PubMed: 30106381

Activity-dependent neuroprotective protein deficiency models synaptic and developmental phenotypes of autism-like syndrome.
The Journal of clinical investigation
Gao AYL | Gozes I | Grigg I | Hacohen-Kleiman G | Karmon G | Korenková V | Le A | McKinney RA | Pasmanik-Chor M | Sragovich S

Evidence 0ad310e2bf

Likewise, mechanistic target of rapamycin (Mtor), which has been linked to cellular regulation, protein translation, autophagy, and the actin cytoskeleton (43–45), was also found to be regulated by ADNP and NAP

Evidence fc203882c1

Additionally, given that ADNP and NAP are linked with autophagy (13), cell adhesion (35), immune response (36), autism (6, 13, 15, 17, 27), and synapse-related processes (6), the analysis included several representative genes pertaining to these processes

Evidence e4f8a2ee64

The measurements showed similar patterns in both tested brain areas, with Adnp deficiency resulting in substantial decreases in spine density (male and female mice) and increases in PSD95-asymmetric shaft synapses (males only, as indicated by increased localization of PSD95 in dendritic shafts rather than spines), which were all rescued by NAP treatment.

Evidence 6ba587a219

In hippocampal CA1 pyramidal cells, all dendritic spine subtypes were reduced in the Adnp+/– mice, except for the thin spines observed in males. The spine loss was rescued by NAP treatment, except for the stubby spines seen in males (Supplemental Figure 1).

Evidence 6d64a95c60

Supplemental Figure 2 shows the cortical spine data indicating a significant genotype effect (P < 0.01) and NAP rescue for all subtypes in males (P < 0.05).

Evidence e2d3c49753

This genotype- and sex-dependent pathology also extended to the cortex, with increased PSD95 shaft synapse density in Adnp+/– males compared with Adnp+/– females (P < 0.01), and was rescued by NAP treatment.

Evidence be17eb6d1f

Importantly, in males, NAP treatment did not affect PSD95 shaft synapse density in either tested region (Supplemental Figures 3 and 4, insets).

Evidence def2f74dcd

Furthermore, NAP treatment increased PSD95 shaft synapse volume in both tested brain regions (Supplemental Figures 3 and 4, insets, P < 0.05).

Evidence cc2f77b80c

In female mice, NAP treatment significantly decreased shaft synapses

Evidence 9651c10ff7

Interestingly, we found that shaft synapse densities (immature synapses) were decreased in the female cortex following NAP treatment (Supplemental Figure 4, P < 0.05).

Evidence a926d24898

Measurements of PSD95 for excitatory shaft synapse volumes (indicative of synaptic maturation) showed significant increases in Adnp+/– mice in both hippocampus and cortex (P < 0.05), but not in the male mouse cortical spines. We observed a further increase with NAP treatment in female mice only, suggesting a compensatory effect (Supplemental Figures 1 and 2, insets).

Evidence 5927d158cb

Interestingly, we detected sex-specific differences in object/ mouse preference in the female mice, which did not prefer mice over objects (potential autistic behavior). The indifference phenotype was ameliorated by NAP treatment (Figure 7D).

Evidence 97f6a74a75

We evaluated the 31 transcripts regulated at both tested ages (see above) and found that the most enriched modified functions were related to nervous system development and activity including synapse assembly, positive regulation of synaptic transmission, glutamatergic, regulation of synapse organization, regulation of cell communication, α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) glutamate receptor clustering, learning or memory, social behavior, regulation of ion transport, vocalization behavior, and nervous system development (Figure 3, Supplemental Tables 11 and 12)

Evidence 8479cb8df7

As with object recognition memory, the deficient social memory of Adnp+/– mice (males and females) was normalized by NAP treatment (Figure 7F).

Evidence b106c33a76

Figure 3, Supplemental Figure 5 (younger age group), Supplemental Figure 6 (older age group), and Supplemental Tables 5 and 8 reveal an overall similar pattern of Adnp genotype–and NAP treatment–regulated human and mouse protein product interactions across ages with Akt1 (the mosaic mutations of which lead to the Proteus syndrome, characterized by the overgrowth of skin, connective tissue, brain, and other tissues; ref. 37) and discs large MAGUK scaffold protein 4 (Dlg4, also known as Psd95), a key regulator of synaptic plasticity (see above) that plays central roles associated with ADNP and NAP function.

Evidence acdaffe217

In the young, developing mouse, specific Adnp genotype– and NAP-regulated hippocampal transcripts included a reduction and rescue of formyl peptide receptor 3 (Fpr-rs3) in males only, in agreement with the previous genotype-associated reduction we observed in the developing embryo (2).

Evidence 4de3b66e3e

Tubulin β 1 class VI (Tubb1) increased in the Adnp+/– female mouse and was rescued by NAP treatment, thus correlating with our genotype-related RNA-seq data (6) (Figure 4A).

Evidence c70b4d9b69

In the 3-month-old hippocampi (Figure 4B), we found significant sex-dependent changes for Adnp+/– gene regulation and NAP rescue in the following genes in male mice: (a) apolipoprotein E (Apoe), the lead gene for Alzheimer’s disease risk, which was shown before to be a major gene regulated by ADNP (10, 13); (b) Gm21949, which is suggested to play a role in calcium-mediated responses, action potential conduction in myelinated cells, and axonal outgrowth and guidance (6); (c) lipase A (Lipa), which is related to lipid metabolism and was previously shown to be regulated by the Adnp genotype in mice (3); (d) autism-associated neuroligin 2 (Nlgn2), a postsynaptic membrane cell adhesion protein that mediates the formation and maintenance of synapses between neurons (12); (e) paired box protein 6 (Pax6), a key regulator in glutamatergic neuronal differentiation (38) and cortical development (39), which was shown before by us to be regulated by ADNP (complete knockout of Adnp rendered Pax6 expression undetectable in the brain primordium, contrasting with increased expression in Adnp+/– embryos [ref. 1] and in subcortical brain domains of 2-month-old male Adnp+/– mice [ref. 3]); and (f) Wolframin endoplasmic reticulum transmembrane glycoprotein (Wfs1), which is associated with neurodegeneration and cellular calcium homeostasis regulation and was previously shown to be regulated by NAP (34).

Evidence 1d46f22320

In the mature cerebral cortex, only histone cluster 1 H3 family member B (Hist1h3b), which was one of the major transcripts downregulated in the hippocampi of 5-month-old Adnp+/–mice compared with Adnp+/+ mice (6, 17), was found here to be downregulated in the female Adnp+/– mouse. This effect was now shown to be reversed by NAP treatment (Figure 4B).

Evidence 65a7d0051a

In male mice, the ATP-binding cassette subfamily F member 3 (Abcf3), bone morphogenetic protein 4 (Bmp4), cadherin 17 (Cdh17), lysine demethylase 5d (Kdm5d), Kruppel-like factor 1 (Klf1), and period circadian regulator 1 (Per1) were upregulated as a consequence of Adnp haploinsufficiency and rescued by NAP

Evidence b98ae60765

In female mice, Akt1 (above) and ionized calcium–binding adapter molecule 1 (Iba1), a marker of microglial activation that crosslinks actin (42), were markedly increased in the Adnp+/– mouse spleen and normalized by NAP treatment, suggesting a potential peripheral inflammation–linked biomarker

Evidence 82d2a72ab2

Importantly, negative geotaxis, a test used to investigate motor coordination and vestibular sensitivity, showed delayed development in Adnp+/– mice and normalization with NAP treatment (Figure 5D).

Evidence f5e2c439da

Specifically, the standing time and step cycle parameters indicated better performance in males, with significant impairments seen in Adnp+/– mice and amelioration with NAP treatment (Figure 6, C and D).

Evidence 518fa3b7b5

We then measured the latency to fall off an inverted cage lid (hanging wire) and found a highly significant impairment (decreased latency) in male Adnp+/– mice and a complete reversal with NAP treatment (Figure 7A). The females were not affected in this behavior, indicating sex differences in motor behavior and development in the haploinsufficient mice

Evidence 727cb7f37e

Likewise, Adnp+/– males, but not females, showed significantly reduced grip strength that was completely reversed by NAP treatment (Figure 7B).

Evidence 9271e97990

Here, we found that object recognition memory was normalized following NAP treatment in Adnp+/– mice (Figure 7C) and that NAP treatment did not change the behavior of normal Adnp+/+ mice (Supplemental Figure 14).

Evidence 6c5835c55c

This was coupled with deficits in olfactory function in the Adnp+/– females, but not males, with the female mice exhibiting impaired odor discrimination that was also restored by NAP treatment (Figure 7E; for more detail, see Supplemental Figure 14).

Evidence 777f26d364

ADNP expression in lymphocytes correlates with inflammation levels (36), disease state, and autophagy (13), as well as intelligence (40).

Evidence 6b7e06501e

Initially, we checked for mouse length (potentially corresponding to stature in an ADNP syndrome child) and discovered shorter lengths as the Adnp+/– mice matured, starting earlier in males (Figure 6, A and B).

Evidence 0609f1af3e

Specifically, in Adnp+/+ male mice, we found that NAP treatment reduced hippocampal stubby, thin, and total spine densities (Supplemental Figure 3, P < 0.05) as well as cortical mushroom, stubby, and total densities (Supplemental Figure 4, P < 0.05).

Evidence 18b7daad0d

Closer inspection suggested a more severe Adnp+/– genotype effect on total spine density in the male cortex compared with the hippocampus (Figure 1A, –1.56-fold reduction compared with Figure 2A, –1.83-fold reduction compared with the Adnp+/+ genotype).

Evidence 85dec25a9d

Further sex comparisons revealed differences in excitatory synapse numbers, with the Adnp+/– male mice showing significantly reduced hippocampal spine density, coupled with increased immature pathologic excitatory shaft synapses compared with Adnp+/– female mice (P < 0.01, Supplemental Table 2)

Evidence 119f1b1c15

Female cortical Hist1h3b was found to be regulated only by the Adnp+/– genotype.

Evidence abb7868b3a

Supplemental Figure 12 shows marked delays in weight gain that were also sex dependent (apparent earlier in females).


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