Behavioral phenotypes in males with XYY and possible role of increased NLGN4Y expression in autism features.

The male sex chromosome disorder, 47,XYY syndrome (XYY), is associated with increased risk for social-emotional difficulties, attention-deficit hyperactivity disorder (ADHD) and autism spectrum disorder (ASD). We hypothesize that increased Y chromosome gene copy number in XYY leads to overexpression of Y-linked genes related to brain development and function, thereby increasing risk for these phenotypes. We measured expression in blood of two Y genes NLGN4Y and RPS4Y in 26 boys with XYY and 11 male controls and evaluated whether NLGN4Y expression correlates with anxiety, ADHD, depression and autistic behaviors (from questionnaires) in boys with XYY. The XYY cohort had increased risk of ASD behaviors on the social responsiveness scale (SRS) and increased attention deficits on the Conners' DSM-IV inattention and hyperactive scales. In contrast, there was no increase in reported symptoms of anxiety or depression by the XYY group. Peripheral expression of two Y genes in boys with XYY vs. typically developing controls was increased twofold in the XYY group. Results from the SRS total and autistic mannerisms scales, but not from the attention, anxiety or depression measures, correlated with peripheral expression of NLGN4Y in boys with XYY. Males with XYY have social phenotypes that include increased risk for autism-related behaviors and ADHD. Expression of NLGN4Y, a gene that may be involved in synaptic function, is increased in boys with XYY, and the level of expression correlates with overall social responsiveness and autism symptoms. Thus, further investigation of NLGN4Y as a plausible ASD risk gene in XYY is warranted.

Cyclin D1 repressor domain mediates proliferation and survival in prostate cancer.

Regulation of the androgen receptor (AR) is critical to prostate cancer (PCa) development; therefore, AR is the first line therapeutic target for disseminated tumors. Cell cycle-dependent accumulation of cyclin D1 negatively modulates the transcriptional regulation of AR through discrete, CDK4-independent mechanisms. The transcriptional corepressor function of cyclin D1 resides within a defined motif termed repressor domain (RD), and it was hypothesized that this motif could be utilized as a platform to develop new strategies for blocking AR function. Here, we demonstrate that expression of the RD peptide is sufficient to disrupt AR transcriptional activation of multiple, prostate-specific AR target genes. Importantly, these actions are sufficient to specifically inhibit S-phase progression in AR-positive PCa cells, but not in AR-negative cells or tested AR-positive cells of other lineages. As expected, impaired cell cycle progression resulted in a suppression of cell doubling. Additionally, cell death was observed in AR-positive cells that maintain androgen dependence and in a subset of castrate-resistant PCa cells, dependent on Akt activation status. Lastly, the ability of RD to cooperate with existing hormone therapies was examined, which revealed that RD enhanced the cellular response to an AR antagonist. Together, these data demonstrate that RD is sufficient to disrupt AR-dependent transcriptional and proliferative responses in PCa, and can enhance efficacy of AR antagonists, thus establishing the impetus for development of RD-based mimetics.

Molecular pathogenesis of spinal and bulbar muscular atrophy.

Studies of the molecular pathogenesis of spinal and bulbar muscular atrophy, as well as of the other polyglutamine repeat diseases, has led to an understanding of the role of protein accumulation in disease pathogenesis. Aggregation of the expanded repeat androgen receptor (AR), rather than playing a pathogenic role, likely reflects the insoluble nature of the misfolded AR. Proteolytic processing of the expanded AR at various stages of its metabolism may contribute to cellular toxicity through the enhancement of AR insolubility, and potentially through the disruption of normal proteolytic degradation processes. The finding that molecular chaperones not only promote solubility, but also enhance the degradation of expanded polyglutamines as well, make them promising targets for therapeutic development. Understanding the role of ligand binding in expanded AR metabolism may provide additional avenues of therapeutic manipulation as well.

Expression of expanded repeat androgen receptor produces neurologic disease in transgenic mice.

Spinal and bulbar muscular atrophy (SBMA) is a motor neuron disease caused by the expansion of a polyglutamine tract within the androgen receptor. This disease is unusual among the polyglutamine diseases in that it involves lower motor and sensory neurons, with relative sparing of other brain structures. We describe the development of transgenic mice, created with a truncated, highly expanded androgen receptor driven by the neurofilament light chain promoter, which develop many of the motor symptoms of SBMA. In addition, transgenic mice created with the prion protein promoter develop widespread neurologic disease, reminiscent of juvenile forms of other polyglutamine diseases. Thus, in these experiments, the distribution of neurologic symptoms depends on the expression level and pattern of the promoter used, rather than on specific characteristics of androgen receptor metabolism or function. The transgenic mice described here develop neuronal intranuclear inclusions (NIIs), a hallmark of SBMA and the other polyglutamine diseases. We have shown these inclusions to be ubiquitinated and to sequester molecular chaperones, components of the 26S proteasome and the transcriptional activator CREB-binding protein. Apart from the presence of NIIs, evidence of neuropathology or neurogenic muscle atrophy was absent, suggesting that the neurologic phenotypes observed in these mice were the result of neuronal dysfunction rather than neuronal degeneration. These mice will provide a useful resource for characterizing specific aspects of motor neuron dysfunction, and for testing therapeutic strategies for this and other polyglutamine diseases.

CREB-binding protein sequestration by expanded polyglutamine.

Spinal and bulbar muscular atrophy (SBMA) is one of eight inherited neurodegenerative diseases known to be caused by CAG repeat expansion. The expansion results in an expanded polyglutamine tract, which likely confers a novel, toxic function to the affected protein. Cell culture and transgenic mouse studies have implicated the nucleus as a site for pathogenesis, suggesting that a critical nuclear factor or process is disrupted by the polyglutamine expansion. In this report we present evidence that CREB-binding protein (CBP), a transcriptional co-activator that orchestrates nuclear response to a variety of cell signaling cascades, is incorporated into nuclear inclusions formed by polyglutamine-containing proteins in cultured cells, transgenic mice and tissue from patients with SBMA. We also show CBP incorporation into nuclear inclusions formed in a cell culture model of another polyglutamine disease, spinocerebellar ataxia type 3. We present evidence that soluble levels of CBP are reduced in cells expressing expanded polyglutamine despite increased levels of CBP mRNA. Finally, we demonstrate that over-expression of CBP rescues cells from polyglutamine-mediated toxicity in neuronal cell culture. These data support a CBP-sequestration model of polyglutamine expansion disease.

Overexpression of Bcl-2 is neuroprotective after experimental brain injury in transgenic mice.

The cell death regulatory protein, Bcl-2, has been suggested to participate in the pathophysiology of various neurological disorders, including traumatic brain injury (TBI). The cognitive function and histopathologic sequelae after controlled cortical impact brain injury were evaluated in transgenic (TG) mice that overexpress human Bcl-2 protein (n = 13) and their wild type (WT) controls (n = 9). Although brain-injured Bcl-2 TG mice exhibited similar posttraumatic deficits in a Morris water maze (MWM) test of spatial memory as their WT counterparts at 1 week postinjury, the preinjury learning ability of Bcl-2 TG mice was impaired significantly compared with their WT littermates (P < 0.05). In contrast, histopathologic analysis revealed significantly attenuated tissue loss in the ipsilateral hemisphere (p < 0.01) and decreased tissue loss in ipsilateral hippocampal area CA3 (P < 0.001) and the dentate gyrus (P < 0.01) in brain-injured Bcl-2 TG mice compared with brain-injured WT mice. Immunohistochemical evaluation of glial fibrillary acidic protein also revealed a significant decrease in reactive astrocytosis in the ipsilateral dorsal thalamus (P < 0.05) and the ventral thalamus (P < 0.01) in brain-injured Bcl-2 TG mice. These results suggest that overexpression of Bcl-2 protein may play a protective role in neuropathologic sequelae after TBI.

Androgen receptor mutation in Kennedy's disease.

Kennedy's disease is an X-linked form of motor neuron disease caused by an expanded polyglutamine repeat in the androgen receptor. While the expansion mutation causes some loss of transcriptional activity by the androgen receptor, the predominant effect of expansion is probably a toxic gain of function, similar to the mechanism of other polyglutamine expansion diseases. Features of the neurodegenerative phenotype of Kennedy's disease have now been reproduced in transgenic animals and neuronal cell culture. Nuclear inclusions of mutant androgen receptor protein are found in these model systems and in autopsy samples from patients with Kennedy's disease.

Caspase-3 cleaves the expanded androgen receptor protein of spinal and bulbar muscular atrophy in a polyglutamine repeat length-dependent manner.

Spinal and bulbar muscular atrophy (SBMA) is one of a group of human inherited neurodegenerative diseases caused by polyglutamine expansion. There is increasing evidence that generation of truncated proteins containing an expanded polyglutamine tract may be an important step in the pathogenesis of these disorders. We have previously demonstrated that the SBMA gene product, the androgen receptor (AR) protein, is toxic when truncated. We now report that in vitro translated full-length AR proteins containing different sized polyglutamine repeats (24, 65 and 97 repeats, respectively) are specifically cleaved by recombinant caspase-3, liberating a polyglutamine containing fragment, and that the susceptibility to cleavage is polyglutamine repeat length-dependent. These findings suggest that AR protein is one of the "death substrates" cleaved by caspase-3 and that caspase-3 might be involved in the pathogenesis of SBMA.

Nonneural nuclear inclusions of androgen receptor protein in spinal and bulbar muscular atrophy.

Spinal and bulbar muscular atrophy is an X-linked motor neuronopathy caused by the expansion of an unstable CAG repeat in the coding region of the androgen receptor (AR) gene. Nuclear inclusions of the mutant AR protein have been shown to occur in the spinal motor neurons of spinal and bulbar muscular atrophy (Li M, Kobayashi Y, Merry D, Tanaka F, Doyu M, Hashizume Y, Fischbeck KH, Sobue G: Nuclear inclusions in spinal and bulbar muscular atrophy. Ann Neurol 1998 (in press)). In this study, we demonstrate the tissue-specific distribution, immunochemical features, and fine structure of nuclear inclusions of spinal and bulbar muscular atrophy. Nuclear inclusions were observed in affected spinal and brainstem motor neurons, but not in other, nonaffected neural tissues. Similar nuclear inclusions occurred in nonneural tissues including scrotal skin, dermis, kidney, heart, and testis, but not in the spleen, liver, and muscle. These inclusions had similar epitope features detectable by antibodies that recognize a small portion of the N-terminus of the AR protein only, and they were ubiquitinated. Electron microscopic immunohistochemistry showed dense aggregates of AR-positive granular material without limiting membrane, both in the neural and nonneural inclusions. These findings indicate that nuclear inclusions of AR protein are present in selected nonneural tissues as well as in neurons that degenerate in spinal and bulbar muscular atrophy, suggesting that a common mechanism underlies in the formation of neural and nonneural nuclear inclusions.

Nuclear inclusions of the androgen receptor protein in spinal and bulbar muscular atrophy.

Spinal and bulbar muscular atrophy (SBMA) is an X-linked motor neuronopathy caused by the expansion of an unstable CAG repeat in the coding region of the androgen receptor (AR) gene. To study AR protein expression in normal and SBMA individuals, we used several antibodies that recognize AR protein, and analyzed neural and nonneural tissues by immunohistochemistry and western blotting. Both the normal and the mutant AR proteins were widely distributed, predominantly, but not exclusively, in the cytoplasm of neurons regardless of the pathological involvement, and predominantly in the nuclei of the nonneural tissues in both normal and SBMA individuals, with different expression levels of AR protein among different tissues. In the motor neurons of SBMA patients, there were AR-immunoreactive ubiquitinated nuclear inclusions that were detected by antibodies that recognize a small portion of the N terminus of the AR protein. Absence of other immunoreactive AR epitopes within the inclusion may be due to altered AR configuration, or masking of AR epitopes by other proteins, or proteolytic cleavage of the AR. Our data show that, in addition to the normal cellular distribution of the AR protein, mutant AR-bearing nuclear inclusions are present in SBMA.

Endoscopic stapling treatment of pharyngeal pouch.

BACKGROUND: We describe our experience of a recently reported endoscopic stapling technique for the treatment of pharyngeal pouch. METHODS: In contrast to other endoscopic procedures, which only divide the common wall between oesophagus and diverticulum, the linear cutting stapler also tightly seals the divided edges of mucosa and muscle. RESULTS: The procedure was performed without complications in three patients with complete resolution of pre-treatment symptoms. CONCLUSIONS: To our knowledge this is the first report of the use of this procedure in Australia. This endoscopic stapling operation appears to be safe, simple and cost-effective and offers advantages over previously used techniques.

Cleavage, aggregation and toxicity of the expanded androgen receptor in spinal and bulbar muscular atrophy.

Spinal and bulbar muscular atrophy (SBMA) is a neurodegenerative disease caused by the expansion of a polyglutamine repeat within the androgen receptor (AR). We have studied the mutant AR in an in vitro system, and find both aggregation and proteolytic processing of the AR protein to occur in a polyglutamine repeat length-dependent manner. In addition, we find the aberrant metabolism of expanded repeat AR to be coupled to cellular toxicity, indicating a likely molecular basis for the toxic gain of AR function that produces neuronal degeneration in SBMA.

A cell culture model for androgen effects in motor neurons.

Androgens are known to alter the morphology, survival, and axonal regeneration of lower motor neurons in vivo. To understand better the molecular mechanisms of androgen action in neurons, we created a model system by stably expressing the human androgen receptor (AR) in motor neuron hybrid cells. Motor neuron hybrid cells express markers consistent with anterior horn cells and can be differentiated into a neuronal phenotype. When differentiated in the presence of androgen, AR-expressing cells, but not control cells, exhibit a dose-dependent change in morphology: androgen-treated cells develop larger cell bodies and broader neuritic processes while continuing to express neuronal markers. In addition, androgen promotes the survival of AR-expressing cells, but not control cells, under low-serum conditions. Our results demonstrate a direct trophic effect of androgens on lower motor neurons, mediated through the AR expressed in this population of neurons.

Evaluation of a commercial enzyme-linked immunosorbent assay for detection of immunoglobulin M antibody in diagnosis of human leptospiral infection.

The PanBio Leptospira immunoglobulin M (IgM) enzyme-linked immunosorbent assay (ELISA) is a commercially available screening test for the diagnosis of acute leptospiral infection. The ability of the test to diagnose early or recent Leptospira interrogans infection was assessed by testing sera with known microagglutination test (MAT) titers to serovars pomona, hardjo, copenhageni, and australis. The IgM ELISA detected all 41 cases of early or recent leptospiral infection (sensitivity, 100%), with a positive ELISA result seen in many cases before MAT antibody titers reached 1:50. Thirty-eight of 41 patients showed seroconversion (fourfold or greater increase in titer by MAT, 2 of 41 patients had a single sample with elevated titer, and 1 patient from whom leptospires were isolated from a blood sample failed to show MAT titers, despite a seroconversion (negative to positive result) in the ELISA. Follow-up sera obtained from 8 of 12 patients (67%) for 3 to 48 months after the acute stage of illness showed persisting IgM antibody. However, the range of levels detected in these samples (maximum ELISA ratio, 2.0) was lower than the range seen when infection was recent. Reactivity in the IgM ELISA was observed for only 1 of 59 serum samples from asymptomatic donors (specificity, 98%) and 16 of 233 serum samples from patients with Ross River virus, brucella, Epstein-Barr virus, cytomegalovirus, mycoplasma, Q-fever, toxoplasma, hepatitis A virus, Treponema pallidum, or Borrelia burgdorferi infection (specificity, 93%), with the majority of these patients showing lower levels of IgM in comparison to those in patients with leptospiral infection. We conclude that this ELISA is sufficiently sensitive for use as an initial screen for leptospiral infections, with subsequent confirmation of positive test results by MAT.

Bcl-2 gene family in the nervous system.

A growing family of genes that share homology with the bcl-2 proto-oncogene is involved in the regulation of cell death. Many of these proteins show widespread expression and are expressed in the nervous system in developing and adult organisms. A physiologic role for Bcl-2 and Bcl-x in neuron survival has been shown. In addition, these proteins have been shown to protect neurons from a wide array of toxic insults. In this review, we discuss the Bcl-2 family of proteins with regard to their structure and interactions. We then discuss the role of apoptotic cell death in the development of the nervous system and as a response to neuronal injury. Lastly, we discuss the evidence for a role for these cell death regulators in neuronal death decisions.

Characterization of an expanded glutamine repeat androgen receptor in a neuronal cell culture system.

Spinal and bulbar muscular atrophy (SBMA) is an inherited form of lower motor neuron degeneration caused by expansion of a CAG repeat in the androgen receptor (AR) gene. To study the mechanism by which this mutation causes neuronal pathology, we stably transfected a motor neuron hybrid cell line with human AR cDNAs containing either 24 or 65 repeats (AR24 and AR65, respectively). Both forms of receptor were able to bind ligand and activate transcription of a reporter construct equally well. Likewise, the subcellular localizations of AR24 and AR65 were similar, in both the presence and the absence of ligand. AR24- and AR65-expressing clones were phenotypically indistinguishable. They survived equally well after differentiation and were equally susceptible to damage by oxidative stress. Our studies thus demonstrate that, in a neuronal system, the expanded repeat AR functions like the normal repeat AR in several important ways. Because levels of AR65 expression were consistently lower than levels of AR24 expression, we propose that the loss of function of AR seen in SBMA may be due to decreased levels of receptor expression rather than to a difference in intrinsic properties. The postulated gain of function responsible for neuronal degeneration remains to be determined.

Spinocerebellar ataxia type-1 and spinobulbar muscular atrophy gene products interact with glyceraldehyde-3-phosphate dehydrogenase.

Spinocerebellar ataxia type1 (SCA1) is one of several neurodegenerative disorders caused by expansions of translated CAG trinucleotide repeats which code for polyglutamine in the respective proteins. Most hypotheses about the molecular defect in these disorders suggest a gain of function, which may involve interactions with other proteins via the expanded polyglutamine tract. In this study we used ataxin-1, the SCA1 gene product, as a bait in the yeast two-hybrid system and identified the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase as an ataxin-1 interacting protein. In addition, the yeast two hybrid data demonstrate that wild type and mutant ataxin-1 form homo and heterodimers. Physical interaction between GAPDH and ataxin-1 was also demonstrated in vitro. To investigate if GAPDH might interact with other glutamine repeat-containing proteins involved in neurodegenerative disorders, we tested its binding to the androgen receptor which is mutated in spinobulbar muscular atrophy. The androgen receptor interacts with GAPDH both in the yeast two-hybrid system and in vitro. The binding of both ataxin-1 and the androgen receptor to GAPDH does not vary with the length of the polyglutamine tract. While provocative, these findings do not address the selective neuronal loss in each of these disorders in light of the wide expression patterns of GAPDH and the respective polyglutamine containing proteins. Nonetheless, such interactions may increase the susceptibility of specific neurons to a variety of insults and initiate degeneration.

Induction of apoptosis by La Crosse virus infection and role of neuronal differentiation and human bcl-2 expression in its prevention.

La Crosse virus causes a highly cytopathic infection in cultured cells and in the murine central nervous system (CNS), with widespread neuronal destruction. In some viral infections of the CNS, apoptosis, or programmed cell death, has been proposed as a mechanism for cytopathology (Y. Shen and T. E. Shenk, Curr. Opin. Genet. Dev. 5:105-111, 1995). To determine whether apoptosis plays a role in La Crosse virus-induced cell death, we performed experiments with newborn mice and two neural tissue culture models. Newborn mice infected with La Crosse virus showed evidence of apoptosis with the terminal deoxynucleotidyl transferase-mediated nicked-end labeling (TUNEL) assay and, concomitantly, histopathological suggestion of neuronal dropout. Infection of tissue culture cells also resulted in DNA fragmentation, TUNEL reactivity, and morphological changes in the nuclei characteristic of apoptotic cells. As in one other system (S. Ubol, P. C. Tucker, D. E. Griffin, and J. M. Hardwick, Proc. Natl. Acad. Sci. USA 91:5202-5206, 1994), expression of the human proto-oncogene bcl-2 was able to protect one neuronal cell line, N18-RE-105, from undergoing apoptosis after La Crosse virus infection and prolonged the survival of infected cells. Nevertheless, expression of bcl-2 did not prevent eventual cytopathicity. However, a human neuronal cell line, NT2N, was resistant to both apoptosis and other types of cytopathicity after infection with La Crosse virus, reaffirming the complexity of cell death. Our results show that apoptosis is an important consequence of La Crosse virus infection in vivo and in vitro.

Stability of an expanded trinucleotide repeat in the androgen receptor gene in transgenic mice.

The expansion of trinucleotide repeat sequences underlies a number of hereditary neurological disorders. To study the stability of a trinucleotide repeat and to develop an animal model of one of these disorders, spinal and bulbar muscular atrophy (SBMA), we have generated transgenic mice carrying either the normal or expanded repeat human androgen receptor (AR) gene. Unlike the disease allele in humans, the AR cDNA containing the expanded repeat in transgenic mice showed no change in repeat length with transmission. Expression of the SBMA AR was found in transgenic mice, but at a lower level than normal endogenous expression. The lack of a physiological pattern of expression may explain why no phenotypic effects of the transgene were observed.

A preliminary evaluation of a prototype western blot confirmatory test kit for syphilis.

A prototype Western blot kit was evaluated as a confirmatory test for syphilis using 131 sera characterized by other serological tests for syphilis. There were 114 treponemal sera (including 94 cases of early syphilis, 83 of which were untreated) and 17 non-treponemal problem sera (11 gave false positive reactions on screening with the TmpA recombinant antigen enzyme immunoassay (EIA), 3 gave false positive fluorescent treponemal antibody absorbed (FTA-abs) tests, and 3 false positive Captia Syphilis G EIA results). Based on the manufacturer's criteria of reactivity in multiple bands for designating a positive result the Western blot test gave a sensitivity of 99.1% (113/114) and a specificity of 88.2% (15/17) when indeterminate reactions were scored positive and 98.2% (112/114) and 100% (17/17) when indeterminate reactions were scored negative. Sensitivity was high in both treated and untreated infection. Corresponding sensitivities for the TPHA and FTA-abs when equivocal reactions were scored negative were 97.5% (111/114) and 99.1% (113/114). The high sensitivity of the FTA-abs in this study is probably due to the large number of untreated primary infections. Our results with the Western blot, confirm earlier studies using 'in-house' test systems and, support a role for a commercial Western blot test in the confirmatory diagnosis of syphilis. Further studies are required to confirm the high specificity and sensitivity of the kit in a larger series including a wider variety of non-treponemal cases as well as patients with untreated and treated infection.