Functional convergence of histone methyltransferases EHMT1 and KMT2C involved in intellectual disability and autism spectrum disorder.

Kleefstra syndrome, caused by haploinsufficiency of euchromatin histone methyltransferase 1 (EHMT1), is characterized by intellectual disability (ID), autism spectrum disorder (ASD), characteristic facial dysmorphisms, and other variable clinical features. In addition to EHMT1 mutations, de novo variants were reported in four additional genes (MBD5, SMARCB1, NR1I3, and KMT2C), in single individuals with clinical characteristics overlapping Kleefstra syndrome. Here, we present a novel cohort of five patients with de novo loss of function mutations affecting the histone methyltransferase KMT2C. Our clinical data delineates the KMT2C phenotypic spectrum and reinforces the phenotypic overlap with Kleefstra syndrome and other related ID disorders. To elucidate the common molecular basis of the neuropathology associated with mutations in KMT2C and EHMT1, we characterized the role of the Drosophila KMT2C ortholog, trithorax related (trr), in the nervous system. Similar to the Drosophila EHMT1 ortholog, G9a, trr is required in the mushroom body for short term memory. Trr ChIP-seq identified 3371 binding sites, mainly in the promoter of genes involved in neuronal processes. Transcriptional profiling of pan-neuronal trr knockdown and G9a null mutant fly heads identified 613 and 1123 misregulated genes, respectively. These gene sets show a significant overlap and are associated with nearly identical gene ontology enrichments. The majority of the observed biological convergence is derived from predicted indirect target genes. However, trr and G9a also have common direct targets, including the Drosophila ortholog of Arc (Arc1), a key regulator of synaptic plasticity. Our data highlight the clinical and molecular convergence between the KMT2 and EHMT protein families, which may contribute to a molecular network underlying a larger group of ID/ASD-related disorders.

Drosophila Courtship Conditioning As a Measure of Learning and Memory.

Many insights into the molecular mechanisms underlying learning and memory have been elucidated through the use of simple behavioral assays in model organisms such as the fruit fly, Drosophila melanogaster. Drosophila is useful for understanding the basic neurobiology underlying cognitive deficits resulting from mutations in genes associated with human cognitive disorders, such as intellectual disability (ID) and autism. This work describes a methodology for testing learning and memory using a classic paradigm in Drosophila known as courtship conditioning. Male flies court females using a distinct pattern of easily recognizable behaviors. Premated females are not receptive to mating and will reject the male's copulation attempts. In response to this rejection, male flies reduce their courtship behavior. This learned reduction in courtship behavior is measured over time, serving as an indicator of learning and memory. The basic numerical output of this assay is the courtship index (CI), which is defined as the percentage of time that a male spends courting during a 10 min interval. The learning index (LI) is the relative reduction of CI in flies that have been exposed to a premated female compared to naïve flies with no previous social encounters. For the statistical comparison of LIs between genotypes, a randomization test with bootstrapping is used. To illustrate how the assay can be used to address the role of a gene relating to learning and memory, the pan-neuronal knockdown of Dihydroxyacetone phosphate acyltransferase (Dhap-at) was characterized here. The human ortholog of Dhap-at, glyceronephosphate O-acyltransferase (GNPT), is involved in rhizomelic chondrodysplasia punctata type 2, an autosomal-recessive syndrome characterized by severe ID. Using the courtship conditioning assay, it was determined that Dhap-at is required for long-term memory, but not for short-term memory. This result serves as a basis for further investigation of the underlying molecular mechanisms.

Mutations in DDX3X Are a Common Cause of Unexplained Intellectual Disability with Gender-Specific Effects on Wnt Signaling.

Intellectual disability (ID) affects approximately 1%-3% of humans with a gender bias toward males. Previous studies have identified mutations in more than 100 genes on the X chromosome in males with ID, but there is less evidence for de novo mutations on the X chromosome causing ID in females. In this study we present 35 unique deleterious de novo mutations in DDX3X identified by whole exome sequencing in 38 females with ID and various other features including hypotonia, movement disorders, behavior problems, corpus callosum hypoplasia, and epilepsy. Based on our findings, mutations in DDX3X are one of the more common causes of ID, accounting for 1%-3% of unexplained ID in females. Although no de novo DDX3X mutations were identified in males, we present three families with segregating missense mutations in DDX3X, suggestive of an X-linked recessive inheritance pattern. In these families, all males with the DDX3X variant had ID, whereas carrier females were unaffected. To explore the pathogenic mechanisms accounting for the differences in disease transmission and phenotype between affected females and affected males with DDX3X missense variants, we used canonical Wnt defects in zebrafish as a surrogate measure of DDX3X function in vivo. We demonstrate a consistent loss-of-function effect of all tested de novo mutations on the Wnt pathway, and we further show a differential effect by gender. The differential activity possibly reflects a dose-dependent effect of DDX3X expression in the context of functional mosaic females versus one-copy males, which reflects the complex biological nature of DDX3X mutations.

Restoring polyamines protects from age-induced memory impairment in an autophagy-dependent manner.

Age-dependent memory impairment is known to occur in several organisms, including Drosophila, mouse and human. However, the fundamental cellular mechanisms that underlie these impairments are still poorly understood, effectively hampering the development of pharmacological strategies to treat the condition. Polyamines are among the substances found to decrease with age in the human brain. We found that levels of polyamines (spermidine, putrescine) decreased in aging fruit flies, concomitant with declining memory abilities. Simple spermidine feeding not only restored juvenile polyamine levels, but also suppressed age-induced memory impairment. Ornithine decarboxylase-1, the rate-limiting enzyme for de novo polyamine synthesis, also protected olfactory memories in aged flies when expressed specifically in Kenyon cells, which are crucial for olfactory memory formation. Spermidine-fed flies showed enhanced autophagy (a form of cellular self-digestion), and genetic deficits in the autophagic machinery prevented spermidine-mediated rescue of memory impairments. Our findings indicate that autophagy is critical for suppression of memory impairments by spermidine and that polyamines, which are endogenously present, are candidates for pharmacological intervention.

Disruption of an EHMT1-associated chromatin-modification module causes intellectual disability.

Intellectual disability (ID) disorders are genetically and phenotypically highly heterogeneous and present a major challenge in clinical genetics and medicine. Although many genes involved in ID have been identified, the etiology is unknown in most affected individuals. Moreover, the function of most genes associated with ID remains poorly characterized. Evidence is accumulating that the control of gene transcription through epigenetic modification of chromatin structure in neurons has an important role in cognitive processes and in the etiology of ID. However, our understanding of the key molecular players and mechanisms in this process is highly fragmentary. Here, we identify a chromatin-modification module that underlies a recognizable form of ID, the Kleefstra syndrome phenotypic spectrum (KSS). In a cohort of KSS individuals without mutations in EHMT1 (the only gene known to be disrupted in KSS until now), we identified de novo mutations in four genes, MBD5, MLL3, SMARCB1, and NR1I3, all of which encode epigenetic regulators. Using Drosophila, we demonstrate that MBD5, MLL3, and NR1I3 cooperate with EHMT1, whereas SMARCB1 is known to directly interact with MLL3. We propose a highly conserved epigenetic network that underlies cognition in health and disease. This network should allow the design of strategies to treat the growing group of ID pathologies that are caused by epigenetic defects.

Oxidative stress-induced modifications of histidyl-tRNA synthetase affect its tRNA aminoacylation activity but not its immunoreactivity.

The aminoacyl-tRNA synthetases are ubiquitously expressed enzymes that catalyze the esterification of amino acids to their cognate tRNAs. Autoantibodies against several aminoacyl-tRNA synthetases are found in autoimmune polymyositis and dermatomyositis patients. Because necrosis is often found in skeletal muscle biopsies of these patients, we hypothesized that cell-death-induced protein modifications may help in breaking immunological tolerance. Since cell death is associated with oxidative stress, the effect of oxidative stress on the main myositis-specific autoantibody target Jo-1 (histidyl-tRNA synthetase; HisRS) was studied in detail. The exposure of Jurkat cells to hydrogen peroxide resulted in the detection of several oxidized methionines and one oxidized tryptophan residue in the HisRS protein, as demonstrated by mass spectrometry. Unexpectedly, the tRNA aminoacylation activity of HisRS appeared to be increased upon oxidative modification. The analysis of myositis patient sera did not lead to the detection of autoantibodies that are specifically reactive with the modified HisRS protein. The results of this study demonstrate that the Jo-1/HisRS autoantigen is modified under oxidative stress conditions. The consequences of these modifications for the function of HisRS and its autoantigenicity are discussed.