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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online March 4, 2004 as doi:10.1096/fj.03-0829fje. |
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Experimental Therapeutics Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, USA;
* Blanchette Rockefeller Neurosciences Institute, Rockville, Maryland, USA
2Correspondence: National Institute of Neurological Disorders and Stroke, Experimental Therapeutics Branch, NIH, Bldg. 10, Room 5C-103, Bethesda, MD 20892, USA. E-mail: Chaset{at}ninds.nih.gov
SPECIFIC AIMS
Degeneration of dopamine neurons in the substantia nigra pars compacta (SNpc) and the aggregation of 
-synuclein into highly insoluble Lewy bodies are hallmarks of Parkinson’s disease (PD). Early conformational changes in the -synuclein molecule appear to contribute to pathogenesis. We examined postmortem PD and undiseased (control) substantia nigra for evidence of pathologic crosslinking of -synuclein by the enzyme tissue transglutaminase (tTG).
PRINCIPAL FINDINGS
1. tTG is increased in PD substantia nigra
Western blot analysis using a tTG-specific mouse monoclonal antibody (mAb; tTG3) revealed that expression of the full-length tTG molecule (78 kDa) was significantly increased in PD substantia nigra lysate (t=3.92; P<0.05) whereas the more variable increase in the expression of the 55 kDa splice variant did not reach statistical significance. Immunohistochemical labeling of tTG using tTG3 showed that cellular expression of tTG in dopamine neurons of SNpc was much stronger in PD than in control cases. In Lewy bodies, however, tTG expression was sporadic and weak. Fluorescent double labeling experiments confirmed these findings, revealing that tTG expression was increased in PD nigra and frequently colocalized with -synuclein in dopamine neurons, although typically not within the Lewy bodies per se.
2. The characteristic isodipeptide crosslink is increased in PD nigral dopamine neurons
Immunohistochemical labeling of the N
-(
-glutamyl)-lysine isodipeptide crosslink, specifically catalyzed by transglutaminase enzymes, was markedly increased in nigral dopamine neurons of PD cases compared with control cases. In contrast with the sparse labeling of tTG in Lewy bodies, there was intense labeling of the isodipeptide crosslink in these inclusions, indicating an abundance of tTG crosslinked protein. Immunofluorescent double labeling (Fig. 1
) confirmed the colocalization of -synuclein (Fig. 1A
) and isodipeptide crosslinks (Fig. 1B
) in somata and Lewy bodies in the majority of nigral dopamine neurons.
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3. The isodipeptide crosslink coprecipitates with -synuclein in the cytosolic fraction of PD substantia nigra
To determine more directly whether nigral -synuclein is crosslinked by tTG, SNpc tissue was fractionated into three lysate components containing cytoplasmic proteins (S1), membrane-associated proteins (S2), and highly hydrophobic and aggregated proteins (P). Proteins containing the isodipeptide crosslink were immunoprecipitated from the cytoplasmic fraction, using the selective N-(-glutamyl)-lysine isodipeptide mAb (Fig. 2
A, B). Western blot revealed a strong -synuclein-positive band migrating at 16 kDa in all PD samples, indicating the presence of the transglutaminase characteristic crosslink in the soluble -synuclein monomer. A significantly lower concentration of the crosslinked -synuclein monomer was eluted from the control nigra (t=7.59, P<0.001; Fig. 2A
). There was a strong positive correlation between the degree of PD progression and the concentration of the crosslinked soluble -synuclein monomer (R2=0.932; P<0.01). With longer chemiluminescence exposure time, -synuclein oligomers containing the tTG crosslink were detected in PD, but not control, samples at 
36, 49, and 64 kDa (Fig. 2B
). When the primary antibody was omitted, only the band corresponding to the isodipeptide antibody IgG was detected (Fig. 2C
).
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4. Uncrosslinked -synuclein monomer is decreased in PD substantia nigra
Examination of the post-immunoprecipitation lysate by Western blot revealed that the concentration of uncrosslinked -synuclein monomer remaining in the nigral PD lysate after extraction of the crosslinked proteins was significantly less than in control lysate (t=2.53; P<0.05; Fig. 2D
).
5. The solubility characteristics of transglutaminase crosslinked proteins extracted from substantia nigra
Despite the fact that the presence of crosslinked -synuclein could not be examined directly by immunoprecipitation of the membrane-associated (S2) and aggregated (P) proteins, Western blot analysis of isodipeptide-containing proteins from the three brain lysate fractions provided additional information. Several bands were evident, including a prominent isodipeptide-reactive band of 52 kDa. Bands were detected at 16, 32, and 45 kDa, consistent with the migration levels of -synuclein species identified in both immunoprecipitation experiments. However, the three latter bands were most prominent in the S2 fraction. Likewise, the putative -synuclein proteins detected in the 16, 32, and 45 kDa bands were in greater concentration in the PD than in control tissue in this fraction.
6. The tTG enzyme coprecipitates with -synuclein monomer in PD substantia nigra
Although tTG is the most likely candidate to mediate -synuclein crosslinking in PD, other transglutaminases catalyze the N-(-glutamyl)-lysine crosslink. The identity of the transglutaminase colocalized with -synuclein was directly tested by immunoprecipitation of -synuclein from SNpc lysate, followed by immunoblotting with the tTG - specific tTG3 mAb. The 78 kDa full-length tTG coprecipitated with -synuclein from samples of PD, but not control, cases.
CONCLUSIONS AND SIGNIFICANCE
The present results demonstrate that expression of tTG and its tTG - characteristic N-(-glutamyl)-lysine crosslink are both increased in the SNpc of PD patients. Immunohistochemical colocalization and immunoprecipitation studies show that -synuclein is a tTG substrate in human nigra and that crosslinked -synuclein is markedly enhanced in PD. The finding that the tTG signature crosslink, but not the tTG protein, is abundant in Lewy bodies suggests that most tTG - catalyzed crosslinking occurs in cytoplasm outside the Lewy bodies prior to incorporation of protein.
Unexpectedly, we identified a novel isoform of the soluble -synuclein monomer that contained a tTG -catalyzed intramolecular crosslink. Although the intramolecularly crosslinked monomer was detected in low concentration in control SNpc, it was highly enriched in PD tissue. Even though the concentration of crosslinked -synuclein appears to be greater in the membrane-associated fraction than in the cytoplasmic fraction, we propose that crosslinking is initiated at the site of the cytoplasmic-soluble monomer. First, a depletion of non-crosslinked, normal -synuclein monomer and a concomitant increase in the concentration in the S1 fraction of the tTG - crosslinked monomer correlated with disease progression, consistent with a net equilibrium shift from cytoplasm to membrane. Second, it was recently discovered that 90% of all -synuclein in Lewy bodies is phosphorylated at Ser 129, yet the steady-state physiological phosphorylation of this residue is only 4%. Phosphorylation of the Ser 129 residue in -synuclein markedly reduces its lipid affinity and has been proposed as the mechanism by which this protein translocates from membranes to cytoplasm. These data provide strong evidence that cytoplasmic phospho-ser -synuclein is the initial locus for tTG-induced crosslinking. We propose that tTG -induced intramolecular crosslinking of the soluble -synuclein monomer is a crucial early step in this process. Our data also suggest that once -synuclein becomes crosslinked by tTG, its solubility characteristics are altered and it is more likely to colocalize with the membrane containing fraction (Fig. 3
).
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The present data offer at least two possible bases for -synuclein-associated pathology in PD. Despite almost normal levels of the soluble -synuclein monomer in PD nigra, the amount of the uncrosslinked, presumably functional, -synuclein was significantly reduced. In previous work we determined that -synuclein translocates between plasma membrane and cytosol in response to cholinergic stimulation and appears to participate in muscarinic receptor endocytosis. This and other findings led us to propose that this translocation is accompanied by a conformational change in the -synuclein molecule. The presence of an internal molecular crosslink is predicted to impair the ability of -synuclein to alter its configuration and decrease its solubility. The increase of the crosslinked -synuclein monomer at the expense of the uncrosslinked normal isoform lends support to the hypothesis that hypofunctionality of -synuclein is a factor in PD pathogenesis.
A second possibility is that the crosslinked monomer itself could be toxic or could induce toxicity indirectly by providing a nucleation site for -synuclein aggregation. The appearance of relatively small oligomers containing the -synuclein crosslink suggests that the aggregation of -synuclein into insoluble inclusions may be seeded by the crosslinked monomer and progress in size through the formation of small oligomers. As such aggregates become larger and more insoluble, they may contribute to neurotoxicity by saturating cellular machinery, such as proteasome and lysosome. The proposed loss-of-function and gain-of-function hypotheses are not mutually exclusive: the complex pathogenesis of PD may include both mechanisms.
The factors leading to the initiation of pathological crosslinking of -synuclein by tTG remain unknown. The enzyme has a dual function as a G-protein coupled to 1B and 1D adrenergic receptors mediating phospholipase C-activated phosphatidylinositol production, as well as being a protein crosslinker. The crosslinking activity of tTG is promoted by calcium and inhibited by GTP. Physiological levels of GTP normally prevent aberrant activity of tTG in response to transient Ca++ currents. The 15–30% reduction in complex I activity that characterizes sporadic PD may contribute to a chronic reduction of ATP and GTP that may promote the crosslinking activity of tTG. Elucidation of the entire cascade leading to aberrant tTG crosslinking of -synuclein in PD may identify the earliest events in PD pathogenesis and point to novel targets for therapeutic interdiction.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0829fje; 
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