Ubiquitinylation of α-Synuclein by Carboxyl Terminus Hsp70-Interacting Protein (CHIP) Is Regulated by Bcl-2-Associated Athanogene 5 (BAG5)

Parkinson's disease (PD) is a common neurodegenerative condition in which abnormalities in protein homeostasis, or proteostasis, may lead to accumulation of the protein α-synuclein (α-syn). Mutations within or multiplications of the gene encoding α-syn are known to cause genetic forms of PD and polymorphisms in the gene are recently established risk factors for idiopathic PD. α-syn is a major component of Lewy bodies, the intracellular proteinaceous inclusions which are pathological hallmarks of most forms of PD. Recent evidence demonstrates that α-syn can self associate into soluble oligomeric species and implicates these α-syn oligomers in cell death. We have previously shown that carboxyl terminus of Hsp70-interacting protein (CHIP), a co-chaperone molecule with E3 ubiquitin ligase activity, may reduce the levels of toxic α-syn oligomers. Here we demonstrate that α-syn is ubiquitinylated by CHIP both in vitro and in cells. We find that the products from ubiquitinylation by CHIP include both monoubiquitinylated and polyubiquitinylated forms of α-syn. We also demonstrate that CHIP and α-syn exist within a protein complex with the co-chaperone bcl-2-associated athanogene 5 (BAG5) in brain. The interaction of CHIP with BAG5 is mediated by Hsp70 which binds to the tetratricopeptide repeat domain of CHIP and the BAG domains of BAG5. The Hsp70-mediated association of BAG5 with CHIP results in inhibition of CHIP E3 ubiquitin ligase activity and subsequently reduces α-syn ubiquitinylation. Furthermore, we use a luciferase-based protein-fragment complementation assay of α-syn oligomerization to investigate regulation of α-syn oligomers by CHIP in living cells. We demonstrate that BAG5 mitigates the ability of CHIP to reduce α-syn oligomerization and that non-ubiquitinylated α-syn has an increased propensity for oligomerization. Thus, our results identify CHIP as an E3 ubiquitin ligase of α-syn and suggest a novel function for BAG5 as a modulator of CHIP E3 ubiquitin ligase activity with implications for CHIP-mediated regulation of α-syn oligomerization

Direct detection of alpha synuclein oligomers in vivo

Background: Rat models of Parkinson’s disease are widely used to elucidate the mechanisms underlying disease etiology or to investigate therapeutic approaches. Models were developed using toxins such as MPTP or 6-OHDA to specifically target dopaminergic neurons resulting in acute neuronal loss in the substantia nigra or by using viral vectors to induce the specific and gradual expression of alpha synuclein in the substantia nigra. The detection of alpha- synuclein oligomers, the presumed toxic species, in these models and others has been possible using only indirect biochemical approaches to date. Here we coinjected AAVs encoding alpha-synuclein fused to the N- or C-terminal half of VenusYFP in rat substantia nigra pars compacta and describe for the first time a novel viral vector rodent model with the unique ability to directly detect and track alpha synuclein oligomers ex vivo and in vivo. Results: Viral coinjection resulted in widespread VenusYFP signal within the nigrostriatal pathway, including cell bodies in the substantia nigra and synaptic accumulation in striatal terminals, suggestive of in vivo alpha-synuclein oligomers formation. Transduced rats showed alpha-synuclein induced dopaminergic neuron loss in the substantia nigra, the appearance of dystrophic neurites, and gliosis in the striatum. Moreover, we have applied in vivo imaging techniques in the living mouse to directly image alpha-synuclein oligomers in the cortex. Conclusion: We have developed a unique animal model that provides a tool for the Parkinson’s disease research community with which to directly detect alpha- synuclein oligomers in vivo and screen therapeutic approaches targeting alpha-synuclein oligomers

Neto1 Is a Novel CUB-Domain NMDA Receptor–Interacting Protein Required for Synaptic Plasticity and Learning

The N-methyl-D-aspartate receptor (NMDAR), a major excitatory ligand-gated ion channel in the central nervous system (CNS), is a principal mediator of synaptic plasticity. Here we report that neuropilin tolloid-like 1 (Neto1), a complement C1r/C1s, Uegf, Bmp1 (CUB) domain-containing transmembrane protein, is a novel component of the NMDAR complex critical for maintaining the abundance of NR2A-containing NMDARs in the postsynaptic density. Neto1-null mice have depressed long-term potentiation (LTP) at Schaffer collateral-CA1 synapses, with the subunit dependency of LTP induction switching from the normal predominance of NR2A- to NR2B-NMDARs. NMDAR-dependent spatial learning and memory is depressed in Neto1-null mice, indicating that Neto1 regulates NMDA receptor-dependent synaptic plasticity and cognition. Remarkably, we also found that the deficits in LTP, learning, and memory in Neto1-null mice were rescued by the ampakine CX546 at doses without effect in wild-type. Together, our results establish the principle that auxiliary proteins are required for the normal abundance of NMDAR subunits at synapses, and demonstrate that an inherited learning defect can be rescued pharmacologically, a finding with therapeutic implications for humans

Clinical correlations with Lewy body pathology in LRRK2-related Parkinson disease

IMPORTANCE: Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of genetic Parkinson disease (PD) known to date. The clinical features of manifesting LRRK2 mutation carriers are generally indistinguishable from those of patients with sporadic PD. However, some PD cases associated with LRRK2 mutations lack Lewy bodies (LBs), a neuropathological hallmark of PD. We investigated whether the presence or absence of LBs correlates with different clinical features in LRRK2-related PD. OBSERVATIONS: We describe genetic, clinical, and neuropathological findings of 37 cases of LRRK2-related PD including 33 published and 4 unpublished cases through October 2013. Among the different mutations, the LRRK2 p.G2019S mutation was most frequently associated with LB pathology. Nonmotor features of cognitive impairment/dementia, anxiety, and orthostatic hypotension were correlated with the presence of LBs. In contrast, a primarily motor phenotype was associated with a lack of LBs. CONCLUSIONS AND RELEVANCE: To our knowledge, this is the first report of clinicopathological correlations in a series of LRRK2-related PD cases. Findings from this selected group of patients with PD demonstrated that parkinsonian motor features can occur in the absence of LBs. However, LB pathology in LRRK2-related PD may be a marker for a broader parkinsonian symptom complex including cognitive impairment

Chaperone-Based Therapies for Disease Modification in Parkinson’s Disease

Parkinson’s disease (PD) is the second most common neurodegenerative disorder and is characterized by the presence of pathological intracellular aggregates primarily composed of misfolded α-synuclein. This pathology implicates the molecular machinery responsible for maintaining protein homeostasis (proteostasis), including molecular chaperones, in the pathobiology of the disease. There is mounting evidence from preclinical and clinical studies that various molecular chaperones are downregulated, sequestered, depleted, or dysfunctional in PD. Current therapeutic interventions for PD are inadequate as they fail to modify disease progression by ameliorating the underlying pathology. Modulating the activity of molecular chaperones, cochaperones, and their associated pathways offers a new approach for disease modifying intervention. This review will summarize the potential of chaperone-based therapies that aim to enhance the neuroprotective activity of molecular chaperones or utilize small molecule chaperones to promote proteostasis.Peer Reviewe

Merging DBS with viral vector or stem cell implantation: “hybrid” stereotactic surgery as an evolution in the surgical treatment of Parkinson's disease

Parkinson's disease (PD) is a complex neurodegenerative disorder that is currently managed using a broad array of symptom-based strategies. However, targeting its molecular origins represents the potential to discover disease-modifying therapies. Deep brain stimulation (DBS), a highly successful treatment modality for PD symptoms, addresses errant electrophysiological signaling pathways in the basal ganglia. In contrast, ongoing clinical trials testing gene and cell replacement therapies propose to protect or restore neuronal-based physiologic dopamine transmission in the striatum. Given promising new platforms to enhance target localization'such as interventional MRI-guided stereotaxy'the opportunity now exists to create hybrid therapies that combine DBS with gene therapy and/or cell implantation. In this mini-review, we discuss approaches used for central nervous system biologic delivery in PD patients in previous trials and propose a new set of strategies based on novel molecular targets. A multifaceted approach, if successful, may not only contribute to our understanding of PD pathology but could introduce a new era of disease modification

CHIP forms a protein complex with BAG5.

<p>(A) Immunoprecipitations with anti-FLAG antibodies were performed from lysates of H4 cells transfected with CHIP with or without FLAG-BAG5 as indicated. Immunoprecipitates were sequentially probed with anti-CHIP (upper), anti-Hsp70 (middle), and anti-FLAG (lower) antibodies. Ten percent of lysates used for immunoprecipitation was loaded as input. The upper CHIP band corresponds to monoubiquitinylated CHIP <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014695#pone.0014695-AlRamahi1" target="_blank">[62]</a>. Molecular weight markers are shown on right. Similar results were found in three separate experiments. (B) PDAs were performed using lysates of H4 cells transfected with CHIP. Proteins that associated with GST alone, GST-BAG5, or GST-BAG5 DARA were probed with anti-CHIP (upper) and anti-Hsp70 (middle) antibodies. Input was 10% of lysates used for PDAs. The presence of equal amounts of GST fusion proteins was confirmed by Ponceau S staining of the membranes (lower). Molecular weight markers are indicated on right. Results are representative of four independent experiments.</p

BAG5 negatively regulates CHIP-mediated reduction of α-syn oligomer levels.

<p>(A) PDAs were performed using GST fusion proteins and lysates from H4 cells transfected with syn-luc1, syn-luc2, CHIP, and Hsp70. Proteins that associated with GST alone or GST-BAG5 were probed with anti-α-syn (upper), anti-CHIP (middle), and anti-Hsp70 (lower) antibodies. Input was 10% of total lysates used for PDAs. Results are representative of three independent experiments. (B) Luciferase activity was measured from H4 cells transfected with syn-luc1 and syn-luc2 plus the vectors indicated on the horizontal axis of the graph. The total DNA used in each condition was equalized using the empty vector pcDNA. Bars correspond to mean (±s.e.m.) luciferase activity normalized to measures obtained for the co-transfection of syn-luc1 and syn-luc2 with pcDNA as control (*P<0.05, ANOVA with Tukey HSD post hoc test versus control). Results shown are from four independent experiments performed in triplicate. (C) Immunoprecipitations with anti-α-syn antibodies or IgG control were performed from mouse brain homogenates. Immunoprecipitates were sequentially probed with anti-α-syn, anti-CHIP, anti-Hsp70, and anti-BAG5 antibodies as indicated. Ten percent of lysates used for immunoprecipitation was loaded as input. A longer exposure of the same blot was required to visualize the input for BAG5. The asterisk (*) indicates cross-reactivity of the secondary antibody with the immunoglobulin heavy chain. Similar results were found in three separate experiments. (D) Immunohistochemistry for α-syn, BAG5, and CHIP was performed on substantia nigra pars compacta samples from patients with a neuropathological diagnosis of DLB. α-syn immunoreactivity (green in upper), BAG5 immunoreactivity (green in lower), and CHIP immunoreactivity (red) in neurons and in α-syn-positive intracytoplasmic inclusions are shown. Co-localization is demonstrated in the merged images (yellow). DAPI staining (blue) demonstrates nuclear localization. Five to ten percent of nigral neurons contained α-syn-positive intracytoplasmic inclusions. At least ten α-syn-positive inclusion-containing neurons were visualized in the substantia nigra pars compacta of each of two patients with DLB. All of the visualized α-syn-positive inclusions were positive for both BAG5 and CHIP.</p

CHIP mediates ubiquitinylation of α-syn in cells.

<p>(A) Immunoprecipitations with anti-luc were performed from lysates of H4 cells transfected with HA-Ub, syn-luc1, and control vector or myc-CHIP as indicated. Immunoprecipitates were sequentially probed with anti-HA (upper) and anti-α-syn (middle) antibodies. Five percent of lysates used for immunoprecipitation was loaded as input and probed with anti-myc or anti-α-syn antibodies (lower). The middle band represents monoubiquitinylated α-syn (UB-α-syn). The asterisks (*) indicate immunoprecipitated bands that remain detectable by the anti-HA antibody following the substitution of all lysines within the α-syn sequence (see (B)). Molecular weight markers are indicated on left in kDa. (B) Immunoprecipitations with anti-luc were performed from lysates of H4 cells transfected with syn-luc1, synKR-luc1, HA-Ub, and myc-CHIP as indicated. Immunoprecipitated proteins were probed with anti-α-syn antibodies (H3C) which recognizes both syn-luc1 and synKR-luc1. The asterisks (*) correspond with the same bands indicated as such in (A). (C) Densitometric quantification of the band representing monoubiquitinylated α-syn when co-transfected with a control vector or myc-CHIP was performed from three independent experiments, one of which is represented in (A). Bars correspond to mean (± S.D.) gray value normalized to measures obtained for co-transfection of syn-luc1 and HA-Ub with control vector. *P<0.05, t-test versus control vector. (D) Proteins immunoprecipitated with anti-luc were probed with anti-α-syn antibodies (Syn-1) with a short or long exposure to film. The asterisks (*) correspond with the same bands seen in (A) and (B). (E) Immunoprecipitations with anti-α-syn were performed from lysates of H4 cells transfected with syn-luc1 and HA-Ub with control vector or myc-CHIP. Immunoprecipitated proteins were sequentially probed with anti-HA (upper) and anti-luc (lower) antibodies. The asterisks (*) correspond to the bands as indicated in (A), (B), and (D). Results are representative of three experiments.</p