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A mammalian nervous-system-specific plasma membrane proteasome complex that modulates neuronal function

Abstract

In the nervous system, rapidly occurring processes such as neuronal transmission and calcium signaling are affected by short-term inhibition of proteasome function. It is unclear how proteasomes are able to acutely regulate such processes, as this action is inconsistent with their canonical role in proteostasis. Here we describe a mammalian nervous-system-specific membrane proteasome complex that directly and rapidly modulates neuronal function by degrading intracellular proteins into extracellular peptides that can stimulate neuronal signaling. This proteasome complex is closely associated with neuronal plasma membranes, exposed to the extracellular space, and catalytically active. Selective inhibition of the membrane proteasome complex by a cell-impermeable proteasome inhibitor blocked the production of extracellular peptides and attenuated neuronal-activity-induced calcium signaling. Moreover, we observed that membrane-proteasome-derived peptides were sufficient to induce neuronal calcium signaling. Our discoveries challenge the prevailing notion that proteasomes function primarily to maintain proteostasis, and highlight a form of neuronal communication that takes place through a membrane proteasome complex.

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Figure 1: 20S proteasome subunits are localized to neuronal PMs.
Figure 2: NMPs are exposed to the extracellular space.
Figure 3: NMPs are tightly associated with PMs.
Figure 4: NMPs are largely 20S proteasomes and in complex with GPM6-family glycoproteins.
Figure 5: NMPs degrade intracellular proteins into extracellular peptides.
Figure 6: NMPs modulate neuronal activity and are required for the release of extracellular peptides.
Figure 7: NMP-derived peptides are sufficient to induce neuronal signaling.
Figure 8: Three proposed models of NMP association with the PM are shown.

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Acknowledgements

We thank S.H. Snyder, X. Dong, J. Nathans, G. Seydoux, M. Caterina, C. Machamer, S. Urban, Z. Li, H. Goldschmidt, K. Hopland, C. Vasavda, A. Dietterich, L. Cairns, and members of the Margolis laboratory (Johns Hopkins University School of Medicine, Baltimore, Maryland, USA) for reagents and valuable input; R. Huganir (Johns Hopkins University School of Medicine, Baltimore, Maryland, USA) for providing anti-NGluR1; and M.E. Greenberg (Harvard Medical School, Boston, Massachusetts, USA) for kindly providing EphB2 antibody. Special thanks to B. Lambrus (Johns Hopkins University School of Medicine, Baltimore, Maryland, USA). This work was funded by institutional funding and the NIH (grant R01 MH102364 to S.S.M.). K.V.R. was supported by the NIGMS (training grant T32 GM007445) and the NSF (Graduate Research Fellowship DGE-1232825).

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K.V.R. and S.S.M. designed experiments. K.V.R. performed all experiments. K.V.R. and S.S.M. analyzed and interpreted the data and wrote the paper.

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Correspondence to Kapil V Ramachandran or Seth S Margolis.

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Integrated supplementary information

Supplementary Figure 1 Proteasomes rapidly regulate neuronal calcium signaling.

Cortical neuronal cultures at 14 days in vitro (DIV14) were transfected using a plasmid that encodes for GCaMP3, a genetically encoded calcium indicator. Bicuculline, a GABA receptor antagonist that relieves inhibition on neuronal circuits and induces regular firing of action potentials, was added to naïve GCaMP3-encoding neurons. Calcium imaging was performed on transfected cultures. Traces of Bicuculline response before and after MG-132 addition are plotted. First black arrowhead indicates when Bicuculline is perfused onto neurons, second arrowhead indicates when MG-132 is spiked into perfusion. Lines above graph (Bicuculline – black, MG-132 – Blue) indicate time window when drug is applied. Quantification of normalized fluorescence intensity (ΔF/F0) measurements of calcium signals over imaging timecourse is presented. Trace is accumulated data from representative neuron over 18 ROIs (regions of interest). Data are presented as mean and s.e.m. of n = 30 neurons from two independent neuronal cultures. *P < 0.01 (two-tailed Student’s t test).

Supplementary Figure 2 Secondary-alone antibody controls do not detect signal by immuno-EM.

(a) Immuno-EM analysis of hippocampal slice preparations using only a secondary gold-conjugated anti-rabbit antibody in the absence of the primary antibody to detect non-specific background staining. Secondary alone controls for anti-goat (b) and anti-mouse (c) are shown. White arrows indicate low background immunogold particle staining observed, note low magnification (11000X). Slices were made from two separate 3 month old mice, >20 slices were generated for immuno-EM analysis, same metrics were used for secondary controls as for slices incubated with primary.

Supplementary Figure 3 20S proteasome subunits are localized to neuronal plasma membranes.

Low magnification (63000X) image of immuno-electron micrographs performed using antibodies against β2 (a), β5 (b), α2 (c), and 19S cap proteasome subunit S2 (d). Labeled ultrastructures: Presynaptic regions (Pre), Postsynaptic regions (Post). Insets shown at higher magnification. Slices were made from two separate 3-month-old mice, >20 slices were generated for immuno-EM analysis.

Supplementary Figure 4 20S proteasome subunits are localized to neuronal plasma membranes.

(a) Immuno-EM analysis using only a secondary gold-conjugated anti-goat antibody in the absence of the primary antibody to detect non-specific background staining (63000X). Labeled ultrastructures: Presynaptic regions (Pre), Postsynaptic regions (Post), Microtubules (MT – black arrowheads), and synaptic vesicles (SV - black arrowheads). Single DIV14 culture, >20 slices generated. Same metrics were used for secondary controls as for slices incubated with primary. (b) Low magnification (63000X) image of Immuno-EM performed using antibodies against the β5 and β2 proteasomal subunits. Arrows correspond to immunogold label distinguished as cytosolic (white) or on membranes (cytosolic face - red, directly on - yellow, extracellular face- green). Labeled ultrastructures: Presynaptic regions (Pre), Postsynaptic regions (Post), Microtubules (MT – black arrowheads), Mitochondria (Mito) and synaptic vesicles (SV - black arrowheads). Multiple punches from single DIV14 culture, >20 slices generated. (c) Quantification of Immuno-EM analysis of HEK293 (HEK293) cells and cortical (Cortical) neurons for the β2 proteasome subunit. Percentage of gold particles in the cytosol (Cyto) and at plasma membranes (Mem) was quantified. Data are presented as mean and s.e.m. >300 gold-particles counted, multiple punches from single DIV14 culture and single HEK293 culture, >20 slices generated. *P < 0.01 (two-tailed Student’s t test).

Supplementary Figure 5 Neuronal membrane proteasomes are exposed to the extracellular space.

(a) Immuno-electron micrographs performed using antibodies against the intracellular domain of Ion Channel Kv1.3 (63000X (left) and 43000X (right)). Arrows correspond to immunogold label distinguished as cytosolic (white). Note close proximity of gold particles to membranes. Similar results were observed and extensively quantified by Gazula et al, 2010. No membrane or extracellular staining was observed using this the antibody raised against Kv1.3. Slices were made from two separate 3 month old mice, >20 slices were generated for immuno-EM analysis. (b) The antibody feeding protocol (Figure 2d) was performed on primary neuronal cultures at DIV14 without primary antibodies, and stained using indicated secondary antibodies alone. Scale bar = 10 μM. Same metrics were used for secondary controls as for slices incubated with primary. (c, d) Primary neuronal cultures at DIV 18 were either untreated (DIV 18), or treated with hydrogen peroxide (H2O2 - 1 mM), Staurosporine (Stauro - 1 μM), or MG-132 (10 μM). Neurons were collected in sample buffer and immunoblotted for caspase-3 cleavage to measure cell death. A separate batch of neurons were treated and incubated with propidium iodide (PI), a DNA intercalating agent that can diffuse into neurons undergoing cellular death. Following PI addition, neurons were immediately imaged to determine whether treatments affected cell death. The percentage of nuclei that were PI positive were counted, compared to the total number of cell bodies, and quantification is shown. Note, in both cases our neuronal cultures are not dying. Data are presented as mean and s.e.m. of n = 2 experiments from independent neuronal cultures. *P < 0.01 (two-tailed Student’s t test). (e) Neuronal cultures were lysed and plasma membranes were pulled down on Concanavalin A-coupled agarose beads (ConA PD). Samples were subjected to immunoblotting using antibodies against indicated proteins. Representative immunoblots are shown.Gazula, V. R. et al. Localization of Kv1.3 channels in presynaptic terminals of brainstem auditory neurons. J Comp Neurol 518, 3205-3220, doi:10.1002/cne.22393 (2010).

Supplementary Figure 6 Neuronal membrane proteasomes are largely made of 20S core proteasome subunits.

Representative western blots of proteasomes purified out of DIV16 neuronal cultures using 20S purification matrices. Purification was done out of either neuronal cytosol (Cyto) or detergent-extracted neuronal plasma membranes (Mem). Inputs (5%) shown to the left. (b,c) Surface biotinylation: Biotinylated proteins from surface biotinylated DIV12 cortical neurons were precipitated on streptavidin affinity beads and subjected to immunoblotting using indicated antibodies. Inputs (10%) are shown to left of streptavidin pulldown (Strep). Flowthrough (FT) that did not bind to streptavidin beads was loaded to the right of Streptavidin pulldown. Note that the combined FT and Strep lanes are roughly equivalent to the Input. This is consistent with the proteasomes being both in the membrane and cytosol. Additionally, surface biotinylation experiments were performed on neurons that had been treated with 1% Formaldehyde to covalently fix protein-protein interactions. Following identical precipitation and preparation to (b), samples were immunoblotted with indicated antibodies (c). Note that actin is pulled down following fixation, unlike conditions without fixation, indicating that surface biotinylation of fixed samples may introduce artifacts of proteins that associate with membrane proteins but are not truly surface exposed. The precipitation of 19S cap proteins with our surface biotinylation under these conditions may indicate an artificial result or the existence of true interactions that are very weak (i.e. they do not occur in non-fixed conditions).

Supplementary Figure 7 Neuronal membrane proteasomes do not exist in heterologous cells and are developmentally regulated in neuronal cultures.

(a) Neuroblastoma-2A cells (N2A), HEK293 cells (HEK), and primary cortical neuronal cultures at DIV14 (Cort) were surface biotinylated. HEK293 cells were used as a heterologous system with non-neural origins and N2A cells were used as a heterologous system with neural origins. Biotinylated proteins were precipitated using streptavidin affinity beads and immunoblotted using indicated antibodies. (b) Human brain tissue was obtained according to IRB protocol, and surface biotinylated. Proteins were purified on streptavidin-agarose beads and subsequently immunoblotted using indicated antibodies. (c) Primary neuronal cultures from DIV5 to DIV8 were surface biotinylated. Biotinylated proteins were precipitated using streptavidin affinity beads and immunoblotted using indicated antibodies. (d) Live primary mouse cortical neuronal cultures at DIV7 or DIV8 were incubated with antibodies against MAP2 or β5 proteasome subunits. Representative images shown. Scale bar = 10 μM. Quantification of immunocytochemistry is shown to the right for the total amount of proteasome signal observed at DIV7 and DIV8. Data are presented as mean and s.e.m. of n = 22 neurons from two independent neuronal cultures for each developmental age. *P < 0.01 (two-tailed Student’s t-test).

Supplementary Figure 8 Neuronal membrane proteasomes are catalytically active and degrade intracellular proteins into extracellular peptides.

(a) Quantification of the 60-minute timecourse of the endpoint proteasome activity assay shown in Figure 4e. Note difference in activity from membrane proteasomes when SDS is added compared to cytosolic proteasomes. Data are presented as mean and s.e.m. of n = 3 experiments from independent proteasome purifications. (b) Following 10 minutes of radiolabel incorporation, media was washed out and replaced with Neurobasal growth media. Media was collected at either the two-minute or 30 minute timepoint following washout. Collected medium was then run through a size-exclusion protocol. An aliquot from each fraction was taken and quantified by liquid scintillation. Samples are normalized to the total amount of radioactivity present in the input sample taken at the two-minute timepoint, following subtraction of the zero-minute timepoint. We observed an increase in the fraction of radioactivity eluting below 500 Da and between 500 and 3000 Da at the 30 minute timepoint compared to the 2 minute timepoint, consistent with a sustained turnover of the intracellular pool of short-lived proteins into amino acids and short peptides. (c) Media from radiolabeled HEK293 cells is collected and purified, as described in Figure 4d, following vehicle treatment or MG-132 treatment. Data are presented as mean and s.e.m. of n = 3 experiments from independent neuronal cultures.

Supplementary Figure 9 Neuronal membrane proteasomes are required for the release of extracellular peptides, and modulate neuronal activity.

(a) Low magnification image of immuno-electron micrographs performed using streptavidin conjugated to gold particles in cortical neurons treated with Biotin-Epoxomicin. Immunogold label shown by arrows in cytosol (red) and on membrane (yellow). Labeled ultrastructures: Presynaptic regions (Pre), Postsynaptic regions (Post), Microtubules (MT). Obtained from multiple punches of a single neuronal culture, >20 slices generated. (b) Immuno-EM analysis using streptavidin conjugated to gold particles in the absence (Vehicle) of the Biotin-Epoxomicin to detect non-specific background staining. Cytosol labeling (red). Same metrics were used for secondary controls as for slices incubated with Biotin-Epoxomicin.

Supplementary Figure 10 Neuronal membrane proteasome–derived peptides are sufficient to induce neuronal signaling.

Purified extracellular peptides were added to naïve GCaMP3-encoding neurons. Representative images (top), quantification of normalized fluorescence intensity measurements of calcium signals over imaging timecourse (bottom). Scale bar = 40 μM. Arrowheads depict peptide addition (white arrowhead) and peptide washout (black arrowhead). Peptides (PK): Peptides pretreated with Proteinase K; Peptides (MG-132): purified media from neurons radiolabeled in the presence of MG-132; Control peptide: random peptides. Data are presented as mean and s.e.m. of n = 8 neurons per treatment from three independent neuronal cultures (total fluorescence in field of view was quantified).*P < 0.01 (one-way ANOVA).

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Ramachandran, K., Margolis, S. A mammalian nervous-system-specific plasma membrane proteasome complex that modulates neuronal function. Nat Struct Mol Biol 24, 419–430 (2017). https://doi.org/10.1038/nsmb.3389

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