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DSCAM gene triplication causes excessive GABAergic synapses in the neocortex in Down syndrome mouse models [1]

['Hao Liu', 'Life Sciences Institute', 'University Of Michigan', 'Ann Arbor', 'Michigan', 'United States Of America', 'Department Of Cell', 'Developmental Biology', 'René N. Caballero-Florán', 'Department Of Pharmacology']

Date: 2023-05

Down syndrome (DS) is caused by the trisomy of human chromosome 21 (HSA21). A major challenge in DS research is to identify the HSA21 genes that cause specific symptoms. Down syndrome cell adhesion molecule (DSCAM) is encoded by a HSA21 gene. Previous studies have shown that the protein level of the Drosophila homolog of DSCAM determines the size of presynaptic terminals. However, whether the triplication of DSCAM contributes to presynaptic development in DS remains unknown. Here, we show that DSCAM levels regulate GABAergic synapses formed on neocortical pyramidal neurons (PyNs). In the Ts65Dn mouse model for DS, where DSCAM is overexpressed due to DSCAM triplication, GABAergic innervation of PyNs by basket and chandelier interneurons is increased. Genetic normalization of DSCAM expression rescues the excessive GABAergic innervations and the increased inhibition of PyNs. Conversely, loss of DSCAM impairs GABAergic synapse development and function. These findings demonstrate excessive GABAergic innervation and synaptic transmission in the neocortex of DS mouse models and identify DSCAM overexpression as the cause. They also implicate dysregulated DSCAM levels as a potential pathogenic driver in related neurological disorders.

Funding: This work was supported by grants from the National Institutes of Health (R21NS094091, R01MH112669, and R01EB028159 to B.Y.; R37NS076752 to LLI), a seed grant from Brain Research Foundation to B.Y., a grant from the Protein Folding Disease Initiative of the University of Michigan to B.Y., startup funds from the University of Michigan Department of Pharmacology to P.M.J., and a University of Michigan Rackham Merit Fellowship and a MI-BRAIN Predoctoral Fellowship to J.M.H.. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2023 Liu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

By combining genetic tools for sparse labeling and whole-cell patch-clamp recording, we found excessive GABAergic innervation and inhibition of cortical PyNs by chandelier cells (ChC) and basket cells in Ts65Dn mice. Genetic normalization of DSCAM levels rescued presynaptic overgrowth and excessive synaptic transmission of GABAergic neurons. Consistently, loss of DSCAM impaired ChC presynaptic growth and reduced inhibition of PyNs. In addition, we found that ChC axon terminal growth and bouton number are coupled and positively correlated with DSCAM levels in both wild-type and Ts65Dn mice. These findings thus highlight the critical role of DSCAM levels in regulating GABAergic synapses in the neocortex. Therefore, dysregulated DSCAM expression levels may be a common contributor to GABAergic dysfunctions in associated neurological diseases.

In this work, we sought to determine the effects of altered DSCAM levels in mouse models of DS, in which the DSCAM gene is triplicated. Previous studies have shown that enhanced GABAergic inhibition impairs cognition in Ts65Dn mice [ 16 – 20 ], the most widely used DS animal model [ 21 , 22 ]. Furthermore, Ts65Dn mice show excessive GABAergic inhibition in the hippocampus [ 16 , 23 – 31 ]. Overproduction of GABAergic neurons caused by triplication of Olig1 and Olig2 contributes to excessive inhibition in the hippocampus [ 29 – 31 ]. However, several lines of evidence suggest heterogeneous etiology in DS brain disorders such that different brain regions exhibit distinct molecular, cellular, and physiological defects. For example, the frequency of miniature inhibitory postsynaptic currents (mIPSCs) is increased in the dentate gyrus, but not the CA1 region, of the hippocampus in Ts65Dn mice [ 25 , 31 ]. In contrast to extensive reports on GABAergic deficits in the hippocampus of DS animal models, very little is known about whether GABAergic signaling is altered in the neocortex. Previous studies showed that the sizes of inhibitory synaptic boutons are enlarged in the neocortex of Ts65Dn mice, suggesting possible alterations in GABAergic synaptic functions in this region [ 32 , 33 ]. In the present study, we show excessive GABAergic innervation of and synaptic transmission to neocortical pyramidal neurons (PyNs) in Ts65Dn and demonstrate that DSCAM overexpression in GABAergic neurons plays a key role in this process.

Down syndrome (DS) is caused by an extra copy of human chromosome 21 (HSA21), and people with DS exhibit a number of medical conditions. A major challenge in DS research is to identify the genes on HSA21 that cause specific cellular and system alterations leading to the symptoms. Down syndrome cell adhesion molecule (DSCAM) is an evolutionarily conserved type I transmembrane protein encoded by a HSA21 gene [ 1 ]. In humans, the DSCAM gene resides in the DS critical region of HSA21 [ 1 ], which is associated with many symptoms of DS. We previously showed that protein levels of the Drosophila homolog of DSCAM, Dscam [ 2 ], determine the sizes of presynaptic terminals in sensory neurons without requiring the ectodomain diversity of the Drosophila Dscam gene [ 3 ]. Moreover, others reported that overexpression of Dscam impairs synaptic targeting and transmission in Drosophila [ 4 , 5 ]. These findings suggest that dysregulated DSCAM levels might contribute to neuronal defects in brain disorders in humans. In fact, altered DSCAM levels have been reported in multiple brain disorders, including DS [ 6 ], autism spectrum disorders (ASDs) [ 7 – 9 ], intractable epilepsy [ 10 ], bipolar disorder [ 11 ], and possibly Fragile X syndrome [ 3 , 4 , 12 , 13 ]. Although recent findings suggest a conserved role of DSCAM in promoting presynaptic growth in vertebrates [ 14 , 15 ], whether dysregulated DSCAM expression results in neuronal defects in brain disorders remains to be empirically determined.

Results

Loss of DSCAM impairs GABAergic synaptic transmission to PyNs To determine whether defective GABAergic bouton numbers caused by loss of DSCAM impairs GABAergic synaptic transmission, patch-clamp recordings in the whole-cell configuration were employed to record inhibitory currents in the PyNs in layers II/III of the ACC from acute neocortical brain slices. Consistent with impaired axonal growth and bouton number in ChCs, we found that the average frequency of mIPSCs was approximately 35% less in DSCAM−/− mice than that in heterozygous littermates (Fig 6A and 6B). In addition, the average amplitude of mIPSCs was reduced by 42%, suggesting that postsynaptic responses were impaired by loss of DSCAM (Fig 6C). Similar changes were observed in the frequency and amplitude of sIPSCs (Fig 6D–6F). PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 6. Loss of DSCAM impairs GABAergic synaptic transmission to neocortical PyNs. (A) Representative traces of mIPSCs from PyNs in layer II/III of the ACC in DSCAM+/− and DSCAM−/− brain slices. (B, C) Quantification of mIPSC frequency (B) and amplitude (C). For each mouse, 3–5 PyNs were recorded. A total of 5 DSCAM+/− and 4 DSCAM−/− mice were analyzed. N: 16 for DSCAM+/−, and 13 for DSCAM−/−. (D) Representative traces of sIPSCs from PyNs in layer II/III of ACC in DSCAM+/− and DSCAM−/− brain slices. (E, F) Quantification of sIPSC frequency (E) and amplitude (F). Approximately 3–5 PyNs were recorded for each mouse. A total of 5 DSCAM+/− and 4 DSCAM−/− mice were analyzed. N: 17 for DSCAM+/−, 14 for DSCAM−/−. Student t test. *: p < 0.05. The data underlying this Figure can be found in https://doi.org/10.5281/zenodo.7714234. ACC, anterior cingulate cortex; DSCAM, Down syndrome cell adhesion molecule; mIPSC, miniature inhibitory postsynaptic current; PyN, pyramidal neuron; sIPSC, spontaneous inhibitory postsynaptic current. https://doi.org/10.1371/journal.pbio.3002078.g006

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3002078

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