scholarly journals Endosomal traffic and glutamate synapse activity are increased in VPS35 D620N mutant knock-in mouse neurons, and resistant to LRRK2 kinase inhibition

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Chelsie A. Kadgien ◽  
Anusha Kamesh ◽  
Austen J. Milnerwood

AbstractVacuolar protein sorting 35 (VPS35) regulates neurotransmitter receptor recycling from endosomes. A missense mutation (D620N) in VPS35 leads to autosomal-dominant, late-onset Parkinson’s disease. Here, we study the basic neurobiology of VPS35 and Parkinson’s disease mutation effects in the D620N knock-in mouse and the effect of leucine-rich repeat kinase 2 (LRRK2) inhibition on synaptic phenotypes. The study was conducted using a VPS35 D620N knock-in mouse that expresses VPS35 at endogenous levels. Protein levels, phosphorylation states, and binding ratios in brain lysates from knock-in mice and wild-type littermates were assayed by co-immunoprecipitation and western blot. Dendritic protein co-localization, AMPA receptor surface expression, synapse density, and glutamatergic synapse activity in primary cortical cultures from knock-in and wild-type littermates were assayed using immunocytochemistry and whole-cell patch clamp electrophysiology. In brain tissue, we confirm VPS35 forms complexes with LRRK2 and AMPA-type glutamate receptor GluA1 subunits, in addition to NMDA-type glutamate receptor GluN1 subunits and D2-type dopamine receptors. Receptor and LRRK2 binding was unaltered in D620N knock-in mice, but we confirm the mutation results in reduced binding of VPS35 with WASH complex member FAM21, and increases phosphorylation of the LRRK2 kinase substrate Rab10, which is reversed by LRRK2 kinase inhibition in vivo. In cultured cortical neurons from knock-in mice, pRab10 is also increased, and reversed by LRRK2 inhibition. The mutation also results in increased endosomal recycling protein cluster density (VPS35-FAM21 co-clusters and Rab11 clusters), glutamate transmission, and GluA1 surface expression. LRRK2 kinase inhibition, which reversed Rab10 hyper-phosphorylation, did not rescue elevated glutamate release or surface GluA1 expression in knock-in neurons, but did alter AMPAR traffic in wild-type cells. The results improve our understanding of the cell biology of VPS35, and the consequences of the D620N mutation in developing neuronal networks. Together the data support a chronic synaptopathy model for latent neurodegeneration, providing phenotypes and candidate pathophysiological stresses that may drive eventual transition to late-stage parkinsonism in VPS35 PD. The study demonstrates the VPS35 mutation has effects that are independent of ongoing LRRK2 kinase activity, and that LRRK2 kinase inhibition alters basal physiology of glutamate synapses in vitro.

2021 ◽  
Author(s):  
Chelsie Kadgien ◽  
Anusha Kamesh ◽  
Jaskaran Khinda ◽  
LiPing Cao ◽  
Jesse Fox ◽  
...  

Abstract Background Vacuolar protein sorting 35 (VPS35) regulates neurotransmitter receptor recycling from endosomes. A missense mutation (D620N) in VPS35 leads to autosomal-dominant, late-onset Parkinson’s disease. Methods Here, we study the basic neurobiology of VPS35 and Parkinson’s disease mutation effects in the D620N knock-in mouse and the effect of leucine-rich repeat kinase 2 (LRRK2) inhibition on synaptic phenotypes. The study was conducted using a VPS35 D620N knock-in mouse that expresses VPS35 at endogenous levels. Protein levels, phosphorylation states, and binding ratios in brain lysates from knock-in mice and wild-type littermates were assayed by co-immunoprecipitation and western blot. Dendritic protein co-localization, AMPA receptor surface expression, synapse density, and glutamatergic synapse activity in primary cortical cultures from knock-in and wild-type littermates were assayed using immunocytochemistry and whole-cell patch clamp electrophysiology. Results In brain tissue, we confirm VPS35 forms complexes with LRRK2 and AMPA-type glutamate receptor GluA1 subunits, in addition to NMDA-type glutamate receptor GluN1 subunits and D2-type dopamine receptors. Receptor and LRRK2 binding was unaltered in D620N knock-in mice, but we confirm the mutation results in reduced binding of VPS35 with WASH complex member FAM21, and increases phosphorylation of the LRRK2 kinase substrate Rab10, which is reversed by LRRK2 kinase inhibition in vivo. In cultured cortical neurons from knock-in mice, pRab10 is also increased, and reversed by LRRK2 inhibition. The mutation also results in increased endosomal recycling protein cluster density (VPS35-FAM21 co-clusters and Rab11 clusters), glutamate transmission, and GluA1 surface expression. LRRK2 kinase inhibition, which reversed Rab10 hyper-phosphorylation, did not rescue elevated glutamate release or surface GluA1 expression in knock-in neurons, but did alter AMPAR traffic in wild-type cells. Conclusions The results improve our understanding of the cell biology of VPS35, and the consequences of the D620N mutation in developing neuronal networks. Together the data support a chronic synaptopathy model for latent neurodegeneration, providing phenotypes and candidate pathophysiological stresses that may drive eventual transition to late-stage parkinsonism in VPS35 PD. The study demonstrates the VPS35 mutation has effects that are independent of ongoing LRRK2 kinase activity, and that LRRK2 kinase inhibition alters basal physiology of glutamate synapses in vitro.


2021 ◽  
Author(s):  
Chelsie Kadgien ◽  
Anusha Kamesh ◽  
Jaskaran Khinda ◽  
Li Ping Cao ◽  
Jesse Fox ◽  
...  

Abstract Vacuolar protein sorting 35 (VPS35) regulates neurotransmitter receptor recycling from endosomes. A missense mutation (D620N) in VPS35 leads to autosomal-dominant, late-onset Parkinson’s disease. Here, we study the basic neurobiology of VPS35 and Parkinson’s disease mutation effects in the D620N knock-in mouse and the effect of leucine-rich repeat kinase 2 (LRRK2) inhibition on synaptic phenotypes. The study was conducted using a VPS35 D620N knock-in mouse that expresses VPS35 at endogenous levels. Protein levels, phosphorylation states, and binding ratios in brain lysates from knock-in mice and wild-type littermates were assayed by co-immunoprecipitation and western blot. Dendritic protein co-localization, AMPA receptor surface expression, synapse density, and glutamatergic synapse activity in primary cortical cultures from knock-in and wild-type littermates were assayed using immunocytochemistry and whole-cell patch clamp electrophysiology. In brain tissue, we confirm VPS35 forms complexes with LRRK2 and AMPA-type glutamate receptor GluA1 subunits, in addition to NMDA-type glutamate receptor GluN1 subunits and D2-type dopamine receptors. Receptor and LRRK2 binding was unaltered in D620N knock-in mice, but we confirm the mutation results in reduced binding of VPS35 with WASH complex member FAM21, and increases phosphorylation of the LRRK2 kinase substrate Rab10, which is reversed by LRRK2 kinase inhibition in vivo. In cultured cortical neurons from knock-in mice, pRab10 is also increased, and reversed by LRRK2 inhibition. The mutation also results in increased endosomal recycling protein cluster density (VPS35-FAM21 co-clusters and Rab11 clusters), glutamate transmission, and GluA1 surface expression. LRRK2 kinase inhibition, which reversed Rab10 hyper-phosphorylation, did not rescue elevated glutamate release or surface GluA1 expression in knock-in neurons, but did alter AMPAR traffic in wild-type cells. The results improve our understanding of the cell biology of VPS35, and the consequences of the D620N mutation in developing neuronal networks. Together the data support a chronic synaptopathy model for latent neurodegeneration, providing phenotypes and candidate pathophysiological stresses that may drive eventual transition to late-stage parkinsonism in VPS35 PD. The study demonstrates the VPS35 mutation has effects that are independent of ongoing LRRK2 kinase activity, and that LRRK2 kinase inhibition alters basal physiology of glutamate synapses in vitro.


2021 ◽  
Author(s):  
Chelsie A Kadgien ◽  
Anusha Kamesh ◽  
Jaskaran Khinda ◽  
Li Ping Cao ◽  
Jesse Fox ◽  
...  

AbstractVacuolar protein sorting 35 (VPS35) regulates receptor recycling from endosomes. A missense mutation in VPS35 (D620N) leads to autosomal-dominant, late-onset Parkinson’s disease. Here, we use a VPS35 D620N knock-in mouse to study the neurobiology of this mutation. In brain tissue, we confirm previous findings that the mutation results in reduced binding of VPS35 with WASH-complex member FAM21, and robustly elevated phosphorylation of the LRRK2 kinase substrate Rab10. In cultured cortical neurons, the mutation results in increased endosomal recycling protein density (VPS35-FAM21 co-clusters and Rab11 clusters), glutamate release, and GluA1 surface expression. LRRK2 kinase inhibition exerted genotype-specific effects on GluA1 surface expression, but did not impact glutamate release phenotypes. These results improve our understanding of the early effects of the D620N mutation on cellular functions that are specific to neurons. These observations provide candidate pathophysiological pathways that may drive eventual transition to late-stage parkinsonism in VPS35 families, and support a synaptopathy model of neurodegeneration.


2015 ◽  
Vol 53 (8) ◽  
pp. 5161-5177 ◽  
Author(s):  
G. A. Smith ◽  
J. Jansson ◽  
E. M. Rocha ◽  
T. Osborn ◽  
P. J. Hallett ◽  
...  

Author(s):  
Ahsan Usmani ◽  
Farbod Shavarebi ◽  
Annie Hiniker

Point mutations in Leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial Parkinson’s disease (PD) and are implicated in a significant portion of apparently sporadic PD. Clinically, LRRK2-driven PD is indistinguishable from sporadic PD, making it an attractive genetic model for the much more common sporadic PD. In this review, we highlight recent advances in understanding LRRK2's subcellular functions using LRRK2-PD models, while also considering some of the limitations of these model systems. Recent developments of particular importance include new evidence of key LRRK2 functions in the endolysosomal system and LRRK2’s regulation of and by Rab GTPases. Additionally, LRRK2's interaction with the cytoskeleton allowed elucidation of the LRRK2 structure and appears relevant to LRRK2 protein degradation and LRRK2 kinase inhibitor therapies. We further discuss how LRRK2's interactions with other PD-driving genes, such as VPS35, GCase, and α-synuclein, may highlight cellular pathways more broadly disrupted in PD.


2021 ◽  
Author(s):  
Federica Albanese ◽  
Daniela Mercatelli ◽  
Luca Finetti ◽  
Giulia Lamonaca ◽  
Sara Pizzi ◽  
...  

Abstract Background: Mutations in leucine-rich repeat kinase 2 (LRRK2) are associated with familiar and sporadic Parkinson’s disease. LRRK2 modulates the autophagy-lysosome pathway (ALP), a clearance process subserving the quality control of cellular proteins and organelles. Since dysfunctional ALP might lead to α-synuclein accumulation and, hence, Parkinson’s disease, LRRK2 kinase modulation of ALP, its age-dependence and relation with pSer129 a-synuclein inclusions in striatal and nigral neurons were investigated in vivo. Methods: Striatal ALP markers were analyzed by Western blotting in 3, 12 and 20-month-old LRRK2 G2019S knock-in mice (bearing enhanced kinase activity), LRRK2 knock-out mice, LRRK2 D1994S knock-in (kinase-dead) mice and wild-type controls. The lysosomotropic agent chloroquine was used to investigate the autophagic flux in vivo. Quantitative Real-time PCR was used to quantify the transcript levels of key ALP genes. The activity of the lysosomal enzyme glucocerebrosidase was measured using enzymatic assay. Immunohistochemistry was used to co-localize LC3B puncta with pSer129 a-synuclein inclusion in striatal MAP-positive and nigral TH-positive neurons. Results: No genotype differences in macroautophagy and chaperone-mediated autophagy markers were observed at 3 months. Conversely, increase of LC3-I, p62, LAMP2 and GAPDH levels, decrease of p-mTOR levels and downregulation of mTOR and TFEB expression was observed in 12-month-old kinase-dead mice. The LC3-II/LC3-I ratio was reduced following administration of chloroquine, suggesting a defective autophagic flux. G2019S knock-in mice showed LAMP2 accumulation and downregulation of ALP key genes MAP1LC3B, LAMP2, mTOR, TFEB and GBA1. Subacute administration of the LRRK2 kinase inhibitor MLi-2 in wild-type and G2019S knock-in mice did not replicate the pattern of kinase-dead mice. Lysosomal glucocerebrosidase activity was increased in 3 and 12-month-old knock-out and kinase-dead mice, and GBA1 expression reduced in 12-month-old G2019S knock-in mice. Immunofluorescence revealed a dissociation between LC3B puncta accumulation and pSer129 a-synuclein inclusions in striatal neurons of kinase-dead and G2019S knock-in mice. Conclusions: We conclude that constitutive LRRK2 kinase silencing results in early deregulation of GCase activity followed by late impairment of macroautophagy and chaperone-mediated autophagy. In G2019S knock-in mice, pSer129 a-synuclein inclusions observed under basal conditions appear unrelated to autophagy impairment.


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