Deletion in chromosome 6 spanning alpha-synuclein and multimerin1 loci in the Rab27a/b double knockout mouse

© The Author(s), 2022. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Pattanayak, R., Underwood, R., Crowley, M. R., Crossman, D. K., Morgan, J. R., & Yacoubian, T. A. Deletion in chromosome 6 spanning alpha-synuclein and multimerin1 loci in the Rab27a/b double knockout mouse. Scientific Reports, 12(1), (2022): 9837, https://doi.org/10.1038/s41598-022-13557-8.We report an incidental 358.5 kb deletion spanning the region encoding for alpha-synuclein (αsyn) and multimerin1 (Mmrn1) in the Rab27a/Rab27b double knockout (DKO) mouse line previously developed by Tolmachova and colleagues in 2007. Western blot and RT-PCR studies revealed lack of αsyn expression at either the mRNA or protein level in Rab27a/b DKO mice. PCR of genomic DNA from Rab27a/b DKO mice demonstrated at least partial deletion of the Snca locus using primers targeted to exon 4 and exon 6. Most genes located in proximity to the Snca locus, including Atoh1, Atoh2, Gm5570, Gm4410, Gm43894, and Grid2, were shown not to be deleted by PCR except for Mmrn1. Using whole genomic sequencing, the complete deletion was mapped to chromosome 6 (60,678,870–61,037,354), a slightly smaller deletion region than that previously reported in the C57BL/6J substrain maintained by Envigo. Electron microscopy of cortex from these mice demonstrates abnormally enlarged synaptic terminals with reduced synaptic vesicle density, suggesting potential interplay between Rab27 isoforms and αsyn, which are all highly expressed at the synaptic terminal. Given this deletion involving several genes, the Rab27a/b DKO mouse line should be used with caution or with appropriate back-crossing to other C57BL/6J mouse substrain lines without this deletion.This study was supported by NIH [R56NS115767 (TAY), RF1NS115767-01A1 (TAY), P50NS108675 (TAY), and NINDS/NIA RF1NS078165 (JRM)]

LRRK2 secretion in exosomes is regulated by 14-3-3

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene cause late-onset Parkinson's disease (PD). Emerging evidence suggests a role for LRRK2 in the endocytic pathway. Here, we show that LRRK2 is released in extracellular microvesicles (i.e. exosomes) from cells that natively express LRRK2. LRRK2 localizes to collecting duct epithelial cells in the kidney that actively secrete exosomes into urine. Purified urinary exosomes contain LRRK2 protein that is both dimerized and phosphorylated. We provide a quantitative proteomic profile of 1673 proteins in urinary exosomes and find that known LRRK2 interactors including 14-3-3 are some of the most abundant exosome proteins. Disruption of the 14-3-3 LRRK2 interaction with a 14-3-3 inhibitor or through acute LRRK2 kinase inhibition potently blocks LRRK2 release in exosomes, but familial mutations in LRRK2 had no effect on secretion. LRRK2 levels were overall comparable but highly variable in urinary exosomes derived from PD cases and age-matched controls, although very high LRRK2 levels were detected in some PD affected cases. We further characterized LRRK2 exosome release in neurons and macrophages in culture, and found that LRRK2-positive exosomes circulate in cerebral spinal fluid (CSF). Together, these results define a pathway for LRRK2 extracellular release, clarify one function of the LRRK2 14-3-3 interaction and provide a foundation for utilization of LRRK2 as a biomarker in clinical trial

14-3-3theta Protects against Neurotoxicity in a Cellular Parkinson's Disease Model through Inhibition of the Apoptotic Factor Bax

Disruption of 14-3-3 function by alpha-synuclein has been implicated in Parkinson's disease. As 14-3-3s are important regulators of cell death pathways, disruption of 14-3-3s could result in the release of pro-apoptotic factors, such as Bax. We have previously shown that overexpression of 14-3-3θ reduces cell loss in response to rotenone and MPP+ in dopaminergic cell culture and reduces cell loss in transgenic C. elegans that overexpress alpha-synuclein. In this study, we investigate the mechanism for 14-3-3θ's neuroprotection against rotenone toxicity. While 14-3-3s can inhibit many pro-apoptotic factors, we demonstrate that inhibition of one factor in particular, Bax, is important to 14-3-3s' protection against rotenone toxicity in dopaminergic cells. We found that 14-3-3θ overexpression reduced Bax activation and downstream signaling events, including cytochrome C release and caspase 3 activation. Pharmacological inhibition or shRNA knockdown of Bax provided protection against rotenone, comparable to 14-3-3θ's neuroprotective effects. A 14-3-3θ mutant incapable of binding Bax failed to protect against rotenone. These data suggest that 14-3-3θ's neuroprotective effects against rotenone are at least partially mediated by Bax inhibition and point to a potential therapeutic role of 14-3-3s in Parkinson's disease

Lysosomal enzyme cathepsin D protects against alpha-synuclein aggregation and toxicity

α-synuclein (α-syn) is a main component of Lewy bodies (LB) that occur in many neurodegenerative diseases, including Parkinson's disease (PD), dementia with LB (DLB) and multi-system atrophy. α-syn mutations or amplifications are responsible for a subset of autosomal dominant familial PD cases, and overexpression causes neurodegeneration and motor disturbances in animals. To investigate mechanisms for α-syn accumulation and toxicity, we studied a mouse model of lysosomal enzyme cathepsin D (CD) deficiency, and found extensive accumulation of endogenous α-syn in neurons without overabundance of α-syn mRNA. In addition to impaired macroautophagy, CD deficiency reduced proteasome activity, suggesting an essential role for lysosomal CD function in regulating multiple proteolytic pathways that are important for α-syn metabolism. Conversely, CD overexpression reduces α-syn aggregation and is neuroprotective against α-syn overexpression-induced cell death in vitro. In a C. elegans model, CD deficiency exacerbates α-syn accumulation while its overexpression is protective against α-syn-induced dopaminergic neurodegeneration. Mutated CD with diminished enzymatic activity or overexpression of cathepsins B (CB) or L (CL) is not protective in the worm model, indicating a unique requirement for enzymatically active CD. Our data identify a conserved CD function in α-syn degradation and identify CD as a novel target for LB disease therapeutics

LRRK2 secretion in exosomes is regulated by 14-3-3

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene cause late-onset Parkinson's disease (PD). Emerging evidence suggests a role for LRRK2 in the endocytic pathway. Here, we show that LRRK2 is released in extracellular microvesicles (i.e. exosomes) from cells that natively express LRRK2. LRRK2 localizes to collecting duct epithelial cells in the kidney that actively secrete exosomes into urine. Purified urinary exosomes contain LRRK2 protein that is both dimerized and phosphorylated. We provide a quantitative proteomic profile of 1673 proteins in urinary exosomes and find that known LRRK2 interactors including 14-3-3 are some of the most abundant exosome proteins. Disruption of the 14-3-3 LRRK2 interaction with a 14-3-3 inhibitor or through acute LRRK2 kinase inhibition potently blocks LRRK2 release in exosomes, but familial mutations in LRRK2 had no effect on secretion. LRRK2 levels were overall comparable but highly variable in urinary exosomes derived from PD cases and age-matched controls, although very high LRRK2 levels were detected in some PD affected cases. We further characterized LRRK2 exosome release in neurons and macrophages in culture, and found that LRRK2-positive exosomes circulate in cerebral spinal fluid (CSF). Together, these results define a pathway for LRRK2 extracellular release, clarify one function of the LRRK2 14-3-3 interaction and provide a foundation for utilization of LRRK2 as a biomarker in clinical trials

14-3-3θ immunoprecipitates with Bax in M17 dopaminergic cells.

<p><b>a)</b> Cell lysates from M17 cells were immunoprecipitated with a polyclonal rabbit antibody against Bax or rabbit IgG, and resulting immunoprecipitants were blotted with a monoclonal mouse antibody against 14-3-3θ in top blot. Lysate lane on right is shown at a different exposure time than the immunoprecipitant lanes from the same gel. Blot was reprobed with anti-Bax antibody to verify Bax pulldown (bottom blot). 14-3-3θ shows specific immunoprecipitation with Bax. <b>b)</b> Cell lysates from M17 cells stably transfected with empty vector or 14-3-3θ tagged with the V5 epitope tag were immunoprecipitated with a polyclonal antibody against Bax and then immunoblotted against 14-3-3θ. Both endogenous 14-3-3θ (lower band marked by arrow) and exogenous, tagged 14-3-3θ (higher band marked by arrowhead) were immunoprecipitated with Bax from cells overexpressing 14-3-3θ, and the total amount of 14-3-3θ immunoprecipitated was increased in 14-3-3θ cells compared to empty vector control cells. Lysate lanes on right were run on a separate gel from the immunoprecipitant lanes. Blot was reprobed with anti-Bax antibody to verify pulldown of Bax (bottom blot).</p

Bax inhibition through alternative means is protective against rotenone toxicity.

<p><b>a)</b> M17 cells were pretreated with BIP (0, 200, or 500 µM) for four hours prior to treatment with rotenone at 1 µM. After 48 hours, cell death was assessed by LDH release into the culture media. LDH release into media was normalized to maximal LDH release for each well. Cells treated with BIP were more resistant to rotenone compared to untreated cells. Error bars reflect SEM. Results reflect three independent experiments with at least two replicates per experiment. ***p<0.001 (Bonferroni's multiple comparison test). <b>b)</b> shRNA targeting Bax showed considerable knockdown of Bax protein expression. Naïve M17 cells were infected with a pLKO.1 lentiviral construct containing Bax-specific shRNA sequence or with an empty pLKO.1 lentiviral construct (with no shRNA sequence; C). Infected cells were selected for in the presence of puromycin. Protein lysates from these infected cells were immunoblotted with a polyclonal antibody against Bax (top blot). Immunoblotting against tubulin (bottom blot) shows comparable protein loading. <b>c)</b> pLKO.1 control or Bax-shRNA M17 cells were treated with rotenone at varying concentrations for 48 hours. Cell death was assessed by LDH release. Bax-knockdown cells showed considerable protection against rotenone compared to control cells at all concentrations tested. Error bars reflect SEM. Results reflect three independent experiments with at least two replicates per experiment. ***p<0.001 (Bonferroni's multiple comparison test). <b>d)</b> shRNA targeting Bax also showed knockdown of Bax protein in both empty vector control and 14-3-3θ stable cell lines. Control and 14-3-3θ stable lines were infected with an empty pLKO.1 virus (C) or with the Bax-specific shRNA lentivirus. Protein lysates from these cells were immunoblotted with a polyclonal antibody against Bax and tubulin. <b>e)</b> Empty vector stable and 14-3-3θ stable cells infected with either empty pLKO.1 or Bax shRNA viruses were treated with rotenone at varying concentrations for 48 hours, and cell death was assessed by LDH release. Knockdown of Bax in 14-3-3θ stable cells provided additional reduction of rotenone toxicity. Error bars reflect SEM. Results reflect four independent experiments with at least two replicates per experiment. *p<0.05, **p<0.01, ***p<0.001 (Bonferroni's multiple comparison test).</p

14-3-3θ mutant that cannot bind Bax is not protective against rotenone.

<p><b>a)</b> Lysates from stable cells overexpressing either full-length 14-3-3θ or a C-terminally deleted mutant 14-3-3θ (aa1-239) were immunoprecipitated with a polyclonal rabbit antibody against Bax and then immunoblotted with a monoclonal mouse antibody against V5. Blot was reprobed with anti-Bax antibody to verify Bax pulldown (bottom blot). Lysate lanes on right in the Bax blot are shown at a different exposure time than the immunoprecipitant lanes from the same gel. Considerably much less mutant 14-3-3θ was immunoprecipitated with Bax compared to full-length 14-3-3θ. <b>b)</b> Vector control, full-length 14-3-3θ, or mutant 14-3-3θ cells were treated with rotenone for 48 hours. Cell death was assessed by LDH release. While full-length 14-3-3θ cells showed decreased cell death in response to rotenone, cells overexpressing mutant 14-3-3θ showed no protection against rotenone compared to vector control cells. Error bars reflect SEM. Results reflect three independent experiments with at least two replicates per experiment. ***p<0.001 (Bonferroni's multiple comparison test). n.s.  =  non-significant.</p

Rotenone-induced Bax activation is reduced in 14-3-3θ-overexpressing cells.

<p><b>a) Less Bax translocated to mitochondria in 14-3-3θ cells in response to rotenone.</b> After treatment with 5 µM rotenone for 24 hours, vector control and 14-3-3θ cell lysates were subfractionated into cytosolic and mitochondrial fractions and immunoblotted with a polyclonal rabbit antibody against Bax. For each fraction, lanes for vector control and 14-3-3θ cells are from the same gel and exposure time but are separated for clarity with regard to quantification. Bax levels were normalized to tubulin for the cytosolic fraction or cyclophilin D for the mitochondrial fraction. Bax levels for rotenone-treated cells are shown as the relative percentage of the corresponding untreated cells. Densitometric quantification included seven separate experiments. Error bars reflect SEM. *p<0.05, **p<0.01 (one sample t-test). <b>b) Total Bax levels were unchanged with rotenone treatment in either cell line.</b> After treatment with 5 µM rotenone for 24 hours, whole cell lysates were immunoblotted with an anti-Bax antibody. <b>c) Fewer 14-3-3θ cells were positive for activated Bax upon rotenone treatment.</b> After treatment without (i-iv) or with rotenone (v-viii) for 16 hours, vector control and 14-3-3θ cells were fixed in 2% paraformaldehyde and immunostained with a monoclonal mouse antibody against the active Bax conformation (6A7) and a goat Alexa 488-conjugated anti-mouse secondary antibody (i, ii, v, vi). Nuclei were stained with Hoechst 33342 (iii, iv, vii, viii). The number of 6A7-positive cells was quantitated with rater blind to experimental conditions. Error bars reflect SEM. **p<0.01, ***p<0.001 (Bonferroni's multiple comparison test). Scale bar  = 50 µm. <b>d) Rotenone-induced Bax oligomerization was reduced in 14-3-3θ cells.</b> Vector control and 14-3-3θ stable cells were treated with 5 µM rotenone for 24 hours. Mitochondrially-enriched fractions were crosslinked and immunoblotted for oligomers with an anti-Bax antibody. Cyclophilin D served as loading control. Densitometric quantification includes three independent experiments. Error bars reflect SEM. ***p<0.001 (Bonferroni's multiple comparison test). n.s.  =  non-significant.</p