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. 2011 Aug 3;31(31):11126-32.
doi: 10.1523/JNEUROSCI.6244-10.2011.

Progranulin Deficiency Decreases Gross Neural Connectivity but Enhances Transmission at Individual Synapses

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Free PMC article

Progranulin Deficiency Decreases Gross Neural Connectivity but Enhances Transmission at Individual Synapses

Lucia Tapia et al. J Neurosci. .
Free PMC article

Abstract

Frontotemporal dementia (FTD) has been linked to mutations in the progranulin gene (GRN) that lead to progranulin (PGRN) haploinsufficiency. Thus far, our understanding of the effects of PGRN depletion in the brain has been derived from investigation of gross pathology, and more detailed analyses of cellular function have been lacking. We report that knocking down PGRN levels in rat primary hippocampal cultures reduces neural connectivity by decreasing neuronal arborization and length as well as synapse density. Despite this, the number of synaptic vesicles per synapse and the frequency of mEPSCs are increased in PGRN knockdown cells, suggesting an increase in the probability of release at remaining synapses. Interestingly, we demonstrate that the number of vesicles per synapse is also increased in postmortem brain sections from FTD patients with PGRN haploinsufficiency, relative to controls. Our observations show that PGRN knockdown severely alters neuronal connectivity in vitro and that the synaptic vesicle phenotype observed in culture is consistent with that observed in the hippocampus of FTD patients.

Figures

Figure 1.
Figure 1.
Dendritic arborization and spine density are reduced in PGRN-deficient neurons. A, Representative Western blot from hippocampal cultures infected with scrambled (Sc) or PGRN siRNA (siRNA), and subsequently infected with empty vector (+V), or a vector expressing human PGRN (+P). B, E, Confocal images of hippocampal neurons infected with scrambled or PGRN siRNA and transiently transfected with dsRed. C, D, F, The number of primary neurites (C), arbor length (D), and the density of dendritic protrusions (F) are significantly decreased in cells expressing PGRN siRNA relative to controls. Subsequent expression of PGRN eliminated differences between Sc and siRNA transfected cells (C, D). F, Histogram of the density of dendritic protrusions. n = 6 to 20 cells per condition from 5–8 separate cultures. **p < 0.01, ***p < 0.001 for one-way ANOVA with Tukey post hoc (C) and for Student's t test (F). D, ***p < 0.0001, F(1,319) = 18.3, two-way ANOVA, treatment. Scale bar, 10 μm.
Figure 2.
Figure 2.
The density of presynaptic clusters is reduced but the number of synaptic vesicles per synapse is increased in PGRN-deficient neurons. A, B, The density of putative synapses defined by colocalization of presynaptic and postsynaptic markers is decreased in PGRN knockdown cultures (n = 9–18 neurons per condition, 5 cultures). B, Confocal images of hippocampal neurons infected with scrambled (Sc) or PGRN siRNA, transiently transfected with PSD-95-RFP, and immunostained for synaptophysin. Scale bar, 5 μm. C, D, The size of synaptophysin puncta is increased in PGRN knockdown neurons. n = 10 images per condition, 3 cultures. D, Confocal images of neurons infected with scrambled or PGRN siRNA (green) and fixed and immunolabeled with anti-synaptophysin (red). Scale bar, 5 μm. E, Overall synaptophysin levels are unchanged in control and PGRN siRNA-expressing cultures. n = 4 blots from 4 separate cultures. F, G, The number of synaptic vesicles per synapse is increased in PGRN knockdown cultures compared to control (n = 41–52 synapses per condition, 3 cultures). Scale bar, 100 nm. ***p < 0.001, one-way ANOVA with Tukey post hoc (A, C, G).
Figure 3.
Figure 3.
Synaptic vesicle recycling and the frequency of mEPSCs are enhanced in PGRN-deficient neurons. A, Representative images of FM4-64 staining of recycling synaptic vesicles in PGRN knockdown and control cultures. Scale bar, 10 μm. There was an increase in the integrated density of FM4-64 puncta (control: 100 ± 8.6%; siRNA: 128.0 ± 3.7%; p = 0.04, n = 3 experiments). B, Representative traces showing mEPSCs. C, Mean mEPSC amplitudes (left) or decay kinetics (middle) are not significantly different in PGRN knockdown cultures; however, mean mEPSC frequency (right) is increased in PGRN knockdown cultures. n = 8–13 cells from 3–4 separate cultures; one-way Kruskal-Wallis ANOVA p = 0.024, KW = 9.46, #p < 0.05 Dunn's post hoc, *p < 0.05 t test direct comparison). D, Cumulative probability plots demonstrate that PGRN siRNA significantly reduces mEPSC IEIs (left; two-way RM-ANOVA #treatment p = 0.0113, F(1,640) = 8.2, ***interaction p = 0.0001, F(40,640) = 6.6). Overexpression of PGRN does not significantly affect IEIs (middle; p = 0.89). The effect of siRNA on mEPSC IEIs is significantly reduced by subsequent PGRN cDNA expression (right; two-way RM-ANOVA #treatment p = 0.034, F(1,840) = 5.1, ***interaction p = 0.0001, F(40,840) = 6.6). E, GluR2 levels are not significantly different in PGRN knockdown cultures. Surface GluR2: 1.26 ± 0.22-fold increase in surface GluR2 levels in PGRN siRNA cells compared to control (p = 0.31). Total GluR2: 1.08 ± 0.26-fold increase in total GluR2 levels in PGRN siRNA cells compared to control (p = 0.78). Shown is a representative blot from n = 3 blots from 3 cultures.
Figure 4.
Figure 4.
The number of synaptic vesicles per synapse is enhanced in the hippocampus of FTD patients with PGRN haploinsufficiency. A, Representative hematoxylin and eosin staining of postmortem hippocampal sections from control and FTD with GRN mutations patients. The line demarcates the CA1 pyramidal cell layer that is virtually ablated in the FTD brain. The box approximates the region of stratum radiatum used for electron micrograph analyses. B, Representative electron micrographs of hippocampal synapses in the stratum radiatum of control and FTD brains. C, The number of synaptic vesicles per synapse in FTD1–FTD3 (107.83 ± 12.50; n = 143 synapses) was significantly higher than in control patients (C1–C3; 45.53 ± 3.2; n = 199 synapses; p = 0.031, Student's t test). Each datum represents the number of vesicles per synapse. Scale bars: A, 1 mm; B, 100 nm.

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