Exome sequencing has markedly enhanced the discovery of genes implicated in Mendelian disorders, particularly for individuals in whom a known clinical entity could not be assigned. This has led to ...the recognition that phenotypic heterogeneity resulting from allelic mutations occurs more commonly than previously appreciated. Here, we report that missense variants in CDC42, a gene encoding a small GTPase functioning as an intracellular signaling node, underlie a clinically heterogeneous group of phenotypes characterized by variable growth dysregulation, facial dysmorphism, and neurodevelopmental, immunological, and hematological anomalies, including a phenotype resembling Noonan syndrome, a developmental disorder caused by dysregulated RAS signaling. In silico, in vitro, and in vivo analyses demonstrate that mutations variably perturb CDC42 function by altering the switch between the active and inactive states of the GTPase and/or affecting CDC42 interaction with effectors, and differentially disturb cellular and developmental processes. These findings reveal the remarkably variable impact that dominantly acting CDC42 mutations have on cell function and development, creating challenges in syndrome definition, and exemplify the importance of functional profiling for syndrome recognition and delineation.
Abstract SMN (Survival Motor Neuron) deficiency is the predominant cause of spinal muscular atrophy (SMA), a severe neurodegenerative disorder that can lead to progressive paralysis and death. ...Although SMN is required in every cell for proper RNA metabolism, the reason why its loss is especially critical in the motor system is still unclear. SMA genetic models have been employed to identify several modifiers that can ameliorate the deficits induced by SMN depletion. Here we focus on WDR79/TCAB1, a protein important for the biogenesis of several RNA species that has been shown to physically interact with SMN in human cells. We show that WDR79 depletion results in locomotion defects in both Drosophila and Caenorhabditis elegans similar to those elicited by SMN depletion. Consistent with this observation, we find that SMN overexpression rescues the WDR79 loss-of-function phenotype in flies. Most importantly, we also found that WDR79 overexpression ameliorates the locomotion defects induced by SMN depletion in both flies and worms. Our results collectively suggest that WDR79 and SMN play evolutionarily conserved cooperative functions in the nervous system and suggest that WDR79/TCAB1 may have the potential to modify SMA pathogenesis.
The dopamine transporter (DAT) is a cell membrane protein whose main function is to reuptake the dopamine (DA) released in the synaptic cleft back into the dopaminergic neurons. Previous studies ...suggested that the activity of DAT is regulated by allosteric proteins such as Syntaxin-1A and is altered by drugs of abuse such as amphetamine (Amph). Because
expresses both DAT (DAT-1) and Syntaxin-1A (UNC-64), we used this model system to investigate the functional and behavioral effects caused by lack of expression of
in cultured dopaminergic neurons and in living animals. Using an inheritable RNA silencing technique, we were able to knockdown
specifically in the dopaminergic neurons. This cell-specific knockdown approach avoids the pleiotropic phenotypes caused by knockout mutations of
and ensures the transmission of dopaminergic specific
silencing to the progeny. We found that, similarly to
knockouts and
silenced lines, animals with reduced
expression in the dopaminergic neurons did not respond to Amph treatment when tested for locomotor behaviors. Our
data demonstrated that in neuronal cultures derived from animals silenced for
, the DA uptake was reduced by 30% when compared to controls, and this reduction was similar to that measured in neurons isolated from animals silenced for
(40%). Moreover, reduced expression of
in the dopaminergic neurons significantly reduced the DA release elicited by Amph. Because in
DAT-1 is the only protein capable to reuptake DA, these data show that reduced expression of
in the dopaminergic neurons decreases the capability of DAT in re-accumulating synaptic DA. Moreover, these results demonstrate that decreased expression of
in the dopaminergic neurons abrogates the locomotor behavior induced by Amph. Taken together these data suggest that Syntaxin-1A plays an important role in both functional and behavioral effects caused by Amph.
In metazoans, the secreted proteome participates in intercellular signalling and innate immunity, and builds the extracellular matrix scaffold around cells. Compared with the relatively constant ...intracellular environment, conditions for proteins in the extracellular space are harsher, and low concentrations of ATP prevent the activity of intracellular components of the protein quality-control machinery. Until now, only a few bona fide extracellular chaperones and proteases have been shown to limit the aggregation of extracellular proteins
. Here we performed a systematic analysis of the extracellular proteostasis network in Caenorhabditis elegans with an RNA interference screen that targets genes that encode the secreted proteome. We discovered 57 regulators of extracellular protein aggregation, including several proteins related to innate immunity. Because intracellular proteostasis is upregulated in response to pathogens
, we investigated whether pathogens also stimulate extracellular proteostasis. Using a pore-forming toxin to mimic a pathogenic attack, we found that C. elegans responded by increasing the expression of components of extracellular proteostasis and by limiting aggregation of extracellular proteins. The activation of extracellular proteostasis was dependent on stress-activated MAP kinase signalling. Notably, the overexpression of components of extracellular proteostasis delayed ageing and rendered worms resistant to intoxication. We propose that enhanced extracellular proteostasis contributes to systemic host defence by maintaining a functional secreted proteome and avoiding proteotoxicity.
Trimethylguanosine synthase 1 (TGS1) is a highly conserved enzyme that converts the 5'-monomethylguanosine cap of small nuclear RNAs (snRNAs) to a trimethylguanosine cap. Here, we show that loss of ...TGS1 in Caenorhabditis elegans, Drosophila melanogaster and Danio rerio results in neurological phenotypes similar to those caused by survival motor neuron (SMN) deficiency. Importantly, expression of human TGS1 ameliorates the SMN-dependent neurological phenotypes in both flies and worms, revealing that TGS1 can partly counteract the effects of SMN deficiency. TGS1 loss in HeLa cells leads to the accumulation of immature U2 and U4atac snRNAs with long 3' tails that are often uridylated. snRNAs with defective 3' terminations also accumulate in Drosophila Tgs1 mutants. Consistent with defective snRNA maturation, TGS1 and SMN mutant cells also exhibit partially overlapping transcriptome alterations that include aberrantly spliced and readthrough transcripts. Together, these results identify a neuroprotective function for TGS1 and reinforce the view that defective snRNA maturation affects neuronal viability and function.
Wilson disease (WD) is caused by mutations in the ATP7B gene that encodes a copper (Cu) transporting ATPase whose trafficking from the Golgi to endo‐lysosomal compartments drives sequestration of ...excess Cu and its further excretion from hepatocytes into the bile. Loss of ATP7B function leads to toxic Cu overload in the liver and subsequently in the brain, causing fatal hepatic and neurological abnormalities. The limitations of existing WD therapies call for the development of new therapeutic approaches, which require an amenable animal model system for screening and validation of drugs and molecular targets. To achieve this objective, we generated a mutant Caenorhabditis elegans strain with a substitution of a conserved histidine (H828Q) in the ATP7B ortholog cua‐1 corresponding to the most common ATP7B variant (H1069Q) that causes WD. cua‐1 mutant animals exhibited very poor resistance to Cu compared to the wild‐type strain. This manifested in a strong delay in larval development, a shorter lifespan, impaired motility, oxidative stress pathway activation, and mitochondrial damage. In addition, morphological analysis revealed several neuronal abnormalities in cua‐1 mutant animals exposed to Cu. Further investigation suggested that mutant CUA‐1 is retained and degraded in the endoplasmic reticulum, similarly to human ATP7B‐H1069Q. As a consequence, the mutant protein does not allow animals to counteract Cu toxicity. Notably, pharmacological correctors of ATP7B‐H1069Q reduced Cu toxicity in cua‐1 mutants indicating that similar pathogenic molecular pathways might be activated by the H/Q substitution and, therefore, targeted for rescue of ATP7B/CUA‐1 function. Taken together, our findings suggest that the newly generated cua‐1 mutant strain represents an excellent model for Cu toxicity studies in WD.
C. elegans CUA‐1 is an ortholog of human ATP7B, mutations in which cause Wilson disease (WD). The copper‐transporting activity of CUA‐1 counteracts copper toxicity by sequestering excess copper ions in lysosomes and promoting their efflux to the extracellular space. The substitution of a conserved histidine (H828Q) in CUA‐1 recapitulates the most common ATP7B variant (H1069Q), causing WD. CUA‐1 (H828Q) undergoes ER‐associated degradation, thereby causing significant copper toxicity in mutant animals.
Spinal muscular atrophy is a devastating disease that is characterized by degeneration and death of a specific subclass of motor neurons in the anterior horn of the spinal cord. Although the gene ...responsible, survival motor neuron 1 (SMN1), was identified 20 years ago, it has proven difficult to investigate its effects in vivo. Consequently, a number of key questions regarding the molecular and cellular functions of this molecule have remained unanswered. We developed a Caenorhabditis elegans model of smn-1 loss-of-function using a neuron-specific RNA interference strategy to knock-down smn-1 selectively in a subclass of motor neurons. The transgenic animals presented a cell-autonomous, age-dependent degeneration of motor neurons detected as locomotory defects and the disappearance of presynaptic and cytoplasmic fluorescent markers in targeted neurons. This degeneration led to neuronal death as revealed by positive reactivity to genetic and chemical cell-death markers. We show that genes of the classical apoptosis pathway are involved in the smn-1-mediated neuronal death, and that this phenotype can be rescued by the expression of human SMN1, indicating a functional conservation between the two orthologs. Finally, we determined that Plastin3/plst-1 genetically interacts with smn-1 to prevent degeneration, and that treatment with valproic acid is able to rescue the degenerative phenotype. These results provide novel insights into the cellular and molecular mechanisms that lead to the loss of motor neurons when SMN1 function is reduced.
Spinal muscular atrophy (SMA) is a degenerative disorder that selectively deteriorates motor neurons due to a deficiency of survival motor neuron protein (SMN). The illness is the leading genetic ...cause of death in infants and is difficult to study in complex biological systems such as humans. A simpler model system, such as the nematode C. elegans, can be used to study potential mechanisms underlying this disease; C. elegans expresses the smn-1 gene, a homologue of SMN; powerful genetic tools in C. elegans research can be used to discover novel genes whose effect on SMN remains unknown or uncharacterized. Currently, conventional screening methods are time-consuming and laborious, as well as being subjective and mostly qualitative. To address these issues, we engineer an automated system capable of performing genetic suppressor screens on C. elegans using microfluidics in combination with custom image analysis software. We demonstrate the utility of this system by isolating 21 alleles that significantly suppress motor neuron degeneration at a screening rate of approximately 300 worms per hour. Many of these mutants also have improved motor function. These isolated alleles can potentially be further studied to understand mechanisms of protection against neurodegeneration. Our system is easily adaptable, providing a means to saturate screens not only implicated in the smn-1 pathway, but also for genes involved in other neurodegenerative phenotypes.
Spinal muscular atrophy (SMA) is a degenerative disorder that selectively deteriorates motor neurons due to a deficiency of survival motor neuron protein (SMN). The illness is the leading genetic ...cause of death in infants and is difficult to study in complex biological systems such as humans. A simpler model system, such as the nematode
C. elegans
, can be used to study potential mechanisms underlying this disease;
C. elegans
expresses the
smn-1
gene, a homologue of SMN; powerful genetic tools in
C. elegans
research can be used to discover novel genes whose effect on SMN remains unknown or uncharacterized. Currently, conventional screening methods are time-consuming and laborious, as well as being subjective and mostly qualitative. To address these issues, we engineer an automated system capable of performing genetic suppressor screens on
C. elegans
using microfluidics in combination with custom image analysis software. We demonstrate the utility of this system by isolating 21 alleles that significantly suppress motor neuron degeneration at a screening rate of approximately 300 worms per hour. Many of these mutants also have improved motor function. These isolated alleles can potentially be further studied to understand mechanisms of protection against neurodegeneration. Our system is easily adaptable, providing a means to saturate screens not only implicated in the
smn-1
pathway, but also for genes involved in other neurodegenerative phenotypes.
A fully automated high-throughput screen using
C. elegans
to investigate genetic mechanisms affecting spinal muscular atrophy (SMA).
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