CRISPR-Cas9 screens in human cells and primary neurons identify modifiers of C9ORF72 dipeptide-repeat-protein toxicity.

Hexanucleotide-repeat expansions in the C9ORF72 gene are the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia (c9ALS/FTD). The nucleotide-repeat expansions are translated into dipeptide-repeat (DPR) proteins, which are aggregation prone and may contribute to neurodegeneration. We used the CRISPR-Cas9 system to perform genome-wide gene-knockout screens for suppressors and enhancers of C9ORF72 DPR toxicity in human cells. We validated hits by performing secondary CRISPR-Cas9 screens in primary mouse neurons. We uncovered potent modifiers of DPR toxicity whose gene products function in nucleocytoplasmic transport, the endoplasmic reticulum (ER), proteasome, RNA-processing pathways, and chromatin modification. One modifier, TMX2, modulated the ER-stress signature elicited by C9ORF72 DPRs in neurons and improved survival of human induced motor neurons from patients with C9ORF72 ALS. Together, our results demonstrate the promise of CRISPR-Cas9 screens in defining mechanisms of neurodegenerative diseases

Regularity of squarefree monomial ideals

We survey a number of recent studies of the Castelnuovo-Mumford regularity of squarefree monomial ideals. Our focus is on bounds and exact values for the regularity in terms of combinatorial data from associated simplicial complexes and/or hypergraphs.Comment: 23 pages; survey paper; minor changes in V.

Semaphorin-Plexin Signaling Guides Patterning of the Developing Vasculature

AbstractMajor vessels of the vertebrate circulatory system display evolutionarily conserved and reproducible anatomy, but the cues guiding this stereotypic patterning remain obscure. In the nervous system, axonal pathways are shaped by repulsive cues provided by ligands of the semaphorin family that are sensed by migrating neuronal growth cones through plexin receptors. We show that proper blood vessel pathfinding requires the endothelial receptor PlexinD1 and semaphorin signals, and we identify mutations in plexinD1 in the zebrafish vascular patterning mutant out of bounds. These results reveal the fundamental conservation of repulsive patterning mechanisms between axonal migration in the central nervous system and vascular endothelium during angiogenesis

Symbolic powers of monomial ideals and Cohen-Macaulay vertex-weighted digraphs

In this paper we study irreducible representations and symbolic Rees algebras of monomial ideals. Then we examine edge ideals associated to vertex-weighted oriented graphs. These are digraphs having no oriented cycles of length two with weights on the vertices. For a monomial ideal with no embedded primes we classify the normality of its symbolic Rees algebra in terms of its primary components. If the primary components of a monomial ideal are normal, we present a simple procedure to compute its symbolic Rees algebra using Hilbert bases, and give necessary and sufficient conditions for the equality between its ordinary and symbolic powers. We give an effective characterization of the Cohen--Macaulay vertex-weighted oriented forests. For edge ideals of transitive weighted oriented graphs we show that Alexander duality holds. It is shown that edge ideals of weighted acyclic tournaments are Cohen--Macaulay and satisfy Alexander dualityComment: Special volume dedicated to Professor Antonio Campillo, Springer, to appea

The Role of the Parkinson's Disease Gene PARK9 in Essential Cellular Pathways and the Manganese Homeostasis Network in Yeast

YPK9 (Yeast PARK9; also known as YOR291W) is a non-essential yeast gene predicted by sequence to encode a transmembrane P-type transport ATPase. However, its substrate specificity is unknown. Mutations in the human homolog of YPK9, ATP13A2/PARK9, have been linked to genetic forms of early onset parkinsonism. We previously described a strong genetic interaction between Ypk9 and another Parkinson's disease (PD) protein α-synuclein in multiple model systems, and a role for Ypk9 in manganese detoxification in yeast. In humans, environmental exposure to toxic levels of manganese causes a syndrome similar to PD and is thus an environmental risk factor for the disease. How manganese contributes to neurodegeneration is poorly understood. Here we describe multiple genome-wide screens in yeast aimed at defining the cellular function of Ypk9 and the mechanisms by which it protects cells from manganese toxicity. In physiological conditions, we found that Ypk9 genetically interacts with essential genes involved in cellular trafficking and the cell cycle. Deletion of Ypk9 sensitizes yeast cells to exposure to excess manganese. Using a library of non-essential gene deletions, we screened for additional genes involved in tolerance to excess manganese exposure, discovering several novel pathways involved in manganese homeostasis. We defined the dependence of the deletion strain phenotypes in the presence of manganese on Ypk9, and found that Ypk9 deletion modifies the manganese tolerance of only a subset of strains. These results confirm a role for Ypk9 in manganese homeostasis and illuminates cellular pathways and biological processes in which Ypk9 likely functions

<i>C-elegans</i> model identifies genetic modifiers of alpha-synuclein inclusion formation during aging

Inclusions in the brain containing alpha-synuclein are the pathological hallmark of Parkinson's disease, but how these inclusions are formed and how this links to disease is poorly understood. We have developed a &lt;i&gt;C-elegans&lt;/i&gt; model that makes it possible to monitor, in living animals, the formation of alpha-synuclein inclusions. In worms of old age, inclusions contain aggregated alpha-synuclein, resembling a critical pathological feature. We used genome-wide RNA interference to identify processes involved in inclusion formation, and identified 80 genes that, when knocked down, resulted in a premature increase in the number of inclusions. Quality control and vesicle-trafficking genes expressed in the ER/Golgi complex and vesicular compartments were overrepresented, indicating a specific role for these processes in alpha-synuclein inclusion formation. Suppressors include aging-associated genes, such as sir-2.1/SIRT1 and lagr-1/LASS2. Altogether, our data suggest a link between alpha-synuclein inclusion formation and cellular aging, likely through an endomembrane-related mechanism. The processes and genes identified here present a framework for further study of the disease mechanism and provide candidate susceptibility genes and drug targets for Parkinson's disease and other alpha-synuclein related disorders

The yeast P5 type ATPase, Spf1, regulates manganese transport into the endoplasmic reticulum

The endoplasmic reticulum (ER) is a large, multifunctional and essential organelle. Despite intense research, the function of more than a third of ER proteins remains unknown even in the well-studied model organism Saccharomyces cerevisiae. One such protein is Spf1, which is a highly conserved, ER localized, putative P-type ATPase. Deletion of SPF1 causes a wide variety of phenotypes including severe ER stress suggesting that this protein is essential for the normal function of the ER. The closest homologue of Spf1 is the vacuolar P-type ATPase Ypk9 that influences Mn2+ homeostasis. However in vitro reconstitution assays with Spf1 have not yielded insight into its transport specificity. Here we took an in vivo approach to detect the direct and indirect effects of deleting SPF1. We found a specific reduction in the luminal concentration of Mn2+ in ∆spf1 cells and an increase following it’s overexpression. In agreement with the observed loss of luminal Mn2+ we could observe concurrent reduction in many Mn2+-related process in the ER lumen. Conversely, cytosolic Mn2+-dependent processes were increased. Together, these data support a role for Spf1p in Mn2+ transport in the cell. We also demonstrate that the human sequence homologue, ATP13A1, is a functionally conserved orthologue. Since ATP13A1 is highly expressed in developing neuronal tissues and in the brain, this should help in the study of Mn2+-dependent neurological disorders

Cocaine Increases Dopamine Release by Mobilization of a Synapsin-Dependent Reserve Pool

Cocaine primarily exerts its behavioral effects by enhancing dopaminergic neurotransmission, amplifying dopamine-encoded sensorimotor integration. The presumed mechanism for this effect is inhibition of the dopamine transporter, which blocks dopamine uptake and prolongs the duration of dopamine in the extracellular space. However, there is growing evidence that cocaine can also augment dopamine release. Here, we directly monitored the actions of cocaine on dopamine release by using electrochemical detection to measure extracellular dopamine in the striatum of anesthetized mice. Cocaine enhanced the levels of striatal dopamine produced by electrical stimulation of dopaminergic neurons. Even after pretreatment with alpha-methyl-p-tyrosine, which depletes the readily releasable pool of dopamine, cocaine was still capable of elevating dopamine levels. This suggests that cocaine enhances dopamine release by mobilizing a reserve pool of dopamine-containing synaptic vesicles. To test this hypothesis, we examined electrically evoked dopamine release in synapsin I/II/III triple knock-out mice, which have impaired synaptic vesicle reserve pools. Knock-out of synapsins greatly reduced the ability of cocaine to enhance dopamine release with long stimulus trains or after depletion of the newly synthesized pool. We therefore conclude that cocaine enhances dopamine release and does so by mobilizing a synapsin-dependent reserve pool of dopamine-containing synaptic vesicles. This capacity to enhance exocytotic release of dopamine may be important for the psychostimulant actions of cocaine

Model Organisms Reveal Insight into Human Neurodegenerative Disease: Ataxin-2 Intermediate-Length Polyglutamine Expansions Are a Risk Factor for ALS

Model organisms include yeast Saccromyces cerevisae and fly Drosophila melanogaster. These systems have powerful genetic approaches, as well as highly conserved pathways, both for normal function and disease. Here, we review and highlight how we applied these systems to provide mechanistic insight into the toxicity of TDP-43. TDP-43 accumulates in pathological aggregates in ALS and about half of FTD. Yeast and fly studies revealed an interaction with the counterparts of human Ataxin-2, a gene whose polyglutamine repeat expansion is associated with spinocerebellar ataxia type 2. This finding raised the hypothesis that repeat expansions in ataxin-2 may associate with diseases characterized by TDP-43 pathology such as ALS. DNA analysis of patients revealed that intermediate-length polyglutamine expansions in ataxin-2 are a risk factor for ALS, such that repeat lengths are greater than normal, but lower than that associated with spinocerebellar ataxia type 2 (SCA2), and are more frequent in ALS patients than in matched controls. Moreover, repeat expansions associated with ALS are interrupted CAA-CAG sequences as opposed to the pure CAG repeat expansions typically associated with SCA2. These studies provide an example of how model systems, when extended to human cells and human patient tissue, can reveal new mechanistic insight into disease