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Research Article
Stephen J. Bonasera
University of Nebraska Medical Center College of Pharmacy
Corresponding Author
ORCiD: https://orcid.org/0000-0001-6808-2821
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Complement 3 (C3) expression is increased in the cerebellum of aging mice that demonstrate locomotor impairments and increased excitatory synapse density. However, C3 regulation of locomotion, as well as C3 roles in excitatory synapse function, remain poorly understood. Here, we demonstrate that constitutive loss of C3 function in mice evokes a locomotor phenotype characterized by decreased speed, increased active state locomotor probability, and gait ataxia. C3 loss does not alter metabolism or body mass composition. No evidence of significant muscle weakness or degenerative arthritis was found in C3 knockout mice to explain decreased gait speeds. In an enriched primary cerebellar granule cell culture model, loss of C3 protein results in increased excitatory synaptic density and increased response to KCl depolarization. Our analysis of excitatory synaptic density in the cerebellar internal granule cell and molecular layers did not demonstrate increased synaptic density in vivo, suggesting the presence of compensatory mechanisms regulating synaptic development. Functional deficits in C3 knockout mice are therefore more likely to result from altered synaptic function and/or connectivity than gross synaptic deficits. Our data demonstrate a novel role for complement proteins in regulation of locomotor function and proper organization of cerebellar neuronal networks.
Keywords
complement 3, gait speed, bone density, microtomography, grip strength, ataxia, cerebellum, internal granule cell, Vglut1, Vgat
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This is a list of supplementary files associated with this preprint. Click to download.
- SupplementalFigure1.png
C3KO mice display slower gait speeds and decreased per-bout distance for movement-in-place. A. Over a 24-hour period C3KO and WT mice have similar overall movement-in-place distances throughout both the light and dark cycles. B. The number of forward movement-in-place bouts across the light and dark cycles is unchanged in C3KO mice. C. No differences in the probability of movement-in-place bouts between WT and C3KO mice. D. No differences in movement-in-place bout duration between WT and C3KO mice. E. Movement-in-place bout speed is significantly reduced in C3KO mice. F. Decreased movement-in-place per-bout distances in C3KO mice. * indicate p<0.01, Bonferroni corrected; error bars are ± 1 standard error of the mean.
- SupplementalFigure2.png
Body composition analysis of C3KO mice revealed minimal differences. WT and C3KO mice demonstrated no genotypic differences in bone mineral density, bone mineral content, bone area, soft tissue ratio, % fat, total tissue mass, or mouse weight by DEXA. The only difference appreciated was a small increase in tissue area of C3KO mice (WT 8.45 ± 0.48, C3KO 90.5 ± 0.60, p=0.044). Data from 8 animals per genotype. Comparisons made by Bonferroni-corrected two-tailed t-tests. * indicates p < 0.05. Error bars are ± one standard deviation of the mean.
- SupplementalFigure3.png
C3KO mice show no deficits in basal (A) or activity-associated (B) metabolic function. C3KO and WT mice were assessed by indirect calorimetry to determine genotypic differences in measures of metabolic function. No differences were noted between genotypes for "V" ̇O2, O2out, DO2, "V" ̇CO2, CO2out, or calculated heat during periods of mouse activity. Data analyzed by multiway ANCOVA controlling for genotype and adiposity.
- SupplementalFigure4.png
Excitatory and inhibitory input onto individual cerebellar granule cells is similar in WT and C3KO cultures at DIV3. Cell specific synapse densities were quantified by generating surfaces for individual pGFP transfected cerebellar granule cells. A. Representative images of WT and C3KO pGFP cerebellar granule cells at DIV3 stained for excitatory and inhibitory synaptic puncta. Far left panel is the maximum projection image from confocal z-stacks (raw), center panel shows the surface and spot detection, far right panel shows output of surfaces with contacting synaptic puncta. Excitatory synaptic puncta (Vglut1) are depicted in red; inhibitory synaptic puncta (VGAT) are depicted in teal. Scale bar represents 20 μm. B. Quantification of synaptic puncta per 100 μm2 of pGFP surface area revealed a trending but not significant increase in excitatory synapse density (WT 1.02±0.21; C3KO 1.60±0.39; p=0.085). C. There were no significant differences in the total surface area of pCGCs between genotypes. D. No differences in the number of primary dendrites were noted between genotypes. Data representative of >20 cells spread across 3 biological replicates per genotype. Differences were calculated by two-tailed Student t-tests. Error bars are ± one standard deviation of the mean.
- SupplementalFigure5.png
Cerebellar neuron cultures generated from DIV6 C3KO mice resemble those from WT mice. To verify that synaptic changes observed in culture were not a reflection of changes in cell distribution, pCGC cultures were fixed and immunostained for cell specific markers of neurons (NeuN), astrocytes (GFAP), and microglia (CD11B). A. Quantification of cell types demonstrated no obvious differences between WT and C3KO cultures (NeuN: WT 82.6±1.0, C3KO 79.3±1.8%; GFAP: WT 0.57±0.36%, C3KO 0.91±0.23%; CD11B: WT 0.37±0.04%, C3KO 0.26±0.04%). Statistics were not performed, as the number of biological replicates used to generate wells of technical replicates were small (WT n=2, C3KO n=3). To ensure that apoptosis rates were not a factor in synaptic density differences, pCGC cultures were immunostained for cleaved caspase-3. B. Quantification of activated caspase-3 demonstrated no significant differences between WT and C3KO cultures (WT 17.9±2.1%, C3KO 19.9±8.4%, p=0.72). C,D. Finally, to validate that synaptic differences were not a result of increased inflammatory signaling, transcript expression of IL1-β and IκBα was measured. Quantification of these transcripts showed no significant differences between WT and C3KO cultures (IkBa: WT 1.12±0.58, C3KO 1.01±0.32 fold change from control, p=0.78; IL1-β: WT 1.02±0.24, C3KO 0.75±0.8 fold change from control, p=0.13). Comparison was made by two-tailed student t-test, n=3 for each group. Error bars are ± one standard deviation of the mean.
- SupplementalFigure6.png
C3KO mice show no difference in Vglut1+ excitatory or GAD65+ inhibitory synapse density in the molecular layer of the cerebellum compared to control mice. 3-month old WT and C3KO mice were processed and imaged for excitatory (Vglut1) and inhibitory (GAD65) synapses. A. Representative images of WT and C3KO molecular layer synapse densities. Scale bar represents 50 μm. B. Quantification of the number of excitatory and inhibitory synaptic puncta in the molecular layer demonstrated no genotypic differences. C. No genotypic differences in average puncta fluorescent intensity. Data from 4 WT and 3 C3KO mice. Error bars are ± one standard deviation of the mean. Statistical analysis was performed using two-tailed Student t-tests.
- SupplementalFigure7.png
C3KO mice show no difference in Vglut2+ excitatory synapse density in the cerebellar molecular layer compared to WT. 3-month old WT and C3KO mice were processed and imaged for excitatory (Vglut2) synapses arising from climbing fibers. A. Representative images of WT and C3KO molecular layer synapse densities. Scale bar represents 50 μm. B. Quantification of the number of excitatory synaptic puncta in the molecular layer demonstrated no genotypic differences. C. No differences in the average puncta fluorescent intensity were observed between groups. Data from 4 WT and 3 C3KO mice. Error bars are ± one standard deviation of the mean. Statistical analysis was performed using two-tailed Student t-tests.
- SupplementalFigure8.png
C3KO mice show no changes in Vglut1+ excitatory or GAD65+ inhibitory synapses in the cerebellar granule cell layer. Cerebellar granule cells receive excitatory input from mossy fibers in a specialized synaptic glomerular structure surrounded by a ring of inhibitory synapses. A. Representative images of WT and C3KO cerebellar granule cell layer stained for Vglut1 and GAD65. Scale bar represents 50μm. B-F. Quantification of several properties of excitatory and inhibitory synapses demonstrated no significant differences between genotypes. B. The number of Vglut1 glomeruli was lower in C3KO mice, but this difference failed to reach significance when adjusted for multiple comparisons. C. Mean glomeruli Vglut1 staining intensity. D. Mean glomeruli Vglut1 volume. E. Mean number of GAD65 synaptic puncta. F. Mean GAD65 puncta staining intensity. Data from 4 WT and 3 C3KO mice. Error bars are ± one standard deviation of the mean. Statistical analysis was performed using two-tailed Student t-tests adjusted for multiple comparisons.
- SupplementalFigure9.png
C3KO mice show no changes in Vglut2+ excitatory synapses in the cerebellar granule cell layer. A. Representative images of WT and C3KO cerebellar granule cell layer stained for Vglut2. Scale bar represents 50μm. B-D. Quantification of several properties of excitatory synapses demonstrated no significant genotypic differences. B. Number of Vglut2+ glomeruli. C. Mean glomeruli Vglut2 staining intensity. D. Mean glomeruli Vglut2 volume. Data from 4 WT and 3 C3KO mice. Error bars are ± one standard deviation of the mean. Statistical analysis was performed using two-tailed Student t-tests adjusted for multiple comparisons.
- SupplementalTable1.csv
WT and C3KO behavioral metrics. Column 1 lists the abbreviation for each behavior, Column 2 lists the overall behavioral assay class, Column 3 provides a brief description of the behavior, Column 4 is an index. Columns 5 and Column 6 list mean values for each behavior, WT and C3KO respectively. Columns 7-22 list results for each test by individual mouse. WT mice are listed in columns 7-14; C3KO mice are listed in columns 15-22. Columns 23, 25, and 27 provide unadjusted p values calculated by Mann-Whitney, Student’s t-test, and fold change (B) methods. Columns 24 and 26 provide p values adjusted by false discovery rate (FDR) for Mann-Whitney (column 41) and Student’s t-test (column 43). Column 28 lists the behavioral fold change.
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Research Article
Stephen J. Bonasera
University of Nebraska Medical Center College of Pharmacy
Corresponding Author
ORCiD: https://orcid.org/0000-0001-6808-2821
Badges
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This manuscript passed our Prescreen assessment
Complete author information
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Peer Review Timeline
CURRENT STATUS: Published
View the published article at Molecular Neurobiology.
Version 1
Posted 11 Mar, 2021
No community comments so far
Reviews received
Received 21 Apr, 2021
Reviewers invited
Invitations sent on 18 Mar, 2021
Editor invited
On 11 Mar, 2021
Editor assigned
On 07 Mar, 2021
First submitted
On 05 Mar, 2021
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PDF Downloads: ...
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License:
This work is licensed under a CC BY 4.0 License. Read Full License
View the published article at Molecular Neurobiology.
Complement 3 (C3) expression is increased in the cerebellum of aging mice that demonstrate locomotor impairments and increased excitatory synapse density. However, C3 regulation of locomotion, as well as C3 roles in excitatory synapse function, remain poorly understood. Here, we demonstrate that constitutive loss of C3 function in mice evokes a locomotor phenotype characterized by decreased speed, increased active state locomotor probability, and gait ataxia. C3 loss does not alter metabolism or body mass composition. No evidence of significant muscle weakness or degenerative arthritis was found in C3 knockout mice to explain decreased gait speeds. In an enriched primary cerebellar granule cell culture model, loss of C3 protein results in increased excitatory synaptic density and increased response to KCl depolarization. Our analysis of excitatory synaptic density in the cerebellar internal granule cell and molecular layers did not demonstrate increased synaptic density in vivo, suggesting the presence of compensatory mechanisms regulating synaptic development. Functional deficits in C3 knockout mice are therefore more likely to result from altered synaptic function and/or connectivity than gross synaptic deficits. Our data demonstrate a novel role for complement proteins in regulation of locomotor function and proper organization of cerebellar neuronal networks.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
Figure 9
This is a list of supplementary files associated with this preprint. Click to download.
- SupplementalFigure1.png
C3KO mice display slower gait speeds and decreased per-bout distance for movement-in-place. A. Over a 24-hour period C3KO and WT mice have similar overall movement-in-place distances throughout both the light and dark cycles. B. The number of forward movement-in-place bouts across the light and dark cycles is unchanged in C3KO mice. C. No differences in the probability of movement-in-place bouts between WT and C3KO mice. D. No differences in movement-in-place bout duration between WT and C3KO mice. E. Movement-in-place bout speed is significantly reduced in C3KO mice. F. Decreased movement-in-place per-bout distances in C3KO mice. * indicate p<0.01, Bonferroni corrected; error bars are ± 1 standard error of the mean.
- SupplementalFigure2.png
Body composition analysis of C3KO mice revealed minimal differences. WT and C3KO mice demonstrated no genotypic differences in bone mineral density, bone mineral content, bone area, soft tissue ratio, % fat, total tissue mass, or mouse weight by DEXA. The only difference appreciated was a small increase in tissue area of C3KO mice (WT 8.45 ± 0.48, C3KO 90.5 ± 0.60, p=0.044). Data from 8 animals per genotype. Comparisons made by Bonferroni-corrected two-tailed t-tests. * indicates p < 0.05. Error bars are ± one standard deviation of the mean.
- SupplementalFigure3.png
C3KO mice show no deficits in basal (A) or activity-associated (B) metabolic function. C3KO and WT mice were assessed by indirect calorimetry to determine genotypic differences in measures of metabolic function. No differences were noted between genotypes for "V" ̇O2, O2out, DO2, "V" ̇CO2, CO2out, or calculated heat during periods of mouse activity. Data analyzed by multiway ANCOVA controlling for genotype and adiposity.
- SupplementalFigure4.png
Excitatory and inhibitory input onto individual cerebellar granule cells is similar in WT and C3KO cultures at DIV3. Cell specific synapse densities were quantified by generating surfaces for individual pGFP transfected cerebellar granule cells. A. Representative images of WT and C3KO pGFP cerebellar granule cells at DIV3 stained for excitatory and inhibitory synaptic puncta. Far left panel is the maximum projection image from confocal z-stacks (raw), center panel shows the surface and spot detection, far right panel shows output of surfaces with contacting synaptic puncta. Excitatory synaptic puncta (Vglut1) are depicted in red; inhibitory synaptic puncta (VGAT) are depicted in teal. Scale bar represents 20 μm. B. Quantification of synaptic puncta per 100 μm2 of pGFP surface area revealed a trending but not significant increase in excitatory synapse density (WT 1.02±0.21; C3KO 1.60±0.39; p=0.085). C. There were no significant differences in the total surface area of pCGCs between genotypes. D. No differences in the number of primary dendrites were noted between genotypes. Data representative of >20 cells spread across 3 biological replicates per genotype. Differences were calculated by two-tailed Student t-tests. Error bars are ± one standard deviation of the mean.
- SupplementalFigure5.png
Cerebellar neuron cultures generated from DIV6 C3KO mice resemble those from WT mice. To verify that synaptic changes observed in culture were not a reflection of changes in cell distribution, pCGC cultures were fixed and immunostained for cell specific markers of neurons (NeuN), astrocytes (GFAP), and microglia (CD11B). A. Quantification of cell types demonstrated no obvious differences between WT and C3KO cultures (NeuN: WT 82.6±1.0, C3KO 79.3±1.8%; GFAP: WT 0.57±0.36%, C3KO 0.91±0.23%; CD11B: WT 0.37±0.04%, C3KO 0.26±0.04%). Statistics were not performed, as the number of biological replicates used to generate wells of technical replicates were small (WT n=2, C3KO n=3). To ensure that apoptosis rates were not a factor in synaptic density differences, pCGC cultures were immunostained for cleaved caspase-3. B. Quantification of activated caspase-3 demonstrated no significant differences between WT and C3KO cultures (WT 17.9±2.1%, C3KO 19.9±8.4%, p=0.72). C,D. Finally, to validate that synaptic differences were not a result of increased inflammatory signaling, transcript expression of IL1-β and IκBα was measured. Quantification of these transcripts showed no significant differences between WT and C3KO cultures (IkBa: WT 1.12±0.58, C3KO 1.01±0.32 fold change from control, p=0.78; IL1-β: WT 1.02±0.24, C3KO 0.75±0.8 fold change from control, p=0.13). Comparison was made by two-tailed student t-test, n=3 for each group. Error bars are ± one standard deviation of the mean.
- SupplementalFigure6.png
C3KO mice show no difference in Vglut1+ excitatory or GAD65+ inhibitory synapse density in the molecular layer of the cerebellum compared to control mice. 3-month old WT and C3KO mice were processed and imaged for excitatory (Vglut1) and inhibitory (GAD65) synapses. A. Representative images of WT and C3KO molecular layer synapse densities. Scale bar represents 50 μm. B. Quantification of the number of excitatory and inhibitory synaptic puncta in the molecular layer demonstrated no genotypic differences. C. No genotypic differences in average puncta fluorescent intensity. Data from 4 WT and 3 C3KO mice. Error bars are ± one standard deviation of the mean. Statistical analysis was performed using two-tailed Student t-tests.
- SupplementalFigure7.png
C3KO mice show no difference in Vglut2+ excitatory synapse density in the cerebellar molecular layer compared to WT. 3-month old WT and C3KO mice were processed and imaged for excitatory (Vglut2) synapses arising from climbing fibers. A. Representative images of WT and C3KO molecular layer synapse densities. Scale bar represents 50 μm. B. Quantification of the number of excitatory synaptic puncta in the molecular layer demonstrated no genotypic differences. C. No differences in the average puncta fluorescent intensity were observed between groups. Data from 4 WT and 3 C3KO mice. Error bars are ± one standard deviation of the mean. Statistical analysis was performed using two-tailed Student t-tests.
- SupplementalFigure8.png
C3KO mice show no changes in Vglut1+ excitatory or GAD65+ inhibitory synapses in the cerebellar granule cell layer. Cerebellar granule cells receive excitatory input from mossy fibers in a specialized synaptic glomerular structure surrounded by a ring of inhibitory synapses. A. Representative images of WT and C3KO cerebellar granule cell layer stained for Vglut1 and GAD65. Scale bar represents 50μm. B-F. Quantification of several properties of excitatory and inhibitory synapses demonstrated no significant differences between genotypes. B. The number of Vglut1 glomeruli was lower in C3KO mice, but this difference failed to reach significance when adjusted for multiple comparisons. C. Mean glomeruli Vglut1 staining intensity. D. Mean glomeruli Vglut1 volume. E. Mean number of GAD65 synaptic puncta. F. Mean GAD65 puncta staining intensity. Data from 4 WT and 3 C3KO mice. Error bars are ± one standard deviation of the mean. Statistical analysis was performed using two-tailed Student t-tests adjusted for multiple comparisons.
- SupplementalFigure9.png
C3KO mice show no changes in Vglut2+ excitatory synapses in the cerebellar granule cell layer. A. Representative images of WT and C3KO cerebellar granule cell layer stained for Vglut2. Scale bar represents 50μm. B-D. Quantification of several properties of excitatory synapses demonstrated no significant genotypic differences. B. Number of Vglut2+ glomeruli. C. Mean glomeruli Vglut2 staining intensity. D. Mean glomeruli Vglut2 volume. Data from 4 WT and 3 C3KO mice. Error bars are ± one standard deviation of the mean. Statistical analysis was performed using two-tailed Student t-tests adjusted for multiple comparisons.
- SupplementalTable1.csv
WT and C3KO behavioral metrics. Column 1 lists the abbreviation for each behavior, Column 2 lists the overall behavioral assay class, Column 3 provides a brief description of the behavior, Column 4 is an index. Columns 5 and Column 6 list mean values for each behavior, WT and C3KO respectively. Columns 7-22 list results for each test by individual mouse. WT mice are listed in columns 7-14; C3KO mice are listed in columns 15-22. Columns 23, 25, and 27 provide unadjusted p values calculated by Mann-Whitney, Student’s t-test, and fold change (B) methods. Columns 24 and 26 provide p values adjusted by false discovery rate (FDR) for Mann-Whitney (column 41) and Student’s t-test (column 43). Column 28 lists the behavioral fold change.
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