Increased SUMO-1 expression in the unilateral rotenone-lesioned mouse model of Parkinson's disease

Neuroscience Letters 544 (2013) 119–124

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Increased SUMO-1 expression in the unilateral rotenone-lesioned mouse model of Parkinson’s disease J. Weetman a , M.B. Wong a , S. Sharry a , A. Rcom-H’cheo-Gauthier a , W.P. Gai b , A. Meedeniya a , D.L. Pountney a,∗ a b

School of Medical Science, Griffith University, Gold Coast, Australia Centre for Neuroscience, Flinders University, Adelaide, Australia

h i g h l i g h t s • • • • •

SUMO-1 modifier in unilateral mouse Parkinson’s disease model (young adult and aged groups). Brain homogenates showed SUMO-1 species increased in lesioned brain hemisphere. Greater relative SUMO-1 increase observed in the young animals. ␣-Synuclein revealed several molecular weight species increased in the lesioned hemisphere. Greater relative increase in ␣-synuclein in the aged group.

a r t i c l e

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Article history: Received 26 July 2012 Received in revised form 12 February 2013 Accepted 31 March 2013 Keywords: ␣-Synuclein SUMO Rotenone Parkinson’s disease

a b s t r a c t Parkinson’s disease (PD) is a neurodegenerative disease resulting from progressive loss of dopaminergic nigrostriatal neurons. ␣-Synuclein protein conformational changes, resulting in cytotoxic/aggregated proteins, have been linked to PD pathogenesis. We investigated a unilateral rotenone-lesioned mouse PD model. Unilateral lesion of the medial forebrain bundle for two groups of male C57 black mice (n = 5); adult (6–12 months) group and aged (1.75–2 years) group, was via stereotactic rotenone injection. After 2 weeks post-lesion, phenotypic Parkinsonian symptoms, resting tremor, postural instability, left-handed bias, ipsiversive rotation and bradykinesia were observed and were more severe in the aged group. We investigated protein expression profiles of the post-translational modifier, SUMO-1, and ␣-synuclein between the treated and control hemisphere, and between adult and aged groups. Western analysis of the brain homogenates indicated that there were statistically significant (p < 0.05) increases in several specific molecular weight species (ranging 12–190 kDa) of both SUMO-1 (0.75–4.3-fold increased) and ␣-synuclein (1.6–19-fold increase) in the lesioned compared to un-lesioned hemisphere, with the adult mice showing proportionately greater increases in SUMO-1 than the aged group. © 2013 Elsevier Ireland Ltd. All rights reserved.

Neurodegenerative diseases display progressive loss of specific neurons, resulting in central nervous system dysfunction [5]. Parkinson’s disease (PD) affects over 6 million people worldwide and epidemiological studies indicate that numbers may double in the next two decades, due to the ageing population and the strong correlation between increased age and incidence of disease [2,9,15,26]. Parkinson’s disease is characterised by motor deficits reflecting a progressive loss of dopaminergic nigrostriatal neurons and the formation of intracellular protein aggregates called Lewy

∗ Corresponding author at: School of Medical Science, Griffith University, Gold Coast Campus, Queensland 4222, Australia. Tel.: +61 7 5552 8970; fax: +61 7 5552 8908. E-mail addresses: d.pountney@griffith.edu.au, [email protected] (D.L. Pountney). 0304-3940/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neulet.2013.03.057

bodies [12]. PD aetiology remains largely unknown, though it can be of either genetic or sporadic origin, with genetic bases accounting for ∼5% of all PD cases, the majority being idiopathic. Causes of idiopathic Parkinson’s may include chronic exposure to certain pesticides, such as rotenone [3], however, no unifying theory has yet been uncovered. In Parkinson’s disease, neurodegeneration is mainly localised to the Substantia Nigra pars compacta (SNpc) in the midbrain, a region more sensitive to oxidative stress [4,16]. Study of the pathophysiology of PD has necessitated the use of animal models, replicating the disease state via genetic modification or toxicological intervention [18]. Rotenone is a pesticide that functions via inhibition of Complex 1 of the mitochondrial respiratory chain [19]. Its use in the replication of PD gains validity via epidemiological correlations between exposure to pesticides and risk of disease development [6,7,25]. Complex 1 inhibitors, such as rotenone, can

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replicate behavioural, anatomical and biochemical characteristics of Parkinson’s disease in animal models [3,27]. Unilateral stereotactic injection of rotenone into the medial forebrain bundle (MFB) permits comparison of protein expression between treated and untreated hemisphere. The injection site allows for diffusion of rotenone to the SNpc [19] whilst avoiding gross physical trauma resulting from the surgery. The choice of acute, targeted rotenone application, over chronic systemic exposure, stems from findings that systemic exposure produces more widespread cell death and ambiguous results [24]. Furthermore, unilateral rotenone injection allows for the isolated treatment of a single hemisphere, permitting direct comparison to a ‘control’ hemisphere within the same animal, eliminating genetic variance. Protein conformational diseases, such as Parkinson’s disease, can be caused by both genetic changes and aberrant behaviour of the protein quality control systems [8]. Thus, the operation of systems that respond to, repair and, if necessary, remove potentially damaging proteins are of equal importance in PD as the proteins themselves. Age-related changes, oxidative stress and pathological mutations can promote the formation of protein aggregates detrimental to neuronal function [17]. Intraneuronal intracytoplasmic protein inclusion bodies (Lewy bodies, LB) have become the classic pathological marker of Parkinson’s disease and their presence reflects the increasing levels of aberrant, misfolded or aggregated protein [22]. A major constituent of LBs is ␣-synuclein, and they also contain SUMO-1 [20], recently shown to be associated with lysosomes in glial ␣-synuclein deposits [28]. SUMO (Small Ubiquitin-like Modifier) proteins are a family of post-translational modifiers that alter cellular distribution, metabolism and function as well as degradation through conjugation, a process known as sumoylation [23]. Interest in SUMO has increased in recent years, with expression knockout experiments showing decreased cellular survival in primary cortical cell lines [1] and the identification of ␣-synuclein as a substrate of SUMO [10]. We have investigated the brain expression profiles of SUMO-1 and ␣-synuclein species in a unilateral rotenone-lesioned mouse model of Parkinson’s disease, including both aged and adult groups, with the expression of several species of both proteins showing increases in the lesioned compared to control hemisphere.

50 mM Tris–HCl pH 7.4/0.32 M sucrose/5 mM EDTA/Sigma protease inhibitor cocktail then stored at −80 ◦ C. Crude mouse brain homogenates were diluted 1:3 in loading buffer and boiled (5 min). 12.5 ␮L of each sample was run in replicate on 12% SDS-PAGE (100 V, 2 h), transferred to nitrocellulose (80 V, 40 min), assessed via Poncean Red staining of proteins, blocked (1 h, 3% BSA, 0.5% skim milk powder), incubated with primary antibody overnight (1:1000; rabbit anti-SUMO-1 [21]); mouse anti-␣-synuclein (Chemikon, aa121-125), washed (4×, TBST), incubated with HRP-conjugated secondary antibody (1:1000; 1 h) washed 4× and developed with West Pico SuperSignal Chemiluminescent Substrate (Thermo Scientific). Membranes were imaged by Alpha Innotech Fluorochem FC2. For re-probing, blots were stripped with 0.2 N NaOH for 30 min, then re-blocked and probed for actin. Bands were quantitated by area under curve relative to actin loading controls (Alpha Innotech software). Significance was assessed via two-tailed, paired t-tests. Lysosomes were isolated from the crude brain tissue homogenates by differential centrifugation and calcium chloride treatment, as described [28]. Homogenate was centrifuged (10,000 × g; 10 min) to remove nuclei and unbroken cells. The pellet was discarded and the supernatant centrifuged at 20,000 × g for 20 min. The resultant pellet was resuspended in 18% (v/v) Percoll/1 mM CaCl2 and then centrifuged (5000 × g; 10 min) to pellet the mitochondria. The supernatant was diluted 1:5 then centrifuged (20,000 × g for 20 min) and the lysosome pellet re-suspended in 20 ␮L. For immunofluorescence, two adult (12-month) male C57 black mice injected with rotenone were perfused transcardially with 0.5% sodium nitrite in 0.1 M phosphate buffered saline followed by Zamboni’s fixative. Free floating tissue cryosections (30 ␮m) were microwaved for 10 min in 1 mM EDTA pH 8.0 for antigen retrieval, blocked (20% normal horse serum) and incubated with primary antibodies: sheep polyclonal anti-SUMO-1 (21; Abcam 22738), mouse anti-␣-synuclein (LB509; Invitrogen) and rabbit anti-cathepsin D (Abcam). Alexafluor secondary antibodies were 1:200 dilution. Imaging was by Olympus FV 1000 confocal microscope.

2. Results

1. Materials and methods Experiments (Griffith University Ethics Committee) comprised 5 aged (1.75–2 years) and 5 adult (6–12 months) C57 male mice. Mice were anaesthetised with isoflurane throughout the surgery. A medial-sagital incision was made and a scope used to determine the location of the physiological markers, Lambda and Bregma used for positioning the injection point as x (1.25 mm) and y (−0.94 mm). A single burr hole allowed for placement of the stereotactic needle, which was then slowly advanced along the z axis 5.35 mm, then retracted to 5.25 mm creating a cavity. A total of 2 ␮L rotenone (1 mg/mL, Sigma Corporation) in 1:1 DMSO:polyethylene glycol was injected in two 1 ␮L applications, 1 min each with 4 min interval. Movement repertoire was observed after 2 weeks and grasp test used to analyse potential unilateral motor deficit. Mice were encouraged to walk along a suspended bar and the number of left paw placements in comparison to total paw placements were counted from video recordings. Animals were sacrificed via injection with Ketamine (320 ␮L) and Xylizil (80 ␮L). Transcardial perfusion with sodium nitrite eliminated blood and extraneous material. The brain was dissected sagitally to separate the two hemispheres and homogenised by handheld homogeniser using 24 strokes per sample 1:5 g/mL

Two groups of male C57 black mice, young adult (6–12 months) and aged (1.75–2 years), were lesioned by unilateral injection of rotenone in the left medial forebrain bundle. We have previously established this unilateral lesion model in Sprague-Dawley rat [19]. Two weeks post-lesion, animals were observed and graded for severity of movement dysfunction. Table 1 shows the observed movement characteristics resulting from unilateral lesioning of both aged and adult animals. Five movement characteristics were investigated; resting tremor, initiation of movement, postural instability, rotational direction and left-handed bias. Resting tremor was involuntary shaking of the paws and ceased with the initiation of voluntary movement. Resting tremor was present in both groups, though more severely in the aged animals. Tremor in both adult and aged animal groups extended beyond the front paws, through the neck, face and hind limbs. Initiation of movement was difficulty initiating a new movement, such as changing direction. This was seen as the animal struggling to move for a period before moving. This movement characteristic was present in both animal groups, with aged animals more severely impaired. Postural instability was observed when the animal stumbled while moving. The aged mice had severe postural instability and were constantly stumbling and/or tipping over. Conversely, the adult mice displayed minimal postural instability, rarely stumbling whilst walking. In the aged mice, the movement deficit was so pronounced that animals failed to complete the grasp test, i.e. were unable to cross the

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Table 1 Movement characteristics of unilaterally-lesioned aged and young adult male C57 black mice. Age groupa Adult (6–12 months) Aged (>1.75 years)

Resting tremorb g

++ +++

Initiation of movementc

Postural instabilityd

Left-hand biase

Rotational directionf

+ +++

+ ++

+ n.d.h

Ipsiversive Ipsiversive

a

A total of 5 mice were lesioned in each age group. Resting tremor, involuntary shaking present in the paws of the mice and ceased with initiation of voluntary movement. c Initiation of movement, difficultly initiating new movement, such as changing direction. d Postural instability, animal stumbled while moving. e Left-handed bias, ratio of left paw placements to total number of paw placements in grasp test. f Rotation direction, favouring of one direction of movement. g +, mild; ++, moderate; +++, severe. h n.d. = not determined. In the aged mice, the movement deficit was so pronounced that animals failed to complete the grasp test. For young adult mice, the left paw accounted for 40% ± 9 of total placements. b

horizontal, suspended bar. The adult mice were able to complete the task and results indicate that they had a right-sided bias, with the left paw accounting for 40% ± 9 of total placements. Animals were sacrificed 2 weeks post-lesioning and brain tissue was prepared for either Western analysis or immunofluorescence. For immunofluorescence, fixation was by perfusion in situ, after which free-floating sections (30 ␮m) were incubated with anti-SUMO-1, anti-␣-synuclein and anti-cathepsin D primary antibodies and imaged by confocal microscopy. Fig. 1A shows typical immunofluorescence (cortex) with perinuclear cytoplasmic ␣synuclein accumulations (arrowhead) in the lesioned hemisphere. Cytoplasmic SUMO-1 granules were frequently associated with ␣synuclein deposits (arrowhead; Fig. 1B and D) in the lesioned hemisphere, but were rare in the non-lesioned hemisphere (Fig. 1E). Normal punctate SUMO-1 staining of the nuclear membrane due to the nuclear pore complex and SUMO-1-nuclear bodies were consistent in all sections. In cells with ␣-synuclein deposits, cytoplasmic SUMO-1 granules were associated with lysosomes (arrows; Fig. 1C) marked by the lysosomal marker, cathepsin D (CatD). To investigate protein expression profiles, Western blot analysis was performed for the post-translational modifier, SUMO-1, and ␣-synuclein. Western blots were subjected to integration of band intensity and were normalised to the band intensities of actin loading controls. Fig. 1F shows representative SUMO-1 and ␣-synuclein Western blots for treated (T) and control (C) hemispheres. Examination of the Western blot data indicates the consistent presence of eight major molecular weight bands immunopositive for SUMO1. These were a 190 kDa, 120 kDa, 90 kDa, 70 kDa, 60 kDa, 40 kDa, 20 kDa and 12 kDa. Fig. 1G shows examples of each of these bands from the five aged animals, for both the treated and control hemispheres. In general, the treated band was more intense than its control counterpart, especially in aged animals. Fig. 1H is a graph of mean integrated intensity of the SUMO-1 bands present for the aged animal group at different molecular weights as a ratio of the actin loading control. In the aged animals, the most significant differences are the 190 kDa and 70 kDa bands, which display a 3.5-fold and 3-fold increase, respectively, at p < 0.001. The 60 kDa band shows a 0.75-fold increase with a significance of p < 0.01. Finally, the 90 kDa, 40 kDa and 20 kDa bands also show a 0.75fold increase, significant at p < 0.05. Whilst the 120 kDa and 12 kDa bands showed an increase in SUMO-1 expression, this increase did not show statistical significance. Fig. 1I shows the mean normalised band integrals for the adult animal group. In the adult mice, the 70 kDa band showed a 4.3-fold increase at a significance of p = 0.001. The 60 kDa band shows a 2.5-fold increase (p = 0.0071). The 12 kDa band showed a significant (p = 0.0044) 2-fold increase, from the treated to control hemispheres. The 90 kDa band showed a significant (p = 0.0159) 3.4-fold increase in the means of the control and treated hemispheres and the 40 kDa band showed a significant (p = 0.0369) almost 2-fold increase in SUMO-1 expression. Whilst there was an increase in the 190 kDa, 120 kDa and 20 kDa

bands, this increase was not significant. There is a clear increase in SUMO-1 in the treated hemisphere of both age groups in all bands from the Western blot data. There are five common statistically significant increased bands between the two age groups, the 90 kDa, 70 kDa, 60 kDa, 40 kDa and 12 kDa bands. The aged mice showed a significant 190 kDa band increase, whereas the increase in the treated hemisphere was not significant in the adult mice for this band. By comparing the full lane integrals for all molecular weight species, we found that SUMO-1 expression has a statistically significant (p = 0.0023), 1.9-fold increase in the mean in the treated hemispheres in the aged animals. In the adult animals, there is a statistically significant (p = 0.0005) 2.67-fold increase in SUMO-1 expression between the treated and control hemispheres. In order to investigate changes in the expression profile for SUMO1 localised in lysosomes, Western blot analysis was performed on enriched lysosomal fractions. Fig. 1J represents the mean of SUMO-1 present within the lysosomal fraction of the crude brain homogenates for both the aged and adult mice. Three bands with significant differences between the control and treated sample groups were at 120 kDa, 90 kDa and 40 kDa, with the 90 kDa band showing the greatest increase in the treated hemisphere. Western blot data for ␣-synuclein indicated the consistent presence of six major molecular weight bands of ␣-synuclein at 120 kDa, 90 kDa, 60 kDa, 40 kDa, 20 kDa and 15 kDa. Fig. 2A shows representative Western blot data for ␣-synuclein in the adult animals, indicating an increase in the expression of ␣-synuclein in the treated hemisphere, when compared to the control. Fig. 2B shows the mean band integrals of ␣-synuclein of the aged animal group at different molecular weights. In the aged animals, the most significant difference is the 20 kDa band, which shows a 19-fold increase, at p < 0.0001, as this band was only faintly present in the control samples. The 120 kDa, 90 kDa and 15 kDa bands all show significant increases (3.9-fold, 4.2-fold and 3.4-fold, respectively) at p ≤ 0.001 from the control to treated hemispheres. The 60 kDa band shows a 2.6-fold increase significant at p = 0.0115. Whilst the 40 kDa band showed an increase in ␣-synuclein expression, this increase did not show statistical significance. Fig. 2C shows mean normalised band integrals for the adult group. In the adult mice, 60 kDa and 20 kDa bands showed an increase at a significance of p < 0.01. The 60 kDa band shows a 2-fold increase (p = 0.0011). The 20 kDa band showed a significant (p = 0.0028) 5.5-fold increase, from the treated to control hemispheres. The 120 kDa, 90 kDa and 40 kDa bands all showed a significant (p = 0.0118, 0.0231 and 0.0202 respectively) increase, 1.6-fold, 2.3-fold and 2.8-fold respectively, in the means of the control and treated hemispheres. There was an increase in the 15 kDa (monomer) band from the control to treated hemisphere, however this was not statistically significant. Overall, there was an increase in ␣-synuclein in the treated hemisphere of both age groups in all bands from, though not all of these increases are statistically significant. Comparing the aged group to the adult mice indicated a higher relative increase in expression of ␣-synuclein in the aged group for

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Fig. 1. SUMO-1 protein expression profiles in the treated and control hemispheres of aged and adult mice. (A–C) Immunofluorescence (rotenone-lesioned hemisphere cortex, 30 ␮m) showing (A) ␣-synuclein perinuclear deposits (green; arrowhead); (B) SUMO-1 cytoplasmic granules (red; arrowhead) associated with ␣-synuclein (green) focal cytoplasmic accumulations in a subset of cells; (C) SUMO-1 granules (green; arrows) associated with lysosomes (red, cathepsin D) in cells with ␣-synuclein (purple) deposits; (D and E) SUMO-1 granules (green) colocalised with ␣-synuclein deposits (red) primarily in lesioned (D), rather than non-lesioned hemisphere (E). Scale bar, 5 ␮m. (F) Representative Western blots for SUMO-1 and ␣-synuclein in crude brain tissue homogenates of the rotenone-treated and control hemispheres of aged mice. (G) Representative Western blot bands for SUMO-1 in crude brain tissue homogenates of the rotenone-treated (A1T, A2T, A3T, A4T, A5T) and control (A1C, A2C, A3C, A4C, A5C) hemispheres of aged mice. (H) Graph of mean band integrals of aged animals. (I) Graph of mean band integrals for adult mice. (J) SUMO-1 expression profiles in isolated lysosomal fractions of the crude brain tissue homogenates of lesioned and non-lesioned hemispheres (mean of aged and adult mice). Overall, the graph indicates an increase in SUMO-1 localisation within lysosomes in response to cellular stress.*p < 0.05 (or 95%); **p < 0.01 (or 99%); ***p < 0.001 (or 99.9%). Error bars indicate the standard error of the mean (SEM). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)

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Fig. 2. ␣-Synuclein protein expression profiles of aged and adult mice. (A) Representative ␣-synuclein Western bands for the treated (Y1T, Y2T, Y3T) and control (Y1C, Y2C, Y3C) hemispheres for the adult mice. (B) Graph of the mean normalised band intensity for aged mice. (C) Integrated band intensities for the adult mice. Error bars indicate the standard error of the mean (SEM). *p < 0.05 (or 95%); **p < 0.01 (or 99%); ***p < 0.001 (or 99.9%); ****p < 0.0001 (or 99.99%).

most bands. Comparing the mean of the total ␣-synuclein lane integral within the aged mice group shows that ␣-synuclein expression has a statistically significant (p = 0.0003) 3.6-fold increase in the mean from the control to treated hemispheres. In the adult animals, there is a statistically significant (p = 0.0002) 2.3-fold increase in ␣-synuclein between the treated and control hemispheres. There was no significant difference between the age groups in total ␣synuclein in the untreated hemispheres. 3. Discussion We examined changes in SUMO-1 and ␣-synuclein protein expression profiles between lesioned and unlesioned brain hemispheres in a unilateral rotenone-lesioned mouse model of PD

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that involved stereotactic rotenone injection into the medial forebrain bundle in adult (6–12 months) and aged (1.75–2 years) animal groups. Movement observations showed that the aged mice exhibited a markedly greater decrease in motor control, when compared to their younger counterparts. The most severe of the motor deficits was the presence of the resting tremor seen in both aged animals and adult mice. Also present were asymmetries of postural movements, more specifically, ipsiversive rotation, turning towards the targeted hemisphere, consistent with dopaminergic loss at the Substantia Nigra. This is supported by work conducted by Gratton and Wise [11] that found stimulation of the SNpc resulted in contralateral rotation, turning away from the targeted hemisphere. The aged mice had more difficulty initiating movement, frequently pausing before they were able to move or change direction. There was an increase in the expression of SUMO-1, from the control to treated hemisphere, in both the adult and aged mice. The increase in SUMO-1 expression between the control to treated hemisphere may indicate an upregulation of SUMO and/or sumoylation in response to cellular stress. As SUMO-1 is a component of Lewy bodies, the increase in SUMO-1 could reflect the formation of Lewy body-like protein aggregates in response to rotenone lesioning. This is consistent with imaging of brain tissue sections that showed association of SUMO-1 granules with ␣-synuclein intracytoplasmic aggregates. Moreover, colocalization of SUMO1 with lysosomes in the lesioned hemisphere is in agreement with our recent findings on human and rat tissue and cell culture models [28]. Recently, SUMO-1 knockdown in transgenic mice has been shown to lead to increased susceptibility to cerebral ischaemia/reperfusion injury, indicating a neuroprotective function of SUMO-1 [14]. Interestingly, Krumova et al. [13] have shown recently that SUMOylation of ␣-synuclein in vitro results in a less aggregation-prone protein and that an ␣-synuclein mutant with impaired SUMOylation showed fewer aggregates in HEK293T cells. Our results show increased SUMO-1 in lysosomes associated with a 90KDa band. Interestingly, a SUMOylated 90 kDa band is also increased in lysosomes in 1321N1 cells subjected to proteasome inhibition [28]. Our results also indicated changes in the protein expression profile of ␣-synuclein, between the treated and control hemispheres. Though the role of ␣-synuclein is not completely understood, there is a well-documented increase in ␣-synuclein high molecular weight species in Parkinson’s disease. Our results consistently showed a change in the protein expression profiles of SUMO-1 and ␣-synuclein in response to rotenone treatment; there was a global increase in expression of both these proteins over the bands present. Our results show a lesser relative increase in SUMO-1 and greater increase in ␣-synuclein in the aged animals, compared to the adult mice, in the treated hemisphere, compared to the control, suggesting a link to ageing. Acknowledgments This study was supported by Australian Research Council, Griffith Health Institute, the Clem Jones Foundation and NHMRC. References [1] D. Anderson, K. Wilkinson, J. Henley, Looking ahead: sumoylation in neuropathological conditions, Drug News Perspect. 22 (2009) 10. [2] N. Archibald, D. Burn, Movement disorders: Parkinson’s disease, Medicine (Baltimore) 36 (2008) 630–635. [3] R. Betarbet, T.B. Sherer, G. MacKenzie, M. Garcia-Osuna, A.V. Panov, J.T. Greenamyre, Chronic systemic pesticide exposure reproduces features of Parkinson’s disease, Nat. Neurosci. 3 (2000) 1301–1306. [4] H. Braak, J.R. Bohl, C.M. Muller, U. Rub, R.A.I. de Vos, K.D. Tredicic, Stanley Fahn Lecture 2005: the staging procedure for the inclusion body pathology

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