Homeostatic scaling is dependent on RIM and its phosphorylation state

As seen before homeostatic scaling cannot be induced when SRPK2 is overexpressed in neurons.

Therefore, we wanted to know, whether phosphorylation sites in RIM1α that were important for the SRPK2 OE effect (increased synaptic release probability) influence the ability of the presynaptic com-partment for homeostatic scaling. We applied 1 µM TTX for 48 hours to RIM1/2 cDKO neurons, that were previously transduced with iGluSnFR and different GFP-RIM1αmutants. We measured presy-naptic homeostatic scaling as before by estimation of∆F/F amplitudes from untreated and TTX treated neurons.

First of all, homeostatic scaling can not be induced in RIM1/2 cDKO neurons (Figure 5.34 A). This is in line with observations from Drosophila, where presynaptic homeostatic scaling in the neuromuscular junction is dependent on functional RIM [Müller et al., 2012]. Next, we tested whether homeostatic scaling can be induced in RIM1/2 cDKO transduced with GFP-RIM1α(S991E). This mutant had the same effect on the synaptic release probability as SRPK2 OE (see Section 5.9.2). Interestingly, we did not see a clear scaling effect, but comparable∆F/F values before and after TTX treatment (Figure 5.34 B). Finally, we in-vestigated the RIM1αmutations S1045A and S1045E for presynaptic homeostatic scaling. As described before, GFP-RIM1α(S1045E) expression in RIM1/2 cDKO neurons had the same effect as SRPK2 OE

Results 88

in otherwise untreated neurons. On the other side, GFP-RIM1α(S1045A) expression failed to induce increased synaptic release with or without elevated SRPK2 levels. Strikingly, GFP-RIM1α(S1045E) failed to induce presynaptic homeostatic scaling (similar as SRPK2 OE and GFP-RIM1α(S991E), Figure 5.34 D), whereas GFP-RIM1α(S1045A) was able to induce a strong homeostatic scaling effect as seen before in WT neurons (Figure 5.34 C).

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Figure 5.34: Homeostatic scaling depended on RIM and its phosphorylation. (A)There is no homeostatic scaling in RIM1/2 cDKO neurons. TTX treated and untreated RIM1/2 cDKO neurons show the same presynaptic neurotransmitter release (average∆F/F of untr.: 0.0166 ± 0.003; TTX: 0.0174 ± 0.003). (B)Expression of GFP-RIM1(S991E) in RIM1/2 cDKO neurons does not lead to any scaling effect after TTX treatment (average∆F/F of untr.: 0.050 ± 0.01; TTX: 0.04 ± 0.004).(C)Rescue of RIM1/2 cDKO neurons with GFP-RIM1(S1045A) allows presy-naptic homeostatic scaling as in WT neurons. Glutamate release is increased by almost 100 % after TTX treatment (average∆F/F of untr.: 0.024 ± 0.001; TTX: 0.049 ± 0.006). (D) This scaling capability is abolished when S1045 is mutated to glutamate, which mimics constant phosphorylation (average∆F/F of untr.: 0.049 ± 0.004; TTX: 0.04

± 0.005). Bar graphs on lower panel illustrate normalized∆F/F to quantify relative changes of homeostatic plasticity.

Red markers in upper graphs indicate means and SEM of paired data. Statistical significance was assessed by ratio paired t-test (* p < 0.05). Number of experiments (n) as indicated in bar graphs.

We also tested the mutations S745A and S745E for the ability of homeostatic presynaptic scaling in similar experiments but did not see any scaling effect with these mutations (performed by Annika Mayer, data not shown). Taken together, the data indicate that neurons that overexpress SRPK2 or RIM1α variants that mimics phosphorylation at sites which lead to increased neurotransmitter release do not scale their neurotransmitter release to adapt to silencing by TTX treatment.

Discussion 89

6 Discussion

Presynaptic neurotransmitter release is of crucial relevance for communication in neuronal networks.

Fusion of synaptic vesicles in response to action potential arrival at the synaptic terminal is orchestrated spatially and temporally precisely to allow for extremely fast synaptic vesicle fusion and synaptic trans-mission. The process of synaptic release is highly plastic and can be potentiated or depressed depending on the current needs for synaptic communication. The multi-domain protein and CAZ member RIM1α functionally regulates different steps during synaptic transmission.

In this study, we demonstrated that RIM1αis dynamically phosphorylated dependent on neuronal activ-ity. By a combination of bioinformatical tools, mass spectrometry experiments and live-cell imaging, our study provides for the first time a systematic analysis of functional relevance of phosphorylation sites in RIM1α. By means of a FM imaging assay and the genetically encoded glutamate sensor iGluSnFR we characterized a reduced synaptic release probability as one of the hallmarks of ablation of RIM1αand RIM2. We demonstrated that this phenotype can be rescued with N-terminally GFP-tagged RIM1αand that this rescue strategy fails when RIM1αis mutated to alanine at the positions T812/814, S991 and S1600. Phospho-mimicry with a glutamate mutation reseted the release probability back to WT levels (or even increased release) in these cases. On the contrary, mutation of S514 to alanine boosted the synaptic release, pointing to positive and negative regulation of release by phosphorylation of RIM1α depending on the site that is phosphorylated. Finally, we identified a novel kinase, SRPK2, in the presy-naptic terminal that regulates sypresy-naptic release in a RIM1/2 dependent manner. The effect of SRPK2 on synaptic release is dependent on its kinase activity and needs the phosphorylation sites S991 and S1045 (and potentially S745) in RIM1 for increasing glutamate release.

6.1 GFP-RIM1α fully rescues reduced synaptic release probability in RIM1α KO and RIM1/2 cDKO neurons

The screening for release relevant phospho-mutants in this work relied on rescue experiments, in which endogenous RIM1αand/or RIM2αwas/were ablated and the fusion protein GFP-RIM1αin the longest

Discussion 90

transcript was expressed via lenti-viral vectors. The experimental design could harbor different prob-lems. Firstly, so far no RIM1αKO phenotype rescue experiment has been described, where a RIM1α fusion protein with a fluorescent tag was used. All complete rescue experiments described to date, used untagged RIM1αvariants with independent expression of a fluorescent marker or without fluorescent marker at all. For example Yang and Calakos (2010) generated an expression construct of RIM1αand GFP with an IRES between the two proteins to rescue mfLTP in hippocampal slices of RIM1αKO mice.

In another study RIM1αwith a point mutation at S413 to alanine was knocked-in to investigate rescue ef-ficacy of this mutation on presynaptic plasticity. Again no fluorescent protein was fused to RIM1α[Kaeser et al., 2008a]. Here, we decided for a fusion protein, because serval follow up experiments need a tagged RIM1αvariant (e.g. FRAP for synaptic persistence of RIM1αmutants). The second issue involved be the splice variant. To date there are no data about the implications of the different splice variants of RIM1αfor its multiple functions in the synaptic terminal. We initially cloned RIM1αin five different splice variants (not shown) and selected the longest transcript we could identify, which only lacks three exons.

Nevertheless, the three missing exons could be of critical function. In 2013, Spangler and colleagues failed to rescue a Liprin1αKO mediated, but RIM1αdependent, release phenotype by overexpressing a shorter RIM1αsplice variant. The question whether, the phenotype could not be rescued because Liprin1αwas essential or whether the used RIM1αsplice variant missed intrinsic properties due to the missing exons remained unresolved. Another possibility is that the exons could be essential for protein interactions, that mediated the RIM1αdependence.

Here, we demonstrated that the strategy to rescue the reduced synaptic release probability of RIM1αKO and RIM1/2 cDKO neurons with a GFP-RIM1αfusion protein in the described splice variant is successful in our hands. In all cases lentiviral expression of GFP-RIM1αwas able to completely reset the release probability back to WT levels. We also saw that the spontaneous release rate (though not statistically significant) changed between WT an KO neurons and that this parameter was also set back to WT values, when GFP-RIM1αwas expressed.

In addition to the functional evidence, we also saw mechanistically proof that GFP-RIM1αis fully func-tional in RIM1αKO and RIM1/2 cDKO neurons. Firstly, it was correctly localized to putative synaptic structures as seen by FM and bassoon co-stainings. Secondly, the long persistence of GFP-RIM1αin synaptic structures, as seen in FRAP experiments, points to an integration or at least an interaction in the CAZ. Finally, in Co-IPs with the GFP-tag of the fusion protein, we were able to identify known interaction partners of RIM1α, which suggests that the overall structure of RIM1αwas intact (performed by Mark E.

Graham, data not shown).

In this study we used two different mouse lines to prepare neurons. We used the originally described RIM1αKO mouse line [Schoch et al., 2002] and the later developed RIM1/2^{f l/f l} mouse line [Kaeser

Discussion 91

et al., 2011] from which RIM1/2 cDKO neurons were prepared. The consequences of RIM1αand RIM1/2 ablation on neurotransmitter release have been studied extensively before. For example RIM1αKO release probability was shown to be reduced by 50 % in different studies [Calakos et al., 2004, Kaeser et al., 2008b]. Hippocampal slices from RIM1/2 cDKO mice exhibited a reduction of EPSC amplitudes by 80 % compared to WT [Kaeser et al., 2011, Kaeser et al., 2012]. However, in another study the reduction of the release probability in RIM1/2 cDKO slices was found to be aprrox. 30 % compared with WT (measured at the calyx of held) [Han et al., 2011].

In general, cells from both mouse lines showed a strong reduction in synaptic evoked release probability in our imaging experiments. In both cases also the spontaneous release and the level of residual dye showed changes, even though we could never detect statistical significance for these parameters. Some-what surprising, the release rate seemed to be more strongly affected in RIM1αKO neurons (reduction by 55.6 % compared to WT), when we applied K⁺stimulation, compared to RIM1/2 cDKO with electrical stimulation (reduction by 43.1 %). In the RIM1αKO cells RIM2αcould compensate for the ablation of RIM1α, at least to a certain extent [Schoch et al., 2006], but in the double knock-out all RIM1/2 isoforms are deleted. Therefore, without a potential compensation one might expect a stronger phenotype. There are several explanations why we did not detect a stronger reduced synaptic release probability in RIM1/2 cDKO neurons than in RIM1αKO neurons. First of all, the phenotype of the RIM1αKO neurons was investigated with FM dyes and potassium stimulation, while the phenotype of the RIM1/2 cDKO neurons was measured with FM dyes and electrical stimulation. Stimulation strength and experimental paradigm influence the visibility and measurement of potential phenotypes. This phenomenon was seen in this study (see Section 5.3.1) and has already been described and published [Nimmervoll et al., 2013]. Sec-ondly, the 5 Hz stimulation paradigm in experiments with electrical field potential stimulation might lead to a facilitation in synapses with low release probability (such a low release probability is expected RIM1/2 cDKO neurons). The facilitation of release would increase the estimated release probability seen in our experiments.

Furthermore, it needs to be considered that a constitutive knock-out of both largeα-RIM isoforms is post-natally lethal [Schoch et al., 2006], therefore RIM1 and 2 were ablated via Cre - recombinase to produce a conditional double knock-out (while the RIM1α KO is constitutive). Nine days after transduction of Cre - recombinase no RIM is detectable anymore in WB from cultured hippocampal RIM1/2^{f l/f l}neurons [Kaeser et al., 2011]. Therefore, we incubated the neurons at least for this time period after transduction with viral Cre-vectors before we performed the experiments. However, viral transduction, even though it is considered to have a high efficacy, probably not always targets 100% of the cultured cells. As a consequence the results might be contaminated by neurons that were not recombined. Additionally, the Cre recombinase results in a loss of newly synthesized RIM, only. Already, produced RIM proteins need

Discussion 92

to be degraded via cellular protein degradation pathways, such as the proteasome. This implicates, that RIM must be accessible for this pathway. When molecules are strongly integrated in the CAZ, a struc-ture which can be stable over days or even weeks [Ziv & Arava, 2014], individual RIM molecules might

“survive” for longer time scales. These minute amounts of RIM molecules can be sufficient to falsify the phenotype, since only a handful of RIM molecules (38 - 39) were estimated to be present at a native synapse [Wilhelm et al., 2014] and probably less at individual active zones.

Nevertheless, the phenotypes of RIM1αKO neurons and RIM1/2 cDKO neurons we observed in this study showed a significant reduction in synaptic release probability in both cases and can be used to investigate the rescue efficacy of GFP-RIM1αvariants with phospho-deficient or phospho-mimetic mu-tations..

Im Dokument Molecular function of RIM1α: (Seite 102-107)