Biology subjects

Toonen, R. F.

Publications and source records attributed to Toonen, R. F..

9 recordsLinked to original sources

bioRxiv2025

One-step induction of human GABAergic neurons promotes presynaptic development & synapse maturation

Human induced pluripotent stem cells (iPSCs) present a powerful approach to study human brain physiology and disease, yet robust, pure GABAergic induction has remained difficult. Here we present improved, single-step, transposon-based GABAergic induction with Ascl1/Dlx2, which yields, unlike lentiviral approaches, exclusively GABAergic neurons and was validated across three independent iPSC lines. Co-seeding with Ngn2-induced excitatory neurons created stable networks of predefined excitation/inhibition ratios, with corresponding synapse ratios. Proteomic and electrophysiological characterization at different developmental time points showed that the single-step induced GABAergic neurons gain a proteomic profile that maps to different cortical interneuron subtypes and display typical GABAergic synaptic properties, producing large, synchronous and picrotoxin-sensitive currents. During early development, synaptic strength increased threefold, which was accompanied by an increase in expression of proteins exclusively enriched for presynaptic SYNGO terms. Synaptic strength continued to increase during late development but with only minor proteomic changes. Taken together, transposon-based GABAergic induction yields exclusively mature GABAergic neurons suitable for studying genes involved in synaptic maturation and to build excitation/inhibition networks for disease modelling.

neuroscience↗

bioRxiv2024

ER Ca2+-levels control neuromodulator secretion by regulating STIM1 and L-type Ca2+-channel activity

AbstractRegulated secretion is typically triggered by (local) increases in intracellular Ca2+, but the source of Ca2+, influx through voltage gated Ca2+ channels or release from the endoplasmic reticulum (ER), has distinct effects, particularly for neuropeptide secretion from dense-core vesicles (DCVs). Here, we show that in primary mouse neurons acute ER Ca2+ depletion by caffeine, cyclopiazonic acid or thapsigargin resulted in minute increases in bulk cytosolic free Ca2+ ([Ca2+]bulk) that did not trigger significant DCV exocytosis. Remarkably, following acute ER Ca2+ depletion, action potential (AP) trains triggered 50-90% less DCV exocytosis as compared to naive neurons. In contrast, synaptic vesicle (SV) exocytosis was similar with/without acute ER Ca2+ depletion. Unexpectedly, acute ER Ca2+ depletion also reduced AP-induced [Ca2+]bulk-increases. L-type Ca2+-channel inhibitor nimodipine produced similar effects: reduced [Ca2+]bulk-increase, DCV-exocytosis but not SV exocytosis, i.e., for all three parameters a phenocopy of ER depletion. Finally, introducing L-type channels lacking STIM1 interaction sites restored DCV exocytosis following ER store depletion. We conclude that in mouse neurons, acute ER Ca2+ release is not effective in releasing neuromodulators. Instead, ER depletion activates a STIM1-dependent negative feedback loop that inhibits L-type Ca2+ channel activity, essential for DCV- but not SV-exocytosis.

neuroscience↗

bioRxiv2024

KIF5A regulates axonal repair and time-dependent axonal transport of SFPQ granules and mitochondria in human motor neurons

Mutations in the microtubule binding motor protein, kinesin family member 5A (KIF5A), cause the fatal motor neuron disease, Amyotrophic Lateral Sclerosis. While KIF5 family members transport a variety of cargos along axons, it is still unclear which cargos are affected by KIF5A mutations. We generated KIF5A null mutant human motor neurons to investigate the impact of KIF5A loss on the transport of various cargoes and its effect on motor neuron function at two different timepoints in vitro. The absence of KIF5A resulted in reduced neurite complexity in young motor neurons (DIV14) and significant defects in axonal regeneration capacity at all developmental stages. KIF5A loss did not affect neurofilament transport but resulted in decreased mitochondria motility and anterograde speed at DIV42. More prominently, KIF5A depletion strongly reduced anterograde transport of SFPQ-associated RNA granules in DIV42 motor neuron axons. We conclude that KIF5A most prominently functions in human motor neurons to promote axonal regrowth after injury as well as to anterogradely transport mitochondria and, to a larger extent, SFPQ-associated RNA granules in a time-dependent manner.

neuroscience↗

bioRxiv2024

Rabphilin-3A negatively regulates neuropeptide release, through its SNAP25 interaction

Neuropeptides and neurotrophins are stored in and released from dense-core vesicles (DCVs). While DCVs and synaptic vesicles (SVs) share fundamental SNARE/SM proteins for exocytosis, a detailed understanding of DCV exocytosis remains elusive. We recently identified the RAB3-RIM1 pathway to be essential for DCV-, but not SV exocytosis, highlighting a significant distinction between the SV- and DCV secretory pathways. Whether RIM1 is the only RAB3 effector that is essential for DCV exocytosis is currently unknown. In this study, we show that rabphilin-3A (RPH3A), a known downstream effector of RAB3A, is a negative regulator of DCV exocytosis. Using live-cell imaging at single vesicle resolution with RPH3A-deficient hippocampal neurons, we show that DCV exocytosis increased 3-fold in the absence of RPH3A. RAB3A-binding deficient RPH3A lost its punctate distribution, but still restored DCV exocytosis to WT levels when re-expressed. SNAP25-binding deficient RPH3A did not rescue DCV exocytosis. In addition, we show that RPH3A did not travel with DCVs, but remained stationary at pre-synapses. RPH3A null neurons also had longer neurites, which was partly restored when ablating all regulated secretion with tetanus neurotoxin. Taken together, these results show that RPH3A negatively regulates DCV exocytosis, potentially also affecting neuron size. Furthermore, RAB3A interaction is required for the synaptic enrichment of RPH3A, but not for limiting DCV exocytosis. Instead, the interaction of RPH3A with SNAP25 is relevant for inhibiting DCV exocytosis.

neuroscience↗

bioRxiv2024

Rab10 regulates neuropeptide release by maintaining Ca2+ homeostasis and protein synthesis

Dense core vesicles (DCVs) transport and release various neuropeptides and neurotrophins that control diverse brain functions, but the DCV secretory pathway remains poorly understood. Here, we tested a prediction emerging from invertebrate studies about the crucial role of the intracellular trafficking GTPase Rab10, by assessing DCV exocytosis at single-cell resolution upon acute Rab10 depletion in mature mouse hippocampal neurons, to circumvent potential confounding effects of Rab10s established role in neurite outgrowth. We observed a significant inhibition of DCV exocytosis in Rab10-depleted neurons, whereas synaptic vesicle exocytosis was unaffected. However, rather than a direct involvement in DCV trafficking, this effect was attributed to two ER-dependent processes, ER-regulated intracellular Ca2+ dynamics and protein synthesis. Gene ontology analysis of differentially expressed proteins upon Rab10 depletion identified substantial alterations in synaptic and ER/ribosomal proteins, including the Ca2+-pump SERCA2. In addition, ER morphology and dynamics were altered, ER Ca2+ levels were depleted and Ca2+ homeostasis was impaired in Rab10-depleted neurons. However, Ca2+ entry using a Ca2+ ionophore still triggered less DCV exocytosis. Instead, leucine supplementation, which enhances protein synthesis, largely rescued DCV exocytosis deficiency. We conclude that Rab10 is required for neuropeptide release by maintaining Ca2+ dynamics and regulating protein synthesis. Furthermore, DCV exocytosis appeared more dependent on (acute) protein synthesis than synaptic vesicle exocytosis.

neuroscience↗

bioRxiv2022

SNARE protein tomosyn regulates dense core vesicle composition but not exocytosis in mammalian neurons

Tomosyn is a large, non-canonical SNARE protein proposed to act as a competitive inhibitor of SNARE complex formation in vesicle exocytosis. In the brain, tomosyn inhibits fusion of synaptic vesicles (SVs), whereas its role in the fusion of neuropeptide-containing dense core vesicles (DCVs) is unknown. Here, we addressed this question using a new mouse model allowing conditional deletion of tomosyn (Stxbp5) and its paralogue tomosyn-2 (Stxbp5l), and an assay that detects DCV exocytosis with single vesicle resolution in primary hippocampal neurons. Surprisingly, loss of both tomosyns did not affect DCV exocytosis but resulted in a strong reduction of intracellular levels of many DCV cargos, most prominently brain-derived neurotrophic factor (BDNF), granin VGF and prohormone convertase PCSK1. Reduced levels of DCV cargos were paralleled by decreased DCV size and impaired mRNA expression of the corresponding genes. We conclude that tomosyns regulate neuropeptide and neurotrophin secretion via control of DCV cargo production, and not at the step of cargo release. Our findings suggest a differential effect of tomosyn on the two main secretory pathways in mammalian neurons and argues against a conserved role of tomosyn as competitive inhibitor of SNARE complex formation.

neuroscience↗

bioRxiv2022

Mapping localization of 21 endogenous proteins in the Golgi apparatus of rodent neurons

The Golgi apparatus is the major sorting hub in the secretory pathway and particularly important for protein sorting in neurons. Knowledge about protein localization in Golgi compartments is largely based on work in cell lines. Here, we systematically compared protein localization of 21 endogenous proteins in the Golgi apparatus of mouse neurons using confocal microscopy and line scan analysis. We localized these proteins by measuring the distance relative to the canonical TGN marker TGN38. Based on this, proteins fell into three groups: upstream of, overlapping with or downstream of TGN38. Seven proteins showed complete overlap with TGN38, while proteins downstream of TGN38 were located at varying distances from TGN38. Proteins upstream of TGN38 were localized in between TGN38 and the cis-/medial Golgi markers Giantin and GM130. This localization was consistent with protein function. Our data provide an overview of the relative localization of endogenous proteins in the Golgi of primary mouse neurons.

cell biology↗

bioRxiv2022

Differential axonal trafficking of Neuropeptide Y-, LAMP1- and RAB7-tagged organelles in vivo

Different organelles traveling through neurons exhibit distinct properties in vitro, but this has not been investigated in the intact brain. We established simultaneous dual color 2- photon microscopy to visualize the trafficking of Neuropeptide Y-, LAMP1- and RAB7- tagged organelles in thalamocortical axons imaged in mouse cortex in vivo. This revealed that LAMP1- and RAB7-tagged organelles move significantly faster than NPY tagged organelles in both anterograde and retrograde direction. NPY travelled more selectively in anterograde direction than LAMP1 and RAB7. By using a synapse marker and a calcium sensor we further investigated the transport dynamics of NPY-tagged organelles. We found that these organelles slow down and pause at synapses. In contrast to previous in vitro studies, a significant increase of transport speed was observed after spontaneous activity and elevated calcium levels in vivo as well as electrically stimulated activity in acute brain slices. Together, we show a remarkable diversity in speeds and properties of three axonal organelle marker in vivo that differ from properties previously observed in vitro.

neuroscience↗

bioRxiv2022

Vti1a/b support distinct aspects of TGN and cis-/medial Golgi organization

Retrograde trafficking towards the trans-Golgi network (TGN) is important for dense core vesicle (DCV) biogenesis. Here, we used Vti1a/b deficient neurons to study the impact of disturbed retrograde trafficking on Golgi organization and cargo sorting. In Vti1a/b deficient neurons, staining intensity of cis-/medial Golgi proteins (e.g., GM130 and giantin) was increased, while intensity of TGN-resident proteins (e.g., TGN38 and TMEM87A) was decreased. Levels and localization of DCV cargo markers and LAMP1 were altered. This phenotype was not caused by reduced Golgi membrane availability or absence of the TGN compartment. The phenotype was partially phenocopied by disturbing sphingolipid homeostasis, but was not rescued by overexpression of sphingomyelin synthases or the sphingolipid synthesis inhibitor myriocin. We conclude that Vti1a/b are important for distinct aspects of TGN and cis-/medial Golgi organization. Our data underline the importance of retrograde trafficking for Golgi organization, DCV cargo sorting and the distribution of proteins of the regulated secretory pathway.

cell biology↗