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.

Figure 1. 20S proteasome subunits are localized to neuronal plasma membranes

(a–f) (left) Western blots of neuronal lysates probed using indicated antibodies. (a–f) (center) Electron micrographs of immunogold labeling (12 nm gold particles) from hippocampal slice preparations using antibodies raised against core catalytic β2 (a, b), β5 (c, d), α2 (e) proteasomal subunits and 19S cap proteasome subunit Rpt5 (f). Representative images shown. White boxes on EM show magnified region (displayed to the right). Several arrows shown corresponding to immunogold label; cytosolic (white); membrane (black). (a–f) (Right) Quantification of gold particles from cytosol (Cyto) and membrane (Mem). N number of micrographs were quantified to get at least 300 gold particles: a) N=49; b) N=47; c) N=43; d) N=54; e) N=54; f) N=92. >300 gold-particles per antibody were counted. Slices were made from two separate 3-month old mice, >20 slices were generated for immuno-EM analysis. Data are presented as mean ± SEM. Source data are available online.

Figure 2. Neuronal membrane proteasomes are exposed to the extracellular space

(a) Electron micrographs of immunogold labeling (12 nm gold particles) from DIV14 primary mouse cortical neuronal cultures using β2 antibody. Representative images shown. Inset shows magnified region. Ultrastructures: Presynaptic (Pre); Postsynaptic (Post); Microtubules (MT); Synaptic vesicles (SV). Arrows: cytosolic (white); membrane (red-cytosolic face), (yellow-overlaying), (green-extracellular face). (N=84 images, >300 gold-particles. Multiple punches from single culture, >20 slices generated). Quantification to right. (b) Quantification depicted for a subset of gold particles near membranes. Each tick mark represents 2 nm from the plasma membrane (PM). Each dot represents a single gold particle, totals shown above (c) Schematic showing three different approaches to determine whether proteasomes were surface-exposed. (d) Antibody Feeding: Live primary mouse DIV14 cortical neuronal cultures were incubated with antibodies against MAP2, N-terminus of GluR1 (GluR1), or β5 proteasome subunits. Representative images shown, scale bar = 10 μM. β5 antibody pre-incubated with the blocking peptide shown below. Quantification of percentage overlap shown (N=2 independent neuronal cultures, n=15 neurons/culture). Significance is calculated between β5 antibody and β5 antibody pre-incubated with blocking peptide. *P<0.01 (two-tailed Student’s t-test). (e) Surface biotinylation: Proteins from surface biotinylated DIV14 cortical neurons were precipitated on streptavidin affinity beads and immunoblotted. Representative immunoblots of input lysates (~3.5% of total, left) and streptavidin pulldown of lysates (Strep) (~11% of total, right). Quantification is of streptavidin signal normalized to input signal (N=4 independent neuronal cultures). *P<0.01 (one-way ANOVA). (f) Protease Protection: Proteinase K (PK) was applied onto DIV14 cultured cortical neurons for indicated times. Cytosolic (Cyto) and membrane (Mem) fractions were immunoblotted. Quantification is below. Significance for each timepoint against the zero minute timepoint is calculated (N=3 independent neuronal cultures). *P<0.01 (two-tailed Student’s t-test). All data are presented as mean ± SEM. Uncropped blots shown in Supplementary Data Set 1. Source data available online.

Figure 3. Neuronal membrane proteasomes are tightly associated with plasma membranes

(a) Primary mouse cortical neuronal cultures at DIV 14 were fractionated into cytosolic (Cyto) and membrane (Mem) components. Membranes were extracted with indicated sequentially increasing concentrations of Digitonin. Samples were analyzed by immunoblotting using antibodies against indicated proteins. Quantification to the right is normalized to input signal levels for each antibody. While 0.25% digitonin extracted cytosolic protein Tubulin, higher concentrations (0.5%, 1.0%) of digitonin were required to extract known hydrophobic proteins such as GluR1. An explanation of percentages loaded on gel is explained in materials and methods. Significance is calculated by comparing signal from the 0.5% digitonin fraction to the 0.25% digitonin fraction for each antibody (N=3 independent neuronal cultures). *P<0.01 (one-way ANOVA). Data are presented as mean ± SEM. (b) Proteasome subunits are tightly bound to membranes. Neuronal cultures at DIV14 were fractionated into cytosolic, peripherally-associated (Periph), and tightly-bound (Bound) proteins. Immunoblots of each fraction using indicated antibodies are shown. Quantification to right, data are presented as mean ± SEM (N=3 independent neuronal cultures). (c) Cultured neurons at DIV14 were phase separated with TX-114 (TX114). Immunoblots shown using indicated antibodies. TX114-free indicates aqueous phase, and TX114-rich contains the TX-114 phase. Quantification to the right, data are presented as mean ± range (N=2 independent neuronal cultures). Uncropped blots are shown in Supplementary Data Set 1. Source data are available online.

Figure 4. Neuronal membrane proteasomes are largely a 20S proteasome and in complex with GPM6 family glycoproteins

(a) Representative immunoblots of proteasomes purified out of neuronal cultures using capped-26S (26S IP) or 20S purification matrices (20S IP). Purification (Pure) was done out of either neuronal cytosol (Cyto) or detergent-extracted neuronal plasma membranes (Mem). (b) Immunoprecipitation with anti-Flag from HEK293 cell lysates previously transfected with plasmids containing Myc/Flag tagged GPM6A and GPM6B, followed by immunoblotting with Myc or proteasome antibodies (α1–7, β2, β5). Inputs (10% of total, left) and immunoprecipitated samples (75% of total, right) are shown. (c) Exogenous expression of GPM6A/B is sufficient to induce surface expression of endogenous proteasomes in HEK293 cells. HEK293 cells were mock transfected (Mock) or transfected with plasmids containing GFP, EphB2, Channelrhodopsin-2 (ChRdp2), GPM6A/B, and GPM6A/B + Myc-tagged β5 (A/B+Myc-β5). Cells were surface biotinylated. Representative immunoblots of input lysates (4% of total, left) and streptavidin pulldowns of lysates (32% of total, right). Quantification shown below is normalized to input signal. β5 western is overexposed in order to see Myc-tagged bands (two arrows, right of immunoblot). Significance is calculated compared to A/B transfected samples (N=3 independent cell cultures and transfections). *P<0.01, one way ANOVA. Data are presented as mean ± SEM. (d) Surface-exposed proteasome expression is unique to nervous system tissues. Tissues from P3 mouse were surface biotinylated. Cortex (Ctx), Hippocampus (Hip), Olfactory bulb (Olf), Hind Brain (Brn), Heart (Ht), Lung (Lg), Kidney (Kid), Liver (Lv), Pancreas (Pnc). Representative immunoblots of input lysates (2% of total, left) and streptavidin pulldowns of lysates (4% of total, right). (e) Representative western blots of input lysates (2.5% of total, left) and streptavidin pulldown (7.5% of total, right) of biotinylated proteins following surface biotinylation of mouse cortex tissue dissected from indicated postnatal ages. Uncropped blots shown in Supplementary Data Set 1. Source data available online.

Figure 5. Neuronal membrane proteasomes degrade intracellular proteins into extracellular peptides

(a) Purified 20S proteasomes from neuronal cytosol (Cyto) or membrane (Mem) were incubated with the fluorogenic proteasome peptide substrate SUC-LLVY-AMC. Endpoint fluorescence with and without incubation with SDS (.02%) is quantified. Significance is shown between SDS-treated and untreated samples (N=3 proteasome purifications, independent neuronal cultures). (b) Schematic for collection and purification of extracellular peptides. Media collected from neurons following radiolabeling was subjected to size exclusion purification, with or without Proteinase K (PK). (c) Representative autoradiograph of lysates from cortical neurons previously radiolabeled with ³⁵S methionine/cysteine for 10 minutes in the presence or absence of MG-132. Quantification of signal normalized to vehicle-treated neurons is shown (right). (d) Rapid efflux of radioactive material out of neuronal cultures into media depends upon proteasome function. Media collected from neurons following radiolabeling with or without MG-132 or ATPγS. Liquid scintillation quantification of media at indicated timepoints is shown normalized to MG-132 at 10-minute timepoint; 2 minute timepoint shown separately on bar graph (right) (Media from N=3 independent neuronal cultures). Significance in line graph is shown for MG-132 treated neurons compared to vehicle alone at each time point. (e) Media collected from neurons following radiolabeling was subjected to size exclusion purification, with or without Proteinase K (PK). The percentage of total radioactivity eluting at different sizes is shown (N=3 independent neuronal cultures and purifications). (f) Release of proteasome-derived peptides in the extracellular space correlates with NMP expression. Experiment performed as described in (d); media collected from either DIV7 or DIV8 neurons, with MG-132 (MG-132) or without (Vehicle). (Media of N=2 independent neuronal cultures) *P<0.05 ((a, e, f) two-tailed Student’s t-test, (e) significance of 500<³⁵S Signal<3000Da compared to <500Da and >3000Da; (d) One-way ANOVA). Data are mean ± SEM (a,c,d,e) or ± range (f). Source data available online.

Figure 6. Neuronal membrane proteasomes are required for release of extracellular peptides and modulate neuronal activity

(a,b) Biotin-epoxomicin does not cross neuronal membranes and covalently modifies proteasome subunits. (a) Neurons treated with biotin-epoxomicin (Bio-Epox) were separated into cytosolic (Cyto) and membrane (Mem) fractions and analyzed by western using streptavidin conjugated to a fluorophore. Immunoblots using indicated antibodies shown below. (b) Immunogold labeling against biotin using streptavidin-Au (black arrows) from neuronal cultures treated with Bio-Epox, with representative images shown. (N=54, obtained from multiple punches of a single neuronal culture, >20 slices generated.) Labeled ultrastructures: Presynaptic regions (Pre), Postsynaptic regions (Post), Microtubules (MT), and synaptic vesicles (SV). Quantification of particles in cytosol and on membrane (right). (c) Specific inhibition of neuronal membrane proteasomes blocks release of extracellular peptides. Media collected from radiolabelled neurons treated with Bio-Epox or without (Vehicle). Liquid scintillation quantification of media at indicated timepoints is shown normalized to Bio-Epox at the 5 minute timepoint; 2 minute timepoint shown separately. Significance is shown for Bio-Epox treated neurons compared to vehicle alone. (N=3 independent neuronal cultures). (d) NMP inhibition modulates speed and intensity of neuronal calcium transients. Bicuculline added (downward black arrowhead) to naïve GCaMP3-encoding neurons. Downward dark blue arrowhead indicates timing of Bio-Epox addition. Representative images (left) and traces of Bicuculline response before and after Bio-Epox addition are plotted (right). Scale bar = 40 μM. Quantification of normalized fluorescence intensity (ΔF/F₀) measurements of calcium signals over imaging timecourse are shown. (e) Average maximum amplitudes are plotted, and include analysis of calcium signaling after treatment with MG-132. Significance compared to Bicuculline stimulation alone. (f) Box-and-whisker plot of all frequencies observed. *P<0.05, one-way ANOVA (E), two-tailed Student’s t-test (B,C). All data are presented as mean ± SEM (D-F, N=2 independent replicate cultures, n=24 neurons per culture, with 18 ROIs (regions of interest) analyzed per neuron). Uncropped blots shown in Supplementary Data Set 1. Source data available online.

Figure 7. Neuronal membrane proteasome-derived peptides are sufficient to induce neuronal signaling

(a) Purified peptides were perfused onto GCaMP3-encoding mouse cortical cultured neurons. Dotted lines indicate time of peptide addition and washout. K⁺ indicates the timing of 55 mM KCl addition to neurons to determine that they still respond properly at the end of the experiment. Line graph shows increase in fluorescence over baseline during time of peptide addition, a decrease following washout and robust increase with KCl addition. Four sample traces from different neurons are plotted. (b, c) Similar to part (a), cultured neurons were incubated with either Peptides (PK) (peptides were pretreated with P K, PK was removed, and then samples dialyzed to remove small molecules) or with Peptides (MG-132) (peptides purified from cells treated with MG-132). (d–h) Indicated drugs were perfused onto neuronal cultures during the times depicted by the dashed lines. Peptides were subsequently added as indicated and described in (a). Concentrations of drugs: BAPTA (2 μM), Thapsigargin (5 μM), Tetrodotoxin (1 μM), Nifedipine (1 μM), APV (2 μM). (i) Quantification of maximum intensity of change from each condition is plotted. *P<0.01 one-way ANOVA. Data are presented as mean ± SEM (N=3 independent replicate cultures, n>15 neurons per treatment, with at least 10 ROIs analyzed per neuron, per condition). Source data available online.

Figure 8. Proposed theoretical models of NMP association with the plasma membrane

Three models of how proteasomes can associate with plasma membranes are shown above. Extracellular and cytoplasmic sides of the plasma membrane are indicated. Symbol key shown below.