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Pathogenic mutations within the TBK1 gene leading to haploinsufficiency are causative of amyotrophic lateral sclerosis (ALS). This gene is linked to autophagy and inflammation, two cellular mechanisms reported to be dysregulated in ALS patients, although its functional role in the pathogenesis could involve other players. We targeted the TBK1 ortholog in zebrafish, an optimal vertebrate model for investigating genetic defects in neurological disorders. We generated zebrafish models with invalidating tbk1 mutations using CRISPR-Cas9 or tbk1 knockdown models using antisense morpholino oligonucleotide (AMO). The early motor phenotype of zebrafish injected with tbk1 AMO beginning at 2 days post fertilization (dpf) is associated with the degeneration of motor neurons. In parallel, CRISPR-induced tbk1 mutants exhibit impaired motor function beginning at 5 dpf and increased lethality beginning at 9 dpf. A metabolomic analysis showed an association between tbk1 loss and severe dysregulation of nicotinamide metabolism, and incubation with nicotinamide riboside rescued the motor behavior of tbk1 mutant zebrafish. Furthermore, a proteomic analysis revealed increased levels of inflammatory markers and dysregulation of programmed cell death pathways. Necroptosis appeared to be strongly activated in TBK1 fish, and larvae treated with the necroptosis inhibitor necrosulfonamide exhibited improved survival. Finally, a combined analysis of mutant zebrafish and TBK1-mutant human motor neurons revealed dysregulation of the KEGG pathway ¡°ALS¡±, with disrupted nuclear-cytoplasmic transport and increased expression of STAT1. These findings point toward a major role for necroptosis in the degenerative features and premature lethality observed in tbk1 mutant zebrafish. Overall, the novel tbk1-deficient zebrafish models offer a great opportunity to better understand the cascade of events leading from the loss of tbk1 expression to the onset of motor deficits, with involvement of a metabolic defect and increased cell death, and for the development of novel therapeutic avenues for ALS and related neuromuscular diseases.

GABA B receptor (GABA B R) activation is known to alleviate pain by reducing neuronal excitability, primarily through inhibition of high voltage©\activated (HVA) calcium (Ca V 2.2) channels and potentiating G protein¨Ccoupled inwardly rectifying potassium (GIRK) channels. Although the analgesic properties of small molecules and peptides have been primarily tested on isolated murine dorsal root ganglion (DRG) neurons, emerging strategies to develop, study, and characterise human pluripotent stem cell (hPSC)©\derived sensory neurons present a promising alternative. In this study, hPSCs were efficiently differentiated into peripheral DRG©\induced sensory neurons (iSNs) using a combined chemical and transcription factor©\driven approach via a neural crest cell intermediate. Molecular characterisation and transcriptomic analysis confirmed the expression of key DRG markers such as BRN3A, ISLET1, and PRPH, in addition to GABA B R and ion channels including Ca V 2.2 and GIRK1 in iSNs. Functional characterisation of GABA B R was conducted using whole©\cell patch clamp electrophysiology, assessing neuronal excitability under current©\clamp conditions in the absence and presence of GABA B R agonists baclofen and ¦Á©\conotoxin Vc1.1. Both baclofen (100?¦ÌM) and Vc1.1 (1?¦ÌM) significantly reduced membrane excitability by hyperpolarising the resting membrane potential and increasing the rheobase for action potential firing. In voltage©\clamp mode, baclofen and Vc1.1 inhibited HVA Ca 2+ channel currents, which were attenuated by the selective GABA B R antagonist CGP 55845. However, modulation of GIRK channels by GABA B Rs was not observed in the presence of baclofen or Vc1.1, suggesting that functional GIRK1/2 channels were not coupled to GABA B Rs in hPSC©\derived iSNs. This study is the first to report GABA B R modulation of membrane excitability in iSNs by baclofen and Vc1.1, highlighting their potential as a future model for studying analgesic compounds.

Alzheimer¡¯s disease (AD) is a devastating neurodegenerative condition that affects memory and cognition, characterized by neuronal loss and currently lacking a cure. Mutations in PSEN1 (Presenilin 1) are among the most common causes of early-onset familial AD (fAD). While changes in neuronal excitability are believed to be early indicators of AD progression, the link between PSEN1 mutations and neuronal excitability remains to be fully elucidated. This study examined iPSC-derived neurons (iNs) from fAD patients with PSEN1 mutations S290C or A246E, alongside CRISPR-corrected isogenic cell lines, to investigate early changes in excitability. Electrophysiological profiling revealed reduced excitability in both PSEN1 mutant iNs compared to their isogenic controls. Neurons bearing S290C and A246E mutations exhibited divergent passive membrane properties compared to isogenic controls, suggesting distinct effects of PSEN1 mutations on neuronal excitability. Additionally, both PSEN1 backgrounds exhibited higher current density of voltage-gated potassium (Kv) channels relative to their isogenic iNs, while displaying comparable voltage-gated sodium (Nav) channel current density. This suggests that the Nav/Kv imbalance contributes to impaired neuronal firing in fAD iNs. Deciphering these early cellular and molecular changes in AD is crucial for understanding disease pathogenesis.