A common polymorphism in serotonin receptor 1B mRNA moderates regulation by miR-96 and associates with aggressive human behaviors.

Non-coding regulatory elements can transduce the human genome's response to environmental stimuli. Thus, there is a possibility that variation in non-coding regulatory elements may underlie some of the diversity in human behavior. However, this idea has remained largely untested due to the difficulty in accurately identifying regulatory elements in the 98% of the human genome that does not encode protein. The recent recognition that small trans-acting RNAs anneal to mRNA and regulate gene expression provides a means to identify and test such variants. Here, we show that microRNA-directed silencing of mRNA can be attenuated by a common human polymorphism. We have identified an element (A-element) within serotonin receptor 1B (HTR1B) mRNA that confers repression by miR-96. The repressive activity of this element is attenuated by a common human variant (G-element) that disrupts a nucleotide critical for its interaction with miR-96. Because deletion of the HTR1B gene leads to an aggressive phenotype in mice, we hypothesized an association between the A/G polymorphism and aggressive phenotypes in a sample of 359 college students. As predicted, individuals homozygous for the ancestral A-element reported more conduct-disorder behaviors than individuals with the G-element. Our studies suggest that such functional variants may be common and may help to refine the search for genes involved in complex behavioral disorders.

Overexpression of HuD, but not of its truncated form HuD I+II, promotes GAP-43 gene expression and neurite outgrowth in PC12 cells in the absence of nerve growth factor.

We have previously shown that the RNA-binding protein HuD binds to a regulatory element in the growth-associated protein (GAP)-43 mRNA and that this interaction involves its first two RNA recognition motifs (RRMs). In this study, we investigated the functional significance of this interaction by overexpression of human HuD protein (pcHuD) or its truncated form lacking the third RRM (pcHuD I+II) in PC12 cells. Morphological analysis revealed that pcHuD cells extended short neurites containing GAP-43-positive growth cones in the absence of nerve growth factor (NGF). These processes also contained tubulin and F-actin filaments but were not stained with antibodies against neurofilament M protein. In correlation with this phenotype, pcHuD cells contained higher levels of GAP-43 without changes in levels of other NGF-induced proteins, such as SNAP-25 and tau. In mRNA decay studies, HuD stabilized the GAP-43 mRNA, whereas HuD I+II did not have any effect either on GAP-43 mRNA stability or on the levels of GAP-43 protein. Likewise, pcHuD I+II cells showed no spontaneous neurite outgrowth and deficient outgrowth in response to NGF. Our results indicate that HuD is sufficient to increase GAP-43 gene expression and neurite outgrowth in the absence of NGF and that the third RRM in the protein is critical for this function.

The identification of an endonuclease that cleaves within an HuR binding site in mRNA.

Messenger RNAs (mRNAs) that contain U-rich elements are targeted for rapid decay. Selective inhibition of this decay results in a rapid increase in steady state level. Thus, this is an important regulatory step in gene expression. Previously, we have found that these mRNAs are selectively stabilized by a specific mRNA binding protein called HuR. The mechanism of action of HuR is not well understood. It has been postulated that HuR stabilizes mRNA by the displacement or inhibition of factors that specifically cleave or deadenyl-ate these mRNAs. In this paper, we report the identification and characterization of a novel endo-nuclease that cleaves within an HuR binding site in p27kip1 mRNA. The specificity of this endonuclease and its inhibition by HuR argue for it playing a role in the postranscriptional regulation of gene expression.

HuR, a novel target of anti-Hu antibodies, is expressed in non-neural tissues.

Paraneoplastic encephalomyelitis (PEM) is characterized by a diverse set of clinical signs that are limited to the nervous system. The serologic hallmark of PEM is the presence of circulating autoantibodies, collectively referred to as 'anti-Hu,' which immunoreact specifically with members of the Elav protein family. Until recently, the ELAV antigens were only detected in neurons, thus strongly supporting a role for anti-Hu antibodies in the selective neural tissue injury in PEM. The identification of HuR, however, a new member with a broad, non-neural pattern of RNA expression, raises several fundamental questions regarding PEM. First, why are non-neural tissues spared in PEM? Second, why is PEM predominantly associated with neuroendocrine tumors? To begin addressing these questions, we sought to determine whether the antibody response to HuR differs from the neural-specific counterparts in patients with PEM, and to characterize the protein expression pattern of this novel antigen in peripheral tissues and tumors. Using sera from 11 patients with Hu-positive PEM, we found that the majority of samples (73%) were weakly or non-reactive for recombinant HuR on Western blot, in contrast to consistently strong immunoreactivity with the neural-specific members HuD and Hel-N1. We also demonstrate that HuR is expressed at the protein level in both non-neural tissues and non-neuroendocrine tumors. These findings suggest that immunoreactive differences among Elav family members may contribute to the neural-restrictive pattern of tissue injury in patients with PEM.

Hu neuronal proteins are expressed in proliferating neurogenic cells.

We have utilized immunochemical techniques to investigate the developmental expression of the Hu proteins, a neuron-specific family of RNA binding proteins in vertebrates. Previous work suggests that these proteins may play an important role in neuronal development and maintenance. For the present study, we developed a monoclonal antibody (MAb 16A11) that binds specifically to an epitope present in gene products of all known Hu genes, including HuD, HuC, and Hel-N1. Using brief pulses (1-2 h) of the DNA precursor analog bromodeoxyuridine (BrdU) in conjunction with MAb 16A11, we observed Hu+/BrdU+ cells in nascent sensory and sympathetic ganglia in vivo, and in populations of cultured neural crest cells. In addition, a few Hu+ cells were ambiguously BrdU+ in the neural tube. We conclude that Hu+ cells first appear in avian neurogenic populations immediately before neuronal birthdays in the peripheral nervous system, and at the time of withdrawal from the mitotic cycle in the central nervous system. Consistent with these conclusions, we have also observed neural crest-derived cells that are both Hu+ and in metaphase of the cell cycle. We suggest that Hu proteins function early in neurogenic differentiation.

Mapping of the gene coding for a paraneoplastic encephalomyelitis antigen (HuD) to human chromosome site 1p34.

A cDNA clone coding for a human brain protein (HuD), which is highly homologous to the Drosophila neuronal determination protein Elav and elicits antibody formation in a high proportion of patients with paraneoplastic encephalomyelitis, was used to isolate a lambda phage recombinant clone, including a large fragment of the relevant human genomic region. The fragment proved to be an efficient probe for the precise subregional mapping of the gene by molecular in situ hybridization onto extended human prometaphase chromosomes. Analysis of banded metaphases with clear hybridization signals pointed unequivocally to the localization of the HuD gene to human chromosome band 1p34.

Cloning and characterization of a Lambert-Eaton myasthenic syndrome antigen.

Lambert-Eaton myasthenic syndrome is a paraneoplastic neuromuscular disorder in which an immune response directed against a small-cell lung tumor crossreacts with antigens in the neuromuscular junction. To isolate and characterize the antigens, we screened a human fetal brain expression library with a high-titer serum from a patient with Lambert-Eaton myasthenic syndrome. This screening resulted in the isolation of a complementary DNA clone encoding an antigen we call myasthenic syndrome antigen B (MysB). Approximately 43% (3 of 7) of Lambert-Eaton myasthenic syndrome sera specifically recognized MysB fusion protein, whereas none of 34 control sera did. The predicted amino acid sequence of this clone shows a high degree of homology to the beta subunit of calcium channel complexes. The MysB pre-messenger RNA is alternatively spliced to yield 3 forms of the protein differing in the domain between two highly conserved alpha-helical segments.

The expression of the Hu (paraneoplastic encephalomyelitis/sensory neuronopathy) antigen in human normal and tumor tissues.

Using immunohistochemistry or Western blot analysis, the authors have studied the expression of the Hu antigen (a neuronal protein identified by the serum of patients with small cell lung cancer and paraneoplastic encephalomyelitis/sensory neuronopathy) in normal human tissues and 115 tumors of different histologic types. In normal tissue, the Hu antigen is highly restricted to the nervous system. In lung tumors, the Hu antigen is restricted in its expression to all small cell carcinomas. A few other neuroendocrine-related tumors, especially neuroblastomas (50%), also express the antigen.

Detection of the anti-Hu antibody in specific regions of the nervous system and tumor from patients with paraneoplastic encephalomyelitis/sensory neuronopathy.

We studied the nervous systems and tumors of five patients with anti-Hu-positive paraneoplastic encephalomyelitis/sensory neuronopathy (PEM/PSN) to determine if the autoantibody found in the serum and CSF was also present in those tissues. Immunohistochemical studies of the nervous system revealed the presence of IgG bound predominantly to the nuclei of most of the neurons and the cytoplasm of some glial cells. IgG was also present to a lesser degree in the neuropil. In brains of patients who died of cancer without the paraneoplastic syndrome, IgG was present in the immediate perivascular areas and to a very limited degree in the neuropil. There was no IgG in neurons, and in only some of the controls a few glial cells showed IgG immunoreactivity in the cytoplasm. The amount of anti-Hu IgG relative to total IgG in various brain regions and tumor was determined by quantitative Western blot analysis. The proportion of anti-Hu IgG was greater in some areas of the brain and tumor than in serum and CSF. Control brains did not contain anti-Hu IgG. There was a limited correlation among (1) the principal clinical symptoms, (2) regions of major tissue injury, and (3) the quantitative anti-Hu IgG distribution. We conclude that although the role of the antibody in the pathogenesis of the disease is still uncertain, its specific localization in the nervous system and tumor suggests an immunologic etiology of this paraneoplastic syndrome.

HuD, a paraneoplastic encephalomyelitis antigen, contains RNA-binding domains and is homologous to Elav and Sex-lethal.

A neuronal antigen (HuD) recognized by the sera of patients with antibody-associated paraneoplastic encephalomyelitis has been isolated by screening a lambda cerebellar expression library. The recombinant antigen provides an unambiguous assay for this rare condition associated with small cell lung cancer. The recombinant antigen has been used to identify specific infiltrating lymphocytes in tumors and affected brain tissues of patients with antibody-associated paraneoplastic encephalomyelitis and sensory neuronopathy. HuD mRNA is uniquely expressed in brain tissue. The HuD protein shows a remarkable homology to the Drosophila proteins Elav and Sex-lethal and is likely to play a role in neuron-specific RNA processing.

Antiserum from a patient with cerebellar degeneration identifies a novel protein in Purkinje cells, cortical neurons, and neuroectodermal tumors.

The serum and cerebrospinal fluid of a patient (NB) with subacute cerebellar degeneration were found to contain a novel antineuronal autoantibody (anti-Nb). Using this antibody, we have identified and characterized antigens present in a subset of neurons in the CNS and in some neuroectodermal tumor lines. Anti-Nb antibody bound to antigens of Mr 150, 120, and 65 kDa in Western blots using extracts of human cerebellar Purkinje cells or human cerebral cortical neurons. Immunohistochemistry demonstrated relatively specific binding of anti-Nb IgG to Purkinje cells in sections of human cerebellum and to some neocortical neurons, especially those in layer VI. Because of the association of cerebellar degeneration with occult malignancies, we screened a number of tumor cell lines for immunoreactivity to anti-Nb antibody; only tumor lines of neuroectodermal origin (melanoma, small-cell lung cancer, and neuroblastoma) expressed the Nb antigen. Anti-Nb antibody thus identifies neuronal and tumor cell antigens that appear to be unique in size and distribution of expression.

Cloning of a leucine-zipper protein recognized by the sera of patients with antibody-associated paraneoplastic cerebellar degeneration.

Antibody-associated paraneoplastic cerebellar degeneration (the Yo syndrome) is an uncommon disorder in which an immune response is specifically directed against tumor tissue and the cerebellum. Screening of a lambda expression library has resulted in the isolation of cDNA clones that encode the major antigen recognized by serum from these patients. The fusion protein produced by the cDNA clones provides the basis of a simple diagnostic assay for this neurological syndrome. The occurrence of leucine-zipper and zinc-finger motifs in the predicted open reading frame suggests that this protein plays a role in the regulation of gene expression.

Anti-Ri: an antibody associated with paraneoplastic opsoclonus and breast cancer.

The serum and cerebrospinal fluid (CSF) of 8 women with ataxia, 6 of whom also had eye movement abnormalities believed to be opsoclonus, were found to contain a highly specific antineuronal antibody we call anti-Ri. Seven of the 8 women also had or developed cancer: carcinoma of the breast in 5, adenocarcinoma in an axillary lymph node in 1, and carcinoma of the fallopian tube in 1. Four patients presented with the neurological disorder; the cancer was diagnosed first in the other 4. Immunohistochemical studies using serum or CSF from all 8 patients revealed a highly specific antibody interaction with central nervous system neuronal nuclei but not with glial or other cells; the titer ranged from 1:5,000 to 1:320,000 in serum and from 1:2,000 to 1:16,000 in CSF. Biotinylated IgG from the patients' serum reacted with the tumors of 3 of 4 patients with anti-Ri antibody but not with breast cancers from patients without anti-Ri antibody. Immunoblots against cerebral cortex neuronal extracts identified protein antigens of 55-kd and 80-kd relative molecular mass. Serum titers by immunoblot ranged from 1:500 to more than 1:40,000 and CSF titers, from 1:10 to 1:2,000. The relative amount of anti-Ri was always higher in CSF than in serum. The antibody was not present in sera from normal individuals; patients with breast cancer without opsoclonus; other patients with opsoclonus; or patients with other paraneoplastic syndromes related to breast, ovarian, or small-cell lung cancer. We conclude that the presence of anti-Ri antibody identifies a subset of patients with paraneoplastic ataxia and eye movement disorders (opsoclonus) who usually suffer from breast or other gynecological cancer; the antibody when present is a useful marker for an underlying malignancy.

Expression of transforming growth factor alpha in human gliomas.

Expression of transforming growth factor alpha (TGF alpha) is frequently associated with the development of human and animal tumors. Using a sensitive immunohistochemical assay, which can be applied on formalin-fixed, paraffin-embedded tissue, we have examined the expression of TGF alpha in 71 human gliomas (63 untreated and 8 recurrent tumors). Tumors were graded by a 3-grade-system: grade I = low grade gliomas, grade II = anaplastic gliomas and grade III = glioblastomas. A strong positive correlation between tumor grade and extent of TGF alpha expression was found (P less than 0.0001). Polymerase chain reaction (PCR) was used to amplify the fourth exon of the TGF alpha gene of 8 glioma DNA specimens and increasing amounts of normal human DNA, which served as a standard. No amplification of the TGF alpha gene copy number in tumors could be detected.

Selective expression of Purkinje-cell antigens in tumor tissue from patients with paraneoplastic cerebellar degeneration.

Paraneoplastic cerebellar degeneration is a rare syndrome that occurs in patients with gynecologic cancer and is characterized by widespread loss of Purkinje cells. To determine whether Purkinje-cell antigens are selectively expressed in the tumors of patients with the syndrome, we examined tumor tissue from 10 patients whose serum contained anti-Purkinje-cell (anti-Yo) antibodies. The origins of the cancers were the breast (five patients), ovary (three), endometrium (one), and fallopian tube (one). We used as controls tumor tissue from 11 patients with ovarian cancer and 10 patients with breast cancer who were neurologically normal. Using immunohistochemical and Western blot analysis, we found that Purkinje-cell antigens were expressed in all the tumors from the 10 patients with paraneoplastic cerebellar degeneration but in none of the tumors from the 21 neurologically normal patients. When IgG from patients with paraneoplastic cerebellar degeneration was affinity-purified to cerebellar Purkinje-cell antigen, immunohistochemical analysis showed that it reacted specifically with the tumor tissue from those patients. We conclude that in patients with paraneoplastic cerebellar degeneration, the anti-Yo antibody results from an immune response to neural antigens expressed by the gynecologic tumors in the patients.

Detection of the anti-Hu antibody in the serum of patients with small cell lung cancer--a quantitative western blot analysis.

We looked for the presence of the anti-Hu antibody in the sera from 50 normal subjects; 44 patients with small cell lung cancer, not associated with paraneoplastic disease; and 25 patients with small cell lung cancer associated with paraneoplastic sensory neuropathy, encephalomyelitis, or both. Using the avidin-biotin immunoperoxidase method and a highly sensitive quantitative Western blot analysis, the anti-Hu antibody was not detected in the 50 normal human sera. Seven of the 44 patients with small cell lung cancer but no paraneoplastic syndrome had detectable levels (average titer, 76 U/ml) of anti-Hu antibody on Western blot. These levels are significantly lower than the average titer of the 25 patients who had small cell lung cancer and paraneoplastic sensory neuropathy or encephalomyelitis (average titer, 4,592 U/ml). In the group with nonparaneoplastic small cell lung cancer (low anti-Hu titer) there was a predominance of women (5 women: 2 men), and all patients had "limited" disease when diagnosed. In the antibody-negative group the sex ratio was 16 women to 21 men and 51% of the patients had "extensive" disease. None of the 7 patients with a low-titer anti-Hu antibody developed a paraneoplastic syndrome by the time of writing. The anti-Hu antibody appears, when present, to be a good marker for small cell lung cancer and, when present at high titer, for small cell lung cancer associated with a paraneoplastic syndrome.

Characterization of a cDNA encoding a 34-kDa Purkinje neuron protein recognized by sera from patients with paraneoplastic cerebellar degeneration.

Paraneoplastic cerebellar degeneration is a neurological disorder of unknown cause occurring in patients with an identified or occult cancer. An autoimmune etiology is likely since autoantibodies directed against the Purkinje cells of the cerebellum have been found in the serum and cerebrospinal fluid of some patients. Two Purkinje cell-specific antigens are recognized by these autoantibodies, a major antigen of 62 kDa (CDR 62, cerebellar degeneration-related 62-kDa protein) and a minor antigen of 34 kDa (CDR 34). Our previous studies have described the isolation and characterization of a human cerebellar cDNA that encodes an epitope recognized by sera from patients with paraneoplastic cerebellar degeneration. We have now established by two independent methods that this gene is uniquely expressed in Purkinje cells of the cerebellum and corresponds to the minor antigen CDR 34. This antigen is also expressed in tumor tissue from a patient with paraneoplastic cerebellar degeneration.