Precisely measured protein lifetimes in the mouse brain reveal differences across tissues and subcellular fractions

Fornasiero, Eugenio F.; Mandad, Sunit; Wildhagen, Hanna; Alevra, Mihai; Rammner, Burkhard; Keihani, Sarva; Opazo, Felipe; Urban, Inga; Ischebeck, Till; Sakib, M. Sadman; Fard, Maryam K.; Kırlı, Koray; Centeno, Tonatiuh Peña; Vidal, Ramón; Rahman, Raza-Ur; Benito, Eva; Fischer, André; Dennerlein, Sven; Rehling, Peter; Feußner, Ivo; Bonn, Stefan; Simons, Mikael; Urlaub, Henning; Rizzoli, Silvio O.

doi:10.1038/s41467-018-06519-0

Cited by 285 publications

(383 citation statements)

References 62 publications

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“…Moreover, three principles govern the replacement of lost OLs and the parallel emergence of precise and profuse remyelination: First, the trigger of OL differentiation is not the death of the ablated OL itself (which was cleared within 24 hours of ablation), but rather the following demyelination, which was delayed by as many as 50 days (average 32 days). This is in line with other OL ablation paradigms, where myelin-which also on the protein level has an extremely long half-life 55 -can survive long after OLs have died 43 . In our experiments, we observed that the OL processes resealed rapidly after cell body ablation ( Supplementary Fig.…”

Section: Discussionsupporting

confidence: 87%

Precision in a sea of profusion: Myelin replacement triggered by single-cell cortical demyelination

Snaidero

Schifferer

Mężydło

et al. 2019

Preprint

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Myelin-rather than being a static insulator of axons-is emerging as an active participant in circuit plasticity. This requires precise regulation of oligodendrocyte numbers and myelination patterns. Here, by devising a laser ablation approach of single oligodendrocytes, followed by in vivo imaging and correlated ultrastructural reconstruction, we show that in mouse cortex demyelination as subtle as loss of a single oligodendrocyte can trigger robust cell replacement and remyelination timed by myelin breakdown. This results in reliable reestablishment of the original myelin pattern along continuously myelinated axons, while in parallel profuse isolated internodes emerge on previously unmyelinated axons. Thus, in mammalian cortex, internodes along partially myelinated cortical axons are typically not re-established, suggesting that the cues that guide 'patchy' myelination are not preserved through cycles of deand remyelination. In contrast, continuous 'obligatory' myelin shows remarkable homeostatic resilience with single axon precision.

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Section: Discussionsupporting

confidence: 87%

Precision in a sea of profusion: Myelin replacement triggered by single-cell cortical demyelination

Snaidero

Schifferer

Mężydło

et al. 2019

Preprint

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“…Because ELKS levels were significantly reduced at the P7 calyx and undetectable at P9, in contrast to published data in other brain areas that reported a ELKS half-life in the range of 11-13 days (Fornasiero et al 2018), our data indicate that at the calyx of Held ELKS has a half-life of a few days (ß3-4 days). These discrepancies can be explained by the facts that our measurements were performed with an IHC method that could result in some amount of the signal not being detected, and also protein half-lives can differ in the various brain regions (Fornasiero et al 2018) and different cellular environments (Dorrbaum et al 2018) (i.e. in vitro vs. in vivo).…”

Section: A Developmental Role For Cast/elks In Presynaptic Growth and Azcontrasting

confidence: 96%

“…In summary, our results in immature calyx of Held show that CAST is the dominant isoform and ELKS levels are increased in the presynaptic terminal in the absence of CAST. Because ELKS levels are dramatically reduced by P7 with undetectable levels by P9, this indicates that its half-life in the calyx of Held is significantly shorter than previous reports in other brain areas of 11-13 days (Fornasiero et al 2018), and allows analysis of CAST/ELKS function in the immature calyx from P7 onwards.…”

Section: Ablation Of Cast In Calyx Of Held Leads To Increased Levelsmentioning

confidence: 63%

“…Although CAST/ELKS levels are characterized in the mature calyx, from P16 onwards (Dong et al 2018), in the immature calyx of Held CAST/ELKS levels were unknown. Furthermore, it was unknown how early ELKS could be ablated in vivo as it is reported that in in vitro cultured neurons CAST/ELKS proteins have a half-life of ß11-13 days (Fornasiero et al 2018). Therefore, we first performed immunohistochemistry (IHC) using antibodies specific for CAST and ELKS, and VGLUT1 on immature, prehearing calyces (P9) in Cast +/+ /Elks +/+ and Cast −/− /Elks +/+ terminals.…”

Section: Ablation Of Cast In Calyx Of Held Leads To Increased Levelsmentioning

confidence: 99%

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Presynaptic development is controlled by the core active zone proteins CAST/ELKS

Radulović

Dong

Goral

et al. 2020

The Journal of Physiology

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Key points CAST/ELKS are positive regulators of presynaptic growth and are suppressors of active zone expansion at the developing mouse calyx of Held. CAST/ELKS regulate all three CaV2 subtype channel levels in the presynaptic terminal and not just CaV2.1. The half‐life of ELKS is on the timescale of days and not weeks. Synaptic transmission was not impacted by the loss of CAST/ELKS. CAST/ELKS are involved in pathways regulating morphological properties of presynaptic terminals during an early stage of circuit maturation. Abstract Many presynaptic active zone (AZ) proteins have multiple regulatory roles that vary during distinct stages of neuronal circuit development. The CAST/ELKS protein family are evolutionarily conserved presynaptic AZ molecules that regulate presynaptic calcium channels, synaptic transmission and plasticity in the mammalian CNS. However, how these proteins regulate synapse development and presynaptic function in a developing neuronal circuit in its native environment is unclear. To unravel the roles of CAST/ELKS in glutamatergic synapse development and in presynaptic function, we used CAST knockout (KO) and ELKS conditional KO (CKO) mice to examine how their loss during the early stages of circuit maturation impacted the calyx of Held presynaptic terminal development and function. Morphological analysis from confocal z‐stacks revealed that combined deletion of CAST/ELKS resulted in a reduction in the surface area and volume of the calyx. Analysis of AZ ultrastructure showed that AZ size was increased in the absence of CAST/ELKS. Patch clamp recordings demonstrated a reduction of all presynaptic CaV2 channel subtype currents that correlated with a loss in presynaptic CaV2 channel numbers. However, these changes did not impair synaptic transmission and plasticity and synaptic vesicle release kinetics. We conclude that CAST/ELKS proteins are positive regulators of presynaptic growth and are suppressors of AZ expansion and CaV2 subtype currents and levels during calyx of Held development. We propose that CAST/ELKS are involved in pathways regulating presynaptic morphological properties and CaV2 channel subtypes and suggest there is developmental compensation to preserve synaptic transmission during early stages of neuronal circuit maturation.

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“…The che-1 mRNA and protein half-lives are short compared to reported mRNA halflives in eukaryotes (Fornasiero et al 2018;Sharova et al 2009) and average protein halflives reported in young C. elegans adults (Dhondt et al 2017;Schwanhausser et al 2011).…”

Section: Copy Number and Lifetime Of Che-1 Mrna And Proteinmentioning

confidence: 76%

Mechanism of life-long maintenance of neuron identity despite molecular fluctuations

Traets

Burght

Jansen

et al. 2020

Preprint

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Cell fate is maintained over long timescales, yet molecular fluctuations can lead to spontaneous loss of this differentiated state. We uncovered a mechanism that explains lifelong maintenance of ASE neuron fate in C. elegans by the terminal selector transcription factor CHE-1. Fluctuations in CHE-1 level are buffered by the reservoir of CHE-1 bound at its target promoters, which ensure continued che-1 expression by preferentially binding the che-1 promoter. We validated this mechanism by showing that che-1 expression was resilient to induced transient CHE-1 depletion, while both expression of CHE-1 targets and ASE function were lost. We identified a 130 bp che-1 promoter fragment responsible for this resilience, with deletion of a homeodomain binding site in this fragment causing stochastic loss of ASE identity long after its determination. Because network architectures that support this mechanism are highly conserved in cell differentiation, it may explain stable cell fate maintenance in many systems. KeywordsNeuronal cell fate, bistability, gene regulatory network, stochastic gene expression, molecular fluctuations, homeodomain proteins, chemotaxis, C. elegans, terminal selector CHE-1, a transcription factor whose expression is transiently induced by the nuclear hormone receptor NHR-67 at the time of determination (Sarin et al. 2009). CHE-1 induces the expression of 500-1000 ASE-specific target genes, such as chemosensory receptors, ion-channels, and neuropeptides, by binding ASE motifs within their promoters (Etchberger et al. 2007). Its continued presence is required for expression of target genes after subtype determination (Etchberger et al. 2009). CHE-1 also upregulates its own expression. This positive feedback loop is necessary for sustaining che-1 expression and ASE cell fate directly after cell determination (Etchberger et al. 2007; Leyva-Diaz and Hobert 2019). Yet, it is unknown whether this positive feedback loop is sufficient for long-term maintenance of ASE fate. The impact of molecular noise, such as variability in CHE-1 protein copy number, on ASE fate maintenance has not been studied. Overall, it is an open question whether a reversible, bistable switch based on positive CHE-1 autoregulation would be sufficiently stable to maintain ASE fate for the animal's lifetime, or if additional mechanisms are necessary to ensure that, once ASE fate is determined, che-1 expression becomes independent of CHE-1 itself and can no longer spontaneously switch off.We show that sufficiently long, transiently induced depletion of CHE-1 caused permanent loss of ASE fate, indicating that it is controlled by a switch that remains reversible long after specification. This raises the question how the switch is protected against molecular noise, which could cause it to spontaneously lose ASE fate. Combining experimental measurements of the key parameters that control the magnitude of noise, i.e. the copy numbers and lifetimes of che-1 mRNA and protein, with stochastic models of the che-1 genetic network, revealed a novel...

show abstract

Precisely measured protein lifetimes in the mouse brain reveal differences across tissues and subcellular fractions

Cited by 285 publications

References 62 publications

Precision in a sea of profusion: Myelin replacement triggered by single-cell cortical demyelination

Precision in a sea of profusion: Myelin replacement triggered by single-cell cortical demyelination

Presynaptic development is controlled by the core active zone proteins CAST/ELKS

Mechanism of life-long maintenance of neuron identity despite molecular fluctuations

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