In vivo Validation of Bimolecular Fluorescence Complementation (BiFC) to Investigate Aggregate Formation in Amyotrophic Lateral Sclerosis (ALS).

Amyotrophic lateral sclerosis (ALS) is a form of motor neuron disease (MND) that is characterized by the progressive loss of motor neurons within the spinal cord, brainstem, and motor cortex. Although ALS clinically manifests as a heterogeneous disease, with varying disease onset and survival, a unifying feature is the presence of ubiquitinated cytoplasmic protein inclusion aggregates containing TDP-43. However, the precise mechanisms linking protein inclusions and aggregation to neuronal loss are currently poorly understood. Bimolecular fluorescence complementation (BiFC) takes advantage of the association of fluorophore fragments (non-fluorescent on their own) that are attached to an aggregation-prone protein of interest. Interaction of the proteins of interest allows for the fluorescent reporter protein to fold into its native state and emit a fluorescent signal. Here, we combined the power of BiFC with the advantages of the zebrafish system to validate, optimize, and visualize the formation of ALS-linked aggregates in real time in a vertebrate model. We further provide in vivo validation of the selectivity of this technique and demonstrate reduced spontaneous self-assembly of the non-fluorescent fragments in vivo by introducing a fluorophore mutation. Additionally, we report preliminary findings on the dynamic aggregation of the ALS-linked hallmark proteins Fus and TDP-43 in their corresponding nuclear and cytoplasmic compartments using BiFC. Overall, our data demonstrates the suitability of this BiFC approach to study and characterize ALS-linked aggregate formation in vivo. Importantly, the same principle can be applied in the context of other neurodegenerative diseases and has therefore critical implications to advance our understanding of pathologies that underlie aberrant protein aggregation.

Identification of a localized nonsense-mediated decay pathway at the endoplasmic reticulum.

Nonsense-mediated decay (NMD) is a translation-dependent RNA quality control mechanism that occurs in the cytoplasm. However, it is unknown how NMD regulates the stability of RNAs translated at the endoplasmic reticulum (ER). Here, we identify a localized NMD pathway dedicated to ER-translated mRNAs. We previously identified NBAS, a component of the Syntaxin 18 complex involved in Golgi-to-ER trafficking, as a novel NMD factor. Furthermore, we show that NBAS fulfills an independent function in NMD. This ER-NMD pathway requires the interaction of NBAS with the core NMD factor UPF1, which is partially localized at the ER in the proximity of the translocon. NBAS and UPF1 coregulate the stability of ER-associated transcripts, in particular those associated with the cellular stress response. We propose a model where NBAS recruits UPF1 to the membrane of the ER and activates an ER-dedicated NMD pathway, thus providing an ER-protective function by ensuring quality control of ER-translated mRNAs.

Of numbers and movement - understanding transcription factor pathogenesis by advanced microscopy.

Transcription factors (TFs) are life-sustaining and, therefore, the subject of intensive research. By regulating gene expression, TFs control a plethora of developmental and physiological processes, and their abnormal function commonly leads to various developmental defects and diseases in humans. Normal TF function often depends on gene dosage, which can be altered by copy-number variation or loss-of-function mutations. This explains why TF haploinsufficiency (HI) can lead to disease. Since aberrant TF numbers frequently result in pathogenic abnormalities of gene expression, quantitative analyses of TFs are a priority in the field. In vitro single-molecule methodologies have significantly aided the identification of links between TF gene dosage and transcriptional outcomes. Additionally, advances in quantitative microscopy have contributed mechanistic insights into normal and aberrant TF function. However, to understand TF biology, TF-chromatin interactions must be characterised in vivo, in a tissue-specific manner and in the context of both normal and altered TF numbers. Here, we summarise the advanced microscopy methodologies most frequently used to link TF abundance to function and dissect the molecular mechanisms underlying TF HIs. Increased application of advanced single-molecule and super-resolution microscopy modalities will improve our understanding of how TF HIs drive disease.

A Tol2 Gateway-Compatible Toolbox for the Study of the Nervous System and Neurodegenerative Disease.

Currently there is a lack in fundamental understanding of disease progression of most neurodegenerative diseases, and, therefore, treatments and preventative measures are limited. Consequently, there is a great need for adaptable, yet robust model systems to both investigate elementary disease mechanisms and discover effective therapeutics. We have generated a Tol2 Gateway-compatible toolbox to study neurodegenerative disorders in zebrafish, which includes promoters for astrocytes, microglia and motor neurons, multiple fluorophores, and compatibility for the introduction of genes of interest or disease-linked genes. This toolbox will advance the rapid and flexible generation of zebrafish models to discover the biology of the nervous system and the disease processes that lead to neurodegeneration.