Common mechanisms of excitatory and inhibitory imbalance in schizophrenia and autism spectrum disorders.

Autism Spectrum Disorders (ASD) and Schizophrenia (SCZ) are cognitive disorders with complex genetic architectures but overlapping behavioral phenotypes, which suggests common pathway perturbations. Multiple lines of evidence implicate imbalances in excitatory and inhibitory activity (E/I imbalance) as a shared pathophysiological mechanism. Thus, understanding the molecular underpinnings of E/I imbalance may provide essential insight into the etiology of these disorders and may uncover novel targets for future drug discovery. Here, we review key genetic, physiological, neuropathological, functional, and pathway studies that suggest alterations to excitatory/inhibitory circuits are keys to ASD and SCZ pathogenesis.

Control of interneuron dendritic growth through NRG1/erbB4-mediated kalirin-7 disinhibition.

Neuregulin 1 (NRG1) is a secreted trophic factor that activates the postsynaptic erbB4 receptor tyrosine kinase. Both NRG1 and erbB4 have been repeatedly associated with schizophrenia, but their downstream targets are not well characterized. ErbB4 is highly abundant in interneurons, and NRG1-mediated erbB4 activation has been shown to modulate interneuron function, but the role for NRG1-erbB4 signaling in regulating interneuron dendritic growth is not well understood. Here we show that NRG1/erbB4 promote the growth of dendrites in mature interneurons through kalirin, a major dendritic Rac1-GEF. Recent studies have shown associations of the KALRN gene with schizophrenia. Our data point to an essential role of phosphorylation in kalirin-7's C terminus as the critical site for these effects. As reduced interneuron dendrite length occurs in schizophrenia, understanding how NRG1-erbB4 signaling modulates interneuron dendritic morphogenesis might shed light on disease-related alterations in cortical circuits.

Distinct roles for the two Rho GDP/GTP exchange factor domains of kalirin in regulation of neurite growth and neuronal morphology.

The actin cytoskeleton, essential for neuronal development, is regulated in part by small GTP binding proteins of the Rho subfamily. Kalirin-9, with two Rho subfamily-specific GDP/GTP exchange factor (GEF) domains, localizes to neurites and growth cones of primary cortical neurons. Kalirin-9 overexpression in cultured cortical neurons induces longer neurites and altered neuronal morphology. Expression of the first GEF domain alone results in drastically shortened axons and excessive growth cones, mediated by Rac1. Expression of the second GEF domain alone induces axonal over-elongation and abundant filopodial neurites, mediated by RhoA. Coordination of the actions of the individual GEF domains through their presence in Kalirin-9, with its Sec14p, spectrin, and Src homology domain 3 motifs, is essential for regulating neurite extension and neuronal morphology.

The neuronal Rho-GEF Kalirin-7 interacts with PDZ domain-containing proteins and regulates dendritic morphogenesis.

Spine function requires precise control of the actin cytoskeleton. Kalirin-7, a GDP/GTP exchange factor for Rac1, interacts with PDZ proteins such as PSD-95, colocalizing with PSD-95 at synapses of cultured hippocampal neurons. PSD-95 and Kalirin-7 interact in vivo and in heterologous expression systems. In primary cortical neurons, transfected Kalirin-7 is targeted to spines and increases the number and size of spine-like structures. A Kalirin-7 mutant unable to interact with PDZ proteins remains in the cell soma, inducing local formation of aberrant filopodial neurites. Kalirin-7 with an inactivated GEF domain reduces the number of spines below control levels. These results provide evidence that PDZ proteins target Kalirin-7 to the PSD, where it regulates dendritic morphogenesis through Rac1 signaling to the actin cytoskeleton.

Isoforms of kalirin, a neuronal Dbl family member, generated through use of different 5'- and 3'-ends along with an internal translational initiation site.

Kalirin is a neuron-specific GDP/GTP exchange factor for Rho subfamily GTP-binding proteins. The major Kalirin transcripts in adult rat brain were identified. Most include a Sec14p-like putative lipid-binding motif followed by nine spectrin-like repeats and a Dbl homology/pleckstrin homology (DH-PH) domain. Kalirin proteins with four different NH(2) termini are generated through the use of five different 5'-ends; three of the proteins differ only at the extreme NH(2) terminus, and one is truncated because translation is initiated at a methionine in the 5th spectrin repeat. Four different 3'-ends yield Kalirin proteins with additional functional domains. Kalirin-7 (7-kilobase pair mRNA) terminates with a PDZ-binding motif, which in Kalirin-8 is replaced by an SH3 domain. Kalirin-9 contains another pair of DH-PH and SH3 domains. Kalirin-12 additionally encodes a putative Ser/Thr protein kinase. Antisera specific for different COOH termini established Kalirin-7 as the most abundant in cortex, with significant amounts of Kalirin-9 and Kalirin-12; Kalirin-7 was less prevalent in cerebellum and olfactory bulb. Kalirin proteins lacking the Sec14p-like domain and first four spectrin-like repeats were much less prevalent. Form-specific antisera demonstrated that different forms of Kalirin were localized to distinct subcellular regions of cultured neurons. Members of the family of Kalirin proteins may subserve different functions at these different locations.

An isoform of kalirin, a brain-specific GDP/GTP exchange factor, is enriched in the postsynaptic density fraction.

Communication between membranes and the actin cytoskeleton is an important aspect of neuronal function. Regulators of actin cytoskeletal dynamics include the Rho-like small GTP-binding proteins and their exchange factors. Kalirin is a brain-specific protein, first identified through its interaction with peptidylglycine-alpha-amidating monooxygenase. In this study, we cloned rat Kalirin-7, a 7-kilobase mRNA form of Kalirin. Kalirin-7 contains nine spectrin-like repeats, a Dbl homology domain, and a pleckstrin homology domain. We found that the majority of Kalirin-7 protein is associated with synaptosomal membranes, but a fraction is cytosolic. We also detected higher molecular weight Kalirin proteins. In rat cerebral cortex, Kalirin-7 is highly enriched in the postsynaptic density fraction. In primary cultures of neurons, Kalirin-7 is detected in spine-like structures, while other forms of Kalirin are visualized in the cell soma and throughout the neurites. Kalirin-7 and its Dbl homology-pleckstrin homology domain induce formation of lamellipodia and membrane ruffling, when transiently expressed in fibroblasts, indicative of Rac1 activation. Using Rac1, the Dbl homology-pleckstrin homology domain catalyzed the in vitro exchange of bound GDP with GTP. Kalirin-7 is the first guanine-nucleotide exchange factor identified in the postsynaptic density, where it is positioned optimally to regulate signal transduction pathways connecting membrane proteins and the actin cytoskeleton.

The novel kinase peptidylglycine alpha-amidating monooxygenase cytosolic interactor protein 2 interacts with the cytosolic routing determinants of the peptide processing enzyme peptidylglycine alpha-amidating monooxygenase.

The cytosolic domain of the peptide-processing integral membrane protein peptidylglycine alpha-amidating monooxygenase (PAM; EC 1.14. 17.3) contains multiple signals determining its subcellular localization. Three PAM cytosolic interactor proteins (P-CIPs) were identified using the yeast two hybrid system (Alam, M. R., Caldwel, B. D., Johnson, R. C., Darlington, D. N., Mains, R. E., and Eipper, B. A. (1996) J. Biol. Chem. 271, 28636-28640); the partial amino acid sequence of P-CIP2 suggested that it was a protein kinase. In situ hybridization and immunocytochemistry show that P-CIP2 is expressed widely throughout the brain; PAM and P-CIP2 are expressed in the same neurons. Based on subcellular fractionation, the 47-kDa P-CIP2 protein is mostly cytosolic. P-CIP2 is a highly selective kinase, phosphorylating the cytosolic domain of PAM, but not the corresponding region of furin or carboxypeptidase D. Although P-CIP2 interacts with stathmin, it does not phosphorylate stathmin. Site-directed mutagenesis, phosphoamino acid analysis, and use of synthetic peptides demonstrate that PAM-Ser(949) is the major site phosphorylated by P-CIP2. Based on both in vitro binding experiments and co-immunoprecipitation from cell extracts, P-CIP2 interacts with PAM proteins containing the wild type cytosolic domain, but not with mutant forms of PAM whose trafficking is disrupted. P-CIP2, through its highly selective phosphorylation of a key site in the cytosolic domain of PAM, appears to play a critical role in the trafficking of this protein.

Holo-cellular retinol-binding protein: distinction of ligand-binding affinity from efficiency as substrate in retinal biosynthesis.

Microsomal enzymes that catalyze the first step in the biosynthesis of retinoic acid from retinal, retinol dehydrogenases (RDHs), access retinol bound to cellular retinol-binding protein (CRBP). This study tested the hypothesis that the RDHs interact with the region in CRBP designated as the "helical cap" by evaluating single site-directed mutations, namely, L29A, I32E, L35A, L35E, L35R, L36A, F57A, R58A, and R58E. UV analysis showed mutants had similar conformations of retinol in their binding pockets. Nevertheless, the mutants bound retinol with affinities 2-5-fold lower than wild type, except for L35 mutants, which had affinities similar to wild type. All mutants' holoforms had more relaxed conformations about their helical caps, judged by sensitivity to partial protease digestion. Mutants showed no significant differences in Km values, but two (L36A, R58A) had increased Vm values and L35 mutants had decreased Vm values. Overall, the data indicate that the residues tested contribute in varying degrees to CRBP rigidity, retinol binding, and RDH recognition/access to bound retinol. The extent of contributions can be distinguished for several residues. For example, L35 mutants had lower kcat values than wild-type CRBP; thus, L35 seems important for RDH access to retinol. F57, on the other hand, a suspected key residue in controlling retinol entrance/exit, does not make a singular contribution to retinol binding. These results suggest a role for the helical cap region as a locus for RDH interaction and as a portal for ligand access to CRBP, and show that the affinity (Kd) of CRBP for retinol alone does not determine the efficiency of holo-CRBP as substrate. These are the first experimental data of enzyme recognition by a specific exterior residue of CRBP (L35).

Enzymatic characteristics of retinal dehydrogenase type I expressed in Escherichia coli.

We expressed RalDH(I) in Escherichia coli and have shown that it functions in vitro with the complex CRBP-retinal (cellular retinol-binding protein) as substrate, either generated in situ from the complex CRBP-retinol and microsomal retinol dehydrogenases or provided directly as CRBP-retinal. Recombinant RalDH(I) had kinetic constants with CRBP-retinal of: Hill coefficient 1.8; K0.5 0.8 microM; and Vm 1.5 nmol/min/mg of protein at 25 degrees C. Apo-CRBP inhibited the reaction with CRBP-retinal with an IC50 of 1.4 microM. Citral inhibited RalDH(I) with an IC50 of approximately 1 microM compared to an IC50 of approximately 12 microM for RalDH(II), but did not serve as substrate for RalDH(I). RalDH(I) did not catalyze efficiently the dehydrogenation of acetaldehyde, but showed higher Vmax/Km values for hexanal, octanal, decanal and benzaldehyde than for either propanal or retinal. These data extend the characterization of RalDH(I), show that apo-CRBP competes with holo-CRBP as substrate for RalDH(I), and expand insight into the pathways of retinoic acid biogenesis from the most abundant substrates in vivo, retinoid-liganded CRBP.

Cloning of a rat cDNA encoding retinal dehydrogenase isozyme type I and its expression in E. coli.

Peptides sequenced from the purified rat liver cytosolic retinal dehydrogenase P1 [Posch, K.C., Burns, R.D. and Napoli, J.L., 1992. Biosynthesis of all-trans-retinoic acid from retinal: recognition of retinal bound to cellular retinol-binding protein (type I) as substrate by a purified cytosolic dehydrogenase. J. Biol. Chem. 267, 19676-19682] were used to design oligonucleotides for cloning its cDNA. The deduced amino acid sequence of P1, now designated retinal dehydrogenase type I or RalDH(I), has close similarity with mouse AHD-2 and rat kidney aldehyde dehydrogenase, but is distinct from rat phenobarbital-inducible aldehyde dehydrogenase (PIADH), the presumed rat liver homolog of mouse AHD-2. Rat kidney (100%) and lung (88%) show relatively high mRNA levels of RalDH(I), liver (34%) and brain (22%) have moderate levels, and testis (8%) has low levels. Retinoid status affects RalDH(I) mRNA levels differently in different tissues. E. coli-expressed RalDH(I) exhibits allosteric kinetics for retinal with a Hill coefficient of 1.7, a K0.5 value of 1.4 microM and a Vmax of 52 nmol min(-1) mg(-1) protein. These data establish the cospecificity of P1 and RalDH(I), show that retinoid status affects expression of its mRNA in a tissue-dependent manner, and illustrate that aldehyde dehydrogenase isozymes with extensive homology can participate in different metabolic paths, e.g., RalDH vs. PIADH.

Cloning of a cDNA encoding an aldehyde dehydrogenase and its expression in Escherichia coli. Recognition of retinal as substrate.

The biosynthesis of the hormone retinoic acid from retinol (vitamin A) involves two sequential steps, catalyzed by retinol dehydrogenases and retinal dehydrogenases, respectively. This report describes the cloning of a cDNA encoding a heretofore unknown aldehyde dehydrogenase from a rat testis library and its expression in Escherichia coli. This enzyme has been designated retinal dehydrogenase, type II, RalDH(II). The deduced amino acid sequence of RalDH(II) had the highest identity with mammalian aldehyde dehydrogenases that feature low Km values (microM) for retinal: human ALDH1 (72.2%), rat retinal dehydrogenase, type I (71.5%), bovine retina (72.7%), and mouse AHD-2 (71.5%). RalDH(II) expressed in E. coli recognizes as substrates free retinal, with a Km of approximately 0.7 microM, and cellular retinol-binding protein-bound retinal, with a Km of approximately 0.2 microM. RalDH(II) also can utilize as substrate retinal generated in situ by microsomal retinol dehydrogenases, from the physiologically most abundant substrate: retinol bound to cellular retinol-binding protein. Rat testis expresses RalDH(II) mRNA most abundantly, followed by (relative to testis): lung (6.7%), brain (6.3%), heart (5.2%), liver (4.4%), and kidney (2.7%). RalDH(II) does not recognize citral, benzaldehyde, acetaldehyde, and propanal efficiently as substrates, but does metabolize octanal and decanal efficiently. These data support a function for RalDH(II) in the pathway of retinoic acid biogenesis.

Distribution of serum amylase isoenzymes in cystic fibrosis homozygotes and heterozygotes.

A simple method has been elaborated for the routine separation and quantitative determination of amylase isoenzymes. The ratio P/S, the quotient of the activity values obtained by densitometric evaluation of the pancreatic and salivary isoenzymes, is used to characterize their distribution. In healthy adults and children the value for P/S is above 1 in 80% of the cases, with a mean of 1.87 +/- 0.23. In 90% of heterozygote CF gene-carriers, the P/S is below 1 with a mean of 0.68 +/- 0.13. In addition to the higher total amylase activity, in MV homozygote patients P/S is less than 0.1, and even 0.001. The phenomenon is explained by a compensatory enhancement of salivary activity. The method is a suitable diagnostic test of the exocrine function of the pancreas and for evaluation of the serum amylase isoenzymes. The P/S value allows to differentiate heterozygote CF gene-carriers from homozygotes and healthy individuals.

Mucoviscidosis: total amylase activity of serum and mixed saliva in homozygous and heterozygous subjects.

Total amylase activity of serum and mixed saliva was studied in homozygotes and heterozygotes for mucoviscidosis and in healthy subjects. Mean serum total activity was 269.0 +/- 113.7 U/l in the homozygotes, exceeding in nearly 50% of the cases the values given in the literature and those observed in the normal controls. The difference against the control group was significant (P less than 0.05). Mean serum total amylase activity of heterozygotes agreed with the mean value for the healthy group (203.5 +/- 79.5 U/l) without a significant difference. Total amylase activity in the saliva of homozygotes (148.700 +/- 65.700 U/l) was higher than in the heterozygotes /118.300 +/- 74.200 U/l) of the healthy children (51.700 +/- 26.500 U/l). The difference between the homozygous and healthy groups was strongly significant (P less than 0.01), and that between the heterozygous group and the combined healthy children and adult groups was also significant (P less than 0.05). In the heterozygotes, salivary amylase activity was slightly elevated but not significantly different from the control group and did not result in a change in serum total amylase activity.