The stress-regulated protein M6a is a key modulator for neurite outgrowth and filopodium/spine formation

Abstract

Neuronal remodeling is a fundamental process by which the brain responds to environmental influences, e.g., during stress. In the hippocampus, chronic stress causes retraction of dendrites in CA3 pyramidal neurons. We have recently identified the glycoprotein M6a as a stress-responsive gene in the hippocampal formation. This gene is down-regulated in the hippocampus of both socially and physically stressed animals, and this effect can be reversed by antidepressant treatment. In the present work, we analyzed the biological function of the M6a protein. Immunohistochemistry showed that the M6a protein is abundant in all hippocampal subregions, and subcellular analysis in primary hippocampal neurons revealed its presence in membrane protrusions (filopodia/spines). Transfection experiments revealed that M6a overexpression induces neurite formation and increases filopodia density in hippocampal neurons. M6a knockdown with small interference RNA methodology showed that M6a low-expressing neurons display decreased filopodia number and a lower density of synaptophysin clusters. Taken together, our findings indicate that M6a plays an important role in neurite/filopodium outgrowth and synapse formation. Therefore, reduced M6a expression might be responsible for the morphological alterations found in the hippocampus of chronically stressed animals. Potential mechanisms that might explain the biological effects of M6a are discussed.

Fig. 1.

Adult rat hippocampus. M6a mRNA localizes to somata of granule and pyramidal neurons, whereas M6a protein is detected in neuronal processes. M6a mRNA expression in the hippocampus as determined by in situ hybridization (A) was found to be localized to the granule cell layer (gcl) of the dentate gyrus and pyramidal neuron cell bodies (stratum pyramidale, pyr) of the CA fields (CA1, CA3). (Scale bar, 1 mm.) In contrast, immunocytochemical detection of M6a protein (B) revealed a distinct absence of M6a expression within the granule cell layer and cell bodies of pyramidal neurons (pyr). M6a expression was rather detected in regions of relatively dense synaptic contact, including the hilus (h), granule cell mossy fiber terminals (mf), and the molecular layer (ml) of the dentate gyrus. Moderate staining was also evident in the stratum oriens (or) and stratum radiatum (rad). (Scale bar, 0.5 mm.)

Fig. 2.

Localization in cultured neurons: both M6a endogenous protein and GFP::M6a fusion protein are present in membrane and filopodia. (A) Immunocytochemical staining of cultured neurons revealed that M6a protein (green) is localized to somatic membrane, along processes and filopodia (B). Staining with the actin marker phalloidin (red) revealed filopodial structures. (C) Hippocampal cultures were transfected with GFP::M6a construct and overexpressed M6a (green) is also sorted to processes, both to dendrites (a) and axons (b) as confirmed with the respective protein markers MAP2 and Tau-1 (red). Fusion protein is restricted to plasma membrane, as shown in a single-frame picture (0.37 μm slice) of the soma (c) and highly enriched in filopodia (d). (Scale bars, 10 μm.)

Fig. 3.

M6a overexpression induces neurite outgrowth and increases filopodium/spine density. Neurons were transfected 1 day after plating with either GFP (control, A) or GFP::M6a constructs (A′), fixed, and stained with antibodies against α-tubulin 2-3 days later. Primary neurites per cell were quantified and mean values +SEM of 40-50 cells per group from three independent experiments are shown in B. Cultures from 7 days in vitro were transfected with GFP::M6a and stained with the membrane dye DiI 2-3 days later. (C and D) Control. (C′ and D′) M6a overexpression. Filopodium/spine density (number of protrusions per 20-μm dendrite length) as shown in D and D′ was quantified and mean +SEM of 65-75 neurons per group from three independent experiments are shown in E as a percentage of control. **, P < 0.001, Mann-Whitney U test, two-tailed. (Scale bars, 10 μm.)

Fig. 4.

Characterization of membrane protrusions induced by M6a overexpression. Neurons between 7 and 9 days in vitro were transfected with GFP::M6a, and fixed 1 or 2 days later. M6a-induced protrusions (green) are observed in dendrites stained with MAP2 (A), are rich in F-actin (B), and contain spinophilin clusters (C). Passing axons, stained with Tau-1, appear to make contact with M6a-promoted protrusions (D). Synaptophysin clusters are observed in contact sites between axons and dendritic protrusions of M6a overexpressing neurons (E). E′ shows higher magnifications of the boxes indicated in E; the arrow points out an example of presynaptic synaptophysin clusters over dendritic filopodia. (Scale bars, 10 μm.)

Fig. 5.

RNA interference reduces M6a mRNA and protein levels, resulting in a lower number of filopodia/spines and synaptophysin immunoreactive clusters. (A) Cultures from 9-10 days in vitro were siRNA-treated with M6a or control sequences. M6a siRNA-treated cultures showed lower M6a transcript levels (measured with real-time RT-PCR, values normalized with the gene β-actin), lower filopodium/spine density (number of protrusions per 20-μm dendritic length, quantified in 75-95 neurons per group from two independent experiments), and lower number of synaptophysin immunopositive puncta (counted in a 20-μm fragment of dendrites measured from the neuronal body for 55-60 cells per group from two independent experiments). Mean values +SEM are expressed as a percentage of control. Representative pictures of control (B-E) and M6a siRNA-treated cells (B′-E′) stained with the antiM6a antibody (B and B′), membrane-labeled with DiI (C and C′) or immunostained with the antibody against synaptophysin (D and E). E and E′ are higher magnifications of the areas indicated in D and D′. Significant differences: **, P < 0.0001, Mann-Whitney U test, two-tailed. (Scale bars, 10 μm.)