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

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

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

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

International audienceNAP (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

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

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

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

International audienceAutophagy 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 beta) 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

ADNP expression in the cerebellum is not subject to regulation by transgene expression in other brain areas.

<p>A] Western blot analysis. Expression of nuclear ADNP in the cerebellum of Tau-Tg mice (1) and non-Tg mice (2) is shown by gel electrophoresis and western blot analysis coupled to quantitative densitometry. Results show an increase in ADNP expression at 3 months of age in both transgenic and non-transgenic mice, followed by a decrease at 5.5 and 9 months of age (each experimental group included 4 mice, 3–4 representatives are shown). B] ADNP quantification in the cerebellum. The ADNP amount in each band was calculated as its percentage from the total amount of all bands, and was then divided by the correlated actin amount of the same sample. ADNP/actin amounts of each group were averaged [Tg: 1-month-old 0.43+0.078, 3-month- old 1.794+0.228, 5.5-month-old 1.009+0.08, 9-month-old 0.998+0.195. Non-Tg: 1-month-old 0.532+0.125, 3-month-old 1.798+0.264, 5.5-month-old 0.903+0.196, 9-month-old 0.406+0.166]. Four mice were used for each experimental point and gel electrophoresis was repeated twice, average results are shown. Statistical analysis is shown in the text.</p

Age-dependent changes in the expression of ADNP: Differences between Tau-Tg and control mice.

<p>A] Tau-Tg mice western blot analyses. Western blot analyses for ADNP were performed on 3-10.5-month-old-mice (3 or 4 replicates/age group, with 4–5 mice/group). One blot is shown, including extracts from 3-10.5-month-old-mice with actin as a control protein (each lane represents 1 mouse and 4 mice are shown per group). B] Age-dependent changes in the expression of ADNP at the protein level in the cerebral cortex: Differences between Tau-Tg and control mice. The ADNP/actin ratio in each immunoreactive band (in A) was calculated as the ADNP percentage from the total amount of all bands, and then divided by the correlating actin amount of the same sample. ADNP/actin ratios for each group were averaged (n = 5/group, average of 3–4 gel replicates). Quantitative densitometry is shown for four ages: 3, 5.5, 9 and 10.5 months (black bars, Tau-Tg; white bars, littermates not expressing the 4R mutated tau as outlined in the method section, *** <i>p</i><0.001, Tau-Tg vs. control mice, see results section). The 10.5-month point shows only the Tau-Tg ADNP expression. Additional experiments <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087383#pone.0087383-Gozes5" target="_blank">[54]</a> compared Tau-Tg and controls revealing no differences at the actin level (densitometry results of 8.4±3.4, vs. 8.3±2.6). In contrast, ADNP levels were significantly decreased in the Tau-Tg 5.5±0.9, vs. controls 11.2±1.35, <i>p</i><0.01 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087383#pone.0087383-Gozes5" target="_blank">[54]</a>. The insert is showing a western blot comparing ADNP expression in Tau-Tg (n = 4) and controls (n = 3), at the age of 3 months, each lane represents 1 mouse. C] Age-dependent changes in the expression of ADNP mRNA in the cerebral cortex: Differences between Tau-Tg and control mice. The ADNP mRNA amount in each sample was calculated using the correlated HPRT mRNA amount of the same sample. ADNP mRNA amounts for each group were averaged. [Tg: 1-month-old 9.422+<u>0.92</u>, 3-month-old 2.788±0.407, 5.5-month-old 1.242±0.314, 9-month-old 1.402±0.642. Non-Tg: 1-month-old 2.<u>78+0.28, 3</u>-month-old 1±0.357, 5.5-month-old 1.222±0.535, 9-month-old 2.18±0.27, n = 5/age group]. RQ  =  relative quantity (<a href="http://de-de.invitrogen.com/etc/medialib/en/filelibrary/Nucleic-Acid-Amplification-Expression-Profiling/PDFs.Par.83765.File.dat/relative-quant-ct.pdf" target="_blank">http://de-de.invitrogen.com/etc/medialib/en/filelibrary/Nucleic-Acid-Amplification-Expression-Profiling/PDFs.Par.83765.File.dat/relative-quant-ct.pdf</a>), (*** <i>p</i><0.01).</p