Mutations in the gene for α-synuclein, a small protein found in presynaptic terminals, were the first to be linked to familial Parkinson disease (PD). Since that initial discovery in 1997, researchers have tried with little success to figure out exactly how the protein can cause the loss of dopaminergic neurons at the root of the movement disorder. The subsequent identification of other PD genes raised hopes that some kind of common pathological pathway might emerge, something akin to the interaction between APP and presenilin-1 and 2, the three genes linked to accelerated production of amyloid-β in familial Alzheimer disease (AD). But while some new kids on the block—parkin, DJ-1, and Pink 1—seem to link to each other in a common pathological pathway in PD (see ARF related news story), α-synuclein has remained a loner. At least until now. In the December 24 Neuron, researchers led by Huaibin Cai at the National Institutes of Health, Bethesda, Maryland, report that the latest PD gene identified, LRRK2, exacerbates α-synuclein pathology in transgenic mice. Though it does not appear that LRRK2 and α-synuclein interact directly, the former seems to perturb the Golgi network, leading to a trafficking defect that promotes retention of α-synuclein in neuronal cell bodies. The findings finally help begin to explain how α-synuclein, and LRRK2, together can cause Parkinson disease.
LRRK2, or Leucine-Rich Repeat Kinase 2, is a large protein with many functional domains, including a kinase domain and a GTPase domain. Mutations in the gene were first linked to PD in 2004, but like with α-synuclein, it is still unclear how the gene causes the disease (see ARF related news story). To investigate, Cai and colleagues made a series of transgenic mice overexpressing wild-type and mutant LRRK2 under the control of an inducible tetracycline operator linked to a calmodulin kinase II (CaMKII) promoter. Joint first authors Xian Lin, Loukia Parisiadou, Xing-Long Gu, and Lizhen Wang also used the same methodology to generate a line of mice overexpressing wild-type or A53T mutant α-synuclein (A53T-Syn). To test if LRRK2 exacerbates α-synuclein pathology, they then made a series of double-transgenic mice.
Lin and colleagues found that the A53T-Syn mice were abnormally small, whereas the LRRK2 animals seemed to thrive. The authors tested four different LRRK2 lines. Two, LRRK2 wild-type (LRRK2WT) and LRRK2 G2019S (carrying the most common PD mutation), overexpressed about eight- to 16-fold, while the other two, LRRK2WT-L and LRRK2-KD, did so only by one- to twofold. LRRK2-KD had the kinase domain deleted.
Beginning at around two months of age, A53T-Syn mice showed signs of locomotor problems, which became more pronounced by six months, with the animals rearing up on their hind legs much more frequently than their wild-type littermates. Mice overexpressing human LRRK2 G2019S had much milder locomotor problems than the A53T-Syn animals. When the researchers examined the latter for the potential cause, they found neuronal damage in the striatum and cortex of 12-month-old animals, as evidenced by expression of cleaved caspase-3 and Jade C, makers of neurodegeneration. This pathology worsened over the next eight months. In contrast, none of the LRRK2 transgenic animals showed any signs of neurodegeneration at any age up to 20 months. Stereology suggested about an 80 percent loss of neurons in the frontal cortex and a 74 percent loss in the dorsal striatum.
Though no neurodegeneration was evident in any of the LRRK2 animals, Lin and colleagues found that LRRK2 did exacerbate synuclein pathology in double-transgenic animals. At one month of age, A53T-Syn mice harbored no sign of neurodegeneration, but offspring of crosses with any of the four LRRK2 transgenic animals did show Jade C and cleaved caspase-3 expression by that age. The numbers of activated astrocytes and microglia were also elevated in the crosses. There seemed to be a dose response since the neurodegeneration seen in the A53T-Syn/LRRK2WT mice was much greater than in the A53T-Syn/LRRK2WT-L, animals, which express less LRRK2. This pathology was further increased in the presence of LRRK2 G2019S.
To delve deeper into problems faced by these animals, the researchers examined them for signs of α-synuclein pathology. The protein is the principal component of Lewy bodies, dense protein aggregates found in the dopaminergic neurons of the substantia nigra and elsewhere in the brains of people with Parkinson disease. In the A53T-Syn animals, α-synuclein was banished from its normal location at nerve terminals and found in the cell bodies, or soma, by about 12 months of age. In contrast, somatic α-synuclein appeared in double-transgenic animals already by one month of age, indicating that LRRK2 hastens the displacement of α-synuclein. Somatic α-synuclein was even found in offspring of wild-type α-synuclein x LRRK2 G2019S crosses. “These results suggest that the LRRK2-induced somatic accumulation of α-synuclein is independent of the presence of PD-related α-synuclein mutation but relies on the expression level of α-synuclein,” write the authors. Notably, in rare human cases, Parkinson disease is caused by simple overexpression of wild-type α-synuclein (see ARF related news story), as is AD by overexpression of wild-type APP.
Next, the scientists addressed the question of how LRRK2 might shift α-synuclein away from nerve terminals. Looking at high-molecular-weight, detergent-insoluble protein, the researchers found that LRRK2 promotes α-synuclein aggregation. The two proteins do not seem to interact directly, as co-immunoprecipitation experiments never captured them together. Phosphorylation of α-synuclein by the LRRK2 seems not to influence aggregation, either, since the researchers actually found a reduction in phosphorylated α-synuclein in double-transgenic animals.
Staining for LRRK2 showed that it did overlap with somatic α-synuclein, however, putting the two proteins in the same intraneuronal vicinity. Lin and colleagues then turned to the Golgi apparatus, since earlier work showed that both α-synuclein (see Cooper et al., 2006) and LRRK2 (see Biskup et al., 2005) may be involved in Golgi biology. At one month of age, this organelle appeared normal in A53T-Syn mice, but the tubular structure was thinner and more fragmented in LRRK2WT and G2019S mice. Crossing either LRRK2 strain with A53T-Syn mice led to severe fragmentation of the cis-Golgi in the offspring, and this fragmentation correlated with the extent of somatic α-synuclein.
“We were very intrigued as to how these proteins affect the Golgi,” Cai told ARF. For example, Cai explored the role of the microtubules, since polarization of the Golgi network is known to depend on retrograde transport along the microtubules, and recently both α- and β-tubulin were shown to bind LRRK2 through its GTPase domain (see Gandhi et al., 2008 and Gillardon et al., 2009). Indeed, the researchers found that LRRK2 co-stained with βIII-tubulin and that free, soluble β-tubulin in LRRK2- and in A53T-Syn/LRRK2WT-overexpressing mice was dramatically reduced.
“These findings are consistent with previous in-vitro assays showing that overexpression of LRRK2 may enhance the polymerization of tubulin in cells, ultimately suggesting that the impairment of microtubule assembly may affect the organization of the microtubule network in the cell, resulting in the fragmentation of the Golgi apparatus,” write the authors. This could, presumably, entrap α-synuclein in the neuronal soma.
Still, some things are left unexplained, not least the relative contributions of α-synuclein and LRRK2 to the different facets of the pathology. As Youren Tong and Jie Shen, from Brigham and Women’s Hospital, Boston, write in an accompanying Neuron preview, “it is somewhat puzzling why overexpression of LRRK2 alone was sufficient to cause disruption of the microtubule network and Golgi apparatus but insufficient to cause neurodegeneration, whereas A53T α-synuclein transgenic mice exhibited much subtler microtubule and Golgi phenotypes but striking neuronal degeneration.”
Cai conceded that much work remains to be done to fully understand the interplay between the two proteins. Another aspect that needs exploration is the function of the LRRK2 kinase domain. The worst α-synuclein pathology occurred in mice that overexpressed LRRK2 with the G2019S mutation, which is difficult to reconcile with the fact that the kinase-deficient LRRK2 also exacerbated α-synuclein toxicity. One thing the researchers did do was look at the effect of knocking out LRRK2. This protected A53T-Syn mice against α-synuclein aggregation, neurodegeneration, gliosis, and Golgi fragmentation, indicating that those pathologies are not simply a non-specific result of LRRK2 overexpression. In support of this, they also saw none of the same pathology when they co-overexpressed LRRK2 with green fluorescent protein or APP as controls.
In their preview, Tong and Shen emphasized that the relevance of these models to PD remains to be proven, since the CaMKII promoter limits overexpression to forebrain neurons, including those in the cortex and striatum. Cai agreed, telling ARF, “we have a lot more work to do and really need to look at dopaminergic neurons.” He said his lab now has mouse lines that drive expression of α-synuclein in those neurons and that he expects that to be more relevant to PD.
One facet of this work that might be relevant to AD is the link between LRRK2 and microtubules. A recent report suggested that the R1628P LRRK2 mutation may be associated with AD (see Zhao et al., 2009), though LRRK2 has not earned a place in AlzGene yet. This gene occupies second place in PDGene. α-synuclein deposits have been found in AD patients; however, Cai suggested that if there is a link between LRRK2 and AD, it may more likely come via the microtubule binding protein tau.—Tom Fagan
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