Giuseppe F. Pontoriero

Co-operative roles for E-cadherin and N-cadherin during lens vesicle separation and lens epithelial cell survival.

The classical cadherins are known to have both adhesive and signaling functions. It has also been proposed that localized regulation of cadherin activity may be important in cell assortment during development. In the context of eye development, it has been suggested that cadherins are important for separation of the invaginated lens vesicle from the surface ectoderm. To test this hypothesis, we conditionally deleted N-cadherin or E-cadherin from the presumptive lens ectoderm of the mouse. Conditional deletion of either cadherin alone did not produce a lens vesicle separation defect. However, these conditional mutants did exhibit common structural deficits, including microphthalmia, severe iris hyperplasia, persistent vacuolization within the fibre cell region, and eventual lens epithelial cell deterioration. To assess the co-operative roles of E-cadherin and N-cadherin within the developing lens, double conditional knockout embryos were generated. These mice displayed distinct defects in lens vesicle separation and persistent expression of another classical cadherin, P-cadherin, within the cells of the persistent lens stalk. Double mutant lenses also exhibited severe defects in lens epithelial cell adhesion and survival. Finally, the severity of the lens phenotype was shown to be sensitive to the number of wild-type E- and N-cadherin alleles. These data suggest that the co-operative expression of both E- and N-cadherin during lens development is essential for normal cell sorting and subsequent lens vesicle separation.

Conditional Deletion of Activating Protein 2α (AP-2α) in the Developing Retina Demonstrates Non-Cell-Autonomous Roles for AP-2α in Optic Cup Development

ABSTRACT Activating protein 2α (AP-2α) is known to be expressed in the retina, and AP-2α-null mice exhibit defects in the developing optic cup, including patterning of the neural retina (NR) and a replacement of the dorsal retinal pigmented epithelium (RPE) with NR. In this study, we analyzed the temporal and spatial retinal expression patterns of AP-2α and created a conditional deletion of AP-2α in the developing retina. AP-2α exhibited a distinct expression pattern in the developing inner nuclear layer of the retina, and colocalization studies indicated that AP-2α was exclusively expressed in postmitotic amacrine cell populations. Targeted deletion of AP-2α in the developing retina did not result in observable retinal defects. Further examination of AP-2α-null mutants revealed that the severity of the RPE defect was variable and, although defects in retinal lamination occur at later embryonic stages, earlier stages showed normal lamination and expression of markers for amacrine and ganglion cells. Together, these data demonstrate that, whereas AP-2α alone does not play an intrinsic role in retinogenesis, it has non-cell-autonomous effects on optic cup development. Additional expression analyses showed that multiple AP-2 proteins are present in the developing retina, which will be important to future studies.

Cell autonomous roles for AP-2α in lens vesicle separation and maintenance of the lens epithelial cell phenotype

In this study, we have created a conditional deletion of AP‐2α in the developing mouse lens (Le‐AP‐2α mutants) to determine the cell‐autonomous requirement(s) for AP‐2α in lens development. Embryonic and adult Le‐AP‐2α mutants exhibited defects confined to lens placode derivatives, including a persistent adhesion of the lens to the overlying corneal epithelium (or lens stalk). Expression of known regulators of lens vesicle separation, including Pax6, Pitx3, and Foxe3 was observed in the Le‐AP‐2α mutant lens demonstrating that these genes do not lie directly downstream of AP‐2α. Unlike germ‐line mutants, Le‐AP‐2α mutants did not exhibit defects in the optic cup, further defining the tissue specific role(s) for AP‐2α in eye development. Finally, comparative microarray analysis of lenses from the Le‐AP‐2α mutants vs. wild‐type littermates revealed differential expression of 415 mRNAs, including reduced expression of genes important for maintaining the lens epithelial cell phenotype, such as E‐cadherin. Developmental Dynamics 237:602–617, 2008.

Decreased expression of a 40-kDa catecholamine-regulated protein in the ventral striatum of schizophrenic brain specimens from the Stanley Foundation Neuropathology Consortium

The majority of heat shock proteins (HSP) act as molecular chaperones protecting cells from deleterious stress. These proteins are able to inhibit the aggregation of partially denatured proteins and refold them into the correct conformation. They have also been shown to be involved in the pathogenesis of many neurodegenerative and psychiatric disorders. Previous reports from our laboratory have described a 40-kDa catecholamine-regulated heat-shock-like protein (CRP40). This study investigates CRP40 expression in ventral striatal specimens obtained from the Stanley Foundation Neuropathology Consortium (SFNC). CRP40 levels were significantly reduced in schizophrenic patients relative to the control group. However, ventral striatal samples of individuals diagnosed with major depression or bipolar disorder did not show significant changes in the expression of the protein. No differences in CRP40 levels were observed due to age, sex or postmortem interval (PMI). Further analysis of the schizophrenic group revealed that unmedicated and medicated patients showed decreases in ventral striatal CRP40 levels relative to the control group. However, the largest reduction in these levels was seen in unmedicated schizophrenic patients. In addition, relative to the unmedicated individuals, the clozapine- and haloperidol-treated groups showed elevations in ventral striatal CRP40 expression, although not significant. An increase in sample size may clarify this observation. Taken together, these results suggest a functional role of CRP40 in the pathogenesis of schizophrenia.

iso-Lactam and reduced amide analogues of the peptidomimetic dopamine receptor modulator 3(R)-[(2(S)-Pyrrolidinylcarbonyl)amino]-2-oxo-1-pyrrolidineacetamide

An analogue of the highly potent gamma-lactam Pro-Leu-Gly-NH(2) peptidomimetic, 3(R)-[(2(S)-pyrrolidinylcarbonyl) amino]-2-oxo-1-pyrrolidineacetamide (2), 4(R)-[[2(S)-pyrrolidinylcarbonyl]amino]-2-oxo-1-pyrrolidineacetamide (3), in which the lactam carbonyl moiety has been placed in a different position with respect to the 3-amino group was synthesized. Also, a series of analogues of 2, compounds 4-6, were synthesized in which each of the amide bonds of 2 were systematically replaced with a reduced amide bond surrogate. The analogues were tested for their ability to enhance the binding of [3H]N-propylnorapomorphine to dopamine receptors in a functional in vitro assay utilizing bovine striatal membranes. Peptidomimetic 3 was shown to be more potent than 2, while 4 and 5 were significantly less effective than 2. Peptidomimetic 6 had a pharmacological profile similar to that of 2.

Knockdown of mortalin within the medial prefrontal cortex impairs normal sensorimotor gating.

The 70‐kDa mitochondrial heat shock protein, mortalin, is a ubiquitously expressed, multifunctional protein that is capable of binding the neurotransmitter, dopamine, within the brain. Dopamine dysregulation has been implicated in many of the abnormal neurological behaviors. Although studies have indicated that mortalin is differentially regulated in response to dopaminergic modulation, research has yet to elucidate the role of mortalin in the regulation of dopaminergic activity. This study seeks to investigate the role of mortalin in the regulation of dopamine‐dependent behavior, specifically as it pertains to schizophrenia (SCZ). Mortalin expression was knocked down through the infusion of antisense oligodeoxynucleotide molecules into the medial prefrontal cortex (mPFC). Rats infused with mortalin antisense oligodeoxynucleotide molecules exhibited significant prepulse inhibition deficits, suggestive of defects in normal sensorimotor gating. Furthermore, mortalin misexpression within the mPFC was coupled to a significant increase in mortalin protein expression within the nucleus accumbens at the molecular level. These findings demonstrate that mortalin plays an essential role in the regulation of dopamine‐dependent behavior and plays an even greater role in the pathogenesis of SCZ. Synapse 64:808–813, 2010.

Cloning, characterization, and functional studies of a human 40-kDa catecholamine-regulated protein: implications in central nervous system disorders

Catecholamine-regulated proteins (CRPs) have been shown to bind dopamine and other structurally related catecholamines; in particular, the 40-kDa CRP (CRP40) protein has been previously cloned and functionally characterized. To determine putative human homologs, BLAST analysis using the bovine CRP40 sequence identified a human established sequence tag (EST) with significant homology (accession #BQ224193). Using this EST, we cloned a recombinant human brain CRP40-like protein, which possessed chaperone activity. Radiolabeled dopamine binding studies with recombinant human CRP40 protein demonstrated the ability of this protein to bind dopamine with low affinity and high capacity. The full-length human CRP40 nucleotide sequence was elucidated (accession #DQ480334) with RNA ligase-mediated rapid amplification of complementary DNA ends polymerase chain reaction, while Northern blot hybridization suggested that human CRP40 is an alternative splice variant of the 70-kDa mitochondrial heat shock protein, mortalin. Human SH-SY5Y neuroblastoma cells treated with the antipsychotic drug, haloperidol, exhibited a significant increase in CRP40 messenger RNA expression compared to untreated control cells, while other dopamine agonists/antagonists also altered CRP40 expression and immunolocalization. In conclusion, these results show that we have cloned a splice variant of mortalin with a novel catecholamine binding function and that this chaperone-like protein may be neuroprotective in dopamine-related central nervous system disorders.

Antipsychotic drug use is correlated with CRP40/mortalin mRNA expression in the dorsolateral prefrontal cortex of human postmortem brain specimens

Heat shock proteins act as intracellular chaperones by assisting with proper protein folding in response to various cellular stresses. In doing so, these proteins protect the cell from unwanted protein aggregation, which in turn, plays an important role in the pathogenesis of numerous disorders. Previous reports from our laboratory have described a 40 kDa catecholamine regulated heat shock-like protein (CRP40), an alternate gene product of the 70 kDa mitochondrial heat shock protein, mortalin. CRP40 shares an intimate association with dopaminergic activity, specifically as it pertains to dopamine dysregulation in schizophrenia. This study investigates human CRP40/mortalin mRNA expression within dorsolateral prefrontal cortex postmortem specimens from normal control, schizophrenic and bipolar patients obtained from the Stanley Medical Research Institute. Real-time polymerase chain reaction was carried out for all patient samples (n=105; n=35 per group) in a blinded manner. No significant alterations in CRP40/mortalin mRNA expression levels were observed between control, bipolar and schizophrenic patients. However, multiple regression demonstrated a distinct positive correlation between CRP40/mortalin mRNA expression and lifetime use of antipsychotic drugs within the schizophrenic patient profile, after controlling for important confounding factors. Thus, the data suggest that human CRP40/mortalin is modulated by dopaminergic activity and may act to protect neurons from excess catecholamine activity in regions of the brain associated with psychosis.