Defects in translational regulation contributing to human cognitive and behavioral disease.

Recent data suggest that the levels of many synaptic proteins may be tightly controlled by the opposing processes of new translation and protein turnover in neurons. Alterations in this balance or in the levels of such dosage-sensitive proteins that result in altered stoichiometry of protein complexes at developing and remodeling synapses may underlie several human cognitive diseases including Fragile X Syndrome, autism spectrum disorders, Angelman syndrome and non-syndromic mental retardation. While a significant amount is known about the transduction of membrane signals to the translational apparatus through kinase cascades acting on general translation factors, much less is understood about how such signals may influence the activity of mRNA-specific regulators, their mechanisms of action and the specific sets of mRNAs they regulate. New approaches to the unbiased in vivo identification of maps of binding sites for these proteins on mRNA is expected to greatly increase our understanding of this crucial level of regulation in neuronal development and function.

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.

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.

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.

Tumor-specific killer cells in paraneoplastic cerebellar degeneration.

Models for immune-mediated tumor regression in mice have defined an essential role for cytotoxic T lymphocytes (CTLs); however, naturally occurring tumor immunity in humans is poorly understood. Patients with paraneoplastic cerebellar degeneration (PCD) provide an opportunity to explore the mechanisms underlying tumor immunity to breast and ovarian cancer. Although tumor immunity and autoimmune neuronal degeneration in PCD correlates with a specific antibody response to the tumor and brain antigen cdr2, this humoral response has not been shown to be pathogenic. Here we present evidence for a specific cellular immune response in PCD patients. We have detected expanded populations of MHC class I-restricted cdr2-specific CTLs in the blood of 3/3 HLA-A2.1+ PCD patients, providing the first description, to our knowledge, of tumor-specific CTLs using primary human cells in a simple recall assay. Cross-presentation of apoptotic cells by dendritic cells also led to a potent CTL response. These results indicate a model whereby immature dendritic cells that engulf apoptotic tumor cells can mature and migrate to draining lymph organs where they could induce a CTL response to tissue-restricted antigens. In PCD, peripheral activation of cdr2-specific CTLs is likely to contribute to the subsequent development of the autoimmune neuronal degeneration.

A post-transcriptional regulatory mechanism restricts expression of the paraneoplastic cerebellar degeneration antigen cdr2 to immune privileged tissues.

Paraneoplastic cerebellar degeneration (PCD) is believed to be an autoimmune disorder initiated by the ectopic expression of a neuron-specific protein in breast and ovarian tumors. PCD antisera was used previously to identify several cerebellar degeneration-related (cdr) genes encoding putative PCD antigens. We have found that the cdr2 gene, which encodes a cytoplasmic leucine zipper protein of unknown function, is expressed in PCD-associated tumors, whereas other cdr genes are not; thus, cdr2 encodes the PCD tumor antigen. To determine whether the expression pattern of cdr2 is consistent with its proposed role in PCD, we have isolated the mouse homolog and examined both the mRNA and protein distribution in adult tissues. We have found that cdr2 mRNA is expressed in almost all tissues, whereas the protein is expressed only in the brain and testis. Within the brain, both the cdr2 mRNA and immunoreactivity are confined primarily to neurons in the cerebellum and brainstem, the regions most affected in PCD. These results suggest first that the tissue-specific expression of cdr2 is regulated at a post-transcriptional level. Moreover, because the brain and testis are considered to be immune-privileged sites, the expression pattern of cdr2 is compatible with the autoimmune model of PCD pathogenesis.

Medication compliance in elderly outpatients using twice-daily dosing and unit-of-use packaging.

OBJECTIVE: To determine the effect of unit-of-use drug packaging of medications on compliance among elderly outpatients treated with complex medication regimens. DESIGN: Nonblind, randomized, clinical trial. SETTING: Geriatric outreach health centers in urban public housing units for independent-living elderly people. PATIENTS: Thirty-one patients (aged > or = 60 y), each taking three or more prescribed medications. Patients were randomly assigned to one of three study groups: group 1 (n = 12), no change in dosing or packaging; group 2 (n = 10), conventional packaging with twice-daily dosing; group 3 (n = 9), unit-of-use packaging with twice-daily dosing. INTERVENTION: A unit-of-use package consisting of a two-ounce plastic cup with a snap-on lid containing all medications to be taken at the time of dosing. MAIN OUTCOME MEASURES: Medication compliance was assessed monthly for six months using tablet counts. RESULTS: Medication compliance was significantly better in group 3 (92.6 percent) using unit-of-use packaging compared with either group 1 (79 percent) or group 2 (82.6 percent) (p = 0.017). Compliance did not differ between groups 1 and 2. CONCLUSIONS: In this small study of elderly outpatients taking three or more medications, unit-of-use packaging and twice-daily dosing improved medication compliance compared with conventional packaging.

Synthesis of phosphatidylinositol in rat liver microsomes is accompanied by the rapid formation of lysophosphatidylinositol.

In mammalian cells, newly synthesized phosphatidylinositol (PI) has a fatty acid composition similar to its precursors, phosphatidic acid and CDP-diacylglycerol (DAG). It is then remodelled by deacylation/reacylation cycles to the predominant form, 1-stearoyl, 2-arachidonoyl PI. Incubation of dipalmitoyl CDP-DAG, [3H]inositol and Mg2+ with rat liver microsomes results in the rapid synthesis of PI, along with the simultaneous formation of multiple species of lysoPI. Analysis of the kinetics of formation of PI and lysoPI reveals no lag in the formation of lysoPI from PI. Moreover, evaluation of the concentration dependencies indicate nearly identical apparent Km values for PI synthesis compared with lysoPI synthesis for the substrates inositol (180 microM) and CDP-DAG (100 microM). The dependence on pH and the requirement for Mg2+ or Mn2+ are nearly identical for PI and lysoPI formation and the labelling of both lipids is similarly inhibited by submicromolar concentrations of calcium and by NEM. These results suggest that the formation of lysoPI is dependent on the initial, rate-limiting synthesis of PI. Pulse-chase analysis of the labelling of these lipids indicates that PI and lysoPI rapidly equilibrate after the initial slow synthesis of PI. In addition, it appears that only newly synthesized PI is involved in lysoPI formation. The extent of lysoPI formation depends upon the fatty acid composition of the added CDP-DAG. A number of experimental approaches demonstrate that lysoPI is not formed when pre-existing microsomal PI is labelled by head group exchange, perhaps because this PI has already undergone remodelling to polyenoic forms. These data suggest that the rapid deacylation of newly synthesized PI may represent the first step in PI remodeling.

Coenzyme A-dependent, ATP-independent acylation of 2-acyl lysophosphatidylinositol in rat liver microsomes.

Phosphatidylinositol (PI) is synthesized from cytidine-diphosphodiacylglycerol (CDP-DAG) and inositol by the enzyme PI synthase. CDP-DAG is itself synthesized from phosphatidic acid and CTP. The observation that PI differs in fatty acid composition from its precursors CDP-DAG and phosphatidic acid led to the proposal that following its synthesis the fatty acids of PI are removed and replaced by others in a process called fatty acid remodelling. Previously, we used rat liver microsomes to study the molecular mechanisms of PI remodelling. Following its synthesis, PI is rapidly deacylated to form lysoPI which is reacylated to form new PI species. PI remodelling occurs predominantly at the 1-position. We demonstrate here that lysoPI can be acylated in the 1-position in an ATP-independent manner. The acylation of 2-acyl lysoPI by the coenzyme A-dependent, ATP-independent mechanism was examined. The acylation exhibits a pH optimum of 7.5, does not require a divalent cation, and is not inhibited by Ca2+ or Mg2+, although Zn2+ is a potent inhibitor. The apparent Km values for coenzyme A and 2-acyl lysoPI are 14 microM and 30 microM, respectively. The acylation of 2-acyl lysoPI incorporates primarily stearic acid into the 1-position of PI, as would be expected based on the fatty acid composition of steady-state PI in rat hepatocytes.

Fatty acid remodelling of phosphatidylinositol under conditions of de novo synthesis in rat liver microsomes.

Phosphatidylinositol (PI) is initially synthesized in mammalian cells with a fatty acid composition similar to that of its precursor, primarily monounsaturated forms of cytidine diphosphodiglyceride (CDP-DAG). However, at the steady state, over 80% of PI exists in the 1-stearoyl, 2-arachidonoyl form. The fatty acid remodelling of PI is due to a number of deacylation/reacylation mechanisms. In the preceding paper we demonstrated that de novo synthesized PI is rapidly deacylated and subsequently reacylated. In this report we present further evidence that cycles of deacylation and reacylation are involved in the remodelling of PI. Incubation of microsomes with CDP-DAG of different fatty acid composition results in quantitative and qualitative differences in lysoPI formation. Additionally, analyses of the resulting lysoPI and PI species reveal that multiple species of fatty acids are incorporated into the 1-position of both PI and lysoPI. Addition of acylation cofactors (fatty acyl CoAs or ATP plus CoA) potentiate reacylation in this system. The addition of stearoyl or myristoyl CoA during de novo synthesis of PI results in the incorporation of these added fatty acids into the I-positive of PI. In addition, some evidence is presented that multiple mechanisms for remodelling of the 1-position of PI may be active in the microsomes, including ATP- and CoA-dependent acylation, ATP-independent, CoA-dependent acylation and CoA-independent mechanisms. Finally, the disappearance of only a subset of lysoPI species upon the addition of acylation cofactors suggests that the reacylation step exhibits some substrate specificity.

The distribution of glycosyl-phosphatidylinositol anchored proteins is differentially regulated by serum and insulin.

Glycosyl-phosphatidylinositol (GPI) anchored proteins are surveyed in two insulin sensitive cell types by surface labeling and phospholipase C-induced release into the medium. Serum starvation selectively increases both the number and intensity of a subset of GPI-anchored proteins. After serum starvation, loss of cell-surface GPI-anchored proteins is induced acutely by either serum re-exposure or insulin, suggesting that hormonal treatment may promote the release of these proteins from the cell surface.

The function of glycosyl phosphoinositides in hormone action.

The molecular events involved in the cellular actions of insulin remain unexplained. Some of the acute actions of the hormone may be due to the intracellular generation of a chemical substance which modulates certain enzyme activities. Such an enzyme-modulating substance has been identified as an inositol phosphate-glycan, produced by the insulin-sensitive hydrolysis of a glycosyl-phosphatidylinositol (glycosyl-PtdIns) precursor. This precursor glycolipid is structurally similar to the glycosyl-phosphoinositide membrane protein anchor. The exposure of fat, liver or muscle cells to insulin results in the hydrolysis of glycosyl-PtdIns, giving rise to the inositol phosphate glycan and diacylglycerol. This hydrolysis reaction is catalysed by a glycosyl-PtdIns-specific phospholipase C. This enzyme has been characterized and purified from a plasma membrane fraction of liver. This reaction also results in the acute release of certain glycosyl-PtdIns-anchored proteins from the cell surface. Elucidation of the functional role of glycosyl-phosphoinositides in the generation of second messengers or the release of proteins may provide further insights into the pleiotropic nature of insulin action.

A model for predicting depression in elderly tenants of public housing.

To assess the prevalence of and risk factors for depression in an older population, the Multilevel Assessment Instrument (MAI) and the Center for Epidemiological Studies Depression Scale (CES-D) were administered to 176 elderly residents of a public housing apartment building in Indianapolis. The 41 patients diagnosed by the CES-D as depressed had significantly lower scores on MAI measures of psychological adjustment, cognitive function, and physical health compared with the nondepressed respondents and were more likely to have had episodes of anxiety and depression in the year before the study. Four variables--respondents' overall physical health as measured by the MAI, days spent sick in bed during the past year, living alone, and educational level--explained some of the variance between the CES-D scores of the depressed and nondepressed patients. A model for predicting current levels of depressive symptomatology based on scores on each of these four variables distinguished the depressed from the nondepressed tenants more than 80 percent of the time.

Self-rated health as a predictor of hospital admission and nursing home placement in elderly public housing tenants.

We assessed the validity of self-rated health in a one-year prospective study of 155 elderly public housing tenants. Compared to studies of elderly community residents, tenants had poorer self-rated health, and higher hospital admission and nursing home placement rates. Poor self-rated health was a risk factor for both outcomes. We conclude that self-rated health may be useful in identifying persons at increased risk for hospital admission and nursing home placement.

Medication use by ambulatory elderly. An in-home survey.

The elderly residents of an urban subsidized apartment building were interviewed in their apartments to determine a comprehensive medication profile. The interviews were conducted by doctor of pharmacy candidates, and 155 residents (81.2%) participated. Compliance was similar to rates previously reported (49.3%), and adverse drug reactions were common (29.1%). Other findings included: compliance did not decrease with advancing age; most elderly individuals could open child-resistant containers (83.4%), read standard container labels (79.7%), and identify teaspoon (97.3%) and tablespoon (88.5%) quantities; inability to open easy-open (flip-off) containers decreased compliance (P = .03); men were more compliant than women (68.4 v 42.4%, P = .006) but were taking fewer medications (3.3 per male v 5.0 per female); only 12.6% of respondents thought they were taking too much medication but when they did compliance diminished (P = .003); medications were infrequently shared (5.6% of respondents reported sharing); and only 6.4% of respondents reported medication expense as a concern.