Neurofilament depletion improves microtubule dynamics via modulation of Stat3/stathmin signaling

In neurons, microtubules form a dense array within axons, and the stability and function of this microtubule network is modulated by neurofilaments. Accumulation of neurofilaments has been observed in several forms of neurodegenerative diseases, but the mechanisms how elevated neurofilament levels destabilize axons are unknown so far. Here, we show that increased neurofilament expression in motor nerves of pmn mutant mice, a model of motoneuron disease, causes disturbed microtubule dynamics. The disease is caused by a point mutation in the tubulin-specific chaperone E (Tbce) gene, leading to an exchange of the most C-terminal amino acid tryptophan to glycine. As a consequence, the TBCE protein becomes instable which then results in destabilization of axonal microtubules and defects in axonal transport, in particular in motoneurons. Depletion of neurofilament increases the number and regrowth of microtubules in pmn mutant motoneurons and restores axon elongation. This effect is mediated by interaction of neurofilament with the stathmin complex. Accumulating neurofilaments associate with stathmin in axons of pmn mutant motoneurons. Depletion of neurofilament by Nefl knockout increases Stat3-stathmin interaction and stabilizes the microtubules in pmn mutant motoneurons. Consequently, counteracting enhanced neurofilament expression improves axonal maintenance and prolongs survival of pmn mutant mice. We propose that this mechanism could also be relevant for other neurodegenerative diseases in which neurofilament accumulation and loss of microtubules are prominent features

Activation of TRESK channels by the inflammatory mediator lysophosphatidic acid balances nociceptive signalling

In dorsal root ganglia (DRG) neurons TRESK channels constitute a major current component of the standing outward current IK$_{SO}$. A prominent physiological role of TRESK has been attributed to pain sensation. During inflammation mediators of pain e.g. lysophosphatidic acid (LPA) are released and modulate nociception. We demonstrate co-expression of TRESK and LPA receptors in DRG neurons. Heterologous expression of TRESK and LPA receptors in Xenopus oocytes revealed augmentation of basal K$^{+}$ currents upon LPA application. In DRG neurons nociception can result from TRPV$_{1}$ activation by capsaicin or LPA. Upon co-expression in Xenopus oocytes LPA simultaneously increased both depolarising TRPV$_{1}$ and hyperpolarising TRESK currents. Patch-clamp recordings in cultured DRG neurons from TRESK[wt] mice displayed increased IK$_{SO}$ after application of LPA whereas under these conditions IK$_{SO}$ in neurons from TRESK[ko] mice remained unaltered. Under current-clamp conditions LPA application differentially modulated excitability in these genotypes upon depolarising pulses. Spike frequency was attenuated in TRESK[wt] neurons and, in contrast, augmented in TRESK[ko] neurons. Accordingly, excitation of nociceptive neurons by LPA is balanced by co-activation of TRESK channels. Hence excitation of sensory neurons is strongly controlled by the activity of TRESK channels, which therefore are good candidates for the treatment of pain disorders

Axonal and dendritic localization of mRNAs for glycogen-metabolizing enzymes in cultured rodent neurons

Background: Localization of mRNAs encoding cytoskeletal or signaling proteins to neuronal processes is known to contribute to axon growth, synaptic differentiation and plasticity. In addition, a still increasing spectrum of mRNAs has been demonstrated to be localized under different conditions and developing stages thus reflecting a highly regulated mechanism and a role of mRNA localization in a broad range of cellular processes. Results: Applying fluorescence in-situ-hybridization with specific riboprobes on cultured neurons and nervous tissue sections, we investigated whether the mRNAs for two metabolic enzymes, namely glycogen synthase (GS) and glycogen phosphorylase (GP), the key enzymes of glycogen metabolism, may also be targeted to neuronal processes. If it were so, this might contribute to clarify the so far enigmatic role of neuronal glycogen. We found that the mRNAs for both enzymes are localized to axonal and dendritic processes in cultured lumbar spinal motoneurons, but not in cultured trigeminal neurons. In cultured cortical neurons which do not store glycogen but nevertheless express glycogen synthase, the GS mRNA is also subject to axonal and dendritic localization. In spinal motoneurons and trigeminal neurons in situ, however, the mRNAs could only be demonstrated in the neuronal somata but not in the nerves. Conclusions: We could demonstrate that the mRNAs for major enzymes of neural energy metabolism can be localized to neuronal processes. The heterogeneous pattern of mRNA localization in different culture types and developmental stages stresses that mRNA localization is a versatile mechanism for the fine-tuning of cellular events. Our findings suggest that mRNA localization for enzymes of glycogen metabolism could allow adaptation to spatial and temporal energy demands in neuronal events like growth, repair and synaptic transmission

Presynaptic localization of Smn and hnRNP R in axon terminals of embryonic and postnatal mouse motoneurons.

Spinal muscular atrophy (SMA) is caused by deficiency of the ubiquitously expressed survival motoneuron (SMN) protein. SMN is crucial component of a complex for the assembly of spliceosomal small nuclear ribonucleoprotein (snRNP) particles. Other cellular functions of SMN are less characterized so far. SMA predominantly affects lower motoneurons, but the cellular basis for this relative specificity is still unknown. In contrast to nonneuronal cells where the protein is mainly localized in perinuclear regions and the nucleus, Smn is also present in dendrites, axons and axonal growth cones of isolated motoneurons in vitro. However, this distribution has not been shown in vivo and it is not clear whether Smn and hnRNP R are also present in presynaptic axon terminals of motoneurons in postnatal mice. Smn also associates with components not included in the classical SMN complex like RNA-binding proteins FUS, TDP43, HuD and hnRNP R which are involved in RNA processing, subcellular localization and translation. We show here that Smn and hnRNP R are present in presynaptic compartments at neuromuscular endplates of embryonic and postnatal mice. Smn and hnRNP R are localized in close proximity to each other in axons and axon terminals both in vitro and in vivo. We also provide new evidence for a direct interaction of Smn and hnRNP R in vitro and in vivo, particularly in the cytosol of motoneurons. These data point to functions of SMN beyond snRNP assembly which could be crucial for recruitment and transport of RNA particles into axons and axon terminals, a mechanism which may contribute to SMA pathogenesis

BDNF/trkB induction of calcium transients through Cav_{v}2.2 calcium channels in motoneurons corresponds to F-actin assembly and growth cone formation on β2-chain laminin (221)

Spontaneous Ca$^{2+}$ transients and actin dynamics in primary motoneurons correspond to cellular differentiation such as axon elongation and growth cone formation. Brain-derived neurotrophic factor (BDNF) and its receptor trkB support both motoneuron survival and synaptic differentiation. However, in motoneurons effects of BDNF/trkB signaling on spontaneous Ca$^{2+}$ influx and actin dynamics at axonal growth cones are not fully unraveled. In our study we addressed the question how neurotrophic factor signaling corresponds to cell autonomous excitability and growth cone formation. Primary motoneurons from mouse embryos were cultured on the synapse specific, β2-chain containing laminin isoform (221) regulating axon elongation through spontaneous Ca$^{2+}$ transients that are in turn induced by enhanced clustering of N-type specific voltage-gated Ca$^{2+}$ channels (Ca$_{v}$2.2) in axonal growth cones. TrkB-deficient (trkBTK$^{-/-}$) mouse motoneurons which express no full-length trkB receptor and wildtype motoneurons cultured without BDNF exhibited reduced spontaneous Ca$^{2+}$ transients that corresponded to altered axon elongation and defects in growth cone morphology which was accompanied by changes in the local actin cytoskeleton. Vice versa, the acute application of BDNF resulted in the induction of spontaneous Ca$^{2+}$ transients and Ca$_{v}$2.2 clustering in motor growth cones, as well as the activation of trkB downstream signaling cascades which promoted the stabilization of β-actin via the LIM kinase pathway and phosphorylation of profilin at Tyr129. Finally, we identified a mutual regulation of neuronal excitability and actin dynamics in axonal growth cones of embryonic motoneurons cultured on laminin-221/211. Impaired excitability resulted in dysregulated axon extension and local actin cytoskeleton, whereas upon β-actin knockdown Ca$_{v}$2.2 clustering was affected. We conclude from our data that in embryonic motoneurons BDNF/trkB signaling contributes to axon elongation and growth cone formation through changes in the local actin cytoskeleton accompanied by increased Ca$_{v}$2.2 clustering and local calcium transients. These findings may help to explore cellular mechanisms which might be dysregulated during maturation of embryonic motoneurons leading to motoneuron disease

Colocalization of Smn and hnRNP R proteins in embryonic motoneurons.

<p>Representative images of cell bodies, axons and growth cones of primary embryonic motoneurons cultured on laminin-111 (A) and laminin-221/211 (B) for 5DIV and stained against Smn and hnRNP R (scale bar: 5 µm). Superimposed colocalizing points are highlighted in white. (C) No differences were observed with respect to colocalization and subcellular distribution of hnRNP R between these two investigated laminin isoforms. Representative images of cell bodies, axons and growth cones of motoneurons cultured on laminin-111 for either 3DIV (D) or 7DIV (E) and labeled against Smn and hnRNP R (scale bar: 5 µm). Both the degree of overlap between Smn and hnRNP R and the subcellular distribution of hnRNP R were regulated over time. The relative ratio of cytosolic versus nuclear hnRNP R immunoreactivity was significantly enhanced by 63% (P = 0.0173, t = 3.914, DF = 4) in motoneuron cell bodies cultured for 7DIV (1.63±0.16, n = 5, N = 46) in comparison to 3DIV (set as ‘1’; n = 5, N = 37). (F) After 7DIV (PCC 0.65±0.02, MOC 0.75±0.01, n = 5, N = 45) colocalization of Smn and hnRNP R in motoneuron cell bodies was higher (PCC P = 0.0112, t = 4.453, DF = 4; MOC P = 0.0086, t = 4.807, DF = 4) than after 3DIV (PCC 0.56±0.03, MOC 0.68±0.02, n = 5, N = 36). In axons the degree of overlap and correlation did not change (PCC P = 0.1504, t = 1.776, DF = 4; MOC P = 0.1449, t = 1.808, DF = 4) over time (3DIV PCC 0.43±0.04, MOC 0.55±0.03, n = 5, N = 36; 7DIV PCC 0.46±0.04, MOC 0.58±0.03, n = 5, N = 46), whereas in axonal growth cones a significant modification of the correlation (PCC P = 0.0467, t = 2.844, DF = 4; MOC P = 0.1565, t = 1.742, DF = 4) of both proteins was detected (3DIV PCC 0.38±0.03, MOC 0.52±0.02, n = 5, N = 37; 7DIV PCC 0.45±0.02, MOC 0.56±0.02, n = 5, N = 34).</p

Coimmunoprecipitation of Smn and hnRNP R in primary motoneurons and native spinal cord.

<p>(A) 1 000 000 primary motoneurons were cultured for 7DIV on laminin-111. Cytosolic and soluble nuclear fractions were subjected to a pull-down with either Smn or hnRNP R antibodies, respectively. Coprecipitation of hnRNP R or Smn, respectively, was determined revealing an interaction of Smn and hnRNP R, particularly in the cytosolic fraction of embryonic mouse motoneurons (eluate lane). Smn was not detectable in the soluble nuclear fraction of motoneurons. HnRNP R was found both in nuclear and cytosolic extracts. For immunoprecipitation experiments a C-terminal antibody directed against hnRNP R (Abcam) was used <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110846#pone.0110846-Glinka1" target="_blank">[29]</a>. Supernatants still contained some Smn or hnRNP R protein, respectively, suggesting that the interaction appears not to be exclusive as demonstrated by immunofluorescence colocalization analysis. No signal was obtained in the washing solution. Successful fractionation was controlled by α tubulin (cytosol) and histone H3 (nucleus) (right panel). (B) Fractionation of spinal cord tissue from E18 mouse embryos revealed a similar result as shown in (A). In the cytosolic fraction hnRNP R IP pulled-down Smn protein and <i>vice versa</i>. Nuclear Smn was not detected in the soluble, but in the corresponding insoluble nuclear fraction (right panel, lower blot). In contrast, nuclear hnRNP R was not found in the insoluble nuclear fraction. Cytosolic and nuclear extracts were validated by α tubulin and histone H3. (C) HEK293T cells were cultured and cytosolic and soluble nuclear fractions were prepared. Smn and hnRNP R were detected in cytosolic extracts as well as in soluble nuclear fractions. The pull down of Smn and hnRNP R, respectively, was successful (eluate lane, IP), but hnRNP R or Smn, respectively, could not be coprecipitated, neither from cytosolic nor from nuclear extracts. Successful fractionation was verified by GAPDH (cytosolic) and histone H3 (nucleus) (right panel).</p

Colocalization of Smn and hnRNP R <i>in</i><i>vivo</i> in E18 motoneurons and axon terminals.

<p>(A) Representative cross section from E18 spinal cord stained against Smn, hnRNP R and ChAT (scale bar: 10 µm). Superimposed colocalizing points are highlighted in white. Smn signals were mainly found in the cytosol, with very few positive spots in the nuclei. HnRNP R immunoreactivity was observed in the nucleus and in the cytosol. Colocalization of Smn and hnRNP R was detected in the cytosol, especially in axonal initiation segments (PCC 0.27±0.03, MOC 0.81±0.01, N = 8). (B) Whole mount preparations from <i>Diaphragm</i> muscles from E18 mouse embryos stained against Smn, hnRNP R, ω-BTX and DAPI (scale bar: 2 µm). Both Smn and hnRNP R immunoreactivity were detected at these defined sites showing partial overlap (PCC 0.24±0.04, MOC 0.54±0.02, N = 6).</p

Direct interaction of hnRNP R and SMN.

<p>(A) Purification scheme of recombinant hnRNP R and SMN expressed as His-tagged proteins in <i>E. coli</i> strain BL21. (B) Affinity purification profile on a fast protein liquid chromatography (FPLC) of hnRNP R and SDS-PAGE of recombinant hnRNP R purification steps visualized by silver staining. (C) Affinity purification profile on a FPLC of SMN and SDS-PAGE of recombinant SMN purification steps visualized by colloidal staining. (D) Coimmunoprecipitation of recombinant SMN and hnRNP R.</p

Smn deficiency in SMA type I axon terminals <i>in</i><i>vivo.</i>

<p>(A, B) Representative motor endplates from E18 <i>Smn^+/+; SMN2tg</i> and <i>Smn^−/−; SMN2tg Diaphragm</i> stained against Smn and SynPhys. Acetylcholine receptors (AChR) and postsynaptic nuclei were visualized by ω-BTX and DAPI, respectively (scale bar: 5 µm). In (A) Smn deficiency is visible by highly reduced immunoreactive signals, as highlighted in the white box, whereas in (B) the number of Smn particles per NMJ is decreased in SMA type I motor endplates, as indicated by white arrowheads. (A, B) In SMA type I axon terminals (n = 3, N = 32) mean Smn signal intensity was significantly reduced (0.43±0.09, P = 0.0220, t = 6.629, DF = 2) in comparison to control motor endplates (set as ‘1’, n = 3, N = 43), whereas SynPhys signals (<i>Smn^−/−; SMN2tg</i> 1.15±0.19, P = 0.5221, t = 0.7694, DF = 2) and the size of the presynaptic compartment (Control 49.48±13.94 µm²; <i>Smn^−/−; SMN2tg</i> 36.56±7.464; P = 0.4596, t = 0.8174, DF = 4) were comparable. (C) Representative images from E18 <i>Smn^+/+; SMN2tg</i> and <i>Smn^−/−; SMN2tg</i> spinal cord cross sections immunolabeled with Smn and ChAT. Quantitative analysis revealed a significant decrease in cytosolic Smn immunoreactivity in SMA type I motoneurons in comparison to <i>Smn^+/+; SMN2tg</i> cells (<i>Smn^+/+; SMN2tg</i> set as ‘1’, n = 6, N = 107; <i>Smn^−/−; SMN2tg</i> 0.46±0.05, n = 6, N = 85; P<0.0001, t = 11.23, DF = 5). ChAT signal intensity was not statistically affected (<i>Smn^−/−; SMN2tg</i> 0.83±0.21; P = 0.4638, t = 0.7928, DF = 5). (D) Representative Western Blot with cytosolic and nuclear fractions from E18 control and <i>Smn^−/−; SMN2tg</i> spinal cord extracts. Histone H3 and α tubulin were used as markers for nuclear and cytosolic fractions, respectively, and as standardization proteins for quantitative analysis. In SMA type I spinal cord extracts cytosolic and nuclear Smn were significantly reduced by 64% (0.36±0.08, N = 10, P<0.0001, t = 8.480, DF = 9) and 86% (0.14±0.03, N = 10, P<0.0001, t = 26.39, DF = 9), respectively, in comparison to <i>Smn^+/+; SMN2tg</i> extracts (set as ‘1’, N = 10).</p