Models for LRRK2-Linked Parkinsonism

Parkinson's disease (PD) is a progressive neurodegenerative movement disorder characterized by the selective loss of dopaminergic neurons and the presence of Lewy bodies. The pathogenesis of PD is not fully understood, but it appears to involve both genetic susceptibility and environmental factors. Treatment for PD that prevents neuronal death progression in the dopaminergic system and abnormal protein deposition in the brain is not yet available. Recently, mutations in the leucine-rich repeat kinase 2 (LRRK2) gene have been identified to cause autosomal-dominant late-onset PD and contribute to sporadic PD. Here, we review the recent models for LRRK2-linked Parkinsonism and their utility in studying LRRK2 neurobiology, pathogenesis, and potential therapeutics

Phosphorylation of p66Shc and forkhead proteins mediates Aβ toxicity

Excessive accumulation of amyloid β-peptide (Aβ) plays an early and critical role in synapse and neuronal loss in Alzheimer's Disease (AD). Increased oxidative stress is one of the mechanisms whereby Aβ induces neuronal death. Given the lessened susceptibility to oxidative stress exhibited by mice lacking p66Shc, we investigated the role of p66Shc in Aβ toxicity. Treatment of cells and primary neuronal cultures with Aβ caused apoptotic death and induced p66Shc phosphorylation at Ser36. Ectopic expression of a dominant-negative SEK1 mutant or chemical JNK inhibition reduced Aβ-induced JNK activation and p66Shc phosphorylation (Ser36), suggesting that JNK phosphorylates p66Shc. Aβ induced the phosphorylation and hence inactivation of forkhead transcription factors in a p66Shc-dependent manner. Ectopic expression of p66ShcS36A or antioxidant treatment protected cells against Aβ-induced death and reduced forkhead phosphorylation, suggesting that p66Shc phosphorylation critically influences the redox regulation of forkhead proteins and underlies Aβ toxicity. These findings underscore the potential usefulness of JNK, p66Shc, and forkhead proteins as therapeutic targets for AD

Striatal Dopamine Transmission Is Subtly Modified in Human A53Tα-Synuclein Overexpressing Mice

Mutations in, or elevated dosage of, SNCA, the gene for α-synuclein (α-syn), cause familial Parkinson's disease (PD). Mouse lines overexpressing the mutant human A53Tα-syn may represent a model of early PD. They display progressive motor deficits, abnormal cellular accumulation of α-syn, and deficits in dopamine-dependent corticostriatal plasticity, which, in the absence of overt nigrostriatal degeneration, suggest there are age-related deficits in striatal dopamine (DA) signalling. In addition A53Tα-syn overexpression in cultured rodent neurons has been reported to inhibit transmitter release. Therefore here we have characterized for the first time DA release in the striatum of mice overexpressing human A53Tα-syn, and explored whether A53Tα-syn overexpression causes deficits in the release of DA. We used fast-scan cyclic voltammetry to detect DA release at carbon-fibre microelectrodes in acute striatal slices from two different lines of A53Tα-syn-overexpressing mice, at up to 24 months. In A53Tα-syn overexpressors, mean DA release evoked by a single stimulus pulse was not different from wild-types, in either dorsal striatum or nucleus accumbens. However the frequency responsiveness of DA release was slightly modified in A53Tα-syn overexpressors, and in particular showed slight deficiency when the confounding effects of striatal ACh acting at presynaptic nicotinic receptors (nAChRs) were antagonized. The re-release of DA was unmodified after single-pulse stimuli, but after prolonged stimulation trains, A53Tα-syn overexpressors showed enhanced recovery of DA release at old age, in keeping with elevated striatal DA content. In summary, A53Tα-syn overexpression in mice causes subtle changes in the regulation of DA release in the striatum. While modest, these modifications may indicate or contribute to striatal dysfunction

The AAA-ATPase VPS4 Regulates Extracellular Secretion and Lysosomal Targeting of α-Synuclein

Many neurodegenerative diseases share a common pathological feature: the deposition of amyloid-like fibrils composed of misfolded proteins. Emerging evidence suggests that these proteins may spread from cell-to-cell and encourage the propagation of neurodegeneration in a prion-like manner. Here, we demonstrated that α-synuclein (αSYN), a principal culprit for Lewy pathology in Parkinson's disease (PD), was present in endosomal compartments and detectably secreted into the extracellular milieu. Unlike prion protein, extracellular αSYN was mainly recovered in the supernatant fraction rather than in exosome-containing pellets from the neuronal culture medium and cerebrospinal fluid. Surprisingly, impaired biogenesis of multivesicular body (MVB), an organelle from which exosomes are derived, by dominant-negative mutant vacuolar protein sorting 4 (VPS4) not only interfered with lysosomal targeting of αSYN but facilitated αSYN secretion. The hypersecretion of αSYN in VPS4-defective cells was efficiently restored by the functional disruption of recycling endosome regulator Rab11a. Furthermore, both brainstem and cortical Lewy bodies in PD were found to be immunoreactive for VPS4. Thus, VPS4, a master regulator of MVB sorting, may serve as a determinant of lysosomal targeting or extracellular secretion of αSYN and thereby contribute to the intercellular propagation of Lewy pathology in PD

Synphilin-1 binds ATP and regulates intracellular energy status.

Recent studies have suggested that synphilin-1, a cytoplasmic protein, is involved in energy homeostasis. Overexpression of synphilin-1 in neurons results in hyperphagia and obesity in animal models. However, the mechanism by which synphilin-1 alters energy homeostasis is unknown. Here, we used cell models and biochemical approaches to investigate the cellular functions of synphilin-1 that may affect energy balance. Synphilin-1 was pulled down by ATP-agarose beads, and the addition of ATP and ADP reduced this binding, indicating that synphilin-1 bound ADP and ATP. Synphilin-1 also bound GMP, GDP, and GTP but with a lower affinity than it bound ATP. In contrast, synphilin-1 did not bind with CTP. Overexpression of synphilin-1 in HEK293T cells significantly increased cellular ATP levels. Genetic alteration to abolish predicted ATP binding motifs of synphilin-1 or knockdown of synphilin-1 by siRNA reduced cellular ATP levels. Together, these data demonstrate that synphilin-1 binds and regulates the cellular energy molecule, ATP. These findings provide a molecular basis for understanding the actions of synphilin-1 in energy homeostasis

Synphilin-1 increases cellular ATP levels.

<p>HEK293T cells were transfected with empty vector, myc-human-synphilin-1, and myc-synphilin-1-mATP (abolishing the five predicted ATP binding sites) for 48 h. Lysates were harvested and subjected to ATP (<b>A</b>) and ADP (<b>B</b>) assays. The experiments were repeated three times. All data are expressed as mean ± SEM. *<i>p</i><0.05 by ANOVA, significant differences between cells expressing wild type synphilin-1 and empty vector; # <i>p</i><0.05 by ANOVA, significant differences between cells expressing wild type synphilin-1 and synphilin-1-mATP variant.</p

Synphilin-1 binds ATP.

<p>myc-Synphilin-1 was affinity-eluted from lysates of transfected HEK293T cells, using ATP-agarose (Jena Bioscience), in the absence or presence of CTP, AMP, ADP, ATP, GMP, GDP, or GTP at 2 mM concentration. Precipitates were resolved by SDS-PAGE and immunoblotted with anti-myc. Equal protein input was controlled by a western blot using anti-myc (bottom). A and B, Shown are representative blots from three repeated experiments. C and D, Quantification of data from A and B. *<i>p</i><0.05 by ANOVA, vs control.</p

Knockdown of synphilin-1 reversed cellular ATP levels to baseline levels.

<p>HEK293T cells were co-transfected with siRNA targeting synphilin-1 and cDNA synphilin-1 constructs at 4∶1 ratio for 72 hours. Cells were harvested for Western blot analysis (A), ATP (B), and ADP assays (C). The experiments were repeated three times. All data are expressed as mean ± SEM. *<i>p</i><0.05 by ANOVA, significant differences between cells expressing vector and synphilin-1; # p<0.05 by ANOVA, significant differences between scramble control siRNA and siRNA targeting synphilin-1 group cells expressing wild type synphilin-1.</p

ATP binding motifs in synphilin-1.

<p><b>A</b>. Predicted ATP binding motifs (KXXXK) of synphilin-1. Underline indicates five predicted ATP binding motifs in two regions (109–349aa and 550–660aa), from N-terminus to C-terminus, named as sites 1 to 5. The ‘Ks’ were mutated to ‘As’ by site mutagenesis. <b>B</b>. HEK293T cells were transfected with empty vector, non-related protein-Lac-Z, myc-synphilin-1, and various altered constructs of myc-synphilin-1 as indicated for 48 h. Lysates were harvested and precipitated with ATP-agarose (A–D) and ADP-agarose (E). The resulting precipitates were resolved by SDS-PAGE and immunoblotted with anti-myc antibodies. The amount of synphilin-1 binding with ATP or ADP was quantified. WT: wild type synphilin-1. 1–5 indicated synphilin-1 constructs with alterations in each of the predicted ATP binding motifs. <b>C</b> and <b>D</b>, HEK293T cells were transfected with empty vector, myc-tagged-Lac-Z, myc-synphilin-1, and myc-synphilin-1-mATP (abolishing the five predicted ATP binding sites) for 48 h. Lysates were subjected to ATP binding assays. <b>D</b>. Quantification data of <b>C</b>. <b>E.</b> Quantification data of synphilin-1 binding with ADP. The experiments were repeated three times with similar results. *<i>p</i><0.05 by ANOVA, vs control.</p

ATP binding regions in synphilin-1.

<p>HEK293T cells were transfected with various HA-tagged synphilin-1 truncated constructs for 48 h. The cells were harvested and the cell lysates were precipitated with ATP-agarose. The resulting precipitates were resolved by SDS-PAGE and immunoblotted with anti-HA antibodies. ATP bound with synphilin-1 predominantly at two regions: 109–349 aa and 550–660 aa. The blots are representative of three separate experiments. Lacz, pcDNA3.1-Lac-Z control construct. A, ATP binding synphilin-1 fragments. B, Loading control of synphilin-1 fragments. C, Diagram of synphilin-1 fragments.</p