ROLE OF SYNAPSIN IN LONG-TERM SYNAPTIC FACILITATION IN APLYSIA

Enhanced expression of the presynaptic protein synapsin has been correlated with certain forms of long-term plasticity and learning and memory. However, the regulation and requirement for enhanced synapsin expression in long-term memory remains unknown. In the present study the technical advantages of the marine mollusc Aplysia were exploited in order to address this issue. In Aplysia, learning-induced enhancement in synaptic strength is modulated by serotonin (5-HT) and treatment with 5-HT in vitro of the sensorimotor synapse induces long-term facilitation (LTF) of synaptic transmission, which lasts for days, as well as the formation of new connections between the sensory and motor neuron. Results from immunofluorescence analysis indicated that 5-HT treatment upregulates synapsin protein levels within sensory neuron varicosities, the presumed site of neurotransmitter release. To investigate the mechanisms underlying increased synapsin expression, the promoter region of the Aplysia synapsin gene was cloned and a cAMP response element (CRE) was identified, raising the possibility that the transcriptional activator cAMP response element-binding protein-1 (CREB1) mediates the 5-HT-induced regulation of synapsin. Results from Chromatin Immunoprecipitation (ChIP) assays indicated that 5-HT treatment enhanced association of CREB1 surrounding the CRE site in the synapsin promoter and led to increased acetylation of histones H3 and H4 and decreased association of histone deacetylase 5 surrounding the CRE site in the synapsin promoter, a sign of transcriptional activation. In addition, sensory neurons injected with an enhanced green fluorescent protein (EGFP) reporter vector driven by the synapsin promoter exhibited a significant increase in EGFP expression following treatment with 5-HT. These results suggest that synapsin expression is regulated by 5-HT in part through transcriptional activation of the synapsin gene and through CREB1 association with the synapsin promoter. Furthermore, RNA interference that blocks 5-HT-induced elevation of synapsin expression also blocked long-term synaptic facilitation. These results indicate that 5-HT-induced regulation of synapsin is necessary for LTF and that synapsin is part of the cascade of synaptic events involved in the consolidation of memory.

Anatomical organization of the memory underlying sensitization in the siphon-withdrawal reflex circuit of {\it Aplysia californica}

Previous studies have shown that short-term sensitization of the Aplysia siphon-withdrawal reflex circuit results in multiple sites of change in synaptic efficacy. In this dissertation I have used a realistic modeling approach (using an integrate-and-fire scheme), in conjunction with electrophysiological experiments, to evaluate the contribution of each site of plasticity to the sensitized response.^ This dissertation contains a detailed description of methodology for the construction of the model circuit, consisting of the LFS motor neurons and ten interneurons known to convey excitatory input to them. The model replicates closely the natural motor neuron firing response to a brief tactile stimulus.^ The various circuit elements have different roles for producing circuit output. For example, the sensory connections onto the motor neuron are important for the production of the phasic response, while the polysynaptic interneuronal connections are important for producing the tonic response.^ The multiple sites of plasticity that produce changes in circuit output also have specialized roles. Presynaptic facilitation of the sensory neuron to LFS connection enhances only the phasic component of the motor neuron firing response. The sensory neuron to interneuron connections primarily enhance the tonic component of the motor neuron firing response. Also, the L29 posttetanic potentiation and the L30 presynaptic inhibition primarily enhance the tonic component of the motor neuron firing response. Finally, the information content at the various sites of plasticity can shift with changes in stimulus intensity. This suggests that while the sites of plasticity encoding memory are fixed, the information content at these sites can be dynamic, shifting in anatomical location with changes in the intensity of the test stimulus.^ These sites of plasticity also produce specific changes in the behavioral response. Sensory-LFS plasticity selectively increases the amplitude of the behavioral response, and has no effect on the duration of the behavioral response. Interneuronal plasticity (L29 and L30) affects both the amplitude and duration of the behavioral response. Other sensory plasticity also affect both the amplitude and duration of the behavioral response, presumably by increasing the recruitment of the interneurons, which provide all of the effect on duration of the behavioral response. ^

Unravelling the multiple roles of FUS in RNA processing and on ALS pathogenesis

Two genes with related functions in RNA biogenesis were recently reported in patients with familial ALS: the FUS/TLS gene at the ALS6 locus and the TARDBP/TDP-43 gene at the ALS10 locus [1, 2]. FUS has been implicated to function in several steps of gene expression, including transcription regulation [3], RNA splicing [4, 5], mRNA transport in neurons [6] and, interestingly, in microRNA (miRNA) processing [7]. The goal of this project is to identify the molecular mechanisms leading to the development of FUS mutations-associated ALS. Specifically, we want to test the hypothesis that these FUS mutations misregulate miRNA levels that in turn affect the expression of genes critical for motor neuron survival. In addition we want to test whether misregulation of the miRNA profile is a common feature in ALS. We have performed immunoprecipitations from total extracts of 293T cells expressing FLAG-tagged FUS to characterize its interactome by mass spectrometry. This proteomic study not only revealed a strong interaction of FUS with splicing factors, but shows that FUS might be involved in many, quite different pathways. To map which parts of the FUS protein contribute to the interaction with splicing factors, we have performed a set of experiments with a series of missense and deletion mutants. With this approach, we will not only gain information on the binding partners of FUS along with a map of the required domains for the interactions, but it will also help to unravel whether certain ALS-associated FUS mutations lead to a loss or gain of function due to gain or loss of interactors. Additionally, we have performed quantitative interactomics using SILAC to identify interactome differences of ALS-associated FUS mutants. To this end we have performed immunoprecipitations of total extract from 293T cells, stably transduced with constructs expressing wild-type FUS-FLAG as well as three different ALS-associated mutants (G156E, R244C, P525L). First results indicate striking differences in the interactome with certain RNA binding proteins. We are now validating these candidates in order to reveal the importance of these differential interactions in the context of ALS.

Combined systemic and local morpholino treatment rescues the phenotype of the SMA Delta 7 mouse model

Spinal muscular atrophy (SMA) is a childhood fatal motor neuron disease caused by mutations in the Survival Motor Neuron 1 (SMN1) gene, currently without effective treatment. One possible therapeutic approach is the use of antisense oligonucleotides (ASOs) to redirect the splicing of a paralogous gene, SMN2, to increase the production of functional SMN protein. A range of ASOs with different chemical properties is suitable for these applications, including a morpholino (MO) variant, which has a particularly excellent safety, and efficacy profile. We used a 25- nt MO oligomer sequence against the ISS-N1 region of SMN2 (HSMN2Ex7D(-10-34)) with superior efficacy to previously described sequences also in transgenic SMA Δ7 mice. The combined local and systemic administration of MO (bare or conjugated to octa-guanidine) is necessary to increase full-length SMN expression, leading to robust neuropathological features improvement and survival rescue. Additionally, several snRNA levels that are dysregulated in SMA mice could be restored by MO treatment. These results demonstrate that MO therapy is efficacious and can result in phenotypic rescue. These data provide important insights for the development of therapeutic strategies in SMA patients.

Effect of Combined Systemic and Local Morpholino Treatment on the Spinal Muscular Atrophy Δ7 Mouse Model Phenotype

BACKGROUND: Spinal muscular atrophy (SMA) is a fatal motor neuron disease of childhood that is caused by mutations in the SMN1 gene. Currently, no effective treatment is available. One possible therapeutic approach is the use of antisense oligos (ASOs) to redirect the splicing of the paralogous gene SMN2, thus increasing functional SMN protein production. Various ASOs with different chemical properties are suitable for these applications, including a morpholino oligomer (MO) variant with a particularly excellent safety and efficacy profile. OBJECTIVE: We investigated a 25-nt MO sequence targeting the negative intronic splicing silencer (ISS-N1) 10 to 34 region. METHODS: We administered a 25-nt MO sequence against the ISS-N1 region of SMN2 (HSMN2Ex7D[-10-34]) in the SMAΔ7 mouse model and evaluated the effect and neuropathologic phenotype. We tested different concentrations (from 2 to 24 nM) and delivery protocols (intracerebroventricular injection, systemic injection, or both). We evaluated the treatment efficacy regarding SMN levels, survival, neuromuscular phenotype, and neuropathologic features. RESULTS: We found that a 25-nt MO sequence against the ISS-N1 region of SMN2 (HSMN2Ex7D[-10-34]) exhibited superior efficacy in transgenic SMAΔ7 mice compared with previously described sequences. In our experiments, the combination of local and systemic administration of MO (bare or conjugated to octaguanidine) was the most effective approach for increasing full-length SMN expression, leading to robust improvement in neuropathologic features and survival. Moreover, we found that several small nuclear RNAs were deregulated in SMA mice and that their levels were restored by MO treatment. CONCLUSION: These results indicate that MO-mediated SMA therapy is efficacious and can result in phenotypic rescue, providing important insights for further development of ASO-based therapeutic strategies in SMA patients.

Splicing changes in SMA mouse motoneurons and SMN-depleted neuroblastoma cells: evidence for involvement of splicing regulatory proteins.

Spinal Muscular Atrophy (SMA) is caused by deletions or mutations in the Survival Motor Neuron 1 (SMN1) gene. The second gene copy, SMN2, produces some, but not enough, functional SMN protein. SMN is essential to assemble small nuclear ribonucleoproteins (snRNPs) that form the spliceosome. However, it is not clear whether SMA is caused by defects in this function that could lead to splicing changes in all tissues, or by the impairment of an additional, less well characterized, but motoneuron-specific SMN function. We addressed the first possibility by exon junction microarray analysis of motoneurons (MNs) isolated by laser capture microdissection from a severe SMA mouse model. This revealed changes in multiple U2-dependent splicing events. Moreover, splicing appeared to be more strongly affected in MNs than in other cells. By testing mutiple genes in a model of progressive SMN depletion in NB2a neuroblastoma cells, we obtained evidence that U2-dependent splicing changes occur earlier than U12-dependent ones. As several of these changes affect genes coding for splicing regulators, this may acerbate the splicing response induced by low SMN levels and induce secondary waves of splicing alterations.

A large animal model of spinal muscular atrophy and correction of phenotype.

OBJECTIVES Spinal muscular atrophy (SMA) is caused by reduced levels of survival motor neuron (SMN) protein, which results in motoneuron loss. Therapeutic strategies to increase SMN levels including drug compounds, antisense oligonucleotides, and scAAV9 gene therapy have proved effective in mice. We wished to determine whether reduction of SMN in postnatal motoneurons resulted in SMA in a large animal model, whether SMA could be corrected after development of muscle weakness, and the response of clinically relevant biomarkers. METHODS Using intrathecal delivery of scAAV9 expressing an shRNA targeting pig SMN1, SMN was knocked down in motoneurons postnatally to SMA levels. This resulted in an SMA phenotype representing the first large animal model of SMA. Restoration of SMN was performed at different time points with scAAV9 expressing human SMN (scAAV9-SMN), and electrophysiology measurements and pathology were performed. RESULTS Knockdown of SMN in postnatal motoneurons results in overt proximal weakness, fibrillations on electromyography indicating active denervation, and reduced compound muscle action potential (CMAP) and motor unit number estimation (MUNE), as in human SMA. Neuropathology showed loss of motoneurons and motor axons. Presymptomatic delivery of scAAV9-SMN prevented SMA symptoms, indicating that all changes are SMN dependent. Delivery of scAAV9-SMN after symptom onset had a marked impact on phenotype, electrophysiological measures, and pathology. INTERPRETATION High SMN levels are critical in postnatal motoneurons, and reduction of SMN results in an SMA phenotype that is SMN dependent. Importantly, clinically relevant biomarkers including CMAP and MUNE are responsive to SMN restoration, and abrogation of phenotype can be achieved even after symptom onset.

ER strikes again: Proteostasis Dysfunction In ALS

The precise contribution of endoplasmic reticulum (ER) chaperone protein disulfide isomerase (PDI) variants in human amyotrophic lateral sclerosis (ALS) patients to the pathogenesis of ALS remained unclear. In the present study, Woehlbier et al (2016) demonstrated that these PDI variants are capable of altering motor neuron morphology, impairing the expression of synaptic proteins, and compromising neuromuscular junction (NMJ) integrity.

STUDYING AGGREGATE FORMATION BY AMYOTROPHIC LATERAL SCLEROSIS-ASSOCIATED MUTANT SOD1 PROTEIN IN DROSOPHILA MODEL

A common pathological hallmark of most neurodegenerative disorders is the presence of protein aggregates in the brain. Understanding the regulation of aggregate formation is thus important for elucidating disease pathogenic mechanisms and finding effective preventive avenues and cures. Amyotrophic Lateral Sclerosis (ALS), also known as Lou Gehrig’s disease, is a selective neurodegenerative disorder predominantly affecting motor neurons. The majority of ALS cases are sporadic, however, mutations in superoxide dismutase 1 (SOD1) are responsible for about 20% of familial ALS (fALS). Mutated SOD1 proteins are prone to misfold and form protein aggregates, thus representing a good candidate for studying aggregate formation. The long-term goal of this project is to identify regulators of aggregate formation by mutant SOD1 and other ALS-associated disease proteins. The specific aim of this thesis project is to assess the possibility of using the well-established Drosophila model system to study aggregation by human SOD1 (hSOD1) mutants. To this end, using wild type and the three mutant hSOD1 (A4V, G85R and G93A) most commonly found among fALS, I have generated 16 different SOD1 constructs containing either eGFP or mCherry in-frame fluorescent reporters, established and tested both cell- and animal-based Drosophila hSOD1 models. The experimental strategy allows for clear visualization of ectopic hSOD1 expression as well as versatile co-expression schemes to fully investigate protein aggregation specifically by mutant hSOD1. I have performed pilot cell-transfection experiments and verified induced expression of hSOD1 proteins. Using several tissue- or cell type-specific Gal4 lines, I have confirmed the proper expression of hSOD1 from established transgenic fly lines. Interestingly, in both Drosophila S2 cells and different fly tissues including the eye and motor neurons, robust aggregate formation by either wild type or mutant hSOD1 proteins was not observed. These preliminary observations suggest that Drosophila might not be a good experimental organism to study aggregation and toxicity of mutant hSOD1 protein. Nevertheless this preliminary conclusion implies the potential existence of a potent protective mechanism against mutant hSOD1 aggregation and toxicity in Drosophila. Thus, results from my SOD1-ALS project in Drosophila will help future studies on how to best employ this classic model organism to study ALS and other human brain degenerative diseases.

Elevated free nitrotyrosine levels, but not protein-bound nitrotyrosine or hydroxyl radicals, throughout amyotrophic lateral sclerosis (ALS)-like disease implicate tyrosine nitration as an aberrant in vivo property of one familial ALS-linked superoxide dismutase 1 mutant

Mutations in superoxide dismutase 1 (SOD1; EC 1.15.1.1) are responsible for a proportion of familial amyotrophic lateral sclerosis (ALS) through acquisition of an as-yet-unidentified toxic property or properties. Two proposed possibilities are that toxicity may arise from imperfectly folded mutant SOD1 catalyzing the nitration of tyrosines [Beckman, J. S., Carson, M., Smith, C. D. & Koppenol, W. H. (1993) Nature (London) 364, 584] through use of peroxynitrite or from peroxidation arising from elevated production of hydroxyl radicals through use of hydrogen peroxide as a substrate [Wiedau-Pazos, M., Goto, J. J., Rabizadeh, S., Gralla, E. D., Roe, J. A., Valentine, J. S. & Bredesen, D. E. (1996) Science 271, 515–518]. To test these possibilities, levels of nitrotyrosine and markers for hydroxyl radical formation were measured in two lines of transgenic mice that develop progressive motor neuron disease from expressing human familial ALS-linked SOD1 mutation G37R. Relative to normal mice or mice expressing high levels of wild-type human SOD1, 3-nitrotyrosine levels were elevated by 2- to 3-fold in spinal cords coincident with the earliest pathological abnormalities and remained elevated in spinal cord throughout progression of disease. However, no increases in protein-bound nitrotyrosine were found during any stage of SOD1-mutant-mediated disease in mice or at end stage of sporadic or SOD1-mediated familial human ALS. When salicylate trapping of hydroxyl radicals and measurement of levels of malondialdehyde were used, there was no evidence throughout disease progression in mice for enhanced production of hydroxyl radicals or lipid peroxidation, respectively. The presence of elevated nitrotyrosine levels beginning at the earliest stages of cellular pathology and continuing throughout progression of disease demonstrates that tyrosine nitration is one in vivo aberrant property of this ALS-linked SOD1 mutant.

A neurotransmitter transporter encoded by the Drosophila inebriated gene

Behavioral and electrophysiological studies on mutants defective in the Drosophila inebriated (ine) gene demonstrated increased excitability of the motor neuron. In this paper, we describe the cloning and sequence analysis of ine. Mutations in ine were localized on cloned DNA by restriction mapping and restriction fragment length polymorphism (RFLP) mapping of ine mutants. DNA from the ine region was then used to isolate an ine cDNA. In situ hybridization of ine transcripts to developing embryos revealed expression of this gene in several cell types, including the posterior hindgut, Malpighian tubules, anal plate, garland cells, and a subset of cells in the central nervous system. The ine cDNA contains an open reading frame of 658 amino acids with a high degree of sequence similarity to members of the Na+/Cl−-dependent neurotransmitter transporter family. Members of this family catalyze the rapid reuptake of neurotransmitters released into the synapse and thereby play key roles in controlling neuronal function. We conclude that ine mutations cause increased excitability of the Drosophila motor neuron by causing the defective reuptake of the substrate neurotransmitter of the ine transporter and thus overstimulation of the motor neuron by this neurotransmitter. From this observation comes a unique opportunity to perform a genetic dissection of the regulation of excitability of the Drosophila motor neuron.

Cloning, expression, and genetic mapping of Sema W, a member of the semaphorin family

The semaphorins comprise a large family of membrane-bound and secreted proteins, some of which have been shown to function in axon guidance. We have cloned a transmembrane semaphorin, Sema W, that belongs to the class IV subgroup of the semaphorin family. The mouse and rat forms of Sema W show 97% amino acid sequence identity with each other, and each shows about 91% identity with the human form. The gene for Sema W is divided into 15 exons, up to 4 of which are absent in the human cDNAs that we sequenced. Unlike many other semaphorins, Sema W is expressed at low levels in the developing embryo but was found to be expressed at high levels in the adult central nervous system and lung. Functional studies with purified membrane fractions from COS7 cells transfected with a Sema W expression plasmid showed that Sema W has growth-cone collapse activity against retinal ganglion-cell axons, indicating that vertebrate transmembrane semaphorins, like secreted semaphorins, can collapse growth cones. Genetic mapping of human SEMAW with human/hamster radiation hybrids localized the gene to chromosome 2p13. Genetic mapping of mouse Semaw with mouse/hamster radiation hybrids localized the gene to chromosome 6, and physical mapping placed the gene on bacteria artificial chromosomes carrying microsatellite markers D6Mit70 and D6Mit189. This localization places Semaw within the locus for motor neuron degeneration 2, making it an attractive candidate gene for this disease.

A single nucleotide in the SMN gene regulates splicing and is responsible for spinal muscular atrophy

SMN1 and SMN2 (survival motor neuron) encode identical proteins. A critical question is why only the homozygous loss of SMN1, and not SMN2, results in spinal muscular atrophy (SMA). Analysis of transcripts from SMN1/SMN2 hybrid genes and a new SMN1 mutation showed a direct relationship between presence of disease and exon 7 skipping. We have reported previously that the exon-skipped product SMNΔ7 is partially defective for self-association and SMN self-oligomerization correlated with clinical severity. To evaluate systematically which of the five nucleotides that differ between SMN1 and SMN2 effect alternative splicing of exon 7, a series of SMN minigenes was engineered and transfected into cultured cells, and their transcripts were characterized. Of these nucleotide differences, the exon 7 C-to-T transition at codon 280, a translationally silent variance, was necessary and sufficient to dictate exon 7 alternative splicing. Thus, the failure of SMN2 to fully compensate for SMN1 and protect from SMA is due to a nucleotide exchange (C/T) that attenuates activity of an exonic enhancer. These findings demonstrate the molecular genetic basis for the nature and pathogenesis of SMA and illustrate a novel disease mechanism. Because individuals with SMA retain the SMN2 allele, therapy targeted at preventing exon 7 skipping could modify clinical outcome.

Role for neuronally derived fractalkine in mediating interactions between neurons and CX3CR1-expressing microglia

A recently identified chemokine, fractalkine, is a member of the chemokine gene family, which consists principally of secreted, proinflammatory molecules. Fractalkine is distinguished structurally by the presence of a CX3C motif as well as transmembrane spanning and mucin-like domains and shows atypical constitutive expression in a number of nonhematopoietic tissues, including brain. We undertook an extensive characterization of this chemokine and its receptor CX3CR1 in the brain to gain insights into use of chemokine-dependent systems in the central nervous system. Expression of fractalkine in rat brain was found to be widespread and localized principally to neurons. Recombinant rat CX3CR1, as expressed in Chinese hamster ovary cells, specifically bound fractalkine and signaled in the presence of either membrane-anchored or soluble forms of fractalkine protein. Fractalkine stimulated chemotaxis and elevated intracellular calcium levels of microglia; these responses were blocked by anti-CX3CR1 antibodies. After facial motor nerve axotomy, dramatic changes in the levels of CX3CR1 and fractalkine in the facial nucleus were evident. These included increases in the number and perineuronal location of CX3CR1-expressing microglia, decreased levels of motor neuron-expressed fractalkine mRNA, and an alteration in the forms of fractalkine protein expressed. These data describe mechanisms of cellular communication between neurons and microglia, involving fractalkine and CX3CR1, which occur in both normal and pathological states of the central nervous system.

Caspase-1 is activated in neural cells and tissue with amyotrophic lateral sclerosis-associated mutations in copper-zinc superoxide dismutase

The mechanism by which mutations in the superoxide dismutase (SOD1) gene cause motor neuron degeneration in familial amyotrophic lateral sclerosis (ALS) is unknown. Recent reports that neuronal death in SOD1-familial ALS is apoptotic have not documented activation of cell death genes. We present evidence that the enzyme caspase-1 is activated in neurons expressing mutant SOD1 protein. Proteolytic processing characteristic of caspase-1 activation is seen both in spinal cords of transgenic ALS mice and neurally differentiated neuroblastoma (line N2a) cells with SOD1 mutations. This activation of caspase-1 is enhanced by oxidative challenge (xanthine/xanthine oxidase), which triggers cleavage and secretion of the interleukin 1β converting enzyme substrate, pro-interleukin 1β, and induces apoptosis. This N2a culture system should be an instructive in vitro model for further investigation of the proapoptotic properties of mutant SOD1.