Numb deficiency in cerebellar Purkinje cells impairs synaptic expression of metabotropic glutamate receptor and motor coordination

Abstract

Protein Numb, first identified as a cell-fate determinant in Drosophila, has been shown to promote the development of neurites in mammals and to be cotransported with endocytic receptors in clathrin-coated vesicles in vitro. Nevertheless, its function in mature neurons has not yet been elucidated. Here we show that cerebellar Purkinje cells (PCs) express high levels of Numb during adulthood and that conditional deletion of Numb in PCs is sufficient to impair motor coordination despite maintenance of a normal cerebellar cyto-architecture. Numb proved to be critical for internalization and recycling of metabotropic glutamate 1 receptor (mGlu1) in PCs. A significant decrease of mGlu1 and an inhibition of long-term depression at the parallel fiber-PC synapse were observed in conditional Numb knockout mice. Indeed, the trafficking of mGlu1 induced by agonists was inhibited significantly in these mutants, but the expression of ionotropic glutamate receptor subunits and of mGlu1-associated proteins was not affected by the loss of Numb. Moreover, transient and persistent forms of mGlu1 plasticity were robustly induced in mutant PCs, suggesting that they do not require mGlu1 trafficking. Together, our data demonstrate that Numb is a regulator for constitutive expression and dynamic transport of mGlu1.

Keywords: Numb; Purkinje cell; cerebellum; mGlu1; trafficking.

Fig. 1.

Impaired motor coordination in Numb-cKO mice. (A) Numb expression at different postnatal stages in control cerebellum. GAPDH was the internal control. (B) Electrophoresis of Numb (214 bp), Numblike (369 bp), and GAPDH (172 bp) amplicons from cerebellar extracts (cere) (n = 5) and individual PCs (n = 7). (C) In situ hybridization with Numb and Numblike riboprobes in cerebellar sagittal sections from P30 control mice (n = 5). Note that Numb (black arrows), but not Numblike (blue arrows), was expressed abundantly in PCs. GCL, granule cell layer; ML, molecular layer; PCL, PC layer. (Scale bars: 100 μm.) (D) Electrophoresis of Numb and calbindin from individual control and Numb-cKO PCs (n = 10). (E) Immunohistochemical staining for calbindin (red), Numb (green), and DAPI (blue) in the cerebellum from control and Numb-cKO mice. Higher magnifications in dashed white boxes indicate that Numb signal is absent from Numb-cKO PCs. (Scale bars: 50 μm.) (F) Numb-cKO mice (P21) displayed normal body size and brain. Average body weights were 17.5 ± 1.7 g (control) and 16.6 ± 1.5 g (cKO) (n = 12 pairs; P > 0.05). (G) Immunostaining for calbindin (calb, red) and EAAT4 (green) shows dendrites and spine formation are normal in Numb-cKO mice compared with control. (Scale bars: 10 μm.) (H) Percentage of steps with hindpaw slips during runs on an elevated horizontal beam (n = 10 pairs). (I) Time spent on the accelerating rotarod for control and Numb-cKO mice (n = 10 pairs). *P < 0.05, **P < 0.01.

Fig. S1.

The expression of Numb in the developing cerebellum. Total protein extracted from mouse cerebella at postnatal stages probed with antibodies against Numb and GAPDH. Signal intensity ratios (Numb/GAPDH) were 18 ± 4% (P0), 31 ± 13% (P3), 37 ± 11% (P5), 48 ± 13% (P7), 98 ± 24% (P10), 168 ± 29% (P14), 194 ± 29% (P21), 208 ± 31% (P30), and 193 ± 29% (P60). n ≥ 3 for each age.

Fig. S2.

PC morphogenesis is normal in Numb-cKO mice. (A) DAPI staining in the cerebellum from control and Numb-cKO mice (P21). The thickness of lobule III, which was 753 ± 35 μm (control; n = 7) or 733 ± 46 μm (Numb-cKO; n = 6), was measured as indicated by dashed lines. (Scale bars: 100 μm.) (B) Calbindin (calb) staining of PCs from control and Numb-cKO mice (P21). The average dendritic tree area was 5.7 ± 0.7 ×10³ μm² (control; n = 16) or 5.6 ± 0.8 ×10³ μm² (Numb-cKO; n = 17). Total dendritic length was 778 ± 86 μm (control; n = 16) or 759 ± 95 μm (Numb-cKO; n = 17). (Scale bars: 10 μm.) (C) Double staining with calbindin and EAAT4 on distal dendrites of PCs from control and Numb-cKO mice (P21). White arrows show spines detected using 2D reconstruction. The number of spines over a 10-μm dendritic fragment was 19.1 ± 1.34 μm (control; n = 15) or 18.8 ± 1.24 μm (Numb-cKO; n = 14). The average spine length was 0.73 ± 0.04 μm (control; n = 15) or 0.72 ± 0.04 μm (Numb-cKO; n = 14). (Scale bars: 1 μm.)

Fig. 2.

Inhibited LTD but normal LTP in Numb-cKO mice. (A) Two consecutive PF EPSCs before (baseline) and after (t = 38 min) LTD induction in control (ctrl) and Numb-cKO PCs. The interval between paired EPSCs was 80 ms. (B) Time courses for percentage changes of EPSC1 amplitude in control (black) and Numb-cKO (gray) mice. Each data point was the average of three successive EPSCs evoked at 0.05 Hz. The arrow indicates LTD induction. (C) Time courses for PPF from the cells shown in B. (D) Example consecutive PF EPSCs before (baseline) and after (t = 38 min) LTP induction. (E) Time courses for percentage changes of EPSC1 amplitude in control (black) and Numb-cKO (gray) mice. (F) Time courses for PPF ratios from the subset of cells shown in E.

Fig. S3.

Induction of PF-LTD in current-clamp configuration. (A) Sample traces of PF EPSPs before and 40 min after a conjunctive stimulation in control mice (P20–P23). Each trace was an average of two consecutive EPSPs. (B) Time course of percentage changes of EPSP amplitude in control mice. (C) Sample traces averaged from two consecutive EPSPs before and 40 min after the conjunction in Numb-cKO mice (P20–P23). (D) Time course of percentage changes in EPSP amplitude in Numb-cKO mice.

Fig. 3.

Synaptic mGlu1 is reduced in Numb-cKO mice. (A) Cerebellar (total) and PSD fractions from control (ctrl) and Numb-cKO mice were probed with antibodies to Numb, calbindin, mGlu1, GluA2, and GluN1. GAPDH and PSD95 were internal controls for total and PSD, respectively. Histograms show percentage changes of proteins in Numb-cKO mice relative to control. Control (n = 4): 100 ± 5% (total, Numb), 100 ± 4% (PSD, Numb); 100 ± 4% (total, calbindin), 100 ± 4% (PSD, calbindin); 100 ± 5% (total, mGlu1), 100 ± 4% (PSD, mGlu1); 100 ± 4% (total, GluA2), 100 ± 6% (PSD, GluA2); 100 ± 7% (total, GluN1), 100 ± 5% (PSD, GluN1). Numb-cKO (n = 4): 51 ± 7% (total, Numb), 50 ± 9% (PSD, Numb); 101 ± 8% (total, calbindin), 99 ± 9% (PSD, calbindin); 96 ± 9% (total, mGlu1), 54 ± 9% (PSD, mGlu1); 104 ± 6% (total, GluA2), 102 ± 6% (PSD, GluA2); 98 ± 8% (total, GluN1), 97 ± 9% (PSD, GluN1). (B) mGlu1 EPSCs produced by a PF burst (Green Inset: 10 pulses, 100 Hz). mGlu1 EPSCs were blocked by its antagonist CPCCOEt (100 μM). Peaks were measured as indicated by black dots. (C) Slow currents were evoked by a pulse (10 psi, 20 ms) of aCSF containing DHPG (100 μM) and were blocked by the mGlu1 antagonist CPCCOEt (100 μM). (D) Representative AMPA EPSCs in control and Numb-cKO PCs. The decay was fit with a single exponential in both cells, and mean time-constants were 12.8 ± 0.9 ms (control; n = 24) and 12.2 ± 1.2 ms (cKO; n = 30). (E) mEPSCs recorded from control (n = 10) and Numb-cKO (n = 10) PCs. mEPSC parameters were frequency, 1.4 ± 0.3 (control) and 1.3 ± 0.3 (cKO); amplitude, 13.2 ± 1.2 pA (control) and 13.8 ± 1.1 pA (cKO). (F) Superposition of PF EPSCs evoked at different intervals in a control cell. (G) PPF as a function of interstimulus interval in control (n = 11) and Numb-cKO (n = 12) cells. **P < 0.01.

Fig. S4.

mGlu1 currents evoked by varying PF pulses and concentrations of DHPG. (A) mGlu1 EPSCs induced by PF burst (Green Inset: five pulses at 100 Hz). The average of peak currents was 157 ± 25 pA (control; n = 17) or 78 ± 13 pA (cKO; n = 16). (B) mGlu1 EPSCs produced by 15 PF pulses at 100 Hz (Green Inset). The average of peak currents was 517 ± 76 pA (control; n = 17) or 255 ± 33 pA (cKO; n = 17). (C) Slow currents evoked by a pulse of aCSF containing DHPG (50 μM) in PCs. The average current was 152 ± 22 pA (control; n = 8) or 84 ± 16 pA (cKO; n = 9). (D) Slow currents evoked by DHPG (150 μM) in PCs. The average currents were 544 ± 122 pA (control; n = 12) and 277 ± 57 pA (cKO; n = 10). *P < 0.05. **P < 0.01.

Fig. S5.

Numb deficiency does not alter DSE at PF–PC synapses. (A) Stimulus protocol with holding potential (hp) of PCs (P17–P20) and stimulation timing (stim). The duration of depolarization to 0 mV was 50 ms. Intervals between control stimuli (1–3) were 20 s. The Δt between depolarization and test stimulus was 5 s. (B) Amplitudes of PF EPSCs derived from one control PC plotted over time for control responses (open circles) and test responses (closed circles). Numbered circles (1–4) correspond to the control and test stimuli in A. Representative EPSCs are shown on the right. (C) Amplitudes of PF EPSCs derived from one Numb-cKO PC (P17–P20) plotted over time for control and test responses. Representative EPSCs are shown on the right. (D) Percentage inhibition of test EPSCs was 33.8 ± 3.9% (control; n = 10) or 35.1 ± 4.6% (Numb-cKO; n = 10), showing no difference between them. **P < 0.01. (E) A brief inhibition of PF EPSPs was induced by a train of 10 stimuli at 50 Hz (indicated by the arrow). PF EPSPs were recorded in PCs from control and Numb-cKO mice (P10–P11) under an elevated temperature (32 °C). (F) Responses of PCs from control (Left) and Numb-cKO (Right) mice to train stimuli at 50 Hz. Vertical bars under traces indicate individual stimulations. (G) PF EPSPs recorded from control (Left) and Numb-cKO (Right) mice at 2 s before the train and 2 s and 30 s following the train. (H) Percentage changes of peak PF EPSPs are plotted in control (n = 7) and in Numb-cKO (n = 6) mice. Arrows mark the start of a brief train.

Fig. S6.

P/Q channel-mediated Ca²⁺ transient is not changed in Numb-cKO mice. (A) A projected z-stack image of a PC filled with Alexa Fluor 594. (Scale bar: 20 μm.) (B) Fluo-4 signals in the dendritic field of PCs from control (ctrl) and Numb-cKO mice (P21). The same microscopic fields are shown at basal (Left) and peak (Center) [Ca²⁺]_i and after application of ω-Agatoxin IVA (Right). (Scale bars: 10 μm.) (C) ω-Agatoxin–sensitive [Ca²⁺]_i in the cells shown in B. Arrows show the initiation of depolarization. (D) Mean amplitudes of changes in Fluo-4 fluorescence (ΔF/F₀) in control and Numb-cKO mice. Ctrl: 3.0 ± 0.5 (n = 17 dendritic regions in 10 PCs). Numb-cKO: 3.1 ± 0.6 (n = 20 dendritic regions in 10 PCs).

Fig. S7.

Normal CF synapse elimination in Numb-cKO mice. Tested PCs were from five control and five Numb-cKO mice between P18 and P25. (A) Sample records of CF EPSCs from control PCs. Three traces in response to different intensities are superimposed. (B) Percentages of control PCs showing number of discrete steps of CF EPSCs (1, 91%; 2, 9%). (C) Sample records of CF EPSCs from Numb-cKO PCs. Three traces in response to different intensities are superimposed. (D) Data from Numb-cKO PCs (1, 88%; 2, 12%).

Fig. 4.

Expressions of mGlu1-associated proteins are normal in Numb-cKO mice. (A) Cerebellar (total) and PSD fraction of control (ctrl) and Numb-cKO mice were probed by immunoblotting with antibodies to TRPC3, IP₃R, STIM1, Homer1, and ERK. GAPDH and PSD95 were loading controls for total and PSD, respectively. Percentage changes of signal intensities were control, 100 ± 4% (total, TRPC3), 100 ± 4% (PSD, TRPC3); 100 ± 5% (total, IP₃R) 100 ± 6% (PSD, IP₃R); 100 ± 8% (total, STIM1), 100 ± 8% (PSD, STIM1); 100 ± 4% (total, Homer1), 100 ± 6% (PSD, Homer1); 100 ± 7% (total, ERK), 100 ± 6% (PSD, ERK); cKO, 98 ± 6% (total, TRPC3), 98 ± 8% (PSD, TRPC3); 96 ± 8% (total, IP₃R), 97 ± 8% (PSD, IP₃R); 104 ± 11% (total, STIM1), 103 ± 11% (PSD, STIM1); 98 ± 5% (total, Homer1), 99 ± 9% (PSD, Homer1); 98 ± 9% (total, ERK), 99 ± 6% (PSD, ERK). n = 4. (B) Percentage changes of transcript copy numbers per cell for the indicated genes. Grm1, 100 ± 9% (n = 13, control) and 105 ± 10% (n = 14, cKO); Gnaq, 100 ± 8% (n = 13, control) and 113 ± 14% (n = 14, cKO); Plcb4, 100 ± 8% (n = 13, control) and 95 ± 14% (n = 14, cKO); Itpr1, 100 ± 11% (n = 13, control) and 112 ± 19% (n = 14, cKO); Trpc3, 100 ± 13% (n = 13, control) and 102 ± 17% (n = 14, cKO); Stim1, 100 ± 12% (n = 13, control) and 92 ± 19% (n = 14, cKO).

Fig. 5.

mGlu1-LTD and mGlu1-STP are normal, but mGlu1 trafficking is inhibited in Numb-cKO mice. (A) mGlu1 EPSCs before and after the conditioning depolarization (depol; 5-s command to 0 mV at the soma) in control (ctrl) and Numb-cKO cells. Peaks of mGlu1 EPSCs and first AMPA EPSCs were measured as indicated by dots and arrows, respectively. (B) Time courses of percentage changes of mGlu1 EPSCs (left y axis) and AMPA EPSCs (right y axis) from control (n = 12) and Numb-cKO (n = 12) cells before and after 5-s depolarization. (C) Representative traces from one control PC showing trials before (pre), and 10, 30, and 180 s after somatic depolarization to 0 mV for 100 ms. (D) Time courses for percentage changes of mGlu1 EPSCs and AMPA EPSCs in control (n = 10) and Numb-cKO (n = 9) cells. (E) Control and Numb-cKO cerebellar slices were stimulated with 100 μM DHPG for 10 min, and mGlu1 in total and PSD fraction was immunoblotted 30 min or 180 min after DHPG challenge. GAPDH was the control. (F) Percentage changes of synaptic mGlu1 intensities were control: 100 ± 5% (0 min), 34 ± 4% (10 min + 30 min), 77 ± 8% (10 min + 180 min); cKO: 100 ± 4% (0 min), 96 ± 8% (10 min + 30 min), 103 ± 8% (10 min + 180 min). n = 4. *P < 0.05. **P < 0.01.