The relevance of MRI blood-sensitive sequences in the diagnostic assessment of late-onset epilepsy.

OBJECTIVE: Sporadic cerebral amyloid angiopathy (CAA) is a degenerative brain small vessel disease of ageing resulting from progressive amyloid deposition in small arteries and arterioles of the cortex and leptomeninges. CAA may be diagnosed by the mean of Boston criteria, particularly with the use of the blood-sensitive T2* MRI sequences (GRE and SWI). Epileptic seizures have rarely been reported in CAA. PATIENTS AND METHODS: We describe two patients with late-onset unprovoked seizures due to CAA. A short literature review on this topic is presented. RESULTS: In our two patients with late-onset unprovoked seizures as the first manifestation of CAA, only GRE and SWI sequences lead to a correct diagnosis. In literature, only 15 patients with CAA presenting with seizures have been reported. In these subjects, data on seizures semiology and prognosis are scarce. CONCLUSIONS: Our report highlights the importance to perform blood-sensitive sequences in all subjects with LOE of otherwise unknown etiology, not to miss a diagnosis of CAA.

Functional convergence spasm: an unexpected finding in a patient with focal epilepsy.

OBJECTIVE: Convergence spasm is a clinical condition characterized by transient episodes of convergence, miosis and accommodation with strabismus and diplopia and it is usually a manifestation of a functional neurological disorder. We describe a patient with a challenging diagnosis of convergence spasm in the setting of occipital lobe epilepsy. CASE REPORT: A 52-year-old woman came for the assessment of focal epilepsy due to left occipital cortical dysplasia. During ocular motility tests, she presented with episodes of short duration (~10-30 seconds) of convergent strabismus. Neuropsychological evaluation showed a severe mixed anxiety-depressive disorder with a tendency toward somatization. RESULTS: Convergence spasm was recorded during video-EEG examination and no ictal activity was present. CONCLUSIONS: To our knowledge, no other report of functional convergence spasm in the context of focal epilepsy associated with cortical dysplasia has been described in literature.

Novel COL4A2 mutation causing familial malformations of cortical development.

OBJECTIVE: This article aimed to describe a novel COL4A2 mutation and the phenotypic features of two family members presenting with epilepsy and cortical development malformations. PATIENTS AND METHODS: The first patient is a 65-year-old woman with hematuria and adult-onset seizures. Brain MRI showed closed lip schizencephaly of right lateral sulcus associated with polymicrogyria of the surrounding cortex and areas of subcortical heterotopia. The second patient is a 40-year-old man, her son. He was born post-term with neonatal distress and psychomotor developmental delay with congenital left leg paresis and strabismus, as well as childhood-onset focal motor seizures. Brain MRI showed a right nucleus-capsular porencephalic cavitation with enlargement of the homolateral ventricle and a focal right occipital cortico-subcortical encephalomalacia. A small heterotopic band was also present in the frontal left subcortical region. RESULTS: We tested both patients with a NGS panel for genetic epilepsies, which evidenced a missense mutation in COL4A2 gene (c.2972G>A, causing the aminoacidic substitution Gly991Glu). CONCLUSIONS: The phenotypic spectrum associated with COL4A2 mutations has not been extensively described in the literature. Testing for COL4A mutations is indicated in patients with malformations of cortical development, particularly in the presence of familial conditions, even in the absence of porencephaly or early hemorrhagic strokes.

Lysosomal accumulation of the hormone-receptor complex during receptor-mediated endocytosis of human choriogonadotropin.

The experiments presented herein were designed to determine the fate of the human choriogonadotropin (hCG) receptor during endocytosis of the receptor-bound hCG. Using several biochemical approaches, it is shown that the receptor is internalized together with the hormone into endocytic vesicles and transferred to lysosomes without ligand dissociation. Once delivered to the lysosomes, the hCG-receptor complex dissociates, and the free hormone is degraded. This pathway appears to prevent receptor recycling and probably promotes receptor degradation.

On the mechanisms involved in the regulation of the cell-surface receptors for human choriogonadotropin and mouse epidermal growth factor in cultured Leydig tumor cells.

The MA-10 cells are a clonal strain of mouse Leydig tumor cells that have receptors for human choriogonadotropin (hCG) and mouse epidermal growth factor (mEGF). Exposure of the cells to hCG results in a reduction in the number of surface hCG receptors, and little or no change in the number of surface mEGF receptors. On the other hand, exposure of the cells to mEGF results in a reduction in the number of both surface mEGF receptors and surface hCG receptors. In order to study these phenomena, we assumed that the number of surface receptors is determined by the rate at which receptors appear at the surface and by the rate of receptor internalization. When these rates were measured, we found that hCG and mEGF reduce their respective surface receptors by increasing the rate of receptor internalization, and that mEGF reduces the surface hCG receptors by decreasing the rate of appearance of the receptor.

On the structure of the luteinizing hormone/chorionic gonadotropin receptor.

In this review we have tried to argue that the evidence indicating that the LH/CG receptor is composed of a single polypeptide is stronger than the evidence indicating that the LH/CG receptor is a more complex structure composed of several subunits. Clearly, however, this issue has not been resolved and probably will not be resolved by performing additional experiments similar to those summarized here. It is our opinion that this issue will be resolved only by 1) reconstitution experiments in which the ability of the purified LH/CG receptor to bind hCG and activate adenylyl cyclase activity is tested; and/or 2) isolation and expression of a full length complementary DNA (cDNA) for the LH/CG receptor and the demonstration of hCG binding and adenylyl cyclase activation by the expressed receptor. Similar experiments will also clarify the proposed structures for the FSH and TSH receptors. As the second decade of work on the LH/CG receptor draws to an end it appears that these experiments are now possible, and hopefully a resolution of the existing controversy will be forthcoming in the near future.

The lutropin/choriogonadotropin receptor ... 4 years later.

A comparison of this review with the review on the LH/CG receptor that we published in this journal 4 yr ago (1) clearly shows that the field has advanced tremendously in this short period of time. Many of the questions that were unanswered then have now been conclusively answered. On the other hand, as is always the case, the new knowledge generated has also resulted in many new questions that are yet to be answered. Hopefully it is clear from this review that the knowledge and experimental tools generated during the last 4 yr have given us new ammunition to address such important issues as the elucidation of the structural determinants of the LH/CG receptor that are involved in the different functions of this receptor, as well as on the molecular bases of receptor activation, inactivation, and regulation. In fact, since we finished writing this review in September of 1992, we are certain that some of the questions left unanswered here will already have an answer in 1993 when this review is published.

Inhibition of the degradation of receptor-bound human choriogonadotropin by leupeptin.

The ability of leupeptin to block the degradation of receptor-bound human choriogonadotropin has been studied. It was found that this compound inhibited hormone degradation and intracellular cathepsin B activity in a parallel fasion, without affecting hormone-stimulated steroidogenesis.

Degradation of the subunits of receptor-bound human choriogonadotropin by Leydig tumor cells.

Biologically active, iodine-labeled derivatives of human choriogonadotropin in which all the iodine is localized either in the alpha or beta subunits have been prepared. It is found that upon binding to Leydig tumor cells these derivatives are ultimately degraded to 3'-monoiodotyrosine. A comparison of the rates of degradation of the derivatives labeled exclusively in the alpha or beta subunits show that the alpha subunit is degraded somewhat faster than the beta subunit. It was also found that NH4Cl, chloroquine and leupeptin inhibited the degradation of both subunits to the same extent. These results show that the Leydig tumor cells degrade both subunits of the receptor-bound human choriogonadotropin, and suggest that the two subunits are degraded by the same mechanism(s).

The low-density lipoprotein pathway of cultured Leydig tumor cells. Utilization of low-density lipoprotein-derived cholesterol for steroidogenesis.

We have previously reported that low-density lipoprotein (LDL) enhances and prolongs steroidogenesis in human choriogonadotropin (CG)-stimulated Leydig tumor cells (MA-10). The studies described herein elucidate the mechanisms by which LDL increases human CG stimulated steroidogenesis. Our results show that the MA-10 cells express the classic LDL pathway. LDL is bound to specific surface binding sites which are regulated by the level of intracellular cholesterol. The cellular processing of bound LDL is temperature-dependent and is inhibited by blocking lysosomal function. By using an LDL derivative in which the core cholesteryl esters have been replaced with [3H]cholesteryl linoleate, we show that LDL cholesterol is rapidly utilized for steroid hormone synthesis. The utilization of LDL cholesterol quantitatively accounts for the LDL-induced augmentation of steroidogenesis. We also show that the addition of LDL to human CG-stimulated MA-10 cells maintains cellular free and esterified cholesterol levels and increases progesterone biosynthesis. The addition of LDL does not, however, affect the cellular utilization of preexisting cholesterol stores for steroidogenesis.

Characterization of the desensitized state of Leydig tumor cells.

A perifusion system has been used to study the in vitro desensitization of isolated Leydig tumor cells. It was observed that the cells become refractory, as measured by decreased rates of steroidogenesis, during continuous perifusions with saturating concentrations of either human choriogonadotropin (CG), cholera toxin, or 8-bromo-cyclic AMP. Furthermore, an initial perifusion of the cells with either human CG, cholera toxin, or 8-bromo-cyclic AMP causes subsequent desensitization towards all three stimuli. Thus, each of these stimuli is equally effective in inducing a state of desensitization in these cells that is manifested by a steroidogenic lesion(s) distal to cyclic AMP formation. It was found that the post-cyclic AMP lesion(s) in the desensitized state occurs prior to the formation of pregnenolone. However, the decreased rates of steroidogenesis do not seem to arise from a depletion of intracellular cholesterol.

The post-endocytotic fate of the gonadotropin receptors is an important determinant of the desensitization of gonadotropin responses.

Internalization of the ligand/receptor complexes is a consequence of the activation of the gonadotropin receptors. Since the recycling or degradation of the internalized receptors results in the maintenance or loss of cell surface receptors respectively and this contributes to the loss of responsiveness, we hypothesized that the fate of the internalized receptors could be an important component of desensitization. We examined this hypothesis using the wild-type and mutants of the human LH (hLHR) receptors and follitropin receptors expressed in MA-10 and KK-1 cells respectively. The receptor mutants were chosen because they are routed mostly to a lysosomal degradation pathway whereas the wild-type receptors are recycled back to the surface. We have shown that agonist stimulation of cells expressing the mutant receptors results in a more pronounced loss of cell surface receptors and agonist responses than stimulation of cells expressing the wild-type receptors. We concluded that receptor recycling promotes the maintenance of cell surface receptors and preserves hormonal responsiveness. This property of the hLHR is likely to be physiologically important because there at least two hLHR-expressing tissues in pregnant women, the maternal corpus luteum and the fetal Leydig cells, where a loss of hormonal responsiveness induced by the elevated levels of human chorionic gonadotropin that occur during pregnancy is not desirable.

The C-terminal tail of the rat lutropin/choriogonadotropin (CG) receptor independently modulates human (h)CG-induced internalization of the cell surface receptor and the lysosomal targeting of the internalized hCG-receptor complex.

The analysis of 21 progressive truncations of the C-terminal tail of the rat LH/CG receptor (rLHR) revealed the presence of a region delineated by residues 628-649 that, when removed, enhanced the degradation of the internalized human (h)CG. The analysis of these truncations also revealed the presence of a region delineated by residues 624-631 that, when removed, enhanced the rate of internalization of hCG. Since there is little overlap between these two regions, we conclude that the structural features of the rLHR that mediate internalization and degradation of the internalized hormone are different. Detailed analyses of cells expressing a truncation at Y637 (designated rLHR-t637) showed that the enhanced degradation of hCG observed in the these cells is due to an increase in the rate of transfer of the internalized hCG-rLHR complex from the endosomes to the lysosomes rather than to the enhanced dissociation of the hCG-rLHR complex in the lysosomes.

Effect of activating and inactivating mutations on the phosphorylation and trafficking of the human lutropin/choriogonadotropin receptor.

The effects of several mutations of the human LH receptor (hLHR) on the phosphorylation, internalization, and turnover of the cell surface receptor were examined. Three gain-of-function mutations associated with Leydig cell hyperplasia (L457R and D578Y) and one associated with Leydig cell adenomas (D578H), one signaling-impaired mutation associated with Leydig cell hypoplasia (I625K), and two laboratory designed signaling-impaired mutations (D405N and Y546F) were used. The signaling-impaired mutations showed a reduction in human CG (hCG)-induced receptor phosphorylation and internalization. Mutation of the phosphorylation sites of these loss-of-function mutants had little or no additional effect on internalization. Cotransfection with G protein-coupled receptor kinase-2 (GRK2) rescued the hCG-induced phosphorylation and internalization of the signaling-impaired mutations but only if the phosphorylation sites were intact. Overexpression of arrestin-3 rescued the rate of internalization regardless of whether or not the phosphorylation sites were intact. Only two of the three constitutively active mutants displayed an increase in basal phosphorylation. Although they all failed to respond to hCG with increased receptor phosphorylation, they all internalized hCG faster than wild-type hLHR (hLHR-wt). Mutation of the phosphorylation sites of these constitutively active mutants lengthened the half-time of internalization of hCG toward that of hLHR-wt. Overexpression of arrestin-3 had little or no effect on the already short half-time of internalization of hCG mediated by these mutants. The data obtained with the signaling-impaired and phosphorylation-deficient mutants of the hLHR support a model whereby receptor phosphorylation and activation play a redundant role in the internalization of hCG. The results obtained with the constitutively active mutants suggest that, when occupied by hCG, these mutants assume a conformation that bypasses many of the steps (i.e. activation, phosphorylation, and/or arrestin binding) involved in internalization.

Studies with insulin and insulin-like growth factor-I show that the increased labeling of phosphatidylinositol-3,4-bisphosphate is not sufficient to elicit the diverse actions of epidermal growth factor on MA-10 Leydig tumor cells.

In a recent publication we showed that addition of mouse epidermal growth factor (mEGF) to MA-10 Leydig tumor cells rapidly leads to an increase in the incorporation of [3H]inositol-derived radioactivity into an unusual lipid that was identified as phosphatidylinositol-3,4-bisphosphate (PI-3,4-P2). Other ligands that are known to bind to MA-10 cells, such as hCG and arginine vasopressin, however, did not elicit this effect. Inasmuch as mEGF modulates the differentiated functions of MA-10 cells in a number of ways, our findings raised the possibility that PI-3,4-P2 may be an intracellular mediator of these actions of mEGF. In an attempt to answer this question, we set out to determine if other ligands increase the labeling of PI-3,4-P2 in MA-10 cells prelabeled with [3H]inositol, and if such ligands mimic the diverse biological actions of mEGF on these cells. The experiments presented herein show that insulin, insulin-like growth factor-I, and transforming growth factor-alpha increase the labeling of PI-3,4-P2 in MA-10 cells, but only transforming growth factor-alpha mimics the actions of mEGF on the differentiated functions of MA-10 cells. We conclude that an increase in the labeling of PI-3,4-P2 is not sufficient to elicit these actions of mEGF.

Reduced gonadotropin responses in a novel clonal strain of Leydig tumor cells established by transfection of MA-10 cells with a mutant gene of the type I regulatory subunit of the cAMP-dependent protein kinase.

Although it is clear that cAMP is an important mediator of the actions of LH/CG in Leydig cells, recent studies from several laboratories have shown that the functions of Leydig cells can also be modulated by hormones and growth factors that do not appear to use cAMP as a second messenger. Thus, in order to increase our understanding of the importance of cAMP as a modulator of the functions of Leydig cells we have used a genetic approach to establish permanent cell lines that express a cAMP-resistant phenotype. MA-10 cells, a clonal strain of cultured Leydig tumor cells that express many of the characteristics of normal Leydig cells, were transfected with an expression vector controlled by the metallothionein promoter and encoding for a mutant form of the regulatory subunit of the type I cAMP-dependent protein kinase. Three stable transfectants that display a Zn+2-dependent decrease in cAMP-dependent protein kinase activity were established. Further characterization of one of the transfectants (designated MA-10(K3)) revealed a parallel reduction in the ability of cAMP and human CG to induce cell rounding, to increase steroid synthesis, or to induce c-fos mRNA. Our initial studies on these mutant cells have already provided novel information about the actions of human CG. These cell lines will also be valuable for further studies on the signaling systems that mediate hormone action in Leydig cells.

Phosphorylation of the lutropin/choriogonadotropin receptor facilitates uncoupling of the receptor from adenylyl cyclase and endocytosis of the bound hormone.

Stably transfected cell lines expressing the wild-type rat LH/CG receptor (rLHR) or a full-length rLHR in which S635, T638, S639, S649 and S653 were simultaneously mutated to alanine residues (designated rLHR-5S/T-->A) were used to probe the importance of receptor phosphorylation on the regulation of receptor functions. The mutant receptor binds hCG with high affinity and transduces the hormonal signal into increases in cAMP and inositol phosphate accumulation comparable in magnitude to those elicited by the wild-type receptor. In contrast to cells expressing rLHR-wt, which respond to hCG or phorbol 12-myristate 13-acetate stimulation with an increase in rLHR phosphorylation, the phosphorylation of rLHR in cells expressing rLHR-5S/T-->A is severely blunted. Likewise, the phorbol 12-myristate 13-acetate-induced desensitization of hCG-induced cAMP accumulation is drastically reduced in cells expressing rLHR-5S/T-->A. In contrast, the hCG-induced desensitization of hCG-induced cAMP accumulation is delayed, but not abolished, in cells expressing rLHR-5S/T-->A. Lastly, the rate of internalization of the receptor-bound hCG is slower in cells expressing rLHR-5S/T-->A than in cells expressing rLHR-wt. These results show that phosphorylation of rLHR is necessary, but not sufficient, for uncoupling of the receptor from adenylyl cyclase and for endocytosis of the receptor-bound hormone.

The regulation of the binding affinity of the luteinizing hormone/choriogonadotropin receptor by sodium ions is mediated by a highly conserved aspartate located in the second transmembrane domain of G protein-coupled receptors.

Sequence alignment shows that there is a highly conserved aspartate in the second transmembrane helix of virtually all G protein-coupled receptors. A previous study on the alpha 2-adrenergic receptor demonstrated that substitution of this acidic residue for the corresponding amide slightly decreases the affinity of the receptor for agonists and completely abolishes the effect of Na+ on the affinity for agonists. Since we have previously shown that Na+ modulates the binding affinity of the LH/CG receptor for ovine LH (oLH) [but not for human CG (hCG)], the experiments described here were designed to determine if the corresponding residue (D383) of the rat LH/CG receptor also mediates this Na+ effect. We used site-directed mutagenesis to create an LH/CG receptor mutant in which D383 was substituted by N. The wild type and mutant receptor [designated rLHR(D383N)] were expressed in human embryonic kidney 293 cells, and the transfected cells were tested for their ability to bind hCG and oLH in medium containing Na+ or an isoosmolar concentration of an appropriate sodium substitute. The results presented here show that this single point mutation of the LH/CG receptor leads to a slight reduction in affinity for hCG and oLH but completely abolishes the effects of Na+ removal on the affinity for oLH. Thus, regardless of the presence or absence of Na+, cells expressing rLHR(D383N) bind oLH with a low affinity comparable to that of the wild type receptor assayed in the presence of Na+. We also measured the ability of hCG and oLH to increase cAMP accumulation in cells expressing the wild type and mutant receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Mutation of a highly conserved acidic residue present in the second intracellular loop of G-protein-coupled receptors does not impair hormone binding or signal transduction of the luteinizing hormone/chorionic gonadotropin receptor.

Sequence alignment shows that there is a highly conserved acidic residue (D or E) at the boundary between the third transmembrane domain and the second intracellular loop of the superfamily of G-protein-coupled receptors. Previous mutagenesis studies demonstrated that substitution of this acidic residue in the beta 2-adrenergic, muscarinic m1, and alpha 2A-adrenergic receptors by the corresponding amide preserved high affinity agonist binding, but significantly reduced or completely abolished activation of the respective effector. To determine whether the corresponding amino acid residue (E441) played a similar role in the functions of the rat LH/CG receptor, we used site-directed mutagenesis to substitute it by D or Q. The wild-type and mutant receptors (E441D or E441Q) were then transfected into human embryonic kidney 293 cells and tested for their ability to bind hCG and respond to it with increased cAMP accumulation. As predicted, the mutant LH/CG receptors were found to bind hCG with high affinity. In contrast to the results summarized above, however, an E441Q or an E441D mutation in the LH/CG receptor results in only a slight increase in the EC50 for cAMP accumulation without decreasing the maximal response attained. The most remarkable effect of these mutations was on localization of the receptor. Thus, while most of the receptors expressed in cells transfected with the E441D mutant could be detected by measuring hormone binding to intact cells, most of the receptors expressed in cells transfected with the E441Q mutant could be detected only upon solubilization of the cells with detergent.

The lutropin/choriogonadotropin receptor is palmitoylated at intracellular cysteine residues.

Most members of the family of G protein-coupled receptors have one or more conserved cysteine residues in their carboxy-terminal cytoplasmic tails which are believed to be consensus sites for palmitoylation. Indeed, a growing number of G protein-coupled receptors (rhodopsin, beta 2-, and alpha 2-adrenergic receptors) have now been shown to have palmitic acid covalently attached to this position. In the case of the beta 2-adrenergic receptor, it was also reported that mutation of the palmitoylated cysteine to glycine greatly diminished the ability of this receptor to interact with and activate Gs. Mutation of this conserved cysteine appears to have little or no effect on the ability of other members of this receptor family (rhodopsin, alpha 2-adrenergic and M2 muscarinic) to activate their cognate G proteins, however. The studies presented here were designed to determine whether another Gs-coupled receptor, the LH/CG receptor, is palmitoylated, and whether this modification is important for receptor function. To facilitate biochemical analysis, we examined these issues using cell lines stably transfected with the wild type LH/CG receptor (LHR-wt) or with a mutant receptor in which the two conserved cysteins were mutated to alanines (designated LHR-C621,622A). Our results show that LHR-wt is palmitoylated but that LHR-C621,622A is not. We also show that LHR-C621,622A is capable of binding human CG (hCG) and transducing the cAMP signal. The main difference that we detected between the wild type and mutant receptor is that the latter is trapped intracellularly and does not appear to mature into the 85 kilodalton protein previously identified as the mature cell surface LH/CG receptor.