Amyotrophic lateral sclerosis (ALS) is a devastating and universally fatal neurodegenerative disease. Mutations in two related RNA-binding proteins, TDP-43 and FUS, that harbor prion-like domains, cause some forms of ALS. There are at least 213 human proteins harboring RNA recognition motifs, including FUS and TDP-43, raising the possibility that additional RNA-binding proteins might contribute to ALS pathogenesis. We performed a systematic survey of these proteins to find additional candidates similar to TDP-43 and FUS, followed by bioinformatics to predict prion-like domains in a subset of them. We sequenced one of these genes, TAF15, in patients with ALS and identified missense variants, which were absent in a large number of healthy controls. These disease-associated variants of TAF15 caused formation of cytoplasmic foci when expressed in primary cultures of spinal cord neurons. Very similar to TDP-43 and FUS, TAF15 aggregated in vitro and conferred neurodegeneration in Drosophila, with the ALS-linked variants having a more severe effect than wild type. Immunohistochemistry of postmortem spinal cord tissue revealed mislocalization of TAF15 in motor neurons of patients with ALS. We propose that aggregationprone RNA-binding proteins might contribute very broadly to ALS pathogenesis and the genes identified in our yeast functional screen, coupled with prion-like domain prediction analysis, now provide a powerful resource to facilitate ALS disease gene discovery. I n the future, personalized genome sequencing will become routine, empowering us to define the genetic basis of many human diseases. Currently, however, complete genome sequencing for individuals to discover rare pathogenic mutations is still too costly and time consuming. Thus, more creative approaches are needed to accelerate the discovery of disease genes. Moreover, even once genes are revealed, the need for innovative approaches to elucidate causality remains critical.ALS, also known as Lou Gehrig's disease, is a devastating adultonset neurodegenerative disease that attacks upper and lower motor neurons (1). A progressive and ultimately fatal muscle paralysis ensues, usually causing death within 2-5 y of disease onset. ALS is mostly sporadic, but ∼10% of cases are familial. Pathogenic mutations in several genes have been linked to familial and sporadic ALS, including SOD1, TARDBP, FUS/TLS, VAPB, OPTN, VCP, and others (2). Two of these genes, TARDBP (TDP-43) and FUS/TLS (FUS) are notable because they encode related RNA-binding proteins that harbor a prion-like domain (3-6). Moreover, both of these proteins have been identified as components of pathological inclusions in neurons of patients with ALS (7-9). Indeed, an emerging concept suggested by the association of FUS and TDP-43 to ALS is that defects in RNA metabolism might contribute to disease pathogenesis. These observations suggested an intriguing possibility: Could TDP-43 and FUS be just the tip of an iceberg? In other words, could other human RNA-binding proteins with properties similar to th...

Variants of UNC13A, a critical gene for synapse function, increase the risk of amyotrophic lateral sclerosis and frontotemporal dementia1–3, two related neurodegenerative diseases defined by mislocalization of the RNA-binding protein TDP-434,5. Here we show that TDP-43 depletion induces robust inclusion of a cryptic exon in UNC13A, resulting in nonsense-mediated decay and loss of UNC13A protein. Two common intronic UNC13A polymorphisms strongly associated with amyotrophic lateral sclerosis and frontotemporal dementia risk overlap with TDP-43 binding sites. These polymorphisms potentiate cryptic exon inclusion, both in cultured cells and in brains and spinal cords from patients with these conditions. Our findings, which demonstrate a genetic link between loss of nuclear TDP-43 function and disease, reveal the mechanism by which UNC13A variants exacerbate the effects of decreased TDP-43 function. They further provide a promising therapeutic target for TDP-43 proteinopathies.

The integrin family of adhesion receptors consists of at least 21 heterodimeric transmembrane proteins that differ in their tissue distribution and ligand specificity. The recently identified ␣8 integrin subunit associates with ␤1 and is predominantly expressed in smooth muscle and other contractile cells in adult tissues, and in mesenchymal and neural cells during development. We now show that ␣8␤1 specifically localizes to focal contacts in cells plated on the extracellular matrix proteins fibronectin or vitronectin. In addition we show that human embryonic kidney cells (293), transfected with ␣8 cDNA, express ␣8␤1 on their surface and use this receptor for adhesion to fibronectin and vitronectin. Furthermore, ␣8␤1 binds to both fibronectin-and vitronectinSepharose and can be specifically eluted from either matrix protein by the arginine-glycine-aspartic acid (RGD)-containing peptide, GRGDSP. Because fibronectin and vitronectin adhesion appeared to be mediated by RGD, we examined additional RGD-containing proteins, including tenascin, fibrinogen, thrombospondin, osteopontin, and denatured collagen type I. We found that only tenascin was able to mediate adhesion of ␣8-transfected 293 cells. By using recombinant fragments of tenascin in adhesion assays, we were able to localize the ␣8␤1 binding domain of tenascin to the RGD-containing, third fibronectin type III repeat. These data strongly suggest that tenascin, fibronectin, and vitronectin are ligands for ␣8␤1 and that this integrin binds to the RGD site in each of these ligands through mechanisms that are distinct and separate from ␣5-and ␣v-containing integrins.Integrins are a class of cell adhesion glycoproteins composed of two noncovalently associated subunits, ␣ and ␤. Each subunit contains a large extracellular domain, a transmembrane domain, and a short cytoplasmic domain. Integrins are known to bind to a wide variety of extracellular matrix proteins, including fibronectin, vitronectin, collagens, and laminins. The specificity of protein binding is determined by particular combinations of ␣ and ␤ subunit pairing. The ligand binding site is formed by the extracellular domain of both subunits and requires the presence of divalent cations. Many integrins interact with ligands through the tripeptide arginine-glycine-aspartic acid (RGD).The ␣8 integrin subunit was originally identified by Bossy et al.(1) in the chick embryo nervous system and was shown to be a partner for ␤1. We have identified human ␣8, cloned and sequenced the cDNA, raised antibodies to the predicted cytoplasmic domain sequence, and determined its distribution in adult mammalian tissues (2). We found that ␣8 is predominantly expressed in a variety of visceral and vascular smooth muscle cells, kidney mesangial cells, and lung myofibroblasts (2).To gain insight into potential functions of ␣8␤1 in vivo, we sought to determine potential ligands. We tested various ligands for their ability to direct ␣8␤1 to focal contacts, to bind to ␣8␤1 by affinity chromatography, and to support adhesion of ␣8...

Rapid eye movement (REM) sleep is an important component of the natural sleep/wake cycle, yet the mechanisms that regulate REM sleep remain incompletely understood. Cholinergic neurons in the mesopontine tegmentum have been implicated in REM sleep regulation, but lesions of this area have had varying effects on REM sleep. Therefore, this study aimed to clarify the role of cholinergic neurons in the pedunculopontine tegmentum (PPT) and laterodorsal tegmentum (LDT) in REM sleep generation. Selective optogenetic activation of cholinergic neurons in the PPT or LDT during non-REM (NREM) sleep increased the number of REM sleep episodes and did not change REM sleep episode duration. Activation of cholinergic neurons in the PPT or LDT during NREM sleep was sufficient to induce REM sleep.rapid eye movement sleep | acetylcholine | optogenetics | mesopontine tegmentum | mouse R apid eye movement (REM) sleep is tightly regulated, yet the mechanisms that control REM sleep remain incompletely understood. Early pharmacological and unit recording studies suggested that ACh was important for REM sleep regulation (1, 2). For example, injection of cholinergic drugs into the dorsal mesopontine tegmentum reliably induced a state very similar to natural REM sleep in cats (3-6). Unit recordings from the cholinergic areas of the mesopontine tegmentum revealed cells that were active during wakefulness and REM sleep, as well as neurons active only during REM sleep (7-13). Electrical stimulation of the laterodorsal tegmentum (LDT) in cats increased the percentage of time spent in REM sleep (14), and activation of the pedunculopontine tegmentum (PPT) in rats induced wakefulness and REM sleep (15). If cholinergic PPT and LDT neurons are necessary for REM sleep to occur, as the early studies suggest, then lesioning the PPT or LDT should decrease REM sleep. In cats, lesions of the PPT and LDT do disrupt REM sleep (16, 17), but lesions in rodents have had little effect on REM sleep or increased REM sleep (18)(19)(20)(21)(22). Additionally, c-fos studies have found very few cholinergic cells activated under high-REM sleep conditions. When c-fos-positive cholinergic neurons in the PPT and LDT are found to correlate with the percentage of REM sleep, they still account for only a few of the total cholinergic cells in the area (23). Juxtacellular recordings of identified cholinergic neurons in the LDT found these cells had wake and REM active firing profiles, with the majority firing the highest during REM sleep (13). These discrepancies have led to alternative theories of REM sleep regulation, where cholinergic neurons do not play a key role (18, 19, 23, 24 and reviewed in 25, 26).The PPT and LDT are made up of heterogeneous populations of cells, including distinct populations of cholinergic, GABAergic, and glutamatergic neurons (27-29). Many GABAergic neurons are active during REM sleep, as indicated by c-fos (23), and both GABAergic and glutamatergic neurons have been found with maximal firing rates during REM sleep in the LDT and medial PPT (13...