Novel evolutionary-conserved role for the activity-dependent neuroprotective protein (ADNP) family that is important for erythropoiesis.

BACKGROUND: ADNP is vital for embryonic development. Is this function conserved for the homologous protein ADNP2? RESULTS: Down-regulation/silencing of ADNP or ADNP2 in zebrafish embryos or mouse erythroleukemia cells inhibited erythroid maturation, with ADNP directly associating with the ß-globin locus control region. CONCLUSION: ADNPs are novel molecular regulators of erythropoiesis. SIGNIFICANCE: New regulators of globin synthesis are suggested. Activity-dependent neuroprotective protein (ADNP) and its homologue ADNP2 belong to a homeodomain, the zinc finger-containing protein family. ADNP is essential for mouse embryonic brain formation. ADNP2 is associated with cell survival, but its role in embryogenesis has not been evaluated. Here, we describe the use of the zebrafish model to elucidate the developmental roles of ADNP and ADNP2. Although we expected brain defects, we were astonished to discover that the knockdown zebrafish embryos were actually lacking blood and suffered from defective hemoglobin production. Evolutionary conservation was established using mouse erythroleukemia (MEL) cells, a well studied erythropoiesis model, in which silencing of ADNP or ADNP2 produced similar results as in zebrafish. Exogenous RNA encoding ADNP/ADNP2 rescued the MEL cell undifferentiated state, demonstrating phenotype specificity. Brg1, an ADNP-interacting chromatin-remodeling protein involved in erythropoiesis through regulation of the globin locus, was shown here to interact also with ADNP2. Furthermore, chromatin immunoprecipitation revealed recruitment of ADNP, similar to Brg1, to the mouse ß-globin locus control region in MEL cells. This recruitment was apparently diminished upon dimethyl sulfoxide (DMSO)-induced erythrocyte differentiation compared with the nondifferentiated state. Importantly, exogenous RNA encoding ADNP/ADNP2 significantly increased ß-globin expression in MEL cells in the absence of any other differentiation factors. Taken together, our results reveal an ancestral role for the ADNP protein family in maturation and differentiation of the erythroid lineage, associated with direct regulation of ß-globin expression.

Thymic stromal lymphopoietin-stimulated CD4+ T cells induce senescence in advanced breast cancer.

Thymic Stromal Lymphopoietin (TSLP) plays a prominent role in inducing type 2 immune response, commonly associated with atopic diseases. TSLP-activated CD4+ T helper 2 cells block early carcinogenesis by inducing terminal differentiation in spontaneous breast and lung cancer models. However, the impact of TSLP induction on advanced cancer with altered cellular phenotypes is unclear. Using an established MMTV-PyMttg breast cancer cell line, we demonstrate that TSLP-stimulated CD4+ T cells possess an antitumor effect in advanced breast cancer. In contrast to early breast cancer suppression, the antitumor immunity mediated by TSLP-stimulated CD4+ T cells in advanced breast cancer is mediated by the induction of a senescent-like phenotype in cancer cells. Inflammatory CD4+ T cells drive breast cancer cells into senescence by releasing interferon-gamma and tumor necrosis factor-alpha, which directly bind to their receptors on cancer cells. Our findings reveal a novel mechanism of TSLP-activated CD4+ T cell immunity against advanced breast cancer, mediated by cellular senescence as a distinct effector mechanism for cancer immunotherapy.

CD4+ T helper 2 cells suppress breast cancer by inducing terminal differentiation.

Cancer immunology research is largely focused on the role of cytotoxic immune responses against advanced cancers. Herein, we demonstrate that CD4+ T helper (Th2) cells directly block spontaneous breast carcinogenesis by inducing the terminal differentiation of the cancer cells. Th2 cell immunity, stimulated by thymic stromal lymphopoietin, caused the epigenetic reprogramming of the tumor cells, activating mammary gland differentiation and suppressing epithelial-mesenchymal transition. Th2 polarization was required for this tumor antigen-specific immunity, which persisted in the absence of CD8+ T and B cells. Th2 cells directly blocked breast carcinogenesis by secreting IL-3, IL-5, and GM-CSF, which signaled to their common receptor expressed on breast tumor cells. Importantly, Th2 cell immunity permanently reverted high-grade breast tumors into low-grade, fibrocystic-like structures. Our findings reveal a critical role for CD4+ Th2 cells in immunity against breast cancer, which is mediated by terminal differentiation as a distinct effector mechanism for cancer immunoprevention and therapy.

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.

Blood-Borne Activity-Dependent Neuroprotective Protein (ADNP) is Correlated with Premorbid Intelligence, Clinical Stage, and Alzheimer's Disease Biomarkers.

Biomarkers for Alzheimer's disease (AD) are vital for disease detection in the clinical setting. Discovered in our laboratory, activity-dependent neuroprotective protein (ADNP) is essential for brain formation and linked to cognitive functions. Here, we revealed that blood borne expression of ADNP and its paralog ADNP2 is correlated with premorbid intelligence, AD pathology, and clinical stage. Age adjustment showed significant associations between: 1) higher premorbid intelligence and greater serum ADNP, and 2) greater cortical amyloid and lower ADNP and ADNP2 mRNAs. Significant increases in ADNP mRNA levels were observed in patients ranging from mild cognitive impairment (MCI) to AD dementia. ADNP2 transcripts showed high correlation with ADNP transcripts, especially in AD dementia lymphocytes. ADNP plasma/serum and lymphocyte mRNA levels discriminated well between cognitively normal elderly, MCI, and AD dementia participants. Measuring ADNP blood-borne levels could bring us a step closer to effectively screening and tracking AD.

ADNP/ADNP2 expression in oligodendrocytes: implication for myelin-related neurodevelopment.

Oligodendrocytes, the myelin-forming cells of the central nervous system, play important roles in brain development and maintenance. Activity-dependent neuroprotective protein (ADNP), an early marker essential for brain formation, interacts with microtubule end-binding proteins (EB1, EB2, and EB3). EB1 and EB3 are highly expressed in neurons (axons and dendritic spines, respectively) and EB1 enhancement of neurite outgrowth is attenuated by EB2. ADNP/EB presence in oligodendrocytes has not been studied so far. Here, we measured messenger RNA (mRNA) levels of ADNP and EB1-EB3 in rat brain oligodendrocytes during culture maturation and in rat brains during development (1, 35, and 75 days) in comparison with rat astrocytes, dorsal root ganglion (DRG) neurons, and the oligodendroglia cell lines (OLN-93 cell line, not expressing the microtubule-associated protein (MAP) tau, and OLN-93 cells stably transfected to express various forms of tau). Results showed that all transcripts studied were expressed in oligodendrocytes. ADNP and EB2 mRNA transcript content peaked at the time of oligodendrocyte maturation (5 days in vitro) and was highest in newborn rat brains compared with mature brains. ADNP2 (the only family member of ADNP), and EB1, although expressed in lower quantities, essentially paralleled ADNP and EB2 expression patterns, respectively. EB3 mRNA, peaking upon oligodendrocyte maturation, showed an apparent second peak of expression (10 days in vitro) and increased in the mature rat brain compared with the newborn brain. DRG cells expressed the highest levels of EB3, when compared with oligodendrocyte precursors and with astrocytes but not when compared with mature oligodendrocytes. Mature oligodendrocytes and oligodendrocyte precursors expressed ~30-40-fold more EB2 vs. EB3, and ~4-7-fold vs. ADNP. DRGs expressed ~5-fold more EB2 vs. EB3 and astrocytes showed an in-between (~20-fold) ratio. Only DRGs expressed similar EB1 and EB3 transcript levels, contrasting with oligodendrocyte and astrocytes (~10-30-fold more EB1). Astrocytes expressed more ADNP than DRGs and oligodendrocyte precursor cells (~2-fold) but not compared with mature oligodendrocytes. EB1 and EB3 were previously found to be associated with tau. Immortalized oligodendrocytes showed an intermediate phenotype of mRNA expression compared with oligodendrocyte precursor cells and mature oligodendrocytes with tau transfection reducing overall ADNP and EB expression. In summary, ADNPs and EBs are highly expressed in oligodendrocytes suggesting an impact on myelin formation in health and disease.

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.