James Eberwine

RNA cargoes associating with FMRP reveal deficits in cellular functioning in Fmr1 null mice

The Fragile X mental retardation-1 (Fmr1) gene encodes a multifunctional protein, FMRP, with intrinsic RNA binding activity. We have developed an approach, antibody-positioned RNA amplification (APRA), to identify the RNA cargoes associated with the in vivo configured FMRP messenger ribonucleoprotein (mRNP) complex. Using APRA as a primary screen, putative FMRP RNA cargoes were assayed for their ability to bind directly to FMRP using traditional methods of assessing RNA-protein interactions, including UV-crosslinking and filter binding assays. Approximately 60% of the APRA-defined mRNAs directly associate with FMRP. By examining a subset of these mRNAs and their encoded proteins in brain tissue from Fmr1 knockout mice, we have observed that some of these cargoes as well as the proteins they encode show discrete changes in abundance and/or differential subcellular distribution. These data are consistent with spatially selective regulation of multiple biological pathways by FMRP.

Expression profile of transcripts in Alzheimer's disease tangle-bearing CA1 neurons

The pathogenesis of neurofibrillary tangles (NFTs) in Alzheimers disease (AD) is poorly understood, but changes in the expression of specific messenger RNAs (mRNAs) may reflect mechanisms underlying the formation of NFTs and their consequences in affected neurons. For these reasons, we compared the relative abundance of multiple mRNAs in tangle‐bearing versus normal CA1 neurons aspirated from sections of AD and control brains. Amplified antisense RNA expression profiling was performed on individual isolated neurons for analysis of greater than 18,000 expressed sequence tagged complementary DNAs (cDNAs) with cDNA microarrays, and further quantitative analyses were performed by reverse Northern blot analysis on 120 selected mRNAs on custom cDNA arrays. Relative to normal CA1 neurons, those harboring NFTs in AD brains showed significant reductions in several classes of mRNAs that are known to encode proteins implicated in AD neuropathology, including phosphatases/kinases, cytoskeletal proteins, synaptic proteins, glutamate receptors, and dopamine receptors. Because cathepsin D mRNA was upregulated in NFT‐bearing CA1 neurons in AD brains, we performed immunohistochemical studies that demonstrated abundant cathepsin D immunoreactivity in the same population of tangle‐bearing CA1 neurons. In addition, levels of mRNAs encoding proteins not previously implicated in AD were reduced in CA1 tangle‐bearing neurons, suggesting that these proteins (eg, activity‐regulated cytoskeleton‐associated protein, focal adhesion kinase, glutaredoxin, utrophin) may be novel mediators of NFT formation or degeneration in affected neurons. Thus, the profile of mRNAs differentially expressed by tangle‐bearing CA1 neurons may represent a “molecular fingerprint” of these neurons, and we speculate that mRNA expression profiles of diseased neurons in AD may suggest new directions for AD research or identify novel targets for developing more effective AD therapies. Ann Neurol 2000;48:77–87

Molecular Characterization of the Dendritic Growth Cone: Regulated mRNA Transport and Local Protein Synthesis

The molecular mechanisms that regulate growth cone guidance of dendrite outgrowth remain to be elucidated. We hypothesized that mRNA localization in dendritic growth cones and their local protein synthesis may be important for growth cone functioning. The appearance of 23 of 31 growth cone mRNAs was developmentally regulated. Also, alteration of growth cone morphology affected the relative levels of three mRNAs. Finally, using single dendrite transfection, it was shown that local protein synthesis occurs in dendrites and growth cones. A heterogeneous population of mRNAs exists in dendritic growth cones of cultured hippocampal neurons whose relative abundances are developmentally regulated and can vary with changes in growth cone physiology. The demonstration of protein synthesis in growth cones suggests that translation of the localized mRNAs may contribute to regulation of growth cone motility and dendrite outgrowth.

Mammalian cell transfection: the present and the future

Transfection is a powerful analytical tool enabling study of the function of genes and gene products in cells. The transfection methods are broadly classified into three groups; biological, chemical, and physical. These methods have advanced to make it possible to deliver nucleic acids to specific subcellular regions of cells by use of a precisely controlled laser-microcope system. The combination of point-directed transfection and mRNA transfection is a new way of studying the function of genes and gene products. However, each method has its own advantages and disadvantages so the optimum method depends on experimental design and objective.

Localization and translation of mRNA in dentrites and axons

The neurons of the brain extend axons and dendrites many hundreds of micrometres away from the cell body. The first electron microscope studies of these processes revealed that many of the structures that are found in the cell body are also present in dendrites. For example, particles resembling ribosomes and membrane structures like those of the endoplasmic reticulum (two structures that are important for protein synthesis) were seen in distal regions of dendrites, near synapses. Subsequent studies focused on identifying messenger RNAs in dendrites and providing evidence of dendritic protein synthesis. Transfection technologies have now been used to analyse translation within dendrites in response to pharmacological stimuli. These studies provide us with clues to the physiological role of the dendrite not just as a signal transducer, but also as a modulator of long-term synaptic efficacy.

PREPARATION OF CDNA FROM SINGLE CELLS AND SUBCELLULAR REGIONS

Phenotypic characterization of cells in conjunction with single-cell mRNA analysis, which yields information regarding expression of multiple genes in individual neurons, facilitates a detailed and comprehensive view of neuronal cell biology. More specifically, the aRNA amplification method has provided an approach to analyze mRNA levels in single cells that have been phenotypically characterized on the basis of electrophysiology, morphology, and/or protein expression. In this way, relative mRNA abundances can be directly assayed from a well-defined population of neurons. The concept of expression profiling led to the development of robotics methods for arraying thousands of cDNAs on microarrays. These cDNA arrays can be screened with labeled aRNA or cDNA to generate a molecular fingerprint of a specific cell type, disease state, or therapeutic efficacy. A broad view of how gene expression is altered in single neurons affected by a particular disease process may provide clues to pathogenetic disease mechanisms or avenues for therapeutic interventions. The use of mRNA profiles to produce diagnostics and therapeutics is called transcript-aided drug design (TADD). When coupled with single-cell resolution, TADD promises to be an important tool in diagnosis of disease states, as well as provide a blueprint on which to develop therapeutic strategies. For example, mRNA abundances in an individual diseased cell may increase, decrease, or remain constant, and thus it is possible that a pharmaceutical alone or in combination with other drugs may be specifically designed to restore mRNA abundances to a normal state. Alternatively, if functional protein levels parallel the mRNA level changes, then drugs targeting the function of the proteins translated from these altered mRNAs may prove to be therapeutic. One promise of such an approach is that information about mRNA abundances that are altered in a diseased cell may provide new therapeutic indications for existing drugs. For example, if the abundance of mRNA for the beta-adrenergic receptor is altered as shown by the microarrays for a particular disease, already available adrenergic receptor agonists or antagonists that had not previously been used in this particular disease paradigm may prove to be therapeutically efficacious. The expression profile of a given cell is a measure of the potential for protein expression. Proteins are generally the functional entities within cells and differences in protein function often result in disease. The ability to monitor the coordinate changes in gene expression, in single phenotypically identified cells, that correlate with disease will provide unique insight into the expressed genetic variability of cells and will likely furnish unforeseen insight into the underlying cellular mechanisms that produce disease etiology.

Stimulus-induced coordinate changes in mRNA abundance in single postsynaptic hippocampal CA1 neurons

The molecular effects of use-dependent changes in synaptic transmission were studied in individual CA1 pyramidal neurons from rat hippocampal slices. Potentiation of excitatory postsynaptic currents was associated with coordinate changes in the relative abundance of several mRNAs 30 min to 3 hr after stimulation. There was a 300% increase in calcium/calmodulin-dependent protein kinase II mRNA levels concordant with a 50% decrease in protein kinase C beta 1 isoform mRNA. A 2-fold increase in zif-268 mRNA was seen, while increases in c-fos and c-jun mRNA levels were inconsistent, gamma-Aminobutyric acid A receptor beta 1 subunit mRNA levels increased 3-fold. Potentiation-induced changes were prevented by N-methyl-D-aspartate receptor blockade. Changes in mRNA abundance in individual cells, with synaptic and glial interactions intact, combine to produce a molecular fingerprint of a potentiated CA1 neuron.

Local translation of classes of mRNAs that are targeted to neuronal dendrites

The functioning of the neuronal dendrite results from a variety of biological processes including mRNA transport to and protein translation in the dendrite. The complexity of the mRNA population in dendrites suggests that specific biological processes are modulated through the regulation of dendritic biology. There are various classes of mRNAs in dendrites whose translation modulates the ability of the dendrite to receive and integrate presynaptic information. Among these mRNAs are those encoding selective transcription factors that function in the neuronal soma and ionotropic glutamate receptors that function on the neuronal membrane. Conclusive evidence that these mRNAs can be translated is reviewed, and identification of the endogenous sites of translation in living dendrites is presented. These data, as well as those described in the other articles resulting from this colloquium, highlight the complexity of dendritic molecular biology and the exquisitely selective and sensitive modulatory role played by the dendrite in facilitating intracellular and intercellular communication.

Immunoprecipitation of mRNA-protein complexes

Immunoprecipitation of mRNA-protein complexes is a method that can be used to study RNA binding protein (RBP)–RNA interactions. In this protocol, an antibody targeting an RBP of interest is used to immunoprecipitate the RBP and any interacting molecules from a cell lysate. Reverse transcription followed by PCR is then used to identify individual mRNAs isolated with the RBP. This method focuses on examining an association between a specific RBP-mRNA complex, and it is best suited for a small scale screening of known or putative binding partners. It can also be used as a second, independent method to verify RBP-mRNA interactions discovered through more universal screening techniques. We describe the immunoprecipitation protocol in practical detail and discuss variations of the method as well as issues associated with it. The procedure takes three days to complete.

Corticosteroid regulation of ion channel conductances and mRNA levels in individual hippocampal CA1 neurons.

Overexposure to corticosteroid hormones is harmful to hippocampal neuronal integrity, likely by perturbation of calcium homeostasis. To identify molecular mechanisms at the single-cell level, we characterized mRNA expression corresponding to voltage- and ligand-gated Ca channels in individual dissociated CA1 neurons in response to long-term corticosterone (CORT) exposure. Predominant mineralocorticoid receptor occupation (ADC-LO group) resulted in low levels of P/Q- and L-type Ca channel mRNAs, high levels of GluR-2 versus GluR-1, and a high ratio of NMDAR-2A to NMDAR-2B mRNA. Corresponding alterations in protein expression were consistent with the restriction of Ca influx. In contrast, additional glucocorticoid receptor occupation (ADC-HI group) altered the expression of these mRNAs in a manner consistent with enhanced Ca influx; interestingly, qualitatively similar alterations were seen in control ADX neurons. Electrophysiological data from the same neurons indicate that Ca current amplitudes also are modulated by CORT, although on a shorter time scale. Finally, principal components analysis (PCA) suggests that neuronal AMPA and NMDA receptor composition may be regulated by MR and GR activation in a complex manner. Therefore, our data implicate molecular events by which CORT may regulate Ca influx into CA1 hippocampal neurons.