Figure 6. Activity-dependent and Camkk2-dependent phosphorylation of MTFR1L by AMPK mediates compartmentalized mitochondria morphology in dendrites of CA1 PNs in vivo.

(A) Western blots (A) of whole cell lysates from mouse hippocampal neurons maintained in culture for 18-21DIV and treated for 5 min with physiological (5mM) extracelllular potassium chloride (KCl) (first column) or high KCl (40mM) inducing membrane depolarization (columns 2 & 3) in the presence (column 3) or absence (columns 1&2) of the Camkk2 inhibitor STO609. These results demonstrate that phosphorylation of AMPKα catalytic subunit on T172 is increased by neuronal depolarization which is blocked by STO609. In turn, AMPK phosphorylation of its substrate MTFR1L on S103 ³³ is increased by depolarization which is Camkk2-dependent since it is blocked by STO609.

(B-J) Rescue experiments showing that phosphomimetic form of Mtfr1l (Mtfr1l^S2D) mimicking phosphorylation by AMPK ³³ is sufficient to rescue compartmentalized mitochondria morphology in basal (B-D), apical oblique (E-G) and apical tufts (H-J) dendrites of CA1 PNs in vivo. CA1 PNs from wild-type (WT) or Camkk2^−/− constitutive knockout mice were IUE with the same mitochondrial markers/cell fills as in Figure 3 (WT and Camkk2^−/−) and a plasmid cDNA expressing phosphomimetic mutant on the two serine residues phosphorylated by AMPK (S103D and S238D) of Mtfr1l (Mtfr1l^S2D) ³³. Data and quantifications from WT and Camkk2^−/− are the same as in Figure 3.

Basal Dendrites (WT: n = 32 dendritic segments, 313 individual mitochondria, mean length = 1.065 μm ± 0.029 (SEM), mean occupancy = 33.01% ± 1.84%; Camkk2^−/−: n = 46 dendritic segments, 449 individual mitochondria, mean length = 4.975 μm ± 0.233 (SEM), mean occupancy = 73.36% ± 2.03%, length increase = 367%, occupancy increase = 122%; Camkk2^−/− + Mtfr1l^S2D: n = 13 dendritic segments, 171 individual mitochondria, mean length = 1.187 μm ± 0.051 (SEM), mean occupancy = 31.89% ± 1.49%)

Apical Oblique Dendrites (WT: n = 28 dendritic segments, 184 individual mitochondria, mean length = 1.569 μm ± 0.104 (SEM), mean occupancy = 42.38% ± 2.98%; Camkk2^−/−: n = 29 dendritic segments, 288 individual mitochondria, mean length = 5.366 μm ± 0.346 (SEM), mean occupancy = 78.64% ± 2.07%, length increase = 242%, occupancy increase = 85.56%; Camkk2^−/− + Mtfr1l^S2D: n = 21 dendritic segments, 223 individual mitochondria, mean length = 2.030 μm ± 0.092 (SEM), mean occupancy = 47.45% ± 2.28%)

Apical Tuft Dendrites (WT: n = 33 dendritic segments, 226 individual mitochondria, mean length = 6.562 μm ± 0.437 (SEM), mean occupancy = 84.19% ± 1.44%; Camkk2^−/−: n = 35 dendritic segments, 199 individual mitochondria, mean length = 11.14 μm ± 0.758 (SEM), mean occupancy = 95.03% ± 1.73%, length increase = 69.77%, occupancy increase = 12.88%; Camkk2^−/− + Mtfr1l^S2D: n = 13 dendritic segments, 110 individual mitochondria, mean length = 5.419 μm ± 0.402 (SEM), mean occupancy = 81.83% ± 2.71%). Scale bar, 5 μm.