Dysregulation of NFM levels leads to uncontrolled axon out growth

NFM is a type IV neuronal intermediate filament, expressed abundantly in the cytosol and along the axons. It is thought to be responsible for regulating radial axon growth (Cleveland et al., 1991; Li et al., 2012). However, its role in regulation of longitudinal axon growth is understudied. In order to establish the role of NFM in axon growth regulation in CGNs, I first established the temporal expression of NFM in the cerebellum at various postnatal ages. I subjected cerebellar lysates from various postnatal ages to immunoblot analysis with an NFM-specific antibody. Surprisingly, I observed that NFM is upregulated and highly

expressed at developmental stages crucial for neurite outgrowth and lengthening of axons, rather than radial axonal growth (Figure 3.32).

Figure 3.32 NFM is expressed in the developing cerebellum of mice: Cerebellar lysates from indicated ages were analyzed by immunoblotting with the NFM and γ–tubulin antibodies where the latter served as loading control.

Owing to its predominant axonal localization and robust expression during cerebellar development, I used cultured CGNs as a model system to investigate if NFM had any effects on axonal length. Taking a gain-of-function approach first, I transfected CGNs at DIV1 with either control or and NFM overexpression plasmids. Upon morphometric analysis of the axonal lengths of the transfected neurons at DIV 5, I observed that excess of NFM led to remarkably increased axonal length when compared to control neurons (Figure 3.33 A, B).

Figure 3.33 NFM promotes axon growth: (A) Representative images of rat CGNs that were transfected at DIV1 with either the control vector or the NFM expression plasmid, and subjected to axon growth assays (Materials and methods section 2.15) at DIV5. Arrows indicate axons. Scale bar equals 100 µm.

(B) Quantifications of the axonal lengths from A. The average axonal length is represented in µms. All measurements and quantifications were done in a blinded manner. A total of 254 neurons from 3 independent experiments were measured (Student’s t-test, ***p<0.001, mean + s.e.m.).

Given that overexpression of NFM results in increased axon growth, I wondered if loss of NFM would have the opposite effect. Hence, I first generated a short hairpin RNAi that specifically targets and knocks down NFM (NFM RNAi#5). In addition to the functional RNAi (NFM RNAi#5), I also generated an NFM-specific non-functional shRNA (NFM RNAi#3), which was unable to knockdown NFM. I validated the shRNAs using HEK293T cells transiently transfected with myc-NFM expression plasmids (Figure 3.34 A). I further

confirmed the efficiency of the RNAi by knocking down endogenous NFM in cultured CGNs (Figure 3.34 B).

Figure 3.34 Knockdown of NFM: (A) Lysates from HEK293T cells, transfected with the myc-NFM plasmid together with control, the functional NFM RNAi#5 or the non-functional NFM RNAi#3 plasmid, were lysed and immunoblotted using the myc and γ–

tubulin antibodies. The latter served as loading control. (B) Representative images of rat CGNs at DIV1 transfected with control vector, the functional NFM RNAi#5 or the non-functional NFM RNAi#3 plasmid together with the transfection marker GFP. Neurons were subjected to immunocytochemistry at DIV5 using the GFP and NFM antibodies. Scale bar equals 100 µm.

I observed that while the functional NFM RNAi#5 robustly knocked down NFM, the non-functional NFM RNAi#3 had no effect, both in HEK293T cells as well as in CGNs.

To determine the effect of loss of NFM on axonal length, I transfected cultured CGNs at DIV1 with either control U6, the functional NFM RNAi#5 or the non-functional NFM RNAi#3 plasmids and subjected the transfected cells to morphometric analyses four days later. Surprisingly, I observed that loss of NFM resulted in a pronounced increase of axon length when compared to the controls. The non-functional NFM RNAi#3 on the other hand had no effect on axon length and hence served as an additional negative control (Figure 3.35 A, B). While loss of NFM resulted in increased axonal length, it had no effect on primary axon branching and dendrite length (Figure 3.35 C, D).

Figure 3.35 Loss of NFM leads to uncontrolled axon growth: (A) Representative images of rat CGNs that were transfected at DIV1 with either control vector, the functional NFM RNAi#5 or the non-functional NFM RNAi#3 plasmids together with the transfection marker GFP and subsequently subjected to axon growth measurements at DIV5. Arrows indicate axons. Scale bar equals 100 µm unless indicated otherwise. (B) Graph represents the quantifications of axon lengths from A. The average axonal lengths are represented in µms. (C) The total dendrite length of the neurons from A was measured, represented in µm. (D) Moreover, the primary axonal branches of the neurons from A were quantified. (B, C, D) All measurements and quantifications were done in a blinded manner. A total of 348 neurons from 3 independent experiments were measured (ANOVA, ***p<0.001, mean + s.e.m.).

In order to rule out possible off-target effects of the NFM RNAi, I performed a rescue experiment, wherein we first generated an RNAi-resistant myc-NFM expression vector (NFM-Res), which harbored four silent mutations in the region targeted by the functional NFM RNAi#5 (Figure 3.36 A). The efficiency of rescue or resistance to knockdown by the myc-NFM-Res expression plasmid was validated by co-transfecting myc-NFM expression plasmids or myc-NFM-Res plasmid together with the functional NFM RNAi#5 in HEK293T cells, followed by immunoblotting analysis. While the NFM RNAi#5 efficiently knocked

generation and validation of the NFM-Res construct, I transfected CGNs at DIV1 with either control U6 vector or NFM RNAi#5 together with either control or NFM-Res plasmids. Four days later at DIV5, morphometric evaluation of the transfected neurons revealed that while loss of NFM resulted in increased axon length, co-expression of the NFM-Rescue plasmid together with the NFM RNAi#5 nullified this effect and restored axon lengths back to baseline levels (Figure 3.36 B, C).

Figure 3.36 NFM knockdown also promotes axon growth: (A) 50 µg of lysates harvested from HEK293T cells, transfected with control, the myc-NFM or myc-NFM-Rescue (Res) plasmid together with control vector or the NFM RNAi#5 plasmid, were immunoblotted with the myc or pan 14-3-3 antibodies.

The latter served as loading control. Schematic on the right shows the NFM RNAi#5 targeting region with the silent mutations indicated in red. (B) Quantifications of rat CGN axonal lengths, that were transfected at DIV1 with control, NFM RNAi#5 together with control or the NFM-Res plasmids, were subjected to axon growth assay at DIV5. All measurements and quantifications were done in a blinded manner. A total of 369 neurons from 3 independent experiments were measured. (ANOVA, ***p<0.001, mean + s.e.m.).

(C) Representative images of transfected neurons from B. Arrows indicate axons. Scale bar equals 100 µm.

Collectively these results indicate that dysregulation of NFM protein levels leads to increased axonal lengths, as both excess and reduced NFM levels results in axon growth promotion.