In this study, we found that overexpression of synphilin-1 attenuated A53T α-synuclein-induced motor abnormalities and decreased astroglial reaction and neuronal degeneration. Moreover, overexpression of synphilin-1 increased beclin-1 and LC3-II levels, promoted formation of aggresome-like structures and diminished the severity of α-synucleinpathy in the brain of double-transgenic mice. Synphilin-1/A53T α-synuclein double-transgenic mice had delayed α-synuclein pathology and survived longer than A53T α-synuclein single-transgenic mice. These results indicate that synphilin-1 plays a neuroprotective role against A53T α-synuclein toxicity in transgenic mice in vivo.
We did not find any features of Parkinsonism in synphilin-1 single-transgenic mice by examining locomotor activity, brain pathology (neuronal degeneration and protein aggregation) and survival. We did find that expression of synphilin-1 significantly increased body weight (described elsewhere). These data are consistent with recent reports showing that there is no loss of dopamine neurons in the substantia nigra in synphilin-1 transgenic mice (14
). The other group recently showed that expression of either wild-type or mutant R621C synphilin-1 induced moderate aggregate formation and subtle neurodegeneration in the mouse brain (15
). This may be due to overexpression of synphilin-1 locally at high concentration via the adenoviral delivery system, or due to the different expression patterns of synphilin-1 in the mouse brain.
Neuronal degeneration is one key feature of Parkinsonism. Consistent with previous reports (23
), A53T single-transgenic mice displayed neuronal degeneration determined by the following indices: an increase in astroglial reaction, prominent axonal injury and activation of apoptotic pathways. In double-transgenic mice, overexpression of synphilin-1 clearly slowed down or protected against A53T α-synuclein-induced neuronal degeneration, ameliorated A53T α-synuclein-induced motor abnormalities and increased the survival of the mice. Previous reports have shown that synphilin-1 displays a protective function against staurosporine and 6-hydroxydopamine toxicity by reducing the hydrolysis of procaspase-3, decreasing poly (ADP-ribose) polymerase cleavage and reducing p53 transcriptional activity and expression (31
). Our results showed that overexpression of synphilin-1 reduced caspase-3 activation in neurons in the brain of double-transgenic mice, indicating that synphilin-1 can reduce some aspects of apoptotic pathway activation and ameliorate α-synuclein toxicity in double-transgenic mice.
Protein aggregation is another hallmark of PD pathogenesis. Synphilin-1 is one of the major constituents of Lewy bodies (12
). Moreover, synphilin-1 has been shown to interact with α-synuclein, the main constituent of Lewy bodies, in a yeast two-hybrid screen and in cellular model systems (5
). Previous studies show that inclusion body formation provides a defense mechanism against build up of a soluble toxic load by channeling this load to an inert location for subsequent handling by autophagy (32
). Inclusions are thought to represent transit stations for protein cargoes destined for degradation to make their final exit from the cell. A recent live cell imaging study suggests that the sequestration of α-synuclein into aggresomes facilitates its clearance from the cell (33
Recent studies show that synphilin-1 contains an ankyrin-like repeat domain that acts as an aggresome-targeting signal. In contrast, formation of multiple small aggregates requires a different segment within synphilin-1, indicating that some aspects of aggregation and aggresome formation determinants can be separated biochemically (10
). Small protein aggregates can be transported in a microtubule-dependent manner to the centrosome, forming an organelle called aggresome (29
). Aggresomes can be seen in mammalian cells after the inhibition of the proteasome (30
). The aggresome serves as a storage compartment for protein aggregates and could be actively involved in their refolding and degradation by autophagic clearance pathways (35
). In our study, there were more neurons with aggresome-like structures in double-transgenic mice than A53T single-transgenic mice at end stage disease. These results suggest that synphilin-1 promotes the formation of aggresome-like structures, consistent with a notion that aggresome represents a protective cellular response to toxic protein (30
). In line with this idea, an in vitro
study shows that aggresomes formed by synphilin-1 and α-synuclein appear to be more prevalent in non-apoptotic cells (11
α-Synuclein degradation depends on the pathways of the UPS and autophagic system (9
). Autophagy appears to be more effective in α-synuclein degradation than the proteasomal pathways (36
). Supporting this possibility is the finding that the autophagy activator, rapamycin, stimulates α-synuclein clearance (39
). In PD and Lewy body diseases, α-synuclein accumulation has been linked to alterations in autophagy and lysosomal functioning (28
). The autophagy pathway is disrupted in patients and in animal models that involve accumulation of α-synuclein (28
). In our study, expression of synphilin-1 increased beclin-1 and LC3-II levels at 7 and 10 months of age in double-transgenic mice, suggesting that synphilin-1 activation of autophagic clearance may contribute to these synphilin-1 protective effects. The decreased accumulation of proteinase K-resistant α-synuclein inclusions in double-transgenic mice may reflect the autophagic clearance of aggresome-like inclusions at a young age. Consistent with this notion is an in vitro
study which shows that induction of autophagy promotes a significant reduction of inclusions in cells expressing α-synuclein/synphilin-1 under conditions of proteasome impairment (9
). We also found abnormal accumulation of ubiquitin in brains of both double-transgenic and A53T single-transgenic mice, suggesting that there may be also impairment of the UPS in these mice. This result is consistent with previous findings that mutant α-synuclein causes inhibition of the UPS (44
Nevertheless, synphilin-1-enhanced autophagic clearance of aggregated proteins appears to be insufficient to clear all of the intracellular accumulation of α-synuclein. Thus, synphilin-1 only can delay α-synuclein pathology but not totally reverse PD-like phenotypes in double-transgenic mice. We detected a slight increase in LC-3 II levels in A53T single-transgenic mice at 7 and 10 months, indicating that there was an alteration in autophagy pathway by A53T α-synuclein expression alone. This is consistent with previous reports showing that mutant α-synuclein stimulated autophagy in an attempt to clear aberrant α-synuclein inclusions (42
). The synphilin-1-induced increase of beclin-1 levels in double-transgenic mice may contribute to neuronal protection by multiple mechanisms. Beclin-1 not only increases autophagy but also binds Bcl2 to block apoptosis (46
). A recent report shows that lentivirus vector delivery of beclin-1 into an α-synuclein mouse model of PD activates autophagy and reduces accumulation of α-synuclein (43
Taken together, the data indicate that transgenic expression of synphilin-1 delayed A53T α-synuclein-induced neuronal degeneration, normalized hyperactivity and increased survival in double-transgenic mice. Synphilin-1 neuroprotection may be related to its role in promoting aggresome formation and increasing autophagic clearance of abnormal α-synuclein in double-transgenic mice. This study elucidates aspects of cellular protective responses to abnormal α-synuclein, which may facilitate identification of therapeutic targets in PD.