Activity-dependent Neuroprotective Protein Syndrome Research Articles

Transient peripheral blood transcriptomic response to ketamine treatment in children with ADNP syndrome

Activity-dependent neuroprotective protein (ADNP) syndrome is a rare neurodevelopmental disorder resulting in intellectual disability, developmental delay and autism spectrum disorder (ASD) and is due to mutations in the ADNP gene. Ketamine treatment has emerged as a promising therapeutic option for ADNP syndrome, showing safety and apparent behavioral improvements in a first open label study. However, the molecular perturbations induced by ketamine remain poorly understood. Here, we investigated the longitudinal effect of ketamine on the blood transcriptome of 10 individuals with ADNP syndrome. Transcriptomic profiling was performed before and at multiple time points after a single low-dose intravenous ketamine infusion (0.5 mg/kg). We show that ketamine triggers immediate and profound gene expression alterations, with specific enrichment of monocyte-related expression patterns. These acute alterations encompass diverse signaling pathways and co-expression networks, implicating upregulation of immune and inflammatory-related processes and down-regulation of RNA processing mechanisms and metabolism. Notably, these changes exhibit a transient nature, returning to baseline levels 24 hours to 1 week after treatment. These findings enhance our understanding of ketamine’s molecular effects and lay the groundwork for further research elucidating its specific cellular and molecular targets. Moreover, they contribute to the development of therapeutic strategies for ADNP syndrome and potentially, ASD more broadly.

Moderate Physical Activity Increases the Expression of ADNP in Rat Brain.

Activity-dependent neuroprotective protein (ADNP) is a neuroprotective protein essential for embryonic development, proper brain development, and neuronal plasticity. Its mutation causes the autism-like ADNP syndrome (also called the Helsmoortel-Van der Aa syndrome), characterized by neural developmental disorders and motor dysfunctions. Similar to the ADNP syndrome, the ADNP haploinsufficient mouse shows low synapse density, leading to motor and cognitive ability delays. Moderate physical activity (PA) has several neuroprotective and cognitive benefits, promoting neuronal survival, differentiation, neurogenesis, and plasticity. Until now, no study has investigated the effect of moderate exercise on ADNP expression and distribution in the rat brain. The aim of the current investigation was to study the effects of moderate exercise on the ADNP expression and neuronal activation measured by the microtubule protein β-Tubulin III. In pursuit of this objective, twenty-four rats were selected and evenly distributed into two categories: sedentary control rats and rats exposed to moderate physical activity on a treadmill over a span of 12 weeks. Our results showed that moderate PA increases the expression of ADNP and β-Tubulin III in the dentate gyrus (DG) hippocampal region and cerebellum. Moreover, we found a co-localization of ADNP and β-Tubulin III in both DG and cerebellum, suggesting a direct association of ADNP with adult neuronal activation induced by moderate PA.

Longitudinal Genotype-Phenotype (Vineland Questionnaire) Characterization of 15 ADNP Syndrome Cases Highlights Mutated Protein Length and Structural Characteristics Correlation with Communicative Abilities Accentuated in Males.

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 1-6years, 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.

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

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.

The cytoplasmic localization of ADNP through 14-3-3 promotes sex-dependent neuronal morphogenesis, cortical connectivity, and calcium signaling.

Defective neuritogenesis is a contributing pathogenic mechanism underlying a variety of neurodevelopmental disorders. Single gene mutations in activity-dependent neuroprotective protein (ADNP) are the most frequent among autism spectrum disorders (ASDs) leading to the ADNP syndrome. Previous studies showed that during neuritogenesis, Adnp localizes to the cytoplasm/neurites, and Adnp knockdown inhibits neuritogenesis in culture. Here, we hypothesized that Adnp is localized in the cytoplasm during neurite formation and that this process is mediated by 14-3-3. Indeed, applying the 14-3-3 inhibitor, difopein, blocked Adnp cytoplasmic localization. Furthermore, co-immunoprecipitations showed that Adnp bound 14-3-3 proteins and proteomic analysis identified several potential phosphorylation-dependent Adnp/14-3-3 binding sites. We further discovered that knockdown of Adnp using in utero electroporation of mouse layer 2/3 pyramidal neurons in the somatosensory cortex led to previously unreported changes in neurite formation beginning at P0. Defects were sustained throughout development, the most notable included increased basal dendrite number and axon length. Paralleling the observed morphological aberrations, ex vivo calcium imaging revealed that Adnp deficient neurons had greater and more frequent spontaneous calcium influx in female mice. GRAPHIC, a novel synaptic tracing technology substantiated this finding, revealing increased interhemispheric connectivity between female Adnp deficient layer 2/3 pyramidal neurons. We conclude that Adnp is localized to the cytoplasm by 14-3-3 proteins, where it regulates neurite formation, maturation, and functional cortical connectivity significantly building on our current understanding of Adnp function and the etiology of ADNP syndrome.

Distinct Impairments Characterizing Different ADNP Mutants Reveal Aberrant Cytoplasmic-Nuclear Crosstalk.

(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.

An open-label study evaluating the safety, behavioral, and electrophysiological outcomes of low-dose ketamine in children with ADNP syndrome.

SummaryActivity-dependent neuroprotective protein (ADNP) syndrome is a rare genetic condition associated with intellectual disability and autism spectrum disorder. Preclinical evidence suggests that low-dose ketamine may induce expression of ADNP and that neuroprotective effects of ketamine may be mediated by ADNP. The goal of the proposed research was to evaluate the safety, tolerability, and behavioral outcomes of low-dose ketamine in children with ADNP syndrome. We also sought to explore the feasibility of using electrophysiological markers of auditory steady-state response and computerized eye tracking to assess biomarker sensitivity to treatment. This study utilized a single-dose (0.5 mg/kg), open-label design, with ketamine infused intravenously over 40 min. Ten children with ADNP syndrome ages 6 to 12 years were enrolled. Ketamine was generally well tolerated, and there were no serious adverse events. The most common adverse events were elation/silliness (50%), fatigue (40%), and increased aggression (40%). Using parent-report instruments to assess treatment effects, ketamine was associated with nominally significant improvement in a wide array of domains, including social behavior, attention deficit and hyperactivity, restricted and repetitive behaviors, and sensory sensitivities, a week after administration. Results derived from clinician-rated assessments aligned with findings from the parent reports. Overall, nominal improvement was evident based on the Clinical Global Impressions - Improvement scale, in addition to clinician-based scales reflecting key domains of social communication, attention deficit and hyperactivity, restricted and repetitive behaviors, speech, thinking, and learning, activities of daily living, and sensory sensitivities. Results also highlight the potential utility of electrophysiological measurement of auditory steady-state response and eye-tracking to index change with ketamine treatment. Findings are intended to be hypothesis generating and provide preliminary support for the safety and efficacy of ketamine in ADNP syndrome in addition to identifying useful endpoints for a ketamine clinical development program. However, results must be interpreted with caution given limitations of this study, most importantly the small sample size and absence of a placebo-control group.

Vineland Adaptive Behavior Scale in a Cohort of Four ADNP Syndrome Patients Implicates Age-Dependent Developmental Delays with Increased Impact of Activities of Daily Living.

Activity-dependent neuroprotective protein (ADNP) is one of the lead genes in autism spectrum disorder/intellectual disability. Heterozygous, de novo ADNP mutations cause the ADNP syndrome. Here, to evaluate natural history of the syndrome, mothers of two ADNP syndrome boys aged 6 and a half and two adults aged 27years (man and woman) were subjected to Vineland III questionnaire assessing adaptive behavior. The boys were assessed again about 2years after the first measurements. The skill measures, presented as standard scores (SS) included domains of communication, daily living, socialization, motor skills and a sum of adaptive behavior composite. The age equivalent (AE) and growth scale values (GSV) encompassing 11 subdomains assess the age level at which the subject's raw score is found at a norm sample median and the individual temporal progression, respectively. The norm referenced standard scores age-matched, mean 100 ± 15 of the two children showed the lowest outcome in communication (SS: 20-30). Daily living skills presented SS of 50-60, with a possible potential loss of some activities as the child ages, especially in interpersonal relationships with people outside of the immediate family (boy A). In contrast, in socialization, both children were at the SS of 38, with some positive increase to SS of ~ 45 (interpersonal relations with family members and coping skills, depending on the particular individual), 2years later. Interestingly, there was an apparent large difference in motor skills (gross and fine) at the young age, with subject B showing a relatively higher level of skills (SS: 70), decreasing to subject A level (SS: 40) 2years later. Together, the adaptive behavior composite suggested a level of SS: 39-48 with B showing a potential increase (SS: 41-44) and A, a substantial decrease (SS: 48-39), suggesting a strong impact of daily living skills. Adults were at SS: 20, which is the lowest possible score. AE showed minor improvements for subject A and B, with all AE values being below 3years. GSVs for subject A showed some improvement with age, especially in interpersonal, play and leisure, and gross motor subdomains. GSV for subject B showed minor improvements in the various subdomains. Notably, all subjects showed a percentile rank < 1 compared with age-matched norms except for subject B as to motor domain (2nd percentile) at the age of 6years. In summary, the results, especially comparing SS and AEs between childhood and adulthood, implied a continuous deterioration of activities compared to the general population, encompassing a slower developmental process coupled to possible neurodegeneration, strongly supporting a great need for disease modifying medicinal procedures.

All in the Family: Living With ADNP Syndrome.

The overarching purpose of this research was to examine the experiences of 1 family living with a child with Helsmoortel-Van Der Aa syndrome or activity-dependent neuroprotective protein (ADNP) syndrome. A retrospective qualitative design was used for this study. Two primary caregivers for a 5-year-old child with ADNP syndrome completed background questionnaires to provide context for semistructured interviews. Each caregiver completed 2 interviews, approximately 2 months apart. Field notes, member checks, and triangulation were used to enhance the credibility of the study. This article summarizes the theme "All in the Family." Having a child with ADNP syndrome affected all aspects of family life. Participants revealed that family dynamics were shaped by experiences stemming from their living arrangements and caregiving responsibilities. Findings from this research highlighted the need for increased support for families faced with ADNP syndrome, as well as the role clinical nurse specialists can play in the lives of caregivers faced with such a rare diagnosis. Furthermore, given the paucity of ADNP syndrome information, the need for more research is warranted.

Sex and the Brain: Novel ADNP Syndrome Mice Are Protected by NAP

In the groundbreaking work by Karmon et al. (1) in the current issue of Biological Psychiatry, the authors developed a novel mouse model of activity-dependent neuroprotective protein (ADNP) syndrome. ADNP syndrome, also known as Helsmoortel–Van Der Aa syndrome, is a rare complex condition diagnosed in children exhibiting delayed development, autism spectrum disorder (ASD) traits, and intellectual disability. ADNP mutations are not limited to ASD; thus, the Gozes group discovered ADNP somatic mutations paralleling Alzheimer’s disease (AD) tauopathy (2), with ADNP playing pivotal roles in maintaining the DNA/chromatin structure (3) and function as well as regulating microtubule vitality (4).

SH3- and actin-binding domains connect ADNP and SHANK3, revealing a fundamental shared mechanism underlying autism.

De novo heterozygous mutations in activity-dependent neuroprotective protein (ADNP) cause autistic ADNP syndrome. ADNP mutations impair microtubule (MT) function, essential for synaptic activity. The ADNP MT-associating fragment NAPVSIPQ (called NAP) contains an MT end-binding protein interacting domain, SxIP (mimicking the active-peptide, SKIP). We hypothesized that not all ADNP mutations are similarly deleterious and that the NAPV portion of NAPVSIPQ is biologically active. Using the eukaryotic linear motif (ELM) resource, we identified a Src homology 3 (SH3) domain-ligand association site in NAP responsible for controlling signaling pathways regulating the cytoskeleton, namely NAPVSIP. Altogether, we mapped multiple SH3-binding sites in ADNP. Comparisons of the effects of ADNP mutations p.Glu830synfs*83, p.Lys408Valfs*31, p.Ser404* on MT dynamics and Tau interactions (live-cell fluorescence-microscopy) suggested spared toxic function in p.Lys408Valfs*31, with a regained SH3-binding motif due to the frameshift insertion. Site-directed-mutagenesis, abolishing the p.Lys408Valfs*31 SH3-binding motif, produced MT toxicity. NAP normalized MT activities in the face of all ADNP mutations, although, SKIP, missing the SH3-binding motif, showed reduced efficacy in terms of MT-Tau interactions, as compared with NAP. Lastly, SH3 and multiple ankyrin repeat domains protein 3 (SHANK3), a major autism gene product, interact with the cytoskeleton through an actin-binding motif to modify behavior. Similarly, ELM analysis identified an actin-binding site on ADNP, suggesting direct SH3 and indirect SHANK3/ADNP associations. Actin co-immunoprecipitations from mouse brain extracts showed NAP-mediated normalization of Shank3-Adnp-actin interactions. Furthermore, NAP treatment ameliorated aberrant behavior in mice homozygous for the Shank3 ASD-linked InsG3680 mutation, revealing a fundamental shared mechanism between ADNP and SHANK3.

Proximity labeling identifies a repertoire of site-specific R-loop modulators

R-loops are three-stranded nucleic acid structures that accumulate on chromatin in neurological diseases and cancers and contribute to genome instability. Using a proximity-dependent labeling system, we identified distinct classes of proteins that regulate R-loops in vivo through different mechanisms. We show that ATRX suppresses R-loops by interacting with RNAs and preventing R-loop formation. Our proteomics screen also discovered an unexpected enrichment for proteins containing zinc fingers and homeodomains. One of the most consistently enriched proteins was activity-dependent neuroprotective protein (ADNP), which is frequently mutated in ASD and causal in ADNP syndrome. We find that ADNP resolves R-loops in vitro and that it is necessary to suppress R-loops in vivo at its genomic targets. Furthermore, deletion of the ADNP homeodomain severely diminishes R-loop resolution activity in vitro, results in R-loop accumulation at ADNP targets, and compromises neuronal differentiation. Notably, patient-derived human induced pluripotent stem cells that contain an ADNP syndrome-causing mutation exhibit R-loop and CTCF accumulation at ADNP targets. Our findings point to a specific role for ADNP-mediated R-loop resolution in physiological and pathological neuronal function and, more broadly, to a role for zinc finger and homeodomain proteins in R-loop regulation, with important implications for developmental disorders and cancers.

Introducing ADNP and SIRT1 as new partners regulating microtubules and histone methylation.

Activity-dependent neuroprotective protein (ADNP) is essential for brain formation and function. As such, de novo mutations in ADNP lead to the autistic ADNP syndrome and somatic ADNP mutations may drive Alzheimer's disease (AD) tauopathy. Sirtuin 1 (SIRT1) is positively associated with aging, the major risk for AD. Here, we revealed two key interaction sites for ADNP and SIRT1. One, at the microtubule end-binding protein (EB1 and EB3) Tau level, with EB1/EB3 serving as amplifiers for microtubule dynamics, synapse formation, axonal transport, and protection against tauopathy. Two, on the DNA/chromatin site, with yin yang 1, histone deacetylase 2, and ADNP, sharing a DNA binding motif and regulating SIRT1, ADNP, and EB1 (MAPRE1). This interaction was linked to sex- and age-dependent altered histone modification, associated with ADNP/SIRT1/WD repeat-containing protein 5, which mediates the assembly of histone modification complexes. Single-cell RNA and protein expression analyses as well as gene expression correlations placed SIRT1-ADNP and either MAPRE1 (EB1), MAPRE3 (EB3), or both in the same mouse and human cell; however, while MAPRE1 seemed to be similarly regulated to ADNP and SIRT1, MAPRE3 seemed to deviate. Finally, we demonstrated an extremely tight correlation for the gene transcripts described above, including related gene products. This correlation was specifically abolished in affected postmortem AD and Parkinson's disease brain select areas compared to matched controls, while being maintained in blood samples. Thus, we identified an ADNP-SIRT1 complex that may serve as a new target for the understanding of brain degeneration.

Drug Repurposing Patent Applications October-December 2020.

Drug Repurposing Patent Applications October-December 2020.

Activity-dependent neuroprotective protein (ADNP)-end-binding protein (EB) interactions regulate microtubule dynamics toward protection against tauopathy.

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 ADNP Syndrome and CP201 (NAP) Potential and Hope.

Activity-dependent neuroprotective protein (ADNP) syndrome, also known as Helsmoortel-Van Der Aa syndrome, is a rare condition, which is diagnosed in children exhibiting signs of autism. Specifically, the disease is suspected when a child is suffering from developmental delay and/or intellectual disability. The syndrome occurs when one of the two copies of the ADNP gene carries a pathogenic sequence variant, mostly a de novo mutation resulting in loss of normal functions. Original data showed that Adnp+/− mice suffer from learning and memory deficiencies, muscle weakness, and communication problems. Further studies showed that the ADNP microtubule-interacting fragment NAP (called here CP201) resolves, in part, Adnp deficiencies and protects against ADNP pathogenic sequence variant abnormalities. With a clean toxicology and positive human adult experience, CP201 is planned for future clinical trials in the ADNP syndrome.

Single Cell ADNP Predictive of Human Muscle Disorders: Mouse Knockdown Results in Muscle Wasting.

Activity-dependent neuroprotective protein (ADNP) mutations are linked with cognitive dysfunctions characterizing the autistic-like ADNP syndrome patients, who also suffer from delayed motor maturation. We thus hypothesized that ADNP is deregulated in versatile myopathies and that local ADNP muscle deficiency results in myopathy, treatable by the ADNP fragment NAP. Here, single-cell transcriptomics identified ADNP as a major constituent of the developing human muscle. ADNP transcript concentrations further predicted multiple human muscle diseases, with concentrations negatively correlated with the ADNP target interacting protein, microtubule end protein 1 (EB1). Reverting back to modeling at the single-cell level of the male mouse transcriptome, Adnp mRNA concentrations age-dependently correlated with motor disease as well as with sexual maturation gene transcripts, while Adnp expressing limb muscle cells significantly decreased with aging. Mouse Adnp heterozygous deficiency exhibited muscle microtubule reduction and myosin light chain (Myl2) deregulation coupled with motor dysfunction. CRISPR knockdown of adult gastrocnemius muscle Adnp in a Cas9 mouse resulted in treadmill (male) and gait (female) dysfunctions that were specifically ameliorated by treatment with the ADNP snippet, microtubule interacting, Myl2—regulating, NAP (CP201). Taken together, our studies provide new hope for personalized diagnosis/therapeutics in versatile myopathies.

Tauopathy in the young autistic brain: novel biomarker and therapeutic target

Given our recent discovery of somatic mutations in autism spectrum disorder (ASD)/intellectual disability (ID) genes in postmortem aged Alzheimer’s disease brains correlating with increasing tauopathy, it is important to decipher if tauopathy is underlying brain imaging results of atrophy in ASD/ID children. We concentrated on activity-dependent neuroprotective protein (ADNP), a prevalent autism gene. The unique availability of multiple postmortem brain sections of a 7-year-old male, heterozygous for ADNP de novo mutation c.2244Adup/p.His559Glnfs*3 allowed exploration of tauopathy, reflecting on a general unexplored mechanism. The tested subject exhibited autism, fine motor delays, severe intellectual disability and seizures. The patient died after multiple organ failure following liver transplantation. To compare to other ADNP syndrome mutations, immortalized lymphoblastoid cell lines from three different patients (including ADNP p.Arg216*, p.Lys408Valfs*31, and p.Tyr719* heterozygous dominant mutations) and a control were subjected to RNA-seq. Immunohistochemistry, high-throughput gene expression profiles in numerous postmortem tissues followed. Comparisons to a control brain and to extensive datasets were used. Live cell imaging investigated Tau-microtubule interaction, protecting against tauopathy. Extensive child brain tauopathy paralleled by multiple gene expression changes was discovered. Tauopathy was explained by direct mutation effects on Tau-microtubule interaction and correction by the ADNP active snippet NAP. Significant pathway changes (empirical P value < 0.05) included over 100 genes encompassing neuroactive ligand–receptor and cytokine–cytokine receptor interaction, MAPK and calcium signaling, axon guidance and Wnt signaling pathways. Changes were also seen in steroid biosynthesis genes, suggesting sex differences. Selecting the most affected genes by the ADNP mutations for gene expression analysis, in multiple postmortem tissues, identified Tau (MAPT)-gene-related expression changes compared with extensive normal gene expression (RNA-seq) databases. ADNP showed relatively reduced expression in the ADNP syndrome cerebellum, which was also observed for 25 additional genes (representing >50% of the tested genes), including NLGN1, NLGN2, PAX6, SMARCA4, and SNAP25, converging on nervous system development and tauopathy. NAP provided protection against mutated ADNP disrupted Tau-microtubule association. In conclusion, tauopathy may explain brain-imaging findings in ADNP syndrome children and may provide a new direction for the development of tauopathy protecting drug candidates like NAP in ASD/ID.

ADNP promotes neural differentiation by modulating Wnt/\u03b2-catenin signaling

ADNP (Activity Dependent Neuroprotective Protein) is a neuroprotective protein whose aberrant expression has been frequently linked to neural developmental disorders, including the Helsmoortel-Van der Aa syndrome (also called the ADNP syndrome). However, its role in neural development and pathology remains unclear. Here, we show that ADNP is required for neural induction and differentiation by enhancing Wnt signaling. Mechanistically, ADNP functions to stabilize β-Catenin through binding to its armadillo domain which prevents its association with key components of the degradation complex: Axin and APC. Loss of ADNP promotes the formation of the degradation complex and β-Catenin degradation via ubiquitin-proteasome pathway, resulting in down-regulation of key neuroectoderm developmental genes. In addition, adnp gene disruption in zebrafish leads to defective neurogenesis and reduced Wnt signaling. Our work provides important insights into the role of ADNP in neural development and the pathology of the Helsmoortel-Van der Aa syndrome caused by ADNP gene mutation.

Microbiota changes associated with ADNP deficiencies: rapid indicators for NAP (CP201) treatment of the ADNP syndrome and beyond

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.