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Human Molecular Genetics Advance Access originally published online on July 17, 2008
Human Molecular Genetics 2008 17(20):3128-3143; doi:10.1093/hmg/ddn210
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© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Parkin deletion causes cerebral and systemic amyloidosis in human mutated tau over-expressing mice

Jose A. Rodríguez-Navarro1, Ana Gómez1, Izaskun Rodal1, Juan Perucho1, Armando Martinez3, Vicente Furió3, Israel Ampuero4, María J. Casarejos1, Rosa M. Solano1, Justo García de Yébenes2 and Maria A. Mena1,*

1 Department of Neurobiology 2 Department of Neurology, Hospital ‘Ramón y Cajal’, Madrid, Spain 3 Department of Pathology, Hospital Universitario San Carlos, Madrid, Spain 4 Banco de tejidos para Investigaciones Neurológicas and CIBERNED, Madrid, Spain

* To whom correspondence should be addressed at: Departamento de Investigación, Hospital Ramón y Cajal, Ctra. de Colmenar, Km. 9, Madrid 28034, Spain. Tel: +34 913368384; Fax: +34 913369016; Email: maria.a.mena{at}hrc.es

Received May 7, 2008; Accepted July 16, 2008


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
Deposition of proteins leading to amyloid takes place in some neurodegenerative diseases such as Alzheimer’s disease and Huntington’s disease. Mutations of tau and parkin proteins produce neurofibrillary abnormalities without deposition of amyloid. Here we report that mature, parkin null, over-expressing human mutated tau (PK–/–/TauVLW) mice have altered behaviour and dopamine neurotransmission, tau pathology in brain and amyloid deposition in brain and peripheral organs. PK–/–/TauVLW mice have abnormal behaviour and severe drop out of dopamine neurons in the ventral midbrain, up to 70%, at 12 months and abundant phosphorylated tau positive neuritic plaques, neuro-fibrillary tangles, astrogliosis, microgliosis and plaques of murine β-amyloid in the hippocampus. PK–/–/TauVLW mice have organomegaly of the liver, spleen and kidneys. The electron microscopy of the liver confirmed the presence of a fibrillary protein deposits with amyloid characteristics. There is also accumulation of mouse tau in hepatocytes. These mice have lower levels of CHIP-HSP70, involved in the proteosomal degradation of tau, increased oxidative stress, measured as depletion of glutathione which, added to lack of parkin, could trigger tau accumulation and amyloidogenesis. This model is the first that demonstrates β-amyloid deposits caused by over-expression of tau and without modification of the amyloid precursor protein, presenilins or secretases. PK–/–/TauVLW mice provide a link between the two proteins more important for the pathogenesis of Alzheimer disease.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
Several sporadic and familial neurodegenerative diseases [Alzheimer’s disease (AD), fronto-temporal dementia (FTD), progressive supranuclear palsy (PSP), cortico basal degeneration (CBD) etc.] present abnormal tau function and pathological deposits of this protein in the human brain. Tau is a protein which stabilizes microtubules and plays a critical role in the neuronal transport system. Most tauopathies have unknown origin and pathogenesis, and they are considered related to posttranslational alterations of tau (1). The familiar tauopathies, described in several hundred families around the world, are related to more than 30 different mutations of the Tau gene, located in chromosome 17. They are clinically characterized by a variable combination of parkinsonism and dementia (2).

The most important sporadic tauopathy in humans is AD, a degenerative dementia characterized pathologically by the extracellular deposition of amyloid and intraneuronal accumulation of neuro-fibrillary tangles, which are immunoreactive to tau (3). The link between tau and β-amyloid peptide is unknown.

Parkinson’s disease (PD) is a disorder of multifactorial aetiology (4). From 10 to 25% of cases present familial aggregation and in the last few years more than 10 genes or loci have been found to be associated with familial parkinsonism transmitted according to mendelian inheritance (5). Deletions or point mutation of the Park-2 gene that codes for parkin, a protein with ubiquitin ligase function, produce PD through the world (6).

The interrelations between tau and parkin are of particular interest. Van de Warrenburg et al. (7) reported a Dutch family with autosomal recessive early-onset parkinsonism and heterozygous missense mutation in combination with a heterozygous exon deletion in the parkin gene. The proband had parkinsonism and ataxia, selective loss of dopaminergic neurons in the substantia nigra pars compacta without Lewy bodies or neuro-fibrillary tangles, neuronal loss in the spinocerebellar system and widespread tau pathology. Morales et al. (8) and Sanchez et al. (9) described a patient with a clinical and pathological phenotype of PSP, and with tau pathology and high levels of phosphorylated tau in his brain due to a single heterozygous C212Y parkin mutation (4,5). In addition, Ros et al. (10) described that in PSP, a disease characterized by important tau pathology, there is an association with the V380L polymorphism of parkin, suggesting that the genetic variants of parkin may modulate the relative risk of tauopathy.

In order to test whether the interaction of tau and parkin is clinically relevant in patients with neurodegenerative diseases, we have crossed a mouse model of tauopathy that over-expresses human mutated tau (TauVLW), with the mutations G272V, P301L and R406W (11), with parkin null mice (PK–/–), produced by a deletion of exon 3 of parkin (12). We and others have previously reported that young double transgenic, PK–/–/TauVLW, mice show mild symptoms of akinesia and dopamine dysfunction, behavioural abnormalities and abnormal deposits of phosphorylated tau in the nervous system (13–15).

In this study, we report that the clinical phenotype observed in young PK–/–/TauVLW increases dramatically with age, the severity of cerebral lesions worsens, and, unexpectedly, the animals develop cerebral and systemic amyloidosis. The amyloidosis takes place without alterations of the proteins usually involved in the production or metabolism of β-amyloid peptide.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
Development and behaviour of PK–/–/TauVLW mice
The development of all groups of mice was normal. PK–/–/TauVLW mice gained less weight than WT during their first 3 months of age and subsequently ceased to grow (Supplementary Material, Table S1). The differences in weight increased with age. At 9 months of age, the ambulatory motor activity, measured as the distance covered in an open field, is reduced in the PK–/–/TauVLW mice (Fig. 1A). The route followed during 10 min by a representative 9-month-old mouse (Fig. 1B) shows graphically that TauVLW and PK–/–/TauVLW mice do not ambulate on the central zone of the open field. This behavioural pattern of avoidance of the central zone is considered characteristic of anxiety. PK–/–/TauVLW run smaller distances in central zone than the other groups (Fig. 1C) and had a decreased number of total entries in the Y maze test, which also suggest that these mice have higher anxiety and decreased ambulatory activity (Fig. 1D). PK–/– and TauVLW mice showed also less total entries than WT confirming the data of alternation in T-maze previously shown for PK–/– mice (12,16). There was a tendency, which did not reach significant levels, towards a reduced mnemonic function, as represented by the alternation index in the Y maze, in PK–/–/TauVLW mice (Fig. 1E).


Figure 1
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  		Figure 1. Motor activity, anxiety, learning, compulsive behaviours and dopamine parameters in the limbic system of 9-month-old WT, PK–/–, TauVLW, and PK–/–/TauVLW mice. (A) Distance covered during 10 min, index of the motor activity, decreased in PK–/–/TauVLW mice. (B) Route followed during 10 min by a representative WT, PK–/–, TauVLW and PK–/–/TauVLW mice in the Actitrack apparatus. (C) Distance run in the central area, far from the walls, which is an anxiety index. (D) The number of total arm entries in the Y-maze test, reflects decreased general activity and increased anxiety of PK–/–/TauVLW mice. (E) Spontaneous alternation behaviour in the Y-maze is a mnemonic index and tends to be decreased in PK–/–/TauVLW mice. (F) Barbering score scale of 6-month-old animals. (G) Barbering score evolution with age in the WT, PK–/–, TauVLW and PK–/–/TauVLW mice. (H) Duration of grooming of 9-month-old male mice over a 10 min period post water mist spray. (I) Typical western blots of DAT and TH in the limbic system of 9-month-old WT, PK–/–, TauVLW and PK–/–/TauVLW mice. (J) Comparative levels of DAT and (K) of TH in limbic system of these four groups of animals. Values are the mean ± SEM (n = 6 animals per experimental group). Statistical analysis was performed by one-way ANOVA followed by Newman–Keuls test. *P < 0.05, **P < 0.01, ***P < 0.001 transgenic versus WT mice; +P < 0.05, ++P < 0.01, +++P < 0.001 PK–/–TauVLW versus PK–/– mice; ^P < 0.05, ^^^P < 0.001 PK–/–TauVLW versus TauVLW mice. The two-way ANOVA followed by Bonferroni post-test showed interaction between parkin deletion and TauVLW overexpression in the number of total arm entries in the Y-maze test (*P < 0.05) and that parkin deletion is the main source of variation for the barbering score and TauVLW overexpression is the main source of variation for the grooming after mist spray.

 
Compulsive behaviour of PK–/–/TauVLW mice and dopamine parameters in the limbic system
PK–/–/TauVLW mice showed motor abnormalities such as myoclonus, uncontrolled movements, loss of balance and postural abnormalities, such as dorsal kyphosis, which increased with age. We present a short videotape of 3-month-old mice in the open field arena as supplementary material. We can see the shortened stride length, the anxious exploration and the compulsive repetition of stereotyped movements.

From 6 months of age, PK–/– mice showed higher barbering scores, an index of long-term grooming, than WT and TauVLW. This abnormal behaviour was so exacerbated in PK–/–/TauVLW mice that they presented even self-injury of the skin of the face and ears (Fig. 1F and G). PK–/–/TauVLW mice spent significantly more time grooming after a water mist spray reaching in the worst cases up to 70% of the observation period (Fig. 1H), following the prototypical syntactic grooming pattern described for the hyper-dopaminergic mutant mice (17).

Key proteins for dopamine synthesis, as TH, and synaptic transport, as dopamine transporter (DAT), were reduced in PK–/–/TauVLW mice ( Fig. 1I–K). The level of reduction, with respect to WT, was comparatively greater for DAT than for TH (73 and 82.5%, respectively of WT) suggesting a more severe disturbance of dopamine reuptake than of synthesis in the limbic system of PK–/–/TauVLW mice.

Loss of dopamine neurons in the midbrain of 12-month-old PK–/–/TauVLW mice
The loss of TH+ neurons, which we observed in young animals (13), greatly increased, up to 70% at 12 months of age in PK–/–/TauVLW mice (Fig. 2A–E). The TH levels in 9-month-old mice (Fig. 2F) are lower than in 3-month-old. These data probe a progressive, age-related, nigrostriatal cell death in PK–/–/TauVLW mice. We observed tau pathology in the form of tau deposition in neurons and tau positive fibres in the substantia nigra of TauVLW mice and more important in PK–/–/TauVLW mice (Fig. 2 G, H and J). Tau immunostaining in neurons from PK–/–/TauVLW mice was observed in the cytoplasm and in the nuclear and perinuclear area (Fig. 2J).


Figure 2
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  		Figure 2. Loss of dopamine neurons, astrogliosis and microgliosis in the midbrain of PK–/–/TauVLW mice. Representative microphotographs of the ventral midbrain of WT (A), PK–/– (B), TauVLW (C) and PK–/–/TauVLW (D) mice immunostained with anti-TH antibody. (A–D) Scale bar = 200 µm. (E) Average number of TH+ neurons per ventral midbrain section of 12-month-old mice expressed as the mean ± SEM (n = 3–5). Representative western blot of TH (F), GFAP (K) and GLUT-5 (N) proteins as markers of DA neurons, astrocytes and microglial cells, and their respective densitometric histograms. β-actin was used as charge control. Values are expressed as the mean ± SEM (n = 6 animals in each experimental group). Representative microphotographs of the substantia nigra immunostained with anti TH (green) and anti tau (red) antibodies (G–J); with anti-TH (green) and anti-GFAP (red) antibodies (L and M) and with isolectin B4-HRP as marker of microglial cells in (O), and double stained with TH in red (P). The nuclei were stained with bis-benzimide (blue). (G, H, L, M, O) Scale bar = 50 µm. (I, J, P) Scale bar=15 µm. (Q) Levels of DARPP-32 in striatum of 9-month-old WT, PK–/–, TauVLW and PK–/–/TauVLW mice. Statistical analysis was performed by one-way ANOVA followed by Newman–Keuls test. *P < 0.05, **P < 0.01, ***P < 0.001 transgenic versus WT mice; +P < 0.05, ++P < 0.01, +++P < 0.001 PK–/–TauVLW versus PK–/– and ^P < 0.05, ^^^P < 0.001 PK–/–TauVLW versus TauVLW mice. The two-way ANOVA showed interaction between parkin deletion and TauVLW overexpression in the number of TH+ cells (**P < 0.01). Parkin deletion was the main source of variation for the changes in the microglial marker (P < 0.05) and tau overexpression was the main source of variation for the changes in GFAP (P < 0.05). Parkin deletion (P < 0.05) and tau overexpression are sources of variation for the changes in TH levels.

 
Astrogliosis and microgliosis in the midbrain of PK–/–/TauVLW mice
We have found increased glial fibrillar acid protein (GFAP) protein levels in the midbrain of 9-month-old PK–/–/TauVLW mice (Fig. 2K). Higher number of reactive astrocytes was observed in the substantia nigra of 12-month-old PK–/–/TauVLW mice co-localizing with TH+ cells in the Figure 2M compared to WT (Fig. 2L).

The levels of GLUT-5, a microglial marker, were also increased in the midbrain of 9-month-old PK–/–/TauVLW mice (Fig. 2N). Activated ameboid shaped microglial cells are shown in the substantia nigra of 12-month-old PK–/–/TauVLW (Fig. 2O), surrounding the TH+ cells (Fig. 2P).

Involvement of striatal efferent neurons in PK–/–/TauVLW mice
Patients with FTDP-17 related to mutations of tau do often present resistance to the treatment with L-DOPA which is attributed to pathology of striatal output neurons. In order to evaluate whether there was pathological involvement of striatal postsynaptic neurons, we have measured the levels of DARPP-32 in these mice. PK–/–/TauVLW mice have significantly reduced levels of DARPP-32 in striatum (Fig. 2Q) suggesting that they have postsynaptic dopamine nigrostriatal pathology.

Tau pathology in PK–/–/TauVLW hippocampus
The level of tau phosphorylation, as measured by the number of neuritic plaques immunoreactive to the AT-8 antibody, which detects Ser 212 phosphorylated tau, was increased around 25% in PK–/–/TauVLW respect to TauVLW, suggesting that the absence of parkin increases the phosphorylation of tau (Fig. 3A–D). We observed neurofibrillary tangles, as shown by Gallyas stain in the hippocampus of TauVLW (Fig. 3E–G) and much more in those of PK–/–/TauVLW (Fig. 4H–J).


Figure 3
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  		Figure 3. Tau pathology in the hippocampus of 12-month-old TauVLW and PK–/–/TauVLW mice. Representative microphotographs of the TauVLW (A) and PK–/–/TauVLW (B) hippocampus immunostained with antibody anti tau phosphorylated in Ser 212 (AT-8). (C) Representative neuritic plaque. (D) The number of senile plaques, immunostained with AT-8, was statistically higher in PK–/–/TauVLW mice. Values are the mean ± SEM (n = 3 mice, 3 slice/mice, twice). Statistical analysis was performed by t-test. ***P < 0.001 PK–/–TauVLW versus TauVLW mice. Representative microphotographs of hippocampus of 12-month-old TauVLW (E–G) and PK–/–/ TauVLW (H–J) Gallyas’s stained. Higher no. of fibres and neuro-fibrillary tangles could be observed in PK–/–/ TauVLW hilus. Scale = 200 µm for (A), (B), (E) and (H); and 50 µm for (C), (F), (I), (G) and (J).

 


Figure 4
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  		Figure 4. Astrogliosis and microgliosis in the hippocampus of 12-month-old TauVLW and PK–/–/TauVLW mice. Representative microphotographs of TauVLW (A–C) and PK–/–/ TauVLW (D–F) hippocampus stained with anti GFAP (A and D) or anti GFAP (green) and anti-tau-5 (red) (B, C, E, F). Scale bar = 50 µm. (G) GFAP+ fluorescence area quantification is significantly higher in the PK–/–/ TauVLW hippocampus (20 fields at 20x of three slices along hippocampus of three mice, n = 3). (H) is a magnification of the square in (F). Scale bar = 25 µm. Representative microphotographs of TauVLW (I) and PK–/–/ TauVLW (J and K) hippocampus stained with isolectine B4 peroxidase-labelled and nuclei with bis-benzimide (blue). Scale bar = 120 µm (I and J), 20 µm (K). The number of microglial cells (isolectin B4+ cells) (L) is significantly higher in the PK–/–/ TauVLW hippocampal hilus region. Statistical analysis was performed by one-way ANOVA followed by Newman–Keuls test. *P < 0.05, ***P<0.001 transgenic versus WT mice, +++P < 0.001 PK–/–TauVLW versus PK–/– and ^^^P < 0.001 PK–/–TauVLW versus TauVLW mice. The two-way ANOVA showed interaction between parkin deletion and TauVLW overexpression in the number of microglial cells (**P < 0.01).

 
Astroglia and microglia in PK–/–/TauVLW hippocampus
We have found tufted astrocytes and astrocytic plaques in TauVLW and more abundantly in PK–/–/TauVLW mice. The fluorescence intensity and the area occupied by GFAP+ cells is greater in PK–/–/TauVLW than in TauVLW hippocampus (Fig. 4 A, D and G). There is co-localization of tau and GFAP immunostaining (Fig. 4 E, F and H), suggesting that there is deposition of tau in astroglia, and infiltration of astrocytes in the neuronal cell layer of the dentate gyrus surrounding neuronal cells with aberrant tau deposition (Fig. 4 E, F and H).

The dentate gyrus of the hipoccampus of TauVLW and PK–/–/TauVLW mice stained with isolectin-B4 HRP-conjugated are shown in the Figure 4I–K. The total number of microglial cells in the hilus is increased in PK–/–/TauVLW mice (Fig. 4L).

β-amyloid deposition in PK–/–/TauVLW brain
After the observation of the prominent tau pathology in the hippocampus of PK–/–/TauVLW, we studied the possible induction of amyloid deposition in the brain of these mice. We have observed amyloid plaques, revealed with antibody anti 1–40 and 1–42 β-amyloid (Aβ) peptides, in the hippocampus of 14-month-old mice while none amyloid plaque were present in WT, PK–/– or TauVLW mice (Fig. 5A–F). The amyloid immunoreactive plaques observed in 14-month-old PK–/–/TauVLW mice are mature (Fig. 5E). We have not found differences in the levels of the amyloid precursor protein (APP) and the peptide Aβ 1–42 by western blot in the limbic system of 9-month-old mice (Supplementary Material, Fig. S1).


Figure 5
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  		Figure 5. β-Amyloid immunoreactivity in the hippocampus of PK–/–/TauVLW mice. Representative microphotographs of the hippocampus of WT (A), PK–/– (B), TauVLW (C), PK–/–/TauVLW (D and E) mice immunostained with antibody anti 1–40 and 1–42 β-amyloid peptides. (E) Magnification of square in (D). Scale = 200 µm for (A–D), 50 µm for (E). (F) The number of amyloid plaques, immunostained with antibody anti 1–40 and 1–42 β-amyloid peptides, was statistically higher in 14-month-old PK–/–/TauVLW mice. Values are the mean ± SEM (n = 4 mice, 3 slice/mice, twice). Statistical analysis was performed by t-test. ***P < 0.001 PK–/–TauVLW versus TauVLW mice.

 
Systemic organomegaly in PK–/–/TauVLW mice
Nine-month-old PK–/–/TauVLW mice had organomegaly involving the liver, kidney and spleen and loss of peripheral fat (Fig. 6 A–D, H and Supplemental Material, Table S1). The weight of the liver reached up to 25% of the total body weight in around 50% of the 9-month-old PK–/–/TauVLW mice. Kidney hypertrophy was universal but less pronounced. Livers of WT, PK–/– and TauVLW were normal, but with some cytoplasmic inclusions in the last group (Fig. 6E–G). PK–/–/TauVLW had severe accumulation of hyaline substances in the Disse space, atrophy and high density of some hepatocytes, and disorganization of the liver structure with extravasation related to the deposits but without the presence of excessive inflammatory infiltrates (Fig. 6H–K).


Figure 6
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  		Figure 6. Weight, appearance and histology of organs from 9-month-old WT, PK–/–, TauVLW and PK–/–/TauVLW mice. (A) Liver (B) kidney and (C) spleen weights corrected by body weight. Values are the mean ± SEM (n = 6 animals per experimental group). Statistical analysis was performed by one-way ANOVA followed by Newman–Keuls test. *P < 0.05, ***P < 0.001 transgenic versus WT mice; +P < 0.05, +++P < 0.001 PK–/–TauVLW versus PK–/– mice, ^P < 0.05, ^^^P < 0.001 PK–/–TauVLW versus TauVLW mice. The two-way ANOVA showed interaction between parkin deletion and TauVLW overexpression in kidney weight (**P < 0.01). Photographs showing the normal appearance of the TauVLW liver (D) and the hypertrophy of PK–/–/TauVLW organs (H) and the body weight differences. Haematoxylin eosin stain of WT (E), PK–/– (F), TauVLW (G), and PK–/–/TauVLW (I) livers, showing the disorganization of liver structure in PK–/–/TauVLW mice. Scale bar = 50 µm. (J) and (K) are magnifications of PK–/–/TauVLW livers stained with toluidine blue. Scale bar = 25 µm.

 
Amyloid deposition in PK–/–/TauVLW mice liver
The electron microscopy of the liver performed in TauVLW animals did not reveal ultrastructural abnormalities (Fig. 7A–C). In PK–/–/TauVLW hepatocytes, however, the characterization of the hyaline deposits revealed the presence of filaments (around 9 nm) and fibrils (25 nm) of amyloid without apparent organization in the Disse space (Fig. 7 D–F, H). Occasionally, these filaments and fibrils accumulated in round-shaped structures. The PK–/–/TauVLW hepatocytes showed electrondense cytoplasms with high number of large mitochondrion. In contrast, TauVLW hepatocytes were rich in glycogen, and in some cases in large intracellular lipidic inclusions and membranous structures similar to pre-autophagosomes (Fig. 7 A–C, G).


Figure 7
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  		Figure 7. Electronic microscopy of TauVLW and PK–/–/TauVLW livers from 9-month-old mice. In TauVLW livers, we observed normal hepatocytes rich in glycogen (X2.600) (A), some of them with big intracellular lipidic inclusions (X3300) (B), and normal sinusoids (X2600) (C). In the magnification in (G), we can observe membranous structures around mitochondrion like pre-autophagosomes. In PK–/–/TauVLW livers, there are fibrillar deposits compatible with amyloid in the Disse space around hepatic capillaries and the hepatocytes are electrondense and irregular (X1600) (D). Kupfer cell with lipidic inclusion rounded by fibrillar deposits (X8300) (E). Intrasinusoidal lymphocyte in contact with endothelium and fibrillar deposit (X5000) (F), magnified x3 in (H).

 
Accumulation of specific proteins in PK–/–/TauVLW livers
In order to determine the composition of the amyloid deposits, we stained WB membranes with Ponceau red (Fig. 8A). Homogenates from PK–/–/TauVLW have at least three protein bands which are much more prominent than those present in homogenates from other types of mice with the same concentration of protein. Some of the proteins increased in PK–/–/TauVLW livers, at least in part, were immunoglobulisms, since the negative control for monoclonal antibodies of the WB revealed with the secondary antibody, an anti mouse IgG, showed high levels of both, heavy (50 KDa) and to a lesser extent light (25 KDa) immunoglobulin chains from livers of the PK–/–/TauVLW animals (Fig. 8B). We confirmed these results by immunocytochemistry (Fig. 8C–F). The hyaline deposits are stained by the HRP-conjugated anti mouse IgG antibody, probing that the liver deposits contain immunoglobulins.


Figure 8
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  		Figure 8. Accumulation of specific proteins in PK–/–/TauVLW livers. (A) Ponceau red staining of SDS–PAGE separated proteins. Higher levels of proteins with 50, 62 and 70 kDa were observed. (B) Representative western blot revealed with secondary antibody anti mouse IgG and DAB and its densitometric histogram in the four experimental groups. Values are expressed as the mean ± SEM (n = 6 animals 9-month-old in each experimental group). Statistical analysis was performed by one-way ANOVA followed by Newman–Keuls test. ***P < 0.001 transgenic versus WT mice; +++P < 0.001 PK–/–TauVLW versus PK–/– mice; ^^^P < 0.001 PK–/–TauVLW versus TauVLW mice. Representative microphotographs of the TauVLW (C and E) and PK–/–/TauVLW (D and F) livers immunostained with secondary antibody anti mouse IgG, showing staining in PK–/–/TauVLW. Scale = 200 µm for (A), (D) and 50 µm for (E and F).

 
Accumulation of tau in livers from PK–/–/TauVLW mice
In order to test if tau was a component of the amyloid deposits, we used a rabbit polyclonal antibody anti-tau, avoiding the secondary staining. In Figure 9A–H, we observed the tau deposits only in the PK–/–/TauVLW livers. These deposits were in the cytoplasm of hepatocytes, resembling Mallory-Denk bodies, but not colocalizing with the amyloid fibres. The levels of mouse tau were four times more in the PK–/–/TauVLW livers (Fig. 9I) than in the other groups. However, there were no changes in the mRNA expression of mouse tau and TauVLW (Fig. 9J and K and data not shown). Both mouse tau and TauVLW were expressed two orders of magnitude more in brain than in liver and there were no differences between TauVLW and PK–/–/TauVLW mice in the expression of TauVLW, neither in brain, nor in the liver (Fig. 9J–K).


Figure 9
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  		Figure 9. Mouse tau accumulation in PK–/–/TauVLW hepatocytes without changes in mRNA expression in PK–/–/TauVLW livers from 9-month-old mice. Representative microphotographs of anti-tau immunohistochemistry with a polyclonal antibody in WT (A and E), PK–/– (B and F), TauVLW (C and G) and PK–/–/TauVLW (D and H) livers. Scale = 50 µm for (A–D) and 15 µm for (E–H). (I) Representative western blot of tau in livers and brains from WT, PK–/–, TauVLW and PK–/–/TauVLW mice revealed with an anti-tau polyclonal antibody and the densitometric histograms of mouse tau. Observe the different scale in liver and brain histograms. Values are mean ± SEM (n = 6 animals per experimental group). Statistical analysis was performed by one-way ANOVA followed by Newman–Keuls test. **P < 0.01, ***P < 0.001 transgenic versus WT mice; ++P < 0.01, +++P < 0.001 PK–/–TauVLW versus PK–/– mice, ^^^P < 0.001 PK–/–TauVLW versus TauVLW mice. The two-way ANOVA showed interaction between parkin deletion and TauVLW overexpression effects in the tau quantity in livers (**P < 0.01). (J) Expression curves of human TauVLW and mouse GAPDH mRNAs in TauVLW and PK–/–/TauVLW mice measured by quantitative PCR. (K) Relative expression of TauVLW/GAPDH comparing brain versus liver expression (100–500-fold more in both, TauVLW and PK–/–/TauVLW brains than in livers) and PK–/–/TauVLW versus TauVLW mice (there are no differences in brain, neither in liver).

 
Deficits in tau degradation and oxidative stress in PK–/–/TauVLW livers
Since tau accumulates in PK–/–/TauVLW livers without changes in its synthesis, we have studied the mechanisms of tau degradation. The complex CHIP/HSP70 and the Sequestosome/p62 have been involved in tau degradation through the proteasome (18–21). The levels of CHIP and HSP70 were lower in PK–/–/TauVLW livers (Fig. 10A and B). In addition, CHIP protein expression is much lower in liver than in brain of mice (Fig. 10D). Contrary, Sequestosome/p62 accumulates in PK–/–/TauVLW livers (Fig. 10C), probably forming part of aggresomes, as it happens in pathologic human livers forming Mallory bodies, similar to Lewy bodies in PD (22,23). Oxidative stress has been proposed as inductor of tau pathology and amyloid formation (22,23). We have measured the GSH levels as redox homeostatic index and we have found that GSH is greatly decreased in the livers of PK–/–/TauVLW mice to 50% of the levels found in WT (Fig. 10E).


Figure 10
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  		Figure 10. Expression of proteins involved in tau degradation in livers and brains of WT, PK–/–, TauVLW and PK–/–/TauVLW 9-month-old mice. Representative western blot and densitometric histograms of CHIP (A), HSP70 (B) and p62 (C) in livers. CHIP and HSP70 expressions are lower, while p62 levels are higher in PK–/–/TauVLW livers. (D) Differential expression of p62 and CHIP in liver and brain of TauVLW and PK–/–/TauVLW mice. Both expressions are lower in the liver than in the brain. (E) GSH levels in WT, PK–/–, TauVLW and PK–/–/TauVLW mice livers. The GSH levels, index of the oxidative status, were measured per duplicate in homogenates of n = 6 mice for group. Statistical analysis was performed by one-way ANOVA followed by Newman–Keuls test. *P < 0.05, **P < 0.01, ***P < 0.001 transgenic versus WT mice; +P < 0.05, +++P < 0.001 PK–/–/TauVLW versus PK–/–; ^P < 0.05 PK–/–TauVLW versus TauVLW mice.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
We have found a mouse model of progressive neurodegeneration that resembles motor, behavioural and pathological hallmarks of parkinsonism and tauopathies, but strikingly, also present amyloid deposits in brain and peripheral organs. We and others have previously described mild symptoms of akinesia and behavioural abnormalities, dopamine deficiency and redistribution of tau in young PK–/–/TauVLW mice (13–15). Surprisingly, however, mature PK–/–/TauVLW mice develop, not only a more severe clinical phenotype and more severe neurochemical abnormalities but they also show, quite unexpectedly, amyloid deposition in brain and other organs. This model could help explain the relation that exists between tau and amyloid deposition, which takes place in AD and other disorders.

Motor and behavioural impairments in PK–/–/TauVLW mice
The abnormalities observed in motor activity and behavioural responses in PK–/–/TauVLW mice are due to cell loss and abnormal function of different neuronal systems including the dopamine systems and the hippocampus. These mice present a progressive shortening of the stride length, decreased ambulatory activity and dorsal kyphosis. PK–/–/TauVLW mice show increased levels of anxiety and compulsive grooming, which result in self-injury in a high percentage of animals. These motor and behavioural abnormalities closely resemble those observed in patients with different kinds of parkinsonisms and tauopathies, as well as chronic users of some recreational drugs (24–32).

We have previously described that 3-month-old PK–/– and PK–/–/TauVLW mice have increased levels of dopamine (135 and 152%, respectively, more than WT) and serotonin (118 and 122%), reduced amphetamine evoked release of dopamine and lower levels of DAT in the limbic system (13). In mouse models, excessive sequential grooming behaviour has been described in the hyper-dopaminergic mutant mice, in which, knocking down the DAT causes an increase in the extracellular levels of DA in the striatum of 170% (17). The treatment with amphetamine or DA agonists after different lesions of the hippocampus and other limbic system nuclei elicited stereotyped behaviour (33,34). The double transgenic PK–/–/TauVLW mice present both, the hippocampal lesions caused by tau accumulation and the elevated DA levels, as well as the reduced levels of DAT in the limbic system. It is interesting to notice that in the limbic system of PK–/–/TauVLW mice, the reduction of DAT is comparatively greater than that of dopamine levels and TH protein, and that this shift in synaptic dopamine could explain, at least in part, the presence of stereotypes. The estrogen deficient mice (aromatase knock-out) are another model that develops compulsive behaviour, with alteration in the levels of enzymes that metabolizes DA (35).

Abnormal anxiety related behaviour has been described with other tests in another PK–/– mice model (36). Our data show a progressive increase of the PK–/– barbering score with age, reaching the level of shaved face in the worst cases. However, 50% of the PK–/–/TauVLW mice present self-injuries at 6 months of age, and only PK–/–/TauVLW animals present obsessive-compulsive grooming after the water mist spray. Probably the hippocampal lesion is necessary for the development of this phenotype (33,34).

Pathological findings in PK–/–/TauVLW mice
Abnormal deposition of tau takes place in several brain regions of PK–/–/TauVLW mice including the hippocampus and the substantia nigra. Pathological tau deposition in PK–/–/TauVLW brain could be enhanced by an excess of dopamine quinones, which facilitates the assembly of tau in insoluble polymers (37,38), and, in addition, by the changes in the subcellular localization of tau that takes place in the absence of parkin. Parkin elimination alters tau aggregation, degradation and solubility in the brain of aged mice (39).

Ageing decreases the number of TH+ cells in the substantia nigra of PK–/– mice. Three-month-old PK–/–/TauVLW mice have 50% of DAergic neurons in the SN, enough to show any symptoms of parkinsonism, but at 12 months of age, only survive a 30% of TH+ cells in the entire ventral midbrain. The surviving dopamine neurons show accumulation of large vesicles in the cytoplasm and around the nucleus. Vesicular traffic deficits could be due to lack of parkin or to TauVLW over-expression. It is interesting to notice that in PK–/–/TauVLW mice, the lesion to the nigrostriatal dopamine system is not limited to the presynaptic neurons but also involves postsynaptic striatal efferent neurons, as shown by the reduction of levels of DARPP-32 protein.

Actin interacts with parkin and parkin helps to maintain the binding of tau to microtubules (40,41). It is likely that, in the absence of parkin, tau does not bind to microtubules, floats in the cytoplasm, it is more easily phosphorylated and deposits (13). We have even observed tau in the nucleus what raises the question about a putative functional consequence of this translocation of tau. Nuclear and perinuclear localization of tau has been reported in patients with autosomal dominant frontotemporal dementia with parkinsonism related to the P301L mutation of tau (42). Elyaman et al. (43) suggested that there is a link between apoptosis, nuclear translocation of GSK-3 and levels of hyperphosphorylated tau. Lefebvre et al. (44) found that nuclear localization of tau was regulated by the balance of glycosylation and phosphorylation of this protein. PK–/–/TauVLW hippocampus have higher number of phosphorylated tau (AT8 positive) neuritic plaques than TauVLW. Twelve-month-old PK–/–/TauVLW mice had 25% more neuritic plaques in the hippocampus than TauVLW in spite of having the same mRNA expression levels of mouse tau and TauVLW. PK–/– mice have 10-fold the no. of plaques than WT mice of 2 years of age (39).

We have previously shown that the phosphorylation ratio of GSK-3 in the Ser 9 is decreased in absence of parkin, which renders GSK-3 more active. In a seminal work using a conditional GSK-3 transgenic model over-expressing TauVLW, pathological tau deposition depends on GSK-3 over-expression; the phenotype is reversed once GSK-3 over-expression has been stopped (45,46). Therefore, higher GSK3 activity could mediate higher tau accumulation in absence of parkin.

In vitro tau ubiquitination has been obtained with the complex CHIP/HSP70, but not with parkin (18–20). However, parkin E3 ubiquitin ligase activity is regulated by CHIP and HSP70 (47), and parkin is protective for substantia nigra dopamine neurons in a tau gene transfer neurodegenerative model (48). This work completely agrees with our finding of parkin implication in tau pathology. In addition to the landmarks of tau pathology such as high number of neuritic plaques, neuro-fibrillary tangles and Gallyas’s positive fibres, the reactive astrogliosis, microgliosis, the hippocampus of PK–/–/TauVLW mice presents deposition of β-amyloid in plaques.

Amyloid deposition in PK–/–/TauVLW mice
The link between tau pathology and β-amyloid accumulation has been largely investigated because it is the pathological characteristic and probably one of the keys to understanding the pathogenesis of AD, the most prevalent neurodegenerative disease world wide. There are still controversies about the bi-directional relation between both pathologies. Several works in transgenic animals supported the upstream role of β-amyloid. Gotz et al. (49) have shown that injection of Aβ into the brain of tau transgenic mice exacerbates tau pathology, whereas Lewis et al. (50) have found enhanced tau pathology in mice expressing APP and tau transgenes compared with single tau transgenic mice. The clearance of Aβ, using Aβ-specific antibodies reduced the tau burden (51), and genetically augmenting tau levels does not modulate the onset or progression of Aβ pathology in a model of transgenic mice (52).

Aβ and tau form soluble complex that may promote self aggregation of both (3). Recently, it has been shown that reducing endogenous tau ameliorates Aβ-induced behavioural deficits in a mouse model of AD (53). Our data agree with the suggestion of mutual interrelation between these two proteins. We have found more deposition of amyloid in brain of PK–/–/TauVLW mice but not differences in the levels of APP or soluble Aβ peptide, suggesting that our findings are not related to overproduction of the pathological peptide or its precursor protein but to increased deposition. To our knowledge, PK–/–/TauVLW mice are the first model that has been developed Aβ pathology without APP or directly related proteins manipulation.

Surprisingly, the amyloid deposits are not restricted to the brain. The peripheral effects of PK–/– deletion can be explained, because parkin mRNA is expressed in liver, kidney and testis (Drosophila PK–/– flies are sterile) at levels higher than in brain (54,55). Moreover, parkin mice knock-out muscle cells are also more sensitive to the toxic effects of intracellular Aβ, parkin over-expression in skeletal muscle cultures provides substantial protection against Aβ toxicity, and accumulation of parkin protein are present in skeletal muscle biopsies taken from patients with inclusion body myositis (56,57).

We have described the accumulation of various proteins in the PK–/–/TauVLW livers. The IgG immunostaining colocalize with the hyaline substance described as amyloidotic substance. Both the light and the heavy chains of immunoglobulins are able to form amyloid deposits mainly in liver and kidney (58–61), and their levels are higher in PK–/–/TauVLW livers. There are at least 25 proteins able to form amyloid fibrils (62). We have focused our attention in tau deposition due to the florid tau pathology in brain, although we cannot rule out the presence of other proteins in the amyloid deposits.

Tau is physiologically expressed in mouse livers (63,64). Mouse tau levels are 4-fold in PK–/–/TauVLW livers than in the other groups, and tau forms cytoplasm inclusions, only detectable in PK–/–/TauVLW hepatocytes. However, mRNA levels of tau or TauVLW do not change. The ubiquitination grade of tau leads to their degradation by proteasome or by lysosomes (65). Tau could be degraded by calpains (66), caspases (67) and by lysosomal cathepsins (68) rendering fragments with different toxicity. The complex CHIP/HSP70 and the Sequestosome/p62 have been involved in tau ubiquitination and degradation through the proteasome. CHIP and HSPs interact with Aβ and influence Aβ metabolism (18–21,69–72). The lower expression of CHIP and HSP70 in PK–/–/TauVLW livers could promote tau accumulation in the absence of parkin. Therefore, the lack of parkin in animals with mutated tau produces a number of cellular changes, such as the decrease of chaperones and the elevation of free radicals, changes that could induce the deposition of other proteins, such as β-amyloid peptide and immunoglobulins in β structures in different organs.

The accumulation of p62 could be a response to tau accumulation. Sequestosome/p62 is a common component of cytoplasmic inclusions in protein aggregation disease, including PD and Alzheimer (22,73–77). A reduction of p62 expression or a decrease of proteosome activity may contribute to accumulation of insoluble/aggregated K63-polyubiquitinated tau (21,78). This protein forms a patch around protein inclusions promoting its degradation by autophagy (79,80). The mouse models in which autophagy are inhibited show a hepatomegaly similar to which we observe in PK–/–/TauVLW mice (81). In the liver, p62 is involved in the mechanism of Mallory body formation (82) and is a component of the hyaline bodies in hepatocellular carcinoma (22,83). The presence of Mallory bodies was described in the liver of a juvenile parkinsonian patient before parkin was discovered (84). Parkin expression is decreased in the 50% of the biopsies of hepatocellular carcinoma, and numerous alterations in the Park-2 gene have been described in different lines of hepatocellular carcinoma (85).

We hope that our study contributes not only to the description of a new model of neurodegenerative disease but also could help explain the basic pathogenic mechanisms which trigger amyloidosis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
Transgenic animals
WT C57BL6/129SV, human-mutated tau over-expressing mice (TauVLW), parkin-null mutant (PK–/–) and double PK–/–/TauVLW littermates mice were obtained as previously described (13). Our experiments were performed on littermates generated by a heterozygote intercross. The animals, therefore, have the same genetic background. The confirmation of the genotype in each mouse was performed by standard PCR techniques of genomic DNA obtained from their tail.

Procedures using laboratory animals were in accordance with the European Union Directives and the Ramón y Cajal University Hospital Animal Care Committee. The mice were housed six per cage, in an enriched environment with tissue paper and cardboard tubes. All efforts were made to minimize the number of animals used and their suffering. Twenty-four mice (6 per group) were used for behavioural studies during aging and were sacrificed at age of 9 months for biochemical studies. In addition, 12 animals 12-month-old, 3 per group, 4 TauVLW and 4 PK–/–/TauVLW mice 14-month-old, were used for histological studies conserved in paraffin and 12 more animals of 12-month-old were conserved frozen for microglial studies. Two TauVLW and two PK–/–/TauVLW 9-month-old mice were used for electron microscopy.

Behavioural studies
Actimeter
Motor activity was analysed in a computerized actimeter (Actitrack, Panlab, Barcelona, Spain). This consisted of a 22.5 x 22.5 cm area, with 16 infrared beams all around, coupled to a computerized control unit. This allows for the analysis of the distance run in the actimeter (ambulation) and the zonal distribution of the movement (anxiety). The anxiety is inversely proportional to the time spent and the distance travelled in the central area far from the walls. The analysis of motor activity was done for a period of 10 min.

Y-maze
The spontaneous alternation behaviour of mice in a Y-maze was used to test short-term memory, motor activity and anxiety. The maze was made of white PVC, with each arm 40 x 21 x 4 cm. The arms converged to a triangular centre, 4 cm per side. Each mouse was placed at the end of an arm and allowed to move freely throughout the maze during a 5 min session. The series of arm entries was recorded visually and with video (taping). General activity was measured as the total number of arm entries, whereas the basic mnemonic function was measured as the percent of spontaneous alternation. Alternation was defined as successive entries into the three arms on overlapping triplet sets. The alternation behaviour (%) was calculated as the ratio of actual alternations to possible alternations (defined as the total number of arm entries minus two), multiplied by 100.

Barbering score
At different ages, each mouse was graded according to extent of facial barbering as illustrated in Figure 1F. A score of 0 was given for no loss of facial hair, 1 for clipped whiskers, 2 for a shaved snout, 3 if they had shaved their entire face, and a score of 4 if they have hurt or injured themselves, similar to previously reported (35).

Grooming after water mist spray
Each mouse was subjected to two squirts of sterile water mist spray and its grooming activity was recorded for 10 min immediately after the mist spray. Grooming duration and frequency of grooming initiations were then analysed blind to the mice genotype.

Genotype by PCR
WT, PK–/–, TauVLW and PK–/–TauVLW mice were genotyped by PCR screening. Genomic DNA was extracted from mouse tail after proteinase K digestion (16 h at 55°C) in lysis buffer (50 mM Tris–HCl pH 8.3; 100 mM NaCl; 5 mM EDTA; 0.8% SDS) and phenol/chloroform/isoamyl alcohol (25:24:1 ratio) extraction, followed by DNA precipitation with 1/10 volume of 3 M sodium acetate (pH 5.2) and 1 volume of absolute ethanol. The pellet was washed with 70% ethanol and allowed to air-dry. The nucleic acids were dissolved in 150 µl of sterile water.

For PCR reaction, 150 ng of genomic DNA was denatured for 3 min at 94°C and subjected to 35 cycles of 1 min at 94°C, 1 min at 53°C and 1 min at 72°C, followed by 5 min of a final extension at 72°C. PCR was performed in a final volume of 40 µl containing 1 unit of Taq DNA polymerase (Promega, 5 U/µl), 1 mM dNTP (4 x 0.25 mM), 2.5 mM MgCl2, 5 mM Tris–HCl (pH 8.0) and 1 µl of specific sense and antisense primers at 50 ng/µl (they are listed in Supplemental Material, Table S2). Twenty microlitre volumes of PCR reaction products were analysed by electrophoresis on a 1.8% agarose gel that was subsequently stained with ethidium bromide for visualization of DNA bands. DNA molecular weight markers (Roche, Spain) were used to provide a size reference for the test reactions.

Western blot
After decapitation, brain parts of 9-month-old mice used for biochemical studies were dissected. The different brain regions were sonicated (VibraCell, level 2 for 30 s) in eight volumes (W/V) of 0.4 N perchloric acid with 0.5 mM Na2S2O5 and 2% EDTA and then centrifuged at 12 000 rpm for 20 min at 4°C. The pellet, with the proteins, was neutralized (W/V = 1/9) with the lysis buffer (0.75% Na2CO3, 2% SDS, 0.25 mM PMSF, 10 mg/ml leupeptin, 2 mg/ml aprotinin, 10 mg/ml pepsin) and then sonicated and centrifuged at 13 400 g for 30 min at 4°C. The supernatant was used for protein determination by BCA assay and for the electrophoresis analysis.

Liver samples were sonicated (VibraCell, level 2 for 30 s) in eight volumes (W/V) of 20 mM Tris–HCl, 10 mM Ack, 1 mM DTT, 1 mM EDTA, 1 mM benzamidine, 0.25% Igepal (NP-40), 0.25 mM PMSF, 10 mg/ml leupeptin, 2 mg/ml aprotinin, 10 mg/ml pepstatin (lysis buffer) and centrifuged at 12 000 rpm for 30 min at 4°C. The supernatant was used for protein determination by BCA assay (Pierce) and for electrophoresis. Samples (20–50 µg) were added to SDS sample loading buffer 2X (10% glycerol, 2% SDS, 0.1% bromophenol blue, 50 mM Tris, pH 6.8, and 5% β-mercaptoetanol), electrophoresed in 10% SDS-polyacrilamide gels and then electroblotted to 0.45 µm nitrocellulose membranes.

For Aβ determinatination, the sample buffer contained 10% glycerol, 8% SDS, 0.1% bromophenol blue, 50 mM Tris, pH 6.8, 10% β-mercaptoetanol and 8 M urea; the samples were electrophoresed in 15% SDS-polyacrilamide gels and electroblotted to 0.2 µm PVDF membranes. For immunolabeling, the blots were blocked with TTBS solution (20 mM Tris–HCl, pH 7.6, 137 mM NaCl plus 0.1% Tween 20) and 5% dry skimmed milk, for 1 h at room temperature. After blocking non-specific binding, the membranes were incubated overnight with specific antibodies in blocking solution at 4°C. Later, the blots were twice washed with blocking solution for 10 min followed by another two washes with TTBS for 5 min each. The blots were developed by chemiluminiscence detection using a commercial kit (ECL-Amersham Biosciences) and quantified by computer-assisted video densitometry; β-actin was used as a control of charge.

Mouse monoclonal anti HSP70 antibody diluted 1:1000 and goat polyclonal anti-p62 (SQSTM-1) (1:500) were from Santa Cruz; rabbit polyclonal anti-parkin antibody diluted 1:500 were from Cell Signaling Technology; rabbit polyclonal anti-β-tubulin (1:5000) was from BabCO; mouse monoclonal anti-CHIP (1:1000) was from Abcam; mouse monoclonal anti-β-actin antibody diluted 1:5000 was from Sigma. Mouse monoclonal anti-tau 7.51 (1:1000) was a generous gift from Dr Avila. Mouse monoclonal anti-GFAP antibody diluted 1:5000, mouse monoclonal anti-tyrosine hydroxylase (TH) antibody (1:5000), mouse monoclonal anti tau-5 antibody (1:5000), polyclonal anti-DARPP-32 (1:5000) and rat monoclonal anti-DAT (1:2500) were from Chemicon (Madrid, Spain); rabbit polyclonal anti-human GLUT-5 (1:250) was from IBL-Hamburg; human monoclonal anti PHF tau (AT-8) antibody from Pierce Endogen (Rockford, IL, USA) was diluted 1:1000; mouse monoclonal anti-tau T14 was from Zymed (San Francisco, CA, USA) (1:5000) and rabbit polyclonal anti tau was from Protein Tech group, (Chicago, IL, USA). Rabbit polyclonal anti-beta amyloid (1–40 and 1–42) from MBL international Corporation (Hamburg, Germany) was diluted 1:1000; Rabbit polyclonal anti-beta amyloid (1–42 fragment) and rabbit polyclonal anti-beta APP were from Abcam and were diluted 1:1000. Goat anti-mouse-HRP and anti-rabbit-HRP secondary antibodies diluted 1:1000 were from Amersham and rabbit anti-goat-HRP was from Santa Cruz, β-actin secondary antibody was an anti-mouse phosphatase alkaline conjugated diluted 1:3000 from Sigma.

RNA isolation and cDNA synthesis
The brains and livers from TauVLW and PK–/–/TauVLW mice were homogenized in 10 volumes TRI Reagent (Gibco, Madrid, Spain) centrifuged at 12.000 x g for 10 min at 4°C. Chloroform was added to the supernatant, mixed well and incubated at RT for 5 min. The mix was centrifuged at 12.000 x g for 10 min at 4°C, the aqueous phase was mixed with isopropanol, it was vortexed for 5–10 s, and incubated at RT for 10 min. After the centrifugation at 12.000 x g for 8 min at 4°C, the supernatant was discarded, and 1 ml of 75% ethanol was added. In order to remove the ethanol, it was centrifuged at 7.500 x g for 5 min, and the RNA pellet was briefly air-dried. The RNA was dissolved in DEPC water and the integrity was verified by electrophoresis.

After the quantification in NanoDrop® ND-1000 Spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA), 2 µg of RNA, 1 µl of 100 µM oligo-dT and water DEPC until 16 µl were incubated for 70°C 5 min in the 2720 Thermal Cycler (Applied Biosystems, Warrington, UK). After cooling on ice, 9 µl of the reaction mixture [5 µl 5x buffer, 2 µl 10 mM dNTPs, 0.5 µl RNA sin (Roche) and 1.5 µl AMV reverse transcriptase (Roche, Madrid, Spain)] were added for each sample. The RT cycles were: 70°C 5 min, 42°C 1 h, and 90°C 3 min in the 2720 Thermal Cycler.

Real time quantitative PCR (RT–qPCR)
Expression levels of tau mRNA were examined by iQTM SybrGreen qPCR (BIO-RAD), on reverse-transcribed total RNA isolated from the mice brains. Amplification reactions were performed in triplicate with 40 ng of sample DNA, 1.5 µM of gene specific primers, and 1X iQTM SybrGreen Supermix (BIO-RAD, Madrid, Spain), in a 25 µl final volume. The primers were designed with Primer Bank (16) and the sequences 5'–3' described in (Supplemental Material, Table S3). RT–qPCR was performed on an iCycler detector (BIO-RAD) with the following thermal profile conditions: 95°C for 10 min, 45 cycles of 95°C for 30 s and 58°C for 1 min. To differentiate specific amplicons from non-specific products using SybrGreen, a DNA dissociation curve was generated after each reaction with the iCycler sequence detection system.

TauVLW and tau mRNA were quantified relative to GAPDH mRNA using the comparative threshold cycle number (Ct) method. The Ct difference ({delta}Ct = Cttau–CtGAPDH) was taken as a relative quantity of the transcript. The amplification efficiency was checked and found to be identical for both genes measured.

Glutathione measurements
Total glutathione (GSx) levels were measured by the method of Tietze (86). A sample (40 µl) of the homogenated liver supernatant in 0.4 N PCA was neutralized with 4 volumes of phosphate buffer (0.2 M NaH2PO4, 0.2 M Na2HPO4, 0.5 M EDTA and pH 7.5). Fifty microlitres of the resulting preparation were mixed with DTNB (0.6 mM), NADPH (0.2 mM) and glutathione reductase (1 unit), and the reaction was monitored in a P96 automatic microtiter reader at 412 nm for 6 min. Oxidized glutathione (GSSG) was measured by the method of Griffith (87). After the neutralization with the phosphate buffer, the sample remaining (120 µl) was mixed with 2-vinylpyridine (1.2 µl) at room temperature for 1 h and the reaction was carried out as described above. Reduced glutathione (GSH) was obtained by subtracting GSSG levels from GSx levels.

Histology
The animals were anaesthetized intraperitoneally with a mixture (5:4:1) of ketamine (50 mg/ml), diazepam (1 mg/ml) and atropine (1 mg/ml), and perfused with 4% paraformaldehyde in PBS. The whole brain and the liver were immersed in paraformaldehyde for 24 h and then included in paraffin, sectioned in the microtome at a thickness of 4 mm, and stained for haematoxylin/eosine (H&E), Nissl's and Gallyas's stain. TH, total tau (tau-5), phosphorylated tau (AT-8), GFAP and β-amyloid (1–40 and 1–42 peptides) antibodies were used for immunohistochemistry. Mouse monoclonal anti TH antibody diluted 1:2000, mouse monoclonal anti tau-5 antibody diluted 1:100 and rabbit polyclonal anti GFAP diluted 1:500 were from Chemicon (Madrid, Spain). Mouse monoclonal anti PHF tau (AT-8) antibody from Pierce Endogen (Rockford, IL, USA) was diluted 1:100. Rabbit polyclonal anti-beta amyloid from MBL international Corporation (Hamburg, Germany) was diluted 1:500. Anti-rabbit and anti-mouse secondary antibodies were from Dako (Denmark).

For immunohistochemical detection of microglia, brains were removed, postfixed with 4% paraformaldehyde in PBS for 24 h at 4°C, transferred to sequential washes in 10, 20 and 30% sucrose in PBS, and frozen on dry ice before sectioning into 10-mm-thick coronal sections on a cryostat. After immunofluorescent visualization of TH, sections were again blocked with 5% normal goat serum and incubated with peroxidase-conjugated isolectin IB4 (Sigma-Aldrich, Madrid, Spain) for 2 h. Finally, the reaction product was detected with DAB chromogen.

The number of TH immunoreactive neurons in the substantia nigra and the number of senile plaques and microglial cells in the hippocampus were counted in an Olympus Bx51 stereological microscope using a Cast Grid software. Dopamine cells, microglial cells and plaques were quantified in 8–9 regularly spaced sections in each mouse brain, covering the entire surface of the antero-posterior extent of the substantia nigra and the hippocampus, respectively. Counting of cells was performed independently by two blind observers. The intensity and area of the fluorescence in marked glial cells were estimated in 20 fields per hippocampus (three slides per animal, n = 3 mice per experimental group, in two independent experiments). The densitometry was made using the ImagePro 3.1 software.

Electron microscopy
The 12-month-old animals were anaesthetized intraperitoneally with a mixture (5:4:1) of ketamine (50 mg/ml), diazepam (1 mg/ml) and atropine (1 mg/ml), and perfused with 3% glutaraldehyde/0.1 mol/l phosphate buffer, pH 7.4. Liver samples were cut in pieces and incubated 24 h with 2.5% glutaraldehyde/0.1 mol/l phosphate buffer, pH 7.4, washed with Sörensen’s phosphate buffer and post-fixed in 2% osmium tetroxide in Sörensen’s phosphate buffer. After dehydratation in acetone and propylene oxide, they were embedded in araldite. Tissue blocks were cut into semi-thin sections to be studied by conventional optic microscopy after toluidine-blue staining. Ultrathin sections were cut (Reichert-Jung Ultracut E) and stained with lead citrate. The samples were studied in an electronic microscope CX-100 with an acceleration potential of 80 kV.

Statistical analysis
The results were statistically evaluated for significance with t-test, one or two-way analysis of variance (ANOVA) followed by the Newman–Keuls or Bonferroni comparison tests as a post hoc evaluation, respectively. Differences were considered statistically significant when P < 0.05.


   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 has been supported in part by grants from the Spanish Ministry of Health, Instituto de Salud Carlos III, FIS2007/07037, CIBER CB06/05/59 and CAM 0202/2006.


   ACKNOWLEDGEMENTS
 
The authors acknowledge Dr J. Benavides, Aventis-Synthelabo, Paris, France; Dr J. Avila, Centro de Biología Molecular Severo Ochoa, CSIC, Madrid, and Dr M. Sánchez, Fundación Jiménez Díaz, Madrid, for generously providing the founders of these colonies, and Dr J. Avila and Dr C. Redondo, Department of Pathology, Hospital Ramon y Cajal, Madrid, for useful suggestions and comments. The authors also thank to Mrs M. Serrano for excellent technical assistance and to Mrs CD Marsden for editorial help.

Conflict of Interest statement. The authors have no conflict of interest to declare.


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

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M. P. Burns, L. Zhang, G. W. Rebeck, H. W. Querfurth, and C. E.-H. Moussa
Parkin promotes intracellular A{beta}1-42 clearance
Hum. Mol. Genet., September 1, 2009; 18(17): 3206 - 3216.
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