The Amyloid Precursor Protein C-Terminal Domain Alters CA1 Neuron Firing, Modifying Hippocampus Oscillations and Impairing Spatial Memory Encoding.

There is a growing consensus that Alzheimer's disease (AD) involves failure of the homeostatic machinery, which underlies the firing stability of neural circuits. What are the culprits leading to neuron firing instability? The amyloid precursor protein (APP) is central to AD pathogenesis, and we recently showed that its intracellular domain (AICD) could modify synaptic signal integration. We now hypothesize that AICD modifies neuron firing activity, thus contributing to the disruption of memory processes. Using cellular, electrophysiological, and behavioral techniques, we show that pathological AICD levels weaken CA1 neuron firing activity through a gene-transcription-dependent mechanism. Furthermore, increased AICD production in hippocampal neurons modifies oscillatory activity, specifically in the γ-frequency range, and disrupts spatial memory task. Collectively, our data suggest that AICD pathological levels, observed in AD mouse models and in human patients, might contribute to progressive neuron homeostatic failure, driving the shift from normal aging to AD.

Fmr1-Deficiency Impacts Body Composition, Skeleton, and Bone Microstructure in a Mouse Model of Fragile X Syndrome.

Fragile X syndrome (FXS) is a neurodevelopmental disorder associated with intellectual disability, hyperactivity, and autism. FXS is due to the silencing of the X-linked FMR1 gene. Murine models of FXS, knock-out (KO) for the murine homolog Fmr1, have been generated, exhibiting CNS-related behavioral, and neuronal anomalies reminiscent of the human phenotypes. As a reflection of the almost ubiquitous expression of the FMR1 gene, FXS is also accompanied by physical abnormalities. This suggests that the FMR1-deficiency could impact skeletal ontogenesis. In the present study, we highlight that Fmr1-KO mice display changes in body composition with an increase in body weight, likely due to both increase of skeleton length and muscular mass along with reduced visceral adiposity. We also show that, while Fmr1-deficiency has no overt impact on cortical bone mineral density (BMD), cortical thickness was increased, and cortical eccentricity was decreased in the femurs from Fmr1-KO mice as compared to controls. Also, trabecular pore volume was reduced and trabecular thickness distribution was shifted toward higher ranges in Fmr1-KO femurs. Finally, we show that Fmr1-KO mice display increased physical activity. Although the precise molecular signaling mechanism that produces these skeletal and bone microstructure changes remains to be determined, our study warrants further investigation on the impact of FMR1-deficiency on whole-body composition, as well as skeletal and bone architecture.

Mesopontine cholinergic inputs to midbrain dopamine neurons drive stress-induced depressive-like behaviors.

Stressful life events are primary environmental factors that markedly contribute to depression by triggering brain cellular maladaptations. Dysregulation of ventral tegmental area (VTA) dopamine neurons has been causally linked to the appearance of social withdrawal and anhedonia, two classical manifestations of depression. However, the relevant inputs that shape these dopamine signals remain largely unknown. We demonstrate that chronic social defeat (CSD) stress, a preclinical paradigm of depression, causes marked hyperactivity of laterodorsal tegmentum (LDTg) excitatory neurons that project to the VTA. Selective chemogenetic-mediated inhibition of cholinergic LDTg neurons prevent CSD-induced VTA DA neurons dysregulation and depressive-like behaviors. Pro-depressant outcomes are replicated by pairing activation of LDTg cholinergic terminals in the VTA with a moderate stress. Prevention of CSD outcomes are recapitulated by blocking corticotropin-releasing factor receptor 1 within the LDTg. These data uncover a neuro-circuitry of depressive-like disorders and demonstrate that stress, via a neuroendocrine signal, profoundly dysregulates the LDTg.

Identification of an acute functional cross-talk between amyloid-ß and glucocorticoid receptors at hippocampal excitatory synapses.

Amyloid-ß is a peptide released by synapses in physiological conditions and its pathological accumulation in brain structures necessary for memory processing represents a key toxic hallmark underlying Alzheimer's disease. The oligomeric form of Amyloid-ß (Aßο) is now believed to represent the main Amyloid-ß species affecting synapse function. Yet, the exact molecular mechanism by which Aßο modifies synapse function remains to be fully elucidated. There is accumulating evidence that glucocorticoid receptors (GRs) might participate in Aßο generation and activity in the brain. Here, we provide evidence for an acute functional cross-talk between Aß and GRs at hippocampal excitatory synapses. Using live imaging and biochemical analysis of post-synaptic densities (PSD) in cultured hippocampal neurons, we show that synthetic Aßo (100 nM) increases GR levels in spines and PSD. Also, in these cultured neurons, blocking GRs with two different GR antagonists prevents Aßo-mediated PSD95 increase within the PSD. By analyzing long-term potentiation (LTP) and long-term depression (LTD) in ex vivo hippocampal slices after pharmacologically blocking GR, we also show that GR signaling is necessary for Aßo-mediated LTP impairment, but not Aßo-mediated LTD induction. The necessity of neuronal GRs for Aßo-mediated LTP was confirmed by genetically removing GRs in vivo from CA1 neurons using conditional GR mutant mice. These results indicate a tight functional interplay between GR and Aß activities at excitatory synapses.

Combination of blood and biphasic calcium phosphate microparticles for the reconstruction of large bone defects in dog: A pilot study.

We previously reported that biphasic calcium phosphate (BCP) microparticles embedded in a blood clot induces ectopic bone formation in mice and repairs a critical femoral defect in rat. The present pilot study aimed to evaluate in dog and in two models of large defects the efficacy of this composite named "blood for reconstruction of bone" (BRB). We show here that BRB is a cohesive biomaterial easy to prepare from dog autologous blood and to mold to fill large bone defects. First in a model of cylindrical femoral condyle defect, the BRB was compared with BCP particles alone. After 8 weeks, this revealed that the amount of mature bone was slightly and significantly higher with BRB than with BCP particles. Second, in a model consisting in a 2 cm-long critical interruptive defect of the ulna, the BRB was compared with autologous bone. After 6 months, we observed that implantation of BRB can induce the complete reconstruction of the defect and that newly formed bone exhibits high regenerative potential. Comparison with the results obtained with autologous bone grafting strongly suggests that the BRB might be an efficient biomaterial to repair large bone defects, as an alternative or in addition to autologous bone. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1842-1850, 2018.

Physiological and pathophysiological control of synaptic GluN2B-NMDA receptors by the C-terminal domain of amyloid precursor protein.

The amyloid precursor protein (APP) harbors physiological roles at synapses and is central to Alzheimer's disease (AD) pathogenesis. Evidence suggests that APP intracellular domain (AICD) could regulate synapse function, but the underlying molecular mechanisms remain unknown. We addressed AICD actions at synapses, per se, combining in vivo AICD expression, ex vivo AICD delivery or APP knock-down by in utero electroporation of shRNAs with whole-cell electrophysiology. We report a critical physiological role of AICD in controlling GluN2B-containing NMDA receptors (NMDARs) at immature excitatory synapses, via a transcription-dependent mechanism. We further show that AICD increase in mature neurons, as reported in AD, alters synaptic NMDAR composition to an immature-like GluN2B-rich profile. This disrupts synaptic signal integration, via over-activation of SK channels, and synapse plasticity, phenotypes rescued by GluN2B antagonism. We provide a new physiological role for AICD, which becomes pathological upon AICD increase in mature neurons. Thus, AICD could contribute to AD synaptic failure.

Cherubism allele heterozygosity amplifies microbe-induced inflammatory responses in murine macrophages.

Cherubism is a rare autoinflammatory bone disorder that is associated with point mutations in the SH3-domain binding protein 2 (SH3BP2) gene, which encodes the adapter protein 3BP2. Individuals with cherubism present with symmetrical fibro-osseous lesions of the jaw, which are attributed to exacerbated osteoclast activation and defective osteoblast differentiation. Although it is a dominant trait in humans, cherubism appears to be recessively transmitted in mice, suggesting the existence of additional factors in the pathogenesis of cherubism. Here, we report that macrophages from 3BP2-deficient mice exhibited dramatically reduced inflammatory responses to microbial challenge and reduced phagocytosis. 3BP2 was necessary for LPS-induced activation of signaling pathways involved in macrophage function, including SRC, VAV1, p38MAPK, IKKα/ß, RAC, and actin polymerization pathways. Conversely, we demonstrated that the presence of a single Sh3bp2 cherubic allele and pathogen-associated molecular pattern (PAMP) stimulation had a strong cooperative effect on macrophage activation and inflammatory responses in mice. Together, the results from our study in murine genetic models support the notion that infection may represent a driver event in the etiology of cherubism in humans and suggest limiting inflammation in affected individuals may reduce manifestation of cherubic lesions.

Subchronic glucocorticoid receptor inhibition rescues early episodic memory and synaptic plasticity deficits in a mouse model of Alzheimer's disease.

The early phase of Alzheimer's disease (AD) is characterized by hippocampus-dependent memory deficits and impaired synaptic plasticity. Increasing evidence suggests that stress and dysregulation of the hypothalamo-pituitary-adrenal (HPA) axis, marked by the elevated circulating glucocorticoids, are risk factors for AD onset. How these changes contribute to early hippocampal dysfunction remains unclear. Using an elaborated version of the object recognition task, we carefully monitored alterations in key components of episodic memory, the first type of memory altered in AD patients, in early symptomatic Tg2576 AD mice. We also combined biochemical and ex vivo electrophysiological analyses to reveal novel cellular and molecular dysregulations underpinning the onset of the pathology. We show that HPA axis, circadian rhythm, and feedback mechanisms, as well as episodic memory, are compromised in this early symptomatic phase, reminiscent of human AD pathology. The cognitive decline could be rescued by subchronic in vivo treatment with RU486, a glucocorticoid receptor antagonist. These observed phenotypes were paralleled by a specific enhancement of N-Methyl-D-aspartic acid receptor (NMDAR)-dependent LTD in CA1 pyramidal neurons, whereas LTP and metabotropic glutamate receptor-dependent LTD remain unchanged. NMDAR transmission was also enhanced. Finally, we show that, as for the behavioral deficit, RU486 treatment rescues this abnormal synaptic phenotype. These preclinical results define glucocorticoid signaling as a contributing factor to both episodic memory loss and early synaptic failure in this AD mouse model, and suggest that glucocorticoid receptor targeting strategies could be beneficial to delay AD onset.

Postnatal soluble FGFR3 therapy rescues achondroplasia symptoms and restores bone growth in mice.

Achondroplasia is a rare genetic disease characterized by abnormal bone development, resulting in short stature. It is caused by a single point mutation in the gene coding for fibroblast growth factor receptor 3 (FGFR3), which leads to prolonged activation upon ligand binding. To prevent excessive intracellular signaling and rescue the symptoms of achondroplasia, we have developed a recombinant protein therapeutic approach using a soluble form of human FGFR3 (sFGFR3), which acts as a decoy receptor and prevents FGF from binding to mutant FGFR3. sFGFR3 was injected subcutaneously to newborn Fgfr3(ach/+) mice-the mouse model of achondroplasia-twice per week throughout the growth period during 3 weeks. Effective maturation of growth plate chondrocytes was restored in bones of treated mice, with a dose-dependent enhancement of skeletal growth in Fgfr3(ach/+) mice. This resulted in normal stature and a significant decrease in mortality and associated complications, without any evidence of toxicity. These results describe a new approach for restoring bone growth and suggest that sFGFR3 could be a potential therapy for children with achondroplasia and related disorders.

Cell-penetrating TAT-FOXO3 fusion proteins induce apoptotic cell death in leukemic cells.

FOXO proteins are Akt-regulated transcription factors involved in the control of cell cycle, DNA repair, stress defense, apoptosis, and tumor suppression. We reported that plasmid-based overexpression of constitutively active FOXO3 in cells from chronic lymphocytic leukemia (CLL) reduced their survival, suggesting that increasing FOXO3 activity in hematologic malignancies may represent a promising therapeutic strategy. The transactivating transcription factor (TAT) protein transduction domain (PTD) derived from the HIV TAT protein was shown to efficiently deliver macromolecular cargo in various cell types. In this study, wild-type FOXO3 and FOXO3 mutated on Akt sites [FOXO3 T32A/S253A/S315A or TM (triple mutant)] were fused to the TAT-PTD. Using biochemical techniques, flow cytometry, and microscopy analysis, we found a rapid and dose-dependent cell penetration into leukemic cells of unlabeled and fluorescein isothiocyanate-labeled TAT-FOXO3 fusion proteins followed by their accumulation within nuclear and cytoplasmic compartments. Treatment with TAT-FOXO3 TM-but not wild-type TAT-FOXO3-proteins induced Jurkat and K562 leukemic cell death and affected cell viability of other hematologic malignancies including primary cells from CLL. Cell transduction with TAT-FOXO3 TM induced apoptotic cell death as shown by morphologic changes, Annexin V/7-AAD (7-amino-actinomycin D) staining, activation of effector caspases, and PARP cleavage, caspase blockade through the use of the inhibitor Z-VAD, and expression of Bim and p27(KIP1). By contrast, TAT-FOXO3 TM blocked cell proliferation of primary T cells, without affecting their viability. Together, our data show that cell penetrating TAT-FOXO3 TM fusion proteins constitute novel potential therapeutic agents in the treatment of lymphoproliferative disorders and hematologic malignancies.

3BP2 adapter protein is required for receptor activator of NFκB ligand (RANKL)-induced osteoclast differentiation of RAW264.7 cells.

The adapter protein 3BP2 (also known as SH3BP2 and Abl SH3-binding protein 2) has been involved in leukocyte signaling and activation downstream immunoreceptors. Genetic studies have further associated 3BP2 mutations to the human disease cherubism and to inflammation and bone dysfunction in mouse. However, how wild type 3BP2 functions in macrophage differentiation remains poorly understood. In this study, using small interfering RNA-mediated silencing of 3BP2 in the RAW264.7 monocytic cell line, we show that 3BP2 was required for receptor activator of NFkappaB ligand (RANKL)-induced differentiation of RAW264.7 cells into multinucleated mature osteoclasts but not for granulocyte macrophage-colony stimulating factor/interleukin-4-induced differentiation into dendritic cells. 3BP2 silencing was associated with impaired activation of multiple signaling events downstream of RANK, including actin reorganization; Src, ERK, and JNK phosphorylation; and up-regulation of osteoclastogenic factors. In addition, 3BP2 knockdown cells induced to osteoclast by RANKL displayed a reduced increase of Src and nuclear factor of activated T cells (NFATc1) mRNA and protein expression. Importantly, 3BP2 interacted with Src, Syk, Vav, and Cbl in monocytic cells, and the introduction of constitutively active mutants of Src and NFATc1 in 3BP2-deficient cells restored osteoclast differentiation. Finally, the expression of a 3BP2 cherubism mutant was found to promote increased Src activity and NFAT-dependent osteoclast formation. Together, this study demonstrates that wild type 3BP2 is a key regulator of RANK-mediated macrophage differentiation into osteoclast through Src and NFATc1 activation.