Fine-tuning of neuronal architecture requires two profilin isoforms

Abstract

Two profilin isoforms (PFN1 and PFN2a) are expressed in the mammalian brain. Although profilins are essential for regulating actin dynamics in general, the specific role of these isoforms in neurons has remained elusive. We show that knockdown of the neuron-specific PFN2a results in a significant reduction in dendrite complexity and spine numbers of hippocampal neurons. Overexpression of PFN1 in PFN2a-deficient neurons prevents the loss of spines but does not restore dendritic complexity. Furthermore, we show that profilins are involved in differentially regulating actin dynamics downstream of the pan-neurotrophin receptor (p75(NTR)), a receptor engaged in modulating neuronal morphology. Overexpression of PFN2a restores the morphological changes in dendrites caused by p75(NTR) overexpression, whereas PFN1 restores the normal spine density. Our data assign specific functions to the two PFN isoforms, possibly attributable to different affinities for potent effectors also involved in actin dynamics, and suggest that they are important for the signal-dependent fine-tuning of neuronal architecture.

Fig. 1.

Pruning of dendrites in shPFN2a-expressing cells. Hippocampal slice cultures were biolytically transfected with shPFN2a (A) or fGFP (B) at 7 DIV, and CA1 neurons were imaged at the indicated time points. Five days posttransfection (dpt), pruning of previously existing apical dendrites was observed in shPFN2a-expressing cells (arrows) that was not detectable in neurons transfected with fGFP. Changes in dendritic structure remained stable up to dpt 9. (C) Dendritic loss was quantified by measuring the first ≈400 μm of the apical dendrite at 1 and 9 dpt. Profilin2a-deficient neurons lost 32% of dendritic length, whereas the length of control cells was unaltered during the imaging period. (***P < 0.001) (Scale bar: A, 50 μm.)

Fig. 2.

Spine density is reduced in shPFN2a-transfected CA1 neurons. (A) Dendritic complexity of basal and apical dendrites was compared between control neurons (n = 25) and shPFN2a-transfected cells (n = 11) using Sholl analysis. (B) High-resolution images of representative basal dendrites of cells in organotypic cultures expressing fGFP or shPFN2a. (C) Spine density of control cells as well as cells expressing shPFN2a. *P < 0.05; **P < 0.005. (Scale bar: 5 μm.)

Fig. 3.

PFN1 cannot rescue the shPFN2a-dependent reduction in dendritic complexity. Sholl analysis (A) and spine density (A1) of CA1 pyramidal neurons in organotypic slice cultures expressing a polycistronic construct containing shPFN2a and PFN2a-mod, an RNAi-resistant PFN2a mutant (PFN2a mod, n = 17). Sholl analysis (B) and spine density (B1) of CA1 pyramidal neurons in organotypic slice cultures expressing a polycistronic construct containing shPFN2a and PFN1 (PFN1, n = 16). Remarkably, coexpression of PFN1 does not prevent the significant reduction in dendritic complexity induced by the knockdown of PFN2a but it prevents the reduction in spine density. *P < 0.05, **P < 0.005.

Fig. 4.

PFN2a but not PFN1 can compensate for p75^NTR-dependent dendritic loss in primary hippocampal neurons. Neurolucida reconstructions of primary hippocampal neurons (21 DIV) expressing fcherry. (A) Control cell expressing fcherry only and a hippocampal neuron transfected with p75^NTR and fcherry. (B) Histograms showing the number of dendritic endings and spine density of neurons expressing only fcherry as a control, p75^NTR, PFN2a, or PFN2a and p75^NTR. (C) Histograms showing the number of dendritic endings and spine density of cells transfected with fcherry as a control, p75^NTR, PFN1, or PFN1 and p75^NTR. *P < 0.05; **P < 0.01; ***P < 0.001. (Scale bar: 100 μm.)

Fig. 5.

Proposed model for the action of both profilin isoforms in different neuronal compartments. Interactions of PFN1, PFN2a, and p75^NTR shape dendritic and spine morphology during processes of synaptic plasticity. The three proteins react with different signaling pathways to regulate actin assembly. PFN1 preferentially binds to PIP2 (26), PFN2a binds with the formin mDia2 (this study), and both interactions stimulate actin assembly, but the actin assembly is directed to different neuronal compartments (single arrows). In contrast, p75^NTR negatively controls the ROCK pathway (14, 15), which modulates spine density and dendritic complexity. Both profilin isoforms and p75^NTR communicate with each other (this study, double arrows), and can thus influence each of the pathways. For reasons of clarity, the known direct interactions between profilins and members of the ROCK signaling cascade (RhoA and ROCK) are not included in this scheme.