Spliceosomal component PRP-40 is a central regulator of microexon splicing.

Microexons (≤27 nt) play critical roles in nervous system development and function but create unique challenges for the splicing machinery. The mechanisms of microexon regulation are therefore of great interest. We performed a genetic screen for alternative splicing regulators in the C. elegans nervous system and identify PRP-40, a core component of the U1 snRNP. RNA-seq reveals that PRP-40 is required for inclusion of alternatively spliced, but not constitutively spliced, exons. PRP-40 is particularly required for inclusion of neuronal microexons, and our data indicate that PRP-40 is a central regulator of microexon splicing. Microexons can be relieved from PRP-40 dependence by artificially increasing exon size or reducing flanking intron size, indicating that PRP-40 is specifically required for microexons surrounded by conventionally sized introns. Knockdown of the orthologous PRPF40A in mouse neuroblastoma cells causes widespread dysregulation of microexons but not conventionally sized exons. PRP-40 regulation of neuronal microexons is therefore a widely conserved phenomenon.

Suppressing Tau Aggregation and Toxicity by an Anti-Aggregant Tau Fragment.

Tau aggregation is a hallmark of a group of neurodegenerative diseases termed Tauopathies. Reduction of aggregation-prone Tau has emerged as a promising therapeutic approach. Here, we show that an anti-aggregant Tau fragment (F3ΔKPP, residues 258-360) harboring the ΔK280 mutation and two proline substitutions (I277P & I308P) in the repeat domain can inhibit aggregation of Tau constructs in vitro, in cultured cells and in vivo in a Caenorhabditis elegans model of Tau aggregation. The Tau fragment reduced Tau-dependent cytotoxicity in a N2a cell model, suppressed the Tau-mediated neuronal dysfunction and ameliorated the defective locomotion in C. elegans. In vitro the fragment competes with full-length Tau for polyanionic aggregation inducers and thus inhibits Tau aggregation. Our combined in vitro and in vivo results suggest that the anti-aggregant Tau fragment may potentially be used to address the consequences of Tau aggregation in Tauopathies.

Glutamatergic nervous system degeneration in a C. elegans TauA152T tauopathy model involves pathways of excitotoxicity and Ca2+ dysregulation.

Mutations in the gene encoding Tau (MAPT-microtubule-associated protein tau) cause a group of neurodegenerative diseases called tauopathies. A recently identified Tau variant, p.A152T, has been reported as a risk factor for frontotemporal dementia-related disorders and Alzheimer disease. However, the mechanism for the pathologies still remain poorly understood. Transgenic Caenorhabditis elegans expressing mutant 2N4R-TauA152T (TauAT) panneuronally show locomotor defects, neurodegeneration and accelerated aging. Here we report that, in TauAT animals, the glutamatergic nervous system is at a high risk of progressive neuronal loss. We present genetic data that this loss occurs predominantly through necrosis. The neuronal loss is caused by several determinants, such as altered adenylyl cyclase (type AC9) pathway, prevalence of excitotoxicity-like conditions, aging-related factors and finally dyshomeostasis of intracellular calcium (Ca2+). The study provides novel insights into the mechanisms involved in selective loss of glutamatergic neurons in a TauAT tauopathy model which could point to new therapeutic targets.

Caenorhabditis elegans models of tauopathy.

One of the hallmarks of the tauopathies, which include the neurodegenerative disorders, such as Alzheimer disease (AD), corticobasal degeneration, frontotemporal dementia, and progressive supranuclear palsy (PSP), is the abnormal accumulation of post-translationally modified, insoluble tau. The result is a loss of neurons, decreased mental function, and complete dependence of patients on others. Aggregation of tau, which under physiologic conditions is a highly soluble protein, is thought to be central to the pathogenesis of these diseases. Indeed one of the strongest lines of evidence is the MAPT gene polymorphisms that lead to the familial forms of tauopathy. Extensive research in animal models over the years has contributed some of the most important findings regarding the pathogenesis of these diseases. Despite this, the precise molecular mechanisms that lead to abnormal tau folding, accumulation, and spreading remain unknown. Owing to the fact that most of the biochemical pathways are conserved, Caenorhabditis elegans provides an alternative approach to identify cellular mechanisms and druggable genes that operate in such disorders. Many human genes implicated in neurodegenerative diseases have counterparts in C. elegans, making it an excellent model in which to study their pathogenesis. In this article, we review some of the important findings gained from C. elegans tauopathy models.-Pir, G. J., Choudhary, B., Mandelkow, E. Caenorhabditiselegans models of tauopathy.

UNC-16/JIP3 regulates early events in synaptic vesicle protein trafficking via LRK-1/LRRK2 and AP complexes.

JIP3/UNC-16/dSYD is a MAPK-scaffolding protein with roles in protein trafficking. We show that it is present on the Golgi and is necessary for the polarized distribution of synaptic vesicle proteins (SVPs) and dendritic proteins in neurons. UNC-16 excludes Golgi enzymes from SVP transport carriers and facilitates inclusion of specific SVPs into the same transport carrier. The SVP trafficking roles of UNC-16 are mediated through LRK-1, whose localization to the Golgi is reduced in unc-16 animals. UNC-16, through LRK-1, also enables Golgi-localization of the µ-subunit of the AP-1 complex. AP1 regulates the size but not the composition of SVP transport carriers. Additionally, UNC-16 and LRK-1 through the AP-3 complex regulates the composition but not the size of the SVP transport carrier. These early biogenesis steps are essential for dependence on the synaptic vesicle motor, UNC-104 for axonal transport. Our results show that UNC-16 and its downstream effectors, LRK-1 and the AP complexes function at the Golgi and/or post-Golgi compartments to control early steps of SV biogenesis. The UNC-16 dependent steps of exclusion, inclusion and motor recruitment are critical for polarized distribution of neuronal cargo.

Tau mutant A152T, a risk factor for FTD/PSP, induces neuronal dysfunction and reduced lifespan independently of aggregation in a C. elegans Tauopathy model.

BACKGROUND: A certain number of mutations in the Microtubule-Associated Protein Tau (MAPT) gene have been identified in individuals with high risk to develop neurodegenerative diseases, collectively called tauopathies. The mutation A152TMAPT was recently identified in patients diagnosed with frontotemporal spectrum disorders, including Progressive Supranuclear Palsy (PSP), Frontotemporal Dementia (FTD), Corticobasal Degeneration (CBD), and Alzheimer disease (AD). The A152TMAPT mutation is unusual since it lies within the N-terminal region of Tau protein, far outside the repeat domain that is responsible for physiological Tau-microtubule interactions and pathological Tau aggregation. How A152TMAPT causes neurodegeneration remains elusive. RESULTS: To understand the pathological consequences of this mutation, here we present a new Caenorhabditis elegans model expressing the mutant A152TMAPT in neurons. While expression of full-length wild-type human tau (Tau(wt), 2N4R) in C. elegans neurons induces a progressive mild uncoordinated locomotion in a dose-dependent manner, mutant tau (Tau(A152T), 2N4R) induces a severe paralysis accompanied by acute neuronal dysfunction. Mutant Tau(A152T) worms display morphological changes in neurons reminiscent of neuronal aging and a shortened life-span. Moreover, mutant A152T overexpressing neurons show mislocalization of pre-synaptic proteins as well as distorted mitochondrial distribution and trafficking. Strikingly, mutant tau-transgenic worms do not accumulate insoluble tau aggregates, although soluble oligomeric tau was detected. In addition, the full-length A152T-tau remains in a pathological conformation that accounts for its toxicity. Moreover, the N-terminal region of tau is not toxic per se, despite the fact that it harbours the A152T mutation, but requires the C-terminal region including the repeat domain to move into the neuronal processes in order to execute the pathology. CONCLUSION: In summary, we show that the mutant Tau(A152T) induces neuronal dysfunction, morphological alterations in neurons akin to aging phenotype and reduced life-span independently of aggregation. This comprehensive description of the pathology due to Tau(A152T) opens up multiple possibilities to identify cellular targets involved in the Tau-dependent pathology for a potential therapeutic intervention.

The Caenorhabditis elegans JIP3 protein UNC-16 functions as an adaptor to link kinesin-1 with cytoplasmic dynein.

Kinesin-1 is a microtubule plus-end-directed motor that transports various cargos along the axon. Previous studies have elucidated the physical and genetic interactions between kinesin-1 and cytoplasmic dynein, a microtubule minus-end-directed motor, in neuronal cells. However, the physiological importance of kinesin-1 in the dynein-dependent retrograde transport of cargo molecules remains obscure. Here, we show that Caenorhabditis elegans kinesin-1 forms a complex with dynein via its interaction with UNC-16, which binds to the dynein light intermediate (DLI) chain. Both kinesin-1 and UNC-16 are required for localization of DLI-1 at the plus ends of nerve process microtubules. In addition, retrograde transport of APL-1 depends on kinesin-1, UNC-16, and dynein. These results suggest that kinesin-1 mediates the anterograde transport of dynein using UNC-16 as a scaffold and that dynein in turn mediates the retrograde transport of cargo molecules in vivo. Thus, UNC-16 functions as an adaptor for kinesin-1-mediated transport of dynein.

The Caenorhabditis elegans Kinesin-3 motor UNC-104/KIF1A is degraded upon loss of specific binding to cargo.

UNC-104/KIF1A is a Kinesin-3 motor that transports synaptic vesicles from the cell body towards the synapse by binding to PI(4,5)P(2) through its PH domain. The fate of the motor upon reaching the synapse is not known. We found that wild-type UNC-104 is degraded at synaptic regions through the ubiquitin pathway and is not retrogradely transported back to the cell body. As a possible means to regulate the motor, we tested the effect of cargo binding on UNC-104 levels. The unc-104(e1265) allele carries a point mutation (D1497N) in the PI(4,5)P(2) binding pocket of the PH domain, resulting in greatly reduced preferential binding to PI(4,5)P(2)in vitro and presence of very few motors on pre-synaptic vesicles in vivo. unc-104(e1265) animals have poor locomotion irrespective of in vivo PI(4,5)P(2) levels due to reduced anterograde transport. Moreover, they show highly reduced levels of UNC-104 in vivo. To confirm that loss of cargo binding specificity reduces motor levels, we isolated two intragenic suppressors with compensatory mutations within the PH domain. These show partial restoration of in vitro preferential PI(4,5)P(2) binding and presence of more motors on pre-synaptic vesicles in vivo. These animals show improved locomotion dependent on in vivo PI(4,5)P(2) levels, increased anterograde transport, and partial restoration of UNC-104 protein levels in vivo. For further proof, we mutated a conserved residue in one suppressor background. The PH domain in this triple mutant lacked in vitro PI(4,5)P(2) binding specificity, and the animals again showed locomotory defects and reduced motor levels. All allelic variants show increased UNC-104 levels upon blocking the ubiquitin pathway. These data show that inability to bind cargo can target motors for degradation. In view of the observed degradation of the motor in synaptic regions, this further suggests that UNC-104 may get degraded at synapses upon release of cargo.