Stephen M. Eacker

Understanding microRNAs in neurodegeneration

Interest in the functions of microRNAs (miRNAs) in the nervous system has recently expanded to include their roles in neurodegeneration. Investigations have begun to reveal the influence of miRNAs on both neuronal survival and the accumulation of toxic proteins that are associated with neurodegeneration, and are providing clues as to how these toxic proteins can influence miRNA expression.

MicroRNA-223 is neuroprotective by targeting glutamate receptors

Stroke is a major cause of mortality and morbidity worldwide. Extracellular glutamate accumulation leading to overstimulation of the ionotropic glutamate receptors mediates neuronal injury in stroke and in neurodegenerative disorders. Here we show that miR-223 controls the response to neuronal injury by regulating the functional expression of the glutamate receptor subunits GluR2 and NR2B in brain. Overexpression of miR-223 lowers the levels of GluR2 and NR2B by targeting 3′-UTR target sites (TSs) in GluR2 and NR2B, inhibits NMDA-induced calcium influx in hippocampal neurons, and protects the brain from neuronal cell death following transient global ischemia and excitotoxic injury. MiR-223 deficiency results in higher levels of NR2B and GluR2, enhanced NMDA-induced calcium influx, and increased miniature excitatory postsynaptic currents in hippocampal neurons. In addition, the absence of MiR-223 leads to contextual, but not cued memory deficits and increased neuronal cell death following transient global ischemia and excitotoxicity. These data identify miR-223 as a major regulator of the expression of GluR2 and NR2B, and suggest a therapeutic role for miR-223 in stroke and other excitotoxic neuronal disorders.

A nuclease that mediates cell death induced by DNA damage and poly(ADP-ribose) polymerase-1

DNA damage-activated nuclease identified Cells that experience stresses and accumulate excessive damage to DNA undergo cell death mediated by a nuclear enzyme known as PARP-1. During this process, apoptosis-inducing factor (AIF) translocates to the nucleus and activates one or more nucleases to cleave DNA. Wang et al. found that macrophage migration inhibitory factor (MIF) is an AIF-associated endonuclease that contributes to PARP-1-induced DNA fragmentation (see the Perspective by Jonas). In mouse neurons in culture, loss of MIF protected neurons from cell death caused by excessive stimulation. Targeting MIF could thus provide a therapeutic strategy against diseases in which PARP-1 activation is excessive. Science, this issue p. 82; see also p. 36 An endonuclease that functions in a disease-associated form of cell death is identified. [Also see Perspective by Jonas] INTRODUCTION Poly(ADP-ribose) (PAR) polymerase-1 (PARP-1) is a nuclear enzyme responding to oxidative stress and DNA damage. Excessive activation of PARP-1 causes an intrinsic caspase-independent cell death program designated parthanatos, which occurs in many organ systems because of toxic or stressful insults, including ischemia-reperfusion injury after stroke and myocardial infarction, inflammatory injury, reactive oxygen species–induced injury, glutamate excitotoxicity, and neurodegenerative diseases. Inhibition or genetic deletion of PARP-1 is profoundly protective against such cellular injury in models of human disease. RATIONALE The molecular mechanisms underlying parthanatos involve release of mitochondrial apoptosis-inducing factor (AIF) and its translocation to the nucleus, which results in chromatinolysis into 20- to 50-kb large DNA fragments—a commitment point for parthanatos. Because AIF itself has no obvious nuclease activity, we propose that AIF recruits a nuclease or a nuclease complex to the nucleus to trigger DNA cleavage and parthanatos. Although the endonuclease G (EndoG) homolog may promote DNA degradation in Caenorhabditis elegans through cooperating with the AIF homolog, our group and others showed that EndoG does not have an essential role in PARP-dependent chromatinolysis and cell death in mammals. Thus, the identity of the nuclease responsible for large DNA fragmentation following AIF entry to the nucleus during parthanatos has been a long-standing mystery. RESULTS Using two sequential unbiased screens, including a human protein array and a small interfering RNA screen, we discovered that macrophage migration inhibitory factor (MIF) binds AIF and is required for parthanatos. Three-dimensional modeling of MIF revealed that the MIF trimer has the same core topology structure as PD-D/E(X)K superfamily nucleases. In the presence of Mg2+ or Ca2+, MIF has both 3′ exonuclease and endonuclease activity. It binds to 5′ unpaired bases of single-stranded DNA with stem loop structure and cleaves its 3′ unpaired bases. These nuclease activities allow MIF to cleave genomic DNA into large fragments. Depletion of MIF markedly reduced chromatinolysis and cell death induced by N-methyl-d-aspartate (NMDA) receptor–activated glutamate excitotoxicity in primary neuronal cultures, DNA damage caused by N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) or focal stroke in mice. Mutating key amino acid residues in the PD-D/E(X)K nuclease domain of MIF eliminated its nuclease activity and prevented parthanatos. Disrupting the AIF and MIF interaction prevented the translocation of MIF from the cytosol to the nucleus and protected against parthanatos. Moreover, depletion of MIF, disruption of AIF and MIF interaction, and eliminating MIF’s nuclease activity has long-lasting histological and behavioral rescue in the focal ischemia model of stroke. CONCLUSION We identified MIF as a PARP-1–dependent AIF-associated nuclease that is required for parthanatos. In response to oxidative stress or DNA damage, PARP-1 activation triggers AIF release from the mitochondria. AIF then recruits MIF to the nucleus where MIF cleaves genomic DNA into large fragments and causes cell death. Depletion of MIF, disruption of AIF and MIF interaction, or blocking MIF nuclease activity inhibited chromatinolysis and parthanatos. Targeting MIF nuclease activity may offer an important therapeutic opportunity for a variety of disorders with excessive PARP-1 activation. Stressors lead to DNA damage, PARP-1 activation, and PAR formation. PAR facilitates the release of AIF from mitochondria where it binds MIF. This complex translocates to the nucleus to bind DNA; the result is DNA fragmentation and cell death. Interference with this cascade by preventing the formation of the AIF-MIF complex or by a nuclease-deficient MIF prevents DNA fragmentation and promotes cell survival. Inhibition or genetic deletion of poly(ADP-ribose) (PAR) polymerase-1 (PARP-1) is protective against toxic insults in many organ systems. The molecular mechanisms underlying PARP-1–dependent cell death involve release of mitochondrial apoptosis-inducing factor (AIF) and its translocation to the nucleus, which results in chromatinolysis. We identified macrophage migration inhibitory factor (MIF) as a PARP-1–dependent AIF-associated nuclease (PAAN). AIF was required for recruitment of MIF to the nucleus, where MIF cleaves genomic DNA into large fragments. Depletion of MIF, disruption of the AIF-MIF interaction, or mutation of glutamic acid at position 22 in the catalytic nuclease domain blocked MIF nuclease activity and inhibited chromatinolysis, cell death induced by glutamate excitotoxicity, and focal stroke. Inhibition of MIF’s nuclease activity is a potential therapeutic target for diseases caused by excessive PARP-1 activation.

The interplay of microRNA and neuronal activity in health and disease

MicroRNAs (miRNAs) are small 19–23 nucleotide regulatory RNAs that function by modulating mRNA translation and/or turnover in a sequence-specific fashion. In the nervous system, miRNAs regulate the production of numerous proteins involved in synaptic transmission. In turn, neuronal activity can regulate the production and turnover of miRNA through a variety of mechanisms. In this way, miRNAs and neuronal activity are in a reciprocal homeostatic relationship that balances neuronal function. The miRNA function is critical in pathological states related to overexcitation such as epilepsy and stroke, suggesting miRNA’s potential as a therapeutic target. We review the current literature relating the interplay of miRNA and neuronal activity and provide future directions for defining miRNA’s role in disease.

NMDA-induced neuronal survival is mediated through nuclear factor I-A in mice

Identification of the signaling pathways that mediate neuronal survival signaling could lead to new therapeutic targets for neurologic disorders and stroke. Sublethal doses of NMDA can induce robust endogenous protective mechanisms in neurons. Through differential analysis of primary library expression and microarray analyses, here we have shown that nuclear factor I, subtype A (NFI-A), a member of the NFI/CAAT-box transcription factor family, is induced in mouse neurons by NMDA receptor activation in a NOS- and ERK-dependent manner. Knockdown of NFI-A induction using siRNA substantially reduced the neuroprotective effects of sublethal doses of NMDA. Further analysis indicated that NFI-A transcriptional activity was required for the neuroprotective effects of NMDA receptor activation. Additional evidence of the neuroprotective effects of NFI-A was provided by the observations that Nfia(-/-) neurons were highly sensitive to NMDA-induced excitotoxicity and were more susceptible to developmental cell death than wild-type neurons and that Nfia(+/-) mice were more sensitive to NMDA-induced intrastriatal lesions than were wild-type animals. These results identify NFI-A as what we believe to be a novel neuroprotective transcription factor with implications in neuroprotection and neuronal plasticity following NMDA receptor activation.

Neuronal Activity Regulates Hippocampal miRNA Expression

Neuronal activity regulates a broad range of processes in the hippocampus, including the precise regulation of translation. Disruptions in proper translational control in the nervous system are associated with a variety of disorders that fall in the autistic spectrum. MicroRNA (miRNA) represent a relatively recently discovered player in the regulation of translation in the nervous system. We have conducted an in depth analysis of how neuronal activity regulates miRNA expression in the hippocampus. Using deep sequencing we exhaustively identify all miRNAs, including 15 novel miRNAs, expressed in hippocampus of the adult mouse. We identified 119 miRNAs documented in miRBase but less than half of these miRNA were expressed at a level greater than 0.1% of total miRNA. Expression profiling following induction of neuronal activity by electroconvulsive shock demonstrates that most miRNA show a biphasic pattern of expression: rapid induction of specific mature miRNA expression followed by a decline in expression. These results have important implications into how miRNAs influence activity-dependent translational control.

Cultured networks of excitatory projection neurons and inhibitory interneurons for studying human cortical neurotoxicity

A method to culture human cortical neurons that yielded a balanced network of excitatory and inhibitory neurons revealed that these cells die in a PARP-dependent manner after neurotoxic insult. Insights into neuronal cell death The study of the mechanisms of cell death in human cortical neurons has been hampered by the lack of human neuronal cultures that exhibit a balanced network of excitatory and inhibitory synapses. Xu et al. now describe a method to culture human neurons with a representative ratio of both excitatory and inhibitory neurons derived from human embryonic stem cells or human inducible pluripotent stem cells. Using this new method, they show that human cortical neurons die in a nitric oxide– and poly(ADP-ribose) polymerase-1–dependent manner. These cultures can be used to study the mechanisms of neurotoxicity in human disorders that involve the demise of cortical neurons. Translating neuroprotective treatments from discovery in cell and animal models to the clinic has proven challenging. To reduce the gap between basic studies of neurotoxicity and neuroprotection and clinically relevant therapies, we developed a human cortical neuron culture system from human embryonic stem cells or human inducible pluripotent stem cells that generated both excitatory and inhibitory neuronal networks resembling the composition of the human cortex. This methodology used timed administration of retinoic acid to FOXG1+ neural precursor cells leading to differentiation of neuronal populations representative of the six cortical layers with both excitatory and inhibitory neuronal networks that were functional and homeostatically stable. In human cortical neuronal cultures, excitotoxicity or ischemia due to oxygen and glucose deprivation led to cell death that was dependent on N-methyl-d-aspartate (NMDA) receptors, nitric oxide (NO), and poly(ADP-ribose) polymerase (PARP) (a cell death pathway called parthanatos that is distinct from apoptosis, necroptosis, and other forms of cell death). Neuronal cell death was attenuated by PARP inhibitors that are currently in clinical trials for cancer treatment. This culture system provides a new platform for the study of human cortical neurotoxicity and suggests that PARP inhibitors may be useful for ameliorating excitotoxic and ischemic cell death in human neurons.

Synaptic Plasticity onto Dopamine Neurons Shapes Fear Learning

Fear learning is a fundamental behavioral process that requires dopamine (DA) release. Experience-dependent synaptic plasticity occurs on DA neurons while an organism is engaged in aversive experiences. However, whether synaptic plasticity onto DA neurons is causally involved in aversion learning is unknown. Here, we show that a stress priming procedure enhances fear learning by engaging VTA synaptic plasticity. Moreover, we took advantage of the ability of the ATPase Thorase to regulate the internalization of AMPA receptors (AMPARs) in order to selectively manipulate glutamatergic synaptic plasticity on DA neurons. Genetic ablation of Thorase in DAT+ neurons produced increased AMPAR surface expression and function that lead to impaired induction of both long-term depression (LTD) and long-term potentiation (LTP). Strikingly, animals lacking Thorase in DAT+ neurons expressed greater associative learning in a fear conditioning paradigm. In conclusion, our data provide a novel, causal link between synaptic plasticity onto DA neurons and fear learning.

High-content genome-wide rnai screen reveals ccr3 as a key mediator of neuronal cell death

Visual Abstract Neuronal loss caused by ischemic injury, trauma, or disease can lead to devastating consequences for the individual. With the goal of limiting neuronal loss, a number of cell death pathways have been studied, but there may be additional contributors to neuronal death that are yet unknown. To identify previously unknown cell death mediators, we performed a high-content genome-wide screening of short, interfering RNA (siRNA) with an siRNA library in murine neural stem cells after exposure to N-methyl-N-nitroso-N′-nitroguanidine (MNNG), which leads to DNA damage and cell death. Eighty genes were identified as key mediators for cell death. Among them, 14 are known cell death mediators and 66 have not previously been linked to cell death pathways. Using an integrated approach with functional and bioinformatics analysis, we provide possible molecular networks, interconnected pathways, and/or protein complexes that may participate in cell death. Of the 66 genes, we selected CCR3 for further evaluation and found that CCR3 is a mediator of neuronal injury. CCR3 inhibition or deletion protects murine cortical cultures from oxygen-glucose deprivation–induced cell death, and CCR3 deletion in mice provides protection from ischemia in vivo. Taken together, our findings suggest that CCR3 is a previously unknown mediator of cell death. Future identification of the neural cell death network in which CCR3 participates will enhance our understanding of the molecular mechanisms of neural cell death.

An image registration pipeline for analysis of transsynaptic tracing in mice

Parkinsons Disease (PD) is a movement disorder characterized by the loss of dopamine neurons in the substantia nigra pars compacta (SNpc) and norepinephrine neurons in the locus coeruleus (LC). To further understand the pathophysiology of PD, the input neurons of the SNpc and LC will be transsynapticly traced in mice using a fluorescent recombinant rabies virus (RbV) and imaged using serial two-photon tomography (STP). A mapping between these images and a brain atlas must be found to accurately determine the locations of input neurons in the brain. Therefore a registration pipeline to align the Allen Reference Atlas (ARA) to these types of images was developed. In the preprocessing step, a brain mask was generated from the transsynaptic tracing images using simple morphological operators. The masks were then registered to the ARA using Large Deformation Diffeomorphic Metric Mapping (LDDMM), an algorithm specialized for calculating anatomically realistic transforms between images. The pipeline was then tested on an STP scan of a mouse brain labeled by an adeno-associated virus (AAV). Based on qualitative evaluation of the registration results, the pipeline was found to be sufficient for use with transsynaptic RbV tracing.