Multimodal Molecular Imaging Demonstrates Myeloperoxidase Regulation of Matrix Metalloproteinase Activity in Neuroinflammation.

Myeloperoxidase (MPO) has paradoxically been found to be able to both activate matrix metalloproteinases (MMPs) as well as inhibit MMPs. However, these regulatory effects have not yet been observed in vivo, and it is unclear which pathway is relevant in vivo. We aim to track MPO regulation of MMP activity in living animals in neuroinflammation. Mice induced with experimental autoimmune encephalomyelitis (EAE), a mouse model of neuroinflammation and multiple sclerosis, were treated with either the MPO-specific inhibitor 4-aminobenzoic acid hydrazide or saline as control. Mice underwent concurrent magnetic resonance imaging (MRI) with the MPO-specific molecular imaging agent MPO-Gd and fluorescence molecular tomography (FMT) with the MMP-targeting agent MMPsense on day 12 after induction. Biochemical and histopathological correlations were performed. Utilizing concurrent MRI and FMT imaging, we found reduced MMP activity in the brain with MPO inhibition, demonstrating MPO activity positively regulates MMP activity in vivo. In vivo MMPSense activation and MMP-9 activity correlated with MPO-Gd+ lesion volume and disease severity. This was corroborated by in vitro assays and histopathological analyses that showed MMP activity and MMP-9+ cells correlated with MPO activity and MPO+ cells. In conclusion, multimodal molecular imaging demonstrates for the first time MPO regulation of MMP activity in living animals. This approach could serve as a model to study the interactions of other biologically interesting molecules in living organisms.

Imaging of Patients with Head and Neck Cancer: From Staging to Surveillance.

In this review, the authors summarize the latest imaging methods and recommendations for each of the various steps in managing patients with head and neck cancer, from staging of disease to posttreatment surveillance. Because staging of head and neck cancers is different for various subsites of the head and neck, imaging is discussed separately for each. A separate discussion of imaging of perineural spread, occult primary tumors, and lymph nodes is followed by a discussion of paradigms for surveillance imaging in the posttreatment neck.

Imaging of the Sella and Parasellar Region in the Pediatric Population.

Masses in the sella and parasellar region comprise about 10% of all pediatric brain tumors but type and frequency differs from those in adults. Imaging is critical for diagnosis and characterization of these lesions. By assessing the site of origin, signal and contrast enhancement characteristics, and the presence or absence of characteristic patterns, differential diagnosis can narrow the possibilities. The clinical presentation is often characteristic for lesion type and should be considered. This article summarizes the characteristic imaging features of the most frequent pediatric tumors and tumor-mimicking lesions in children in this region.

The Role of Imaging for Trigeminal Neuralgia: A Segmental Approach to High-Resolution MRI.

High-resolution MRI affords exquisite anatomic detail and allows radiologists to scrutinize the entire course of the trigeminal nerve (cranial nerve [CN] V). This article focuses first on the normal MRI appearance of the course of CN V and how best to image each segment. Special attention is then devoted to the role of MRI in presurgical evaluation of patients with neurovascular conflict and in identifying secondary causes of trigeminal neuralgia, including multiple sclerosis. Fundamental concepts in postsurgical imaging after neurovascular decompression are also addressed. Finally, how imaging has been used to better understand the etiology of trigeminal neuralgia is discussed.

Myeloperoxidase Nuclear Imaging for Epileptogenesis.

PURPOSE: To determine if myeloperoxidase (MPO) is involved in epileptogenesis and if molecular nuclear imaging can be used to noninvasively map inflammatory changes in epileptogenesis. MATERIALS AND METHODS: The animal and human studies were approved by the institutional review boards. Pilocarpine-induced epileptic mice were treated with 4-aminobenzoic acid hydrazide (n = 46), a specific irreversible MPO inhibitor, or saline (n = 42). Indium-111-bis-5-hydroxytryptamide-diethylenetriaminepentaacetate was used to image brain MPO activity (n = 6 in the 4-aminobenzoic acid hydrazide and saline groups; n = 5 in the sham group) by using single photon emission computed tomography/computed tomography. The role of MPO in the development of spontaneous recurrent seizures was assessed by means of clinical symptoms and biochemical and histopathologic data. Human brain specimens from a patient with epilepsy and a patient without epilepsy were stained for MPO. The Student t test, one-way analysis of variance, and Mann-Whitney and Kruskal-Wallis tests were used. Differences were regarded as significant if P was less than .05. RESULTS: MPO and leukocytes increased in the brain during epileptogenesis (P < .05). Blocking MPO delayed spontaneous recurrent seizures (99.6 vs 142 hours, P = .016), ameliorated the severity of spontaneous recurrent seizures (P < .05), and inhibited mossy fiber sprouting (Timm index, 0.31 vs 0.03; P = .003). Matrix metalloproteinase activity was upregulated during epileptogenesis in an MPO-dependent manner (1.44 vs 0.94 U/mg, P = .049), suggesting that MPO acts upstream of matrix metalloproteinases. MPO activity was mapped during epileptogenesis in vivo in the hippocampal regions. Resected temporal lobe tissue from a human patient with refractory epilepsy but not the temporal lobe tissue from a patient without seizures demonstrated positive MPO immunostaining, suggesting high translational potential for this imaging technology. CONCLUSION: The findings of this study highlight an important role for MPO in epileptogenesis and show MPO to be a potential therapeutic target and imaging biomarker for epilepsy.

Regulation of synaptic structure and function by FMRP-associated microRNAs miR-125b and miR-132.

MicroRNAs (miRNAs) are noncoding RNAs that suppress translation of specific mRNAs. The miRNA machinery interacts with fragile X mental retardation protein (FMRP), which functions as translational repressor. We show that miR-125b and miR-132, as well as several other miRNAs, are associated with FMRP in mouse brain. miR-125b and miR-132 had largely opposing effects on dendritic spine morphology and synaptic physiology in hippocampal neurons. FMRP knockdown ameliorates the effect of miRNA overexpression on spine morphology. We identified NMDA receptor subunit NR2A as a target of miR-125b and show that NR2A mRNA is specifically associated with FMRP in brain. In hippocampal neurons, NR2A expression is negatively regulated through its 3' UTR by FMRP, miR-125b, and Argonaute 1. Regulation of NR2A 3'UTR by FMRP depends in part on miR-125b. Because NMDA receptor subunit composition profoundly affects synaptic plasticity, these observations have implications for the pathophysiology of fragile X syndrome, in which plasticity is altered.

Regulation of postsynaptic RapGAP SPAR by Polo-like kinase 2 and the SCFbeta-TRCP ubiquitin ligase in hippocampal neurons.

The ubiquitin-proteasome pathway (UPP) regulates synaptic function, but little is known about specific UPP targets and mechanisms in mammalian synapses. We report here that the SCF(beta-TRCP) complex, a multisubunit E3 ubiquitin ligase, targets the postsynaptic spine-associated Rap GTPase activating protein (SPAR) for degradation in neurons. SPAR degradation by SCF(beta-TRCP) depended on the activity-inducible protein kinase Polo-like kinase 2 (Plk2). In the presence of Plk2, SPAR physically associated with the SCF(beta-TRCP) complex through a canonical phosphodegron. In hippocampal neurons, disruption of the SCF(beta-TRCP) complex by overexpression of dominant interfering beta-TRCP or Cul1 constructs prevented Plk2-dependent degradation of SPAR. Our results identify a specific E3 ubiquitin ligase that mediates degradation of a key postsynaptic regulator of synaptic morphology and function.

Activity-induced Polo-like kinase 2 is required for homeostatic plasticity of hippocampal neurons during epileptiform activity.

Homeostatic plasticity mechanisms stabilize the activity of a neuron or neuronal circuit during prolonged periods of increased network activity and have been proposed to function in the prevention of epilepsy. How homeostatic plasticity is achieved at the molecular level during hyperactivity states in general, and during epileptiform activity in particular, is unclear. Using organotypic hippocampal slice cultures as a model system, we found that the protein kinase Polo-like kinase 2 (Plk2) was induced during prolonged epileptiform activity and was required for the activity-dependent reduction in membrane excitability of pyramidal neurons. Disruption of Plk2 function by dominant-negative or RNA interference not only blocked the downregulation of membrane excitability during epileptiform activity, but also unmasked a slow and progressive potentiation in synaptic strength that prevented the ability of the slice to undergo long-term potentiation. Thus, Plk2 function is required to prevent escalating potentiation and maintain synapses in a plastic state during epileptiform activity in hippocampal slice cultures.

Critical role of CDK5 and Polo-like kinase 2 in homeostatic synaptic plasticity during elevated activity.

Homeostatic plasticity keeps neuronal spiking output within an optimal range in the face of chronically altered levels of network activity. Little is known about the underlying molecular mechanisms, particularly in response to elevated activity. We report that, in hippocampal neurons experiencing heightened activity, the activity-inducible protein kinase Polo-like kinase 2 (Plk2, also known as SNK) was required for synaptic scaling-a principal mechanism underlying homeostatic plasticity. Synaptic scaling also required CDK5, which acted as a "priming" kinase for the phospho-dependent binding of Plk2 to its substrate SPAR, a postsynaptic RapGAP and scaffolding molecule that is degraded following phosphorylation by Plk2. RNAi knockdown of SPAR weakened synapses, and overexpression of a SPAR mutant resistant to Plk2-dependent degradation prevented synaptic scaling. Thus, priming phosphorylation of the Plk2 binding site in SPAR by CDK5, followed by Plk2 recruitment and SPAR phosphorylation-degradation, constitutes a molecular pathway for neuronal homeostatic plasticity during chronically elevated activity.

Control of dendritic arborization by the phosphoinositide-3'-kinase-Akt-mammalian target of rapamycin pathway.

The molecular mechanisms that determine the size and complexity of the neuronal dendritic tree are unclear. Here, we show that the phosphoinositide-3' kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR) signaling pathway promotes the growth and branching of dendrites in cultured hippocampal neurons. Constitutively active mutants of Ras, PI3K, and Akt, or RNA interference (RNAi) knockdown of lipid phosphatase PTEN (phosphatase and tensin homolog deleted on chromosome Ten), induced growth and elaboration of dendrites that was blocked by mTOR inhibitor rapamycin and/or by overexpression of eIF-4E binding protein 1 (4E-BP1), which inhibits translation of 5' capped mRNAs. The effect of PI3K on dendrites was lost in more mature neurons (>14 d in vitro). Dendritic complexity was reduced by inhibition of PI3K and by RNAi knockdown of mTOR or p70 ribosomal S6 kinase (p70S6K, an effector of mTOR). A rapamycin-resistant mutant of mTOR "rescued" the morphogenetic effects of PI3K in the presence of rapamycin. By regulating global and/or local protein translation, and as a convergence point for multiple signaling pathways, mTOR could play a central role in the control of dendrite growth and branching during development and in response to activity.

Bax/Bak-dependent release of DDP/TIMM8a promotes Drp1-mediated mitochondrial fission and mitoptosis during programmed cell death.

Mitochondrial morphology within cells is controlled by precisely regulated rates of fusion and fission . During programmed cell death (PCD), mitochondria undergo extensive fragmentation and ultimately caspase-independent elimination through a process known as mitoptosis . Though this increased fragmentation is due to increased fission through the recruitment of the dynamin-like GTPase Drp1 to mitochondria , as well as to a block in mitochondrial fusion , cellular mechanisms underlying these processes remain unclear. Here, we describe a mechanism for the increased mitochondrial Drp1 levels and subsequent stimulation of mitochondrial fission seen during PCD. We observed Bax/Bak-mediated release of DDP/TIMM8a, a mitochondrial intermembrane space (IMS) protein , into the cytoplasm, where it binds to and promotes the mitochondrial redistribution of Drp1, a mediator of mitochondrial fission. Using both loss- and gain-of-function assays, we also demonstrate that the Drp1- and DDP/TIMM8a-dependent mitochondrial fragmentation observed during PCD is an important step in mitoptosis, which in turn is involved in caspase-independent cell death. Thus, following Bax/Bak-mediated mitochondrial outer membrane permeabilization (MOMP), IMS proteins released comprise not only apoptogenic factors such as cytochrome c involved in caspase activation but also DDP/TIMM8a, which activates Drp1-mediated fission to promote mitochondrial fragmentation and subsequently elimination during PCD.

Polo-like kinases in the nervous system.

Polo like kinases (Plks) are key regulators of the cell cycle, but little is known about their functions in postmitotic cells such as neurons. Recent findings indicate that Plk2 and Plk3 are dynamically regulated in neurons by synaptic activity at the mRNA and protein levels. In COS cells, Plk2 and Plk3 interact with spine-associated Rap guanosine triphosphatase-activating protein (SPAR), a regulator of actin dynamics and dendritic spine morphology, leading to its degradation through the ubiquitin-proteasome system. Induction of Plk2 in hippocampal neurons eliminates SPAR protein, depletes a core postsynaptic scaffolding molecule (PSD-95), and causes loss of mature dendritic spines and synapses. These findings implicate neuronal Plks as mediators of activity-dependent change in molecular composition and morphology of synapses. Induction of Plks might provide a homeostatic mechanism for global dampening of synaptic strength following heightened neuronal activity ('synaptic scaling'). Synapse-specific actions of induced Plks are also possible, particularly in light of the discovery of phosphoserine/threonine peptide motifs as binding targets of the polo box domain, which could allow for 'priming' phosphorylation by upstream kinases that could 'tag' Plk substrates only in specific synapses.

Frequency-dependent modulation of retinogeniculate transmission by serotonin.

The relay of visual information converging in the lateral geniculate nucleus (LGN) en route to the visual cortex is modulated by projections from brainstem nuclei. The release of serotonin, one mediator of these effects, has been shown to act at a presynaptic site to inhibit neurotransmitter release at the retinogeniculate synapse, the connection between retinal ganglion cells and thalamocortical relay neurons in the LGN. To understand how serotonergic inhibition of synaptic transmission influences the transfer of information at this synapse, we examined the EPSCs and firing responses of relay neurons to 5-carboxytryptamine (5-CT), a 5-HT1 receptor agonist that preferentially activates the presynaptic over postsynaptic modulatory effects of serotonin. Bath application of 5-CT inhibits synaptic strength, relieves synaptic depression, and reduces the total synaptic charge transferred at the retinogeniculate synapse in mouse LGN brain slices. In contrast, 5-CT does not significantly alter the membrane potential response of relay neurons to trains of intracellular current injections. Here we show that presynaptic serotonergic modulation results in a frequency-dependent inhibition of relay neuron firing. At low-frequency stimulation, 5-CT markedly reduces charge transfer at the retinogeniculate synapse, thus inhibiting relay neuron firing. However, inhibition of firing by 5-CT is diminished during high-frequency stimulation, because relief from synaptic depression partially offsets the reduction in charge transfer. Thus, presynaptic serotonergic inhibition plays a powerful role in modulating the frequency range of visual information transmitted via the retinogeniculate synapse such that high-frequency inputs are more reliably transmitted than low-frequency inputs.

Intra- and intermolecular domain interactions of the C-terminal GTPase effector domain of the multimeric dynamin-like GTPase Drp1.

Mammalian Drp1 is a dynamin-like GTPase required for mitochondrial fission. Although it exists primarily as a cytosolic homo-tetramer in vivo, it can also self-assemble into higher order structures on the mitochondrial outer membrane, where it is required for proper mitochondrial division. Functional studies and sequence comparisons have revealed four different structural domains in Drp1, comprising N-terminal GTP-binding, middle, insert B, and C-terminal GTPase effector (GED) domains. Here we describe an intramolecular interaction within Drp1 between the GED and the N-terminal GTP-binding and middle domains. A point mutation (K679A) within the C-terminal GED domain inhibits this intramolecular association, without affecting the formation of Drp1 tetramers or the intermolecular associations among isolated C-terminal domains. Mutant Drp1 K679A exhibits impaired GTPase activity, and when overexpressed in mammalian cells it decreases mitochondrial division. Sedimentation experiments indicate that the K679A mutation either increases Drp1 complex formation or, more likely, decreases complex disassembly as compared with wild-type Drp1. Taken together, these data suggest that the C-terminal GED domain is important for stimulation of GTPase activity, formation and stability of higher order complexes, and efficient mitochondrial division.

Interaction of the deafness-dystonia protein DDP/TIMM8a with the signal transduction adaptor molecule STAM1.

The Mohr-Tranebjaerg-Jensen deafness-dystonia-optic atrophy protein DDP/TIMM8a is translated on cytoplasmic ribosomes but targeted ultimately to the mitochondrial intermembrane space, where it is involved in mitochondrial protein import. STAM1 is a cytoplasmic signal-transducing adaptor molecule implicated in cytokine signaling. We report here a direct interaction between DDP and STAM1, identified by yeast two-hybrid screening and confirmed by co-immunoprecipitation, fusion protein "pull downs," and nuclear redistribution assays. DDP coordinates Zn(2+), and Zn(2+) was found to stimulate the DDP-STAM1 interaction in vitro. Endogenous STAM1 localizes predominantly to early endosomes, and we found no evidence that STAM1 is imported into mitochondria in vitro. Thus, the DDP-STAM1 interaction likely occurs in the cytoplasm or at the mitochondrial outer membrane. The DDP-STAM1 interaction requires a coiled-coil region in STAM1 that overlaps with the immunoreceptor tyrosine-based activation motif (ITAM), a region previously shown to be important for interaction with Jak2/3 and hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs). Thus, DDP binding may alter the interactions of STAM1 with several cytoplasmic proteins involved in cell signaling and endosomal trafficking.