Investigation of an amyotrophic lateral sclerosis-associated profilin 1 mutant

Amyotrophic lateral sclerosis (ALS) is a devastating disease that has no cure. Mutations in profilin 1 were identified as a cause of familial ALS in 2012. We investigated the impact of expressing mutant profilin 1 in cultured primary neurons and in spinal cords during mouse development. Our data from studies using transfected primary hippocampal mouse neurons show that profilin 1 C71G expression results in increased dendritic spine density. These results show that expression of profilin 1 C71G alters cell morphology. We developed a novel mouse model with expression of PFN1 C71G targeted to α-motor neurons in the spinal cord during development. Adult mice had no transgene expression. When the embryos of these mice were analysed, they showed evidence of reduced neuronal outgrowth and abnormal brain development. Adult mice had no transgene expression. These mice presented with motor deficits and a reduction in the number of motor neurons in the spinal cord. Further analysis of these motor neurons in these mice revealed reduced levels of TDP and ChAT. Although profilin 1 C71G was only expressed during development, adult mice presented with some ALS-associated pathology and motor symptoms. This study highlights the effect of profilin 1 during neurodevelopment and the impact that this may have in amyotrophic lateral sclerosis

A novel microfluidic device-based neurite outgrowth inhibition assay reveals the neurite outgrowth-promoting activity of tropomyosin Tpm3.1 in hippocampal neurons

Overcoming neurite inhibition is integral for restoring neuronal connectivity after CNS injury. Actin dynamics are critical for neurite growth cone formation and extension. The tropomyosin family of proteins is a regarded as master regulator of actin dynamics. This study investigates tropomyosin isoform 3.1 (Tpm3.1) as a potential candidate for overcoming an inhibitory substrate, as it is known to influence neurite branching and outgrowth. We designed a microfluidic device that enables neurons to be grown adjacent to an inhibitory substrate, Nogo-66. Results show that neurons, overexpressing hTpm3.1, have an increased propensity to overcome Nogo-66 inhibition. We propose Tpm3.1 as a potential target for promoting neurite growth in an inhibitory environment in the central nervous system

Functional characterisation of filamentous actin probe expression in neuronal cells

<div><p>Genetically encoded filamentous actin probes, Lifeact, Utrophin and F-tractin, are used as tools to label the actin cytoskeleton. Recent evidence in several different cell types indicates that these probes can cause changes in filamentous actin dynamics, altering cell morphology and function. Although these probes are commonly used to visualise actin dynamics in neurons, their effects on axonal and dendritic morphology has not been systematically characterised. In this study, we quantitatively analysed the effect of Lifeact, Utrophin and F-tractin on neuronal morphogenesis in primary hippocampal neurons. Our data show that the expression of actin-tracking probes significantly impacts on axonal and dendrite growth these neurons. Lifeact-GFP expression, under the control of a pBABE promoter, caused a significant decrease in total axon length, while another Lifeact-GFP expression, under the control of a CAG promoter, decreased the length and complexity of dendritic trees. Utr261-EGFP resulted in increased dendritic branching but Utr230-EGFP only accumulated in cell soma, without labelling any neurites. Lifeact-7-mEGFP and F-tractin-EGFP in a pEGFP-C1 vector, under the control of a CMV promoter, caused only minor changes in neuronal morphology as detected by Sholl analysis. The results of this study demonstrate the effects that filamentous actin tracking probes can have on the axonal and dendritic compartments of neuronal cells and emphasise the care that must be taken when interpreting data from experiments using these probes.</p></div

Soluble LILRA3 promotes neurite outgrowth and synapses formation through a high-affinity interaction with Nogo 66

Inhibitory proteins, particularly Nogo 66, a highly conserved 66-amino-acid loop of Nogo A (an isoform of RTN4), play key roles in limiting the intrinsic capacity of the central nervous system (CNS) to regenerate after injury. Ligation of surface Nogo receptors (NgRs) and/or leukocyte immunoglobulin-like receptor B2 (LILRB2) and its mouse orthologue the paired immunoglobulin-like receptor B (PIRB) by Nogo 66 transduces inhibitory signals that potently inhibit neurite outgrowth. Here, we show that soluble leukocyte immunoglobulin-like receptor A3 (LILRA3) is a high-affinity receptor for Nogo 66, suggesting that LILRA3 might be a competitive antagonist to these cell surface inhibitory receptors. Consistent with this, LILRA3 significantly reversed Nogo-66-mediated inhibition of neurite outgrowth and promoted synapse formation in primary cortical neurons through regulation of the ERK/MEK pathway. LILRA3 represents a new antagonist to Nogo-66-mediated inhibition of neurite outgrowth in the CNS, a function distinct from its immune-regulatory role in leukocytes. This report is also the first to demonstrate that a member of LILR family normally not expressed in rodents exerts functions on mouse neurons through the highly homologous Nogo 66 ligand.12 page(s

Sholl analysis of dendritic complexity of mouse hippocampal neurons transfected with filamentous actin tracking probes.

<p><b>Dendritic complexity was</b> measured as number of intersections per shell as a function of distance from the soma. A Neurons transfected with Lifeact-GFP(1) displayno significant changes in dendritic compliexity compared to EGFP control. Lifeact-GFP(2) transfected neuron consistently had the lowest number of intersections per shell from 0–60 μm from the soma. Lifeact-EGFP transfected neurons showed an increase in dendritic complexity from 0–20 μm and a decrease from 20–60 μm compared to EGFP control. Lifeact-7-mEGFP transfected neurons showed an increase in dendritic complexity from 0–30 μm and a significant decrease from 30–40 μm. Utr261-EGFP also showed an increase in dendritic complexity from 0–30 μm. F-tractin-EGFP transfection resulted in increased dendritic complexity at 0–20 μm from the cell soma. Between 17 and 47 cells, collected from at least 3 biological replicates, were analysed per construct. Error bars represent standard error of the mean. Significance was determined by two-way ANOVA with Tukey's test for multiple corrections. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Significant difference between EGFP and each actin tracking probe is indicated by the following symbols: Lifeact-GFP(2) = #, Lifeact-EGFP = §, Lifeact-7-mEGFP = £, Utr-261-EGFP = ⁺, F-tractin-EGFP = ‡.</p

Effects of filamentous actin tracking probes on the morphology of primary mouse hippocampal neurons [EGFP control and Lifeact-GFP(1)].

<p>Representative images of neurons transfected with EGFP (A-D) or Lifeact-GFP(1) (E-H). (A, E) expressed probes; (B, F) axonal marker Tau1; (C, G) pan-neuronal β3-tubulin; (D, H) merged images. Scale bar = 50μm.</p

Effects of filamentous actin tracking probes on the morphology of primary mouse hippocampal neurons [Lifeact-GFP(2) and Lifeact-EGFP].

<p>Representative images of neurons transfected with Lifeact-GFP(2) (A-D) or Lifeact-EGFP (E-H). (A, E) expressed probes; (B, F) axonal marker Tau1; (C, G) pan-neuronal β3-tubulin; (D, H) merged images. Scale bar = 50μm.</p

Quantitative analysis of axonal morphology at DIV3 after transfection with EGFP, Lifeact-GFP(1), Lifeact-GFP(2), Lifeact-EGFP, Lifeact-7-mEGFP, Utr261-EGFP and F-tractin-EGFP expressing constructs.

<p>Neurons transfected with Lifeact-GFP(1) show decreased total axon length (A), decreased primary axon total length (B) and decreased total length of primary axon branches (D), compared to EGFP control. Neurons transfected with Lifeact-EGFP and Utr261-EGFP show also show decreased primary axon total length (B). Neurons transfected with Lifeact-GFP(2), Lifeact-7-mEGFP and F-tractin-EGFP did not show any significant changes in axonal morphology when compared to EGFP control (A-F). Between 17 and 47 cells, collected from at least 3 biological replicates, were analysed per construct. Error bars represent standard error of the mean. Significance was determined by Kruskal-Wallis Test (non-parametric one-way ANOVA) and Dunn's multiple corrections test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.</p

Summary of quantitative morphological analysis.

<p>Summary of quantitative morphological analysis.</p

Quantitative analysis of dendritic morphology at DIV3 after transfection with EGFP, Lifeact-GFP(1), Lifeact-GFP(2), Lifeact-EGFP, Lifeact-7-mEGFP, Utr261-EGFP and F-tractin-EGFP expressing constructs.

<p>Compared to EGFP control, neurons transfected with Lifeact-GFP(2) showed significant decreases in the total length of dendritic trees (B), the mean length of primary dendritic shaft (C), the mean length of primary dendritic branches (E) and the mean length of dendritic trees (H). Neurons transfected with Utr261-EGFP showed an increase in the number of primary dendritic branches (D) and mean number of primary dendritic branches per dendritic tree per cell (G), compared to EGFP control. Neurons transfected with Lifeact-GFP(1), Lifeact-EGFP, Lifeact-7-mEGFP and F-tractin-EGFP did not show any significant changes in dendritic morphology when compared to EGFP control (A-H) Between 17 and 47 cells, collected from at least 3 biological replicates, were analysed per construct. Error bars represent standard error of the mean. Significance was determined by Kruskal-Wallis Test (non-parametric one-way ANOVA) and Dunn's multiple corrections test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.</p