Proteolytic degradation and potential role of onconeural protein cdr2 in neurodegeneration.

Cerebellar degeneration-related protein 2 (cdr2) is expressed in the central nervous system, and its ectopic expression in tumor cells of patients with gynecological malignancies elicits immune responses by cdr2-specific autoantibodies and T lymphocytes, leading to neurological symptoms. However, little is known about the regulation and function of cdr2 in neurodegenerative diseases. Because we found that cdr2 is highly expressed in the midbrain, we investigated the role of cdr2 in experimental models of Parkinson's disease (PD). We found that cdr2 levels were significantly reduced after stereotaxic injection of 1-methyl-4-phenylpyridinium (MPP(+)) into the striatum. cdr2 levels were also decreased in the brains of post-mortem PD patients. Using primary cultures of mesencephalic neurons and MN9D cells, we confirmed that MPP(+) reduces cdr2 in tyrosine hydroxylase-positive dopaminergic neuronal cells. The MPP(+)-induced decrease of cdr2 was primarily caused by calpain- and ubiquitin proteasome system-mediated degradation, and cotreatment with pharmacological inhibitors of these enzymes or overexpression of calcium-binding protein rendered cells less vulnerable to MPP(+)-mediated cytotoxicity. Consequently, overexpression of cdr2 rescued cells from MPP(+)-induced cytotoxicity, whereas knockdown of cdr2 accelerated toxicity. Collectively, our findings provide insights into the novel regulatory mechanism and potentially protective role of onconeural protein during dopaminergic neurodegeneration.

Developing global insight into RNA regulation.

Systematic dissection of the activity of RNA-binding proteins (RBPs) has begun to yield global insight into how they work. The paradigm we have used has been the study of Nova, a neuron-specific RBP targeted in an autoimmune neurologic disorder associated with cancer. We have developed a combination of biochemical, genetic, and bioinformatic methods to generate a global understanding of Nova's role as a splicing regulator. Genome-wide identification and validation of Nova target RNAs have yielded unexpected insights into the protein's mechanism of action, its role in neurobiology, and the unique roles RBPs have in the biology of the neuronal synapse. These studies provide us with a paradigm for understanding the role of RBPs in neurons and in disease and, more generally, with the hope that it will be feasible to develop a comprehensive understanding of posttranscriptional regulation.

FMRP RNA targets: identification and validation.

The Fragile X Syndrome is caused by the loss of function of the FMR1 gene (Pieretti et al. 1991. Cell 66, 817-822; O'Donnell & Warren 2002. Annu Rev Neurosci 25, 315-338]. Identification of the RNA targets to which FMRP binds is a key step in understanding the function of the protein and the cellular defects caused by its absence (Darnell et al. 2004 Ment Retard Dev Disabil Res Rev 10, 49-52). Here we discuss the current understanding of FMRP as an RNA-binding protein, the different approaches that have been taken to identify FMRP RNA targets and the relevance of some of these approaches to FMRP biology. In addition, we present evidence that point mutations in the K-homology (KH)1 or KH2 domains of FMRP abrogate its polyribosome association in transfected neuroblastoma cells but that the deletion of the RGG box does not. This suggests that RNA binding by the RGG box of FMRP may mediate other aspects of cellular mRNA metabolism such as mRNA localization or that it may have a role downstream of polyribosome association.

Effective antigen cross-presentation by prostate cancer patients' dendritic cells: implications for prostate cancer immunotherapy.

Despite the potency with which dendritic cells (DCs) are able to utilize the exogenous MHC I antigen cross-presentation pathway to cross-present antigen for the activation of killer T cells in model systems, concern about defects in immune function in cancer patients has led to uncertainty regarding whether immune cells derived from patients can effectively be used to generate tumor vaccines. We have undertaken a careful analysis of the potency of using DCs obtained from prostate cancer patients to cross-present antigen derived from human prostate tumor cells for the activation of antigen-specific T cells. Such DCs can be matured ex vivo into functionally active cells and are capable of cross-presenting influenza antigen derived from internalized apoptotic prostate tumor cells. Importantly, we demonstrate effective stimulation of both CD4+ and CD8+ T cells, as evident by production of IFN-gamma, and the ability of CD8+ T cells to differentiate into effector CTLs. These results, defining conditions in which prostate cancer patient DCs can efficiently utilize the cross-presentation pathway and in which apoptotic tumor can serve as a source of antigen for DCs to activate T cells, demonstrate that this system warrants clinical study as a potential immunotherapy.

Neuronal signaling through alternative splicing: some exons CaRRE.

Alternative splicing represents a mechanism by which a single gene can be used to create proteins with different functions. Neurons use alternative splicing to produce channels with different sequences and biophysical or regulatory properties. O'Donovan and Darnell discuss a mechanism by which neurons can alter channel splicing in response to neuronal activity through a signal generated by calcium and calcium/calmodulin-dependent kinase activity.

Fragile X mental retardation protein targets G quartet mRNAs important for neuronal function.

Loss of fragile X mental retardation protein (FMRP) function causes the fragile X mental retardation syndrome. FMRP harbors three RNA binding domains, associates with polysomes, and is thought to regulate mRNA translation and/or localization, but the RNAs to which it binds are unknown. We have used RNA selection to demonstrate that the FMRP RGG box binds intramolecular G quartets. This data allowed us to identify mRNAs encoding proteins involved in synaptic or developmental neurobiology that harbor FMRP binding elements. The majority of these mRNAs have an altered polysome association in fragile X patient cells. These data demonstrate that G quartets serve as physiologically relevant targets for FMRP and identify mRNAs whose dysregulation may underlie human mental retardation.

Microarray identification of FMRP-associated brain mRNAs and altered mRNA translational profiles in fragile X syndrome.

Fragile X syndrome results from the absence of the RNA binding FMR protein. Here, mRNA was coimmunoprecipitated with the FMRP ribonucleoprotein complex and used to interrogate microarrays. We identified 432 associated mRNAs from mouse brain. Quantitative RT-PCR confirmed some to be >60-fold enriched in the immunoprecipitant. In parallel studies, mRNAs from polyribosomes of fragile X cells were used to probe microarrays. Despite equivalent cytoplasmic abundance, 251 mRNAs had an abnormal polyribosome profile in the absence of FMRP. Although this represents <2% of the total messages, 50% of the coimmunoprecipitated mRNAs with expressed human orthologs were found in this group. Nearly 70% of those transcripts found in both studies contain a G quartet structure, demonstrated as an in vitro FMRP target. We conclude that translational dysregulation of mRNAs normally associated with FMRP may be the proximal cause of fragile X syndrome, and we identify candidate genes relevant to this phenotype.

Dendritic cell maturation is required for the cross-tolerization of CD8+ T cells.

In vivo models have shown that tissue-restricted antigen may be captured by bone marrow-derived cells and cross-presented for the tolerization of CD8+ T cells. Although these studies have shown peripheral tolerization of CD8+ T cells, the mechanism of antigen transfer and the nature of the antigen-presenting cell (APC) remain undefined. We report here the establishment of an in vitro system for the study of cross-tolerance and show that dendritic cells (DCs) phagocytose apoptotic cells and tolerize antigen-specific CD8+ T cells when cognate CD4+ T helper cells are absent. Using this system, we directly tested the "two-signal" hypothesis for the regulation of priming versus tolerance. We found that the same CD83+ myeloid-derived DCs were required for both cross-priming and cross-tolerance. These data suggested that the current model for peripheral T cell tolerance, "signal 1 in the absence of signal 2", requires refinement: the critical checkpoint is not DC maturation, but instead the presence of a third signal, which is active at the DC-CD4+ T cell interface.

Paraneoplastic neurologic disease antigens: RNA-binding proteins and signaling proteins in neuronal degeneration.

Studies of the disorders known as paraneoplastic neurologic degenerations exemplify the successful application of modern molecular biological techniques to diseases, yielding, even for these extremely rare disorders, wide-ranging insight into basic neurobiology, tumor immunity, and autoimmune neurologic disease. Immune responses to paraneoplastic neurologic degeneration antigens, also called onconeural antigens, have been exploited to clone and characterize a number of neuron-specific proteins, including several RNA-binding proteins and new kinds of signaling molecules. The biology and functions of these proteins are reviewed, and a model in which their functions are related to the pathogenesis of autoimmune neurologic disease is discussed.

The splice of life: alternative splicing and neurological disease.

Splicing of pre-messenger RNA is regulated differently in the brain compared with other tissues. Recognition of aberrations in splicing events that are associated with neurological disease has contributed to our understanding of disease pathogenesis in some cases. Neuron-specific proteins involved in RNA splicing and metabolism are also affected in several neurological disorders. These findings have begun to bridge what we know about the mechanisms regulating neuron-specific splicing and our understanding of neural function and disease.

Involvement of the TRAP220 component of the TRAP/SMCC coactivator complex in embryonic development and thyroid hormone action.

The TRAP220 component of the TRAP/SMCC complex, a mammalian homologof the yeast Mediator that shows diverse coactivation functions, interacts directly with nuclear receptors. Ablation of the murine Trap220 gene revealed that null mutants die during an early gestational stage with heart failure and exhibit impaired neuronal development with extensive apoptosis. Primary embryonic fibroblasts derived from null mutants show an impaired cell cycle regulation and a prominent decrease of thyroid hormone receptor function that is restored by ectopic TRAP220 but no defect in activation by Gal4-RARalpha/RXRalpha, p53, or VP16. Moreover, haploinsufficient animals show growth retardation, pituitary hypothyroidism, and widely impaired transcription in certain organs. These results indicate that TRAP220 is essential for a wide range of physiological processes but also that it has gene- and activator-selective functions.

A brain-enriched polypyrimidine tract-binding protein antagonizes the ability of Nova to regulate neuron-specific alternative splicing.

The Nova paraneoplastic antigens are neuron-specific RNA binding proteins that participate in the control of alternative splicing. We have used the yeast two-hybrid system to isolate Nova interacting proteins and identify an RNA binding protein that is closely related to the polypyrimidine tract-binding protein (PTB). The expression of this protein, brPTB, is enriched in the brain, where it is expressed in glia and neurons. brPTB interacts with Nova proteins in cell lines and colocalizes with Nova within neuronal nuclei. We previously found that Nova binds to a pyrimidine-rich RNA element present upstream of an alternatively spliced exon, E3A, in glycine receptor alpha2 (GlyRalpha2) pre-mRNA, and this binding is implicated in Nova-dependent regulation of splicing. Cotransfection assays with a GlyRalpha2 minigene demonstrate that brPTB antagonizes the action of Nova to increase utilization of GlyRalpha2 E3A. brPTB binds to a 90-nt GlyRalpha2 RNA adjacent to the Nova binding site, but with an affinity that is more than 10-fold lower than Nova. When a putative binding site for brPTB on the GlyRalpha2 RNA is mutated, binding is abolished and the inhibitory effect on Nova-dependent exon selection disappears. These results suggest that brPTB is a tissue-restricted RNA binding protein that interacts with and inhibits the ability of Nova to activate exon selection in neurons.

The tetranucleotide UCAY directs the specific recognition of RNA by the Nova K-homology 3 domain.

The Nova family of proteins are target antigens in the autoimmune disorder paraneoplastic opsoclonus-myoclonus ataxia and contain K-homology (KH)-type RNA binding domains. The Nova-1 protein has recently been shown to regulate alternative splicing of the alpha2 glycine receptor subunit pre-mRNA by binding to an intronic element containing repeats of the tetranucleotide UCAU. Here, we have used selection-amplification to demonstrate that the KH3 domain of Nova recognizes a single UCAY element in the context of a 20-base hairpin RNA; the UCAY tetranucleotide is optimally presented as a loop element of the hairpin scaffold and requires protein residues C-terminal to the previously defined KH domain. These results suggest that KH domains in general recognize tetranucleotide motifs and that biological RNA targets of KH domains may use either RNA secondary structure or repeated sequence elements to achieve high affinity and specificity of protein binding.

Cdr2, a target antigen of naturally occuring human tumor immunity, is widely expressed in gynecological tumors.

The paraneoplastic neurological disorders provide perhaps the best known example of naturally occurring tumor immunity in humans. For example, patients with paraneoplastic cerebellar degeneration (PCD) appear to suppress the growth of occult breast or ovarian tumors that express a neuronal antigen termed cdr2. PCD patients harbor cdr2-specific CTLs in their peripheral blood, and these cells are likely mediators of the tumor suppression. Whereas cdr2 therefore appears to be the target of an effective immune response in patients with PCD, the general relevance to cancer patients has been unclear, due in part to reports indicating that cdr2 is not expressed in tumors obtained from neurologically normal patients. We have reexamined this question, and we find that cdr2 is widely expressed in such tumors, indicating that cdr2 is in fact an important tumor antigen in the general population of breast and ovarian cancer patients.

Nova-1 regulates neuron-specific alternative splicing and is essential for neuronal viability.

We have combined genetic and biochemical approaches to analyze the function of the RNA-binding protein Nova-1, the paraneoplastic opsoclonus-myoclonus ataxia (POMA) antigen. Nova-1 null mice die postnatally from a motor deficit associated with apoptotic death of spinal and brainstem neurons. Nova-1 null mice show specific splicing defects in two inhibitory receptor pre-mRNAs, glycine alpha2 exon 3A (GlyRalpha2 E3A) and GABA(A) exon gamma2L. Nova protein in brain extracts specifically bound to a previously identified GlyRalpha2 intronic (UCAUY)3 Nova target sequence, and Nova-1 acted directly on this element to increase E3A splicing in cotransfection assays. We conclude that Nova-1 binds RNA in a sequence-specific manner to regulate neuronal pre-mRNA alternative splicing; the defect in splicing in Nova-1 null mice provides a model for understanding the motor dysfunction in POMA.

Sequence-specific RNA binding by a Nova KH domain: implications for paraneoplastic disease and the fragile X syndrome.

The structure of a Nova protein K homology (KH) domain recognizing single-stranded RNA has been determined at 2.4 A resolution. Mammalian Nova antigens (1 and 2) constitute an important family of regulators of RNA metabolism in neurons, first identified using sera from cancer patients with the autoimmune disorder paraneoplastic opsoclonus-myoclonus ataxia (POMA). The structure of the third KH domain (KH3) of Nova-2 bound to a stem loop RNA resembles a molecular vise, with 5'-Ura-Cyt-Ade-Cyt-3' pinioned between an invariant Gly-X-X-Gly motif and the variable loop. Tetranucleotide recognition is supported by an aliphatic alpha helix/beta sheet RNA-binding platform, which mimics 5'-Ura-Gua-3' by making Watson-Crick-like hydrogen bonds with 5'-Cyt-Ade-3'. Sequence conservation suggests that fragile X mental retardation results from perturbation of RNA binding by the FMR1 protein.

Detection and treatment of activated T cells in the cerebrospinal fluid of patients with paraneoplastic cerebellar degeneration.

Patients with paraneoplastic cerebellar degeneration (PCD) offer the opportunity to explore the mechanisms underlying tumor immunity and immune-mediated neuronal degeneration. Cytotoxic T lymphocytes (CTLs) specific for the PCD onconeural antigen cdr2 found in the blood of patients with PCD are likely to be effectors of PCD tumor immunity. Here, we suggest a role for CTLs in the autoimmune destruction of Purkinje neurons. More than 75% of the cells obtained from the cerebrospinal fluid (CSF) of PCD patients were CD3+ alphabeta T cells. In patients with active/progressive disease, 20% to 40% of CSF cells were activated T cells, and the CD4+ helper cells were Th1-type cells. Three PCD patients were given tacrolimus, a specific inhibitor of activated T cells, which markedly reduced these cells in the CSF. Tacrolimus also reduced the number of activated cdr2-specific CTLs in the peripheral blood, but did not lead to tumor recurrence. We suggest that activated cdr2-specific CTLs in the CSF contribute to Purkinje degeneration in PCD, and that tacrolimus therapy may benefit patients with paraneoplastic neurological disease and other T cell-mediated autoimmune neurological disorders.

The cytoplasmic Purkinje onconeural antigen cdr2 down-regulates c-Myc function: implications for neuronal and tumor cell survival.

Paraneoplastic cerebellar degeneration (PCD) is a disorder in which breast or ovarian tumors express an onconeural antigen termed cdr2, which normally is expressed in cerebellar Purkinje neurons. This leads to an immune response to cdr2 that is associated with tumor immunity and autoimmune cerebellar degeneration. We have found that cdr2, a cytoplasmic protein harboring a helix-leucine zipper (HLZ) motif, interacts specifically with the HLZ motif of c-Myc. Both proteins colocalize in the cytoplasm of adult cerebellar Purkinje neurons, and coimmunoprecipitate from tumor cell lines and cerebellar extracts. cdr2 down-regulates c-Myc-dependent transcription in cotransfection assays, and redistributes Myc protein in the cytoplasm. Disease antisera from six of six PCD patients specifically blocked the interaction between cdr2 and c-Myc in vitro. These data indicate that cdr2 normally sequesters c-Myc in the neuronal cytoplasm, thereby down-regulating c-Myc activity, and suggest a mechanism whereby inhibition of cdr2 function by autoantibodies in PCD may contribute to Purkinje neuronal death.

Mammalian ELAV-like neuronal RNA-binding proteins HuB and HuC promote neuronal development in both the central and the peripheral nervous systems.

Hu proteins are mammalian embryonic lethal abnormal visual system (ELAV)-like neuronal RNA-binding proteins that contain three RNA recognition motifs. Although Drosophila ELAV is required for the correct differentiation and survival of neurons, the roles played by the Hu genes in the mammalian nervous system remain largely unknown. To explore the in vivo functions of mouse Hu proteins, we overexpressed them in rat pheochromocytoma PC12 cells, where they induced neuronal phenotype in the absence of nerve growth factor. We have characterized the functions of various forms of mHuB and mHuC bearing point mutations or deletions. Mutants of mHuC that had amino acid exchanges in the RNP1 domain of the first or second RNA recognition motifs (RRMs) lost biologic activity as well as RNA-binding activity. In addition, the mutants containing only the third RRM failed to induce the neuronal phenotype in PC12 cells and inhibited the biologic activity of cotransfected wild-type mHuB and mHuC, thus acting as a dominant-negative form. However, these mutants could not suppress the nerve growth factor-induced differentiation of PC12 cells. Further, we misexpressed wild-type and dominant-negative Hu in E9.5 mouse embryos, by using electroporation into the neural tube at the level of the rhombencephalon. mHuB and mHuC induced the ectopic expression of neuronal markers, whereas the dominant-negative forms of mHuB and mHuC suppressed the differentiation of central nervous system motor neurons. From these results, we suggest that Hu proteins are required for neuronal differentiation in the mammalian nervous system.