Integrating non-mammalian model organisms in the diagnosis of rare genetic diseases in humans.

Next-generation sequencing technology has rapidly accelerated the discovery of genetic variants of interest in individuals with rare diseases. However, showing that these variants are causative of the disease in question is complex and may require functional studies. Use of non-mammalian model organisms - mainly fruitflies (Drosophila melanogaster), nematode worms (Caenorhabditis elegans) and zebrafish (Danio rerio) - enables the rapid and cost-effective assessment of the effects of gene variants, which can then be validated in mammalian model organisms such as mice and in human cells. By probing mechanisms of gene action and identifying interacting genes and proteins in vivo, recent studies in these non-mammalian model organisms have facilitated the diagnosis of numerous genetic diseases and have enabled the screening and identification of therapeutic options for patients. Studies in non-mammalian model organisms have also shown that the biological processes underlying rare diseases can provide insight into more common mechanisms of disease and the biological functions of genes. Here, we discuss the opportunities afforded by non-mammalian model organisms, focusing on flies, worms and fish, and provide examples of their use in the diagnosis of rare genetic diseases.

Morgan's legacy: fruit flies and the functional annotation of conserved genes.

In 1915, "The Mechanism of Mendelian Heredity" was published by four prominent Drosophila geneticists. They discovered that genes form linkage groups on chromosomes inherited in a Mendelian fashion and laid the genetic foundation that promoted Drosophila as a model organism. Flies continue to offer great opportunities, including studies in the field of functional genomics.

Glial lipid droplets and ROS induced by mitochondrial defects promote neurodegeneration.

Reactive oxygen species (ROS) and mitochondrial defects in neurons are implicated in neurodegenerative disease. Here, we find that a key consequence of ROS and neuronal mitochondrial dysfunction is the accumulation of lipid droplets (LD) in glia. In Drosophila, ROS triggers c-Jun-N-terminal Kinase (JNK) and Sterol Regulatory Element Binding Protein (SREBP) activity in neurons leading to LD accumulation in glia prior to or at the onset of neurodegeneration. The accumulated lipids are peroxidated in the presence of ROS. Reducing LD accumulation in glia and lipid peroxidation via targeted lipase overexpression and/or lowering ROS significantly delays the onset of neurodegeneration. Furthermore, a similar pathway leads to glial LD accumulation in Ndufs4 mutant mice with neuronal mitochondrial defects, suggesting that LD accumulation following mitochondrial dysfunction is an evolutionarily conserved phenomenon, and represents an early, transient indicator and promoter of neurodegenerative disease.

A drosophila genetic resource of mutants to study mechanisms underlying human genetic diseases.

Invertebrate model systems are powerful tools for studying human disease owing to their genetic tractability and ease of screening. We conducted a mosaic genetic screen of lethal mutations on the Drosophila X chromosome to identify genes required for the development, function, and maintenance of the nervous system. We identified 165 genes, most of whose function has not been studied in vivo. In parallel, we investigated rare variant alleles in 1,929 human exomes from families with unsolved Mendelian disease. Genes that are essential in flies and have multiple human homologs were found to be likely to be associated with human diseases. Merging the human data sets with the fly genes allowed us to identify disease-associated mutations in six families and to provide insights into microcephaly associated with brain dysgenesis. This bidirectional synergism between fly genetics and human genomics facilitates the functional annotation of evolutionarily conserved genes involved in human health.

Characterization and antiviral susceptibility of SARS-CoV-2 Omicron BA.2.

The recent emergence of SARS-CoV-2 Omicron (B.1.1.529 lineage) variants possessing numerous mutations has raised concerns of decreased effectiveness of current vaccines, therapeutic monoclonal antibodies and antiviral drugs for COVID-19 against these variants1,2. The original Omicron lineage, BA.1, prevailed in many countries, but more recently, BA.2 has become dominant in at least 68 countries3. Here we evaluated the replicative ability and pathogenicity of authentic infectious BA.2 isolates in immunocompetent and human ACE2-expressing mice and hamsters. In contrast to recent data with chimeric, recombinant SARS-CoV-2 strains expressing the spike proteins of BA.1 and BA.2 on an ancestral WK-521 backbone4, we observed similar infectivity and pathogenicity in mice and hamsters for BA.2 and BA.1, and less pathogenicity compared with early SARS-CoV-2 strains. We also observed a marked and significant reduction in the neutralizing activity of plasma from individuals who had recovered from COVID-19 and vaccine recipients against BA.2 compared to ancestral and Delta variant strains. In addition, we found that some therapeutic monoclonal antibodies (REGN10987 plus REGN10933, COV2-2196 plus COV2-2130, and S309) and antiviral drugs (molnupiravir, nirmatrelvir and S-217622) can restrict viral infection in the respiratory organs of BA.2-infected hamsters. These findings suggest that the replication and pathogenicity of BA.2 is similar to that of BA.1 in rodents and that several therapeutic monoclonal antibodies and antiviral compounds are effective against Omicron BA.2 variants.

De novo variants in FRYL are associated with developmental delay, intellectual disability, and dysmorphic features.

FRY-like transcription coactivator (FRYL) belongs to a Furry protein family that is evolutionarily conserved from yeast to humans. The functions of FRYL in mammals are largely unknown, and variants in FRYL have not previously been associated with a Mendelian disease. Here, we report fourteen individuals with heterozygous variants in FRYL who present with developmental delay, intellectual disability, dysmorphic features, and other congenital anomalies in multiple systems. The variants are confirmed de novo in all individuals except one. Human genetic data suggest that FRYL is intolerant to loss of function (LoF). We find that the fly FRYL ortholog, furry (fry), is expressed in multiple tissues, including the central nervous system where it is present in neurons but not in glia. Homozygous fry LoF mutation is lethal at various developmental stages, and loss of fry in mutant clones causes defects in wings and compound eyes. We next modeled four out of the five missense variants found in affected individuals using fry knockin alleles. One variant behaves as a severe LoF variant, whereas two others behave as partial LoF variants. One variant does not cause any observable defect in flies, and the corresponding human variant is not confirmed to be de novo, suggesting that this is a variant of uncertain significance. In summary, our findings support that fry is required for proper development in flies and that the LoF variants in FRYL cause a dominant disorder with developmental and neurological symptoms due to haploinsufficiency.

Loss of the endoplasmic reticulum protein Tmem208 affects cell polarity, development, and viability.

Nascent proteins destined for the cell membrane and the secretory pathway are targeted to the endoplasmic reticulum (ER) either posttranslationally or cotranslationally. The signal-independent pathway, containing the protein TMEM208, is one of three pathways that facilitates the translocation of nascent proteins into the ER. The in vivo function of this protein is ill characterized in multicellular organisms. Here, we generated a CRISPR-induced null allele of the fruit fly ortholog CG8320/Tmem208 by replacing the gene with the Kozak-GAL4 sequence. We show that Tmem208 is broadly expressed in flies and that its loss causes lethality, although a few short-lived flies eclose. These animals exhibit wing and eye developmental defects consistent with impaired cell polarity and display mild ER stress. Tmem208 physically interacts with Frizzled (Fz), a planar cell polarity (PCP) receptor, and is required to maintain proper levels of Fz. Moreover, we identified a child with compound heterozygous variants in TMEM208 who presents with developmental delay, skeletal abnormalities, multiple hair whorls, cardiac, and neurological issues, symptoms that are associated with PCP defects in mice and humans. Additionally, fibroblasts of the proband display mild ER stress. Expression of the reference human TMEM208 in flies fully rescues the loss of Tmem208, and the two proband-specific variants fail to rescue, suggesting that they are loss-of-function alleles. In summary, our study uncovers a role of TMEM208 in development, shedding light on its significance in ER homeostasis and cell polarity.

Rare de novo gain-of-function missense variants in DOT1L are associated with developmental delay and congenital anomalies.

Misregulation of histone lysine methylation is associated with several human cancers and with human developmental disorders. DOT1L is an evolutionarily conserved gene encoding a lysine methyltransferase (KMT) that methylates histone 3 lysine-79 (H3K79) and was not previously associated with a Mendelian disease in OMIM. We have identified nine unrelated individuals with seven different de novo heterozygous missense variants in DOT1L through the Undiagnosed Disease Network (UDN), the SickKids Complex Care genomics project, and GeneMatcher. All probands had some degree of global developmental delay/intellectual disability, and most had one or more major congenital anomalies. To assess the pathogenicity of the DOT1L variants, functional studies were performed in Drosophila and human cells. The fruit fly DOT1L ortholog, grappa, is expressed in most cells including neurons in the central nervous system. The identified DOT1L variants behave as gain-of-function alleles in flies and lead to increased H3K79 methylation levels in flies and human cells. Our results show that human DOT1L and fly grappa are required for proper development and that de novo heterozygous variants in DOT1L are associated with a Mendelian disease.

Bi-allelic variants in INTS11 are associated with a complex neurological disorder.

The Integrator complex is a multi-subunit protein complex that regulates the processing of nascent RNAs transcribed by RNA polymerase II (RNAPII), including small nuclear RNAs, enhancer RNAs, telomeric RNAs, viral RNAs, and protein-coding mRNAs. Integrator subunit 11 (INTS11) is the catalytic subunit that cleaves nascent RNAs, but, to date, mutations in this subunit have not been linked to human disease. Here, we describe 15 individuals from 10 unrelated families with bi-allelic variants in INTS11 who present with global developmental and language delay, intellectual disability, impaired motor development, and brain atrophy. Consistent with human observations, we find that the fly ortholog of INTS11, dIntS11, is essential and expressed in the central nervous systems in a subset of neurons and most glia in larval and adult stages. Using Drosophila as a model, we investigated the effect of seven variants. We found that two (p.Arg17Leu and p.His414Tyr) fail to rescue the lethality of null mutants, indicating that they are strong loss-of-function variants. Furthermore, we found that five variants (p.Gly55Ser, p.Leu138Phe, p.Lys396Glu, p.Val517Met, and p.Ile553Glu) rescue lethality but cause a shortened lifespan and bang sensitivity and affect locomotor activity, indicating that they are partial loss-of-function variants. Altogether, our results provide compelling evidence that integrity of the Integrator RNA endonuclease is critical for brain development.

Pathogenic SATB2 missense variants affecting p.Gly392 have variable functional implications and result in diverse clinical phenotypes.

SATB2-associated syndrome (SAS) is caused by pathogenic variants in SATB2, which encodes an evolutionarily conserved transcription factor. Despite the broad range of phenotypic manifestations and variable severity related to this syndrome, haploinsufficiency has been assumed to be the primary molecular explanation.In this study, we describe eight individuals with SATB2 variants that affect p.Gly392 (four women, age range 2-16 years; p.Gly392Arg, p.Gly392Glu and p.Gly392Val). Of these, individuals with p.Gly392Arg substitutions were found to have more severe neurodevelopmental phenotypes based on an established rubric scoring system when compared with individuals with p.Gly392Glu, p.Gly392Val and other previously reported causative SATB2 missense variants. Consistent with the observations at the phenotypic level, using human cell-based and model organism functional data, we documented that while all three described p.Gly392 variants affect the same residue and seem to all have a partial loss-of-function effect, some effects on SATB2 protein function appear to be variant-specific. Our results indicate that genotype-phenotype correlations in SAS are more complex than originally thought, and variant-specific genotype-phenotype correlations are needed.

Visualization of intracellular ATP dynamics in different nephron segments under pathophysiological conditions using the kidney slice culture system.

ATP depletion plays a central role in the pathogenesis of kidney diseases. Recently, we reported spatiotemporal intracellular ATP dynamics during ischemia reperfusion (IR) using GO-ATeam2 mice systemically expressing an ATP biosensor. However, observation from the kidney surface did not allow visualization of deeper nephrons or accurate evaluation of ATP synthesis pathways. Here, we established a novel ATP imaging system using slice culture of GO-ATeam2 mouse kidneys, evaluated the ATP synthesis pathway, and analyzed intracellular ATP dynamics using an ex vivo IR-mimicking model and a cisplatin nephropathy model. Proximal tubules (PTs) were found to be strongly dependent on oxidative phosphorylation (OXPHOS) using the inhibitor oligomycin A, whereas podocytes relied on both OXPHOS and glycolysis using phloretin an active transport inhibitor of glucose. We also confirmed that an ex vivo IR-mimicking model could recapitulate ATP dynamics in vivo; ATP recovery in PTs after reoxygenation varied depending on anoxic time length, whereas ATP in distal tubules (DTs) recovered well even after long-term anoxia. After cisplatin administration, ATP levels in PTs decreased first, followed by a decrease in DTs. An organic cation transporter 2 inhibitor, cimetidine, suppressed cisplatin uptake in kidney slices, leading to better ATP recovery in PTs, but not in DTs. Finally, we confirmed that a mitochondria protection reagent (Mitochonic Acid 5) delayed the cisplatin-induced ATP decrease in PTs. Thus, our novel system may provide new insights into the energy dynamics and pathogenesis of kidney disease.

The microRNA processor DROSHA is a candidate gene for a severe progressive neurological disorder.

DROSHA encodes a ribonuclease that is a subunit of the Microprocessor complex and is involved in the first step of microRNA (miRNA) biogenesis. To date, DROSHA has not yet been associated with a Mendelian disease. Here, we describe two individuals with profound intellectual disability, epilepsy, white matter atrophy, microcephaly and dysmorphic features, who carry damaging de novo heterozygous variants in DROSHA. DROSHA is constrained for missense variants and moderately intolerant to loss-of-function (o/e = 0.24). The loss of the fruit fly ortholog drosha causes developmental arrest and death in third instar larvae, a severe reduction in brain size and loss of imaginal discs in the larva. Loss of drosha in eye clones causes small and rough eyes in adult flies. One of the identified DROSHA variants (p.Asp1219Gly) behaves as a strong loss-of-function allele in flies, while another variant (p.Arg1342Trp) is less damaging in our assays. In worms, a knock-in that mimics the p.Asp1219Gly variant at a worm equivalent residue causes loss of miRNA expression and heterochronicity, a phenotype characteristic of the loss of miRNA. Together, our data show that the DROSHA variants found in the individuals presented here are damaging based on functional studies in model organisms and likely underlie the severe phenotype involving the nervous system.

TNPO2 variants associate with human developmental delays, neurologic deficits, and dysmorphic features and alter TNPO2 activity in Drosophila.

Transportin-2 (TNPO2) mediates multiple pathways including non-classical nucleocytoplasmic shuttling of >60 cargoes, such as developmental and neuronal proteins. We identified 15 individuals carrying de novo coding variants in TNPO2 who presented with global developmental delay (GDD), dysmorphic features, ophthalmologic abnormalities, and neurological features. To assess the nature of these variants, functional studies were performed in Drosophila. We found that fly dTnpo (orthologous to TNPO2) is expressed in a subset of neurons. dTnpo is critical for neuronal maintenance and function as downregulating dTnpo in mature neurons using RNAi disrupts neuronal activity and survival. Altering the activity and expression of dTnpo using mutant alleles or RNAi causes developmental defects, including eye and wing deformities and lethality. These effects are dosage dependent as more severe phenotypes are associated with stronger dTnpo loss. Interestingly, similar phenotypes are observed with dTnpo upregulation and ectopic expression of TNPO2, showing that loss and gain of Transportin activity causes developmental defects. Further, proband-associated variants can cause more or less severe developmental abnormalities compared to wild-type TNPO2 when ectopically expressed. The impact of the variants tested seems to correlate with their position within the protein. Specifically, those that fall within the RAN binding domain cause more severe toxicity and those in the acidic loop are less toxic. Variants within the cargo binding domain show tissue-dependent effects. In summary, dTnpo is an essential gene in flies during development and in neurons. Further, proband-associated de novo variants within TNPO2 disrupt the function of the encoded protein. Hence, TNPO2 variants are causative for neurodevelopmental abnormalities.

Expression pattern of Runt-related transcription factor (RUNX) family members and the role of RUNX1 during kidney development.

Runt-related transcription factor (RUNX) family members play critical roles in the development of multiple organs. Mammalian RUNX family members, consisting of RUNX1, RUNX2, and RUNX3, have distinct tissue-specific expression and function. In this study, we examined the spatiotemporal expression patterns of RUNX family members in developing kidneys and analyzed the role of RUNX1 during kidney development. In the developing mouse kidney, RUNX1 protein was strongly expressed in the ureteric bud (UB) tip and weakly expressed in the distal segment of the renal vesicle (RV), comma-shaped body (CSB), and S-shaped body (SSB). In contrast, RUNX2 protein was restricted to the stroma, and RUNX3 protein was only expressed in immune cells. We also analyzed the expression of RUNX family members in the cynomolgus monkey kidney. We found that expression patterns of RUNX2 and RUNX3 were conserved between rodents and primates, whereas RUNX1 was only expressed in the UB tip, not in the RV, CSB, or SSB of cynomolgus monkeys, suggesting a species differences. We further evaluated the roles of RUNX1 using two different conditional knockout mice: Runx1f/f:HoxB7-Cre and Runx1f/f:R26-CreERT2 and found no abnormalities in the kidney. Our findings showed that RUNX1, which is mainly expressed in the UB tip, is not essential for kidney development.

Dominant missense variants in SREBF2 are associated with complex dermatological, neurological, and skeletal abnormalities.

PURPOSE: We identified 2 individuals with de novo variants in SREBF2 that disrupt a conserved site 1 protease (S1P) cleavage motif required for processing SREBP2 into its mature transcription factor. These individuals exhibit complex phenotypic manifestations that partially overlap with sterol regulatory element binding proteins (SREBP) pathway-related disease phenotypes, but SREBF2-related disease has not been previously reported. Thus, we set out to assess the effects of SREBF2 variants on SREBP pathway activation. METHODS: We undertook ultrastructure and gene expression analyses using fibroblasts from an affected individual and utilized a fly model of lipid droplet (LD) formation to investigate the consequences of SREBF2 variants on SREBP pathway function. RESULTS: We observed reduced LD formation, endoplasmic reticulum expansion, accumulation of aberrant lysosomes, and deficits in SREBP2 target gene expression in fibroblasts from an affected individual, indicating that the SREBF2 variant inhibits SREBP pathway activation. Using our fly model, we discovered that SREBF2 variants fail to induce LD production and act in a dominant-negative manner, which can be rescued by overexpression of S1P. CONCLUSION: Taken together, these data reveal a mechanism by which SREBF2 pathogenic variants that disrupt the S1P cleavage motif cause disease via dominant-negative antagonism of S1P, limiting the cleavage of S1P targets, including SREBP1 and SREBP2.

Loss-of-function in RBBP5 results in a syndromic neurodevelopmental disorder associated with microcephaly.

PURPOSE: Epigenetic dysregulation has been associated with many inherited disorders. RBBP5 (HGNC:9888) encodes a core member of the protein complex that methylates histone 3 lysine-4 and has not been implicated in human disease. METHODS: We identify 5 unrelated individuals with de novo heterozygous variants in RBBP5. Three nonsense/frameshift and 2 missense variants were identified in probands with neurodevelopmental symptoms, including global developmental delay, intellectual disability, microcephaly, and short stature. Here, we investigate the pathogenicity of the variants through protein structural analysis and transgenic Drosophila models. RESULTS: Both missense p.(T232I) and p.(E296D) variants affect evolutionarily conserved amino acids located at the interface between RBBP5 and the nucleosome. In Drosophila, overexpression analysis identifies partial loss-of-function mechanisms when the variants are expressed using the fly Rbbp5 or human RBBP5 cDNA. Loss of Rbbp5 leads to a reduction in brain size. The human reference or variant transgenes fail to rescue this loss and expression of either missense variant in an Rbbp5 null background results in a less severe microcephaly phenotype than the human reference, indicating both missense variants are partial loss-of-function alleles. CONCLUSION: Haploinsufficiency of RBBP5 observed through de novo null and hypomorphic loss-of-function variants is associated with a syndromic neurodevelopmental disorder.

Effect of bisphosphonate and denosumab treatment on TBS in Japanese breast cancer patients with AIBL.

INTRODUCTION: Bisphosphonates and denosumab increase bone mineral density (BMD) for osteoporosis treatment in patients with aromatase inhibitor-associated bone loss (AIBL). This study aimed to directly compare bisphosphonates with denosumab in treating patients with AIBL and to determine the effect of denosumab on the trabecular bone score (TBS). MATERIALS AND METHODS: Thirty-nine patients with AIBL receiving osteoporosis treatment (21 in the bisphosphonates group and 18 in the denosumab group) were retrospectively evaluated for changes in lumbar spine and femoral BMD, lumbar spine bone quality (assessed by TBS), and blood bone metabolic markers. The Mann-Whitney and Wilcoxon tests were used for statistical evaluation. RESULTS: After 24 months of treatment, the lumbar spine BMD change rate was 5.82 ± 1.10% with bisphosphonates and 10.49 ± 1.20% with denosumab, with the change rate of denosumab significantly increasing over that of bisphosphonates. The change rate in femoral BMD was 2.69 ± 1.16% with bisphosphonates and 2.95 ± 1.26% with denosumab, with no significant difference between the two groups. The rate of decrease in tartrate-resistant acid phosphatase isoform 5b was significantly higher in the denosumab group. The change rate in TBS at 24 months of treatment was 0.53 ± 1.26% in the bisphosphonates group and 1.08 ± 1.33% in the denosumab group, with no significant difference between the two groups. After 24 months, TBS remained stable. CONCLUSION: Both bisphosphonates and denosumab may increase BMD, improve bone metabolism, and inhibit bone quality loss in patients with AIBL.

Proteinuria frequency and subsequent renal dysfunction in bevacizumab-treated patients: a single center, retrospective, observational study.

BACKGROUND: Proteinuria is a common adverse event observed during treatment with antivascular endothelial growth factor (VEGF) antibodies. Proteinuria is a risk factor for renal dysfunction and cardiovascular complications in patients with chronic kidney disease. However, the association between anti-VEGF antibody-induced proteinuria and renal dysfunction or cardiovascular complications remains unclear. METHODS: This retrospective, observational study included patients with cancer that were treated with bevacizumab (BV) at Kyoto University Hospital (Kyoto, Japan) between January 2006 and March 2018. Adverse event rates were compared between patients who developed qualitative ≥ 2 + proteinuria and those who developed < 1 + proteinuria. Adverse events were defined as renal dysfunction (i.e., ≥ 57% decrease in the eGFR, compared to the rate at the initial treatment) and hospitalization due to BV-associated cardiovascular complications and other adverse events. RESULTS: In total, 734 patients were included in this analysis. Renal dysfunction was more common in patients with ≥ 2 + proteinuria than in those with < 1 + proteinuria (13/199, 6.5% vs. 12/535, 2.3%). Seven of these 13 patients with ≥ 2 + proteinuria had transient reversible renal dysfunction. Only four (2.0%) patients had BV-associated renal dysfunction. Of the 734 patients, six patients, 16 patients, and 13 patients were hospitalized because of the adverse events of cardiovascular complications, thromboembolisms, and cerebrovascular complications, respectively. No relationship was observed between these adverse events and proteinuria. CONCLUSION: BV treatment-induced proteinuria was not associated with renal dysfunction or other adverse events. Continuing BV with caution is a possible treatment option, even after proteinuria develops, in patients with cancer and a limited prognosis.

Environment-based object values learned by local network in the striatum tail.

Basal ganglia contribute to object-value learning, which is critical for survival. The underlying neuronal mechanism is the association of each object with its rewarding outcome. However, object values may change in different environments and we then need to choose different objects accordingly. The mechanism of this environment-based value learning is unknown. To address this question, we created an environment-based value task in which the value of each object was reversed depending on the two scene-environments (X and Y). After experiencing this task repeatedly, the monkeys became able to switch the choice of object when the scene-environment changed unexpectedly. When we blocked the inhibitory input from fast-spiking interneurons (FSIs) to medium spiny projection neurons (MSNs) in the striatum tail by locally injecting IEM-1460, the monkeys became unable to learn scene-selective object values. We then studied the mechanism of the FSI-MSN connection. Before and during this learning, FSIs responded to the scenes selectively, but were insensitive to object values. In contrast, MSNs became able to discriminate the objects (i.e., stronger response to good objects), but this occurred clearly in one of the two scenes (X or Y). This was caused by the scene-selective inhibition by FSI. As a whole, MSNs were divided into two groups that were sensitive to object values in scene X or in scene Y. These data indicate that the local network of striatum tail controls the learning of object values that are selective to the scene-environment. This mechanism may support our flexible switching behavior in various environments.

TM2D genes regulate Notch signaling and neuronal function in Drosophila.

TM2 domain containing (TM2D) proteins are conserved in metazoans and encoded by three separate genes in each model organism species that has been sequenced. Rare variants in TM2D3 are associated with Alzheimer's disease (AD) and its fly ortholog almondex is required for embryonic Notch signaling. However, the functions of this gene family remain elusive. We knocked-out all three TM2D genes (almondex, CG11103/amaretto, CG10795/biscotti) in Drosophila and found that they share the same maternal-effect neurogenic defect. Triple null animals are not phenotypically worse than single nulls, suggesting these genes function together. Overexpression of the most conserved region of the TM2D proteins acts as a potent inhibitor of Notch signaling at the γ-secretase cleavage step. Lastly, Almondex is detected in the brain and its loss causes shortened lifespan accompanied by progressive motor and electrophysiological defects. The functional links between all three TM2D genes are likely to be evolutionarily conserved, suggesting that this entire gene family may be involved in AD.