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

Die Rolle von RNA-bindenden Proteinen in Motoneuronerkrankungen

Motoneuron diseases form a heterogeneous group of pathologies characterized by the progressive degeneration of motoneurons. More and more genetic factors associated with motoneuron diseases encode proteins that have a function in RNA metabolism, suggesting that disturbed RNA metabolism could be a common underlying problem in several, perhaps all, forms of motoneuron diseases. Recent results suggest that SMN interacts with hnRNP R and TDP-43 in neuronal processes, which are not part of the classical SMN complex. This point to an additional function of SMN, which could contribute to the high vulnerability of spinal motoneurons in spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). The current study elucidates functional links between SMN, the causative factor of SMA (spinal muscular atrophy), hnRNP R, and TDP-43, a genetic factor in ALS (amyotrophic lateral sclerosis). In order to characterize the functional interaction of SMN with hnRNP R and TDP-43, we produced recombinant proteins and investigated their interaction by co-immunoprecipitation. These proteins bind directly to each other, indicating that no other co-factors are needed for this interaction. SMN potentiates the ability of hnRNP R and TDP-43 to bind to ß-actin mRNA. Depletion of SMN alters the subcellular distribution of hnRNP R in motoneurons both in SMN-knockdown motoneurons and SMA mutant mouse (delta7 SMA). 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 and ALS. ALS and FTLD (frontotemporal lobar degeneration) are linked by several lines of evidence with respect to clinical and pathological characteristics. Both sporadic and familial forms are a feature of the ALS-FTLD spectrum, with numerous genes having been associated with these pathological conditions. Both diseases are characterized by the pathological cellular aggregation of proteins. Interestingly, some of these proteins such as TDP-43 and FUS have also common relations not only with ALS-FTLD but also with SMA. Intronic hexanucleotide expansions in C9ORF72 are common in ALS and FTLD but it is unknown whether loss of function, toxicity by the expanded RNA or dipeptides from non ATG-initiated translation is responsible for the pathophysiology. This study tries to characterize the cellular function of C9ORF72 protein. To address this, lentiviral based knockdown and overexpression of C9ORF72 was used in isolated mouse motoneurons. The results clearly show that survival of these motoneurons was not affected by altered C9ORF72 levels, whereas adverse effects on axon growth and growth cone size became apparent after C9ORF72 suppression. Determining the protein interactome revealed several proteins in complexes with C9ORF72. Interestingly, C9ORF72 is present in a complex with cofilin and other actin binding proteins that modulate actin dynamics. These interactions were confirmed both by co-precipitation analyses and in particular by functional studies showing altered actin dynamics in motoneurons with reduced levels of C9ORF72. Importantly, the phosphorylation of cofilin is enhanced in C9ORF72 depleted motoneurons and patient derived lymphoblastoid cells with reduced C9ORF72 levels. These findings indicate that C9ORF72 regulates axonal actin dynamics and the loss of this function could contribute to disease pathomechanisms in ALS and FTLD.Motoneuronerkrankungen bilden eine heterogene Gruppe von Pathologien, die durch die progressive Degeneration von Motoneuronen charakterisiert sind. Zunehmend werden genetische Faktoren in Assoziation mit Motoneuronerkrankungen identifiziert, die eine Funktion im RNA Metabolismus besitzen, was dafür spricht, dass ein gestörter RNA Metabolismus ein gemeinsames zugrunde liegendes Problem in mehreren, vielleicht allen, Formen von Motoneuronerkrankungen sein könnte. Neuere Ergebnisse legen nahe, dass SMN mit hnRNP R und TDP-43 in neuronalen Prozessen interagiert, die nicht Teil der klassischen Rolle des SMN Komplexes sind. Dies deutet auf eine zusätzliche Funktion von SMN hin, die zur hohen Störanfälligkeit von spinalen Motoneuronen in spinaler Muskelatrophie (SMA) und amyotropher Lateralsklerose (ALS) beitragen könnte. Die vorliegende Arbeit beleuchtet funktionelle Beziehungen zwischen SMN, dem auslösenden Faktor der SMA, und hnRNP R, sowie TDP-43, einem weiteren genetischen Faktor bei ALS. Um die funktionelle Interaktion von SMN mit hnRNP R und TDP-43 zu charakterisieren, wurden rekombinante Proteine hergestellt und ihre Interaktion durch co-Immunpräzipitation untersucht. Diese Proteine binden direkt an einander, was darauf hindeutet, dass für diese Interaktion keine weiteren co-Faktoren erforderlich sind. SMN potenziert die Fähigkeit von hnRNP R und TDP-43, β-Aktin mRNA zu binden. Depletion von SMN verändert die subzelluläre Verteilung von hnRNP R in Motoneuronen sowohl in SMN-knock-down Motoneuronen, als auch in der SMA Mausmutante (delta7 SMA). Diese Daten deuten auf Funktionen von SMN jenseits der snRNP Assemblierung hin, die entscheidend für die Rekrutierung und den Transport von RNA Partikel in Axonen und Axon Terminalen sein könnten, einem Mechanismus, der zur Pathogenese von SMA und ALS beitragen könnte. ALS und FTLD (fronto-temporale Lobus Degeneration) sind aufgrund mehrerer Nachweislinien bezüglich klinischer und pathologischer Charakteristika vernetzt. Sowohl sporadische als auch familiäre Formen sind Merkmal des ALS-FTLD Spektrums, wobei zahlreiche Gene mit diesen pathologischen Erscheinungen assoziiert wurden. Beide Krankheiten sind durch pathologische zelluläre Proteinaggregation charakterisiert. Interessanterweise haben einige dieser Proteine, wie TDP-43 und FUS, einen gemeinsamen Bezug nicht nur mit ALS-FTLD, sondern auch mit SMA. Intronische Hexanukleotid-Expansionen in C9ORF72 sind häufig in ALS und FTLD, es ist jedoch unbekannt, ob Funktionsverlust, Toxizität aufgrund der verlängerten RNA, oder Dipeptide von non-ATG initiierter Translation für die Pathophysiologie verantwortlich sind. Die vorliegende Arbeit versucht die zelluläre Funktion von C9ORF72 Protein zu charakterisieren. Hierfür wurde lentiviraler knock-down und Überexpression von C9ORF72 in isolierten Motoneuronen eingesetzt. Die Ergebnisse zeigen deutlich, dass das Überleben dieser Motoneurone durch veränderte C9ORF72 Konzentrationen nicht beeinflusst wurde, wohingegen negative Auswirkungen auf Axonwachstum und Wachstumskegelgröße nach C9ORF72 Suppression deutlich wurden. Die Bestimmung des Protein Interaktoms identifizierte mehrere Proteinkomplexe mit C9ORF72. Interessanterweise liegt C9ORF72 in einem Komplex mit Cofilin und anderen Aktin-bindenden Protein vor, welche die Aktin Dynamik modulieren. Diese Interaktionen wurden sowohl durch Analyse von co-Präzipitationen als auch besonders durch funktionelle Studien bestätigt, die eine veränderte Aktin Dynamik in Motoneuronen mit reduzierter C9ORF72 Konzentration zeigten. Wichtig ist die Beobachtung, dass die Phosphorylierung von Cofilin in C9ORF72 depletierten Motoneuronen und in Lymphoblastoid-Zellen mit reduzierter C9ORF72 Konzentration verstärkt ist. Diese Ergebnisse zeigen, dass C9ORF72 die axonale Aktin Dynamik reguliert und dass der Verlust dieser Funktion zu Krankheits-Pathomechanismen in ALS und FTLD beitragen könnte

The Role of RNA Binding Proteins in Motoneuron Diseases

Motoneuron diseases form a heterogeneous group of pathologies characterized by the progressive degeneration of motoneurons. More and more genetic factors associated with motoneuron diseases encode proteins that have a function in RNA metabolism, suggesting that disturbed RNA metabolism could be a common underlying problem in several, perhaps all, forms of motoneuron diseases. Recent results suggest that SMN interacts with hnRNP R and TDP-43 in neuronal processes, which are not part of the classical SMN complex. This point to an additional function of SMN, which could contribute to the high vulnerability of spinal motoneurons in spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). The current study elucidates functional links between SMN, the causative factor of SMA (spinal muscular atrophy), hnRNP R, and TDP-43, a genetic factor in ALS (amyotrophic lateral sclerosis). In order to characterize the functional interaction of SMN with hnRNP R and TDP-43, we produced recombinant proteins and investigated their interaction by co-immunoprecipitation. These proteins bind directly to each other, indicating that no other co-factors are needed for this interaction. SMN potentiates the ability of hnRNP R and TDP-43 to bind to ß-actin mRNA. Depletion of SMN alters the subcellular distribution of hnRNP R in motoneurons both in SMN-knockdown motoneurons and SMA mutant mouse (delta7 SMA). 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 and ALS. ALS and FTLD (frontotemporal lobar degeneration) are linked by several lines of evidence with respect to clinical and pathological characteristics. Both sporadic and familial forms are a feature of the ALS-FTLD spectrum, with numerous genes having been associated with these pathological conditions. Both diseases are characterized by the pathological cellular aggregation of proteins. Interestingly, some of these proteins such as TDP-43 and FUS have also common relations not only with ALS-FTLD but also with SMA. Intronic hexanucleotide expansions in C9ORF72 are common in ALS and FTLD but it is unknown whether loss of function, toxicity by the expanded RNA or dipeptides from non ATG-initiated translation is responsible for the pathophysiology. This study tries to characterize the cellular function of C9ORF72 protein. To address this, lentiviral based knockdown and overexpression of C9ORF72 was used in isolated mouse motoneurons. The results clearly show that survival of these motoneurons was not affected by altered C9ORF72 levels, whereas adverse effects on axon growth and growth cone size became apparent after C9ORF72 suppression. Determining the protein interactome revealed several proteins in complexes with C9ORF72. Interestingly, C9ORF72 is present in a complex with cofilin and other actin binding proteins that modulate actin dynamics. These interactions were confirmed both by co-precipitation analyses and in particular by functional studies showing altered actin dynamics in motoneurons with reduced levels of C9ORF72. Importantly, the phosphorylation of cofilin is enhanced in C9ORF72 depleted motoneurons and patient derived lymphoblastoid cells with reduced C9ORF72 levels. These findings indicate that C9ORF72 regulates axonal actin dynamics and the loss of this function could contribute to disease pathomechanisms in ALS and FTLD

Bacteria–Cancer Interface: Awaiting the Perfect Storm

Epidemiological evidence reveal a very close association of malignancies with chronic inflammation as a result of persistent bacterial infection. Recently, more studies have provided experimental evidence for an etiological role of bacterial factors disposing infected tissue towards carcinoma. When healthy cells accumulate genomic insults resulting in DNA damage, they may sustain proliferative signalling, resist apoptotic signals, evade growth suppressors, enable replicative immortality, and induce angiogenesis, thus boosting active invasion and metastasis. Moreover, these cells must be able to deregulate cellular energetics and have the ability to evade immune destruction. How bacterial infection leads to mutations and enriches a tumour-promoting inflammatory response or micro-environment is still not clear. In this review we showcase well-studied bacteria and their virulence factors that are tightly associated with carcinoma and the various mechanisms and pathways that could have carcinogenic properties

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

Subcellular distribution of Smn and hnRNP R in isolated embryonic motoneurons.

<p>(A) Motoneurons showed reduced Smn protein levels upon lentiviral knockdown of Smn. Uninfected or GFP-infected mouse embryonic motoneurons were used as controls. Levels of calnexin and hnRNP R were not affected. For this experiment a C-terminal antibody directed against hnRNP R was used as reported recently <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110846#pone.0110846-Glinka1" target="_blank">[29]</a>. This antibody recognizes distinct hnRNP R isoforms. (B) Representative images of motoneurons cultured for 7DIV and labeled against Smn (scale bar: 10 µm). GFP-transfected controls revealed immunoreactive signals for Smn in the cytosol, in neuronal processes and in Gem-like nuclear structures. Upon lentiviral Smn knockdown both cytosolic Smn immunoreactivity (Uninfected set as ‘1’, n = 4, N = 51; GFP 1.02±0.04, n = 4, N = 60; sh-Smn 0.34±0.02, n = 4, N = 74; P<0.001, t = 19.19) and number of Gems per nucleus (Uninfected 1.03±0.18, n = 4, N = 51; GFP 0.97±0.15, n = 4, N = 60; sh-Smn 0.08±0.02, n = 4, N = 74; P<0.01, t = 4.929) were significantly reduced in comparison to uninfected cells. (C) Subcellular distribution of hnRNP R in soma, axon and growth cone of primary motoneurons cultured for 5DIV and costained against synaptophysin (SynPhys) and neurofilament (NF-L) (scale bar: 10 µm (upper row), 5 µm). (D) Lentiviral knockdown of hnRNP R led to a dose-dependent reduction of hnRNP R levels. Calnexin and Smn protein were not altered significantly. (E) HnRNP R knockdown was also detected by immunofluorescence validating the used antiserum peptide ICN 1-18 (GFP 1.00±0.04, n = 8, N = 100; sh-hnRNP R 0.48±0.04, n = 6, N = 63; P<0.0001, t = 8.719, DF = 12) (scale bar: 10 µm).</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