A multi-lab experimental assessment reveals that replicability can be improved by using empirical estimates of genotype-by-lab interaction.

The utility of mouse and rat studies critically depends on their replicability in other laboratories. A widely advocated approach to improving replicability is through the rigorous control of predefined animal or experimental conditions, known as standardization. However, this approach limits the generalizability of the findings to only to the standardized conditions and is a potential cause rather than solution to what has been called a replicability crisis. Alternative strategies include estimating the heterogeneity of effects across laboratories, either through designs that vary testing conditions, or by direct statistical analysis of laboratory variation. We previously evaluated our statistical approach for estimating the interlaboratory replicability of a single laboratory discovery. Those results, however, were from a well-coordinated, multi-lab phenotyping study and did not extend to the more realistic setting in which laboratories are operating independently of each other. Here, we sought to test our statistical approach as a realistic prospective experiment, in mice, using 152 results from 5 independent published studies deposited in the Mouse Phenome Database (MPD). In independent replication experiments at 3 laboratories, we found that 53 of the results were replicable, so the other 99 were considered non-replicable. Of the 99 non-replicable results, 59 were statistically significant (at 0.05) in their original single-lab analysis, putting the probability that a single-lab statistical discovery was made even though it is non-replicable, at 59.6%. We then introduced the dimensionless "Genotype-by-Laboratory" (GxL) factor-the ratio between the standard deviations of the GxL interaction and the standard deviation within groups. Using the GxL factor reduced the number of single-lab statistical discoveries and alongside reduced the probability of a non-replicable result to be discovered in the single lab to 12.1%. Such reduction naturally leads to reduced power to make replicable discoveries, but this reduction was small (from 87% to 66%), indicating the small price paid for the large improvement in replicability. Tools and data needed for the above GxL adjustment are publicly available at the MPD and will become increasingly useful as the range of assays and testing conditions in this resource increases.

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.

Novel ADNP Syndrome Mice Reveal Dramatic Sex-Specific Peripheral Gene Expression With Brain Synaptic and Tau Pathologies.

BACKGROUND: ADNP is essential for embryonic development. As such, de novo ADNP mutations lead to an intractable autism/intellectual disability syndrome requiring investigation. METHODS: Mimicking humans, CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 editing produced mice carrying heterozygous Adnp p.Tyr718∗ (Tyr), a paralog of the most common ADNP syndrome mutation. Phenotypic rescue was validated by treatment with the microtubule/autophagy-protective ADNP fragment NAPVSIPQ (NAP). RESULTS: RNA sequencing of spleens, representing a peripheral biomarker source, revealed Tyr-specific sex differences (e.g., cell cycle), accentuated in females (with significant effects on antigen processing and cellular senescence) and corrected by NAP. Differentially expressed, NAP-correctable transcripts, including the autophagy and microbiome resilience-linked FOXO3, were also deregulated in human patient-derived ADNP-mutated lymphoblastoid cells. There were also Tyr sex-specific microbiota signatures. Phenotypically, Tyr mice, similar to patients with ADNP syndrome, exhibited delayed development coupled with sex-dependent gait defects. Speech acquisition delays paralleled sex-specific mouse syntax abnormalities. Anatomically, dendritic spine densities/morphologies were decreased with NAP amelioration. These findings were replicated in the Adnp+/- mouse, including Foxo3 deregulation, required for dendritic spine formation. Grooming duration and nociception threshold (autistic traits) were significantly affected only in males. Early-onset tauopathy was accentuated in males (hippocampus and visual cortex), mimicking humans, and was paralleled by impaired visual evoked potentials and correction by acute NAP treatment. CONCLUSIONS: Tyr mice model ADNP syndrome pathology. The newly discovered ADNP/NAP target FOXO3 controls the autophagy initiator LC3 (microtubule-associated protein 1 light chain 3), with known ADNP binding to LC3 augmented by NAP, protecting against tauopathy. NAP amelioration attests to specificity, with potential for drug development targeting accessible biomarkers.

Therapeutic Potential of Vasoactive Intestinal Peptide and its Derivative Stearyl-Norleucine-VIP in Inflammation-Induced Osteolysis.

The common use of dental and orthopedic implants calls for special attention to the immune response leading to peri-prosthetic bone loss and implant failure. In addition to the well-established microbial etiology for oral implant failure, wear debris and in particular titanium (Ti) particles (TiP) in the implant vicinity are an important trigger of inflammation and activation of bone resorption around oral and orthopedic implants, presenting an unmet medical need. Here, we employed bacterial-derived lipopolysaccharides (LPS) to model infection and TiP to model aseptic inflammation and osteolysis. We assessed inflammation in vitro by measuring IL1ß, IL6 and TNFα mRNA expression in primary macrophages, osteoclastogenesis in RANKL-induced bone marrow derived pre-osteoclasts and osteolysis in vivo in a mouse calvarial model. We also assessed the trans-epithelial penetrability and safety of the tested compound in rats. Our results show that a lipophilic super-active derivative of vasoactive intestinal peptide (VIP), namely stearyl-norleucine-VIP (SNV) presented superior anti-inflammatory and anti-osteoclastogenic effects compared to VIP in vitro. In the bacterial infection model (LPS), SNV significantly reduced IL1ß expression, while VIP increased IL6 expression. In the aseptic models of osteolysis, SNV showed greater suppression of in vitro osteoclastogenesis than VIP, and significantly inhibited inflammation-induced osteolysis in vivo. We also observed that expression levels of the VIP receptor VPAC-2, but not that of VPAC-1, dramatically decreased during osteoclast differentiation. Importantly, SNV previously shown to have an increased stability compared to VIP, showed here significant trans-epithelial penetration and a clean toxicological profile, presenting a novel drug candidate that could be applied topically to counter both aseptic and infection-related bone destruction.

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.

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.

Age and Sex-Dependent ADNP Regulation of Muscle Gene Expression Is Correlated with Motor Behavior: Possible Feedback Mechanism with PACAP.

The activity-dependent neuroprotective protein (ADNP), a double-edged sword, sex-dependently regulates multiple genes and was previously associated with the control of early muscle development and aging. Here we aimed to decipher the involvement of ADNP in versatile muscle gene expression patterns in correlation with motor function throughout life. Using quantitative RT-PCR we showed that Adnp+/- heterozygous deficiency in mice resulted in aberrant gastrocnemius (GC) muscle, tongue and bladder gene expression, which was corrected by the Adnp snippet, drug candidate, NAP (CP201). A significant sexual dichotomy was discovered, coupled to muscle and age-specific gene regulation. As such, Adnp was shown to regulate myosin light chain (Myl) in the gastrocnemius (GC) muscle, the language acquisition gene forkhead box protein P2 (Foxp2) in the tongue and the pituitary-adenylate cyclase activating polypeptide (PACAP) receptor PAC1 mRNA (Adcyap1r1) in the bladder, with PACAP linked to bladder function. A tight age regulation was observed, coupled to an extensive correlation to muscle function (gait analysis), placing ADNP as a muscle-regulating gene/protein.

Immune-modulatory Properties of the Octapeptide NAP in Campylobacter jejuni Infected Mice Suffering from Acute Enterocolitis.

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.

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.

The autism/neuroprotection-linked ADNP/NAP regulate the excitatory glutamatergic synapse.

Activity-dependent neuroprotective protein (ADNP), essential for brain formation, was discovered as a leading de novo mutated gene causing the autism-like ADNP syndrome. This syndrome is phenotypically characterized by global developmental delays, intellectual disabilities, speech impediments, and motor dysfunctions. The Adnp haploinsufficient mouse mimics the human ADNP syndrome in terms of synapse density and gene expression patterns, as well as in developmental, motor, and cognitive abilities. Peripheral ADNP was also discovered as a biomarker for Alzheimer's disease and schizophrenia, with nasal administration of the ADNP snippet peptide NAP (enhancing endogenous ADNP activity) leading to partial cognitive and functional protection at the cellular, animal and clinical settings. Here, a novel formulation for effective delivery of NAP is provided with superior brain penetration capabilities. Also provided are methods for treating pertinent clinical implications such as autism, cognitive impairments, olfactory deficits, and muscle strength using the formulation in the Adnp haploinsufficient mouse. Results showed a dramatically specific increase in brain/body bioavailability with the new formulation, without breaching the blood brain barrier. Additional findings included improvements using daily intranasal treatments with NAP, at the behavioral and brain structural levels, diffusion tensor imaging (DTI), translatable to clinical practice. Significant effects on hippocampal and cerebral cortical expression of the presynaptic Slc17a7 gene encoding vesicular excitatory glutamate transporter 1 (VGLUT1) were observed at the RNA and immunohistochemical levels, explaining the DTI results. These findings tie for the first time a reduction in presynaptic glutamatergic synapses with the autism/Alzheimer's/schizophrenia-linked ADNP deficiency coupled with amelioration by NAP (CP201).

Activity-dependent neuroprotective protein (ADNP) is an alcohol-responsive gene and negative regulator of alcohol consumption in female mice.

Neuroadaptations in the brain reward system caused by excessive alcohol intake, lead to drinking escalation and alcohol use disorder phenotypes. Activity-dependent neuroprotective protein (ADNP) is crucial for brain development, and is implicated in neural plasticity in adulthood. Here, we discovered that alcohol exposure regulates Adnp expression in the mesolimbic system, and that Adnp keeps alcohol drinking in moderation, in a sex-dependent manner. Specifically, Sub-chronic alcohol treatment (2.5 g/kg/day for 7 days) increased Adnp mRNA levels in the dorsal hippocampus in both sexes, and in the nucleus accumbens of female mice, 24 h after the last alcohol injection. Long-term voluntary consumption of excessive alcohol quantities (~10-15 g/kg/24 h, 5 weeks) increased Adnp mRNA in the hippocampus of male mice immediately after an alcohol-drinking session, but the level returned to baseline after 24 h of withdrawal. In contrast, excessive alcohol consumption in females led to long-lasting reduction in hippocampal Adnp expression. We further tested the regulatory role of Adnp in alcohol consumption, using the Adnp haploinsufficient mouse model. We found that Adnp haploinsufficient female mice showed higher alcohol consumption and preference, compared to Adnp intact females, whereas no genotype difference was observed in males. Importantly, daily intranasal administration of the ADNP-snippet drug candidate NAP normalized alcohol consumption in Adnp haploinsufficient females. Finally, female Adnp haploinsufficient mice showed a sharp increase in alcohol intake after abstinence, suggesting that Adnp protects against relapse in females. The current data suggest that ADNP is a potential novel biomarker and negative regulator of alcohol-drinking behaviors.

Anti-inflammatory Effects of the Octapeptide NAP in Human Microbiota-Associated Mice Suffering from Subacute Ileitis.

The octapeptide NAP is well known for its neuroprotective properties. We here investigated whether NAP treatment could alleviate pro-inflammatory immune responses during experimental subacute ileitis. To address this, mice with a human gut microbiota were perorally infected with one cyst of Toxoplasma gondii (day 0) and subjected to intraperitoneal synthetic NAP treatment from day 1 until day 8 postinfection (p.i.). Whereas placebo (PLC) control animals displayed subacute ileitis at day 9 p.i., NAP-treated mice exhibited less pronounced pro-inflammatory immune responses as indicated by lower numbers of intestinal mucosal T and B lymphocytes and lower interferon (IFN)-γ concentrations in mesenteric lymph nodes. The NAP-induced anti-inflammatory effects were not restricted to the intestinal tract but could also be observed in extra-intestinal including systemic compartments, given that pro-inflammatory cytokines were lower in liver, kidney, and lung following NAP as compared to PLC application, whereas at day 9 p.i., colonic and serum interleukin (IL)-10 concentrations were higher in the former as compared to the latter. Remarkably, probiotic commensal bifidobacterial loads were higher in the ileal lumen of NAP as compared to PLC-treated mice with ileitis. Our findings thus further support that NAP might be regarded as future treatment option directed against intestinal inflammation.

The octapetide NAP alleviates intestinal and extra-intestinal anti-inflammatory sequelae of acute experimental colitis.

The octapeptide NAP has been shown to exert neuroprotective properties and reduce neuro-inflammatory responses. The aim of the present study was to investigate if NAP provides anti-inflammatory effects in acute murine colitis. To address this, C57BL/6 j mice were challenged with 3.5% dextran sulfate sodium from day 0 until day 6 to induce colitis, either treated intraperitoneally with NAP or placebo (NaCl 0.9%) from day 1 until day 6 post-induction (p.i.) and subjected to in depth macroscopic, microscopic and immunological evaluations. Whereas NAP application did not alleviate macroscopic (i.e. clinical) sequelae of colitis, lower numbers of apoptotic, but higher counts of proliferating/regenerating colonic epithelial cells could be observed in NAP as compared to placebo treated mice at day 7 p.i. Furthermore, lower numbers of adaptive immune cells such as T lymphocytes and regulatory T cells were abundant in the colonic mucosa and lamina propria upon NAP versus placebo treatment that were accompanied by less colonic secretion of pro-inflammatory mediators including IFN-γ and nitric oxide at day 7 p.i. In mesenteric lymph nodes, pro-inflammatory IFN-γ, TNF and IL-6 concentrations were increased in placebo, but not NAP treated mice at day 7 p.i., whereas interestingly, elevated anti-inflammatory IL-10 levels could be observed in NAP treated mice only. The assessed anti-inflammatory properties of NAP were not restricted to the intestinal tract, given that in extra-intestinal compartments such as the kidneys, IFN-γ levels increased in placebo, but not NAP treated mice upon colitis induction. NAP induced effects were accompanied by distinct changes in intestinal microbiota composition, given that colonic luminal loads of bifidobacteria, regarded as anti-inflammatory, "health-promoting" commensal species, were two orders of magnitude higher in NAP as compared to placebo treated mice and even naive controls. In conclusion, NAP alleviates intestinal and extra-intestinal pro-inflammatory sequelae of acute experimental colitis and may provide novel treatment options of intestinal inflammatory diseases in humans.

Risperidone and NAP protect cognition and normalize gene expression in a schizophrenia mouse model.

Mutated disrupted in schizophrenia 1 (DISC1), a microtubule regulating protein, leads to schizophrenia and other psychiatric illnesses. It is hypothesized that microtubule stabilization may provide neuroprotection in schizophrenia. The NAP (NAPVSIPQ) sequence of activity-dependent neuroprotective protein (ADNP) contains the SxIP motif, microtubule end binding (EB) protein target, which is critical for microtubule dynamics leading to synaptic plasticity and neuroprotection. Bioinformatics prediction for FDA approved drugs mimicking SxIP-like motif which displace NAP-EB binding identified Risperidone. Risperidone or NAP effectively ameliorated object recognition deficits in the mutated DISC1 mouse model. NAP but not Risperidone, reduced anxiety in the mutated mice. Doxycycline, which blocked the expression of the mutated DISC1, did not reverse the phenotype. Transcripts of Forkhead-BOX P2 (Foxp2), a gene regulating DISC1 and associated with human ability to acquire a spoken language, were increased in the hippocampus of the DISC1 mutated mice and were significantly lowered after treatment with NAP, Risperidone, or the combination of both. Thus, the combination of NAP and standard of care Risperidone in humans may protect against language disturbances associated with negative and cognitive impairments in schizophrenia.

New horizons in schizophrenia treatment: autophagy protection is coupled with behavioral improvements in a mouse model of schizophrenia.

Autophagy plays a key role in the pathophysiology of schizophrenia as manifested by a 40% decrease in BECN1/Beclin 1 mRNA in postmortem hippocampal tissues relative to controls. This decrease was coupled with the deregulation of the essential ADNP (activity-dependent neuroprotector homeobox), a binding partner of MAP1LC3B/LC3B (microtubule-associated protein 1 light chain 3 ß) another major constituent of autophagy. The drug candidate NAP (davunetide), a peptide fragment from ADNP, enhanced the ADNP-LC3B interaction. Parallel genetic studies have linked allelic variation in the gene encoding MAP6/STOP (microtubule-associated protein 6) to schizophrenia, along with altered MAP6/STOP protein expression in the schizophrenic brain and schizophrenic-like behaviors in Map6-deficient mice. In this study, for the first time, we reveal significant decreases in hippocampal Becn1 mRNA and reversal by NAP but not by the antipsychotic clozapine (CLZ) in Map6-deficient (Map6(+/-)) mice. Normalization of Becn1 expression by NAP was coupled with behavioral protection against hyperlocomotion and cognitive deficits measured in the object recognition test. CLZ reduced hyperlocomotion below control levels and did not significantly affect object recognition. The combination of CLZ and NAP resulted in normalized outcome behaviors. Phase II clinical studies have shown NAP-dependent augmentation of functional activities of daily living coupled with brain protection. The current studies provide a new mechanistic pathway and a novel avenue for drug development.

Novel tubulin and tau neuroprotective fragments sharing structural similarities with the drug candidate NAP (Davuentide).

NAP (NAPVSIPQ, davunetide) is a microtubule stabilizing peptide drug candidate. Here, we set out to identify NAP-like peptides that provide neuroprotection and reduce tau pathology. NAP-like peptides were derived using publically available search engines, which identified sequence homologies in the microtubule subunit tubulin and in the microtubule associated protein, tau. NATLSIHQ (NAT) and STPTAIPQ were derived from tubulin, and TAPVPMPD (TAP) was derived from tau. All peptides provided neuroprotection against the Alzheimer's disease (AD) toxin, the amyloid-ß 1-42 peptide, although NAT and TAP were much more potent than STPTAIPQ. NAT also protected astrocytes, while STPTAIPQ was active only at micromolar concentrations. Because NAT and TAP were much more potent than STPTAIPQ in neuroprotection, those peptides were also tested for inhibition of tau-like aggregation (the second protein hallmark pathology of AD). Both NAT and TAP inhibited tau-like aggregation, with NAT being active over a very broad concentration range. NAT also protected in vivo in a frontotemporal dementia transgenic mouse model (Tau-Tg), when tested at the age of ~10 months. Results showed significantly decreased levels of the NAP parent protein, activity-dependent neuroprotective protein in the cerebral cortex of the Tau-Tg which was increased back to normal levels by NAT treatment. This was coupled to protection of Brain-Body weight ratio in the compromised Tau-Tg. With AD being the major tauopathy and with tau taking part in frontotemporal dementia, novel NAP derivatives that reduce tauopathy and provide neuroprotection are of basic and clinical interest.

Novel marker for the onset of frontotemporal dementia: early increase in activity-dependent neuroprotective protein (ADNP) in the face of Tau mutation.

Tauopathy, a major pathology in Alzheimer's disease, is also found in ~50% of frontotemporal dementias (FTDs). Tau transcript, a product of a single gene, undergoes alternative splicing to yield 6 protein species, each with either 3 or 4 microtubule binding repeat domains (tau 3R or 4R, associated with dynamic and stable microtubules, respectively). While the healthy human brain shows a 1/1 ratio of tau 3R/4R, this ratio may be dramatically changed in the FTD brain. We have previously discovered that activity-dependent neuroprotective protein (ADNP) is essential for brain formation in the mouse, with ADNP+/- mice exhibiting tauopathy, age-driven neurodegeneration and behavioral deficits. Here, in transgenic mice overexpressing a mutated tau 4R species, in the cerebral cortex but not in the cerebellum, we showed significantly increased ADNP expression (~3-fold transcripts) in the cerebral cortex of young transgenic mice (~disease onset), but not in the cerebellum, as compared to control littermates. The transgene-age-related increased ADNP expression paralleled augmented dynamic tau 3R transcript level compared to control littermates. Blocking mutated tau 4R transgene expression resulted in normalization of ADNP and tau 3R expression. ADNP was previously shown to be a member of the SWItch/Sucrose NonFermentable (SWI/SNF) chromatin remodeling complex. Here, Brahma (Brm), a component of the SWI/SNF complex regulating alternative splicing, showed a similar developmental expression pattern to ADNP. Immunoprecipitations further suggested Brm-ADNP interaction coupled to ADNP - polypyrimidine tract-binding protein (PTB)-associated splicing factor (PSF)-binding, with PSF being a direct regulator of tau transcript splicing. It should be noted that although we have shown a correlation between levels of ADNP and tau isoform expression three months of age, we are not presenting evidence of a direct link between the two. Future research into ADNP/tau relations is warranted.

NAP (davunetide) modifies disease progression in a mouse model of severe neurodegeneration: protection against impairments in axonal transport.

NAP (davunetide) is a novel neuroprotective compound with mechanism of action that appears to involve microtubule (MT) stabilization and repair. To evaluate, for the first time, the impact of NAP on axonal transport in vivo and to translate it to neuroprotection in a severe neurodegeneration, the SOD1-G93A mouse model for amyotrophic lateral sclerosis (ALS) was used. Manganese-enhanced magnetic resonance imaging (MRI), estimating axonal transport rates, revealed a significant reduction of the anterograde axonal transport in the ALS mice compared to healthy control mice. Acute NAP treatment normalized axonal transport rates in these ALS mice. Tau hyperphosphorylation, associated with MT dysfunction and defective axonal transport, was discovered in the brains of the ALS mice and was significantly reduced by chronic NAP treatment. Furthermore, in healthy wild type (WT) mice, NAP reversed axonal transport disruption by colchicine, suggesting drug-dependent protection against axonal transport impairment through stabilization of the neuronal MT network. Histochemical analysis showed that chronic NAP treatment significantly protected spinal cord motor neurons against ALS-like pathology. Sequential MRI measurements, correlating brain structure with ALS disease progression, revealed a significant damage to the ventral tegmental area (VTA), indicative of impairments to the dopaminergic pathways relative to healthy controls. Chronic daily NAP treatment of the SOD1-G93A mice, initiated close to disease onset, delayed degeneration of the trigeminal, facial and hypoglossal motor nuclei as was significantly apparent at days 90-100 and further protected the VTA throughout life. Importantly, protection of the VTA was significantly correlated with longevity and overall, NAP treatment significantly prolonged life span in the ALS mice.

Protection against tauopathy by the drug candidates NAP (davunetide) and D-SAL: biochemical, cellular and behavioral aspects.

Activity-dependent neuroprotective protein (ADNP) is essential for brain formation and partial deficiency in ADNP results in cognitive deficits coupled with tauopathy and neuronal cell death. Our previous results indicated that a peptide snippet from ADNP, NAPVSIPQ (NAP, generic name, davunetide) can restore in part ADNP deficiencies. NAP interacts with tubulin and this interaction is displaced by the NAP related peptide that is derived from activity-dependent neurotrophic factor (ADNF), SALLRSIPA (SAL) and its all D-amino acid peptide derivative (D-SAL, also known as AL-309). Both NAP and D-SAL were shown to protect neurons against amyloid beta toxicity however the mechanism of protection is still under investigation. In addition, NAP protects against tau hyperphosphorylation associated with ADNP deficiency, in vivo. To investigate whether the mechanism of in vitro neuroprotection relates to the in vivo protection against tauopathy and to draw potential additional parallelism between NAP and D-SAL, we asked if: 1]NAP and D-SAL protect against amyloid beta related tau hyperphosphorylation in vitro; and 2] D-SAL protects against haploinsufficiency in ADNP, inhibiting tauopathy in vivo. Assessment of NAP and D-SAL neuroprotection in primary cortical neuro-glial cultures treated with amyloid beta showed that both peptides reduced toxin-related neuronal damage and protected against tau hyperphosphorylation. In vivo, chronic D-SAL administration protected against tau hyperphosphorylation associated with ADNP deficiency (ADNP+/- mice), showing for the first time protection against deficits in odor discrimination and in social recognition. These studies associate neuroprotection in vivo and in vitro and provide a broad base for future drug development based on NAP and D-SAL against multiple neurodegenerative conditions.

NAP (davunetide) enhances cognitive behavior in the STOP heterozygous mouse--a microtubule-deficient model of schizophrenia.

NAP (generic name, davunetide) is an active fragment of activity-dependent neuroprotective protein (ADNP). ADNP-/- embryos exhibit CNS dysgenesis and die in utero. ADNP+/- mice survive but demonstrate cognitive dysfunction coupled with microtubule pathology. NAP treatment ameliorates, in part, ADNP-associated dysfunctions. The microtubule, stable tubule-only polypeptide (STOP) knockout mice were shown to provide a reliable model for schizophrenia. Here, STOP-/- as well as STOP+/- showed schizophrenia-like symptoms (hyperactivity) that were ameliorated by chronic treatment with the antipsychotic drug, clozapine. Daily intranasal NAP treatment significantly decreased hyperactivity in the STOP+/- mice and protected visual memory.