Targeting the Brain with Single-Domain Antibodies: Greater Potential Than Stated So Far?

Treatments for central nervous system diseases with therapeutic antibodies have been increasingly investigated over the last decades, leading to some approved monoclonal antibodies for brain disease therapies. The detection of biomarkers for diagnosis purposes with non-invasive antibody-based imaging approaches has also been explored in brain cancers. However, antibodies generally display a low capability of reaching the brain, as they do not efficiently cross the blood-brain barrier. As an alternative, recent studies have focused on single-domain antibodies (sdAbs) that correspond to the antigen-binding fragment. While some reports indicate that the brain uptake of these small antibodies is still low, the number of studies reporting brain-penetrating sdAbs is increasing. In this review, we provide an overview of methods used to assess or evaluate brain penetration of sdAbs and discuss the pros and cons that could affect the identification of brain-penetrating sdAbs of therapeutic or diagnostic interest.

GABAB1e promotes the malignancy of human cancer cells by targeting the tyrosine phosphatase PTPN12.

Neurotransmitter receptors are involved in cancer progression. Among them, the heterodimeric GABAB receptor, activated by the main inhibitory neurotransmitter GABA, is composed of the transmembrane GABAB1 and GABAB2 subunits. The oncogenic role of the isoform GABAB1e (GB1e) containing only the extracellular domain of GABAB1 remains unclear. We revealed that GB1e is largely expressed in human breast cancer (BrCa) cell lines as well as in BrCa tissues where it is upregulated. Moreover, GB1e promoted the malignancy of BrCa cells both in vitro and in vivo. We propose that GB1e favors EGFR signaling by interacting with PTPN12 to disrupt the interaction between EGFR and PTPN12, and phosphorylation of Y230 and Y404 on GB1e is required in this process. Our data highlight that the GABBR1 gene through the expression of the GB1e isoform might play an important oncogenic role in BrCa and that GB1e is of interest for the treatment of some cancers.

Allosteric nanobodies uncover a role of hippocampal mGlu2 receptor homodimers in contextual fear consolidation.

Antibodies have enormous therapeutic and biotechnology potential. G protein-coupled receptors (GPCRs), the main targets in drug development, are of major interest in antibody development programs. Metabotropic glutamate receptors are dimeric GPCRs that can control synaptic activity in a multitude of ways. Here we identify llama nanobodies that specifically recognize mGlu2 receptors, among the eight subtypes of mGluR subunits. Among these nanobodies, DN10 and 13 are positive allosteric modulators (PAM) on homodimeric mGlu2, while DN10 displays also a significant partial agonist activity. DN10 and DN13 have no effect on mGlu2-3 and mGlu2-4 heterodimers. These PAMs enhance the inhibitory action of the orthosteric mGlu2/mGlu3 agonist, DCG-IV, at mossy fiber terminals in the CA3 region of hippocampal slices. DN13 also impairs contextual fear memory when injected in the CA3 region of hippocampal region. These data highlight the potential of developing antibodies with allosteric actions on GPCRs to better define their roles in vivo.

Major ligand-induced rearrangement of the heptahelical domain interface in a GPCR dimer.

G protein-coupled receptors (GPCRs) are major players in cell communication. Although they form functional monomers, increasing evidence indicates that GPCR dimerization has a critical role in cooperative phenomena that are important for cell signal integration. However, the structural bases of these phenomena remain elusive. Here, using well-characterized receptor dimers, the metabotropic glutamate receptors (mGluRs), we show that structural changes at the dimer interface are linked to receptor activation. We demonstrate that the main dimer interface is formed by transmembrane α helix 4 (TM4) and TM5 in the inactive state and by TM6 in the active state. This major change in the dimer interface is required for receptor activity because locking the TM4-TM5 interface prevents activation by agonist, whereas locking the TM6 interface leads to a constitutively active receptor. These data provide important information on the activation mechanism of mGluRs and improve our understanding of the structural basis of the negative cooperativity observed in these GPCR dimers.

Biased signaling through G-protein-coupled PROKR2 receptors harboring missense mutations.

Various missense mutations in the gene coding for prokineticin receptor 2 (PROKR2), a G-protein-coupled receptor, have been identified in patients with Kallmann syndrome. However, the functional consequences of these mutations on the different signaling pathways of this receptor have not been studied. We first showed that the wild-type PROKR2 can activate different G-protein subtypes (Gq, Gs, and Gi/o) and recruit ß-arrestins in transfected HEK-293 cells. We then examined, for each of these signaling pathways, the effects of 9 mutations that did not significantly impair cell surface targeting or ligand binding of the receptor. Four mutant receptors showing defective Gq signaling (R85C, R85H, R164Q, and V331M) could still recruit ß-arrestins on ligand activation, which may cause biased signaling in vivo. Conversely, the R80C receptor could activate the 3 types of G proteins but could not recruit ß-arrestins. Finally, the R268C receptor could recruit ß-arrestins and activate the Gq and Gs signaling pathways but could not activate the Gi/o signaling pathway. Our results validate the concept that mutations in the genes encoding membrane receptors can bias downstream signaling in various ways, possibly leading to pathogenic and, perhaps in some cases, protective (e.g., R268C) effects.

Variations in PROKR2, but not PROK2, are associated with hypopituitarism and septo-optic dysplasia.

CONTEXT: Loss-of-function mutations in PROK2 and PROKR2 have been implicated in Kallmann syndrome (KS), characterized by hypogonadotropic hypogonadism and anosmia. Recent data suggest overlapping phenotypes/genotypes between KS and congenital hypopituitarism (CH), including septo-optic dysplasia (SOD). OBJECTIVE: We screened a cohort of patients with complex forms of CH (n = 422) for mutations in PROK2 and PROKR2. RESULTS: We detected 5 PROKR2 variants in 11 patients with SOD/CH: novel p.G371R and previously reported p.A51T, p.R85L, p.L173R, and p.R268C-the latter 3 being known functionally deleterious variants. Surprisingly, 1 patient with SOD was heterozygous for the p.L173R variant, whereas his phenotypically unaffected mother was homozygous for the variant. We sought to clarify the role of PROKR2 in hypothalamopituitary development through analysis of Prokr2(-/-) mice. Interestingly, these revealed predominantly normal hypothalamopituitary development and terminal cell differentiation, with the exception of reduced LH; this was inconsistent with patient phenotypes and more analogous to the healthy mother, although she did not have KS, unlike the Prokr2(-/-) mice. CONCLUSIONS: The role of PROKR2 in the etiology of CH, SOD, and KS is uncertain, as demonstrated by no clear phenotype-genotype correlation; loss-of-function variants in heterozygosity or homozygosity can be associated with these disorders. However, we report a phenotypically normal parent, homozygous for p.L173R. Our data suggest that the variants identified herein are unlikely to be implicated in isolation in these disorders; other genetic or environmental modifiers may also impact on the etiology. Given the phenotypic variability, genetic counseling may presently be inappropriate.

PROKR2 variants in multiple hypopituitarism with pituitary stalk interruption.

CONTEXT: Pituitary stalk interruption represents a frequent feature of congenital hypopituitarism, but only rare cases have been assigned to a known genetic cause. OBJECTIVE: Using a candidate gene approach, we tested several genes as potential causes of hypopituitarism with pituitary stalk interruption. We hypothesized that ectopic posterior pituitary may be a consequence of defective neuronal axon projections along the pituitary stalk or defective angiogenesis of hypophyseal portal circulation. Considering the role of the prokineticin 2 pathway in angiogenesis and neuronal migration, we screened PROK2 and PROKR2 genes. DESIGN: PROK2 and PROKR2 and all genes previously known to be involved in hypopituitarism with pituitary stalk interruption (LHX4, HESX1, OTX2, and SOX3) were screened in 72 index cases with pituitary stalk interruption syndrome from the GENHYPOPIT database. In vitro studies were performed to assess the functional consequences of allelic variants. RESULTS: We identified two heterozygous PROKR2 mutations (p.Leu173Arg and p.Arg85His) previously reported in isolated hypogonadotroph hypogonadism and a novel PROKR2 variant (p.Ala51Thr) that, in contrast with both other mutations, did not impair receptor signaling activity. Three allelic variants of HESX1 were identified: the heterozygous p.Phe156Ser and the homozygous p.Arg109X mutations were functionally deleterious, whereas p.Ser67Thr was found as a rare allelic variant in association with p.Arg85His PROKR2 mutation in the same patient. CONCLUSIONS: We report PROKR2 variants in congenital hypopituitarism with pituitary stalk interruption, suggesting a potential role of the prokineticin pathway in pituitary development.

Interdomain movements in metabotropic glutamate receptor activation.

Many cell surface receptors are multimeric proteins, composed of several structural domains, some involved in ligand recognition, whereas others are responsible for signal transduction. In most cases, the mechanism of how ligand interaction in the extracellular domains leads to the activation of effector domains remains largely unknown. Here we examined how the extracellular ligand binding to the venus flytrap (VFT) domains of the dimeric metabotropic glutamate receptors activate the seven transmembrane (7TM) domains responsible for G protein activation. These two domains are interconnected by a cysteine-rich domain (CRD). We show that any of the four disulfide bridges of the CRD are required for the allosteric coupling between the VFT and the 7TM domains. More importantly, we show that a specific association of the two CRDs corresponds to the active state of the receptor. Indeed, a specific crosslinking of the CRDs with intersubunit disulfide bridges leads to fully constitutively active receptors, no longer activated by agonists nor by allosteric modulators. These data demonstrate that intersubunit movement at the level of the CRDs represents a key step in metabotropic glutamate receptor activation.

GABAB receptor activation protects neurons from apoptosis via IGF-1 receptor transactivation.

The G-protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs) play key roles in cell-cell communication. Several studies revealed important synergisms between these two types of receptors, with some of the actions of either receptor being mediated through transactivation of the other. Among the large GPCR family, GABA(B) receptor is activated by the neurotransmitter GABA, and is expressed in most neurons where it mediates slow and prolonged inhibition of synaptic transmission. Here we show that this receptor is involved in the regulation of life and death decisions of cerebellar granule neurons (CGNs). We show that specific activation of GABA(B) receptor can protect neurons from apoptosis through a mechanism that involves transactivation of the IGF-1 receptor (IGF-1R). Further work demonstrated that this cross talk was dependent on G(i/o)-protein, PLC, cytosolic Ca(2+), and FAK1 but independent of PKC, while IGF-1R-induced signaling involved Src kinase, PI3 kinase, and Akt activation. These results reveal a new function for this important GPCR and further highlight the importance of functional cross-talk networks between GPCRs and RTKs. Our results reveal GABA(B) receptor as a potential drug target for the treatment of neurodegenerative disorders.

A comparative phenotypic study of kallmann syndrome patients carrying monoallelic and biallelic mutations in the prokineticin 2 or prokineticin receptor 2 genes.

CONTEXT: Both biallelic and monoallelic mutations in PROK2 or PROKR2 have been found in Kallmann syndrome (KS). OBJECTIVE: The objective of the study was to compare the phenotypes of KS patients harboring monoallelic and biallelic mutations in these genes. DESIGN AND PATIENTS: We studied clinical and endocrine features that reflect the functioning of the pituitary-gonadal axis, and the nonreproductive phenotype, in 55 adult KS patients (42 men and 13 women), of whom 41 had monoallelic mutations and 14 biallelic mutations in PROK2 or PROKR2. RESULTS: Biallelic mutations were associated with more frequent cryptorchidism (70% vs. 34%, P < 0.05) and microphallus (90% vs. 28%, P < 0.001) and lower mean testicular volume (1.2 +/- 0.4 vs. 4.5 +/- 6.0 ml; P < 0.01) in male patients. Likewise, the testosterone level as well as the basal FSH level and peak LH level under GnRH-stimulation were lower in males with biallelic mutations (0.2 +/- 0.1 vs. 0.7 +/- 0.8 ng/ml; P = 0.05, 0.3 +/- 0.1 vs. 1.8 +/- 3.0 IU/liter; P < 0.05, and 0.8 +/- 0.8 vs. 5.2 +/- 5.5 IU/liter; P < 0.05, respectively). Nonreproductive, nonolfactory anomalies were rare in both sexes and were never found in patients with biallelic mutations. The mean body mass index of the patients (23.9 +/- 4.2 kg/m(2) in males and 26.3 +/- 6.6 kg/m(2) in females) did not differ significantly from that of gender-, age-, and treatment-matched KS individuals who did not carry a mutation in PROK2 or PROKR2. Finally, circadian cortisol levels evaluated in five patients, including one with biallelic PROKR2 mutations, were normal in all cases. CONCLUSION: Male patients carrying biallelic mutations in PROK2 or PROKR2 have a less variable and on average a more severe reproductive phenotype than patients carrying monoallelic mutations in these genes. Nonreproductive, nonolfactory clinical anomalies associated with KS seem to be restricted to patients with monoallelic mutations.

PROKR2 missense mutations associated with Kallmann syndrome impair receptor signalling activity.

Kallmann syndrome (KS) combines hypogonadism due to gonadotropin-releasing hormone deficiency, and anosmia or hyposmia, related to defective olfactory bulb morphogenesis. In a large series of KS patients, ten different missense mutations (p.R85C, p.R85H, p.R164Q, p.L173R, p.W178S, p.Q210R, p.R268C, p.P290S, p.M323I, p.V331M) have been identified in the gene encoding the G protein-coupled receptor prokineticin receptor-2 (PROKR2), most often in the heterozygous state. Many of these mutations were, however, also found in clinically unaffected individuals, thus raising the question of their actual implication in the KS phenotype. We reproduced each of the ten mutations in a recombinant murine Prokr2, and tested their effects on the signalling activity in transfected HEK-293 cells, by measuring intracellular calcium release upon ligand-activation of the receptor. We found that all mutated receptors except one (M323I) had decreased signalling activities. These could be explained by different defective mechanisms. Three mutations (L173R, W178S, P290S) impaired cell surface-targeting of the receptor. One mutation (Q210R) abolished ligand-binding. Finally, five mutations (R85C, R85H, R164Q, R268C, V331M) presumably impaired G protein-coupling of the receptor. In addition, when wild-type and mutant receptors were coexpressed in HEK-293 cells, none of the mutant receptors that were retained within the cells did affect cell surface-targeting of the wild-type receptor, and none of the mutant receptors properly addressed at the plasma membrane did affect wild-type receptor signalling activity. This argues against a dominant negative effect of the mutations in vivo.

Human G(salpha) mutant causes pseudohypoparathyroidism type Ia/neonatal diarrhea, a potential cell-specific role of the palmitoylation cycle.

Pseudohypoparathyroidism type Ia (PHP-Ia) results from the loss of one allele of G(salpha), causing resistance to parathyroid hormone and other hormones that transduce signals via G(s). Most G(salpha)mutations cause the complete loss of protein expression, but some cause loss of function only, and these have provided valuable insights into the normal function of G proteins. Here we have analyzed a mutant G(salpha) (alphas-AVDT) harboring AVDT amino acid repeats within its GDP/GTP binding site, which was identified in unique patients with PHP-Ia accompanied by neonatal diarrhea. Biochemical and intact cell analyses showed that alphas-AVDT is unstable but constitutively active as a result of rapid GDP release and reduced GTP hydrolysis. This instability underlies the PHP-Ia phenotype. alphas-AVDT is predominantly localized in the cytosol, but in rat and mouse small intestine epithelial cells (IEC-6 and DIF-12 cells) alphas-AVDT was found to be localized predominantly in the membrane where adenylyl cyclase is present and constitutive increases in cAMP accumulation occur in parallel. The likely cause of this membrane localization is the inhibition of an activation-dependent decrease in alphas palmitoylation. Upon the overexpression of acyl-protein thioesterase 1, however, alphas-AVDT translocates from the membrane to the cytosol, and the constitutive accumulation of cAMP becomes attenuated. These results suggest that PHP-Ia results from the instability of alphas-AVDT and that the accompanying neonatal diarrhea may result from its enhanced constitutive activity in the intestine. Hence, palmitoylation may control the activity and localization of G(salpha) in a cell-specific manner.