Jelena Mojsilovic-Petrovic

A yeast functional screen predicts new candidate ALS disease genes

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 aggregation-prone 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.

Evaluating the role of the FUS/TLS-related gene EWSR1 in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease affecting motor neurons. Mutations in related RNA-binding proteins TDP-43, FUS/TLS and TAF15 have been connected to ALS. These three proteins share several features, including the presence of a bioinformatics-predicted prion domain, aggregation-prone nature in vitro and in vivo and toxic effects when expressed in multiple model systems. Given these commonalities, we hypothesized that a related protein, EWSR1 (Ewing sarcoma breakpoint region 1), might also exhibit similar properties and therefore could contribute to disease. Here, we report an analysis of EWSR1 in multiple functional assays, including mutational screening in ALS patients and controls. We identified three missense variants in EWSR1 in ALS patients, which were absent in a large number of healthy control individuals. We show that disease-specific variants affect EWSR1 localization in motor neurons. We also provide multiple independent lines of in vitro and in vivo evidence that EWSR1 has similar properties as TDP-43, FUS and TAF15, including aggregation-prone behavior in vitro and ability to confer neurodegeneration in Drosophila. Postmortem analysis of sporadic ALS cases also revealed cytoplasmic mislocalization of EWSR1. Together, our studies highlight a potential role for EWSR1 in ALS, provide a collection of functional assays to be used to assess roles of additional RNA-binding proteins in disease and support an emerging concept that a class of aggregation-prone RNA-binding proteins might contribute broadly to ALS and related neurodegenerative diseases.

Hypoxia-inducible factor-1 (HIF-1) is involved in the regulation of hypoxia-stimulated expression of monocyte chemoattractant protein-1 (MCP-1/CCL2) and MCP-5 (Ccl12) in astrocytes

BackgroundNeuroinflammation has been implicated in various brain pathologies characterized by hypoxia and ischemia. Astroglia play an important role in the initiation and propagation of hypoxia/ischemia-induced inflammation by secreting inflammatory chemokines that attract neutrophils and monocytes into the brain. However, triggers of chemokine up-regulation by hypoxia/ischemia in these cells are poorly understood. Hypoxia-inducible factor-1 (HIF-1) is a dimeric transcriptional factor consisting of HIF-1α and HIF-1β subunits. HIF-1 binds to HIF-1-binding sites in the target genes and activates their transcription. We have recently shown that hypoxia-induced expression of IL-1β in astrocytes is mediated by HIF-1α. In this study, we demonstrate the role of HIF-1α in hypoxia-induced up-regulation of inflammatory chemokines, human monocyte chemoattractant protein-1 (MCP-1/CCL2) and mouse MCP-5 (Ccl12), in human and mouse astrocytes, respectively.MethodsPrimary fetal human astrocytes or mouse astrocytes generated from HIF-1α+/+ and HIF-1α+/- mice were subjected to hypoxia (<2% oxygen) or 125 μM CoCl2 for 4 h and 6 h, respectively. The expression of HIF-1α, MCP-1 and MCP-5 was determined by semi-quantitative RT-PCR, western blot or ELISA. The interaction of HIF-1α with a HIF-1-binding DNA sequence was examined by EMSA and supershift assay. HIF-1-binding sequence in the promoter of MCP-1 gene was cloned and transcriptional activation of MCP-1 by HIF-1α was analyzed by reporter gene assay.ResultsSequence analyses identified HIF-1-binding sites in the promoters of MCP-1 and MCP-5 genes. Both hypoxia and HIF-1α inducer, CoCl2, strongly up-regulated HIF-1α expression in astrocytes. Mouse HIF-1α+/- astrocytes had lower basal levels of HIF-1α and MCP-5 expression. The up-regulation of MCP-5 by hypoxia or CoCl2 in HIF-1α+/+ and HIF-1α+/- astrocytes was correlated with the levels of HIF-1α in cells. Both hypoxia and CoCl2 also up-regulated HIF-1α and MCP-1 expression in human astrocytes. EMSA assay demonstrated that HIF-1 activated by either hypoxia or CoCl2 binds to wild-type HIF-1-binding DNA sequence, but not the mutant sequence. Furthermore, reporter gene assay demonstrated that hypoxia markedly activated MCP-1 transcription but not the mutated MCP-1 promoter in transfected astrocytes.ConclusionThese findings suggest that both MCP-1 and MCP-5 are HIF-1 target genes and that HIF-1α is involved in transcriptional induction of these two chemokines in astrocytes by hypoxia.

Protecting Motor Neurons from Toxic Insult by Antagonism of Adenosine A2a and Trk Receptors

The death of motor neurons in amyotrophic lateral sclerosis (ALS) is thought to result from the interaction of a variety of factors including excitotoxicity, accumulation of toxic proteins, and abnormal axonal transport. Previously, we found that the susceptibility of motor neurons to excitotoxic insults can be limited by inhibiting signals evoked by brain-derived neurotrophic factor (BDNF) activation of the receptor tyrosine kinase B (TrkB). Here we show that this can be achieved by direct kinase inhibition or by blockade of a transactivation pathway that uses adenosine A2a receptors and src-family kinases (SFKs). Downstream signaling cascades (such as mitogen-activated protein kinase and phosphatidylinositol-3 kinase) are inhibited by these blockers. In addition to protecting motor neurons from excitotoxic insult, these agents also prevent toxicity that follows from the expression of mutant proteins (G85R superoxide dismutase 1; G59S p150glued) that cause familial motor neuron disease. TrkB, adenosine A2a receptors, and SFKs associate into complexes in lipid raft and nonlipid raft membranes and the signaling from lipids rafts may be particularly important because their disruption by cholesterol depletion blocks the ability of BDNF to render motor neurons vulnerable to insult. The neuroprotective versatility of Trk antagonism suggests that it may have broad utility in the treatment of ALS patients.

FOXO3a is broadly neuroprotective in vitro and in vivo against insults implicated in motor neuron diseases

Aging is a risk factor for the development of adult-onset neurodegenerative diseases. Although some of the molecular pathways regulating longevity and stress resistance in lower organisms are defined (i.e., those activating the transcriptional regulators DAF-16 and HSF-1 in Caenorhabditis elegans), their relevance to mammals and disease susceptibility are unknown. We studied the signaling controlled by the mammalian homolog of DAF-16, FOXO3a, in model systems of motor neuron disease. Neuron death elicited in vitro by excitotoxic insult or the expression of mutant SOD1, mutant p150glued, or polyQ-expanded androgen receptor was abrogated by expression of nuclear-targeted FOXO3a. We identify a compound [Psammaplysene A (PA)] that increases nuclear localization of FOXO3a in vitro and in vivo and show that PA also protects against these insults in vitro. Administration of PA to invertebrate model systems of neurodegeneration similarly blocked neuron death in a DAF-16/FOXO3a-dependent manner. These results indicate that activation of the DAF-16/FOXO3a pathway, genetically or pharmacologically, confers protection against the known causes of motor neuron diseases.

GluR1 Controls Dendrite Growth through Its Binding Partner, SAP97

Activity-dependent dendrite elaboration influences the pattern of interneuronal connectivity and network function. In the present study, we examined the mechanism by which the GluR1 subunit of AMPA receptors controls dendrite morphogenesis. GluR1 binds to SAP97, a scaffolding protein that is a component of the postsynaptic density, via its C-terminal 7 aa. We find that elimination of this interaction in vitro or in vivo (by deleting the C-terminal 7 aa of GluR1, GluR1Δ7) does not influence trafficking, processing, or cell surface GluR1 expression but does prevent translocation of SAP97 from the cytosol to membranes. GluR1 and SAP97 together at the plasma membrane promotes dendrite branching in an activity-dependent manner, although this does not require physical association. Our findings suggest that the C-terminal 7 aa of GluR1 are essential for bringing SAP97 to the plasma membrane, where it acts to translate the activity of AMPA receptors into dendrite growth.

Role of GluR1 in activity-dependent motor system development

Activity-dependent specification of neuronal architecture during early postnatal life is essential for refining the precision of communication between neurons. In the spinal cord under normal circumstances, the AMPA receptor subunit GluR1 is expressed at high levels by motor neurons and surrounding interneurons during this critical developmental period, although the role it plays in circuit formation and locomotor behavior is unknown. Here, we show that GluR1 promotes dendrite growth in a non-cell-autonomous manner in vitro and in vivo. The mal-development of motor neuron dendrites is associated with changes in the pattern of interneuronal connectivity within the segmental spinal cord and defects in strength and endurance. Transgenic expression of GluR1 in adult motor neurons leads to dendrite remodeling and supernormal locomotor function. GluR1 expression by neurons within the segmental spinal cord plays an essential role in formation of the neural network that underlies normal motor behavior.

Embryonic motor neuron dendrite growth is stunted by inhibition of nitric oxide-dependent activation of soluble guanylyl cyclase and protein kinase G

We have examined the participation of a neuronal nitric oxide synthase (nNOS) signaling pathway in the elaboration of motor neuron dendrites during embryonic life. During chick embryogenesis, nNOS is expressed by interneurons that surround the motor neuron pools in the ventral horn. Pseudorabies virus tracing suggests that these cells, while juxtaposed to motor neurons are not synaptically connected to them. The downstream effectors, soluble guanylyl cyclase (sGC) and protein kinase G (PKG), are found in motor neurons as well as several other populations of spinal cord cells. To determine the functional significance of the nNOS/sGC/PKG signaling pathway, pharmacological inhibitors were applied to chick embryos and the effects on motor neuron dendrites monitored. Inhibition of nNOS activity led to a lasting reduction in the overall size and degree of branching of the dendritic tree. These alterations in dendritic architecture were also seen when the activity of sGC or PKG was blocked. Our results suggest that normal motor neuron dendrite elaboration depends, in part, on the activity‐dependent generation of NO by ventral horn interneurons, which then activates sGC and PKG in motor neurons.

The Proline/Arginine Dipeptide from hexanucleotide repeat expanded C9ORF72 inhibits the proteasome

Abstract An intronic hexanucleotide repeat expansion (HRE) mutation in the C9ORF72 gene is the most common cause of familial ALS and frontotemporal dementia (FTD) and is found in ∼7% of individuals with apparently sporadic disease. Several different diamino acid peptides can be generated from the HRE by noncanonical translation (repeat-associated non-ATG translation, or RAN translation), and some of these peptides can be toxic. Here, we studied the effects of two arginine containing RAN translation products [proline/arginine repeated 20 times (PR20) and glycine/arginine repeated 20 times (GR20)] in primary rat spinal cord neuron cultures grown on an astrocyte feeder layer. We find that PR20 kills motor neurons with an LD50 of 2 µM, but in contrast to the effects of other ALS-causing mutant proteins (i.e., SOD or TDP43), PR20 does not evoke the biochemical signature of mitochondrial dysfunction, ER stress, or mTORC down-regulation. PR20 does result in a time-dependent build-up of ubiquitylated substrates, and this is associated with a reduction of flux through both autophagic and proteasomal degradation pathways. GR20, however, does not have these effects. The effects of PR20 on the proteasome are likely to be direct because (1) PR20 physically associates with proteasomes in biochemical assays, and (2) PR20 inhibits the degradation of a ubiquitylated test substrate when presented to purified proteasomes. Application of a proteasomal activator (IU1) blocks the toxic effects of PR20 on motor neuron survival. This work suggests that proteasomal activators have therapeutic potential in individuals with C9ORF72 HRE.

Signaling events in axons and/or dendrites render motor neurons vulnerable to mutant superoxide dismutase toxicity

The survival of dorsal root ganglion and sympathetic neurons is promoted whether nerve growth factor (NGF) activates TrkA receptors on the cell body or the axon. Yet other aspects of neurotrophic factor actions (i.e., ability to promote axon growth, selection of neurochemical phenotype and engagement of signaling modules) differ as a function of the location of the ligand–receptor interaction. The extent to which these observations are relevant to CNS neurons is unknown. This may be particularly relevant to neurodegenerative diseases such as amyotrophic lateral sclerosis, where beneficial axon–target interactions are disturbed early in the disease process. Here we characterize the growth of pure motor neurons in compartment cultures and show that brain-derived neurotrophic factor (BDNF) stimulation of the cell body or axons/dendrites promotes survival. Expression of G37R mutant superoxide dismutase (SOD) in motor neurons will lead to death and this depends on BDNF activation of TrkB on axons and/or dendrites. BDNF action depends upon endocytosis of the BDNF–TrkB complex and de novo protein synthesis. These results highlight the importance of signaling events occurring in axons/dendrites in mutant SOD toxicity.