STOP Codon Mutations at Sites of Natural Caspase Cleavage Are Implicated in Autism and Alzheimer’s Disease: The Case of ADNP

Gozes, Illana; Shazman, Shula

doi:10.3389/fendo.2022.867442

Cited by 14 publications

(7 citation statements)

References 45 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Similarly, brain tauopathy was also revealed in postmortem analysis of a young ADNP syndrome boy (Grigg et al, 2020). Most of the previously reported mutations in the ADNP syndrome as well as in Alzheimer's disease (Ivashko‐Pachima et al, 2022), are STOP or frameshift STOP mutations, with high impact effects on ADNP function (Gozes & Shazman, 2022).…”

Section: Introductionmentioning

confidence: 99%

A novel davunetide (NAPVSIPQQ to NAPVSIPQE) point mutation in activity‐dependent neuroprotective protein (ADNP) causes a mild developmental syndrome

Gozes

Shazman

2023

Eur J of Neuroscience

Self Cite

View full text Add to dashboard Cite

NAP (NAPVSIPQ, drug candidate name, davunetide) is the neuroprotective fragment of activity-dependent neuroprotective protein (ADNP). Recent studies identified NAPVSIP as a Src homology 3 (SH3) domain-ligand association site, responsible for controlling signalling pathways regulating the cytoskeleton. Furthermore, the SIP motif in NAP/ADNP was identified as crucial for direct microtubule end-binding protein interaction facilitating microtubule dynamics and Tau microtubule interaction, at the microtubule end-binding protein site EB1 and EB3. Most de novo ADNP mutations reveal heterozygous STOP or frameshift STOP aberrations, driving the autistic/intellectual disability-related ADNP syndrome. Here, we report for the first time on a de novo missense mutation, resulting in ADNP containing NAPVISPQE instead of NAPVSIPQQ, in a child presenting developmental hypotonia, possibly associated with inflammation affecting food intake in early life coupled with fear of peer interactions and suggestive of a novel case of the ADNP syndrome. In silico modelling showed that the mutation Q (polar side chain) to E (negative side chain) affected the electrostatic characteristics of ADNP (reducing, while scattering the electrostatic positive patch). Comparison with the most prevalent pathogenic ADNP mutation, p.Tyr719*, indicated a further reduction in the electrostatic patch. Previously, exogenous NAP partially ameliorated deficits associated with ADNP p.Tyr719* mutations in transfected cells and in CRISPR/Cas9 genome edited cell and mouse models. These findings stress the importance of the NAP sequence in ADNP and as a future putative therapy for the ADNP syndrome.

show abstract

Section: Introductionmentioning

confidence: 99%

A novel davunetide (NAPVSIPQQ to NAPVSIPQE) point mutation in activity‐dependent neuroprotective protein (ADNP) causes a mild developmental syndrome

Gozes

Shazman

2023

Eur J of Neuroscience

Self Cite

View full text Add to dashboard Cite

show abstract

“…These results indicate processing of ADNP by proteases 33 . Recently, a computational analysis identified ADNP cleavage sites mediated by caspases and proprotein convertases 34 . Amino acid residues 734–738 of ADNP, residing at the C-terminal part of the protein, contain the motif DDSDS, which is potentially recognized by caspase-3 and caspase-7.…”

Section: Discussionmentioning

confidence: 99%

Tracing the invisible mutant ADNP protein in Helsmoortel-Van der Aa syndrome patients

D’Incal,

Cappuyns,

Choukri

et al. 2024

Sci Rep

View full text Add to dashboard Cite

Heterozygous de novo mutations in the Activity-Dependent Neuroprotective Homeobox (ADNP) gene underlie Helsmoortel-Van der Aa syndrome (HVDAS). Most of these mutations are situated in the last exon and we previously demonstrated escape from nonsense-mediated decay by detecting mutant ADNP mRNA in patient blood. In this study, wild-type and ADNP mutants are investigated at the protein level and therefore optimal detection of the protein is required. Detection of ADNP by means of western blotting has been ambiguous with reported antibodies resulting in non-specific bands without unique ADNP signal. Validation of an N-terminal ADNP antibody (Aviva Systems) using a blocking peptide competition assay allowed to differentiate between specific and non-specific signals in different sample materials, resulting in a unique band signal around 150 kDa for ADNP, above its theoretical molecular weight of 124 kDa. Detection with different C-terminal antibodies confirmed the signals at an observed molecular weight of 150 kDa. Our antibody panel was subsequently tested by immunoblotting, comparing parental and homozygous CRISPR/Cas9 endonuclease-mediated Adnp knockout cell lines and showed disappearance of the 150 kDa signal, indicative for intact ADNP. By means of both a GFPSpark and Flag-tag N-terminally fused to a human ADNP expression vector, we detected wild-type ADNP together with mutant forms after introduction of patient mutations in E. coli expression systems by site-directed mutagenesis. Furthermore, we were also able to visualize endogenous ADNP with our C-terminal antibody panel in heterozygous cell lines carrying ADNP patient mutations, while the truncated ADNP mutants could only be detected with epitope-tag-specific antibodies, suggesting that addition of an epitope-tag possibly helps stabilizing the protein. However, western blotting of patient-derived hiPSCs, immortalized lymphoblastoid cell lines and post-mortem patient brain material failed to detect a native mutant ADNP protein. In addition, an N-terminal immunoprecipitation-competent ADNP antibody enriched truncating mutants in overexpression lysates, whereas implementation of the same method failed to enrich a possible native mutant protein in immortalized patient-derived lymphoblastoid cell lines. This study aims to shape awareness for critical assessment of mutant ADNP protein analysis in Helsmoortel-Van der Aa syndrome.

show abstract

“…These results agree with the findings of the present study. Interestingly, high ketamine doses were originally associated with increased apoptosis, prompting the use of NAP as a protective agent [ 43 ], with ADNP and NAP (davunetide) directly in involved in protection against apoptosis [ 72 ]. The protocol used here included the same incubation period and low concentrations of ketamine, suggested before to increase ADNP [ 42 ]; however, based on the current study’s results, ketamine’s direct association with ADNP may invoke additional pathways.…”

Section: Discussionmentioning

confidence: 99%

NAP (Davunetide): The Neuroprotective ADNP Drug Candidate Penetrates Cell Nuclei Explaining Pleiotropic Mechanisms

Ganaiem,

Gildor,

Shazman

et al. 2023

Cells

Self Cite

View full text Add to dashboard Cite

(1) Background: Recently, we showed aberrant nuclear/cytoplasmic boundaries/activity-dependent neuroprotective protein (ADNP) distribution in ADNP-mutated cells. This malformation was corrected upon neuronal differentiation by the ADNP-derived fragment drug candidate NAP (davunetide). Here, we investigated the mechanism of NAP nuclear protection. (2) Methods: CRISPR/Cas9 DNA-editing established N1E-115 neuroblastoma cell lines that express two different green fluorescent proteins (GFPs)—labeled mutated ADNP variants (p.Tyr718* and p.Ser403*). Cells were exposed to NAP conjugated to Cy5, followed by live imaging. Cells were further characterized using quantitative morphology/immunocytochemistry/RNA and protein quantifications. (3) Results: NAP rapidly distributed in the cytoplasm and was also seen in the nucleus. Furthermore, reduced microtubule content was observed in the ADNP-mutated cell lines. In parallel, disrupting microtubules by zinc or nocodazole intoxication mimicked ADNP mutation phenotypes and resulted in aberrant nuclear–cytoplasmic boundaries, which were rapidly corrected by NAP treatment. No NAP effects were noted on ADNP levels. Ketamine, used as a control, was ineffective, but both NAP and ketamine exhibited direct interactions with ADNP, as observed via in silico docking. (4) Conclusions: Through a microtubule-linked mechanism, NAP rapidly localized to the cytoplasmic and nuclear compartments, ameliorating mutated ADNP-related deficiencies. These novel findings explain previously published gene expression results and broaden NAP (davunetide) utilization in research and clinical development.

show abstract

STOP Codon Mutations at Sites of Natural Caspase Cleavage Are Implicated in Autism and Alzheimer’s Disease: The Case of ADNP

Cited by 14 publications

References 45 publications

A novel davunetide (NAPVSIPQQ to NAPVSIPQE) point mutation in activity‐dependent neuroprotective protein (ADNP) causes a mild developmental syndrome

A novel davunetide (NAPVSIPQQ to NAPVSIPQE) point mutation in activity‐dependent neuroprotective protein (ADNP) causes a mild developmental syndrome

Tracing the invisible mutant ADNP protein in Helsmoortel-Van der Aa syndrome patients

NAP (Davunetide): The Neuroprotective ADNP Drug Candidate Penetrates Cell Nuclei Explaining Pleiotropic Mechanisms

Contact Info

Product

Resources

About