Cassandre Labelle-Dumais

COL4A1 and COL4A2 Mutations and Disease: Insights into Pathogenic Mechanisms and Potential Therapeutic Targets

Heterotrimers composed of collagen type IV alpha 1 (COL4A1) and alpha 2 (COL4A2) constitute one of the most abundant components of nearly all basement membranes. Accordingly, mutations in COL4A1 or COL4A2 are pleiotropic and contribute to a broad spectrum of disorders, including myopathy, glaucoma and hemorrhagic stroke. Here, we summarize the contributions of COL4A1 and COL4A2 mutations in human disease, integrate knowledge gained from model organisms and evaluate the implications for pathogenic mechanisms and therapeutic approaches.

COL4A2 Mutations Impair COL4A1 and COL4A2 Secretion and Cause Hemorrhagic Stroke

Collagen, type IV, alpha 1 (COL4A1) and alpha 2 (COL4A2) form heterotrimers and are abundant components of basement membranes, including those of the cerebral vasculature. COL4A1 mutations are an increasingly recognized cause of multisystem disorders, including highly penetrant cerebrovascular disease and intracerebral hemorrhage (ICH). Because COL4A1 and COL4A2 are structurally and functionally associated, we hypothesized that variants in COL4A2 would also cause ICH. We sequence COL4A2 in 96 patients with ICH and identify three rare, nonsynonymous coding variants in four patients that are not present in a cohort of 144 ICH-free individuals. All three variants change evolutionarily conserved amino acids. Using a cellular assay, we show that these putative mutations cause intracellular accumulation of COL4A1 and COL4A2 at the expense of their secretion, which supports their pathogenecity. Furthermore, we show that Col4a2 mutant mice also have completely penetrant ICH and that mutations in mouse and human lead to retention of COL4A1 and COL4A2 within the endoplasmic reticulum (ER). Importantly, two of the three putative mutations found in patients trigger ER stress and activate the unfolded protein response. The identification of putative COL4A2 mutations that might contribute to ICH in human patients provides insight into the pathogenic mechanisms of this disease. Our data suggest that COL4A2 mutations impair COL4A1 and COL4A2 secretion and can also result in cytotoxicity. Finally, our findings suggest that, collectively, mutations in COL4A1 and COL4A2 contribute to sporadic cases of ICH.

COL4A1 Mutations Cause Ocular Dysgenesis, Neuronal Localization Defects, and Myopathy in Mice and Walker-Warburg Syndrome in Humans

Muscle-eye-brain disease (MEB) and Walker Warburg Syndrome (WWS) belong to a spectrum of autosomal recessive diseases characterized by ocular dysgenesis, neuronal migration defects, and congenital muscular dystrophy. Until now, the pathophysiology of MEB/WWS has been attributed to alteration in dystroglycan post-translational modification. Here, we provide evidence that mutations in a gene coding for a major basement membrane protein, collagen IV alpha 1 (COL4A1), are a novel cause of MEB/WWS. Using a combination of histological, molecular, and biochemical approaches, we show that heterozygous Col4a1 mutant mice have ocular dysgenesis, neuronal localization defects, and myopathy characteristic of MEB/WWS. Importantly, we identified putative heterozygous mutations in COL4A1 in two MEB/WWS patients. Both mutations occur within conserved amino acids of the triple-helix-forming domain of the protein, and at least one mutation interferes with secretion of the mutant proteins, resulting instead in intracellular accumulation. Expression and posttranslational modification of dystroglycan is unaltered in Col4a1 mutant mice indicating that COL4A1 mutations represent a distinct pathogenic mechanism underlying MEB/WWS. These findings implicate a novel gene and a novel mechanism in the etiology of MEB/WWS and expand the clinical spectrum of COL4A1-associated disorders.

COL4A1 Mutations in Patients with Sporadic Late-Onset Intracerebral Hemorrhage

Mutations in the type IV collagen alpha 1 gene (COL4A1) cause dominantly inherited cerebrovascular disease. We seek to determine the extent to which COL4A1 mutations contribute to sporadic, nonfamilial, intracerebral hemorrhages (ICHs).

Netrin-1 receptor-deficient mice show enhanced mesocortical dopamine transmission and blunted behavioural responses to amphetamine.

The mesocorticolimbic dopamine (DA) system is implicated in neurodevelopmental psychiatric disorders including schizophrenia but it is unknown how disruptions in brain development modify this system and increase predisposition to cognitive and behavioural abnormalities in adulthood. Netrins are guidance cues involved in the proper organization of neuronal connectivity during development. We have hypothesized that variations in the function of DCC (deleted in colorectal cancer), a netrin‐1 receptor highly expressed by DA neurones, may result in altered development and organization of mesocorticolimbic DA circuitry, and influence DA function in the adult. To test this hypothesis, we assessed the effects of reduced DCC on several indicators of DA function. Using in‐vivo microdialysis, we showed that adult mice that develop with reduced DCC display increased basal DA levels in the medial prefrontal cortex and exaggerated DA release in response to the indirect DA agonist amphetamine. In contrast, these mice exhibit normal levels of DA in the nucleus accumbens but significantly blunted amphetamine‐induced DA release. Concomitantly, using conditioned place preference, locomotor activity and prepulse inhibition paradigms, we found that reduced DCC diminishes the rewarding and behavioural‐activating effects of amphetamine and protects against amphetamine‐induced deficits in sensorimotor gating. Furthermore, we found that adult DCC‐deficient mice exhibit altered dendritic spine density in layer V medial prefrontal cortex pyramidal neurones but not in nucleus accumbens medium spiny neurones. These findings demonstrate that reduced DCC during development results in a behavioural phenotype opposite to that observed in developmental models of schizophrenia and identify DCC as a critical factor in the development of DA function.

Manitoba-oculo-tricho-anal (MOTA) syndrome is caused by mutations in FREM1

Background Manitoba-oculo-tricho-anal (MOTA) syndrome is a rare condition defined by eyelid colobomas, cryptophthalmos and anophthalmia/microphthalmia, an aberrant hairline, a bifid or broad nasal tip, and gastrointestinal anomalies such as omphalocele and anal stenosis. Autosomal recessive inheritance had been assumed because of consanguinity in the Oji-Cre population of Manitoba and reports of affected siblings, but no locus or cytogenetic aberration had previously been described. Methods and results This study shows that MOTA syndrome is caused by mutations in FREM1, a gene previously mutated in bifid nose, renal agenesis, and anorectal malformations (BNAR) syndrome. MOTA syndrome and BNAR syndrome can therefore be considered as part of a phenotypic spectrum that is similar to, but distinct from and less severe than, Fraser syndrome. Re-examination of Frem1bat/bat mutant mice found new evidence that Frem1 is involved in anal and craniofacial development, with anal prolapse, eyelid colobomas, telecanthus, a shortened snout and reduced philtral height present in the mutant mice, similar to the human phenotype in MOTA syndrome. Conclusions The milder phenotypes associated with FREM1 deficiency in humans (MOTA syndrome and BNAR syndrome) compared to that resulting from FRAS1 and FREM2 loss of function (Fraser syndrome) are also consistent with the less severe phenotypes resulting from Frem1 loss of function in mice. Together, Fraser, BNAR and MOTA syndromes constitute a clinically overlapping group of FRAS–FREM complex diseases.

Peri-Pubertal Emergence of UNC-5 Homologue Expression by Dopamine Neurons in Rodents

Puberty is a critical period in mesocorticolimbic dopamine (DA) system development, particularly for the medial prefrontal cortex (mPFC) projection which achieves maturity in early adulthood. The guidance cue netrin-1 organizes neuronal networks by attracting or repelling cellular processes through DCC (deleted in colorectal cancer) and UNC-5 homologue (UNC5H) receptors, respectively. We have shown that variations in netrin-1 receptor levels lead to selective reorganization of mPFC DA circuitry, and changes in DA-related behaviors, in transgenic mice and in rats. Significantly, these effects are only observed after puberty, suggesting that netrin-1 mediated effects on DA systems vary across development. Here we report on the normal expression of DCC and UNC5H in the ventral tegmental area (VTA) by DA neurons from embryonic life to adulthood, in both mice and rats. We show a dramatic and enduring pubertal change in the ratio of DCC:UNC5H receptors, reflecting a shift toward predominant UNC5H function. This shift in DCC:UNC5H ratio coincides with the pubertal emergence of UNC5H expression by VTA DA neurons. Although the distribution of DCC and UNC5H by VTA DA neurons changes during puberty, the pattern of netrin-1 immunoreactivity in these cells does not. Together, our findings suggest that DCC:UNC5H ratios in DA neurons at critical periods may have important consequences for the organization and function of mesocorticolimbic DA systems.

Nuclear receptor NR5A2 is required for proper primitive streak morphogenesis.

NR5A2, also known as liver receptor homologue 1 (LRH‐1) and fetoprotein transcription factor (FTF), is an orphan nuclear receptor involved in the regulation of cholesterol metabolism and steroidogenesis in the adult. NR5A2 was also shown to be expressed during early mouse embryogenesis. Consistent with its early expression pattern, a targeted disruption of this gene leads to embryonic lethality around the gastrulation period. To characterize the embryonic phenotype resulting from NR5A2 loss of function, we undertook morphological and marker gene analyses and showed that NR5A2−/− embryos display growth retardation, epiblast disorganization, a mild embryonic–extraembryonic constriction, as well as abnormal thickening of the proximo‐posterior epiblast. We demonstrated that, although initial specification of the anterior–posterior axis occurred in the absence of NR5A2, primitive streak formation was impaired and neither embryonic nor extraembryonic mesoderm was generated. Moreover, although the visceral endoderm does not show major morphological abnormalities in NR5A2−/− embryos, a decrease in the expression level of HNF4 and GATA4 was observed. Aggregation experiments demonstrated that, in the presence of wild‐type tetraploid cells, NR5A2 mutant cells in the epiblast are capable of undergoing normal gastrulation. Therefore, our results suggest a requirement for NR5A2 in extraembryonic tissues and identify a novel role of this gene in proper primitive streak morphogenesis. Developmental Dynamics 235:3359–3369, 2006.

Post-pubertal emergence of a dopamine phenotype in netrin-1 receptor-deficient mice

During the pubertal period the mesocortical dopamine (DA) system undergoes substantial reorganization of neuronal connectivity and functional refinement. Netrins are guidance cues involved in the organization of neuronal circuitry. We have previously shown that adult mice that develop with reduced levels of the netrin‐1 receptor [deleted in colorectal cancer (DCC)] display selective reorganization of mesocortical DA circuitry, show enhanced mesocortical DA function and exhibit a behavioural phenotype opposite to that observed in animal models of schizophrenia. Here we assess whether the dcc behavioural and DA phenotypes are present prior to the maturation of the mesocortical DA system by comparing dcc‐heterozygous and wild‐type mice at the post‐weaning and peri‐pubertal periods on various indices of DA function. At both the post‐weaning and peri‐pubertal ages, but unlike in adulthood, dcc‐heterozygous and wild‐type mice show no differences in the number of midbrain DA neurones or in tyrosine hydroxylase protein levels in the medial prefrontal cortex. Furthermore, the elevated baseline concentration of mesocortical DA and DA metabolites observed in adult dcc‐heterozygous mice is not present in either post‐weanling or peri‐pubertal mice. Interestingly, post‐weanling, but not peri‐pubertal, dcc‐heterozygous mice show greater baseline concentrations of DA metabolites in the nucleus accumbens, opposite to what was observed in adulthood. Finally, neither post‐weanling nor peri‐pubertal dcc‐heterozygous mice demonstrate the blunted amphetamine‐induced locomotor response observed in adulthood. Thus, these findings show that the ‘protective’dcc phenotype has a post‐pubertal emergence and indicate that DCC may play a role in the normal maturation of the mesocorticolimbic DA system.

Impaired Progesterone Production in Nr5a2+/− Mice Leads to a Reduction in Female Reproductive Function

Abstract NR5A2 is an orphan nuclear receptor involved in cholesterol metabolism and embryogenesis. The high level of expression of NR5A2 in the ovary and its involvement in the regulation of steroidogenic gene expression also suggest a role for this transcription factor in female reproductive function. In vivo evidence for a role for NR5A2 in fertility, however, is still lacking. In order to address this possibility, we used Nr5a2+/− mice to demonstrate that heterozygosity for a null mutation of Nr5a2 leads to a decreased fertility in females. Our results indicate that although Nr5a2+/− mice display normal follicular development, ovulation, and estrogen production, they exhibit altered luteal function. More specifically, we show that the reduced reproductive ability of Nr5a2+/− females arises from a reduction in circulating progesterone concentrations and can be rescued by exogenous progesterone supplementation. This study therefore provides the first in vivo evidence for a role of NR5A2 in reproductive function and steroidogenesis.