Skip Navigation


Human Molecular Genetics Advance Access originally published online on May 12, 2006
Human Molecular Genetics 2006 15(13):2045-2058; doi:10.1093/hmg/ddl129
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
15/13/2045    most recent
ddl129v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (15)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Menéndez, J.
Right arrow Articles by de Yébenes, J.G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Menéndez, J.
Right arrow Articles by de Yébenes, J.G.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Suppression of Parkin enhances nigrostriatal and motor neuron lesion in mice over-expressing human-mutated tau protein

J. Menéndez1,{dagger}, J.A. Rodríguez-Navarro1,{dagger}, R.M. Solano1, M.J. Casarejos1, I. Rodal2, R. Guerrero3, M.P. Sánchez3, J. Avila4, M.A. Mena1,* and J.G. de Yébenes5

1 Servicio de Neurobiología, Departamento de Investigación (-1D) Hospital Ramón y Cajal, 2 Banco de Tejidos para Investigaciones Neurológicas, 3 Laboratorio de Neurología, Fundación Jiménez Díaz, 4 Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid, Consejo Superior de Investigaciones Científicas, Spain and 5 Servicio de Neurología Hospital Ramón y Cajal Ctra. de Colmenar, Km. 9, Madrid 28034, Spain

* To whom correspondence should be addressed. Tel: +34 913368384; fax: +34 913369016; Email: maria.a.mena{at}hrc.es

Received January 27, 2006; Accepted May 10, 2006


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Abnormal deposition of protein tau takes place in the brain of patients with several neurodegenerative diseases. Few of these patients present frontotemporal dementia with parkinsonism and amyotrophy (FTDPA-17), an autosomal dominant tauopathy related to mutations of the gene that codes for protein tau, localized in chromosome 17. The great majority of patients with tauopathies such as Alzheimer's disease, sporadic frontotemporal dementia or progressive supranuclear palsy do not show a Mendelian pattern of inheritance. We have occasionally seen tauopathies in patients with parkin mutations and, therefore, hypothesized that the protein tau interacts with parkin. We have tested that hypothesis in mice with combined genetic modifications of tau (over-expression of human tau with three mutations known to produce FTDPA-17) and parkin (deleted) proteins. Homozygote parkin null or over-expressing mutated-human tau mice have subtle behavioral and molecular abnormalities but do not express a clinical phenotype of neurodegenerative disease. Mice with combined homozygous mutations of these two genes show progressively abnormal walking already noticeable at 3 months of age, loss of dopamine and dopamine markers in striatum, nuclear tau immunoreactive deposits in motor neurons of the spinal cord, abnormal expression of glial markers and enhanced levels of pro-apoptotic proteins; findings that were absent or less pronounced in homozygote animals with deletions of parkin or over-expression of tau. The double transgenic mice do not express normal mechanisms of adaptation to stress such as increased levels of GSH and Hsp-70. In addition, they have reduced levels of CHIP–Hsc70, a complex known to attenuate aggregation of tau and to enhance ubiquitination of phosphorylated tau. We have found high levels of phosphorylated tau in parkin–/–+tauVLW mice and a relative decrease of the inactivated pSer9 to total GSK-3 levels. Our data reveal that there are interactions between tau and parkin that could be relevant for the pathogenesis and treatment of tauopathies. Similarly, we hope that the double transgenic parkin–/–+tauVLW mice could be useful for testing of compounds with putative therapeutic value in human tauopathies.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Tau is a protein that stabilizes microtubules and plays a critical role in the neuronal transport system (1–3). Abnormal tau function and pathological deposits of this protein in the human brain have been described in a number of sporadic and familial neurological diseases, that produce different clinical phenotypes (4–7). Alzheimer's disease is a mostly sporadic disorder with abnormal deposition of highly phosphorylated tau into the neurons. Frontotemporal dementias include a complex of different tauopathies, some sporadic, some familial, mostly related to different mutations of the tau gene, characterized by dementia with prominent deficits of the frontal lobe, such as apathy, disinhibition, change of personality, and often by parkinsonism (7). Progressive supranuclear palsy (PSP) and cortico basal degeneration are mostly sporadic tauopathies (7). Familial tauopathies, described in more than 100 families around the world, are clinically characterized by a variable combination of parkinsonism and dementia with prominent involvement of the frontal lobes, and are related to 34 known different mutations of the Tau gene, located in chromosome 17 (8–10). Most tauopathies, however, are of unknown origin and pathogenesis, and they are considered related to post-translational alteration of tau.

Tau interacts with many proteins in the brain. The best known interactions are those of tau and tubulin in the microtubules. In vitro experiments have revealed tau-mediated fibrillization of alpha-synuclein protein at low concentrations (11), but accumulations of tau and amyloid beta-protein occur independently in the human entorhinal cortex (12). Fe65, one of the ligands of the amyloid precursor protein (APP) cytodomain, is associated with tau in vivo and in vitro (13). Great interest has been developed in the proteins that play a role in the phosphorylation of tau. Glycogen synthase kinase-3 (GSK-3) is one of them. Its function could be inhibited by compounds presently used in the clinic, such as lithium (14,15), or in the process of development. In addition, other proteins, such as the 14-3-3 protein, mediate the phosphorylation of protein tau by serum- and glucocorticoid-induced protein kinase 1 (16).

The interrelations between tau and parkin are of particular interest. Van de Warremburg et al. (17) reported a family with parkin mutations and tau pathology in brain. Morales et al. (18) described a patient with autosomal recessive parkinsonism because of a single heterozygous C212Y parkin mutation, who had a clinical and pathological phenotype of PSP. That patient had tau pathology and high levels of phosphorylated tau in his brain (19). As he had the high-risk genotype H1/H1 for development of tauopathies, it was concluded that, in individuals with haplotypes of risk of tau, a partial deficit of parkin function could favor the development of tau pathology. More recently, Yang et al. (20) have found that parkin is essential for the stabilization of tubulin in microtubules. The absence of parkin could, therefore, make unstable the binding of tau to tubulin and increase the levels of tau in the neuronal cytosol, making tau more susceptible to phosphorylation.

In order to test whether the interaction of tau and parkin is relevant in patients with neurodegenerative diseases, we have investigated a mouse model of tauopathy that over-expresses tau and has null parkin function produced by a deletion of exon 3 for parkin. The parkin null animals do not have a deficit of monoamines in basal ganglia (21) but rather increased striatal levels and increased intracellular monoamino oxidase (MAO)-mediated metabolism of dopamine. The enhanced production of free radical via MAO did not produce a massive loss of nigrostriatal dopamine neurons because it was compensated by increased levels of glutathione (GSH). Mice over-expressing mutated tau have also been reported (22,23). Mutated tau is hyperphosphorylated, as phosphatase PP2A is unable to bind to it (24). Mice over-expressing mutated tau appear to behave normally though they have lysosomal abnormalities and hyperphosphorylated tau, but no obvious cellular damage. As both parkin and tau are proteins that play important roles in parkinsonism, we have investigated the effect of combining over-expression of tau with suppression of parkin on: (i) nigrostriatal dopamine and other monoamine neurons, anterior horn cells of the spinal cord; (ii) the homeostasis of important free radical chelating agents such as glutathione; (iii) the expression of glial antigens; and (iv) the expression of proapoptotic, heat shock proteins, c-terminal Hsc interacting protein (CHIP) and GSK-3. Also, we have tested the effect of the absence of parkin on tau pathology. We have found that the animals with mutations of both parkin and tau have histological and biochemical deficits that were not present in any of the groups with homozygous mutations of either gene, further supporting the concept of a functional interaction between tau and parkin.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Breeding, development and behavior
Breeding of the animals was performed according to the following schema. The numbers in brackets represent the expected/found frequencies of genotypes of the offspring expressed in percentage.

Parkin null mice.

Formula
Tau mutant mice.

Formula
Parkin null + tau mutant mice.

Formula

All the animals used in these experiments were homozygote. For simplicity, mice expressing human-mutated tau are indicated as tauVLW. The four groups of animals developed normally. Therefore, we confirm normal development and growth of parkin-null mutant (parkin–/–) and human-mutated tau over-expressing mice (tauVLW), as previously reported (21,23), the double mutated mice (parkin–/–+tauVLW) weighed less than the mice of the same age from other experimental groups. No abnormal behavior was observed in any of the experimental groups though some double mutant mice dragged their hindlimbs at the end of the observation period.

Motor activity was slightly reduced in tauVLW and in parkin–/–+tauVLW mice but the differences were not statistically significant for any of the parameters evaluated in animals from 3 to 5 months (data not shown).

Footprints, as and index of the length of the stride, are presented (Fig. 1). Double mutant parkin–/–+tauVLW mice of 3 and 8 months of age had shorter steps and the abnormality progressively increases with age.


Figure 1291
View larger version (24K):
[in this window]
[in a new window]
  		Figure 1. Footprint analysis. (A) Experimental set-up used to study the footprints left by the mice walking on the 60 cm paper sheet. (B) Examples of footprints left by the hind limbs of WT, parkin–/–, tauVLW, and parkin–/–+tauVLW mice walking on the paper. (C) Quantification of the hind limb stride of the footprint patterns of the WT, parkin–/–, tauVLW, and parkin–/–+tauVLW 3 months old and (D) 8 months old mice. The values are expressed as mean±SEM of the each animal mean step length (n=6 animals in each 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 parkin–/–+ tauVLW versus parkin–/– and {wedge}P<0.05, {wedge}{wedge}{wedge}P<0.001 parkin–/–+tauVLW versus tauVLW mice. The two-way ANOVA analysis shown interaction (***P<0.001) between the two transgenic mice lines only in the 8 months group.

 
Macroscopic and histologic appearance of the nervous system
The brain and spinal cord appeared macroscopically normal in the four experimental groups and no evidence of cortical atrophy was observed. The findings in hippocampus have been described elsewhere (25). Essentially, the cellular structure was normal but there were changes in the cellular localization of tau in neurons of the CA1 area in the double parkin–/–+tauVLW mice. No immunoreactivity to human tau was observed in wild-type (WT) and parkin–/– mice, as expected. There was normal axonal tau immunoreactivity in tauVLW mice, but the immunostaining against human tau was perinuclear in parkin–/–+tauVLW mice. Here, we also confirm perinuclear localization of tau in anterior horn cells of the spinal cord (Fig. 2C) of the double parkin–/–+tauVLW mice, but not in other experimental groups. The number of motor neurons was significantly reduced in double mutant mice to 60% of the other experimental groups (Fig. 2A and B).


Figure 1292
View larger version (59K):
[in this window]
[in a new window]
  		Figure 2. Representative microphotographs of anterior horn cells of the spinal cord. (A) Nissl stain of the spinal cord motor neurons; parkin–/–+tauVLW mice have a reduced number of motor neurons (B) and present aggregates of tau, immunoreactive to AT100 antibody, in the nuclear and perinuclear area that are absent in tauVLW mice (C). Bar: 20 µm. Magnification 400x. Statistical analysis was performed by one-way ANOVA followed by Newman–Keuls test. ***P<0.001 transgenic versus WT mice; +++P<0.001 parkin–/–+tauVLW versus parkin–/– and {wedge}{wedge}{wedge}P<0.001 parkin–/–+tauVLW versus tauVLW mice. The two-way ANOVA analysis shown interaction (***P<0.001) between the two transgenic mice lines.

 
Both parkin–/– and tauVLW mice had normal appearance of the substantia nigra and normal levels of tyrosine hydroxylase (TH) neurons in their brain stem (Fig. 3). Mice with mutations of the two genes had a reduction of the area of the substantia nigra and around 50% depletion of TH+ neurons (Fig. 3).


Figure 1293
View larger version (73K):
[in this window]
[in a new window]
  		Figure 3. Representative microphotographs of the midbrain and TH+ cells in the substantia nigra. Low power, (2x, A–D, scale bar=50 µm) and high power (10x, E–H, scale bar =30 µm) view of the brain stem of WT (A and E), parkin–/– (B and F) tauVLW (C and G) and parkin–/–+tauVLW (D and H) immunostaining to TH antibody. (I) Average number of TH+ neurons per brain stem section expressed as the mean±SEM (n=3–5). Statistical analysis was performed by one-way ANOVA followed by Newman–Keuls test. ***P<0.001 transgenic versus WT mice; +++P<0.001 parkin–/–+ tauVLW versus parkin–/– and {wedge}{wedge}{wedge}P<0.001 parkin–/–+tauVLW versus tauVLW mice.

 
Monoamines and their metabolites in double transgenic parkin–/–+tauVLW mice
Striatum
As previously reported, parkin–/– mice do not show reduced dopamine levels in striatum (Fig. 4), but they have high levels of monoamine metabolites including MAO-derived metabolites such as di-hydroxyphenyl acetic acid (DOPAC), homovanillic acid (HVA) and 5-hydroxy-indole acetic acid (5-HIAA). Tau over-expressing mice, whose levels of monoamines have not been previously reported, had striatal levels of dopamine similar to those of the WT and slightly elevated levels of metabolites, though not as much as in parkin–/– mice. The double transgenic parkin–/–+tauVLW was characterized, instead, by reduced levels of dopamine and DOPAC, in comparison with WT, parkin–/– and tauVLW mice.


Figure 1294
View larger version (36K):
[in this window]
[in a new window]
  		Figure 4. Monoamine metabolism in striatum of WT, parkin–/–, tauVLW and parkin–/–+tauVLW mice. Results are expressed in nanograms per gram of fresh tissue and as percentage of WT controls. Values are the mean±SEM (n=5–6). 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 parkin–/–+tauVLW versus parkin–/– mice and {wedge}P<0.05, {wedge}{wedge}{wedge}P<0.001 parkin–/–+tauVLW versus tauVLW mice.

 
No changes were found in noradrenaline (NA) levels in any of the experimental groups. With regards to serotonin (5-HT) the levels were similar in all groups. The levels of the MAO-mediated 5-HT metabolite, 5-HIAA were high in all experimental groups and significant differences were observed in the parkin–/–+tauVLW and in parkin–/– mice (Fig. 4).

Limbic system
The changes observed in the limbic system (Table 1) were different from that observed in the striatum. Dopamine levels were significantly increased in parkin–/–+tauVLW and parkin–/– mice and dopamine metabolites were increased in the three experimental groups. The levels of NA, which in the WT controls are around twice the levels found in the striatum, were unchanged in parkin–/– mice and in tauVLW mice, but were elevated in parkin–/–+tauVLW mice. 5-HT levels were unchanged in the three groups of transgenic animals but 5-HIAA was increased in parkin–/– mice and in parkin–/–+tauVLW mice.


View this table:
[in this window]
[in a new window]
  		Table 1. Monoamine metabolism in limbic system of WT, parkin–/–, tauVLW and parkin–/–+tauVLW mice

 
Diencephalon and brain stem
No significant differences in the levels of monoamines and metabolites were found in any of these areas (data not shown).

Protein markers of dopamine neurons
Protein markers of dopamine neurons in striatum are presented in Figure 5. Parkin–/–+tauVLW mice had a reduction of both TH and dopamine transporter (DAT), confirming the abnormalities of nigrostriatal dopamine neurons. No significant changes of these markers were found in any other groups. The density of dopaminergic terminals in striatum, as shown by immunohistochemical staining against TH is presented in Figure 5A.


Figure 1295
View larger version (55K):
[in this window]
[in a new window]
  		Figure 5. Dopamine markers expression in mice striatum. Representative microphotographs of the TH+ cells in the striatum, scale bar=100 µm (A). Representative western blot of TH (B) and DAT (C) proteins and their respective densitometric histograms. ß-actin was used as charge control. Values are expressed as the mean±SEM (n=6 animals in each experimental group). Statistical analysis was performed by one-way ANOVA followed by Newman–Keuls test. *P<0.05 parkin–/–+tauVLW mice versus WT mice; +P<0.05 parkin–/–+tauVLW mice versus parkin–/– mice.

 
Glial and neuronal markers in parkin–/–+tauVLW striatum
Glial and neuronal markers are presented in Figure 6. GFAP is reduced in parkin–/–+tauVLW mice but is not in the other groups. GLUT-5, a marker of resting and reactive microglia, was increased with respect to WT in two groups of mice, parkin–/–, and tauVLW. Neuronal enolase is unchanged. Our data suggest a reduction of astrocytes in striatum of parkin–/–+tauVLW mice without changes of this cellular population in other transgenic animals.


Figure 1296
View larger version (31K):
[in this window]
[in a new window]
  		Figure 6. Glial and neuronal markers expression in mice striatum. Representative western blot of GFAP (A), resting and reactive microglia (GLUT-5) (B) and enolase (C) proteins and their respective densitometric histograms. ß-actin was used as charge control. Values are expressed as the mean±SEM (n=6 animals in each experimental group). Statistical analysis was performed by one-way ANOVA followed by Newman–Keuls test. **P<0.01, ***P<0.001 transgenic mice versus WT controls; +P<0.05 parkin–/–+tauVLW mice versus parkin–/– mice; {wedge}{wedge}P<0.01 parkin–/–+tauVLW mice versus tauVLW mice.

 
Alteration of GSH homeostasis in parkin–/–+tauVLW mice
Parkin–/– mice have high levels of GSH in striatum (21). Here we confirm (Fig. 7) that finding. We have also observed that tauVLW mice and parkin–/–+tauVLW mice have striatal levels of GSH similar to WT. Parkin–/–+tauVLW mice had reduced levels of GSH with respect to parkin–/– mice. Oxidized glutathione (GSSG) was highest in tauVLW mice. Parkin–/–+tauVLW mice had striatal levels of GSSG similar to those of WT but reduced with respect to tauVLW mice. The ratios of GSH/GSSG, which indicate indirectly the reserve of free radical scavengers, were highest in parkin–/– mice, intermediate in parkin–/–+tauVLW mice and lowest in tauVLW mice.


Figure 1297
View larger version (19K):
[in this window]
[in a new window]
  		Figure 7. GSH homeostasis in striatum of WT, parkin–/–, tauVLW and parkin–/–+tauVLW mice. The GSH and GSSG levels were measured in homogenates of brain regions from 3-month-old male mice. Values are expressed as the mean±SEM (n=6 animals in each 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<0.001 parkin–/–+tauVLW mice versus parkin–/– mice and {wedge}P<0.05 parkin–/–+tauVLW mice versus tauVLW mice.

 
In the limbic system, where the metabolism of dopamine, as measured by the ratio DOPAC/DA, is lower than in the striatum, the levels of GSH, GSSG and GSH/GSSG were unchanged. No difference of these parameters was found in brain stem or diencephalon.

Expression of tau in striatum
Levels of tau, as measured by immunoreactivity to 7.51 antibody, which labels both endogenous mouse tau + human-mutated tau, were reduced in parkin–/– mice with respect to WT. As expected, immunoreactivity to 7.51 antibody was increased, with respect to WT in tauVLW and parkin–/–+tauVLW mice, but there was no difference between these two groups (Fig. 8). T14 immunoreactive tau, which labels human mutated tau, was only detected in tauVLW and parkin–/–+tauVLW mice and, similarly to 7.51, there was no difference between these two experimental groups.


Figure 1298
View larger version (16K):
[in this window]
[in a new window]
  		Figure 8. Striatal levels of tau immunoreactive to antibodies 7.51 (total tau) and T14 (human-mutated tau) in WT, parkin–/–, tauVLW, and parkin–/–+tauVLW mice. Representative western blots of 7.51 (A) and T14 (B) are presented. b-actin was used as charge control. Values are expressed as the mean±SEM (n=6 animals in each experimental group). Statistical analysis was performed by one-way ANOVA followed by Newman–Keuls test. *P<0.05, ***P<0.001 transgenic mice versus WT mice; +++P<0.001 parkin–/–+tauVLW mice versus parkin–/– mice.

 
Expression of heat shock and cell death proteins
Heat shock protein Hsp-70 was increased in parkin–/– and, furthermore, in tauVLW mice (Fig. 9), but not in parkin–/–+tauVLW. As heat shock proteins are non-selective reactants of stress it is conceivable that both parkin–/– and tauVLW express neuroprotective proteins in response to the molecular challenges produced by the mutations but parkin–/–+tauVLW mice lack that protective mechanism. This is further confirmed by our findings that CHIP/Hsc70, a complex that ubiquitinates phosphorylated tau and enhances cell survival (26), is significantly reduced in parkin–/–+tauVLW mice without significant changes in the other experimental groups. In agreement with this hypothesis, the ratio Bcl-2/Bax, an index of anti-/pro-apoptotic proteins, was reduced in parkin–/– and tauVLW mice and, furthermore, in double transgenic.


Figure 1299
View larger version (32K):
[in this window]
[in a new window]
  		Figure 9. Bcl-2, Bax, Hsp-70 and CHIP proteins expression in striatum of WT, parkin–/–, tauVLW, and parkin–/–+tauVLW mice. Representative western blots of Bcl-2/Bax (A), Hsp-70 (B) and CHIP proteins (C) are presented. ß-actin was used as charge control. Values are expressed as the mean±SEM (n=6 animals in each 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 mice versus WT mice; +P<0.05, ++P<0.01 parkin–/–+tauVLW mice versus parkin–/– mice; {wedge}P<0.05 parkin–/–+tauVLW mice versus tauVLW mice.

 
Expression of GSK-3ß and P-Ser GSK-3ß
The levels of GSK-3ß were increased in parkin–/– mice but not in any of the other experimental groups (Fig. 10). The ratio of inactivated P-Ser9 to total GSK-3ß was reduced in parkin–/– and parkin–/–+tauVLW mice (Fig. 10) suggesting a relative increase of activity-enhanced GSK-3.


Figure 12910
View larger version (30K):
[in this window]
[in a new window]
  		Figure 10. Striatal levels of GSK-3ß, P-Ser-GSK-3ß and P-Ser-GSK-3ß/GSK-3ß of WT, parkin–/–, tauVLW, and parkin–/– +tauVLW mice. Representative western blots of GSK-3ß (A), P-Ser GSK-3ß (B) proteins and P-Ser GSK-3ß/GSK-3ß (C) are presented. ß-actin was used as charge control. Values are expressed as the mean±SEM (n=6 animals in each experimental group). Statistical analysis was performed by one-way ANOVA followed by Newman–Keuls test. *P<0.05, **P<0.01 transgenic mice versus WT mice.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
We have investigated the role of suppressing parkin and over-expressing mutated human tau in mice with single and combined mutations of these Park-2 and Tau genes. We have observed that young adult double mutant mice have evidence for nigrostriatal cell damage including a reduction of TH+ neurons in the substantia nigra, dopamine levels and dopamine cell markers in striatum, as well as a reduction in the expression of astroglial markers. These double mutant mice, in addition, have motor neuron loss.

The phenotypic diversity of clinical signs and symptoms observed in humans with mutations of tau includes dementia, parkinsonism and motor neuron disease. Cognitive deficits have not been observed in these animals though they have histological changes, mostly consistent with nuclear immunoreactivity to tau, in hippocampus (25). Similarly, no evidence of clear motor deficits (amyotrophy or paraplegia) is observed in 3-month-old animals, in spite of histological and biochemical evidence of severe nigrostriatal damage. It is of interest to take into consideration that patients with parkinsonism do have a long phase of presymtomatic nigrostriatal dysfunction and that motor signs are only detected when there is a massive dropout of nigrostrial dopamine cells, usually greater than 50%. Otherwise our findings are consistent with the spectrum of deficits observed in patients with mutations of tau. It is important to notice that the phenotype of tauopathy was not present in mice with tau mutations and normal parkin function, suggesting that parkin plays a very important role in modulating the toxic role of tau. The abnormalities of gait, as measured by footprint analysis, appear in early adulthood and increased with age in the double transgenic mice, at least up to 8 months of age.

We have investigated the mechanisms of cell death in parkin–/–+tauVLW mice. Proteins involved in apoptosis were shifted towards cell death in both parkin–/– and tauVLW mice and, furthermore, in parkin–/–+tauVLW mice. Hsp-70, which is neuroprotective, was increased in both groups of mice with mutations of one gene but not in the group with the mutation of both genes. The complex CHIP–Hsc70, which attenuates tau aggregation (27) and increases ubiquitination of phosphorylated tau (26), was reduced in the double transgenic group. The ratio pSer9 to total GSK-3 levels was reduced in parkin–/– and parkin–/–+tauVLW mice suggesting an excess of activity enhanced GSK-3 in parkin–/–+tauVLW mice, which could be responsible, at least in part, for the hyperphosphorylation of tau observed in these animals. GSH was increased in parkin–/–, but not in any of the other groups of animals, and with respect to WT, GSSG was increased in tauVLW mice but not in the other groups. Our data suggest that parkin–/– and tauVLW mice have dopaminergic nigrostriatal stress that they are able to compensate by different mechanisms. However, the combined effect of the lack of parkin and over-expression of human tau, with three mutations known to produce pathology, damages the nigrostriatal dopamine neurons even in young animals of 3-months of age.

We have found a pronounced effect of the double mutation in nigrostriatal dopamine neurons, demonstrated by the reduction of striatal levels of dopamine and its metabolite DOPAC in these animals as well as by the decrease of the expression of dopamine markers such as TH and DAT proteins. No significant reductions of the levels of dopamine and its metabolites in other brain regions of the double mutant or in any brain area of the single mutant, parkin–/– or tauVLW, was observed. In fact the levels of dopamine and its metabolites are increased in parkin–/– mice, as previously reported (27), and confirmed here. The explanation for this selective toxicity is probably related to the different turnover of dopamine in the different dopamine systems. For instance, the ratio DOPAC/DA, which could be considered an indirect index of dopamine turnover, is 0.4 in striatum and 0.06 in the limbic system in our animals. As the conversion of dopamine to DOPAC produces free radicals, one molecule of H2O2 per molecule of dopamine, our data indicates that the special vulnerability of the nigrostriatal system may be related to its enhanced metabolism of dopamine and its excessive production of free radicals, as both parameters are almost 7-fold those of the limbic system. We have previously suggested that high levels of DOPAC in parkin–/– mice indicate an accelerated metabolism of dopamine via mono-amino oxidase. High MAO B activity in parkin–/– has been recently confirmed (28,29). Enhanced MAO-related dopamine metabolism could be compensated by levels of GSH as it is found in parkin–/– mice. Direct measurement of sprouting of dopamine nerve terminals is beyond the scope of this study, but there is a discrepancy between cellular changes observed in the substantia nigra and those found in striatum. We support the hypothesis that there should be sprouting. Sprouting of nigrostriatal dopamine terminals have been postulated in patients with Parkinson's disease and in animal models of this disease as an explanation to the relatively mild clinical deficits observed after severe nigral lesions.

Parkin–/–+tauVLW mice, lacking the high protective levels of GSH found in parkin–/– mice, are not protected against the abnormal dopamine metabolism related to the suppression of the parkin function. Therefore, their nigrostriatal dopamine neurons may be damaged and, eventually, disappear. We do not know why this effect is restricted, in animals of 3 months, to the nigrostriatal dopamine neurons and not to other dopamine, noradrenaline or 5-HT neurons in the brain, but it is important to realize that the turnover of dopamine in the nigrostriatal neurons, as measured by the ratio DOPAC/DA, is much greater in the nigrostriatal dopamine neurons than in other monoamine systems in the brain. Furthermore, other parameters, such as high levels of Fe2+ and low levels of GSH, make the nigrostriatal dopamine system more susceptible.

In addition to the inability to compensate for oxidative stress, other mechanisms appear to be involved in cell death in parkin–/–+tauVLW mice. These mechanisms may be related to the shift of anti-/pro-apoptotic proteins, to the reduced expression of Hsp-70 and to the changes in cellular distribution 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 (30). Elyaman et al. (31) have suggested that there is a link between apoptosis, nuclear translocation of GSK-3 and levels of hyperphosphorylated tau. Lefebvre et al. (32) found that nuclear localization of tau was regulated by the balance of glycosylation and phosphorylation of this protein. Recent studies suggested that the levels of hyperphosphorylated tau modulate nuclear functions such as the expression of the estrogen receptor-alpha in hippocampal neurons of patients with Alzheimer's disease (33).

The interactions between parkin and tau may be interesting in the pathogenesis of neurodegenerative disorders. Tau pathology is present in the brain of patients with autosomal recessive parkinsonism related to mutations of the gene Park-2 (17) and it has been suggested that tau accumulation in the brains of these patients is related to the fact that parkin is a protein with ubiquitine ligase function and that tau is a substrate of parkin. More recently, parkin has been described as a protein that stabilizes the microtubules (20). By doing so, it helps the binding of tau to the microtubular transport system, which is critical for neuronal survival and function. In the absence of parkin, the binding of tau to microtubules may be impaired and its cellular distribution and function may change. Guerrero et al. (25) have found abnormal distribution of tau immunoreactivity in hippocampal neurons of parkin–/–+tauVLW mice. In these animals tau is localized around the nucleus, instead of its normal distribution in the axon. We have also here observed that tau appears in the nucleus and perinuclear area of motor neurons of the spinal cord. Inability of tau to bind to microtubules may produce two deleterious effects: (i) impairment of the function of the transport system, of critical importance for cell survival and function, and (ii) increased levels of free cytosolic tau, more susceptible to phosphorylation by kinases. When the levels of phosphorylated tau increase, the self aggregation of tau is enhanced. We would like to hypothesize, according to our data, that in neurons from WT animals, tau phosphorylated by GSK-3 is ubiquitinated by the help of CHIP/Hsc70 and further processed in the proteasome. In neurons from parkin–/–+tauVLW, however, the combined effect of enhanced GSK3 activity and the reduction of CHIP increases the levels of nuclear tau, less susceptible to proteosomal degradation.

The interaction between parkin and tau may be clinically relevant. We have created these double transgenic mice to investigate the putative potentiation of toxic effects of mutations of these two genes because we have observed one patient with a single C212Y mutation of parkin, and with the H1/H1 haplotype of tau, who developed a neurological disorder that fulfilled clinical and pathological criteria for PSP (18). This patient had abnormal tau deposits in midbrain, diencephalon, cortex and other areas of his nervous system and high levels of phosphorylated tau. Heterozygotes for parkin mutations and carriers of the H1 haplotype are usually asymptomatic. It was possible, therefore, that the disease was unrelated to the mutations but it could not be excluded that it was triggered by an interaction of both proteins. It is already known that carriers of H1/H1 genotype have a greater risk of sporadic PSP, which is extremely rare in subjects with H2 haplotype (34). More recently (35) we have found that the polymorphism VAL380LEU of parkin is more frequent in patients with PSP, suggesting that the presence of Leu in such a position has a neuroprotective effect. This further suggest that the risk of a tauopathy, even in sporadic cases, may be determined by genetic factors, including polymorphisms of parkin. Our data suggest that the mechanism of the disease in individuals with apparently sporadic disorders could be related to the effects of mutations of multiple genes, transmitted with Mendelian inheritance, but insufficient to produce a clinical phenotype in the absence of additional mutations or the coexistence of environmental factors. If this proves to be the case, the identification of genetic risks factors, and not only of mutations, may be critical for the investigation and treatment of disease.

Several studies are under way to experimentally modify the phosphorylation and self-aggregation of tau with promising results in vitro (15,36). If these treatments prove helpful, they should be administered to patients with initial clinical syndromes since neuroprotection, even with the best possible treatment, may be useless in patients with fully established disease, in whom there is already massive neuronal loss and tissue degeneration of certain neuronal areas at the time the diagnosis is made. We hope that our study helps to identify factors of risk of tauopathies in patients and to provide a useful model for testing putative treatments of tau-related disorders.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Transgenic animals
WT C57BL6/129SV and parkin-null mutant (parkin–/–) littermates mice were obtained from Aventis Pharma SA laboratory (Vitry-sur-Seine, France) (21). Human-mutated tau over-expressing mice (tauVLW) were obtained from Dr J. Avila. The tauVLW line over-expresses a human four-repeat tau isoform with two N-terminal exons carrying three mutations (G272V, P301L and R406W) linked to frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17). The mice were generated by pronuclear injection of tauVLW gene driven by the mouse Thy- promoter. All transgenic tauVLW mice used in the experiments described here were homozygotes and the insertion was corroborated using TT1 and TT2 primers to amplify a 470-bp product specifically from the transgene and not from endogenous murine DNA, whereas as an internal control for DNA, TT1 and THY were used to amplify a 450-bp product specifically from murine genomic DNA but not from the transgene (23). The double mutant parkin–/–+tauVLW was obtained by breeding the two previously described transgenic mice. 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. All efforts were made to minimize the number of animals used and their suffering. Twenty-four 3-month-old male mice, divided into four experimental groups of six WT animals, homozygote parkin null (parkin–/–), over-expressing human-mutated tau (tauVLW) and the double mutant (parkin–/–+tauVLW) were used for biochemical and behavior, and three additional animals of each group for histological studies. Six additional animals per group were used for longitudinal evaluation of motor behavior aged 5 and 8 months.

Motor assessment
Young adult male mice (3–5-month-old) were used for evaluation of motor activity in open field observation and by computerized analysis of movements in an actimeter (Actitrack, Panlab, Barcelona, Spain). This consisted of a box of 45x45 cm of area, with infra-red beams all around, spaced 2.5 cm, coupled to a computerized control unit. We have analyzed the following parameters: (i) distance run in the actimeter (ambulation); (ii) time spent in slow movements (speed <5 cm/s); (iii) resting time (inactivity); and (iv) number of head entries into square holes (exploratory activity). The analysis of motor activity was done for periods of 10 min. The experimental set-up used to study the footprint patterns by the mice walking on the 60 cm paper sheet is shown in Figure 1A.

Brain regions and tissue preparation
After decapitation, brain parts used for biochemical studies were dissected, according to Carlsson and Lindqvist (37) and the areas that contain a rich density of dopamine terminals, limbic system and striatum, or DA cell bodies (midbrain) were used for further analysis. The brain parts were frozen on dry ice. Spinal cords were dissected out, immersed in 4% paraformaldehyde for 24 h and used for histological studies. 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 13 400 g for 20 min at 4°C. The supernatant was used for monoamines and their metabolites as well as for glutathione determination.

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 electrophoresis analysis.

Histological studies
The animals used for these studies were anesthetized 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. The whole brain and the spinal cord were immersed in paraformaldehyde for 24 h and then included in paraffin, sectioned in the microtome at a thickness of 4 µm, and stained for haematoxylin/eosine (H&E), Nissl's stain, solocrom for myelin, tyrosine hydroxylase (TH) and tau (AT100) immunoreactivity. Rabbit polyclonal anti TH antibody, from Chemicon, (Madrid, Spain) was diluted 1:2000 and human monoclonal anti PHF tau antibody from Pierce Endogen (Rockford, IL, USA) was diluted 1:100. Anti-mouse and anti-rabbit secondary antibodies were from Dako (Denmark).

The number of TH immunoreactive neurons in the substantia nigra was counted in an Olympus Bx51 microscope using Cast Grid software. Dopamine cells were quantified stereologically in three regularly spaced sections covering the entire surface of the anterio-posterior extent of the substantia nigra. Similarly, the number of anterior horn cells of the spinal cord were counted in Nissl-stained sections. Motoneurons were quantified stereologically on three regularly spaced sections covering the ventral horn of the lumbar extent of the spinal cord.

Determination of monoamines and their metabolites
The brain regions were frozen on dry ice and the levels of DA and its metabolites, 3-methoxy-tyramine (3-MT), 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), noradrenaline (NA) and its metabolite, 4-hydroxy-3-methoxy-phenyl-glycol (MHPG), serotonin (5-HT) and its metabolite, 5-hydroxy-indole-acetic acid (5-HIAA), were measured by HPLC with an ESA coulochem detector, according to Mena et al. (38). Briefly, the tissue was sonicated in 8 vol (w/v) of 0.4 N perchloric acid (PCA) with 0.5 mM Na2S2O5 and 2% EDTA and then centrifuged for 10 min. Monoamine levels were determined from 20 µl of the resulting supernatant.

The chromatographic conditions were: a column Nucleosil 5C18; the mobile phase, a citrate/acetate buffer 0.1 M, pH 3.9 with 10% methanol, 1 mM EDTA and 1.2 mM heptane sulfonic acid; and the detector voltage conditions: D1 (+0.05), D2 (–0.39) and the guard cell (+0.40).

Glutathione measurements
Total glutathione (GSx) levels were measured by the method of Tietze (39). A sample (40 µl) of the homogenated brain regions supernatant in 0.4 N PCA was neutralized with four 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 (40). 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 earlier. Reduced glutathione (GSH) was obtained by subtracting GSSG levels from GSx levels.

Western blot
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% ß-mercaptoethanol), electrophoresed in 10% SDS–polyacrylamide gels and then electroblotted to 0.45 µM nitrocellulose 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, blots were washed twice with blocking solution for 10 min followed by another two washes with TTBS for 5 min each. The blots were developed by chemiluminescence detection using a commercial kit (ECL-Amersham Biosciences) and quantified by computer-assisted video densitometry. ß-actin was used as a control of charge.

Antibodies
Mouse monoclonal anti-glial fibrillar acid protein (GFAP) antibody diluted 1:5000, mouse monoclonal anti-tyrosine hydroxylase (TH) antibody (1:5000) and rat monoclonal anti-dopamine transporter (DAT) (1:2500) were from Chemicon; mouse monoclonal anti-Bcl2 (1:500), rabbit polyclonal anti-Bax (1:750) and mouse monoclonal anti Hsp-70 (1:100) were from Santa Cruz; rabbit polyclonal anti-ß-tubulin (1:10 000) was from Covance; rabbit polyclonal anti-human GLUT-5 (1:250) was from IBL-Hamburg; rabbit anti-rat neuron specific enolase (NSE) (1:1000) was from Polysciences; mouse monoclonal anti-CHIP (1:1000) was from Abcam; rabbit polyclonal anti-phospho GSK 3{alpha}/ß (21/9) (1:1000) was from Cell Signaling; mouse monoclonal anti GSK 3{alpha}/ß (1:1000) was from BD Biosciences; mouse monoclonal anti-human tau T14 (1:1000) was from Zymed; mouse monoclonal anti-tau 7.51 (1:1000) was a generous gift from Dr Avila; mouse monoclonal anti-ß-actin antibody diluted 1:5000 was from Sigma.

Anti-mouse and anti-rabbit secondary antibodies diluted 1:1000 were from Amersham. ß-actin secondary antibody was an anti-mouse phosphatase alkaline conjugate diluted 1:3000 from Sigma.

Antibodies used for immunohistochemistry were the following: rabbit anti-tyrosine hydroxylase polyclonal antibody from Chemicon, diluted 1:500 and anti-human PHF-tau monoclonal antibody (AT100) from Pierce Endogen, Rockford, IL, USA, diluted 1:500.

Statistical analysis
The results were statistically evaluated with different tests. Significant differences between the four experimental groups were analyzed with one and two way analysis of variance (ANOVA) followed by the Newman–Keuls multiple comparison test or Bonferroni test, respectively, as a post hoc evaluation. Differences were considered statistically significant when P<0.05.


   ACKNOWLEDGEMENTS
 
This work was supported by the Spanish Government Grants, FIS 2002/PI20265, 2004/PI40360 and RED CIEN 03/06. J.M. and J.A.R. are recipients of FIS predoctoral fellowships. The authors thank Dr Alexis Brice and Aventis Corporation for providing parkin-KO mice and R. Villaverde for her kind technical assistance.

Conflict of Interest statement. None declared.


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


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Delacourte A. and Buee L. (1997) Normal and pathological Tau proteins as factors for microtubule assembly. Int. Rev. Cytol. 171:167–224.[Web of Science][Medline]

  2. Kosik K.S., Joachim C.L., Selkoe D.J. (1986) Microtubule-associated protein tau (tau) is a major antigenic component of paired helical filaments in Alzheimer disease. Proc. Natl Acad. Sci. USA 83:4044–4048.[Abstract/Free Full Text]

  3. Baas P.W. and Qiang L. (2005) Neuronal microtubules: when the MAP is the roadblock. Trends Cell. Biol. 15:183–187.[CrossRef][Medline]

  4. Avila J., Perez M., Lucas J.J., Gomez-Ramos A., Santa Maria I., Moreno F., Smith M., Perry G., Hernandez F. (2004) Assembly in vitro of tau protein and its implications in Alzheimer's disease. Curr. Alzheimer Res. 1:97–101.[Medline]

  5. Goedert M. (2005) Tau gene mutations and their effects. Mov. Disord. 20:S45–S52.

  6. Mott R.T., Dickson D.W., Trojanowski J.Q., Zhukareva V., Lee V.M., Forman M., Van Deerlin V., Ervin J.F., Wang D.S., Schmechel D.E., et al. (2005) Neuropathologic, biochemical, and molecular characterization of the frontotemporal dementias. J. Neuropathol. Exp. Neurol. 64:420–428.[Medline]

  7. Santacruz K., Lewis J., Spires T., Paulson J., Kotilinek L., Ingelsson M., Guimaraes A., DeTure M., Ramsden M., McGowan E., et al. (2005) Tau suppression in a neurodegenerative mouse model improves memory function. Science 309:476–481.[Abstract/Free Full Text]

  8. Goedert M. and Jakes R. (2005) Mutations causing neurodegenerative tauopathies. Biochim. Biophys. Acta 1739:240–250.[Medline]

  9. Baba Y., Tsuboi Y., Baker M.C., Uitti R.J., Hutton M.L., Dickson D.W., Farrer M., Putzke J.D., Woodruff B.K., Ghetti B. (2005) The effect of tau genotype on clinical features in FTDP-17. Parkinsonism Relat. Disord. 11:205–208.[Web of Science][Medline]

  10. Forman M.S. (2004) Genotype–phenotype correlations in FTDP-17: does form follow function? Exp. Neurol. 187:229–234.[CrossRef][Web of Science][Medline]

  11. Mamah C.E., Lesnick T.G., Lincoln S.J., Strain K.J., de Andrade M., Bower J.H., Ahlskog J.E., Rocca W.A., Farrer M.J., Maraganore D.M. (2005) Interaction of alpha-synuclein and tau genotypes in Parkinson's disease. Ann. Neurol. 57:439–443.[CrossRef][Web of Science][Medline]

  12. Katsuno T., Morishima-Kawashima M., Saito Y., Yamanouchi H., Ishiura S., Murayama S., Ihara Y. (2005) Independent accumulations of tau and amyloid beta-protein in the human entorhinal cortex. Neurology 64:687–692.[Abstract/Free Full Text]

  13. Barbato C., Canu N., Zambrano N., Serafino A., Minopoli G., Ciotti M.T., Amadoro G., Russo T., Calissano P. (2005) Interaction of Tau with Fe65 links tau to APP. Neurobiol. Dis. 18:399–408.[Medline]

  14. Feyt C., Kienlen-Campard P., Leroy K., N'Kuli F., Courtoy P.J., Brion J.P., Octave J.N. (2005) Lithium chloride increases the production of amyloid-beta peptide independently from its inhibition of glycogen synthase kinase 3. J. Biol. Chem. 280:33220–33227.[Abstract/Free Full Text]

  15. Noble W., Planel E., Zehr C., Olm V., Meyerson J., Suleman F., Gaynor K., Wang L., LaFrancois J., Feinstein B. (2005) Inhibition of glycogen synthase kinase-3 by lithium correlates with reduced tauopathy and degeneration in vivo. Proc. Natl Acad. Sci. USA 102:6990–6995.[Abstract/Free Full Text]

  16. Chun J., Kwon T., Lee E.J., Kim C.H., Han Y.S., Hong S.K., Hyun S., Kang S.S. (2004) 14-3-3 Protein mediates phosphorylation of microtubule-associated protein tau by serum- and glucocorticoid-induced protein kinase 1. Mol. Cells 18:360–368.[Web of Science][Medline]

  17. van de Warrenburg B.P., Lammens M., Lucking C.B., Denefle P., Wesseling P., Booij J., Praamstra P., Quinn N., Brice A., Horstink M.W. (2001) Clinical and pathologic abnormalities in a family with parkinsonism and parkin gene mutations. Neurology 56:555–557.[Abstract/Free Full Text]

  18. Morales B., Martinez A., Gonzalo I., Vidal L., Ros R., Gomez-Tortosa E., Rabano A., Ampuero I., Sanchez M., Hoenicka J. (2002) Steele–Richardson–Olszewski syndrome in a patient with a single C212Y mutation in the parkin protein. Mov. Disord. 17:1374–1380.[CrossRef][Web of Science][Medline]

  19. Sanchez M.P., Gonzalo I., Avila J., De Yebenes J.G. (2002) Progressive supranuclear palsy and tau hyperphosphorylation in a patient with a C212Y parkin mutation. J. Alzheimers Dis. 4:399–404.[Medline]

  20. Yang F., Jiang Q., Zhao J., Ren Y., Sutton M.D., Feng J. (2005) Parkin stabilizes microtubules through strong binding mediated by three independent domains. J. Biol. Chem. 280:17154–17162.[Abstract/Free Full Text]

  21. Itier J.M., Ibanez P., Mena M.A., Abbas N., Cohen-Salmon C., Bohme G.A., Laville M., Pratt J., Corti O., Pradier L., et al. (2003) Parkin gene inactivation alters behaviour and dopamine neurotransmission in the mouse. Hum. Mol. Genet. 12:2277–2291.[Abstract/Free Full Text]

  22. Hernandez F., Borrell J., Guaza C., Avila J., Lucas J.J. (2002) Spatial learning deficit in transgenic mice that conditionally over-express GSK-3beta in the brain but do not form tau filaments. J. Neurochem. 83:1529–1533.[CrossRef][Web of Science][Medline]

  23. Lim F., Hernandez F., Lucas J.J., Gomez-Ramos P., Moran M.A., Avila J. (2001) FTDP-17 mutations in tau transgenic mice provoke lysosomal abnormalities and Tau filaments in forebrain. Mol. Cell. Neurosci. 18:702–714.[CrossRef][Web of Science][Medline]

  24. Goedert M., Satumtira S., Jakes R., Smith M.J., Kamibayashi C., White C.L. III, Sontag E. (2000) Reduced binding of protein phosphatase 2A to tau protein with frontotemporal dementia and parkinsonism linked to chromosome 17 mutations. J. Neurochem. 75:2155–2162.[CrossRef][Web of Science][Medline]

  25. Guerrero R., Navarro P., Gallego E., Avila J., de Yébenes J.G., Sánchez M.P. (2006) Parkin and tau interaction analysis in parkin deficient and mutant transgenic mice. Submitted for publication.

  26. Shimura H., Schwartz D., Gygi S.P., Kosik K.S. (2004) CHIP–Hsc70 complex ubiquitinates phosphorylated tau and enhances cell survival. J. Biol. Chem. 279:4869–4876.[Abstract/Free Full Text]

  27. Sahara N., Murayama M., Mizoroki T., Urushitani M., Imai Y., Takahashi R., Murata S., Tanaka K., Takashima A. (2005) In vivo evidence of CHIP up-regulation attenuating tau aggregation. J. Neurochem. 94:1254–1263.[CrossRef][Web of Science][Medline]

  28. Casarejos M.J., Solano R.M., Menendez J., Rodriguez-Navarro J.A., Correa C., Garcia de Yebenes J., Mena M.A. (2005) Differential effects of l-DOPA on monoamine metabolism, cell survival and glutathione production in midbrain neuronal-enriched cultures from parkin knockout and wild-type mice. J. Neurochem. 94:1005–1014.[CrossRef][Medline]

  29. Serrano A., Menendez J., Casarejos M.J., Solano R.M., Gallego E., Sanchez M., Mena M.A., Garcia de Yebenes J. (2005) Effects of cinnarizine, a calcium antagonist that produces human parkinsonism, in parkin knock out mice. Neuropharmacology 49:208–219.[Medline]

  30. Adamec E., Murrell J.R., Takao M., Hobbs W., Nixon R.A., Ghetti B., Vonsattel J.P. (2002) P301L tauopathy: confocal immunofluorescence study of perinuclear aggregation of the mutated protein. J. Neurol. Sci. 200:85–93.[CrossRef][Web of Science][Medline]

  31. Elyaman W., Terro F., Wong N.S., Hugon J. (2002) In vivo activation and nuclear translocation of phosphorylated glycogen synthase kinase-3beta in neuronal apoptosis: links to tau phosphorylation. Eur. J. Neurosci. 15:651–660.[CrossRef][Web of Science][Medline]

  32. Lefebvre T., Ferreira S., Dupont-Wallois L., Bussiere T., Dupire M.J., Delacourte A., Michalski J.C., Caillet-Boudin M.L. (2003) Evidence of a balance between phosphorylation and O-GlcNAc glycosylation of Tau proteins–a role in nuclear localization. Biochim. Biophys. Acta 1619:167–176.[Medline]

  33. Hu X.Y., Qin S., Lu Y.P., Ravid R., Swaab D.F., Zhou J.N. (2003) Decreased estrogen receptor-alpha expression in hippocampal neurons in relation to hyperphosphorylated tau in Alzheimer patients. Acta Neuropathol. (Berl.) 106:213–220.[CrossRef][Medline]

  34. Hoenicka J., Vidal L., Morales B., Ampuero I., Jimenez-Jimenez F.J., Berciano J., del Ser T., Jimenez A., Ruiz P.G., de Yebenes J.G. (2002) Molecular findings in familial Parkinson disease in Spain. Arch. Neurol. 59:966–970.[Abstract/Free Full Text]

  35. Ros R., Ampuero I., de Yébenes J.G. (2006) Parkin polymorphisms in progressive supranuclear palsy. Submitted for publication.

  36. Taniguchi S., Suzuki N., Masuda M., Hisanaga S., Iwatsubo T., Goedert M., Hasegawa M. (2005) Inhibition of heparin-induced tau filament formation by phenothiazines, polyphenols, and porphyrins. J. Biol. Chem. 280:7614–7623.[Abstract/Free Full Text]

  37. Carlsson A. and Lindqvist M. (1973) Effect of ethanol on the hydroxylation of tyrosine and tryptophan in rat brain in vivo. J. Pharm. Pharmacol. 25:437–440.[Web of Science][Medline]

  38. Mena M.A., Garcia de Yebenes M.J., Tabernero C., Casarejos M.J., Pardo B., Garcia de Yebenes J. (1995) Effects of calcium antagonists on the dopamine system. Clin. Neuropharmacol. 18:410–426.[Web of Science][Medline]

  39. Tietze F. (1969) Enzymatic method for quantitative determination of nanogram amounts of total and oxidized glutathione: application to mammalian blood and other tissue. Anal. Biochem. 27:502–522.[CrossRef][Web of Science][Medline]

  40. Griffith O.W. (1980) Determination of glutathione and glutathione disulfide using glutathione reductase and 2-vinylpyridine. Anal. Biochem. 106:207–212.[CrossRef][Web of Science][Medline]


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 NeuroscientistHome page
M. A. Mena and J. Garcia de Yebenes
Glial Cells as Players in Parkinsonism: The "Good," the "Bad," and the "Mysterious" Glia
Neuroscientist, December 1, 2008; 14(6): 544 - 560.
[Abstract] [PDF]

Home page
 Hum Mol GenetHome page
J. A. Rodriguez-Navarro, A. Gomez, I. Rodal, J. Perucho, A. Martinez, V. Furio, I. Ampuero, M. J. Casarejos, R. M. Solano, J. G. de Yebenes, et al.
Parkin deletion causes cerebral and systemic amyloidosis in human mutated tau over-expressing mice
Hum. Mol. Genet., October 15, 2008; 17(20): 3128 - 3143.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
R. M. Solano, M. J. Casarejos, J. Menendez-Cuervo, J. A. Rodriguez-Navarro, J. Garcia de Yebenes, and M. A. Mena
Glial Dysfunction in Parkin Null Mice: Effects of Aging
J. Neurosci., January 16, 2008; 28(3): 598 - 611.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
15/13/2045    most recent
ddl129v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (15)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Menéndez, J.
Right arrow Articles by de Yébenes, J.G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Menéndez, J.
Right arrow Articles by de Yébenes, J.G.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?