Serum Response Factor (SRF)-cofilin-actin signaling axis modulates mitochondrial dynamics

Abstract

Aberrant mitochondrial function, morphology, and transport are main features of neurodegenerative diseases. To date, mitochondrial transport within neurons is thought to rely mainly on microtubules, whereas actin might mediate short-range movements and mitochondrial anchoring. Here, we analyzed the impact of actin on neuronal mitochondrial size and localization. F-actin enhanced mitochondrial size and mitochondrial number in neurites and growth cones. In contrast, raising G-actin resulted in mitochondrial fragmentation and decreased mitochondrial abundance. Cellular F-actin/G-actin levels also regulate serum response factor (SRF)-mediated gene regulation, suggesting a possible link between SRF and mitochondrial dynamics. Indeed, SRF-deficient neurons display neurodegenerative hallmarks of mitochondria, including disrupted morphology, fragmentation, and impaired mitochondrial motility, as well as ATP energy metabolism. Conversely, constitutively active SRF-VP16 induced formation of mitochondrial networks and rescued huntingtin (HTT)-impaired mitochondrial dynamics. Finally, SRF and actin dynamics are connected via the actin severing protein cofilin and its slingshot phosphatase to modulate neuronal mitochondrial dynamics. In summary, our data suggest that the SRF-cofilin-actin signaling axis modulates neuronal mitochondrial function.

Fig. 1.

Altered mitochondrial morphology, distribution, and activity in SRF-deficient neurons. (A and B) Mitochondria (arrows) in P14 corpus callosal axons of WT neurons are electron-dense. (C) Higher magnification reveals a double membrane of a mitochondrion indicated by the dashed area in A. (B) In contrast, in axons of SRF-deficient neurons, mitochondria are vacuolized and frequently bottle-shaped (higher magnification flipped 90° in D). WT (E and F) and Srf mutant (G and H) P14 cortices were labeled for mitochondria (Tom20, red) and neurites (Smi32, green). In WT neurons, mitochondria were present in neurites (arrows in E), whereas abundance was reduced in SRF-deficient neurons (arrows in G). (I) Number of vacuolized mitochondria (vac. mito.) in an axon cross-section is depicted. ko, knockout; wt, wild type. (J) Basal ATP content in mitochondria of P14 Srf mutant brains is reduced compared with WT. (K) ATP production rate is decreased in mitochondria derived from SRF-deficient hippocampi compared with WT. (L) Percentage of mitochondria found in neurite segments ranging from 0 to ≥60 μm. In neurite parts >60 μm away from the cell body, mitochondria were reduced in Srf mutants compared with WT. Numbers in bars represent numbers of independent animals. (Scale bar: A–D, 1 μm; E–H, 10 μm.)

Fig. 2.

SRF deficiency modulates mitochondrial size, localization, and shape in vitro. (A and B) Primary mouse hippocampal neurons express pDSRed2Mito, allowing for mitochondrial visualization (red, Insets). (A) In WT neurons, mitochondria of variable size were present throughout the neurite. (B) In neurons lacking SRF, mitochondria accumulated in the cell body and only rarely entered neurites. (Insets) pDSRed2Mito signal of the entire neuron is shown. A WT neuron (C) and Srf mutant neuron (D) expressing pDSRed2Mito were used for time-lapse videomicroscopy. (E and F) Dashed areas show the position of individual frames. (E) In WT neurons, mitochondria move between frames taken every 5 s. (F) In contrast, on SRF ablation, almost no mitochondrial movement was observed. Size and distribution of synaptophysin were comparable between WT (G) and mutant (H) neurons. EM pictures of WT (I and K) and Srf mutant (J and L) hippocampal neurons in culture. (I and K) In WT neurons, double membranes and cristae structures are discernible. In Srf mutant neurons, balloon-shaped inclusions were observed (arrows in J and L). K and L are higher magnifications of dashed areas in I and J. (M) Quantification of mitochondrial object size in WT (wt) and SRF-deficient neurons. ko, knockout. (N) Percentage of neurite length occupied by mitochondria (mitochron.) is decreased in SRF-deficient neurons compared with WT. (O) Velocity of mitochondrial transport in WT and Srf mutant neurons. (P) Percentage of mitochondrial objects engaged in transport is depicted. (*P < 0.05; **P < 0.01; ***P < 0.001.) Numbers in bars represent numbers of independent experiments. (Scale bar: A–D, G, and H, 10 μm; E and F, 3 μm; I and J, 1 μm; K and L, 100 μm.)

Fig. 3.

SRF-VP16 induces mitochondrial network formation and alters transport parameters. In WT neurons, constitutively active SRF-VP16–induced mitochondrial networks (B, F, and J) compared with SRF-ΔMADS-VP16–expressing neurons (A, E, and I) are shown. (C, G, and K) In an Srf mutant neuron, mitochondria accumulate around the cell body. (D, H, and L) SRF-VP16 expression in Srf mutant neurons increased mitochondrial object size and neurite occupancy. (M–P) Kymographs depict distance traveled by mitochondria (white bars) within 5 min. SRF-VP16 in WT neurons increased the number and velocity of moving mitochondria (N) compared with an SRF-ΔMADS-VP16–expressing neuron (M). On SRF ablation, mitochondria fail to move (O), a phenotype alleviated by SRF-VP16 (P). SRF-VP16 but not CREB-VP16 changed object size (Q) and occupancy (R). ko, knockout; wt, wild type. (S and T) SRF-VP16 increased the velocity and percentage of mitochondria being transported. (*P < 0.05.) Numbers in bars represent numbers of independent experiments. (Scale bar: A–L, 10 μm.)

Fig. 4.

SRF-VP16 modulates neurodegeneration-inflicted mitochondrial dynamics. (A–H) In primary neurons, GFP-HTT130, an aggregation-competent huntingtin mutant, induced mitochondrial fragmentation and decreased occupancy (C and G) compared with the control GFP-HTT23 (A and E) in an SRF-ΔMADS-VP16–expressing neuron. (D and H) SRF-VP16 inhibited mitochondrial fragmentation and increased mitochondrial occupancy in the presence of aggregation-competent HTT130. (I–P) SRF (N) is down-regulated in the hippocampal CA1 region of adult huntingtin transgenic mice compared with a nuclear SRF stain in WT mice (M). Antiubiquitin staining depicts huntingtin aggregates in HTT transgenic mice (L) but not in controls (K). (Q) Lysates of hippocampi derived from WT and HTT transgenic mice were blotted for the indicated antibodies. Full-length SRF (approximately 67 kDa), in contrast to other gene regulators (c-Jun, MRTF-A, RelA), is down-regulated in huntingtin transgenic mice. SRF-VP16 but not CREB-VP16 modulated mitochondrial (mito.) dynamics inflicted by HTT130 quantified by object size (R) and occupancy (S). (T) Mitochondrial transport velocity known to be decreased by aggregation-competent HTT130 is almost doubled by SRF-VP16 but not by the SRF control construct (SRF-ΔMADS-VP16). FCCP decreases size (U) and occupancy of mitochondria (V) in WT neurons. SRF-VP16 enhanced mitochondrial size (U) and neurite occupancy (V) in the presence of FCCP. (*P < 0.05; **P < 0.01; ***P < 0.001.) Numbers in bars represent numbers of independent experiments. (Scale bar: A–H, 10 μm; I–P, 20 μm.)

Fig. 5.

Actin tread-milling modulates neurite and growth cone mitochondrial dynamics. WT neurons were treated with latrunculin (B and G) or expressed actin mutant proteins favoring G-actin (R62D; C and H) or F-actin (G15S, D and I; S14C, E and J). Latrunculin shortened mitochondrial length (B and G) compared with controls (A and F). Actin R62D (C and H) decreased mitochondrial size and occupancy compared with controls (A and F). In contrast, actin G15S (D and I) but not S14C (E and J) increased mitochondrial size and occupancy. Growth cone stimulated with the attractive guidance cue BDNF (L and Q) harbored more mitochondria than a nontreated growth cone (K and P). (M and R) In contrast, ephrin-A5, a guidance cue disrupting F-actin, reduced growth cone mitochondrial number. Actin R62D (N and S) reduced mitochondrial number in growth cones compared with controls (K and P). (O and T) Actin G15S-enhanced mitochondrial number in growth cones. Individual growth cones labeled with a dashed line are indicated. Quantification of object size (U) and occupancy (V) was modulated by actin mutants and latrunculin. Quantification of mitochondria in growth cones, without (W) and with (X) normalization for growth cone area, is shown. (*P < 0.05; **P < 0.01; ***P < 0.001.) Numbers in bars represent numbers of independent experiments. (Scale bar: A–T, 10 μm.)

Fig. 7.

Cofilin S3E affects mitochondrial dynamics and counteracts SRF-VP16. In primary hippocampal neurons (A–H), SRF-VP16 enhanced mitochondrial size and number in neurites (B and F). (C and G) Cofilin S3E reduced mitochondrial size and neurite occupancy. (D and H) Coexpression of cofilin S3E and SRF-VP16 prevented SRF-VP16’ s potential to enhance mitochondrial size and number in neurites. In Srf mutant neurons expressing SRF-ΔMADS-VP16 (K and O), P-cofilin levels were up-regulated compared with WT (I and M). SRF-VP16 reduced P-cofilin levels in WT (J and N) and more pronouncedly in Srf mutant (L and P) neurons. Cofilin S3E reduced mitochondrial (mito.) size (Q) and neurite occupancy (R) and counteracted SRF-VP16’s impact on mitochondria. Quantification of phosphocofilin (S) and total cofilin (T) levels in individual neurons is shown. (*P < 0.05; **P < 0.01; ***P < 0.001.) Numbers in bars represent numbers of independent experiments. ko, knockout; n.s., not significant; wt, wild type. (Scale bar: A–P, 10 μm.)

Fig. 6.

Active cofilin and slingshot rescue mitochondrial motility and neurite growth impaired by SRF ablation. In primary neurons, SRF ablation resulted in mitochondrial fragmentation (B and F) compared with WT (A and E) as before (Fig. 2). (C and G) Overexpression of cofilin S3A in WT neurons only slightly enhanced mitochondrial size and neurite occupancy (compare with Q and R). (D and H) Active cofilin S3A rescued impaired mitochondrial dynamics induced by SRF deficiency and elevated neurite growth. (I–P) In WT neurons, active Ssh l (K and O) in comparison to the control Ssh s (I and M) increased mitochondrial size and neurite occupancy, although not statistically significantly (compare with U and V). (L and P) Active Ssh enhanced mitochondrial size and occupancy as well as neurite growth in Srf mutant neurons. In addition, Ssh l (P) suppressed cofilin phosphorylation in SRF-deficient neurons (compare N with P). Cofilin S3A rescued mitochondrial (mito.) size (Q) and neurite occupancy (R) in Srf mutants. (S and T) SRF-deficient neurons expressing cofilin S3A achieved neurite length comparable to WT neurons (S). av., average; ko, knockout; wt, wild type. (T) Cofilin S3A also enhanced neurite growth in WT neurons. Active Ssh l rescued mitochondrial size (U), neurite occupancy (V), and neurite length (W) in Srf mutant neurons. (X) Ssh reduced P-cofilin in WT and more strongly in Srf mutants. (*P < 0.05; **P < 0.01; ***P < 0.001.) Numbers in bars represent numbers of independent experiments. (Scale bar: A–P, 10 μm.)

Fig. 8.

Summary of SRF-cofilin-actin signaling in neuronal mitochondrial dynamics. (A) Summary of the SRF-cofilin-actin interplay. (B–D) Compared with the control (C), F-actin, SRF-VP16, cofilin S3A, and slingshot (summarized in B) enhanced mitochondrial size and neurite occupancy. (D) In contrast, G-actin, SRF deficiency, huntingtin, and cofilin S3E decreased both parameters. BDNF (B) increased, whereas ephrin-A5 (D) decreased, mitochondrial number in growth cones. (E) (Top) SRF GOF via SRF-VP16 decreased P-cofilin levels and enhanced mitochondrial size. (Middle) SRF loss of function (LOF) enhanced P-cofilin and reduced mitochondrial size. (Bottom) In sum, this suggests that fine-tuning of cofilin activity by WT SRF is important toward modulation of mitochondrial size.

Fig. P1.

(A) Individual mitochondria labeled in red are found in a neurite. In addition, many mitochondria are found in the neuronal cell body. (B) Distribution of mitochondria (red) in a WT neuron. (Inset) Mitochondria of various shapes are localized in the growth cone, neurite, and cell body. (C) Summary of experiments resulting in reduced mitochondrial size and clearance of mitochondria from growth cones and neurites, including G-actin elevation (via actin R62D), loss of SRF, expression of aggregation-competent huntingtin or inactive cofilin, application of the actin-depolymerization toxin latrunculin, and stimulation of neurons with ephrin-A5. (D) Summary of experiments resulting in enhanced mitochondrial size and increased numbers of mitochondria in growth cones and neurites, including F-actin elevation (via actin G15S), SRF-VP16 activity, expression of active cofilin S3A or its activating phosphatase slingshot, and stimulation of neurons with BDNF.