MPPs and HtrA1 appear to interact directly.

<p>(<b>A</b>) MPPs displayed differential activity in affecting The pattern of extracellular HtrA1 complexes (arrowheads) from HeLa cell low-serum conditioned medium is differentially altered by treatment with MPPs (25 mM, 37°C, 1 hr), as demonstrated when probed with polyclonal anti-HtrA1 antibody. (<b>B</b>) MPPs may induce conformational changes upon binding to HtrA1. The accessibility of N-terminal and C-terminal epitopes in the presence of MPPs was determined by ELISA and compared to DMSO controls. Error bars indicate the standard deviation. (<b>C</b>) Extracellular HtrA1 (arrows) could be precipitated from HEK-HtrA1 conditioned medium using HEMIN-conjugated agarose beads but not control, unconjugated agarose beads. (<b>D</b>) Competitive binding experiments using conditioned medium from HEK-HtrA1 or HeLa cells pre-incubated with MPPs revealed reduced recovery of HtrA1 (arrows) in the presence of competitor compounds when probed with polyclonal anti-HtrA1 antibody. (<b>E</b>) Degradation of Fibulin 5 in fixed HeLa cells treated with HtrA1 conditioned medium was enhanced in the presence of TPP, ZPP and PPP-IX. RMA: Rosmarinic acid, TPP: tin protoporphyrin IX, ZPP: zinc protoporphyrin IX, PPP-IX: protoporphyrin IX, HEM: HEMIN, CPP: cobalt protoporphyrin IX, CM: conditioned medium.</p

Formation of oligomer complexes by secreted HtrA1 protein in cell culture.

<p>(<b>A</b>) Serum rich (10%) conditioned medium from HeLa cells contained a higher level of HtrA1 complexes than low serum (0.2%) conditioned medium. Medium was collected at the indicated time points and subjected to non-reducing (- β-ME) or reducing (+ β-ME) SDS-PAGE. Immunoblotting with polyclonal anti-HtrA1 antibody detected full length HtrA1 (arrow) and HtrA1-containing complexes (arrowheads) under non-reducing conditions. Full length HtrA1 could be detected under non-reducing conditions. (<b>B</b>) Serum rich and low serum conditioned media subjected to native PAGE revealed two prominent bands when probed with monoclonal anti-HtrA1 antibody (bracket). A dilution series is shown for clarity. (<b>C</b>) A schematic diagram of the human HtrA1 expressed from the full-length construct, tagged with an HA epitope (red) at the N-terminus and V5/hexa-His tag (blue) at the C-terminus. S denotes the signal peptide. (<b>D</b>) Exogenous HtrA1 is stably expressed in HEK293 cells. HtrA1 could be detected in conditioned medium from HeLa cells and stably transfected HEK-HtrA1 cells, but not that from HEK293 cells or medium that has not been exposed to cells, when probed with monoclonal anti-HtrA1 antibody (middle panel). Probing with anti-V5 antibody (right panel) detected specific bands (arrows, arrowheads) only in HEK-HtrA1 conditioned medium, although a non-specific band was detected in all samples (asterisk). Coomassie blue staining is included as a loading control (left panel). (<b>E</b>) Serum rich (10%) conditioned medium contained a higher level of exogenous HtrA1 complexes than serum free conditioned medium. Exogenous HtrA1 was captured from conditioned medium via the hexa-His tag, using Ni-NTA columns. Full-length HtrA1 (arrow) could be detected in the input conditioned medium (control) and column-captured fraction (bound) under both serum conditions. HtrA1 complexes (arrowheads) were only detected under serum rich conditions. CM: conditioned medium.</p

PPP-IX binds to the protease domain of HtrA1 and disease-associated mutations eliminate binding.

<p>(<b>A</b>) PPP-IX competed with HEMIN for endogenous HtrA1 binding. HeLa cell lysate was subjected to HEMIN pull down. Less HtrA1 was recovered in the presence of PPP-IX. Akt was used as a control and was not purified from the same pull down. (<b>B</b>) The HtrA1 protease domain was sufficient for interaction with MPPs. A schematic diagram of the deletion constructs tagged with HA and V5/hexa-His epitopes is shown (top). Cell lysates from HEK293 expressing variant HtrA1 proteins were subjected to HEMIN pull down and eluates (H) were examined for tagged protein with the anti-HA antibody (bottom left). The amount of recovered protein compared to input (I) is shown (bottom right). (<b>C</b>) PPP-IX competed with HEMIN for binding to the HtrA1 protease domain in a dose-dependent manner, with less HtrA1 protein recovery as PPP-IX concentration increased, revealed by immunoblotting for anti-HA. Akt was not similarly purified. The amount of recovered protein compared to input is shown (bottom). (<b>D</b>) Disease associated mutations reduced the interaction between HtrA1 and MPPs. The position of engineered single amino acid mutations in variant HtrA1 constructs is shown in the schematic diagram (top). Variant HtrA1 protein pulled down from transfected cell lysates with HEMIN agarose (H) was probed for HtrA1 (left panel) and the amount of recovered protein compared to input (I) was calculated (right panel). R274Q and V297 M significantly reduced the binding of HtrA1 to HEMIN (*: p<0.01). HtrA family member HtrA2 is not purified, demonstrating that the interaction is specific to HtrA1.</p

The formation of HtrA1 extracellular complexes can be affected by small molecules.

<p>(<b>A</b>) A schematic of the chemical intervention strategy for selecting molecules capable of affecting extracellular HtrA1 complex formation. “Hit compounds” were identified when the HtrA1 complex pattern was altered in comparison to DMSO treatment. (<b>B</b>) Conditioned serum from HEK-HtrA1 and HeLa cells was probed with anti-HA and anti-HtrA1 antibody respectively after treatment with the seven hit compounds. All compounds increased HtrA1 complex abundance (arrowheads) compared to the DMSO treated control. DMSO treatment did not alter complex formation. CBD: (S)-(-)-Carbidopa, RMA: Rosmarinic acid, ZPP: zinc protoporphyrin IX, TPP: tin protoporphyrin IX, ACTD: Actinomycin D, YM: YM 90709, AZ: AZ 10417808. (<b>C</b>) Schematic diagrams of the chemical structure of the non-MPP hit compounds. Dashed lines indicate a conserved chemical moiety in Carbidopa and Rosmarinic acid. (<b>D</b>) Schematic diagram of the chemical structures of the protoporphyrin IX-based metalloporphyrins used.</p

Muscle strength and total activity are decreased in <i>Htra2</i>-deficient mice.

<p>(<i>A–D</i>) The hind limb suspension test was performed on HTRA2-deficient neonates. HTRA2 KO mice performed poorly compared to WT littermates in hang time (<i>A</i>) and number of pull attempts (<i>C</i>) from P7. NesKO neonates performed poorly compared to NesWT littermates in hang time (<i>B</i>) and number of pull attempts (<i>D</i>) from P8 (n = 28 (HTRA2 WT), 19 (HTRA2 KO), 10 (NesWT), 16 (NesKO)). (<i>E–F</i>) The weanling observation total activity score was reduced in P19–21 HTRA2 KO animals (<i>E</i>) compared to WT littermates, and NesKO animals (<i>F</i>) compared to NesWT littermates (n = 19 (HTRA2 WT), 12 (HTRA2 KO), 7 (NesWT), 4 (NesKO)). (<i>G–H</i>) Grip strength was reduced in HTRA2 KO (<i>G</i>) and NesKO (<i>H</i>) animals compared to respective WT littermates (n = 17 (HTRA2 WT), 9 (HTRA2 KO) 11 (NesWT), 5 (NesKO)). Data represents Mean ± SEM (#: 0.05≤p≤0.10, *: p≤0.05, **: p≤0.001, ***: p≤0.0001 by independent t-tests).</p

Brain-specific disruption of OPA1 processing.

<p>(<i>A</i>) Levels of autophagy markers are unchanged in P33 HTRA2 KO brain. The LC3β-II to LC3β-I ratio is unchanged in P33 brain and there is no accumulation of P62 (n = 3). Actin is included as a loading control. (<i>B</i>) Processing of OPA1 was altered in HTRA2 KO brain at P25 but not in other tissues, with increased abundance of S-OPA1 (filled arrowhead) and decreased L-OPA1 (open arrowhead). MFN2 was not altered when compared to WT. VDAC was used as a loading control. Calculating the ratio of S-OPA1 to L-OPA1 for tissues from P25–P30 HTRA2 KO mice confirmed changes in OPA1 processing were present in the brain but not in other tissues when compared to WT (***: p<0.0001 by t-test, n≥3 per genotype). (<i>C</i>) Altered processing could be detected in HTRA2 KO brain from P9 onwards. PHB2 was used as a loading control. Calculating the ratio of S-OPA1 to L-OPA1 for P9–P10 brains confirmed the change in processing (***: p<0.0001 by t-test, n≥6 per genotype). (<i>D</i>) Processing changes were also detected in NesKO brain at P25. PHB2 was used as a loading control. Calculating the ratio of S-OPA1 to L-OPA1 for brain from NesKO and NesWT mice at P24–P26 confirmed OPA1 processing was significantly altered (**: p<0.01 by t-test, n≥3 per genotype). KO denotes HTRA2 KO, WT denotes HTRA2 WT, nKO denotes NesKO and nWT denotes NesWT.</p

Loss of HTRA2 resulted in accumulation of large, structurally abnormal mitochondria in cerebellar granule cells.

<p>(<i>A–B</i>) TUNEL staining did not reveal any increase in cell death in the cerebellum of HTRA2 KO mice at P20. (<i>C–F</i>) Increased numbers of swollen mitochondria were detected in HTRA2 KO animals compared to WT littermates at both P20 (<i>C–D</i>) and P32 (<i>E–F</i>). (<i>G–K</i>) Five structural classes of mitochondria were detected in transmission electron micrographs of the cerebellar granule layer: N, normal (<i>G</i>), NV, normal-vesicular (<i>H</i>), V, vesicular (<i>I</i>), VS, vesicular-swollen (<i>J</i>) and S, swollen (<i>K</i>). (<i>L</i>) Mitochondria were assigned to one of the five structural classes and quantitation revealed an increase in swollen mitochondria at both P20 and P32. (<i>M</i>) The sizes of the mitochondria were measured at both P20 and P32 for both WT and HTRA2 KO animals, revealing increased proportions of large mitochondria in HTRA2 KO animals. (<i>N</i>) Calculating the cumulative probability of mitochondrial size revealed that the distributions of size were significantly altered at both P20 and P32 (*:p<0.05, **: p<0.001 by the Kolmogorov-Smirnov comparison, <i>n</i> = 1166 (P20 WT), 939 (P20 KO), 674 (P32 WT) and 1121 (P32 KO)). Scale bars: A–B: 100 µm, C–F: 1 µm, G–K: 500 nm.</p

Neural deletion of <i>Htra2</i> is sufficient to generate neurological phenotypes.

<p>(<i>A</i>) Exons 2 to 4 of <i>Htra2</i> were flanked with loxP sites, with a FRT flanked neo cassette 3′ to exon 4. Expression of <i>FlpE</i> causes deletion of the selection cassette. Cre-mediated deletion causes excision of exons 2 to 4. Small arrows beneath the allele constructs denote the position of genotyping primers. (<i>B</i>) PCR from genomic DNA can distinguish WT (+, arrow, 279 bp), KO (–, filled arrowhead, 358 bp) and floxed (f, empty arrowhead, 313 bp) alleles of <i>Htra2</i>. (<i>C</i>) Western blot analysis confirmed loss of HTRA2 protein (arrow) in all tissues of HTRA2 KO mice and reduction in brain of NesKO mice (arrowheads denote non-specific bands). The levels of HTRA2 protein in NesKO spleen and thymus were comparable with NesWT. Cx: cortex, Mb: midbrain, Hb: hindbrain. PHB2 was used as a loading control. (<i>D</i>) HTRA2 KO mice and NesKO mice were smaller than WT littermates by comparison. The size of the thymus and spleen was reduced although brain was relatively normal in size (representative animals shown at P30, scale bar: 1 cm.). (<i>E</i>) Body weight of HTRA2 KO and NesKO mice did not increase beyond P18 (<i>n</i> = 56 (HTRA2 WT), 62 (HTRA2 KO), 35 (NesWT), 25 (NesKO), error bars indicate SEM).</p