N-Cofilin Can Compensate for the Loss of ADF in Excitatory Synapses

Actin plays important roles in a number of synaptic processes, including synaptic vesicle organization and exocytosis, mobility of postsynaptic receptors, and synaptic plasticity. However, little is known about the mechanisms that control actin at synapses. Actin dynamics crucially depend on LIM kinase 1 (LIMK1) that controls the activity of the actin depolymerizing proteins of the ADF/cofilin family. While analyses of mouse mutants revealed the importance of LIMK1 for both pre- and postsynaptic mechanisms, the ADF/cofilin family member n-cofilin appears to be relevant merely for postsynaptic plasticity, and not for presynaptic physiology. By means of immunogold electron microscopy and immunocytochemistry, we here demonstrate the presence of ADF (actin depolymerizing factor), a close homolog of n-cofilin, in excitatory synapses, where it is particularly enriched in presynaptic terminals. Surprisingly, genetic ablation of ADF in mice had no adverse effects on synapse structure or density as assessed by electron microscopy and by the morphological analysis of Golgi-stained hippocampal pyramidal cells. Moreover, a series of electrophysiological recordings in acute hippocampal slices revealed that presynaptic recruitment and exocytosis of synaptic vesicles as well as postsynaptic plasticity were unchanged in ADF mutant mice. The lack of synaptic defects may be explained by the elevated n-cofilin levels observed in synaptic structures of ADF mutants. Indeed, synaptic actin regulation was impaired in compound mutants lacking both ADF and n-cofilin, but not in ADF single mutants. From our results we conclude that n-cofilin can compensate for the loss of ADF in excitatory synapses. Further, our data suggest that ADF and n-cofilin cooperate in controlling synaptic actin content

Preserved Morphology and Physiology of Excitatory Synapses in Profilin1-Deficient Mice

Profilins are important regulators of actin dynamics and have been implicated in activity-dependent morphological changes of dendritic spines and synaptic plasticity. Recently, defective presynaptic excitability and neurotransmitter release of glutamatergic synapses were described for profilin2-deficient mice. Both dendritic spine morphology and synaptic plasticity were fully preserved in these mutants, bringing forward the hypothesis that profilin1 is mainly involved in postsynaptic mechanisms, complementary to the presynaptic role of profilin2. To test the hypothesis and to elucidate the synaptic function of profilin1, we here specifically deleted profilin1 in neurons of the adult forebrain by using conditional knockout mice on a CaMKII-cre-expressing background. Analysis of Golgi-stained hippocampal pyramidal cells and electron micrographs from the CA1 stratum radiatum revealed normal synapse density, spine morphology, and synapse ultrastructure in the absence of profilin1. Moreover, electrophysiological recordings showed that basal synaptic transmission, presynaptic physiology, as well as postsynaptic plasticity were unchanged in profilin1 mutants. Hence, loss of profilin1 had no adverse effects on the morphology and function of excitatory synapses. Our data are in agreement with two different scenarios: i) profilins are not relevant for actin regulation in postsynaptic structures, activity-dependent morphological changes of dendritic spines, and synaptic plasticity or ii) profilin1 and profilin2 have overlapping functions particularly in the postsynaptic compartment. Future analysis of double mutant mice will ultimately unravel whether profilins are relevant for dendritic spine morphology and synaptic plasticity

The German National Registry of Primary Immunodeficiencies (2012-2017)

Introduction: The German PID-NET registry was founded in 2009, serving as the first national registry of patients with primary immunodeficiencies (PID) in Germany. It is part of the European Society for Immunodeficiencies (ESID) registry. The primary purpose of the registry is to gather data on the epidemiology, diagnostic delay, diagnosis, and treatment of PIDs. Methods: Clinical and laboratory data was collected from 2,453 patients from 36 German PID centres in an online registry. Data was analysed with the software Stata® and Excel. Results: The minimum prevalence of PID in Germany is 2.72 per 100,000 inhabitants. Among patients aged 1–25, there was a clear predominance of males. The median age of living patients ranged between 7 and 40 years, depending on the respective PID. Predominantly antibody disorders were the most prevalent group with 57% of all 2,453 PID patients (including 728 CVID patients). A gene defect was identified in 36% of patients. Familial cases were observed in 21% of patients. The age of onset for presenting symptoms ranged from birth to late adulthood (range 0–88 years). Presenting symptoms comprised infections (74%) and immune dysregulation (22%). Ninety-three patients were diagnosed without prior clinical symptoms. Regarding the general and clinical diagnostic delay, no PID had undergone a slight decrease within the last decade. However, both, SCID and hyper IgE- syndrome showed a substantial improvement in shortening the time between onset of symptoms and genetic diagnosis. Regarding treatment, 49% of all patients received immunoglobulin G (IgG) substitution (70%—subcutaneous; 29%—intravenous; 1%—unknown). Three-hundred patients underwent at least one hematopoietic stem cell transplantation (HSCT). Five patients had gene therapy. Conclusion: The German PID-NET registry is a precious tool for physicians, researchers, the pharmaceutical industry, politicians, and ultimately the patients, for whom the outcomes will eventually lead to a more timely diagnosis and better treatment

Dendritic spine morphology.

<p>Shown are the mean values (±SEM).</p>*<p>:P<0.05.</p

Deletion of profilin1 in Pfn1^{flx/flx,CaMKII-cre} mice.

<p>(<b>A</b>) Immunoblot analysis in different brain regions from an adult Pfn1^flx/flx control and an adult Pfn1^{flx/flx,CaMKII-cre} mutant (P70), revealing efficient deletion of profilin1 in the forebrain of mutants. In all three forebrain tissues (cortex (CX), striatum (STR), hippocampus (HIP)), profilin1 expression was almost undetectable in mutants. In contrast, profilin1 expression level was unchanged in the cerebellum (CB), in which cre is not expressed. Identical results were obtained when investigating profilin1 expression levels in two other Pfn1^{flx/flx,CaMKII-cre} mice. (<b>B</b>) Immunoblot analysis of hippocampal synaptosomes, demonstrating the absence of profilin1 from synaptic structures in mutants. (<b>C</b>) No changes in profilin2 expression were found in the cortex, hippocampus, or striatum of three individual profilin1-deficient mice. (<b>D</b>) Normal profilin2 content in hippocampal synaptosomes from two individual Pfn1^{flx/flx,CaMKII-cre} mice. Expression of β tubulin was examined to control protein load in A–D.</p

Normal presynaptic function of hippocampal CA3-CA1 synapses in Pfn1^{flx/flx,CaMKII-cre} mice.

<p>(<b>A</b>) Basal synaptic transmission, as deduced from input-output curves, was normal in Schaffer-collateral-CA1 synapses of Pfn1^{flx/flx,CaMKII-cre} mice (n = 15 for controls and 10 for mutants). (<b>B</b>) In Pfn1^{flx/flx,CaMKII-cre} mice, no changes were found in paired-pulse ratios (PPR; n = 14 for controls, n = 17 for mutants) at various inter-stimulus intervals (ISI; 10–200 ms). Cumulative curves of amplitudes (<b>C</b>) and inter-event intervals (IEI) of mEPSCs (<b>D</b>) were virtually equal between genotypes (n = 8 in each group). Insets in C and D depict mean values.</p

Unimpaired synaptic plasticity in the absence of profilin1.

<p>(<b>A</b>) In Pfn1^{flx/flx,CaMKII-cre} mice, no difference was found in LTD induced by low frequency stimulation (1 Hz) of 15 min duration (n = 9 for controls, n = 12 for mutants) when analyzing the last 10 min of the recordings. LTP induced by either a single 100 Hz tetanus of 1 s duration (<b>B</b>) or by theta-burst stimulation (TBS) (<b>C</b>) was also not different between genotypes (1×100 Hz: n = 10 for controls and 8 for mutants; TBS: n = 10 for both groups). ns: not significant.</p

ADF is dispensable for synaptic plasticity.

<p>(<b>A</b>) Long-term depression induced by low frequency stimulation (1 Hz for 15 min) with paired pulses (interpulse interval 50 ms) showed no difference between CTR and ADF-KO (last 10 min of recording; CTR: 0.725±0.042, n = 8; ADF-KO: 0.706±0.038, n = 9; P = 0.739). (<b>B</b>) Likewise, long-term potentiation induced by a single tetanic stimulation with 100 Hz for 1 s was indistinguishable between CTR and ADF-KO (last 10 min of recording; CTR: 1.205±0.035, n = 12; ADF-KO: 1.221±0.028, n = 12; P = 0.732).</p

Basal synaptic transmission and presynaptic physiology were independent of ADF.

<p>(<b>A</b>) Input-output curves were not different between CTR and ADF-KO (n = 12 for CTR; n = 16 for ADF-KO), indicating unchanged basal synaptic transmission in ADF-KO. (<b>B</b>) Paired pulse ratio (PPR) at different interstimulus intervals (10 to 200 ms) was unaltered in ADF-KO (n = 12 for CTR; n = 18 for ADF-KO). (<b>C</b>) Exemplary traces showing miniature excitatory postsynaptic currents (mEPSC) recorded from a CTR (upper trace) and an ADF-KO pyramidal cell (lower trace). (<b>D+E</b>) Both mEPSC amplitudes and interevent intervals (IEI) were unaltered in ADF-KO as deduced from the cumulative curves and mean values (insets; mEPSC amplitudes: CTR: 7.29±0.33 pA, n = 14 cells from 7 mice; ADF-KO: 7.09±0.42, n = 9/3; P = 0.699; IEI: CTR: 0.831±0.049 s; ADF-KO: 0.712±0.031 s; P = 0.089) were unaltered in ADF-KO. (<b>F</b>) Additionally, presynaptic short-term vesicle depression induced by 10 Hz stimulation for 2 min was similar in CTR and ADF-KO.</p

N-cofilin compensates the loss of ADF at synapses.

<p>(<b>A</b>) Immunoblot analyses show enrichment of synaptic markers (PSD-95, synaptophysin) in synaptosomes. Equal protein load was verified by Coomassie staining of SDS-PAGEs. Compared to total protein lysates (homogenate) and the organelle- and nuclei-containing fraction P1 PSD-95 and synaptophysin signals were increased in synaptosomes. (<b>B</b>) Immunoblots showing that synaptic n-cofilin levels were increased in synaptosomes from ADF-KO. (<b>C</b>) Immunoblot analysis demonstrating equal cytosolic actin levels in CTR, n-cofilin mutants (n-Cof^{flx/flx,CaMKII-cre}), ADF-KO and double mutants (ACC) that lack both ADF and n-cofilin. Conversely, actin levels were increased in microsomal and synaptosomal preparations from ACC mice. (<b>D</b>) Quantification of microsomal actin levels. In microsomes from ACC mice, actin levels were significantly increased to 266.8±15.8% of CTR levels (n = 4; P<0.001) and significantly higher compared to n-Cof^{flx/flx,CaMKII-cre} mice (P<0.001) or ADF-KO (P<0.001). No significant increase in actin levels was found in microsomes from ADF-KO or n-cofilin mutants. (<b>E</b>) Quantification of actin levels in synaptosomes. Actin levels in synaptosomes were significantly increased in n-cofilin mutants (+10.1±2.1%; n = 4; P<0.05) and ACC mice (+27.8±5.2%; n = 4; P<0.01). In ACC mice, synaptosomal actin levels were significantly higher than in synaptosomes from n-cofilin mutants (P<0.05) or ADF-KO (P<0.01). No increase was found in synaptosomes from ADF-KO.</p