Differential regulation of MAP2 by phosphorylation events in proline-rich versus C-terminal domains.

MAP2 is a critical cytoskeletal regulator in neurons. The phosphorylation of MAP2 (MAP2-P) is well known to regulate core functions of MAP2, including microtubule (MT)/actin binding and facilitation of tubulin polymerization. However, site-specific studies of MAP2-P function in regions outside of the MT-binding domain (MTBD) are lacking. We previously identified a set of MAP2 phosphopeptides which are differentially expressed and predominantly increased in the cortex of individuals with schizophrenia relative to nonpsychiatric comparison subjects. The phosphopeptides originated not from the MTBD, but from the flanking proline-rich and C-terminal domains of MAP2. We sought to understand the contribution of MAP2-P at these sites on MAP2 function. To this end, we isolated a series of phosphomimetic MAP2C constructs and subjected them to cell-free tubulin polymerization, MT-binding, actin-binding, and actin polymerization assays. A subset of MAP2-P events significantly impaired these functions, with the two domains displaying different patterns of MAP2 regulation: proline-rich domain mutants T293E and T300E impaired MT assembly and actin-binding affinity but did not affect MT-binding, while C-terminal domain mutants S426E and S439D impaired all three functions. S443D also impaired MT assembly with minimal effects on MT- or actin-binding. Using heterologous cells, we also found that S426E but not T293E had a lower capability for process formation than the wild-type protein. These findings demonstrate the functional utility of MAP2-P in the proline-rich and C-terminal domains and point to distinct, domain-dependent regulations of MAP2 function, which can go on to affect cellular morphology.

MAP2 is differentially phosphorylated in schizophrenia, altering its function.

Schizophrenia (Sz) is a highly polygenic disorder, with common, rare, and structural variants each contributing only a small fraction of overall disease risk. Thus, there is a need to identify downstream points of convergence that can be targeted with therapeutics. Reduction of microtubule-associated protein 2 (MAP2) immunoreactivity (MAP2-IR) is present in individuals with Sz, despite no change in MAP2 protein levels. MAP2 is phosphorylated downstream of multiple receptors and kinases identified as Sz risk genes, altering its immunoreactivity and function. Using an unbiased phosphoproteomics approach, we quantified 18 MAP2 phosphopeptides, 9 of which were significantly altered in Sz subjects. Network analysis grouped MAP2 phosphopeptides into three modules, each with a distinct relationship to dendritic spine loss, synaptic protein levels, and clinical function in Sz subjects. We then investigated the most hyperphosphorylated site in Sz, phosphoserine1782 (pS1782). Computational modeling predicted phosphorylation of S1782 reduces binding of MAP2 to microtubules, which was confirmed experimentally. We generated a transgenic mouse containing a phosphomimetic mutation at S1782 (S1782E) and found reductions in basilar dendritic length and complexity along with reduced spine density. Because only a limited number of MAP2 interacting proteins have been previously identified, we combined co-immunoprecipitation with mass spectrometry to characterize the MAP2 interactome in mouse brain. The MAP2 interactome was enriched for proteins involved in protein translation. These associations were shown to be functional as overexpression of wild type and phosphomimetic MAP2 reduced protein synthesis in vitro. Finally, we found that Sz subjects with low MAP2-IR had reductions in the levels of synaptic proteins relative to nonpsychiatric control (NPC) subjects and to Sz subjects with normal and MAP2-IR, and this same pattern was recapitulated in S1782E mice. These findings suggest a new conceptual framework for Sz-that a large proportion of individuals have a "MAP2opathy"-in which MAP function is altered by phosphorylation, leading to impairments of neuronal structure, synaptic protein synthesis, and function.

Correction: Genetic risk for schizophrenia and psychosis in Alzheimer disease.

A number of collaborators were not acknowledged for their contribution to this published article. The acknowledgements that were missing in this published article can now be found in the associated correction.

Genetic risk for schizophrenia and psychosis in Alzheimer disease.

Psychotic symptoms, defined as the occurrence of delusions or hallucinations, are frequent in Alzheimer disease (AD), affecting ~40 to 60% of individuals with AD (AD with psychosis (AD+P)). In comparison with AD subjects without psychosis, AD+P subjects have more rapid cognitive decline and poor outcomes. Prior studies have estimated the heritability of psychosis in AD at 61%, but the underlying genetic sources of this risk are not known. We evaluated a Discovery Cohort of 2876 AD subjects with (N=1761) or without psychosis (N=1115). All subjects were genotyped using a custom genotyping array designed to evaluate single-nucleotide polymorphisms (SNPs) with evidence of genetic association with AD+P and include SNPs affecting or putatively affecting risk for schizophrenia and AD. Results were replicated in an independent cohort of 2194 AD subjects with (N=734) or without psychosis (N=1460). We found that AD+P is associated with polygenic risk for a set of novel loci and inversely associated with polygenic risk for schizophrenia. Among the biologic pathways identified by the associations of schizophrenia SNPs with AD+P are endosomal trafficking, autophagy and calcium channel signaling. To the best of our knowledge, these findings provide the first clear demonstration that AD+P is associated with common genetic variation. In addition, they provide an unbiased link between polygenic risk for schizophrenia and a lower risk of psychosis in AD. This provides an opportunity to leverage progress made in identifying the biologic effects of schizophrenia alleles to identify novel mechanisms protecting against more rapid cognitive decline and psychosis risk in AD.

Genome-wide association analysis of age-at-onset in Alzheimer's disease.

The risk of Alzheimer's disease (AD) is strongly determined by genetic factors and recent genome-wide association studies (GWAS) have identified several genes for the disease risk. In addition to the disease risk, age-at-onset (AAO) of AD has also strong genetic component with an estimated heritability of 42%. Identification of AAO genes may help to understand the biological mechanisms that regulate the onset of the disease. Here we report the first GWAS focused on identifying genes for the AAO of AD. We performed a genome-wide meta-analysis on three samples comprising a total of 2222 AD cases. A total of ~2.5 million directly genotyped or imputed single-nucleotide polymorphisms (SNPs) were analyzed in relation to AAO of AD. As expected, the most significant associations were observed in the apolipoprotein E (APOE) region on chromosome 19 where several SNPs surpassed the conservative genome-wide significant threshold (P<5E-08). The most significant SNP outside the APOE region was located in the DCHS2 gene on chromosome 4q31.3 (rs1466662; P=4.95E-07). There were 19 additional significant SNPs in this region at P<1E-04 and the DCHS2 gene is expressed in the cerebral cortex and thus is a potential candidate for affecting AAO in AD. These findings need to be confirmed in additional well-powered samples.

Genome-wide association study of Alzheimer's disease with psychotic symptoms.

Psychotic symptoms occur in ~40% of subjects with Alzheimer's disease (AD) and are associated with more rapid cognitive decline and increased functional deficits. They show heritability up to 61% and have been proposed as a marker for a disease subtype suitable for gene mapping efforts. We undertook a combined analysis of three genome-wide association studies (GWASs) to identify loci that (1) increase susceptibility to an AD and subsequent psychotic symptoms; or (2) modify risk of psychotic symptoms in the presence of neurodegeneration caused by AD. In all, 1299 AD cases with psychosis (AD+P), 735 AD cases without psychosis (AD-P) and 5659 controls were drawn from Genetic and Environmental Risk in AD Consortium 1 (GERAD1), the National Institute on Aging Late-Onset Alzheimer's Disease (NIA-LOAD) family study and the University of Pittsburgh Alzheimer Disease Research Center (ADRC) GWASs. Unobserved genotypes were imputed to provide data on >1.8 million single-nucleotide polymorphisms (SNPs). Analyses in each data set were completed comparing (1) AD+P to AD-P cases, and (2) AD+P cases with controls (GERAD1, ADRC only). Aside from the apolipoprotein E (APOE) locus, the strongest evidence for association was observed in an intergenic region on chromosome 4 (rs753129; 'AD+PvAD-P' P=2.85 × 10(-7); 'AD+PvControls' P=1.11 × 10(-4)). SNPs upstream of SLC2A9 (rs6834555, P=3.0 × 10(-7)) and within VSNL1 (rs4038131, P=5.9 × 10(-7)) showed strongest evidence for association with AD+P when compared with controls. These findings warrant further investigation in larger, appropriately powered samples in which the presence of psychotic symptoms in AD has been well characterized.

Targeting the post-synaptic proteome has therapeutic potential for psychosis in Alzheimer Disease.

Individuals with Alzheimer Disease who develop psychotic symptoms (AD + P) experience more rapid cognitive decline and have reduced indices of synaptic integrity relative to those without psychosis (AD-P). We sought to determine whether the postsynaptic density (PSD) proteome is altered in AD + P relative to AD-P, analyzing PSDs from dorsolateral prefrontal cortex of AD + P, AD-P, and a reference group of cognitively normal elderly subjects. The PSD proteome of AD + P showed a global shift towards lower levels of all proteins relative to AD-P, enriched for kinases, proteins regulating Rho GTPases, and other regulators of the actin cytoskeleton. We computationally identified potential novel therapies predicted to reverse the PSD protein signature of AD + P. Five days of administration of one of these drugs, the C-C Motif Chemokine Receptor 5 inhibitor, maraviroc, led to a net reversal of the PSD protein signature in adult mice, nominating it as a novel potential treatment for AD + P.

Trio and Kalirin as unique enactors of Rho/Rac spatiotemporal precision.

Rac1 and RhoA are among the most widely studied small GTPases. The classic dogma surrounding their biology has largely focused on their activity as an "on/off switch" of sorts. However, the advent of more sophisticated techniques, such as genetically-encoded FRET-based sensors, has afforded the ability to delineate the spatiotemporal regulation of Rac1 and RhoA. As a result, there has been a shift from this simplistic global view to one incorporating the precision of spatiotemporal modularity. This review summarizes emerging data surrounding the roles of Rac1 and RhoA as cytoskeletal regulators and examines how these new data have led to a revision of the traditional dogma which placed Rac1 and RhoA in antagonistic pathways. This more recent evidence suggests that rather than absolute activity levels, it is the tight spatiotemporal regulation of Rac1 and RhoA across multiple roles, from oppositional to complementary, that is necessary to execute coordinated cytoskeletal processes affecting cell structure, function, and migration. We focus on how Kalirin and Trio, as dual GEFs that target Rac1 and RhoA, are uniquely designed to provide the spatiotemporally-precise shifts in Rac/Rho balance which mediate changes in neuronal structure and function, particularly by way of cytoskeletal rearrangements. Finally, we review how alterations in Trio and/or Kalirin function are associated with cellular abnormalities and neuropsychiatric disease.

Web-accessible interactive software of 3D anatomy representing pathophysiological conditions to enhance the patient-consent process for procedures.

Conveying to a patient the exact physical nature of a disease or procedure can be difficult. By establishing an access website, and using existing 3D viewer software along with our expanding set of anatomical models, we can provide an interface to manipulate realistic, 3D models of common anatomical ailments, chosen from a database frequently updated at the request of the medical community. Physicians will be able to show patients exactly what their condition looks like internally, and explain in better detail how a procedure will be performed.

Identification of residues in the V domain of CD80 (B7-1) implicated in functional interactions with CD28 and CTLA4.

The CD80 (B7-1) molecule is a 45-60-kD member of the immunoglobulin superfamily that is expressed on a variety of cell types of haematopoietic origin. CD80 can provide a critical costimulatory signal to T cells by interacting with the T cell surface molecule CD28. CD80 also binds to the CD28-related molecule CTLA4, which is expressed on activated T cells, Recently, additional ligands of CD28 and CTLA4 have been described in mice and humans. One of them, CD86 (B-70 or B7-2) was characterized at the molecular level. Although similar in predicted structure to CD80, it is distantly related in amino acid sequence. In this study, human CD80 mutants were generated and tested for their ability to maintain the interaction with CD28 leading to adhesion and enhanced IL-2 production. Two hydrophobic residues in the V-like domain of CD80 were identified as critical for binding to CD28 and are also important for the interaction with CTLA4. These residues are adjacent to the epitope of the BB1 antibody, which inhibits CD28-CD80 interactions. One of these residues, Y87, is conserved in all CD80 and CD86 cloned from various species. These results being to unravel the structural requirements for binding to CD28 and CTLA4.

Structural analysis of the human immunodeficiency virus-binding domain of CD4. Epitope mapping with site-directed mutants and anti-idiotypes.

The CD4 molecule, a differentiation marker expressed primarily by T lymphocytes, plays an important role in lymphocyte activation. CD4 is also the receptor for HIV. A number of recent studies have localized the high affinity binding site of the HIV envelope glycoprotein, gp120, to the NH2-terminal (V1) domain of CD4, a region with sequence and predicted structural homology with Ig kappa chain V domains (V kappa). In this report, we show that V1 bears structural similarities with V kappa regions through detailed epitope mapping of 26 CD4 mAbs. The binding sites of these mAbs were initially defined relative to one another by crossblocking analysis and were then localized to specific domains of CD4 in blocking studies with truncated, soluble CD4 proteins. The epitopes within the V1 domain were mapped in detail with a panel of 17 substitution mutants, and the specificities of several mAbs that appear to recognize very similar epitopes were examined in crossblocking studies with anti-idiotype antibodies. The location of the epitopes is consistent with a V kappa-like structure of V1. Most of the epitopes lie within regions of predicted exposed loops. A number of these epitopes span discontinuous residues in the linear sequence that lies in close proximity in an Ig fold. Alignment of CD4 V1 with the Ig V kappa chains places these epitopes within stretches corresponding to the complimentarity-determining regions. This epitope analysis is relevant for a vaccine strategy for HIV based on anti-idiotype antibodies to CD4 mAbs and for studies with CD4 antibodies on the role of CD4 in T lymphocyte activation.

Long-term effects of the concomitant use of memantine with cholinesterase inhibition in Alzheimer disease.

BACKGROUND: Patients using cholinesterase inhibitors (ChEIs) have a delay in nursing home (NH) admission compared with those who were not using the medication. There are no long-term studies of the effects of memantine in combination with ChEIs use in Alzheimer disease (AD). This study was conducted to examine the effects of ChEIs and memantine on time to death and time to NH admission. METHODS: Time to NH admission and death was examined in 943 probable AD patients who had at least a 1-year follow-up evaluation. Of these patients, 140 (14.9%) used both ChEIs and memantine, 387 (41%) [corrected] used only ChEIs, and 416 (44.1%) [corrected] used neither. The mean (SD) follow-up time was 62.3 (35.8) months. The analysis was conducted with multivariable Cox proportional hazard models controlling for critical covariates (ie, age, education level, gender, severity of the dementia, hypertension, diabetes mellitus, heart disease, psychiatric symptoms and use of psychotropic medications). RESULTS: Compared with those who never used cognitive enhancers, patients who used ChEIs had a significant delay in NH admission (HR: 0.37, 95% CI 0.27 to 0.49); this effect was significantly augmented with the addition of memantine (HR: 0.29, 95% CI 0.11 to 0.72) (memantine+ChEI vs ChEI alone). ChEIs alone, or in combination with memantine had no significant association on time to death. CONCLUSIONS: This observational study revealed that the addition of the NMDA receptor antagonist memantine to the treatment of AD with ChEI significantly altered the treated history of AD by extending time to nursing home admission.

The hydrolytic water molecule in trypsin, revealed by time-resolved Laue crystallography.

Crystals of bovine trypsin were acylated at the reactive residue, serine 195, to form the transiently stable p-guanidinobenzoate. Hydrolysis of this species was triggered in the crystals by a jump in pH. The hydrolysis was monitored by three-dimensional Laue crystallography, resulting in three x-ray diffraction structures, all from the same crystal and each representing approximately 5 seconds of x-ray exposure. The structures were analyzed at a nominal resolution of 1.8 angstroms and were of sufficient quality to reproduce subtle features in the electron-density maps for each of the structures. Comparison of the structures before and after the pH jump reveals that a water molecule has positioned itself to attack the acyl group in the initial step of the hydrolysis of this transient intermediate.

Mutagenesis and Laue structures of enzyme intermediates: isocitrate dehydrogenase.

Site-directed mutagenesis and Laue diffraction data to 2.5 A resolution were used to solve the structures of two sequential intermediates formed during the catalytic actions of isocitrate dehydrogenase. Both intermediates are distinct from the enzyme-substrate and enzyme-product complexes. Mutation of key catalytic residues changed the rate determining steps so that protein and substrate intermediates within the overall reaction pathway could be visualized.

The catalytic pathway of cytochrome p450cam at atomic resolution.

Members of the cytochrome P450 superfamily catalyze the addition of molecular oxygen to nonactivated hydrocarbons at physiological temperature-a reaction that requires high temperature to proceed in the absence of a catalyst. Structures were obtained for three intermediates in the hydroxylation reaction of camphor by P450cam with trapping techniques and cryocrystallography. The structure of the ferrous dioxygen adduct of P450cam was determined with 0.91 angstrom wavelength x-rays; irradiation with 1.5 angstrom x-rays results in breakdown of the dioxygen molecule to an intermediate that would be consistent with an oxyferryl species. The structures show conformational changes in several important residues and reveal a network of bound water molecules that may provide the protons needed for the reaction.

Altering genotype and phenotype by DNA-mediated gene transfer.

Transformation, or DNA-mediated gene transfer, permits the introduction of new genetic information into a cell and frequently results in a change in phenotype. The transforming DNA is ultimately integrated into a recipient cell chromosome. No unique chromosomal locations are apparent, different lines contain the transforming DNA on different chromosomes. Expression of transformed genes frequently results in the synthesis of new polypeptide products which restore appropriate mutant cells to the wild-type phenotype. Thus transformation provides an in vivo assay for the functional role of DNA sequence organization about specific genes. Transforming genes coding for selectable functions, such as adenine phosphoribosyltransferase or thymidine kinase, have now been isolated by utilizing transformation in concert with molecular cloning. Finally, transformation may provide a general approach to the analysis of complex heritable phenotypes by permitting the distinction between phenotypic changes without concomitant changes in DNA and functional genetic rearrangements.

Temporal and spatial control of the adenovirus proteinase by both a peptide and the viral DNA.

The adenovirus proteinase (AVP) uses both an 11-amino acid peptide (pVIc) and the viral DNA as cofactors to increase its catalytic rate constant 6000-fold. The crystal structure of an AVP-pVIc complex at 2.6-A resolution reveals a new protein fold of an enzyme that is the first member of a new class of cysteine proteinases, which arose via convergent evolution.

Isolation of human anti-idiotypic antibodies by phage display for clinical immune response assays.

The clinical development of therapeutic proteins requires assays that measure the pharmacokinetic (PK) profile of, and the potential immune response (IR) to, the protein agent. Each assay requires reagents that are highly specific for the therapeutic protein. For therapeutic monoclonal antibodies, anti-CDR-specific, or anti-idiotypic (anti-id), antibodies are an ideal class of reagents suitable for these assays because of their high specificity and affinity to the drug antibody. We generated anti-ids to two human antibodies by antibody phage display using the MorphoSys HuCAL GOLD Fab library. To selectively target the CDR regions, serum and a framework-matched mAb were included as competitors during the phage selection process. Panels of CDR-specific Fabs, with low to sub-nM affinities, were isolated against both targets. The CDR specificity of these Fabs was shown by their lack of binding to a framework-matched control mAb and by competition of this binding with the soluble antigens of the respective therapeutic mAb targets. The candidate anti-id Fabs were able to detect both immobilized and soluble target Ab without being affected by serum, a requirement for both PK assay and the IR bridging assay format. Combinations of the Fabs for PK detection assays were identified by pairwise binding studies, although the pair for one target mAb lacks the desired sensitivity for PK assays. To evaluate their potential as anti-drug antibodies (ADAs), the best Fabs for one of the targets were converted and produced as the required bivalent human mAbs. In comparison to rodent mAbs and primate polyclonal serum, the phage display derived human mAbs were equally effective as reference standards. Our results demonstrate that competition-based phage selection can be an effective method for the isolation of anti-idiotypic antibodies for PK and IR assay development, and in this latter case, overcome limitations of current methods using rodent derived anti-ids.

Purification and characterization of human H-ras proteins expressed in Escherichia coli.

The full-length normal and T24 mutant human H-ras proteins and two truncated derivatives of the T24 mutant were expressed efficiently in Escherichia coli. The proteins accumulated to 1 to 5% of total cellular protein, and each was specifically recognized by anti-ras monoclonal antibodies. The two full-length proteins as well as a carboxyl-terminal truncated derivative (deleted for 23 amino acid residues) were soluble upon cell lysis and were purified to 90% homogeneity without the use of denaturants. In contrast, an amino-terminal truncated ras derivative (deleted for 22 amino acid residues) required treatment with urea for its solubilization. The guanine nucleotide binding activity of these four proteins was assessed by a combination of ligand binding on proteins blots, immunoprecipitation, and standard filter binding procedures. The full-length proteins showed similar binding kinetics and a stoichiometry approaching 1 mol of GTP bound per mol of protein. The showed similar binding kinetics and a stoichiometry approaching 1 mol of GTP bound per mol of protein. The carboxyl-terminal truncated protein also bound GTP, but to a reduced extent, whereas the amino-terminal truncated protein did not have binding activity. Apparently, the carboxyl-terminal domain of ras, although important for transforming function, does not play a critical role in GTP binding.

DNA methylation as a putative mechanism for reduced dendritic spine density in the superior temporal gyrus of subjects with schizophrenia.

Reduced dendritic spine density (DSD) in cortical layer 3 of the superior temporal gyrus (STG), and multiple other brain regions, is consistently observed in postmortem studies of schizophrenia (SZ). Elucidating the molecular mechanisms of this intermediate phenotype holds promise for understanding SZ pathophysiology, identifying SZ treatment targets and developing animal models. DNA methylation (DNAm), the addition of a methyl group to a cytosine nucleotide, regulates gene transcription and is a strong candidate for such a mechanism. We tested the hypothesis that DNAm correlates with DSD in the human STG and that this relationship is disrupted in SZ. We used the Illumina Infinium HumanMethylation450 Beadchip Array to quantify DNAm on a genome-wide scale in the postmortem STG from 22 SZ subjects and matched non-psychiatric control (NPC) subjects; DSD measures were available for 17 of the 22 subject pairs. We found DNAm to correlate with DSD at more sites than expected by chance in NPC, but not SZ, subjects. In addition, we show that the slopes of the linear DNAm-DSD correlations differed between SZ and NPC subjects at more sites than expected by chance. From these data, we identified 2 candidate genes for mediating DSD abnormalities in SZ: brain-specific angiogenesis inhibitor 1-associated protein 2 (BAIAP2) and discs large, Drosophila, homolog of, 1 (DLG1). Together, these data suggest that altered DNAm in SZ may be a mechanism for SZ-related DSD reductions.