Functional characterization of Neurofilament Light b splicing and misbalance in zebrafish

Neurofilaments (NFs), a major cytoskeletal component of motor neurons, play a key role in their differentiation, establishment and maintenance of their morphology and mechanical strength. The de novo assembly of these neuronal intermediate filaments requires the presence of the neurofilament light subunit, NEFL, which expression is reduced in motor neurons in Amyotrophic Lateral Sclerosis (ALS). This study used zebrafish as a model to characterize the NEFL homologue neflb, which encodes two different isoforms via splicing of the primary transcript (neflbE4 and neflbE3). In vivo imaging showed that neflb is crucial for proper neuronal development, and that disrupting the balance between its two isoforms specifically affects NF assembly and motor axon growth, with resulting motor deficits. This equilibrium is also disrupted upon partial depletion of TDP-43, a RNA binding protein that is mislocalized into cytoplasmic inclusions in ALS. The study supports interaction of NEFL expression and splicing with TDP-43 in a common pathway, both biologically and pathogenetically.


INTRODUCTION
A major cytoskeletal structure of the axon is composed by fibrillary networks of neurofilaments (NFs), which are expressed exclusively in mature neurons in both the central and peripheral nervous system [1] [2] [3]. NFs are formed by the assembly of three main proteins classified by their respective size: the neurofilament light (NEFL), middle and high protein (NEFM and NEFH) [4]. All NF proteins possess a conserved central alphahelical rod domain, necessary for the dimer formation during the first step of assembly, and unique head and tail domains that protrude from the filament core [4]. The assembly properties of these factors depend on their stoichiometry [5], [6] and post-translational modifications [7], [8]. In fact, NEFM and NEFH cannot form long filaments by themselves, but each can co-assemble to form filaments in combination with NEFL both in vitro [9] and in cultured cells [10], [11]. NFs are especially abundant in motor neurons (MNs) where they play a key role in the organization of the neuronal cytoarchitecture and are involved in perikarya differentiation, protrusions (dendrites and axons) maturation and synaptic functions [12].
In CMT, 30 mutations have been described in residues throughout NEFL [17] and cause a variable clinical phenotypes [18] from the complete absence of NEFL [19] to the production of aggregate-prone mutants [20], [21]. In particular, some of these mutants display altered phosphorylation patterns that suppress the filament assembly process, underlying the importance of this post-translational modification for NF assembly [21].
In ALS, the abnormal deposition of hyper-phosphorylated forms of NFs has been detected in MNs [22]- [24].The importance NEFL in ALS is supported by the following evidence. First, high levels of NEFL protein are detected in cerebrospinal fluid (CSF) and blood of all ALS cases [25] as early as 12 months before the onset of the disease [26].
Consequently, NEFL is considered the most relevant biomarker in ALS and its level is used in ALS prognosis as it strictly correlates with disease severity [27]- [29]. Second, perturbation of NEFL mRNA steady state in ALS spinal motor neurons could be a key pathogenic mechanism [30]. Indeed, disrupting the stoichiometry of NF subunits leads to NF aggregation reminiscent of ALS pathology [31]- [34]. The evidence of a pathological dysregulation of NEFL expression in ALS is strengthened by the existence of a direct interaction between NEFL mRNA 3'UTR region with SOD1, TDP-43, and RGNEF, , which are all genetic causes of ALS [35]- [39]. Importantly, the question of whether deficits in NEFL RNA regulation can induce motor neuron degeneration has not been tested in vivo. In order to address this hypothesis, we used zebrafish (Danio rerio), a vertebrate model used to easily study normal and pathological events in the nervous system [40]- [42]. A number of genetic models in zebrafish have been established for a range of neurodegenerative disorders, including ALS [43]- [49].
In this study, we examined the consequences of altering the expression of the NEFL orthologue (neflb) in zebrafish and established a direct link between neflb mRNA splicing modulations with an ALS-like phenotype (atrophy of motor axons and paralysis). We refined our understanding of this misbalance by ectopic expression of the two neflb isoforms, and also also explored neflb expression in a TDP-43 knockdown model.

MATERIAL AND METHODS
Zebrafish lines and microinjections. Wild-type and transgenic zebrafish embryos were raised at 28°C in embryo medium: 0.6 g/L aquarium salt (Instant Ocean, Blacksburg, VA) in reverse osmosis water containing 0.01 mg/L methylene blue. AB wild-type fish, and the transgenic lines Tg(Mnx1:eGFP) [50], Tg(elavl3:Gal4)zf349 -referred to as Tg(HuC:Gal4) - [51], Tg(Mnx1:Gal4) [52], Tg(5xUAS:RFP) nkuasrfp1a -referred to as Tg(UAS:RFP) - [53] have been used in this study. Zebrafish husbandry was performed according to approved guidelines. All procedures for zebrafish experimentation were approved by the Institutional Ethics Committee at the Research Center of the ICM and by French and European legislation.
neflb-eGFP constructs/cloning: pUCminus containing N-terminally eGFP-tagged zebrafish cDNAs of both neflb splice variants (neflbE3 and neflbE4) were purchased from Cliniscience. eGFP-neflbE3 and eGFP-neflbE4 were removed by restriction enzymes, and subcloned by ligation into pCS2 for ubiquitous expression in SW13 cells and p5e-10xUAS [54] for in-vivo expression in zebrafish motoneurons using the Tg(Mnx1:Gal4) trigger line. analyzed using the Manual Tracking plugin of ImageJ software, and the swim duration, swim distance, and maximum swim velocity of each embryo were calculated as previously described [44].

FACS.
Embryo dissociation was performed as previously described [55]. We then characterized the motor phenotype of each condition at 48 hpf by Touch Evoked Escape Response (TEER). Following a light touch, both uninjected and St Ctrol Mo injected embryos escaped and swam away from the center of the Petri dish all the way to the edges of the plate, whereas embryos injected with neflb SV Mo did not (Figure 2C), swimming distance being reduced by 89% (Figure 2F).
Although no developmental abnormalities were observed among conditions (Figure 2G), neflb SV Mo injection resulted in a very strong and specific motor phenotype (88% of embryos) characterized by the inability to swim ( Figure 2H). As no differences were observed between uninjected and St Ctrol embryos, only the later were used further in this study.
To determine whether the motor deficit observed was associated with cell death, Acridine Orange vital staining was used to detect apoptotic neurons. No major signal was detected in neflb SV Mo embryos compared to controls ( Figure 2I). There were also no significant differences in spinal cord thickness among the groups ( Figure 2L). Thus, we concluded that the abnormal expression of neflbE3 isoform doesn't affect neurons development.
The motor phenotype associated with the alteration of neflbE3 expression correlates with atrophy of motor axons. Previous work has shown a direct link between motor behavior deficits and disorganization of motor neuron morphology [44], [58]- [60]. Thus, we examined the somitic axonal projections of the motor neurons. Axonal projections from motor neurons exit the spinal cord grouped as one nerve fascicle per somite, then grow along the somitic muscle all the way to its most ventral part and then back up around it, while branching and connecting with muscle fibers. Mirror innervation of the dorsal part of the somitic muscle is achieved through the same process.
As shown in Figure 3A i, all somitic muscles appeared properly innervated in control embryos. However, neflb SV morphants displayed shortened and disorganized motor neuron nerve fascicles with major branching abnormalities (Figure 3A ii). Filament tracer plugin of the Imaris software was used to model the nerve fascicles in 3D and analyze the morphological defects ( Figure 3A i' and ii'). In neflb SV morphants, the somitic nerve fascicle main length was decreased by 46% as compared to controls ( Figure 3C).
Furthermore, axons were less branched ( Figure 3D) and innervating a reduced area on muscle (Figure 3E), confirming the strong and specific phenotype of the neflb SV morphants.
To refine our understanding of this phenotype, we performed in vivo time-lapse imaging over the first ten hours (starting from 16hpf) of muscle innervation by motor axons, in control and nefl SV morphants in parallel. As shown in Supplementary Movie 1 and in Figure 3E , motor axonal projections in nefl SV morphants appear to initiate at the same developmental stage as in the controls (Figure E a-b'), and they sprout in the right direction, ventrally towards the somitic muscles. However, as shown in Supplementary Movie 1, motor axons constantly grew back and forth, and never reached normal length ( Figure 3E a''-b'', Figure 3F).
neflbE4 participates in motor neuron axonal growth, whereas neflbE3 fails to polymerize normally and forms aggregates. In order to assess the assembly properties of neflbE4 and neflbE3 in vivo, we generated both UAS:eGFP-neflbE4 and UAS:eGFP- NEFM and NEFH and is a core protein necessary for the assembly of NF proteins into filamentous structures [61]. In order to determine the assembly competency of neflb variants, eGFP-neflbE4 or eGFP-neflbE3 were expressed alone or in combination with mouse NEFM or NEFH ( Figure 5A).
Neither neflbE4 nor neflbE3 assembled into filamentous structures on their own ( Figure   5A i-i'). Although eGFP-neflbE4 and eGFP-neflbE3 partially co-localized with NEFM suggesting an interaction, only eGFP-neflbE3 could form higher order structures (filaments and bundles) with NEFM ( Figure 5A ii'). Finally, neither of them formed filaments in presence of NEFH ( Figure 5A iii-iii').
Because zebrafish are poikilothermic organisms living at 28°C, neflb could have different assembly properties according to temperature, as previously shown for the lamprey Nefl [62]. Therefore, assembly of neflbE3 with NEFM was verified not only at 37°C, but also at 28°C (Figure 5B). At 28°C, neflbE3 was still able to form bundles of filament with NEFM, although less efficiently than at 37°C. Thus, the basic mechanisms of assembly and bundling of neflbE3 are operant at both temperatures. Only recently, the NEFL orthologue neflb has been characterized in zebrafish, and shown to be expressed in neurons [57]. Here, we described the existence of two mRNA splice variants of this gene that we called neflbE4 and neflbE3, which are differently expressed during zebrafish development. neflbE3 is the first isoform to be produced ubiquitously before being completely replaced by neflbE4, which is a neuron-specific isoform.

TDP
Interestingly, when neflbE3 isoform expression was forced to persist beyond its normal developmental window, the fish displayed a strongly compromised swimming motor behavior, which correlated with aberrant motor axons growth and ramification.
Importantly, these affected motor neurons present an abnormal assembly of neflbE3 protein consistent with the formation of abnormal bundles and aggregates when expressed together with NEFM in SW13 vim , human cell line lacking intermediate filaments commonly used to study de novo NF assembly.

NF stoichiometry:
The requirement for NEFL in the assembly of the other subunits, NEFM and NEFH, has been widely described [6], [63], [64]. Thus, Nefl KO mice exhibit motor neurons with no NFs and axons of smaller caliber [65], as well as functional consequences [66]. Furthermore NEFL mRNA is reduced in ALS motor neurons in autopsy specimens and is associated with altered NF triplet protein stoichiometry and neurofilamentous aggregation [30].
The relevance of disruption of NF stoichiometry has also been implicated in conditions of NEFL overexpression. Transgenic overexpression of Nefl in mice was sufficient to generate NF accumulation in motor neuronal perikarya and proximal axons, accompanied by axonal and dendritic degeneration [32].
On the contrary, the additional expression of NEFL was beneficial in a transgenic mouse overexpressing NEFH [67], reducing NF accumulation and axonal transport defects in a dose-dependent manner. Taken together, these results reinforce the importance of the TDP-43 and splicing: mRNA mis-splicing is a common pathological mechanism widely recognized in ALS. In particular, the effects of TDP-43 on RNA metabolism have been extensively studied due to the pivotal role that this protein plays in ALS physiopathology [68]- [72]. TDP-43 mutations are known to induce ALS clinical traits through mRNA missplicing in patients [73], [74]. Depletion of TDP-43 in cell culture can cause the abnormal splicing of cryptic exons and lead to toxicity [75]. Also, mice expressing TDP-43 mutated in its low-complexity domain suffered from neurodegeneration that correlated with the consistent skipping of constitutive exons normally spliced by wild-type TDP-43 (named skiptic exons) [72], [76]. In 2007, the direct stability-mediated effect of TDP-43 on NEFL mRNA was described [77]. In this study, we explored the influence of TDP-43 on neflb gene expression in zebrafish using an ALS model generated by Tardbp KD [43].

NEFL Splicing in human:
In terms of mammalian NEFL, two possible splice variants are predicted for this transcript (Ensembl, ENSG00000277586) [78], [79]. This prediction is corroborated by the presence of multiple NEFL bands on Western blots of neuronal tissues, but they remain poorly characterized [80]. All together, the possible existence of a physiological or pathological splicing regulation of human NEFL and relevance to disease should be explored in light of the results of this study in zebrafish.