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EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online July 21, 2005 as doi:10.1096/fj.05-3845fje. |
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-synuclein in the substantia nigra
* Interdisciplinary Center for Neurosciences (IZN), Department of Neuroanatomy, University of Heidelberg, Heidelberg, Germany; and
European Molecular Biology Laboratory (EMBL), Monterotondo, Italy
1 Correspondence: Interdisciplinary Center for Neurosciences (IZN), Department of Neuroanatomy, University of Heidelberg, Im Neuenheimer Feld 307, D-69120 Heidelberg, Germany. E-mail: O.von_Bohlen{at}web.de
SPECIFIC AIMS
The neurotrophin receptors trkB and trkC and their ligands are prominently expressed in the adult substantia nigra pars compacta (SNpc), yet their physiological roles in the nigrostriatal system are largely obscure. Using mice carrying haploin-sufficiencies for trkB or/and trkC, we investigated the effect of trkB and trkC signaling for the maintenance and metabolic welfare of dopaminergic (DAergic) neurons in the SNpc of mice aged 21–23 months and their striatal projections. We were interested in the cellular deposition of 
-synuclein, a hallmark of Parkinson’s disease (PD). Our findings indicate that unimpaired trkB and trkC signaling is crucial for the survival of SNpc DAergic neurons and maintenance of their projections to the striatum in aged mice. This provides the first evidence for a physiological role of trkB and trkC neurotrophin receptors for the maintenance of adult SNpc DAergic neurons and prevention of -synuclein deposits.
PRINCIPAL FINDINGS
To investigate a putative effect of aging and trkB/trkC receptors on the SNpc and their DAergic projection to the striatum, we analyzed adult (6–8 months old) and aged (21–23 months old) heterozygous mice with targeted deletions of the trkB and/or trkC receptor genes (trkB(+/–), trkC(+/–), trkB/trkC(+/–)/(+/–) mice) and their age-matched control littermates (trkB/trkC(+/+)/(+/+)). In contrast to the homozygous mutants, the haploin-sufficient mice survive into adulthood.
1. Reduced levels of trkB and/or trkC receptors cause prominent neuron losses in the aged SNpc
Analysis of cell numbers in the SNpc of trkB/trkC(+/+)/(+/+) mice indicate there is significant neuron loss in the SNpc during aging. Losses in total neuronal numbers of 21- to 23-month-old mice amounted to 13.5% vs. 6- to 8-month-old littermates (Fig. 1
A). Compared with aged control mice, aged trkB(+/–) mice revealed a further reduction to 15% in cell numbers; neuron losses in the SNpc of aged trkB/trkC(+/–)/(+/–) amounted to 21.3% (Fig. 1A
).
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To determine whether cell losses seen in the aged haploin-sufficient mice included losses of DAergic neurons, we analyzed numbers of tyrosine hydroxylase (TH) immunopositive SNpc neurons in normal adult and aged mice. Age-related reductions in total cell numbers in the SNpc (–13.5%) were paralleled by a 16.6% loss of DAergic neurons.
Slight but insignificant differences were noted between adult wild-type and adult mutant littermates (Fig. 1B
). Comparisons of numbers of TH-immunopositive neurons in the SNpc of aged heterozygous trk mutants and respective age-matched control mice revealed significant reductions in DAergic cell numbers, ranging from –14.4% (aged trkC(+/–)) and –17.8% (aged trkB(+/–)) to –26.0% (aged trkB/trkC(+/–)/(+/–)) (Fig. 1C
). Compared with adult trkB(+/–) mice, aged trkB(+/–) mice displayed a reduction of 27.8%; aged trkC(+/–) mice showed a 25.7% reduction compared with adult trkC(+/–) mice. The strongest effect was found in heterozygous double knockout animals. Compared with adult trkB/C(+/–)/(+/–) mice, the aged trkB/C(+/–)/(+/–) mice showed a reduction of 34.5%. Together, these data indicate that trkB and trkC haploin-sufficiencies cause prominent reductions in total neuron numbers and numbers of TH-positive neurons in the SNpc of aged mice. These losses usually occur between adulthood and old age. Thus, neurotrophin signaling through trkB and trkC appears to be indispensable for assuring unimpaired survival of DAergic neuronal cell bodies in the SNpc during aging.
2. Reduced levels of trkB and/or trkC receptors compromises the DAergic innervation of the striatum
We next investigated possible consequences of these changes for the DAergic innervation of the striatum, the target of SNpc DAergic projections. There is a significant age-related (6–8 vs. 21–23 months) decline in the density of TH-positive fibers in normal (trkB/trkC(+/+)/(+/+)) mice. In adult mutant animals vs. age-matched controls, slight but insignificant reductions in the densities of TH-ir fibers were noted in trkB(+/–) and trkC(+/–) mice. A significant reduction (–8.8%) was only found in the heterozygous double mutant mice (Fig. 1D
).
Another significant reduction in the fiber densities was found in aged trkB or/and trkC heterozygous knockout mice compared with adult and age-matched (21 to 23 months old) controls (Fig. 1E
). The most pronounced reduction was found in aged trkB/trkC(+/–)/(+/–) mice (–26% compared with age-matched controls; –39% compared with adult trkB/trkC(+/–)/(+/–) mice). Together, these data indicate that losses of TH-ir fibers in the striatum of trkB or/and trkC heterozygous mutant mice parallel losses of TH-positive neuronal cell bodies in the SNpc.
3. Reduced levels of trkB and/or trkC receptors cause abnormal accumulation of -synuclein in the remaining cells of the SNpc
In addition to the prominent degeneration of DAergic neurons in the SNpc seen in human PD, abnormal accumulation of -synuclein in neuronal cell bodies of the SNpc are morphological hallmarks of PD. Similar to what has been described for -synucleinopathies, -synuclein accumulating in the neuronal cell bodies of the SNpc of aged trkB or/and trkC mutants mice was found by using immunohistochemistry. Adult and aged control mice revealed no or very rare deposits of -synuclein in the SNpc. Adult heterozygous trkB, trkC, and trkB/C mice also display only rare deposits of -synuclein in the SNpc. A marked and significant increase in profiles with cytoplasmatic -synuclein accumulation in the SNpc was found in the aged heterozygous mutant mice; the highest increase was found in the aged trkB/trkC(+/–)/(+/–) mice. Double labeling experiments using antibodies directed against TH and -synuclein revealed that in the aged double heterozygous knockout mice, 95.2% of the -synuclein accumulating cell bodies in the SNpc were TH-positive whereas only 4.8% of the -synuclein accumulating cells were TH-negative. In aged trkC(+/–) mice, 3% of the -synuclein accumulating cells were TH-negative and in aged trkB(+/–) mice 3.9% of the -synuclein accumulating cells were TH-negative. These non-TH-positive -synuclein-accumulating cells may represent non-DAergic cells or DAergic neurons that have lost their DAergic phenotype. Together, these data show that losses of TH-positive neurons in mice lacking one allele of trkB or/and trkC are accompanied by massive accumulations of -synuclein in the remaining TH-ir cell bodies in the SNpc. Concerning -synuclein immunohistochemistry, we found diffuse cytoplasmatic staining or irregularly shaped inclusions of moderate intensity but failed to detect a discrete staining typical of the "pale bodies" or Lewy bodies (LBs), respectively. Neither aged heterozygous trkB/trkC(+/–)/(+/–) mice nor age-matched littermates (trkB/trkC(+/+)/(+/+)) displayed thioflavin S-positive inclusions in the SNpc.
CONCLUSIONS AND SIGNIFICANCE
Both BDNF and NT-3 promote survival of DAergic neurons, lending support to speculations that impaired signaling through their cognate receptors may be harmful for DAergic neurons. TrkB and trkC receptors as well as their ligands are prominently expressed in the adult SN. Degeneration of SN DAergic neurons and their striatal projections as well as synuclein-positive aggregates in the remaining neurons of the SN are hallmarks of PD. Reduced expression of the neurotrophin BDNF in the SN of PD patients has been reported. Moreover, mutations in the BDNF gene have been found to play a role in the development of familial PD. It has been suggested that pathogenic -synuclein mutations are linked to a reduced production of the trkB ligand BDNF. It may therefore be hypothesized that reduced neurotrophin signaling may be causally linked to the impairment of DAergic nigrostriatal system during aging and in PD. Aged mice carrying mutations in neurotrophin receptors display losses of DAergic neurons in the SNpc and striatal DAergic fibers that exceed those seen during normal aging. Thus, reduced neurotrophin signaling may contribute to a PD-like phenotype in aged mice. Our data support the notion that dramatic losses of DAergic neurons in the SNpc and striatal DAergic fibers result from deficits in trkB- and trkC-mediated neurotrophin signaling.
Not only in human PD, but also in the MPTP mouse model of PD, there is a reduction in DAergic functions and an abnormal accumulation of -synuclein in neurons in the SNpc. Kirik and co-workers (2002) detailed that overexpression of -synuclein in the rat nigrostriatal system induces -synuclein-positive cytoplasmic inclusions similar to those seen in brains of PD patients. These irregular deposits were accompanied by a loss of 30–80% of the nigral DAergic neurons and a 10–50% reduction of TH-ir fibers in the striatum; however, significant motor impairment developed only in those animals where DAergic neuron loss exceeded a critical threshold of 50–60%.
In support of the view that the phenotype of the trkB and trkC heterozygous mutant mice may be related to PD, we have shown an abnormal accumulation of -synuclein in neurons. However, we failed to detect typical LBs. Therefore, it is conceivable that the trkB and trkC heterozygous mutant phenotypes resemble a presymptomatic phase of PD, a stage corresponding to the Braak’s stage III of idiopathic PD. PD stage III is characterized by involvement of the SNpc but a lack of LBs in this area. This assumption is also consistent with observations reported by Wakabayashi and co-workers (1998), who demonstrated that -synuclein accumulation of the type 1 is found in incidental LBs disease, considered a presymptomatic phase of PD.
Aggregates of -synuclein in LBs and -synuclein-containing inclusions can be recognized by thioflavin S staining. In contrast, small punctate aggregates are thioflavin S negative, indicating a nonfibrillar conformation. Since we were unable to detect thioflavin S-positive inclusions in the aged heterozygous trk double mutants, the -synuclein-positive aggregates in the SNpc are unlikely to represent fibrillar inclusions. This further strengthens the view that the morphological phenotype of the aged mutant mice resembles a presymptomatic phase of PD. It has been suggested that the small spherical aggregates are formed before the formation of fibrillar inclusions. They may represent the cellular equivalents of protofibrils, which, rather than the fibril itself, may be the pathogenic substrate in PD.
The neurotoxins MPTP and 6-OHDA reproduced specific features of PD in animal models. These neurotoxins act on the mitochondrial complex I. Markham and colleagues (2004) showed that BDNF increases rat brain mitochondrial respiratory coupling at complex I. These findings indicate that neurotrophic stimulation can change the efficacy of coupling at complex I. Thus, reduced neurotrophin signaling may induce mitochondrial dysfunction and may lead us to discover pathways to cell death and to the formation of small punctate -synuclein aggregates in the surviving SNpc neurons (Fig. 2
).
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In conclusion, we have discovered a phenotype of the nigrostriatal system in aged heterozygous trkB and/or trkC mutant mice that resembles a preclinical stage of PD. The phenotype includes losses of TH-positive neurons in the SNpc and TH-positive fibers in the striatum as well as deposits of -synuclein in DAergic cell bodies. These data substantiate the view that neurotrophin signaling is physiologically relevant for the maintenance of the DAergic nigrostriatal system and that its deterioration may contribute to elicit a PD-like phenotype.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4006fje; doi: 10.1096/fj.05-3845fje
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compared with aged wild-type littermates. One symbol indicates P < 0.05, two symbols indicate P < 0.01. Error bars display SD.