Skip to main content
Journal ArticleOPEN ACCESS

The Shank3 interaction partner ProSAPiP1 regulates postsynaptic SPAR levels and the maturation of dendritic spines in hippocampal neurons

Frontiers in Synaptic Neuroscience (2016) 8(MAY)
DOI: 10.3389/fnsyn.2016.00013
9Citations
Citations of this article
26Readers
Mendeley users who have this article in their library.

Abstract

The postsynaptic density or PSD is a submembranous compartment containing a wide array of proteins that contribute to both morphology and function of excitatory glutamatergic synapses. In this study, we have analyzed functional aspects of the Fezzin ProSAP-interacting protein 1 (ProSAPiP1), an interaction partner of the well-known PSD proteins Shank3 and SPAR. Using lentiviral-mediated overexpression and knockdown of ProSAPiP1, we found that this protein is dispensable for the formation of both pre- and postsynaptic specializations per se. We further show that ProSAPiP1 regulates SPAR levels at the PSD and the maturation of dendritic spines. In line with previous findings on the ProSAPiP1 homolog PSD-Zip70, we conclude that Fezzins essentially contribute to the maturation of excitatory spine synapses.

Figures

  • FIGURE 1 | Synaptic distribution of green fluorescent protein-ProSAP-interacting protein 1 (GFP-ProSAPiP1) in primary hippocampal neurons. (A) GFP-ProSAPiP1 is clearly visible at the expected molecular weight (∼ 125 kDa) in infected primary hippocampal culture at DIV28, whereas no signal is observed in the uninfected control (Uninf.). Post-synaptic density (PSD) fraction from rat hippocampus was loaded as reference for endogenous ProSAPiP1, which is present in all lanes (∼ 85 kDa). β3-Tubulin and β-Actin serve as loading control. (A,B) Protein levels of endogenous ProSAPiP1 were significantly increased after overexpression of GFP-ProSAPiP1 at DIV28. (B) Statistical analysis was performed using unpaired two-sided t-test. ∗p < 0.05; n = 3 lysates from three independent cultures for each condition. (C,D) Both ProSAPiP1 puncta intensity and ProSAPiP1 cluster size were significantly increased after overexpression of GFP-ProSAPiP1 in primary hippocampal culture at DIV28. Statistical analysis was performed using unpaired two-sided t-test. ∗p < 0.05; n = 10 neurons from two independent cultures. (E) Exemplary hippocampal neurons, infected with GFP-ProSAPiP1 (green) and immunostained for Bassoon (red) on DIV14 and DIV28 as indicated. Statistical analysis shows a significant increase of synaptic GFP-ProSAPiP1 signals from DIV14 to DIV28 (42.9% on DIV14; 70.3% on DIV28) and was performed using unpaired two-sided t-test. ∗∗∗p < 0.001; n = 10 neurons from two independent cultures. (F) On DIV28, 62.1% of GFP-ProSAPiP1 signals co-localized with VGluT1 and 31.1% with VGAT, respectively. (G) Primary hippocampal cultures were infected with either FUGW empty vector (Vector) or GFP-ProSAPiP1 (both green) and stained for Bassoon (red) on DIV28 as indicated. No significant difference was observed. Scale bar: 10 µm. n = 15 neurons from three independent cultures.
  • FIGURE 2 | Analysis of presynaptic specializations after ProSAPiP1 knockdown in mature primary hippocampal neurons. (A) RNAi based knockdown of ProSAPiP1 in primary hippocampal culture (RNAi) leads to a significant reduction of endogenous ProSAPiP1, whereas there are no differences between the uninfected (Uninf.) and the scrambled control (Scr). PSD from rat hippocampus was loaded as reference for endogenous ProSAPiP1. β3-Tubulin and β-Actin serve as loading control. Statistical analysis was performed using One-way ANOVA. ∗p < 0.05; n = 3 lysates from three independent cultures for each condition. (B) Infected (GFP, green) primary hippocampal cultures were stained on DIV28 for Bassoon (red), VGluT1 (red) or VGAT (red) as indicated. No significant differences were observed in the density of Bassoon, VGluT1 or VGAT positive puncta per 10 µm dendrite length between Scr and RNAi. Scale bar: 10 µm. Statistical analysis was performed using unpaired two-sided t-test. n = 15 neurons from three independent cultures.
  • FIGURE 3 | Analysis of Shank3 and SPAR after ProSAPiP1 overexpression and knockdown in mature primary hippocampal neurons. (A) Primary hippocampal cultures were infected with either FUGW empty vector (Vector) or GFP-ProSAPiP1 and stained for Shank3 (red) or SPAR (red) on DIV28 as indicated. The intensity of SPAR-positive puncta was significantly increased. (B) Primary hippocampal cultures were infected with either Scr or RNAi and stained for Shank3 (red) or SPAR (red) on DIV28 as indicated. A significant reduction was observed in both density and intensity of SPAR positive puncta between Scr and RNAi. Scale bar: 10 µm. Statistical analysis was performed using unpaired two-sided t-test. ∗p < 0.05; ∗∗p < 0.01. n = 15 neurons from three independent cultures.
  • FIGURE 4 | Analysis of dendritic spines after ProSAPiP1 overexpression and knockdown in mature primary hippocampal neurons. (A,B) For spine analysis, neurons infected with Vector, GFP-ProSAPiP1, Scr or RNAi were additionally infected with RFP-tagged LifeAct visualizing F-actin on DIV24 and processed for analysis on DIV28. (A) Spine density and the percentage of filopodia were significantly decreased in neurons overexpressing GFP-ProSAPiP1 as indicated. (B) Spine density remained unchanged between Scr and RNAi while the percentage of mushroom-like spines was significantly decreased after knockdown of ProSAPiP1 as indicated. (A,B) IF, immunofluorescence; RM, reconstructed model. Scale bar: 10 µm. Statistical analysis was performed using unpaired two-sided t-test. ∗p < 0.05; ∗∗∗p < 0.001. n = 15 neurons from three independent cultures.

Author supplied keywords

References Powered by Scopus

Strong association of de novo copy number mutations with autism

Science
2235Citations
N/AReaders
Get full text

Autistic-like behaviours and hyperactivity in mice lacking ProSAP1/Shank2

Nature
516Citations
N/AReaders
Get full text

Meta-analysis of SHANK Mutations in Autism Spectrum Disorders: A Gradient of Severity in Cognitive Impairments

PLoS Genetics
475Citations
N/AReaders
Get full text
View more at Scopus

Cited by Powered by Scopus

Prosapip1-Dependent Synaptic Adaptations in the Nucleus Accumbens Drive Alcohol Intake, Seeking, and Reward

Neuron
43Citations
N/AReaders
Get full text

22q13 deletion syndrome: communication disorder or autism? Evidence from a specific clinical and neurophysiological phenotype

Translational Psychiatry
27Citations
N/AReaders
Get full text

Structural basis for PDZ domain interactions in the post-synaptic density scaffolding protein Shank3

Journal of Neurochemistry
17Citations
N/AReaders
Get full text
View more at Scopus

Register to see more suggestions

Mendeley helps you to discover research relevant for your work.

Already have an account?

Cite

CITATION STYLE

APA

Reim, D., Weis, T. M., Halbedl, S., Delling, J. P., Grabrucker, A. M., Boeckers, T. M., & Schmeisser, M. J. (2016). The Shank3 interaction partner ProSAPiP1 regulates postsynaptic SPAR levels and the maturation of dendritic spines in hippocampal neurons. Frontiers in Synaptic Neuroscience, 8(MAY). https://doi.org/10.3389/fnsyn.2016.00013

Readers over time

‘16‘17‘18‘19‘20‘21‘22‘25036912

Readers' Seniority

Tooltip

PhD / Post grad / Masters / Doc 15

83%

Researcher 3

17%

Readers' Discipline

Tooltip

Neuroscience 8

36%

Agricultural and Biological Sciences 7

32%

Biochemistry, Genetics and Molecular Bi... 5

23%

Medicine and Dentistry 2

9%

Article Metrics

Tooltip
Mentions
Blog Mentions: 1
News Mentions: 1
Social Media
Shares, Likes & Comments: 2

Save time finding and organizing research with Mendeley

Sign up for free
0