Developmental and Injury-induced Changes in DNA Methylation in Regenerative versus Non-regenerative Regions of the Vertebrate Central Nervous System.

BACKGROUND: Because some of its CNS neurons (e.g., retinal ganglion cells after optic nerve crush (ONC)) regenerate axons throughout life, whereas others (e.g., hindbrain neurons after spinal cord injury (SCI)) lose this capacity as tadpoles metamorphose into frogs, the South African claw-toed frog, Xenopus laevis, offers unique opportunities for exploring differences between regenerative and non-regenerative responses to CNS injury within the same organism. An earlier, three-way RNA-seq study (frog ONC eye, tadpole SCI hindbrain, frog SCI hindbrain) identified genes that regulate chromatin accessibility among those that were differentially expressed in regenerative vs non-regenerative CNS [11]. The current study used whole genome bisulfite sequencing (WGBS) of DNA collected from these same animals at the peak period of axon regeneration to study the extent to which DNA methylation could potentially underlie differences in chromatin accessibility between regenerative and non-regenerative CNS. RESULTS: Consistent with the hypothesis that DNA of regenerative CNS is more accessible than that of non-regenerative CNS, DNA from both the regenerative tadpole hindbrain and frog eye was less methylated than that of the non-regenerative frog hindbrain. Also, consistent with observations of CNS injury in mammals, DNA methylation in non-regenerative frog hindbrain decreased after SCI. However, contrary to expectations that the level of DNA methylation would decrease even further with axotomy in regenerative CNS, DNA methylation in these regions instead increased with injury. Injury-induced differences in CpG methylation in regenerative CNS became especially enriched in gene promoter regions, whereas non-CpG methylation differences were more evenly distributed across promoter regions, intergenic, and intragenic regions. In non-regenerative CNS, tissue-related (i.e., regenerative vs. non-regenerative CNS) and injury-induced decreases in promoter region CpG methylation were significantly correlated with increased RNA expression, but the injury-induced, increased CpG methylation seen in regenerative CNS across promoter regions was not, suggesting it was associated with increased rather than decreased chromatin accessibility. This hypothesis received support from observations that in regenerative CNS, many genes exhibiting increased, injury-induced, promoter-associated CpG-methylation also exhibited increased RNA expression and association with histone markers for active promoters and enhancers. DNA immunoprecipitation for 5hmC in optic nerve regeneration found that the promoter-associated increases seen in CpG methylation were distinct from those exhibiting changes in 5hmC. CONCLUSIONS: Although seemingly paradoxical, the increased injury-associated DNA methylation seen in regenerative CNS has many parallels in stem cells and cancer. Thus, these axotomy-induced changes in DNA methylation in regenerative CNS provide evidence for a novel epigenetic state favoring successful over unsuccessful CNS axon regeneration. The datasets described in this study should help lay the foundations for future studies of the molecular and cellular mechanisms involved. The insights gained should, in turn, help point the way to novel therapeutic approaches for treating CNS injury in mammals.

Neurophysiological and Behavioral Analysis in Xenopus.

Because of its resilience to hypoxia and trauma, the frog has long been a favored preparation of neurophysiologists. Its use has led to the discovery of many fundamental properties of neurons and neural circuits. Neurophysiologists were originally attracted to Xenopus embryos, tadpoles, and frogs because of their ready availability, their external development, and the anatomical accessibility and relatively simple neural circuitry of the Xenopus visual, locomotory, and vocalization systems. Nowadays, the sequencing of Xenopus genomes and the panoply of tools for manipulating gene expression have created new opportunities for neurophysiologists to address the molecular underpinnings of how neurons generate behaviors in a vertebrate. Here, we introduce protocols for harnessing the power of Xenopus for performing electrophysiological studies of neural circuitry in the developing optic tectum and spinal cord, as well as in vocalization, and for studying the ontogeny of locomotory behavior.

Comparative gene expression profiling between optic nerve and spinal cord injury in Xenopus laevis reveals a core set of genes inherent in successful regeneration of vertebrate central nervous system axons.

BACKGROUND: The South African claw-toed frog, Xenopus laevis, is uniquely suited for studying differences between regenerative and non-regenerative responses to CNS injury within the same organism, because some CNS neurons (e.g., retinal ganglion cells after optic nerve crush (ONC)) regenerate axons throughout life, whereas others (e.g., hindbrain neurons after spinal cord injury (SCI)) lose this capacity as tadpoles metamorphose into frogs. Tissues from these CNS regions (frog ONC eye, tadpole SCI hindbrain, frog SCI hindbrain) were used in a three-way RNA-seq study of axotomized CNS axons to identify potential core gene expression programs for successful CNS axon regeneration. RESULTS: Despite tissue-specific changes in expression dominating the injury responses of each tissue, injury-induced changes in gene expression were nonetheless shared between the two axon-regenerative CNS regions that were not shared with the non-regenerative region. These included similar temporal patterns of gene expression and over 300 injury-responsive genes. Many of these genes and their associated cellular functions had previously been associated with injury responses of multiple tissues, both neural and non-neural, from different species, thereby demonstrating deep phylogenetically conserved commonalities between successful CNS axon regeneration and tissue regeneration in general. Further analyses implicated the KEGG adipocytokine signaling pathway, which links leptin with metabolic and gene regulatory pathways, and a novel gene regulatory network with genes regulating chromatin accessibility at its core, as important hubs in the larger network of injury response genes involved in successful CNS axon regeneration. CONCLUSIONS: This study identifies deep, phylogenetically conserved commonalities between CNS axon regeneration and other examples of successful tissue regeneration and provides new targets for studying the molecular underpinnings of successful CNS axon regeneration, as well as a guide for distinguishing pro-regenerative injury-induced changes in gene expression from detrimental ones in mammals.

Comparisons of SOCS mRNA and protein levels in Xenopus provide insights into optic nerve regenerative success.

In vertebrates from fishes to mammals, optic nerve injury induces increased expression ofSuppressor of Cytokine Signaling 3(SOCS3) mRNA, a modulator of cytokine signaling that is known to inhibit CNS axon regeneration. Unlike amniotes, however, anamniotes successfully regenerate optic axons, despite this increase. To address this seeming paradox, we examined the SOCS3 response to optic nerve injury in the frog,Xenopus laevis, at both the mRNA and protein levels. Far from being only transiently induced, SOCS3 mRNA expression increased throughout regeneration in retinal ganglion cells, but immunostaining and Western blots indicated that this increase was reflected at the protein level in regenerating optic axons but not in ganglion cell bodies. Polysome profiling provided additional evidence that SOCS3 protein levels were regulated post-translationally by demonstrating that the mRNA was efficiently translated in the injured eye. In tumor cells, another member of theSOCS gene family,SOCS2, is known to mediate SOCS3 degradation by targeting it for proteasomal degradation. Unlike the SOCS2 response in mammalian optic nerve injury, SOCS2 expression increased inXenopusretinal ganglion cells after injury, at both the mRNA and protein levels; it was, however, largely absent from both uninjured and regenerating optic axons. We propose a similar degradation mechanism may be spatially restricted inXenopusto keep SOCS3 protein levels sufficiently in check within ganglion cell bodies, where SOCS3 would otherwise inhibit transcription of genes needed for regeneration, but allow them to rise within the axons, where SOCS3 has pro-regenerative effects.

Tracing Central Nervous System Axon Regeneration in Xenopus.

Axonal tracing allows visualizing connectivity between neurons, providing useful information about structure, neuronal location, and function of the nervous system. Identifying regenerating axons and their neuron cell bodies present the particular challenges of labeling the projections of interest while unambiguously demonstrating regrowth of those axons that have been damaged. In the developing brain, an additional labeling challenge arises, as new connections are being made throughout the duration of an experiment. Various strategies have been used to label regenerating axons, including transgenic animals expressing neuron-specific fluorescent proteins, and application of a single labeling molecule after axotomy and regeneration. However, the single label approach is limited in its application to the developing brain, primarily because it leads to the conclusion that every axon that is labeled has regenerated. Double-labeling overcomes these obstacles by identifying regenerating cells as those that are labeled with two different tracing molecules. Moreover, the use of dextran amines, which are only taken up by injured axons and transported retrogradely, provides further confidence of labeling regenerating axons and neuron cell bodies. The procedure described herein provides a straightforward method for using fluorescently labeled dextran amines to identify regenerating supraspinal neurons in Xenopus, but can be applied to other areas of the central and peripheral nervous system as well.

A novel role for the nuclear localization signal in regulating hnRNP K protein stability in vivo.

hnRNP K is a highly conserved nucleocytoplasmic shuttling protein, which associates with RNAs through synergistic binding via its three KH domains. hnRNP K is required for proper nuclear export and translational control of its mRNA targets, and these processes are controlled by hnRNP K's movement between subcellular compartments. Whereas the nuclear export and localization of hnRNP K that is associated with mRNP complexes has been well studied, the trafficking of hnRNP K that is unbound to mRNA has yet to be elucidated. To that end, we expressed an EGFP-tagged RNA binding-defective form of hnRNP K in intact Xenopus embryos, and found it was rapidly degraded in vivo. Deleting hnRNP K's nuclear localization signal (NLS), which contains two prospective ubiquitination sites, rescued the protein from degradation. These data demonstrate a novel activity for the NLS of hnRNP K in regulating the protein's stability in vivo when it is unbound to nucleic acids.

Post-transcriptional regulation mediated by specific neurofilament introns in vivo.

Neurons regulate genes post-transcriptionally to coordinate the supply of cytoskeletal proteins, such as the medium neurofilament (NEFM), with demand for structural materials in response to extracellular cues encountered by developing axons. By using a method for evaluating functionality of cis-regulatory gene elements in vivo through plasmid injection into Xenopus embryos, we discovered that splicing of a specific nefm intron was required for robust transgene expression, regardless of promoter or cell type. Transgenes utilizing the nefm 3'-UTR but substituting other nefm introns expressed little or no protein owing to defects in handling of the messenger (m)RNA as opposed to transcription or splicing. Post-transcriptional events at multiple steps, but mainly during nucleocytoplasmic export, contributed to these varied levels of protein expression. An intron of the ß-globin gene was also able to promote expression in a manner identical to that of the nefm intron, implying a more general preference for certain introns in controlling nefm expression. These results expand our knowledge of intron-mediated gene expression to encompass neurofilaments, indicating an additional layer of complexity in the control of a cytoskeletal gene needed for developing and maintaining healthy axons.

Using Xenopus Embryos to Study Transcriptional and Posttranscriptional Gene Regulatory Mechanisms of Intermediate Filaments.

Intermediate filament genes exhibit highly regulated, tissue-specific patterns of expression during development and in response to injury. Identifying the responsible cis-regulatory gene elements thus holds great promise for revealing insights into fundamental gene regulatory mechanisms controlling tissue differentiation and repair. Because much of this regulation occurs in response to signals from surrounding cells, characterizing them requires a model system in which their activity can be tested within the context of an intact organism conveniently. We describe methods for doing so by injecting plasmid DNAs into fertilized Xenopus embryos. A prokaryotic element for site-specific recombination and two dual HS4 insulator elements flanking the reporter gene promote penetrant, promoter-typic expression that persists through early swimming tadpole stages, permitting the observation of fluorescent reporter protein expression in live embryos. In addition to describing cloning strategies for generating these plasmids, we present methods for coinjecting test and reference plasmids to identify the best embryos for analysis, for analyzing reporter protein and RNA expression, and for characterizing the trafficking of expressed reporter RNAs from the nucleus to polysomes. Thus, this system can be used to study the activities of cis-regulatory elements of intermediate filament genes at multiple levels of transcriptional and posttranscriptional control within an intact vertebrate embryo, from early stages of embryogenesis through later stages of organogenesis and tissue differentiation.

Phosphorylation of heterogeneous nuclear ribonucleoprotein K at an extracellular signal-regulated kinase phosphorylation site promotes neurofilament-medium protein expression and axon outgrowth in Xenopus.

Post-transcriptional control of cytoskeletal genes fine-tunes the supply of structural materials to growing axons in response to extracellular cues. In Xenopus, heterogeneous nuclear ribonucleoprotein K (hnRNPK) plays a crucial role in the nuclear export and translation of multiple cytoskeletal-related mRNAs required for axon outgrowth, and as a substrate of multiple kinases, is thus a likely molecular target of cell signaling pathways regulating such outgrowth. To study the role of hnRNPK's phosphorylation by extracellular signal-regulated kinase (ERK) in Xenopus axon outgrowth, we identified the only ERK1 phosphorylation site on Xenopus hnRNPK (S257; homologous with S284 of human hnRNPK) using an in vitro phosphorylation assay and tested its function in vivo by expressing phosphomimetic (S257D) and phosphodeficient (S257A) forms of hnRNPK in Xenopus embryos. Although neither form altered hnRNPK nuclear export, only the phosphomimetic form significantly rescued both neurofilament protein expression and axon outgrowth from hnRNPK knockdown. This finding represents a previously unidentified function of phosphorylation at this phylogenetically conserved site and implicates hnRNPK as an intracellular molecular target of ERK-mediated signaling in axon outgrowth.

Microtubule-associated protein tau promotes neuronal class II ß-tubulin microtubule formation and axon elongation in embryonic Xenopus laevis.

Compared with its roles in neurodegeneration, much less is known about microtubule-associated protein tau's normal functions in vivo, especially during development. The external development and ease of manipulating gene expression of Xenopus laevis embryos make them especially useful for studying gene function during early development. To study tau's functions in axon outgrowth, we characterized the most prominent tau isoforms of Xenopus embryos and manipulated their expression. None of these four isoforms were strictly analogous to those commonly studied in mammals, as all constitutively contained exon 10, which is preferentially removed from mammalian fetal tau isoforms, as well as exon 8, which in mammals is rare. Nonetheless, like mammalian tau, Xenopus tau exhibited alternative splicing of exon 4a, which in mammals distinguishes 'big' tau of peripheral neurons, and exon 6. Strongly suppressing tau expression with antisense morpholino oligonucleotides only modestly compromised peripheral nerve outgrowth of intact tadpoles, but severely disrupted neuronal microtubules containing class II ß-tubulins while leaving other microtubules largely unperturbed. Thus, the relatively mild dependence of axon development on tau likely resulted from having only a single class of microtubules disrupted by its loss. Also, consistent with its greater expression in long peripheral axons, boosting expression of 'big' tau increased neurite outgrowth significantly and enhanced tubulin acetylation more so than did the smaller isoform. These data demonstrate the utility of Xenopus as a tool to gain new insights into tau's functions in vivo.

A method for using direct injection of plasmid DNA to study cis-regulatory element activity in F0 Xenopus embryos and tadpoles.

The ability to express exogenous reporter genes in intact, externally developing embryos, such as Xenopus, is a powerful tool for characterizing the activity of cis-regulatory gene elements during development. Although methods exist for generating transgenic Xenopus lines, more simplified methods for use with F0 animals would significantly speed the characterization of these elements. We discovered that injecting 2-cell stage embryos with a plasmid bearing a ϕC31 integrase-targeted attB element and two dual ß-globin HS4 insulators flanking a reporter transgene in opposite orientations relative to each other yielded persistent expression with sufficiently high penetrance for characterizing the activity of the promoter without having to coinject integrase RNA. Expression began appropriately during development and persisted into swimming tadpole stages without perturbing the expression of the cognate endogenous gene. Coinjected plasmids having the same elements but expressing different reporter proteins were reliably coexpressed within the same cells, providing a useful control for variations in injections between animals. To overcome the high propensity of these plasmids to undergo recombination, we developed a method for generating them using conventional cloning methods and DH5α cells for propagation. We conclude that this method offers a convenient and reliable way to evaluate the activity of cis-regulatory gene elements in the intact F0 embryo.

c-Jun N-terminal kinase phosphorylation of heterogeneous nuclear ribonucleoprotein K regulates vertebrate axon outgrowth via a posttranscriptional mechanism.

c-Jun N-terminal kinase (JNK) mediates cell signaling essential for axon outgrowth, but the associated substrates and underlying mechanisms are poorly understood. We identified in Xenopus laevis embryos a novel posttranscriptional mechanism whereby JNK regulates axonogenesis by phosphorylating a specific site on heterogeneous nuclear ribonucleoprotein K (hnRNP K). Both JNK inhibition and hnRNP K knockdown inhibited axon outgrowth and translation of hnRNP K-regulated cytoskeletal RNAs (tau and neurofilament medium), effects that were alleviated by expressing phosphomimetic, but not phosphodeficient, forms of hnRNP K. Immunohistochemical and biochemical analyses indicated that JNK phosphorylation of hnRNP K occurred within the cytoplasm and was necessary for the translational initiation of hnRNP K-targeted RNAs but not for hnRNP K intracellular localization or RNA binding. Thus, in addition to its known roles in transcription and cytoskeletal organization, JNK acts posttranscriptionally through hnRNP K to regulate translation of proteins crucial for axonogenesis.

Heterogeneous nuclear ribonucleoprotein K, an RNA-binding protein, is required for optic axon regeneration in Xenopus laevis.

Axotomized optic axons of Xenopus laevis, in contrast to those of mammals, retain their ability to regenerate throughout life. To better understand the molecular basis for this successful regeneration, we focused on the role of an RNA-binding protein, heterogeneous nuclear ribonucleoprotein (hnRNP) K, because it is required for axonogenesis during development and because several of its RNA targets are under strong post-transcriptional control during regeneration. At 11 d after optic nerve crush, hnRNP K underwent significant translocation into the nucleus of retinal ganglion cells (RGCs), indicating that the protein became activated during regeneration. To suppress its expression, we intravitreously injected an antisense Vivo-Morpholino oligonucleotide targeting hnRNP K. In uninjured eyes, it efficiently knocked down hnRNP K expression in only the RGCs, without inducing either an axotomy response or axon degeneration. After optic nerve crush, staining for multiple markers of regenerating axons showed no regrowth of axons beyond the lesion site with hnRNP K knockdown. RGCs nonetheless responded to the injury by increasing expression of multiple growth-associated RNAs and experienced no additional neurodegeneration above that normally seen with optic nerve injury. At the molecular level, hnRNP K knockdown during regeneration inhibited protein, but not mRNA, expression of several known hnRNP K RNA targets (NF-M, GAP-43) by compromising their efficient nuclear transport and disrupting their loading onto polysomes for translation. Our study therefore provides evidence of a novel post-transcriptional regulatory pathway orchestrated by hnRNP K that is essential for successful CNS axon regeneration.

hnRNP K post-transcriptionally co-regulates multiple cytoskeletal genes needed for axonogenesis.

The RNA-binding protein, hnRNP K, is essential for axonogenesis. Suppressing its expression in Xenopus embryos yields terminally specified neurons with severely disorganized microtubules, microfilaments and neurofilaments, raising the hypothesis that hnRNP K post-transcriptionally regulates multiple transcripts of proteins that organize the axonal cytoskeleton. To identify downstream candidates for this regulation, RNAs that co-immunoprecipitated from juvenile brain with hnRNP K were identified on microarrays. A substantial number of these transcripts were linked to the cytoskeleton and to intracellular localization, trafficking and transport. Injection into embryos of a non-coding RNA bearing multiple copies of an hnRNP K RNA-binding consensus sequence found within these transcripts largely phenocopied hnRNP K knockdown, further supporting the idea that it regulates axonogenesis through its binding to downstream target RNAs. For further study of regulation by hnRNP K of the cytoskeleton during axon outgrowth, we focused on three validated RNAs representing elements associated with all three polymers - Arp2, tau and an α-internexin-like neurofilament. All three were co-regulated post-transcriptionally by hnRNP K, as hnRNP K knockdown yielded comparable defects in their nuclear export and translation but not transcription. Directly knocking down expression of all three together, but not each one individually, substantially reproduced the axonless phenotype, providing further evidence that regulation of axonogenesis by hnRNP K occurs largely through pleiotropic effects on cytoskeletal-associated targets. These experiments provide evidence that hnRNP K is the nexus of a novel post-transcriptional regulatory module controlling the synthesis of proteins that integrate all three cytoskeletal polymers to form the axon.

Metamorphosis and the regenerative capacity of spinal cord axons in Xenopus laevis.

Throughout the vertebrate subphylum, the regenerative potential of central nervous system axons is greatest in embryonic stages and declines as development progresses. For example, Xenopus laevis can functionally recover from complete transection of the spinal cord as a tadpole but is unable to do so after metamorphosing into a frog. Neurons of the reticular formation and raphe nucleus are among those that regenerate axons most reliably in tadpole and that lose this ability after metamorphosis. To identify molecular factors associated with the success and failure of spinal cord axon regeneration, we pharmacologically manipulated thyroid hormone (TH) levels using methimazole or triiodothyronine, to either keep tadpoles in a permanently larval state or induce precocious metamorphosis, respectively. Following complete spinal cord transection, serotonergic axons crossed the lesion site and tadpole swimming ability was restored when metamorphosis was inhibited, but these events failed to occur when metamorphosis was prematurely induced. Thus, the metamorphic events controlled by TH led directly to the loss of regenerative potential. Microarray analysis identified changes in hindbrain gene expression that accompanied regeneration-permissive and -inhibitory conditions, including many genes in the permissive condition that have been previously associated with axon outgrowth and neuroprotection. These data demonstrate that changes in gene expression occur within regenerating neurons in response to axotomy under regeneration-permissive conditions in which normal development has been suspended, and they identify candidate genes for future studies of how central nervous system axons can successfully regenerate in some vertebrates.

Post-transcriptional control of neurofilaments: New roles in development, regeneration and neurodegenerative disease.

Neurofilament (NF) protein expression is coupled to axon development and the maintenance of neuronal homeostasis. Here, we present evidence that this tight regulation depends critically on post-transcriptionally regulated changes in NF mRNA transport, translation and stability. Recent studies have shown that post-transcriptional mechanisms modulate increases in NF gene transcription during axon regeneration to yield the final pattern of NF protein expression. Other recent work has found that post-transcriptional control of NFs shares elements with that of other axonal proteins and that its dysregulation contributes to amyotrophic lateral sclerosis. Such studies herald a novel approach to understanding how neurons coordinate the expressions of functionally related proteins and provide new insights into how the dysregulation of this control can lead to disease.

Transcriptional and translational dynamics of light neurofilament subunit RNAs during Xenopus laevis optic nerve regeneration.

Neurofilaments (NFs), which comprise one of three cytoskeletal polymers of vertebrate axons, are heteropolymers of multiple NF subunit proteins. During Xenopus laevis optic nerve regeneration, NF subunit composition undergoes progressive changes that correlate with regenerative success. Understanding the relative contributions of transcriptional and post-transcriptional gene regulatory mechanisms to these changes should therefore provide insights into the control of the axonal growth program. Previously, we examined this issue with respect to the medium neurofilament protein (NF-M). Because the integrity of NF heteropolymers depends upon maintaining properly balanced expression among multiple subunits, we have now extended this analysis to include the four light NF subunits - peripherin, the light NF triplet protein (NF-L), and two additional alpha-internexin-like proteins. Within 3 days after an optic nerve crush injury to one eye, primary transcript levels of NF subunits increased in both eyes. Levels of mRNA, however, increased in only the operated eye and did so later than did increases in primary transcript, indicating that mRNA levels are under significant post-transcriptional regulation. As measured by polysome profiling, the translational efficiencies of individual NF subunit mRNAs also shifted throughout regeneration, with operated eye mRNAs being generally more translationally active than those of unoperated eyes. Also, in operated eyes, the precise mix of efficiently and poorly translated messages throughout regeneration varied independently for each subunit, indicating that their translations are fine-tuned separately. These results suggest a model whereby traumatic disruption of visual circuitry leads to increased expression of NF primary transcripts in both eyes. These increases are subsequently modulated post-transcriptionally to accommodate shifting demands at each phase of regeneration for NF heteropolymers of differing composition in regrowing axons.

A crucial role for hnRNP K in axon development in Xenopus laevis.

We report that hnRNP K, an RNA-binding protein implicated in multiple aspects of post-transcriptional gene control, is essential for axon outgrowth in Xenopus. Its intracellular localization was found to be consistent with one of its known roles as an mRNA shuttling protein. In early embryos, it was primarily nuclear, whereas later it occupied both the nucleus and cytoplasm to varying degrees in different neuronal subtypes. Antisense hnRNP K morpholino oligonucleotides (MOs) microinjected into blastomeres suppressed hnRNP K expression from neural plate stages through to at least stage 40. Differentiating neural cells in these embryos expressed several markers for terminally differentiated neurons but failed to make axons. Rescue experiments and the use of two separate hnRNP K MOs were carried out to confirm that these effects were specifically caused by knockdown of hnRNP K expression. For insights into the involvement of hnRNP K in neuronal post-transcriptional gene control at the molecular level, we compared effects on expression of the medium neurofilament protein (NF-M), the RNA for which binds hnRNP K, with that of peripherin, another intermediate filament protein, the RNA for which does not bind hnRNP K. hnRNP K knockdown compromised NF-M mRNA nucleocytoplasmic export and translation, but had no effect on peripherin. Because eliminating NF-M from Xenopus axons attenuates, but does not abolish, their outgrowth, hnRNP K must target additional RNAs needed for axon development. Our study supports the idea that translation of at least a subset of RNAs involved in axon development is controlled by post-transcriptional regulatory modules that have hnRNP K as an essential element.

Dynamic endogenous association of neurofilament mRNAs with K-homology domain ribonucleoproteins in developing cerebral cortex.

The low, middle, and high molecular mass neurofilament subunit proteins (NF-L, NF-M, and NF-H) co-polymerize to form neurofilaments (NFs). During development, NF subunit expression is highly regulated, and in neurodegenerative disease, aberrant regulation of this expression can lead to the formation of harmful aggregates. NF expression in both development and disease is under significant post-transcriptional control, but the specific ribonucleoproteins (RNPs) involved are only poorly understood. Previously, mass spectrometry on affinity purified proteins from rat brain identified three K-homology (KH) domain RNPs - hnRNP K, hnRNP E1, hnRNP E2 - as being capable of binding NF-M RNA. In the current study, to determine whether these RNPs associate with NF mRNAs endogenously, we performed a co-immunoprecipitation assay on homogenates of postnatal and developing rat cerebral cortex. We found that all three NF mRNAs indeed associated endogenously with these RNPs and that the degree of this association changed during postnatal development, a period when NF expression is under significant post-transcriptional control. The degree of these associations changed independently of the abundance of either the RNPs or the NF messages, indicating that the RNA-protein interactions themselves are directly regulated. This study is consistent with a model whereby these RNPs and NF mRNAs are components of a dynamic post-transcriptional regulatory module that influences the cytoskeletal compositions of neurons.

A living cell-based biosensor utilizing G-protein coupled receptors: principles and detection methods.

This study explores the feasibility of using a bullfrog fibroblast cell line (FT cells) expressing G protein coupled receptors (GPCRs) as the basis for a living cell-based biosensor. We have fabricated gold microelectrode arrays on a silicon dioxide substrate that supports long term, robust growth of the cells at room temperature and under ambient atmospheric conditions. Activation of an endogenous GPCR to ATP was monitored with an optical method that detects rises in intracellular calcium and with an electrochemical method that monitors the increased secretion of pre-loaded norepinephrine on a MEMS device. FT cells were also transfected to express reporter genes driven by several different promoters, raising the possibility that they could be modified genetically to express novel GPCRs as well. The ability to harness GPCRs for BioMEMS applications by using cells that are easy to grow on MEMS devices and to modify genetically opens the way for a new generation of devices based on these naturally selective and highly sensitive chemoreceptors.