Integrative genetic analysis illuminates ALS heritability and identifies risk genes.

Amyotrophic lateral sclerosis (ALS) has substantial heritability, in part shared with fronto-temporal dementia (FTD). We show that ALS heritability is enriched in splicing variants and in binding sites of 6 RNA-binding proteins including TDP-43 and FUS. A transcriptome wide association study (TWAS) identified 6 loci associated with ALS, including in NUP50 encoding for the nucleopore basket protein NUP50. Independently, rare variants in NUP50 were associated with ALS risk (P = 3.71.10-03; odds ratio = 3.29; 95%CI, 1.37 to 7.87) in a cohort of 9,390 ALS/FTD patients and 4,594 controls. Cells from one patient carrying a NUP50 frameshift mutation displayed a decreased level of NUP50. Loss of NUP50 leads to death of cultured neurons, and motor defects in Drosophila and zebrafish. Thus, our study identifies alterations in splicing in neurons as critical in ALS and provides genetic evidence linking nuclear pore defects to ALS.

tRNA overexpression rescues peripheral neuropathy caused by mutations in tRNA synthetase.

Heterozygous mutations in six transfer RNA (tRNA) synthetase genes cause Charcot-Marie-Tooth (CMT) peripheral neuropathy. CMT mutant tRNA synthetases inhibit protein synthesis by an unknown mechanism. We found that CMT mutant glycyl-tRNA synthetases bound tRNAGly but failed to release it, resulting in tRNAGly sequestration. This sequestration potentially depleted the cellular tRNAGly pool, leading to insufficient glycyl-tRNAGly supply to the ribosome. Accordingly, we found ribosome stalling at glycine codons and activation of the integrated stress response (ISR) in affected motor neurons. Moreover, transgenic overexpression of tRNAGly rescued protein synthesis, peripheral neuropathy, and ISR activation in Drosophila and mouse CMT disease type 2D (CMT2D) models. Conversely, inactivation of the ribosome rescue factor GTPBP2 exacerbated peripheral neuropathy. Our findings suggest a molecular mechanism for CMT2D, and elevating tRNAGly levels may thus have therapeutic potential.

Novel vertebrate- and brain-specific driver of neuronal outgrowth.

During the process of neuronal outgrowth, developing neurons produce new projections, neurites, that are essential for brain wiring. Here, we discover a relatively late-evolved protein that we denote Ac45-related protein (Ac45RP) and that, surprisingly, drives neuronal outgrowth. Ac45RP is a paralog of the Ac45 protein that is a component of the vacuolar proton ATPase (V-ATPase), the main pH regulator in eukaryotic cells. Ac45RP mRNA expression is brain specific and coincides with the peak of neurogenesis and the onset of synaptogenesis. Furthermore, Ac45RP physically interacts with the V-ATPase V0-sector and colocalizes with V0 in unconventional, but not synaptic, secretory vesicles of extending neurites. Excess Ac45RP enhances the expression of V0-subunits, causes a more elaborate Golgi, and increases the number of cytoplasmic vesicular structures, plasma membrane formation and outgrowth of actin-containing neurites devoid of synaptic markers. CRISPR-cas9n-mediated Ac45RP knockdown reduces neurite outgrowth. We conclude that the novel vertebrate- and brain-specific Ac45RP is a V0-interacting constituent of unconventional vesicular structures that drives membrane expansion during neurite outgrowth and as such may furnish a tool for future neuroregenerative treatment strategies.

FUS-mediated regulation of acetylcholine receptor transcription at neuromuscular junctions is compromised in amyotrophic lateral sclerosis.

Neuromuscular junction (NMJ) disruption is an early pathogenic event in amyotrophic lateral sclerosis (ALS). Yet, direct links between NMJ pathways and ALS-associated genes such as FUS, whose heterozygous mutations cause aggressive forms of ALS, remain elusive. In a knock-in Fus-ALS mouse model, we identified postsynaptic NMJ defects in newborn homozygous mutants that were attributable to mutant FUS toxicity in skeletal muscle. Adult heterozygous knock-in mice displayed smaller neuromuscular endplates that denervated before motor neuron loss, which is consistent with 'dying-back' neuronopathy. FUS was enriched in subsynaptic myonuclei, and this innervation-dependent enrichment was distorted in FUS-ALS. Mechanistically, FUS collaborates with the ETS transcription factor ERM to stimulate transcription of acetylcholine receptor genes. Co-cultures of induced pluripotent stem cell-derived motor neurons and myotubes from patients with FUS-ALS revealed endplate maturation defects due to intrinsic FUS toxicity in both motor neurons and myotubes. Thus, FUS regulates acetylcholine receptor gene expression in subsynaptic myonuclei, and muscle-intrinsic toxicity of ALS mutant FUS may contribute to dying-back motor neuronopathy.

Increased GABAB receptor signaling in a rat model for schizophrenia.

Schizophrenia is a complex disorder that affects cognitive function and has been linked, both in patients and animal models, to dysfunction of the GABAergic system. However, the pathophysiological consequences of this dysfunction are not well understood. Here, we examined the GABAergic system in an animal model displaying schizophrenia-relevant features, the apomorphine-susceptible (APO-SUS) rat and its phenotypic counterpart, the apomorphine-unsusceptible (APO-UNSUS) rat at postnatal day 20-22. We found changes in the expression of the GABA-synthesizing enzyme GAD67 specifically in the prelimbic- but not the infralimbic region of the medial prefrontal cortex (mPFC), indicative of reduced inhibitory function in this region in APO-SUS rats. While we did not observe changes in basal synaptic transmission onto LII/III pyramidal cells in the mPFC of APO-SUS compared to APO-UNSUS rats, we report reduced paired-pulse ratios at longer inter-stimulus intervals. The GABAB receptor antagonist CGP 55845 abolished this reduction, indicating that the decreased paired-pulse ratio was caused by increased GABAB signaling. Consistently, we find an increased expression of the GABAB1 receptor subunit in APO-SUS rats. Our data provide physiological evidence for increased presynaptic GABAB signaling in the mPFC of APO-SUS rats, further supporting an important role for the GABAergic system in the pathophysiology of schizophrenia.

Haploinsufficiency of MeCP2-interacting transcriptional co-repressor SIN3A causes mild intellectual disability by affecting the development of cortical integrity.

Numerous genes are associated with neurodevelopmental disorders such as intellectual disability and autism spectrum disorder (ASD), but their dysfunction is often poorly characterized. Here we identified dominant mutations in the gene encoding the transcriptional repressor and MeCP2 interactor switch-insensitive 3 family member A (SIN3A; chromosome 15q24.2) in individuals who, in addition to mild intellectual disability and ASD, share striking features, including facial dysmorphisms, microcephaly and short stature. This phenotype is highly related to that of individuals with atypical 15q24 microdeletions, linking SIN3A to this microdeletion syndrome. Brain magnetic resonance imaging showed subtle abnormalities, including corpus callosum hypoplasia and ventriculomegaly. Intriguingly, in vivo functional knockdown of Sin3a led to reduced cortical neurogenesis, altered neuronal identity and aberrant corticocortical projections in the developing mouse brain. Together, our data establish that haploinsufficiency of SIN3A is associated with mild syndromic intellectual disability and that SIN3A can be considered to be a key transcriptional regulator of cortical brain development.

ATP6AP1 deficiency causes an immunodeficiency with hepatopathy, cognitive impairment and abnormal protein glycosylation.

The V-ATPase is the main regulator of intra-organellar acidification. Assembly of this complex has extensively been studied in yeast, while limited knowledge exists for man. We identified 11 male patients with hemizygous missense mutations in ATP6AP1, encoding accessory protein Ac45 of the V-ATPase. Homology detection at the level of sequence profiles indicated Ac45 as the long-sought human homologue of yeast V-ATPase assembly factor Voa1. Processed wild-type Ac45, but not its disease mutants, restored V-ATPase-dependent growth in Voa1 mutant yeast. Patients display an immunodeficiency phenotype associated with hypogammaglobulinemia, hepatopathy and a spectrum of neurocognitive abnormalities. Ac45 in human brain is present as the common, processed ∼40-kDa form, while liver shows a 62-kDa intact protein, and B-cells a 50-kDa isoform. Our work unmasks Ac45 as the functional ortholog of yeast V-ATPase assembly factor Voa1 and reveals a novel link of tissue-specific V-ATPase assembly with immunoglobulin production and cognitive function.

Identification of domains within the V-ATPase accessory subunit Ac45 involved in V-ATPase transport and Ca2+-dependent exocytosis.

The vacuolar (H(+))-ATPase (V-ATPase) is crucial for maintenance of the acidic microenvironment in intracellular organelles, whereas its membrane-bound V(0)-sector is involved in Ca(2+)-dependent membrane fusion. In the secretory pathway, the V-ATPase is regulated by its type I transmembrane and V(0)-associated accessory subunit Ac45. To execute its function, the intact-Ac45 protein is proteolytically processed to cleaved-Ac45 thereby releasing its N-terminal domain. Here, we searched for the functional domains within Ac45 by analyzing a set of deletion mutants close to the in vivo situation, namely in transgenic Xenopus intermediate pituitary melanotrope cells. Intact-Ac45 was poorly processed and accumulated in the endoplasmic reticulum of the transgenic melanotrope cells. In contrast, cleaved-Ac45 was efficiently transported through the secretory pathway, caused an accumulation of the V-ATPase at the plasma membrane and reduced dopaminergic inhibition of Ca(2+)-dependent peptide secretion. Surprisingly, removal of the C-tail from intact-Ac45 caused cellular phenotypes also found for cleaved-Ac45, whereas C-tail removal from cleaved-Ac45 still allowed its transport to the plasma membrane, but abolished V-ATPase recruitment into the secretory pathway and left dopaminergic inhibition of the cells unaffected. We conclude that domains located in the N- and C-terminal portions of the Ac45 protein direct its trafficking, V-ATPase recruitment and Ca(2+)-dependent-regulated exocytosis.

Gene expression profiling of pituitary melanotrope cells during their physiological activation.

The pituitary melanotrope cells of the amphibian Xenopus laevis are responsible for the production of the pigment-dispersing peptide α-melanophore-stimulating hormone, which allows the animal to adapt its skin color to its environment. During adaptation to a dark background the melanotrope cells undergo remarkable changes characterized by dramatic increases in cell size and secretory activity. In this study we performed microarray mRNA expression profiling to identify genes important to melanotrope activation and growth. We show a strong increase in the expression of the immediate early gene (IEG) c-Fos and of the brain-derived neurotrophic factor gene (BDNF). Furthermore, we demonstrate the involvement of another IEG in the adaptation process, Nur77, and conclude from in vitro experiments that the expression of both c-Fos and Nur77 are partially regulated by the adenylyl cyclase system and calcium ions. In addition, we found a steady up-regulation of Ras-like product during the adaptation process, possibly evoked by BDNF/TrkB signaling. Finally, the gene encoding the 105-kDa heat shock protein HSPh1 was transiently up-regulated in the course of black-background adaptation and a gene product homologous to ferritin (ferritin-like product) was >100-fold up-regulated in fully black-adapted animals. We suggest that these latter two genes are induced in response to cellular stress and that they may be involved in changing the mode of mRNA translation required to meet the increased demand for de novo protein synthesis. Together, our results show that microarray analysis is a valuable approach to identify the genes responsible for generating coordinated responses in physiologically activated cells.

A proteome map of the pituitary melanotrope cell activated by black-background adaptation of Xenopus laevis.

Upon transfer of Xenopus laevis from a white to a black background, the melanotrope cells in the pituitary pars intermedia secrete alpha-melanocyte-stimulating hormone, which stimulates dispersion of melanin pigment in skin melanophores. This adaptive behavior is under the control of neurotransmitters and neuropeptides of hypothalamic origin. The alpha-melanocyte-stimulating hormone-producing cells and their hypothalamic control system provide an interesting model to study proteins required for biosynthetic and secretory processes involved in peptide hormone production and for brain-pituitary signaling. We present a 2-D PAGE-based proteome map of melanotrope cells from black-adapted animals, identifying 204 different proteins by MS analysis.

An isoform of the vacuolar (H(+))-ATPase accessory subunit Ac45.

The vacuolar (H(+))-ATPase (V-ATPase) is the main regulator of intraorganellar pH and in neuroendocrine cells is controlled by its accessory subunit, Ac45. Here, we report the discovery of the first isoform of a V-ATPase accessory subunit, namely an Ac45-like protein, denoted Ac45LP. Phylogenetic analysis revealed a lineage-dependent evolutionary history: Ac45 is absent in birds, and Ac45LP is absent in placental mammals, whereas all other tetrapod species contain both genes. In contrast to Ac45, Ac45LP is not proteolytically cleaved, a prerequisite for proper Ac45 routing. Intriguingly, Xenopus Ac45LP mRNA was expressed in developing neural tissue and in neural crest cells. In adult Xenopus, Ac45 mRNA is widely expressed mostly in neuroendocrine tissues, while Ac45LP mRNA expression was found to be restricted to the kidney and the lung. This novel Ac45LP may provide additional possibilities for V-ATPase regulation during neurodevelopment as well as in kidney and lung cells.

A comprehensive overview of the vertebrate p24 family: identification of a novel tissue-specifically expressed member.

The members of the p24 protein family have an important but unclear role in transport processes in the early secretory pathway. The p24 family consists of four subfamilies (alpha, beta, gamma, and delta), whereby the exact composition of the family varies among species. Despite more than 15 years of p24 research, the vertebrate p24 family is still surprisingly ill characterized. Here, we describe the human, mouse, Xenopus, and zebrafish orthologues of 10 p24 family members and a new member that we term p24gamma(5). Of these eleven p24 family members, nine are conserved throughout the vertebrate lineage, whereas two (p24gamma(4) and p24delta(2)) occur in some but not all vertebrates. We further show that all p24 proteins are widely expressed in mouse, except for p24alpha(1) and p24gamma(5) that display restricted expression patterns. Thus, we present for the first time a comprehensive overview of the phylogeny and expression of the vertebrate p24 protein family.

Physiological manipulation of cellular activity tunes protein and ultrastructural profiles in a neuroendocrine cell.

To study in vivo the dynamics of the biosynthetic and secretory processes in a neuroendocrine cell, we use the proopiomelanocortin-producing intermediate pituitary melanotrope cells of Xenopus laevis. The activity of these cells can be simply manipulated by adapting the animal to a white or a black background, resulting in inactive and hyperactive cells respectively. Here, we applied differential display proteomics and field emission scanning electron microscopy (FESEM) to examine the changes in architecture accompanying the gradual transition of the inactive to the hyperactive melanotrope cells. The proteomic analysis showed differential expression of neuroendocrine secretory proteins, endoplasmic reticulum (ER)-resident chaperones, and housekeeping and metabolic proteins. The FESEM study revealed changes in the ultrastructure of the ER and Golgi and the number of secretory granules. We conclude that activation of neuroendocrine cells tunes their molecular machineries and organelles to become professional secretors.

Reduced Aph-1b expression causes tissue- and substrate-specific changes in gamma-secretase activity in rats with a complex phenotype.

The gamma-secretase enzyme complex displays intramembrane catalytic activity toward many type I transmembrane proteins, including the Alzheimer-linked amyloid-beta-protein precursor (APP) and the neuregulin receptor ErbB4. Active gamma-secretase is a tetrameric protein complex consisting of presenilin-1 (or -2), nicastrin, PEN-2, and Aph-1a (or -1b). We have recently discovered that pharmacogenetically bred apomorphine-susceptible Wistar rats (APO-SUS) have only one or two copies of the Aph-1b gene (termed I/I and II/II rats, respectively), whereas their phenotypic counterparts (APO-UNSUS) have three copies (III/III). As a result, APO-SUS rats display reduced Aph-1b expression and a complex phenotype reminiscent of neurodevelopmental disorders. Here we determined in the I/I and III/III rats the gamma-secretase cleavage activity toward the three APP superfamily members, p75 neurotrophin receptor, ErbB4, and neuregulin-2, and found that the cleavage of only a subset of the substrates was changed. Furthermore, the observed differences were restricted to tissues that normally express relatively high Aph-1b compared with Aph-1a levels. Thus, we provide in vivo evidence that subtle alterations in gamma-secretase subunit composition may lead to a variety of affected (neuro)developmental signaling pathways and, consequently, a complex phenotype.

Gene dosage effect on gamma-secretase component Aph-1b in a rat model for neurodevelopmental disorders.

A combination of genetic factors and early life events is thought to determine the vulnerability of an individual to develop a complex neurodevelopmental disorder like schizophrenia. Pharmacogenetically selected, apomorphine-susceptible Wistar rats (APO-SUS) display a number of behavioral and pathophysiological features reminiscent of such disorders. Here, we report microarray analyses revealing in APO-SUS rats, relative to their counterpart APO-UNSUS rats, a reduced expression of Aph-1b, a component of the gamma-secretase enzyme complex that is involved in multiple (neuro)developmental signaling pathways. The reduced expression is due to a duplicon-based genomic rearrangement event resulting in an Aph-1b dosage imbalance. The expression levels of the other gamma-secretase components were not affected. However, gamma-secretase cleavage activity was significantly changed, and the APO-SUS/-UNSUS Aph-1b genotypes segregated with a number of behavioral phenotypes. Thus, a subtle imbalance in the expression of a single, developmentally important protein may be sufficient to cause a complex phenotype.