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.

BACKGROUND: Helsmoortel-Van der Aa syndrome is a neurodevelopmental disorder in which patients present with autism, intellectual disability, and frequent extra-neurological features such as feeding and gastrointestinal problems, visual impairments, and cardiac abnormalities. All patients exhibit heterozygous de novo nonsense or frameshift stop mutations in the Activity-Dependent Neuroprotective Protein (ADNP) gene, accounting for a prevalence of 0.2% of all autism cases worldwide. ADNP fulfills an essential chromatin remodeling function during brain development. In this study, we investigated the cerebellum of a died 6-year-old male patient with the c.1676dupA/p.His559Glnfs*3 ADNP mutation.
RESULTS: The clinical presentation of the patient was representative of the Helsmoortel-Van der Aa syndrome. During his lifespan, he underwent two liver transplantations after which the child died because of multiple organ failure. An autopsy was performed, and various tissue samples were taken for further analysis. We performed a molecular characterization of the cerebellum, a brain region involved in motor coordination, known for its highest ADNP expression and compared it to an age-matched control subject. Importantly, epigenome-wide analysis of the ADNP cerebellum identified CpG methylation differences and expression of multiple pathways causing neurodevelopmental delay. Interestingly, transcription factor motif enrichment analysis of differentially methylated genes showed that the ADNP binding motif was the most significantly enriched. RNA sequencing of the autopsy brain further identified downregulation of the WNT signaling pathway and autophagy defects as possible causes of neurodevelopmental delay. Ultimately, label-free quantification mass spectrometry identified differentially expressed proteins involved in mitochondrial stress and sirtuin signaling pathways amongst others. Protein-protein interaction analysis further revealed a network including chromatin remodelers (ADNP, SMARCC2, HDAC2 and YY1), autophagy-related proteins (LAMP1, BECN1 and LC3) as well as a key histone deacetylating enzyme SIRT1, involved in mitochondrial energy metabolism. The protein interaction of ADNP with SIRT1 was further biochemically validated through the microtubule-end binding proteins EB1/EB3 by direct co-immunoprecipitation in mouse cerebellum, suggesting important mito-epigenetic crosstalk between chromatin remodeling and mitochondrial energy metabolism linked to autophagy stress responses. This is further supported by mitochondrial activity assays and stainings in patient-derived fibroblasts which suggest mitochondrial dysfunctions in the ADNP deficient human brain.
CONCLUSIONS: This study forms the baseline clinical and molecular characterization of an ADNP autopsy cerebellum, providing novel insights in the disease mechanisms of the Helsmoortel-Van der Aa syndrome. By combining multi-omic and biochemical approaches, we identified a novel SIRT1-EB1/EB3-ADNP protein complex which may contribute to autophagic flux alterations and impaired mitochondrial metabolism in the Helsmoortel-Van der Aa syndrome and holds promise as a new therapeutic target.

Activity-dependent neuroprotective protein (ADNP) is essential for neurodevelopment and de novo mutations in ADNP cause the ADNP syndrome. From brain pathologies point of view, tauopathy has been demonstrated at a young age, implying stunted development coupled with early/accelerated neurodegeneration. Given potential genotype-phenotype differences and age-dependency, we have assessed here a cohort of 15 individuals (1-27-year-old), using 1-3 longitudinal parent (caretaker) interview/s (Vineland 3 questionnaire) over several years. Our results indicated developmental delays, or even developmental arrests, coupled with potential spurts of development at early ages. Severe outcomes correlated with the truncating high impact mutation, in other words, the remaining mutated protein length as well as with the tested individual age, corroborating the hypothesis of developmental delays coupled with accelerated aging. A significant correlation was noted between mutated protein length and communication, implying a high impact of ADNP on communicative skills. Additionally, correlations were discovered between the two previously described epi-genetic signatures in ADNP emphasizing aberrant acquisition of motor behaviors, with truncating mutations around the nuclear localization signal being mostly affected. Finally, all individuals seem to acquire an age equivalent of 1-6 years, requiring disease modification treatment, such as the ADNP-derived drug candidate, NAP (davunetide), which has recently shown efficacy in women suffering from the neurodegenerative disorder, progressive supranuclear palsy (PSP), a late-onset tauopathy.

(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.

(1) Background: Activity-dependent neuroprotective protein (ADNP) is essential for neuronal structure and function. Multiple de novo pathological mutations in ADNP cause the autistic ADNP syndrome, and they have been further suggested to affect Alzheimer\'s disease progression in a somatic form. Here, we asked if different ADNP mutations produce specific neuronal-like phenotypes toward better understanding and personalized medicine. (2) Methods: We employed CRISPR/Cas9 genome editing in N1E-115 neuroblastoma cells to form neuron-like cell lines expressing ADNP mutant proteins conjugated to GFP. These new cell lines were characterized by quantitative morphology, immunocytochemistry and live cell imaging. (3) Results: Our novel cell lines, constitutively expressing GFP-ADNP p.Pro403 (p.Ser404* human orthologue) and GFP-ADNP p.Tyr718* (p.Tyr719* human orthologue), revealed new and distinct phenotypes. Increased neurite numbers (day 1, in culture) and increased neurite lengths upon differentiation (day 7, in culture) were linked with p.Pro403*. In contrast, p.Tyr718* decreased cell numbers (day 1). These discrete phenotypes were associated with an increased expression of both mutant proteins in the cytoplasm. Reduced nuclear/cytoplasmic boundaries were observed in the p.Tyr718* ADNP-mutant line, with this malformation being corrected by the ADNP-derived fragment drug candidate NAP. (4) Conclusions: Distinct impairments characterize different ADNP mutants and reveal aberrant cytoplasmic-nuclear crosstalk.

The 1102-amino-acid activity-dependent neuroprotective protein (ADNP) was originally discovered by expression cloning through the immunological identification of its 8-amino-acid sequence NAPVSIPQ (NAP), constituting the smallest active neuroprotective fragment of the protein. ADNP expression is essential for brain formation and cognitive function and is dysregulated in a variety of neurodegenerative diseases (Alzheimer\'s disease, Parkinson\'s disease, and schizophrenia). ADNP has been found to be mutated in autism, with an estimated prevalence of 0.17% (together, these autism cases now constitute ADNP syndrome cases) and our recent results showed somatic mutations in ADNP in Alzheimer\'s disease brains correlating with tauopathy. Furthermore, Adnp haploinsufficiency in mice causes an age-dependent reduction in cognitive functions coupled with tauopathy-like features such as an increased formation of tangle-like structures, defective axonal transport, and Tau hyperphosphorylation. ADNP and its derived peptides, NAP and SKIP, directly interact with end-binding proteins (EBs), which decorate plus-tips of the growing axonal cytoskeleton-microtubules (MTs). Functionally, NAP and SKIP are neuroprotective and stimulate axonal transport. Clinical trials have suggested the potential efficacy of NAP (davunetide, CP201) for improving cognitive performance/functional activities of daily living in amnestic mild cognitive impairment (aMCI) and schizophrenia patients, respectively. However, NAP was not found to be an effective treatment (though well-tolerated) for progressive supranuclear palsy (PSP) patients. Here we review the molecular mechanism of NAP activity on MTs and how NAP modulates the MT-Tau-EBs crosstalk. We offer a molecular explanation for the different protective potency of NAP in selected tauopathies (aMCI vs. PSP) expressing different ratios/pathologies of the alternatively spliced Tau mRNA and its resulting protein (aMCI expressing similar quantities of the dynamic Tau 3-MT binding isoform (Tau3R) and the Tau 4-MT binding isoform (Tau4R) and PSP enriched in Tau4R pathology). We reveal the direct effect of truncated ADNPs (resulting from de novo autism and newly discovered Alzheimer\'s disease-related somatic mutations) on MT dynamics. We show that the peptide SKIP affects MT dynamics and MT-Tau association. Since MT impairment is linked with neurodegenerative and neurodevelopmental conditions, the current study implicates a paucity/dysregulation of MT-interacting endogenous proteins, like ADNP, as a contributing mechanism and provides hope for NAP and SKIP as MT-modulating drug candidates.

The activity-dependent neuroprotective protein (ADNP) syndrome is an autistic-like disorder, instigated by mutations in ADNP. This syndrome is characterized by developmental delays, impairments in speech, motor function, abnormal hearing, and intellectual disabilities. In the Adnp-haploinsufficient mouse model, many of these impediments are evident, appearing in a sex-dependent manner. In zebra finch songbird (ZF; Taeniopygia guttata), an animal model used for song/language studies, ADNP mRNA most robust expression is observed in the cerebrum of young males, potentially corroborating with male ZF exclusive singing behavior and developed cerebral song system. Herein, we report a similar sex-dependent ADNP expression profile, with the highest expression in the cerebrum (qRT-PCR) in the brain of another songbird, the domesticated canary (Serinus canaria domestica). Additional analyses for the mRNA transcripts of the ADNP regulator, vasoactive intestinal peptide (VIP), sister gene ADNP2, and speech-related Forkhead box protein P2 (FoxP2) revealed multiple sex and brain region-dependent positive correlations between the genes (including ADNP). Parallel transcript expression patterns for FoxP2 and VIP were observed alongside specific FoxP2 increase in males compared with females as well as VIP/ADNP2 correlations. In spatial view, a sexually independent extensive form of expression was found for ADNP in the canary cerebrum (RNA in situ hybridization). The songbird cerebral mesopallium area stood out as a potentially high-expressing ADNP tissue, further strengthening the association of ADNP with sense integration and auditory memory formation, previously implicated in mouse and human.

Human infections with the food-borne zoonotic pathogen Campylobacter jejuni are progressively rising and constitute serious global public health and socioeconomic burdens. Hence, application of compounds with disease-alleviating properties are required to combat campylobacteriosis and post-infectious sequelae. In our preclinical intervention study applying an acute C. jejuni induced enterocolitis model, we surveyed the anti-pathogenic and immune-modulatory effects of the octapeptide NAP which is well-known for its neuroprotective and anti-inflammatory properties. Therefore, secondary abiotic IL-10-/- mice were perorally infected with C. jejuni and intraperitoneally treated with synthetic NAP from day 2 until day 5 post-infection. NAP-treatment did not affect gastrointestinal C. jejuni colonization but could alleviate clinical signs of infection that was accompanied by less pronounced apoptosis of colonic epithelial cells and enhancement of cell regenerative measures on day 6 post-infection. Moreover, NAP-treatment resulted in less distinct innate and adaptive pro-inflammatory immune responses that were not restricted to the intestinal tract but could also be observed in extra-intestinal and even systemic compartments. NAP-treatment further resulted in less frequent translocation of viable pathogens from the intestinal tract to extra-intestinal including systemic tissue sites. For the first time, we here provide evidence that NAP application constitutes a promising option to combat acute campylobacteriosis.

Activity-dependent neuroprotective protein (ADNP) and its protein snippet NAP (drug candidate CP201) regulate synapse formation and cognitive as well as behavioral functions, in part, through microtubule interaction. Given potential interactions between the microbiome and brain function, we now investigated the potential effects of the ADNP-deficient genotype, mimicking the ADNP syndrome on microbiota composition in the Adnp+/- mouse model. We have discovered a surprising robust sexually dichotomized Adnp genotype effect and correction by NAP (CP201) as follows. Most of the commensal bacterial microbiota tested were affected by the Adnp genotype and corrected by NAP treatment in a male sex-dependent manner. The following list includes all the bacterial groups tested-labeled in bold are male Adnp-genotype increased and corrected (decreased) by NAP. (1) Eubacteriaceae (EubV3), (2) Enterobacteriaceae (Entero), (3) Enterococcus genus (gEncocc), (4) Lactobacillus group (Lacto), (5) Bifidobacterium genus (BIF), (6) Bacteroides/Prevotella species (Bac), (7) Clostridium coccoides group (Coer), (8) Clostridium leptum group (Cluster IV, sgClep), and (9) Mouse intestinal Bacteroides (MIB). No similarities were found between males and females regarding sex- and genotype-dependent microbiota distributions. Furthermore, a female Adnp+/- genotype associated decrease (contrasting male increase) was observed in the Lactobacillus group (Lacto). Significant correlations were discovered between specific bacterial group loads and open-field behavior as well as social recognition behaviors. In summary, we discovered ADNP deficiency associated changes in commensal gut microbiota compositions, a sex-dependent biomarker for the ADNP syndrome and beyond. Strikingly, we discovered rapidly detected NAP (CP201) treatment-dependent biomarkers within the gut microbiota.

Focusing on microtubule heterogeneity and brain specificity allowed for initial discoveries of multiple tubulin isotypes four decades ago. Methods evolved from using radioactive labelling and single cell cultures to monoclonal antibodies recognizing discrete forms of tubulin in single neurons. With the advantage of molecular cloning and fluorescent protein tagging, essential components for microtubule dynamics/stability and function were identified, including activity-dependent neuroprotective protein, ADNP and its peptide snippet, NAP (drug candidate, davunetide/CP201). ADNP/NAP through the SxIP motif interact with microtubule end binding proteins EB1 and EB3 to increase microtubule dynamics, axonal transport and dendritic spine formation. Recent transcriptomic analysis of the young mouse brain at the single cell level enabled characterization of cell-type specific cytoskeleton related gene signatures (e.g., tubulin transcripts, microtubule-associated protein Tau, Mapt and microtubule end binding protein, EB3, Mapre3) at unprecedented detail. Here, we review these findings with a methodological perspective to highlight how cutting-edge techniques have allowed us to disentangle cytoskeleton dynamics in health and disease.