone-specific decreases in

Conclusions

This study has shown that retinal alterations in a mouse model of HD occur at similar times to other neuronal deficits. The retinal phenotype is progressive, with the cone pathway targeted first. The deficit in the cone pathway arises due to loss of key proteins involved in cone phototransduction. In addition to these alterations, the retina shows signs of neuronal remodelling, gliosis and limited cell death. While the function of the rod system is initially preserved in this mouse model of HD,

Acknowledgments

The authors would like to thank Drs Terence Pang and Thibault Renoir for providing some of the R6/1 and wild type tissue samples used in this work.

References (44)

  • A.J. Tobin et al.

    Huntington's disease: the challenge for cell biologists

    Trends Cell Biol.

    (2000)
  • M.M. Ward

    Localization and possible function of P2Y(4) receptors in the rodent retina

    Neuroscience

    (2008)
  • A.E. Weymouth et al.

    Rodent electroretinography: methods for extraction and interpretation of rod and cone responses

    Prog. Retin. Eye Res.

    (2008)
  • G. Abou-Sleymane

    Polyglutamine expansion causes neurodegeneration by altering the neuronal differentiation program

    Hum. Mol. Genet.

    (2006)
  • P. Avasthi

    Trafficking of membrane proteins to cone but not rod outer segments is dependent on heterotrimeric kinesin-II

    J. Neurosci.

    (2009)
  • J.C. Blanks et al.

    Selective lectin binding of the developing mouse retina

    J. Comp. Neurol.

    (1983)
  • M. Caramins

    Genetically confirmed clinical Huntington's disease with no observable cell loss

    J. Neurol. Neurosurg. Psychiatry

    (2003)
  • B. Chang

    The nob2 mouse, a null mutation in Cacna1f: anatomical and functional abnormalities in the outer retina and their consequences on ganglion cell visual responses

    Vis. Neurosci.

    (2006)
  • J. Chua

    Functional remodeling of glutamate receptors by inner retinal neurons occurs from an early stage of retinal degeneration

    J. Comp. Neurol.

    (2009)
  • S. Engelender

    Huntingtin-associated protein 1 (HAP1) interacts with the p150Glued subunit of dynactin

    Hum. Mol. Genet.

    (1997)
  • M.A. Fox et al.

    Synaptotagmin I and II are present in distinct subsets of central synapses

    J. Comp. Neurol.

    (2007)
  • U. Greferath

    Rod bipolar cells in the mammalian retina show protein kinase C-like immunoreactivity

    J. Comp. Neurol.

    (1990)
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    This work was supported by the National Health and Medical Research Council (NHMRC) of Australia (NHMRC grant #350434 to ELF) and Retina Australia. AJH is an Australian Research Council (ARC) Future Fellow.

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