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Review
. 2023 Jul 14:16:1115880.
doi: 10.3389/fnmol.2023.1115880. eCollection 2023.

An intellectual-disability-associated mutation of the transcriptional regulator NACC1 impairs glutamatergic neurotransmission

James A Daniel  1 , Sofia Elizarova  1 , Ali H Shaib  2 , Abed A Chouaib  3 , Helge M Magnussen  4 , Jianlong Wang  5 , Nils Brose  1 , JeongSeop Rhee  1 , Marilyn Tirard  1
Affiliations
Review

An intellectual-disability-associated mutation of the transcriptional regulator NACC1 impairs glutamatergic neurotransmission

James A Daniel et al. Front Mol Neurosci. .

Abstract

Advances in genome sequencing technologies have favored the identification of rare de novo mutations linked to neurological disorders in humans. Recently, a de novo autosomal dominant mutation in NACC1 was identified (NM_052876.3: c.892C > T, NP_443108.1; p.Arg298Trp), associated with severe neurological symptoms including intellectual disability, microcephaly, and epilepsy. As NACC1 had never before been associated with neurological diseases, we investigated how this mutation might lead to altered brain function. We examined neurotransmission in autaptic glutamatergic mouse neurons expressing the murine homolog of the human mutant NACC1, i.e., Nacc1-R284W. We observed that expression of Nacc1-R284W impaired glutamatergic neurotransmission in a cell-autonomous manner, likely through a dominant negative mechanism. Furthermore, by screening for Nacc1 interaction targets in the brain, we identified SynGAP1, GluK2A, and several SUMO E3 ligases as novel Nacc1 interaction partners. At a biochemical level, Nacc1-R284W exhibited reduced binding to SynGAP1 and GluK2A, and also showed greatly increased SUMOylation. Ablating the SUMOylation of Nacc1-R284W partially restored its interaction with SynGAP1 but did not restore binding to GluK2A. Overall, these data indicate a role for Nacc1 in regulating glutamatergic neurotransmission, which is substantially impaired by the expression of a disease-associated Nacc1 mutant. This study provides the first functional insights into potential deficits in neuronal function in patients expressing the de novo mutant NACC1 protein.

Keywords: NAC1/BTBD14B; SUMO; SynGAP1; neuron; synapse.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Expression of Nacc1-R284W decreases EPSC amplitude compared to Nacc1-WT in Nacc1+/− glutamatergic neurons. Autaptic glutamatergic hippocampal neurons from Nacc1+/− mice were transduced with lentivirus inducing expression of either EGFP (black), Nacc1-WT (blue), or Nacc1-R284W (red). Synaptic function was then examined using patch clamp recording. (A) Overlaid representative eEPSC traces taken from a single cell from each transduction condition. (B) Bar charts showing the eEPSC amplitudes measured from single autaptic neurons transduced as above. (C) Representative overlaid traces of the current evoked by the application of 500 mM sucrose to evoke fusion of synaptic vesicles (SVs) in the RRP. (D) Bar charts showing the average total charge transferred by the release of the RRP in individual neurons transduced as above. (E) Bar charts showing the charge transferred by the eEPSC in individual neurons. (F) Bar charts showing the probability of vesicular release (Pvr) of individual neurons transduced as above. Pvr was calculated by dividing the charge transfer during an evoked EPSC by the charge transfer during the sucrose response, and then expressed as a percentage. (G) Bar charts showing the amplitude of spontaneous miniature EPSCs (mEPSCs) recorded from individual transduced neurons in the presence of 300 nM TTX. (H) Bar charts showing the frequency (Hz) of mEPSCs recorded from individual transduced neurons. (I) Overlaid representative traces of the current induced by perfusion with 100 μM glutamate. (J) Bar charts showing the amplitude of the peak current generated by glutamate application in transduced neurons. (K) Overlaid representative traces of the current induced by perfusion with 10 μM kainic acid (KA). (L) Bar charts showing the amplitude of the peak current generated by KA application in transduced neurons. In each chart, dots represent values recorded for individual cells, while the bar height represents the median of all cells. The number of cells analyzed for each condition is displayed beneath the condition label on each charts. Data were compared using a Kruskal-Wallis test by ranks followed by Dunn’s multiple comparisons test. * Denotes a p value of between 0.01 and 0.05, ** denotes a p value of between 0.001 and 0.01.
Figure 2
Figure 2
Nacc1 interacts with the synaptic proteins SynGAP1 and GluK2A. (A) Anti-HA (left panel) and anti-SynGAP1 (right panel) Western blot analysis of input and eluate fractions from an anti-SynGAP1 affinity purification (IP: anti-SynGAP1) in N2A cells expressing SynGAP1 and HA-Nacc1 alone or in combination. Black arrowheads indicate unmodified HA-Nacc1 (left) or SynGAP1 (right). (B) Anti-GluK2A/3 (left panel) and anti-HA (right panel) Western blot analysis of input and eluate fractions from an anti-HA affinity purification (IP: anti-HA) in N2A cells expressing GluK2A and HA-Nacc1 alone or in combination. A black arrowhead indicates GluK2A in the left blot. On the right, a black arrowhead indicates unmodified HA-Nacc1. Molecular weight markers (kDa) are indicated to the left of the blots. Individual lanes of single Western blot membranes are shown in A and B, since several lanes contained samples not relevant to the figure. The full blots are available in Supplementary Figure S8. Images are representative of at least 3 independent experiments.
Figure 3
Figure 3
Nacc1 is conjugated to SUMO in vivo and in vitro and Nacc1-R284W exhibits increased SUMO conjugation in neurons. (A) Western blot analysis using anti-Nacc1 antibody of input and HA peptide eluate fractions from an anti-HA affinity purification (IP: anti-HA) of WT (WT) and His6-HA-SUMO1 knock-in (KI) mouse brain. The black arrowhead indicates the position of non-modified Nacc1, the white arrowhead indicates Nacc1 modified with one SUMO1 peptide (appearing as doublet), and the white arrow indicate Nacc1 modified with two SUMO1 peptides. (B) Anti-Nacc1 Western blot analysis of input and eluate fractions from an anti-HA affinity purification (IP: anti-HA) in N2A cells expressing HA-SUMO1 plus either EGFP, Nacc1-WT, Nacc1-K167R, Nacc1-K485R, or Nacc1-2KR. The black arrowhead indicates unmodified Nacc1, the white arrowheads indicate Nacc1 conjugated to a single SUMO1 peptide (appearing as a doublet), and the white arrow indicates Nacc1 conjugated to two SUMO1 peptides. (C) Anti-Nacc1 Western blot analysis of input and affinity purification eluate fractions (IP: anti-HA) of N2A cells expressing HA-SUMO2 plus either EGFP, Nacc1-WT, Nacc1-K167R, Nacc1-K485R, or Nacc1-2KR. The black arrowhead indicates unmodified Nacc1 and the white arrowheads indicate Nacc1 conjugated to a single SUMO2 peptide (appearing as a doublet). Molecular weight markers (kDa) are indicated to the left of the blots. (D) Anti-HA Western blot analysis of lysate from primary hippocampal neuron infected with lentivirus mediating expression of EGFP, HA-Nacc1-WT, or HA-Nacc1-R284W. The black arrowhead indicates non-modified Nacc1, the white arrowheads indicate Nacc1 modified with one SUMO peptide, and the white arrow indicates Nacc1 modified with two SUMO peptides. (E) Bar charts showing a quantification of the ratio of unmodified Nacc1 to the SUMO-Nacc1 band at 90 kDa (upper chart), or to the SUMO-Nacc1 band at 105 kDa (lower chart). Data were normalized to the total protein loading for each well. Data were compared using a Welch t-test. * Denotes a p value of between 0.01 and 0.05, *** denotes a p value of between 0.0001 and 0.001. Images are representative of at least 3 independent experiments. Corresponding anti-HA blots for the experiments shown in (B) and (C) are presented in Supplementary Figure S9. Molecular weights in kDa are indicated next to all blots.
Figure 4
Figure 4
Nacc1-R284W exhibits impaired binding to SynGAP1 and GluK2A. (A) Representative anti-HA Western blot analysis of input and eluate fractions from an anti-HA affinity purification (IP: anti-HA) in N2A cells expressing one of four HA-Nacc1 constructs, as indicated above the blot. Black arrowhead indicates unmodified Nacc1, white arrowhead indicates Nacc1 conjugated to a SUMO protein. (B) Anti-GluK2A (top) and anti-HA (bottom) Western blot analysis of input and eluate fractions from an anti-HA affinity purification (IP: anti-HA) in N2A cells expressing GluK2A alone or in combination with one of four HA-Nacc1 constructs as indicated on top of the blot/images. A black arrowhead indicates GluK2A (top panel) or non-SUMO modified Nacc1 (bottom panel). (C) Bar charts showing quantification of the amount of GluK2A enriched after anti-HA-Nacc1 affinity purification using the various HA-Nacc1 mutants. The amount of GluK2A present in the eluate sample after anti-HA affinity purification was normalized firstly to the amount of GluK2A in the corresponding input sample, then to the amount of HA-Nacc1 enriched in the corresponding sample after affinity purification, and then finally expressed as a proportion of the amount of enrichment observed in the HA-Nacc1-WT sample. Data were compared using a Brown-Forsythe ANOVA followed by a Dunnett’s T3 multiple comparisons test. * Denotes a p value of between 0.01 and 0.05, ** denotes a p value of between 0.001 and 0.01. In each chart, dots represent values for a biological replicate and the bar height represents the median of all cells. N = 3 independent experiments. (D) Anti-HA (top panel) and anti-SynGAP1 (bottom panel) Western blot analysis of input and eluate fractions from an anti-SynGAP1 affinity purification (IP: anti-SynGAP1) of N2A cells expressing SynGAP1 alone or in combination with the various Nacc1 constructs as indicated on top of the blot. A black arrowhead indicates unmodified Nacc1 (top panel) or SynGAP1 (bottom panel). (E) Bar chart showing quantification of the amount of HA-Nacc1-R284W enriched by anti-SynGAP1 affinity purification, compared to HA-Nacc1-WT. The amount of each HA-Nacc1 construct present in the eluate sample after anti-SynGAP1 affinity purification was normalized firstly to the amount of HA-Nacc1 in the corresponding input sample, then to the amount of SynGAP1 enriched in the corresponding sample after affinity purification, and then finally expressed as a proportion of the amount of enrichment observed in the HA-Nacc1-WT sample. In each chart, dots represent values for a biological replicate and the bar height represents the median of all cells. Data were compared using a Welch’s t-test. ** Denotes a p value of between 0.001 and 0.01. N = 5 independent experiments. (F) Bar chart showing quantification of the amount of Nacc1-R284W-2KR enriched by anti-SynGAP1 affinity purification, compared to Nacc1-WT. Data normalization was performed as described for (E). In each chart, dots represent values for a biological replicate and the bar height represents the median of all cells. NS denotes no significant difference. N = 3 independent experiments. Molecular weights in kDa are indicated next to all blots.
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