A blueprint for translational regenerative medicine.

The past few decades have produced a large number of proof-of-concept studies in regenerative medicine. However, the route to clinical adoption is fraught with technical and translational obstacles that frequently consign promising academic solutions to the so-called "valley of death." Here, we present a proposed blueprint for translational regenerative medicine. We offer principles to help guide the selection of cells and materials, present key in vivo imaging modalities, and argue that the host immune response should be considered throughout design and development. Last, we suggest a pathway to navigate the often complex regulatory and manufacturing landscape of translational regenerative medicine.

RNA sequencing of synaptic and cytoplasmic Upf1-bound transcripts supports contribution of nonsense-mediated decay to epileptogenesis.

The nonsense mediated decay (NMD) pathway is a critical surveillance mechanism for identifying aberrant mRNA transcripts. It is unknown, however, whether the NMD system is affected by seizures in vivo and whether changes confer beneficial or maladaptive responses that influence long-term outcomes such the network alterations that produce spontaneous recurrent seizures. Here we explored the responses of the NMD pathway to prolonged seizures (status epilepticus) and investigated the effects of NMD inhibition on epilepsy in mice. Status epilepticus led to increased protein levels of Up-frameshift suppressor 1 homolog (Upf1) within the mouse hippocampus. Upf1 protein levels were also higher in resected hippocampus from patients with intractable temporal lobe epilepsy. Immunoprecipitation of Upf1-bound RNA from the cytoplasmic and synaptosomal compartments followed by RNA sequencing identified unique populations of NMD-associated transcripts and altered levels after status epilepticus, including known substrates such as Arc as well as novel targets including Inhba and Npas4. Finally, long-term video-EEG recordings determined that pharmacologic interference in the NMD pathway after status epilepticus reduced the later occurrence of spontaneous seizures in mice. These findings suggest compartment-specific recruitment and differential loading of transcripts by NMD pathway components may contribute to the process of epileptogenesis.

microRNA targeting of the P2X7 purinoceptor opposes a contralateral epileptogenic focus in the hippocampus.

The ATP-gated ionotropic P2X7 receptor (P2X7R) modulates glial activation, cytokine production and neurotransmitter release following brain injury. Levels of the P2X7R are increased in experimental and human epilepsy but the mechanisms controlling P2X7R expression remain poorly understood. Here we investigated P2X7R responses after focal-onset status epilepticus in mice, comparing changes in the damaged, ipsilateral hippocampus to the spared, contralateral hippocampus. P2X7R-gated inward currents were suppressed in the contralateral hippocampus and P2rx7 mRNA was selectively uploaded into the RNA-induced silencing complex (RISC), suggesting microRNA targeting. Analysis of RISC-loaded microRNAs using a high-throughput platform, as well as functional assays, suggested the P2X7R is a target of microRNA-22. Inhibition of microRNA-22 increased P2X7R expression and cytokine levels in the contralateral hippocampus after status epilepticus and resulted in more frequent spontaneous seizures in mice. The major pro-inflammatory and hyperexcitability effects of microRNA-22 silencing were prevented in P2rx7(-/-) mice or by treatment with a specific P2X7R antagonist. Finally, in vivo injection of microRNA-22 mimics transiently suppressed spontaneous seizures in mice. The present study supports a role for post-transcriptional regulation of the P2X7R and suggests therapeutic targeting of microRNA-22 may prevent inflammation and development of a secondary epileptogenic focus in the brain.

Overexpression of 14-3-3ζ Increases Brain Levels of C/EBP Homologous Protein CHOP.

Recent studies demonstrated that overexpression of the molecular chaperone 14-3-3ζ protects the brain against endoplasmic reticulum (ER) stress and prolonged seizures. The 14-3-3 targets responsible for improved neuronal survival after seizures remain unknown. Here we explored the mechanism, finding that protein levels of the ER-stress-associated transcription factor C/EBP homologous protein (CHOP) were significantly higher in 14-3-3ζ-overexpressing mice. Since previous studies by us demonstrated that loss of CHOP increased vulnerability to seizure damage, we explored whether elevated CHOP levels result from 14-3-3ζ overexpression and contribute to the protection. Pull-down experiments suggested that 14-3-3ζ could bind CHOP as well as sequester a CHOP-targeting microRNA. However, 14-3-3ζ overexpression remained protective against seizure-induced hippocampal injury in mice lacking CHOP. These studies reveal a novel function for 14-3-3ζ in regulating CHOP levels but show that this is not required for protection against seizure-induced neuronal death.

Hsp27 binding to the 3'UTR of bim mRNA prevents neuronal death during oxidative stress-induced injury: a novel cytoprotective mechanism.

Neurons face a changeable microenvironment and therefore need mechanisms that allow rapid switch on/off of their cytoprotective and apoptosis-inducing signaling pathways. Cellular mechanisms that control apoptosis activation include the regulation of pro/antiapoptotic mRNAs through their 3'-untranslated region (UTR). This region holds binding elements for RNA-binding proteins, which can control mRNA translation. Here we demonstrate that heat shock protein 27 (Hsp27) prevents oxidative stress-induced cell death in cerebellar granule neurons by specific regulation of the mRNA for the proapoptotic BH3-only protein, Bim. Hsp27 depletion induced by oxidative stress using hydrogen peroxide (H2O2) correlated with bim gene activation and subsequent neuronal death, whereas enhanced Hsp27 expression prevented these. This effect could not be explained by proteasomal degradation of Bim or bim promoter inhibition; however, it was associated with a specific increase in the levels of bim mRNA and with its binding to Hsp27. Finally, we determined that enhanced Hsp27 expression in neurons exposed to H2O2 or glutamate prevented the translation of a reporter plasmid where bim-3'UTR mRNA sequence was cloned downstream of a luciferase gene. These results suggest that repression of bim mRNA translation through binding to the 3'UTR constitutes a novel cytoprotective mechanism of Hsp27 during stress in neurons.

Mitochondrial localization of the forkhead box class O transcription factor FOXO3a in brain.

FOXO3a is member of the Forkhead box class O transcription factors, which functions in diverse pathways to regulate cellular metabolism, differentiation, and apoptosis. FOXO3a shuttles between the cytoplasm and nucleus and may be activated in neurons by stressors, including seizures. A subset of nuclear transcription factors may localize to mitochondria, but whether FOXO3a is present within brain mitochondria is unknown. Here, we report that purified mitochondrial fractions from rat, mouse, and human hippocampus, as well as HT22 hippocampal cells, contain FOXO3a protein. Immunogold electron microscopy supported the presence of FOXO3a within brain mitochondria, and chromatin immunoprecipitation analysis suggested FOXO3a was associated with mitochondrial DNA. Over-expression of a mitochondrially targeted FOXO3a fusion protein in HT22 cells, but not primary hippocampal neurons, conferred superior protection against glutamate toxicity than FOXO3a alone. Mitochondrial FOXO3a levels were reduced in the damaged region of the mouse hippocampus after status epilepticus, while mitochondrial fractions from the hippocampus of patients with temporal lobe epilepsy displayed higher levels of FOXO3a than controls. These results support mitochondria as a site of FOXO3a localization, which may contribute to the overall physiological and pathophysiological functions of this transcription factor.