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 centenarianderived 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.

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 ␣-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-␤ 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.

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 Nacetylglucosamine (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 biopsyderived 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.

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.

Alzheimer's disease (AD) is characterized by massive neuron loss in distinct brain regions, extracellular accumulations of the amyloid precursor protein-fragment amyloid-b (Ab) and intracellular tau fibrils containing hyperphosphorylated tau. Experimental evidence suggests a relation between presenilin (PS) mutations, Ab formation, and tau phosphorylation in triggering cell death; however, how Ab and PS affect taudependent 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, Ab amplified degeneration in the presence of wt tau but not of PHP tau. The data indicate that PS1 and Ab inversely modulate tau-dependent neurodegeneration at distinct steps. They indicate that the mode by which PHP tau causes neurotoxicity is downstream of Ab and that tau phosphorylation is the limiting factor in Ab-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.

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.

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.

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