Vitamin B12 modulates Parkinson’s disease LRRK2 kinase activity through allosteric regulation and confers neuroprotection

Missense mutations in Leucine-Rich Repeat Kinase 2 (LRRK2) cause the majority of familial and some sporadic forms of Parkinson’s disease (PD). The hyperactivity of LRRK2 kinase induced by the pathogenic mutations underlies neurotoxicity, promoting the development of LRRK2 kinase inhibitors as therapeutics. Many potent and specific small molecule LRRK2 inhibitors have been reported with promise. However, nearly all inhibitors are ATP competitive – some with unwanted side effects and unclear clinical outcome - alternative types of LRRK2 inhibitors are lacking. Herein we find 5’-deoxyadenosylcobalamin (AdoCbl), a physiological form of the essential micronutrient vitamin B12 as a mixed-type allosteric inhibitor of LRRK2 kinase activity. Multiple assays show that AdoCbl directly binds LRRK2, leading to the alterations of protein conformation and ATP binding in LRRK2. STD-NMR analysis of a LRRK2 homologous kinase reveals the contact sites in AdoCbl that interface with the kinase domain. Furthermore, we provide evidence that AdoCbl modulates LRRK2 activity through disruption of LRRK2 dimerization. Treatment with AdoCbl inhibits LRRK2 kinase activity in cultured cells and brain tissue, and importantly prevents neurotoxicity in primary rodent cultures as well as in transgenic C. elegans and D. melanogaster expressing LRRK2 disease variants. Finally, AdoCbl alleviates deficits in dopamine release sustainability caused by LRRK2 disease variants in mouse models. Our study uncovers vitamin B12 as a novel class of LRRK2 kinase modulator with a distinct mechanism, which can be harnessed to develop new LRRK2-based PD therapeutics in the futur

Activation of FADD-Dependent Neuronal Death Pathways as a Predictor of Pathogenicity for LRRK2 Mutations

Background Despite the plethora of sequence variants in LRRK2, only a few clearly segregate with PD. Even within this group of pathogenic mutations, the phenotypic profile can differ widely. Objective We examined multiple properties of LRRK2 behavior in cellular models over-expressing three sequence variants described in Greek PD patients in comparison to several known pathogenic and non-pathogenic LRRK2 mutations, to determine if specific phenotypes associated with pathogenic LRRK2 can be observed in other less-common sequence variants for which pathogenicity is unclear based on clinical and/or genetic data alone. Methods The oligomerization, activity, phosphorylation, and interaction with FADD was assessed in HEK293T cells over-expressing LRRK2; while the induction of neuronal death was determined by quantifying apoptotic nuclei in primary neurons transiently expressing LRRK2. Results One LRRK2 variant, A211V, exhibited a modest increase in kinase activity, whereas only the pathogenic mutants G2019S and I2020T displayed significantly altered auto-phosphorylation. We observed an induction of detergent-insoluble high molecular weight structures upon expression of pathogenic LRRK2 mutants, but not the other LRRK2 variants. In contrast, each of the variants tested induced apoptotic death of cultured neurons similar to pathogenic LRRK2 in a FADD-dependent manner. Conclusions Overall, despite differences in some properties of LRRK2 function such as kinase activity and its oligomerization, each of the LRRK2 variants examined induced neuronal death to a similar extent. Furthermore, our findings further strengthen the notion of a convergence on the extrinsic cell death pathway common to mutations in LRRK2 that are capable of inducing neuronal death

Activation of FADD-Dependent Neuronal Death Pathways as a Predictor of Pathogenicity for LRRK2 Mutations.

Despite the plethora of sequence variants in LRRK2, only a few clearly segregate with PD. Even within this group of pathogenic mutations, the phenotypic profile can differ widely.We examined multiple properties of LRRK2 behavior in cellular models over-expressing three sequence variants described in Greek PD patients in comparison to several known pathogenic and non-pathogenic LRRK2 mutations, to determine if specific phenotypes associated with pathogenic LRRK2 can be observed in other less-common sequence variants for which pathogenicity is unclear based on clinical and/or genetic data alone.The oligomerization, activity, phosphorylation, and interaction with FADD was assessed in HEK293T cells over-expressing LRRK2; while the induction of neuronal death was determined by quantifying apoptotic nuclei in primary neurons transiently expressing LRRK2.One LRRK2 variant, A211V, exhibited a modest increase in kinase activity, whereas only the pathogenic mutants G2019S and I2020T displayed significantly altered auto-phosphorylation. We observed an induction of detergent-insoluble high molecular weight structures upon expression of pathogenic LRRK2 mutants, but not the other LRRK2 variants. In contrast, each of the variants tested induced apoptotic death of cultured neurons similar to pathogenic LRRK2 in a FADD-dependent manner.Overall, despite differences in some properties of LRRK2 function such as kinase activity and its oligomerization, each of the LRRK2 variants examined induced neuronal death to a similar extent. Furthermore, our findings further strengthen the notion of a convergence on the extrinsic cell death pathway common to mutations in LRRK2 that are capable of inducing neuronal death

α-Synuclein oligomers potentiate neuroinflammatory NF-κB activity and induce Cav3.2 calcium signaling in astrocytes

Abstract Background It is now realized that Parkinson’s disease (PD) pathology extends beyond the substantia nigra, affecting both central and peripheral nervous systems, and exhibits a variety of non-motor symptoms often preceding motor features. Neuroinflammation induced by activated microglia and astrocytes is thought to underlie these manifestations. α-Synuclein aggregation has been linked with sustained neuroinflammation in PD, aggravating neuronal degeneration; however, there is still a lack of critical information about the structural identity of the α-synuclein conformers that activate microglia and/or astrocytes and the molecular pathways involved. Methods To investigate the role of α-synuclein conformers in the development and maintenance of neuroinflammation, we used primary quiescent microglia and astrocytes, post-mortem brain tissues from PD patients and A53T α-synuclein transgenic mice that recapitulate key features of PD-related inflammatory responses in the absence of cell death, i.e., increased levels of pro-inflammatory cytokines and complement proteins. Biochemical and -omics techniques including RNAseq and secretomic analyses, combined with 3D reconstruction of individual astrocytes and live calcium imaging, were used to uncover the molecular mechanisms underlying glial responses in the presence of α-synuclein oligomers in vivo and in vitro. Results We found that the presence of SDS-resistant hyper-phosphorylated α-synuclein oligomers, but not monomers, was correlated with sustained inflammatory responses, such as elevated levels of endogenous antibodies and cytokines and microglial activation. Similar oligomeric α-synuclein species were found in post-mortem human brain samples of PD patients but not control individuals. Detailed analysis revealed a decrease in Iba1Low/CD68Low microglia and robust alterations in astrocyte number and morphology including process retraction. Our data indicated an activation of the p38/ATF2 signaling pathway mostly in microglia and a sustained induction of the NF-κB pathway in astrocytes of A53T mice. The sustained NF-κB activity triggered the upregulation of astrocytic T-type Cav3.2 Ca2+ channels, altering the astrocytic secretome and promoting the secretion of IGFBPL1, an IGF-1 binding protein with anti-inflammatory and neuroprotective potential. Conclusions Our work supports a causative link between the neuron-produced α-synuclein oligomers and sustained neuroinflammation in vivo and maps the signaling pathways that are stimulated in microglia and astrocytes. It also highlights the recruitment of astrocytic Cav3.2 channels as a potential neuroprotective mediator against the α-synuclein-induced neuroinflammation. Graphical Abstrac

Disrupting FADD-dependent signaling in primary neurons is neuroprotective.

<p>a) Primary cortical neurons were co-transfected with an EGFP-pCMS reporter vector, and the LRRK2 mutant together with the FADD dominant negative (HA-tagged leucine-zipper FADD death domain; lzDD) or empty pcDNA vector as a control. The percentage of GFP-positive neurons containing apoptotic nuclear features (defined as above) was determined 72h following transfection. The presence of the dimeric FADD death domain significantly reduced neuronal apoptotic death in cultures expressing each of the LRRK2 variants. b) A representative image of primary neurons expressing Flag-K544E-LRRK2 with the lz-DD fragment, co-stained for ant-GFP and anti-HA (red). *** p<0.001 compared to WT LRRK2; ## p<0.01 compared to pcDNA; ### p<0.001 compared to pcDNA.</p

Rare LRRK2 PD-related sequence variants fail to significantly alter its oligomerization or solubility to a similar extent as pathogenic mutants.

<p>a) HEK293T cells expressing GFP-tagged I2020T-LRRK2 were extracted with 0.1% Triton X-100 for 5 min, fixed and processed for anti-GFP immunostaining with DAPI counterstaining. b) HEK293T cells were transfected and extracted as in (a), and the resulting soluble and insoluble fractions were subjected to SEC. Alternate fractions were analyzed by SDS-PAGE for the presence of GFP-tagged LRRK2. HEK293T cells transiently over-expressing Flag-LRRK2 were (c) lysed normally to produce whole-cell extracts, or (d, e) subjected to mild in situ extraction as in (a). c) Detergent-free total cell extracts of HEK293T cells subjected to SEC were analyzed by dot blot using anti-Flag antibodies to show any shift in elution towards lower or higher molecular weight complexes. Representative blots are shown (of n = 2), demonstrating in a complimentary way the differential oligomeric behavior of the different LRRK2 mutants. The approximate elution point of the native molecular weight standards thyroglobulin and apoferritin are shown as a reference only. d) The amount of LRRK2 (in ng; determined by ELISA using recombinant full-length LRRK2 as a standard) present in the total fraction, or in either the soluble or insoluble fractions was determined by ELISA. Similar levels of expression were observed for each of the mutants tested. The increased ratio of insoluble:soluble LRRK2 for the I2020T mutant (e) reflects its re-organization to extraction-resistant filamentous structures.,None of the other sequence variants significantly altered this profile in comparison to WT-LRRK2, although there was a non-significant trend for reduced solubility for the T1410M variant. ** p<0.01 compared to WT-LRRK2.</p

Kinase dependent apoptotic death of primary neurons expressing pathogenic or rare mutations in LRRK2.

<p><b>a)</b> Primary rat cortical neurons were co-transfected with WT or mutant LRRK2 as indicated together with pcms-EGFP, and fixed and processed for anti-LRRK2 immunofluorescence together with Hoechst to label nuclei. Representative images are shown depicting anti-LRRK2 in red. <b>b)</b> Primary neurons as in (a) except treated with the LRRK2 kinase inhibitor MLi-2 (10nM). <b>c)</b> For quantification of apoptotic neurons, a minimum of 100 GFP-positive neurons were counted from each of three parallel coverslips per condition in a blinded fashion. Three independent transfections were conducted. Apoptotic neurons were defined as having two or more condensed nuclear fragments within a GFP-expressing neuronal profile. ** p<0.01, compared to WT; *** p<0.001, compared to WT; ### p<0.001, compared to the corresponding vehicle-treated culture.</p

Interaction with the death adaptor protein FADD is a common feature of LRRK2 sequence variants that elicit neurotoxicity.

<p>HEK293T cells were transiently transfected with Flag-LRRK2 and V5-FADD at a ratio of 4:1, and after 3 days of expression clarified cell extracts were incubated overnight with M2-Flag resin (Sigma), followed by washing and elution in 2X SDS buffer. The eluates and input lysates were separated by SDS-PAGE and the membranes probed with anti-V5 (rabbit) or anti-Flag antibodies (rabbit). (a) A representative image of the Western immunoblot is shown to demonstrate the interaction between LRRK2 and FADD. (b) Quantification of band intensity from three independent experiments. The value of the V5 immunoreactive band was normalized to the amount of Flag-LRRK2 immunoprecipitated in each sample. * p<0.05 compared to WT-LRRK2. To more precisely quantify the change in interaction between LRRK2 and FADD, we developed an ELISA-based approach. c) Lysates from cells expressing V5-FADD and either EGFP or Flag-WT-LRRK2 were incubated in Flag-coated ELISA plates. In the left plot, LRRK2 (clone UDD3) was detected only in lysates transfected with Flag-LRRK2. To confirm that V5-FADD does not bind non-specifically to the ELISA plate independently of the presence of LRRK2, we processed parallel ELISA wells using V5 as the detection antibody (right plot), confirming that V5-FADD can only be detected with co-expressed with LRRK2. d) To determine the linearity of the signal, we incubated increasing amounts of lysate from cells co-expressing Flag-tagged WT-LRRK2. Using V5 as the detection antibody to detect the presence of FADD, we observe an increase in chemiluminescence signal between 5 and 20 μg of cell extract. In experiments quantifying the interaction of FADD with mutant LRRK2, we assessed 15 μg of cell extract loaded in each well. e) The presence of LRRK2 was determined by sandwich ELISA using anti-LRRK2 (clone UDD3) as the detection antibody. Lysates from cells co-expressing LRRK2 and V5-FADD were analyzed by SDS-PAGE to confirm FADD expression in all samples. f) While each of the three pathogenic mutant forms of LRRK2 showed slightly increased interaction with FADD using this approach, only the G2019S-LRRK2 was significantly greater than WT LRRK2. Similarly, each of the three rare LRRK2 sequence variants showed significantly greater interaction with FADD. * p<0.05 compared to WT-LRRK2.</p

A motif within the armadillo repeat of Parkinson&apos;s-linked LRRK2 interacts with FADD to hijack the extrinsic death pathway

In experimental models, both in vivo and cellular, over-expression of Parkinson&apos;s linked mutant leucine-rich repeat kinase 2 (LRRK2) is sufficient to induce neuronal death. While several cell death associated proteins have been linked to LRRK2, either as protein interactors or as putative substrates, characterization of the neuronal death cascade remains elusive. In this study, we have mapped for the first time the domain within LRRK2 that mediates the interaction with FADD, thereby activating the molecular machinery of the extrinsic death pathway. Using homology modeling and molecular docking approaches, we have identified a critical motif within the N-terminal armadillo repeat region of LRRK2. Moreover, we show that co-expression of fragments of LRRK2 that contain the FADD binding motif, or deletion of this motif itself, blocks the interaction with FADD, and is neuroprotective. We further demonstrate that downstream of FADD, the mitochondrial proteins Bid and Bax are recruited to the death cascade and are necessary for neuronal death. Our work identifies multiple novel points within neuronal death signaling pathways that could potentially be targeted by candidate therapeutic strategies and highlight how the extrinsic pathway can be activated intracellularly in a pathogenic context