Brain-derived neurotrophic factor expression in serotonergic neurons improves stress resilience and promotes adult hippocampal neurogenesis.

The neurotrophin brain-derived neurotrophic factor (BDNF) stimulates adult neurogenesis, but also influences structural plasticity and function of serotonergic neurons. Both, BDNF/TrkB signaling and the serotonergic system modulate behavioral responses to stress and can lead to pathological states when dysregulated. The two systems have been shown to mediate the therapeutic effect of antidepressant drugs and to regulate hippocampal neurogenesis. To elucidate the interplay of both systems at cellular and behavioral levels, we generated a transgenic mouse line that overexpresses BDNF in serotonergic neurons in an inducible manner. Besides displaying enhanced hippocampus-dependent contextual learning, transgenic mice were less affected by chronic social defeat stress (CSDS) compared to wild-type animals. In parallel, we observed enhanced serotonergic axonal sprouting in the dentate gyrus and increased neural stem/progenitor cell proliferation, which was uniformly distributed along the dorsoventral axis of the hippocampus. In the forced swim test, BDNF-overexpressing mice behaved similarly as wild-type mice treated with the antidepressant fluoxetine. Our data suggest that BDNF released from serotonergic projections exerts this effect partly by enhancing adult neurogenesis. Furthermore, independently of the genotype, enhanced neurogenesis positively correlated with the social interaction time after the CSDS, a measure for stress resilience.

Stress-Related Dysfunction of Adult Hippocampal Neurogenesis-An Attempt for Understanding Resilience?

Newborn neurons in the adult hippocampus are regulated by many intrinsic and extrinsic cues. It is well accepted that elevated glucocorticoid levels lead to downregulation of adult neurogenesis, which this review discusses as one reason why psychiatric diseases, such as major depression, develop after long-term stress exposure. In reverse, adult neurogenesis has been suggested to protect against stress-induced major depression, and hence, could serve as a resilience mechanism. In this review, we will summarize current knowledge about the functional relation of adult neurogenesis and stress in health and disease. A special focus will lie on the mechanisms underlying the cascades of events from prolonged high glucocorticoid concentrations to reduced numbers of newborn neurons. In addition to neurotransmitter and neurotrophic factor dysregulation, these mechanisms include immunomodulatory pathways, as well as microbiota changes influencing the gut-brain axis. Finally, we discuss recent findings delineating the role of adult neurogenesis in stress resilience.

Targeting the histone demethylase LSD1 prevents cardiomyopathy in a mouse model of laminopathy.

LMNA mutations in patients are responsible for a dilated cardiomyopathy. Molecular mechanisms underlying the origin and development of the pathology are unknown. Herein, using mouse pluripotent embryonic stem cells (ESCs) and a mouse model both harboring the p.H222P Lmna mutation, we found early defects in cardiac differentiation of mutated ESCs and dilatation of mutated embryonic hearts at E13.5, pointing to a developmental origin of the disease. Using mouse ESCs, we demonstrated that cardiac differentiation of LmnaH222P/+ was impaired at the mesodermal stage. Expression of Mesp1, a mesodermal cardiogenic gene involved in epithelial-to-mesenchymal transition of epiblast cells, as well as Snai1 and Twist expression, was decreased in LmnaH222P/+ cells compared with WT cells in the course of differentiation. In turn, cardiomyocyte differentiation was impaired. ChIP assay of H3K4me1 in differentiating cells revealed a specific decrease of this histone mark on regulatory regions of Mesp1 and Twist in LmnaH222P/+ cells. Downregulation or inhibition of LSD1 that specifically demethylated H3K4me1 rescued the epigenetic landscape of mesodermal LmnaH222P/+ cells and in turn contraction of cardiomyocytes. Inhibition of LSD1 in pregnant mice or neonatal mice prevented cardiomyopathy in E13.5 LmnaH222P/H222P offspring and adults, respectively. Thus, LSD1 appeared to be a therapeutic target to prevent or cure dilated cardiomyopathy associated with a laminopathy.

Prominent Postsynaptic and Dendritic Exocytosis of Endogenous BDNF Vesicles in BDNF-GFP Knock-in Mice.

Brain-derived neurotrophic factor (BDNF) is a secreted messenger molecule that is crucial for neuronal function and induction of synaptic plasticity. Although altered availability of BDNF underlies many neurological deficits and neurodegenerative disorders, secretion dynamics of endogenous BDNF are unexplored. We generated a BDNF-GFP knock-in (KiBE) mouse, in which GFP-labeled BDNF is expressed under the control of the unaltered endogenous mouse BDNF gene regulatory elements. This KiBE mouse model enables for the first time live cell imaging analysis of endogenous BDNF dynamics. We show that BDNF-GFP release and biological activity in vivo are unaffected by the GFP tag, since homozygous KiBE mice, which lack wild-type BDNF, are healthy and have a normal life expectancy. STED superresolution microscopy shows that 70% of BDNF-GFP vesicles in KiBE mouse neurites are localized in dendrites, being typically 200 nm away from synaptic release sites. Live cell imaging in hippocampal slices also reveals prominent targeting of endogenous BDNF-GFP vesicles to dendrites. Fusion pore opening and cargo release of dendritic BDNF vesicles start within 30 s after a strong depolarizing stimulus and continue for > 100 s thereafter, revealing an astonishingly delayed and prolonged release of endogenous BDNF.

Neural stem cell lineage-specific cannabinoid type-1 receptor regulates neurogenesis and plasticity in the adult mouse hippocampus.

Neural stem cells (NSCs) in the adult mouse hippocampus occur in a specific neurogenic niche, where a multitude of extracellular signaling molecules converges to regulate NSC proliferation as well as fate and functional integration. However, the underlying mechanisms how NSCs react to extrinsic signals and convert them to intracellular responses still remains elusive. NSCs contain a functional endocannabinoid system, including the cannabinoid type-1 receptor (CB1). To decipher whether CB1 regulates adult neurogenesis directly or indirectly in vivo, we performed NSC-specific conditional inactivation of CB1 by using triple-transgenic mice. Here, we show that lack of CB1 in NSCs is sufficient to decrease proliferation of the stem cell pool, which consequently leads to a reduction in the number of newborn neurons. Furthermore, neuronal differentiation was compromised at the level of dendritic maturation pointing towards a postsynaptic role of CB1 in vivo. Deteriorated neurogenesis in NSC-specific CB1 knock-outs additionally resulted in reduced long-term potentiation in the hippocampal formation. The observed cellular and physiological alterations led to decreased short-term spatial memory and increased depression-like behavior. These results demonstrate that CB1 expressed in NSCs and their progeny controls neurogenesis in adult mice to regulate the NSC stem cell pool, dendritic morphology, activity-dependent plasticity, and behavior.

ESC-Derived BDNF-Overexpressing Neural Progenitors Differentially Promote Recovery in Huntington's Disease Models by Enhanced Striatal Differentiation.

Huntington's disease (HD) is characterized by fatal motoric failures induced by loss of striatal medium spiny neurons. Neuronal cell death has been linked to impaired expression and axonal transport of the neurotrophin BDNF (brain-derived neurotrophic factor). By transplanting embryonic stem cell-derived neural progenitors overexpressing BDNF, we combined cell replacement and BDNF supply as a potential HD therapy approach. Transplantation of purified neural progenitors was analyzed in a quinolinic acid (QA) chemical and two genetic HD mouse models (R6/2 and N171-82Q) on the basis of distinct behavioral parameters, including CatWalk gait analysis. Explicit rescue of motor function by BDNF neural progenitors was found in QA-lesioned mice, whereas genetic mouse models displayed only minor improvements. Tumor formation was absent, and regeneration was attributed to enhanced neuronal and striatal differentiation. In addition, adult neurogenesis was preserved in a BDNF-dependent manner. Our findings provide significant insight for establishing therapeutic strategies for HD to ameliorate neurodegenerative symptoms.

Epigenetic Regulation of Cardiac Differentiation of Embryonic Stem Cells and Tissues.

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated by transcription factors which bind genomic regulatory regions including enhancers and promoters of cardiac constitutive genes. DNA is wrapped around histones that are subjected to chemical modifications. Modifications of histones further lead to repressed, activated or poised gene transcription, thus bringing another level of fine tuning regulation of gene transcription. Embryonic Stem cells (ES cells) recapitulate within embryoid bodies (i.e., cell aggregates) or in 2D culture the early steps of cardiac development. They provide in principle enough material for chromatin immunoprecipitation (ChIP), a technology broadly used to identify gene regulatory regions. Furthermore, human ES cells represent a human cell model of cardiogenesis. At later stages of development, mouse embryonic tissues allow for investigating specific epigenetic landscapes required for determination of cell identity. Herein, we describe protocols of ChIP, sequential ChIP followed by PCR or ChIP-sequencing using ES cells, embryoid bodies and cardiac specific embryonic regions. These protocols allow to investigating the epigenetic regulation of cardiac gene transcription.

Amyloid-Beta Induced Changes in Vesicular Transport of BDNF in Hippocampal Neurons.

The neurotrophin brain derived neurotrophic factor (BDNF) is an important growth factor in the CNS. Deficits in transport of this secretory protein could underlie neurodegenerative diseases. Investigation of disease-related changes in BDNF transport might provide insights into the cellular mechanism underlying, for example, Alzheimer's disease (AD). To analyze the role of BDNF transport in AD, live cell imaging of fluorescently labeled BDNF was performed in hippocampal neurons of different AD model systems. BDNF and APP colocalized with low incidence in vesicular structures. Anterograde as well as retrograde transport of BDNF vesicles was reduced and these effects were mediated by factors released from hippocampal neurons into the extracellular medium. Transport of BDNF was altered at a very early time point after onset of human APP expression or after acute amyloid-beta(1-42) treatment, while the activity-dependent release of BDNF remained unaffected. Taken together, extracellular cleavage products of APP induced rapid changes in anterograde and retrograde transport of BDNF-containing vesicles while release of BDNF was unaffected by transgenic expression of mutated APP. These early transport deficits might lead to permanently impaired brain functions in the adult brain.

PSA-NCAM positive neural progenitors stably expressing BDNF promote functional recovery in a mouse model of spinal cord injury.

BACKGROUND: Neural stem cells for the treatment of spinal cord injury (SCI) are of particular interest for future therapeutic use. However, until now, stem cell therapies are often limited due to the inhibitory environment following the injury. Therefore, in this study, we aimed at testing a combinatorial approach with BDNF (brain-derived neurotrophic factor) overexpressing early neural progenitors derived from mouse embryonic stem cells. BDNF is a neurotrophin, which both facilitates neural differentiation of stem cells and favors regeneration of damaged axons. METHODS: Mouse embryonic stem cells, modified to stably express BDNF-GFP, were differentiated into PSA-NCAM positive progenitors, which were enriched, and SSEA1 depleted by a sequential procedure of magnetic-activated and fluorescence-activated cell sorting. Purified cells were injected into the lesion core seven days after contusion injury of the spinal cord in mice, and the Basso mouse scale (BMS) test to evaluate motor function was performed for 5 weeks after transplantation. To analyze axonal regeneration the anterograde tracer biotinylated dextran amine was injected into the sensorimotor cortex two weeks prior to tissue analysis. Cellular differentiation was analyzed by immunohistochemistry of spinal cord sections. RESULTS: Motor function was significantly improved in animals obtaining transplanted BDNF-GFP-overexpressing cells as compared to GFP-expressing cells and vehicle controls. Stem cell differentiation in vivo revealed an increase of neuronal and oligodendrocytic lineage differentiation by BDNF as evaluated by immunohistochemistry of the neuronal marker MAP2 (microtubule associated protein 2) and the oligodendrocytic markers ASPA (aspartoacylase) and Olig2 (oligodendrocyte transcription factor 2). Furthermore, axonal tracing showed a significant increase of biotin dextran amine positive corticospinal tract fibers in BDNF-GFP-cell transplanted animals caudally to the lesion site. CONCLUSIONS: The combinatorial therapy approach by transplanting BDNF-overexpressing neural progenitors improved motor function in a mouse contusion model of SCI. Histologically, we observed enhanced neuronal and oligodendrocytic differentiation of progenitors as well as enhanced axonal regeneration.

Impaired 2-AG Signaling in Hippocampal Glutamatergic Neurons: Aggravation of Anxiety-Like Behavior and Unaltered Seizure Susceptibility.

BACKGROUND: Postsynaptically generated 2-arachidonoylglycerol activates the presynaptic cannabinoid type-1 receptor, which is involved in synaptic plasticity at both glutamatergic and GABAergic synapses. However, the differential function of 2-arachidonoylglycerol signaling at glutamatergic vs GABAergic synapses in the context of animal behavior has not been investigated yet. METHODS: Here, we analyzed the role of 2-arachidonoylglycerol signaling selectively in hippocampal glutamatergic neurons. Monoacylglycerol lipase, the primary degrading enzyme of 2-arachidonoylglycerol, is expressed at presynaptic sites of excitatory and inhibitory neurons. By adeno-associated virus-mediated overexpression of monoacylglycerol lipase in glutamatergic neurons of the mouse hippocampus, we selectively interfered with 2-arachidonoylglycerol signaling at glutamatergic synapses of these neurons. RESULTS: Genetic modification of monoacylglycerol lipase resulted in a 50% decrease in 2-arachidonoylglycerol tissue levels without affecting the content of the second major endocannabinoid anandamide. A typical electrophysiological read-out for 2-arachidonoylglycerol signaling is the depolarization-induced suppression of excitation and of inhibition. Elevated monoacylglycerol lipase levels at glutamatergic terminals selectively impaired depolarization-induced suppression of excitation, while depolarization-induced suppression of inhibition was not significantly changed. At the behavioral level, mice with impaired hippocampal glutamatergic 2-arachidonoylglycerol signaling exhibited increased anxiety-like behavior but showed no alterations in aversive memory formation and seizure susceptibility. CONCLUSION: Our data indicate that 2-arachidonoylglycerol signaling selectively in hippocampal glutamatergic neurons is essential for the animal's adaptation to aversive situations.

A view of bivalent epigenetic marks in two human embryonic stem cell lines reveals a different cardiogenic potential.

Human embryonic stem (HUES) cells are derived from early individual embryos with unique genetic printing. However, how their epigenetic status might affect their potential to differentiate toward specific lineages remains a puzzling question. Using chromatin immunoprecipitation (ChIP)-polymerase chain reaction and ChIP-on-chip, the status of bivalent domains on gene promoters (ie, histone 3 on lysine 4 and histone 3 on lysine 27 trimethylation) was monitored for both undifferentiated and bone morphogenetic protein 2 (BMP2)-induced cardiac-committed cells. A marked difference in the epigenetic profile of HUES cell lines was observed and this was correlated to the pattern of gene expression induced by BMP2 as well as to their potential to generate cardiac progenitors and differentiated myocytes. Thus, the epigenetic H3trimeK4 and H3trimeK27 prints generating bivalent domains on promoters, could be used to predict a preference in their differentiation toward a specific lineage.

Embryonic stem cells stably expressing BDNF-GFP exhibit a BDNF-release-dependent enhancement of neuronal differentiation.

Brain-derived neurotrophic factor (BDNF) is known to be a crucial regulator of neuronal survival and synaptic plasticity in the mammalian brain. Furthermore, BDNF positively influences differentiation of embryonic neural precursors, as well as that of neural stem cells from adult neurogenic niches. To study the impact of cell-released BDNF on neural differentiation of embryonic stem cells (ESCs), which represent an attractive source for cell transplantation studies, we have generated mouse ESC clones overexpressing BDNF-GFP by use of knock-in technology. After neural differentiation in vitro, we observed that ESC clones overexpressing BDNF-GFP gave rise to an increased number of neurons as compared to control ESCs. Neurons derived from BDNF-GFP-expressing ESCs harbored a more complex dendritic morphology and differentiated into the GABAergic lineage more than controls. Moreover, we show that ESC-derived neurons released BDNF-GFP in an activity-dependent manner and displayed similar electrophysiological properties as cortical neurons. Thus, our study describes the generation of ESCs stably overexpressing BDNF-GFP, which are ideally suited to investigate the ameliorating effects of BDNF in cell transplantation studies of various neuropathological conditions.

Rejuvenating senescent and centenarian human cells by reprogramming through the pluripotent state.

Direct reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) provides a unique opportunity to derive patient-specific stem cells with potential applications in tissue replacement therapies and without the ethical concerns of human embryonic stem cells (hESCs). However, cellular senescence, which contributes to aging and restricted longevity, has been described as a barrier to the derivation of iPSCs. Here we demonstrate, using an optimized protocol, that cellular senescence is not a limit to reprogramming and that age-related cellular physiology is reversible. Thus, we show that our iPSCs generated from senescent and centenarian cells have reset telomere size, gene expression profiles, oxidative stress, and mitochondrial metabolism, and are indistinguishable from hESCs. Finally, we show that senescent and centenarian-derived pluripotent stem cells are able to redifferentiate into fully rejuvenated cells. These results provide new insights into iPSC technology and pave the way for regenerative medicine for aged patients.

Cardiac commitment of primate embryonic stem cells.

Primate nonhuman and human embryonic stem (ES) cells provide a powerful model of early cardiogenesis. Furthermore, engineering of cardiac progenitors or cardiomyocytes from ES cells offers a tool for drug screening in toxicology or to search for molecules to improve and scale up the process of cardiac differentiation using high-throughput screening technology, as well as a source of cell therapy of heart failure. Spontaneous differentiation of ES cells into cardiomyocytes is, however, limited. Herein, we describe a simple protocol to commit both rhesus and human ES cells toward a cardiac lineage and to sort out early cardiac progenitors. Primate ES cells are challenged for 4 d with the cardiogenic morphogen bone morphogenetic protein 2 (BMP2) and sorted out using anti-SSEA-1 antibody-conjugated magnetic beads. Cardiac progenitor cells can be generated and isolated in 4 d using this protocol.

Differentiation in vivo of cardiac committed human embryonic stem cells in postmyocardial infarcted rats.

Human embryonic stem (HES) cells can give rise to cardiomyocytes in vitro. However, whether undifferentiated HES cells also feature a myocardial regenerative capacity after in vivo engraftment has not been established yet. We compared two HES cell lines (HUES-1 and I6) that were specified toward a cardiac lineage by exposure to bone morphogenetic protein-2 (BMP2) and SU5402, a fibroblast growth factor receptor inhibitor. Real-time polymerase chain reaction (PCR) revealed that the cardiogenic inductive factor turned on expression of mesodermal and cardiac genes (Tbx6, Isl1, FoxH1, Nkx2.5, Mef2c, and alpha-actin). Thirty immunosuppressed rats underwent coronary artery ligation and, 2 weeks later, were randomized and received in-scar injections of either culture medium (controls) or BMP2 (+/-SU5402)-treated HES cells. After 2 months, human cells were detected by anti-human lamin immunostaining, and their cardiomyocytic differentiation was evidenced by their expression of cardiac markers by reverse transcription-PCR and immunofluorescence using an anti-beta myosin antibody. No teratoma was observed in hearts or any other organ of the body. The ability of cardiac-specified HES cells to differentiate along the cardiomyogenic pathway following transplantation into infarcted myocardium raises the hope that these cells might become effective candidates for myocardial regeneration.

Inverse and distinct modulation of tau-dependent neurodegeneration by presenilin 1 and amyloid-beta in cultured cortical neurons: evidence that tau phosphorylation is the limiting factor in amyloid-beta-induced cell death.

Alzheimer's disease (AD) is characterized by massive neuron loss in distinct brain regions, extracellular accumulations of the amyloid precursor protein-fragment amyloid-beta (A beta) and intracellular tau fibrils containing hyperphosphorylated tau. Experimental evidence suggests a relation between presenilin (PS) mutations, A beta formation, and tau phosphorylation in triggering cell death; however, how A beta and PS affect tau-dependent degeneration is unknown. Using herpes simplex virus 1-mediated gene-transfer of fluorescent-tagged tau constructs in primary cortical neurons, we demonstrate that tau expression exerts a neurotoxic effect that is increased with a construct mimicking disease-like hyperphosphorylation [pseudohyperphosphorylated (PHP) tau]. Live imaging revealed that PHP tau expression is associated with increased perikarya suggesting the development of a 'ballooned' phenotype as a specific feature of tau-mediated cell death. Transgenic expression of PS1 suppressed tau-induced neurodegeneration. In contrast, A beta amplified degeneration in the presence of wt tau but not of PHP tau. The data indicate that PS1 and A beta inversely modulate tau-dependent neurodegeneration at distinct steps. They indicate that the mode by which PHP tau causes neurotoxicity is downstream of A beta and that tau phosphorylation is the limiting factor in A beta-induced cell death. Suppression of tau expression or inhibition of tau phosphorylation at disease-relevant sites may provide an effective therapeutic strategy to prevent neurodegeneration in Alzheimer's disease.

O-glycosylation of the tail domain of neurofilament protein M in human neurons and in spinal cord tissue of a rat model of amyotrophic lateral sclerosis (ALS).

Mammalian neurofilaments (NFs) are modified by post-translational modifications that are thought to regulate NF assembly and organization. Whereas phosphorylation has been intensely studied, the role of another common modification, the attachment of O-linked N-acetylglucosamine (GlcNAc) to individual serine and threonine residues, is hardly understood. We generated a novel monoclonal antibody that specifically recognizes an O-glycosylated epitope in the tail domain of NF-M and allows determination of the glycosylation state at this residue. The antibody displays strong species preference for human NF-M, shows some reactivity with rat but not with mouse or bovine NF-M. By immunohistochemistry and Western blot analysis of biopsy-derived human temporal lobe tissue we show that immunoreactivity is highly enriched in axons parallel to hyperphosphorylated NFs. Treatment of cultured neurons with the GlcNAcase inhibitor PUGNAc causes a 40% increase in immunoreactivity within 1 h, which is completely reversible and parallels the total increase in cellular O-GlcNAc modification. Treatment with the mitogen-activated protein kinase kinase inhibitor PD-98059 leads to a similar increase in immunoreactivity. In spinal cord tissue of a transgenic rat model for amyotrophic lateral sclerosis, immunoreactivity is strongly decreased compared with wild-type animals while phosphorylation is increased. The data suggest that hyperphosphorylation and tail domain O-glycosylation of NFs are synchronously regulated in axons of human neurons in situ and that O-glycosylation of NF-M is highly dynamic and closely interweaved with phosphorylation cascades and may have a pathophysiological role.

Functional interactions of tau and their relevance for Alzheimer's disease.

Abnormalities in tau modification and tau aggregation are a characteristic histopathological hallmark of Alzheimer's disease (AD). However it is less clear, how tau pathology is linked to other factors, e.g. the formation of amyloid plaques, mutations in the presenilin gene, or the ApoE genotype that all have been shown to contribute to the disease. In this article, data reporting tau's interactions with other factors that may be relevant for AD and that have been obtained from various types of in vitro experiments, cell culture experiments and animal models are summarized and brought into a mechanistic framework. Evidences for functional links of these interactions to neurodegenerative events characteristic for AD are discussed and weighed.