Ablation of TFR1 in Purkinje Cells Inhibits mGlu1 Trafficking and Impairs Motor Coordination, But Not Autistic-Like Behaviors

Abstract

Group 1 metabotropic glutamate receptors (mGlu1/5s) are critical to synapse formation and participate in synaptic LTP and LTD in the brain. mGlu1/5 signaling alterations have been documented in cognitive impairment, neurodegenerative disorders, and psychiatric diseases, but underlying mechanisms for its modulation are not clear. Here, we report that transferrin receptor 1 (TFR1), a transmembrane protein of the clathrin complex, modulates the trafficking of mGlu1 in cerebellar Purkinje cells (PCs) from male mice. We show that conditional knock-out of TFR1 in PCs does not affect the cytoarchitecture of PCs, but reduces mGlu1 expression at synapses. This regulation by TFR1 acts in concert with that by Rab8 and Rab11, which modulate the internalization and recycling of mGlu1, respectively. TFR1 can bind to Rab proteins and facilitate their expression at synapses. PC ablation of TFR1 inhibits parallel fiber-PC LTD, whereas parallel fiber-LTP and PC intrinsic excitability are not affected. Finally, we demonstrate that PC ablation of TFR1 impairs motor coordination, but does not affect social behaviors in mice. Together, these findings underscore the importance of TFR1 in regulating mGlu1 trafficking and suggest that mGlu1- and mGlu1-dependent parallel fiber-LTD are associated with regulation of motor coordination, but not autistic behaviors.SIGNIFICANCE STATEMENT Group 1 metabotropic glutamate receptor (mGlu1/5) signaling alterations have been documented in cognitive impairment, neurodegenerative disorders, and psychiatric diseases. Recent work suggests that altered mGlu1 signaling in Purkinje cells (PCs) may be involved in not only motor learning, but also autistic-like behaviors. We find that conditional knock-out of transferrin receptor 1 (TFR1) in PCs reduces synaptic mGlu1 by tethering Rab8 and Rab11 in the cytosol. PC ablation of TFR1 inhibits parallel fiber-PC LTD, whereas parallel fiber-PC LTP and PC intrinsic excitability are intact. Motor coordination is impaired, but social behaviors are normal in TFR1^flox/flox;pCP2-cre mice. Our data reveal a new regulator for trafficking and synaptic expression of mGlu1 and suggest that mGlu1-dependent LTD is associated with motor coordination, but not autistic-like behaviors.

Keywords: autism; metabotropic glutamate receptor; motor coordination; purkinje cell; trafficking; transferrin receptor.

Figure 1.

Gene targeting strategy for the generation of TFR1^flox/flox mice. A LoxP was inserted into intron 2. Another loxP along with a FRT-flanked PGK/Neo cassette was inserted into intron 3. 5′ and 3′ probes used for Southern blot analysis of genomic DNA appear in black bars. The loxP and FRT elements are shown as filled and open triangles, respectively.

Figure 2.

TFR1 is expressed at parallel fiber–PC synapses. A, Immunohistochemical staining for calbindin (calb, red) and TFR1 (green) in PCs from P2, P7, and P14 control mice. Scale bars, 20 μm. B, Electrophoresis of TFR1, calbindin, and GAPDH amplicons in individual PCs from control mice at P7 (n = 5) and P14 (n = 6). C, Synaptosomes were stained with antibodies against vGluT1 and TFR1 (white arrows). Scale bar, 5 μm. D, Synaptosomes were stained with antibodies against EAAT4 and TFR1 (white arrows). Scale bar, 5 μm.

Figure 3.

PC morphogenesis is normal in TFR1^flox/flox;pCP2-cre mice. A, Electrophoresis of TFR1 (154 bp), calbindin (184 bp), and GAPDH (220 bp) amplicons from individual TFR1^flox/flox (control, n = 10) and TFR1^flox/flox;pCP2-cre (cKO, n = 10) PCs. B, Immunohistochemical staining for calbindin (red) and TFR1 (green) in the cerebellum from control and cKO mice. Arrowheads show TFR1 signal was absent in PCs of cKO mice. ML, Molecular layer; PCL, Purkinje cell layer; GCL, granule cell layer. Scale bars, 20 μm. C, cKO mice (P21) displayed normal body weight and brain size. Average body weights were 17.3 ± 1.7 g (control) and 16.5 ± 1.5 g (cKO; n = 14 pairs; p = 0.38). n.s., Not significant. D, Nissl staining in the cerebellum from control and cKO mice (P21). Scale bars, 100 μm. E, Immunostaining for calbindin (calb, red) and EAAT4 (green) showing that dendrites and spine formation were normal in cKO mice. Scale bars, 10 μm.

Figure 4.

Synaptic mGlu1 is reduced in TFR1^flox/flox;pCP2-cre mice. A, Cerebellar (total) and PSD fractions from control and cKO mice were probed with antibodies to TFR1, calbindin (calb), GluA2, and mGlu1. GAPDH and PSD95 were internal controls for total and PSD, respectively. Histograms show percentage changes of proteins in cKO mice relative to control (n = 4 pairs). TFR1: 73 ± 7% (total; p = 0.014) and 44 ± 6% (PSD; p = 0.0067). calb: 101 ± 6% (total; p = 0.69) and 99 ± 7% (PSD; p = 0.45). GluA2: 96 ± 5% (total; p = 0.44) and 99 ± 6% (PSD; p = 0.37). mGlu1: 90 ± 6% (total; p = 0.31) and 40 ± 7% (PSD; p = 0.0076). B, mEPSCs recorded from control (n = 10) and cKO (n = 10) PCs. Averages of frequency were 2.3 ± 0.2 Hz (control) and 2.2 ± 0.2 Hz (cKO; p = 0.56). Averages of amplitude were 21 ± 1.3 pA (control) and 21 ± 1.2 pA (cKO; p = 0.67). C, Sample recordings of climbing fiber–EPSCs from control and cKO PCs. Three traces evoked by different intensities are superimposed. The peak averages of climbing fiber–EPSCs were 867 ± 89 pA (control, n = 23) and 880 ± 98 pA (cKO, n = 25; p = 0.25). Right, Percentages of discrete steps (1 or 2) of climbing fiber–EPSCs. For control: 90% (1) and 9% (2), n = 23. For cKO: 88% (1) and 11% (2), n = 25. D, Example mGlu1-EPSCs in response to burst stimulations (gray inset) in control and cKO cells. mGlu1-EPSCs were blocked by its antagonist CPCCOEt (100 μm). E, Slow currents were evoked by a pulse (10 psi, 20 ms) of aCSF containing DHPG (100 μm) and blocked by mGlu1 antagonist CPCCOEt (100 μm). *p < 0.05. **p < 0.01. n.s., Not significant.

Figure 5.

Encocannabinoid-dependent suppression of PC synaptic transmission is unchanged by TFR1 deletion. A, Superimposed parallel fiber–EPSCs by two successive parallel fiber stimulations in the absence (basal) and presence of DHPG in control and cKO mice. The bar graph shows the percentage changes of first EPSC amplitudes relative to basal values. The scatterd points show changes in PPD. B, Examples of climbing fiber–EPSCs in response to paired stimuli in control and cKO mice. Records obtained before (basal) and during the presence of DHPG are superimposed. The bar graph shows percentage changes of first EPSC amplitudes relative to basal values. Control: 58 ± 8% (n = 10). cKO: 60 ± 5% (n = 10). p = 0.34. The right panel shows changes in PPD. Control: 0.57 ± 0.04 (basal) and 0.76 ± 0.07 (DHPG), n = 10. cKO: 0.59 ± 0.05 (basal) and 0.80 ± 0.08 (DHPG), n = 10. p = 0.52. n.s., Not significant.

Figure 6.

GABAergic synaptic transmission onto PCs is not changed by TFR1 deletion. A, mIPSCs recorded from control (n = 9) and cKO (n = 9) PCs. Averages of frequency were 7.3 ± 0.3 Hz (control) and 6.7 ± 0.3 Hz (cKO; p = 0. 26). Averages of amplitude were 85 ± 8 pA (control) and 76 ± 10 pA (cKO; p = 0.18). n.s., Not significant. B, Superimposed eIPSCs were recorded from a single PC of control and cKO mice in response to stimulations at 15, 25, and 35 μA. Each trace is derived from averaging IPSCs of three successive traces recorded every 20 s. The right panel shows the relationship between eIPSC amplitude and a series of stimulation intensity for control (n = 12) and cKO (n = 11) mice. C, Immunofluorescence using antibodies to gephyrin and vGAT in control and cKO mice (P21). Nuclei were stained with DAPI (blue). Both gephyrin and vGAT showed a dotted appearance in the molecular layer and were mostly overlapped, suggesting the putative GABAergic synapses. The density of double-labeled puncta was 5.4 ± 0.2/100 μm² in TFR1^flox/flox mice (n = 6) and 5.2 ± 0.2/100 μm² in TFR1^flox/flox;pCP2-cre mice (n = 6). Scale bars, 50 μm.

Figure 7.

mGlu1 trafficking is inhibited in TFR1^flox/flox;pCP2-cre mice. A, mGlu1 in total and PSD fractions was immunoblotted 30 min or 180 min after DHPG challenge. β-actin was the control. B, HEK293 cells were transfected with myc-mGlu1α and GFP-TFR1. mGlu1α and TFR1 were visualized by fluorescent antibody and GFP signals, respectively. C, Histogram showing the ratios of the Triton X-100-insoluble (I) and Triton X-100-soluble (S) fraction in HEK293 cells. TFR1, 35 ± 11% (mGlu1) and 115 ± 23% (mGlu1+TFR1), p = 0.0023. mGlu1, 134 ± 17% (mGlu1) and 273 ± 26% (mGlu1+TFR1), p = 0.0073. Rab8, 7 ± 3% (mGlu1) and 31 ± 10% (mGlu1+TFR1), p = 0.0047. Rab11, 5 ± 3% (mGlu1) and 15 ± 4% (mGlu1+TFR1), p = 0.0051. The experiment was repeated for four times. D, Precleared brain lysates from WT mice were immunoprecipitated with rat anti-TFR1 antibody and immunoprecipitates were probed with antibodies to Rab11, Rab8, mGlu1, and TFR1. The experiment was performed three times. Rat IgG was used as the negative control. E, Expression of Rab proteins in PSD fraction was decreased in cKO mice. β-actin and PSD-95 were used as loading controls of total and PSD fractions, respectively. Percentage changes of Rab proteins in PSD fraction from cKO mice were 47 ± 6% (Rab8; p = 0.0075) and 35 ± 5% (Rab11; p = 0.0068). n = 4 pairs (P21). F, Working model showing that TFR1 facilitates the trafficking of mGlu1 from the intracellular reserve pool to the membrane by binding to Rab11 and decreases the endocytosis of mGlu1 by recruiting Rab8. *p < 0.05, **p < 0.01. n.s., Not significant.

Figure 8.

LTD is deficient whereas LTP is normal in TFR1^flox/flox;pCP2-cre mice. A, Example parallel fiber–EPSCs before (baseline) and after (t = 38 min) LTD stimulation in control and cKO PCs. B, Time course of percentage changes of EPSC1 amplitudes in control and cKO animals. Each data point represents the average of three successive EPSCs evoked at 0.05 Hz. The upward arrow shows LTD tetnus. **p < 0.01. C, Time course of PPF (EPSC2/EPSC1) from the cells shown in B. D, Example EPSCs before (baseline) and after (t = 38 min) LTP stimulation. E, Time course of percentage changes of EPSC amplitude in control and cKO mice. Each data point represents the average of three successive EPSCs evoked at 0.05 Hz. F, Time courses of PPF ratios from the subset of cells shown in E. G, Schema showing SFV injection into the vermis cerebellum. Eighteen hours after injection, eGFP signals were observed in scattered PCs (right). The amplification shows the affected spines (arrowheads) in distal dendrites. Scale bar, 20 μm. H, Representative traces of EPSCs recorded from cKO PCs expressing eGFP or eGFP+mGlu1 (mGlu1) before (baseline) and after (t = 38 min) LTD induction. I, Time course of percentage changes of EPSC amplitudes in cKO PCs transduced by eGFP or mGlu1. J, Time course of PPFs shown in I.

Figure 9.

LTD and STP of mGlu1 are unaltered in TFR1^flox/flox;pCP2-cre mice. A, Example mGlu1-EPSCs before and after mGlu1-LTD induction in control and cKO cells. Current amplitudes were measured at the peak of slow EPSCs. B, Time course of percentage changes in mGlu1-EPSC amplitudes from control (n = 12) and cKO (n = 12) cells induced by 5 s depolarization at the time indicated by the arrow. C, Representative mGlu1 traces showing four different trials before (pre) and 10, 30, 60, and 180 s after a transient depolarization from one cell. Right, Time course of percentage changes of mGlu1-EPSCs in control (n = 10) and cKO (n = 9) cells.

Figure 10.

Normal intrinsic plasticity in TFR1^flox/flox;pCP2-cre mice. A, Recording configuration for whole-cell recording. Inset, Example AP. B, AP threshold. C, AP amplitude. D, AP half-width. E, AP AHP. F, Example traces of intrinsic PC excitability as apparent from AP firing evoked by 400 pA current injections. G, No difference in evoked firing frequency relative to various levels of current injections. Inset barplot, Average slope of firing rate per current step. H, Example of traces for intrinsic plasticity with current injections of 400 pA. I, LTP induction protocol induced enhanced spike output in both control and cKO PCs. n.s., Not significant.

Figure 11.

Normal spontaneous spike firing in TFR1^flox/flox;pCP2-cre mice. A, Example spontaneous spikes of PCs recorded from anterior lobules (I–III) in control and cKO mice. B, Example spontaneous spikes of PCs recorded from posterior lobules (IX–X) in control and cKO mice. C, A 100 Hz parallel fiber tetanization caused increased spontaneous spike firing in cell attached recordings in control and cKO PCs. For these recordings, only cells that showed regular simple spike firing were used. Arrows indicate the time point of tetanization. n.s., Not significant.

Figure 12.

Impaired motor learning but normal social interactions were seen in TFR1^flox/flox;pCP2-cre mice. A, Percentage of steps with hindpaw slips during runs on an elevated horizontal beam. TFR1^flox/flox: 16 ± 2%. TFR1^flox/flox;pCP2-cre: 34 ± 6%. n = 10 pairs; p = 0.023. B, Time spent on the accelerating rotarod for control and cKO mice. TFR1^flox/flox: 229 ± 13 s; TFR1^flox/flox;pCP2-cre: 184 ± 17 s for session 7 (p = 0.019); TFR1^flox/flox: 249 ± 12 s; TFR1^flox/flox;pCP2-cre: 195 ± 12 s for session 8. n = 10 pairs. p = 0.014. C, Configuration of a three-chamber social interaction evaluated by relative time spent in each chamber. D, Example movement traces of a control mouse and a cKO mouse. E, Summary of spent time in S1, center, and empty chambers of control (n = 12) and cKO (n = 11) mice showing that both genotypes preferred to spend time in the room with S1 compared with the empty room. E′, Summary of sniffing time onto S1 of control and cKO mice. F, Configuration of a three-chamber after the introduction of S2. G, Example movement traces of a control mouse and a cKO mouse mice in the three-chamber with S1 and S2. H, Summary of spent time in S1, center, and S2 chambers of control (n = 12) and cKO (n = 11) mice showing that both genotypes preferred to spend time in the room with S2 compared with S1. H′, Summary of sniffing time onto S2 of control and cKO mice. I, In the open-field test, control (n = 17) and cKO mice (n = 15) showed no differences in time spent in the inner (control: 8 ± 4%; cKO: 5 ± 1%), middle (control: 20 ± 4%; cKO: 25 ± 3%), or outer (control: 72 ± %; cKO: 70 ± 5%) zones. *p < 0.05, **p < 0.01. n.s., Not significant.