Mossy fiber expression of αSMA in human hippocampus and its relevance to brain evolution and neuronal development

Abstract

α-Smooth muscle actin (αSMA) is best characterized as the building block of thin filaments in smooth muscle cells. We observed a clear αSMA immunolabeling in adult human hippocampal mossy fibers (MF), prompting us to explore this novel pattern in phylogenic and ontogenic perspectives in the present study. αSMA immunolabeling occurred distinctively at the hippocampal MF terminals in humans from infancy to elderly. Hippocampal MF αSMA immunolabeling was not observed in mice and rats, visible in CA3 in guinea pigs and cats, and prominent in CA3 and dentate hilus in Rhesus monkeys. MF αSMA immunolabeling in human hippocampus emerged and refined from the last gestational trimester to early infancy. A transient overall neuronal labeling of ɑSMA was observed in prenatal human brains. ɑSMA expression was detected in human and rat primary neuronal cultures. The specificity of ɑSMA antibodies was confirmed by ACTA2 small interfering RNA (siRNA) silencing in SH-SY5Y cells. With this validation, we detected a higher αSMA protein level in dentate gyrus lysates relative to other human brain areas. Taken together, αSMA is distinctly expressed in human hippocampal mossy fibers. This pattern is related to hippocampal evolution among mammals and involves a refinement of neuronal αSMA expression during brain development.

Keywords: Actin isoforms; Brain development; Mammalian evolution; Synaptoplasticity; Trisynaptic circuit.

Fig. 1

Immunolabeling of α-smooth muscle actin (αSMA) in hippocampal mossy fibers (MF) with densitometry relative to age. Antibody information, counterstain, ages of the brain donors, scale bar and quantitative methodology are included in the figure. (A) Concurrent ɑSMA immunolabeling by the goat antibody in human temporal lobe and adult rat brain sections, with enlarged panels illustrating the antibody labeling in human (A1) but not rat (A2, A3) hippocampal MF. (B) Mossy fiber ɑSMA immunolabeling by a rabbit antibody, enlarged panels showing simultaneous labeling in vascular smooth muscle cells (B1), the transition pattern of MF labeling at the border of CA3/CA2 (B2), and a lack of fiber-like labeling in the prosubiculum (B3). (C) Distribution of ɑSMA labeled MF across the anteroposterior axis of the hippocampal formation, with sections passing the head, body and tail of the hippocampus indicated. (D) Densitometry of αSMA labeling in the MF field (hilus and CA3) relative to other temporal lobe areas. The specific optic density (o.d.) in the areas of interest are calculated using a threshold o.d. obtained from the white matter region or from an adjacent section immunostained by excluding the primary antibody. (E, F) Graphic and statistical analysis of data from 28 postmortem brains, donor’s ages 1–76 years. *: Significantly different according to posthoc test. Additional abbreviations: FG: fusiform gyrus; PHG: parahippocampal gyrus; Ent: entorhinal cortex; Sub: subiculum, Pro-S: prosubiculum, Pre-S: presubiculum; Para-S: parasubiculum; ML: molecular layer; GCL: granule cell layer; s.o.: stratum oriens; s.p.: stratum pyramidale; s.r.: stratum radiatum; s.l.m.: stratum lacunosum-moleculare; mf: mossy fiber; WM: white matter.

Fig. 2

Double immunofluorescent characterization of αSMA labeling in the human hippocampal mossy fibers and vascular cells. Information about antibody combination, fluorescent indicators, image panel arrangement and scale bars are provided. A complete colocalization of ɑSMA and β-secretase 1 (BACE1) labeling is seen at the mossy fiber terminals (mf) (A, A1, A2, A3). Note the unlabeled mossy cells (*) in the hilus. (B, B1, B2, B3) αSMA labeled MF terminals occur in close proximity to the thorny excrescences of sortilin labeled hilar mossy cells (*) and CA3 pyramidal neurons. (A3, B3) αSMA immunolabeling of the nucleated pericytes at capillaries and small vascular profiles. (C) Typical ɑSMA labeling in the smooth muscle cells in the tunica media (TM) in a section of the basal artery. The internal elastic laminae (IEL) exhibits autofluorescence through the blue and green fluorescence channels. Other abbreviations are as defined in Fig. 1.

Fig. 3

Evolutionary trend of αSMA labeling in hippocampal mossy fibers among mammals. Shown are low power and enlarged images of αSMA immunolabeling (rabbit antibody) with hematoxylin counterstain in temporal lobe sections from the species as indicated, along with BACE1 labeling in adjacent sections for comparison. αSMA labeling of the MF is not recognizable in mouse (A) and in rat as illustrated in Fig. 1A. However, lightly stained MF terminals (pointed by arrowheads) are seen in the CA3 area but not in the dentate hilus in guinea pig (C) and cat (E). In monkey (G) and human (I) temporal lobe sections, the MF terminals in both CA3 and the hilus are clearly labeled by the ɑSMA antibody. In comparison, BACE1 labeled MF are present in CA3 and the hilus in all species (B, D, F, H, J). Small and large blood vessels (pointed by arrows) are labeled by the αSMA antibody in all species. The quantitative data of aSMA labeling densities from the species are illustrated as panels (A1, C1, E1, G1 and I1), with the specific optic density (o.d.) calculated by subtracting the background cutoff measured outside the section covered area in the same image. Scale bars are indicated in the panels. Abbreviations are as defined in Fig. 1.

Fig. 4

Developmental trend of neuronal and hippocampal mossy fiber αSMA immunolabeling in human brains. The left and right panel groups show low magnification images and enlarged fields of temporal lobe sections immunolabeled by the rabbit and goat antibodies to αSMA. Gestational weeks (GW) and ages of brain donors are indicated on the left. αSMA immunolabeling occurs in the cortical grey matter, heavier in layers II/III relative to IV-VI, and occurs in the hippocampal cellular layers in the 30GW, 36 GW cases (A, B, C, D), with labeled cellular profiles seen in the enlarged panels (A1, A2, B1, B2, C1, C2, D1, D2). This cellular labeling is reduced in the newborn case (E1, E2, F1, F2), sparsely seen in the infant case (G1, G2, H1, H2), and not present in the youth case (I1, I2, J1, J2). αSMA labeling of the MF emerges in the dentate gyrus and CA3 in mix with local cellular labeling in the fetal and newborn brains and appears as heavier and distinct terminal labeling against little cellular labeling in the infant and youth cases. The two antibodies exhibit noticeable differences regarding the cellular labeling, with the goat antibody having a high background reactivity over the sections. Quantitative values (mean ± S.D. %) from the 28 GW-28 days (d), 6 months (m) to 1 year (y) and 8–22 y groups, respectively, are the following: 100 ± 29.7, 61.2 ± 29.4 and 47.4 ± 15.5 for rabbit antibody labeling in the temporal neocortex; 59.7 ± 16.0, 70.1 ± 15.9 and 100 ± 19.9 for rabbit antibody labeling in MF field; 100 ± 17.3, 59.6 ± 9.9 and 59.0 ± 29.3 for goat antibody labeling in the temporal neocortex; 85.6 ± 13.4, 76.7 ± 5.9 and 100 ± 12.6 for goat antibody labeling in MF field. Statistics are as indicated in the graphs. Abbreviations are as defined in Fig. 1.

Fig. 5

Immunohistochemical labeling of β-secretase 1 (BACE) and zinc transporter 3 (ZnT3) in temporal lobe sections from prenatal, infant and youth human brains. Image panels show the developmental pattern of BACE1 (A, B, C, D, D1, E, F, G, H) and ZnT3 (I, J, K, L, M, N, O, P) immunolabeling among the temporal lobe sections at low magnification, with enlarged panels (D1, E1, L1, N1) show the details of MF labeling in the dentate gyrus and CA3. BACE1 labeling appears to increase progressively from the prenatal to infant cases, while ZnT3 labeling appears to increase mainly after birth. A clearly ZnT3 labeled band is located in the inner molecular layer (iML) of the dentate gyrus (L1, N1). Quantitative values (mean ± S.D. %) from the 28 GW-28 days (d), 6 months (m) to 1 year (y) and 8–22 y groups, respectively, are the following: 44.3 ± 9.6, 52.6 ± 16.0 and 82.4 ± 8.5 for BACE1 labeling in the temporal neocortex; 80.4 ± 17.9, 96.3 ± 3.6 and 100 ± 10.2 for BACE1 labeling in the MF field; 39.3 ± 23.2, 92.1 ± 13.9 and 92.7 ± 7.8 for ZnT3 labeling in the temporal neocortex; 45.2 ± 20.4, 93.7 ± 20.4 and 100 ± 8.1 for ZnT3 labeling in the MF field. Statistical reports are included in the graphs. Additional abbreviations are as defined in main Fig. 1.

Fig. 6

Immunofluorescent characterization of αSMA expression in cultured human and rat primary cortical neurons. Shown are double immunofluorescence for αSMA and neuronal markers with DAPI counterstain in morphologically differentiated neurons fixed following 10 and 5 days in vitro (DIV). αSMA immunofluorescence is colocalized with that of microtubule associated proteins 2 (MAP2) (A, C), β-tubulin (B) and neuron-specific nuclear antigen (NeuN) (D) in the somata and processes of the primary neurons. Scale bars are indicated in the left panels.

Fig. 7

Verification of ɑSMA antibody specificity with ACTA2 gene silencing method and assessment of ɑSMA protein levels in human dentate gyrus relative to control regions. (A) Western blot detection of αSMA, β-tubulin, F-actin and microtubule associated protein 2 (MAP2) in lysates from a representative set of cultured SY-SH5Y cells in the untreated (blank) (−/−), ACTA2 siRNA treated (+/−) and negative control siRNA treated (−/+) groups. The αSMA signal is greatly reduced in the ACTA2 siRNA treated cells, whereas that of the β-tubulin, F-actin and MAP2 proteins are comparable between the cell groups. (B) Quantitation from 3 separate culture experiments. (C) Microdissection of the dentate gyrus (DG), temporal neocortical (TC), frontal neocortical (FC) and basal artery (BA) samples from postmortem human brain. (D) Representative membrane images showing the optimization of the loading amounts of human frontal cortical and BA extracts to blot αSMA protein. (E, F) Representative immunoblot images showing the detection of αSMA relative to reference proteins in the regional brain and BA samples., with the amount of sample loading indicated. Note the increased signal in DG relative to FC, TC and cerebellar cortical (CBL) lysates (E, G), and similar levels of collagen IV and neuron-specific nuclear antigen (NeuN) in these regional samples (F). (H, I) Immunoblot images and quantification showing a higher αSMA protein level in the DG than TC lysates from an additional set of brains (n = 4) blotted with the rabbit and goat αSMA antibodies, respectively. Also note that the levels of GAPDH are reduced or become undetectable in the BA extracts with the dilution of sample loading (D, E, F, H). *: Significantly different per posthoc test.