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, 13 (11), 1420-1437
eCollection

Adhesion Regulating Molecule 1 Mediates HAP40 Overexpression-Induced Mitochondrial Defects

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Adhesion Regulating Molecule 1 Mediates HAP40 Overexpression-Induced Mitochondrial Defects

Zih-Ning Huang et al. Int J Biol Sci.

Abstract

Striatal neuron death in Huntington's disease is associated with abnormal mitochondrial dynamics and functions. However, the mechanisms for this mitochondrial dysregulation remain elusive. Increased accumulation of Huntingtin-associated protein 40 (HAP40) has been shown to be associated with Huntington's disease. However, the link between increased HAP40 and Huntington's disease remains largely unknown. Here we show that HAP40 overexpression causes mitochondrial dysfunction and reduces cell viability in the immortalized mouse striatal neurons. HAP40-associated mitochondrial dysfunction is associated with reduction of adhesion regulating molecule 1 (ADRM1) protein. Consistently, depletion of ADRM1 by shRNAs impaired mitochondrial functions and increased mitochondrial fragmentation in mouse striatal cells. Moreover, reducing ADRM1 levels enhanced activity of fission factor dynamin-related GTPase protein 1 (Drp1) via increased phosphorylation at serine 616 of Drp1 (Drp1Ser616). Restoring ADRM1 protein levels was able to reduce HAP40-induced ROS levels and mitochondrial fragmentation and improved mitochondrial functions and cell viability. Moreover, reducing Drp1 activity by Drp1 inhibitor, Mdivi-1, ameliorates both HAP40 overexpression- and ADRM1 depletion-induced mitochondrial dysfunction. Taken together, our studies suggest that HAP40-mediated reduction of ADRM1 alters the mitochondrial fission activity and results in mitochondrial fragmentation and mitochondrial dysfunction.

Keywords: ADRM1; Drp1.; HAP40; Huntington's disease; mitochondrial dynamics.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1
Figure 1
Overexpression of HAP40 impairs mitochondrial functions. (A) Immunoblot detection of STHdhQ7/Q7 striatal cells transfected with FLAG or FLAG-HAP40 plasmids. (B) Quantification of JC-1 associated red/green fluorescent intensity ratio in STHdhQ7/Q7 striatal cells transfected with the indicated plasmids. Flow cytometric analysis of mitochondrial membrane potential by JC-1 staining in STHdhQ7/Q7 striatal cells in the presence of FLAG or FLAG-HAP40. (C) The ATP content measured 48 hrs after transfection with the indicated plasmids. The ATP content of cells expressing FLAG only was arbitrarily set to be 1. (D) The total cell number of the STHdhQ7/Q7 striatal cells transfected with the FLAG or FLAG-HAP40 plasmid measured by the MTT assay. Cell number was measured 48 hours after transfection. The percentage number of cells expressing FLAG only was arbitrarily set to be 100%. (E) The normalized ATP content of STHdhQ7/Q7 striatal cells transfected with the FLAG or FLAG-HAP40 plasmid. (F) Flow cytometric analysis of ROS in STHdhQ7/Q7 striatal cells in the presence of FLAG or FLAG-HAP40 plasmid. Quantification of percentage of the cells with positive ROS in STHdhQ7/Q7 striatal cells transfected with indicated plasmids. Data are from three independent experiments and presented as mean normalized units ± SEM. Data showing significant differences with P < 0.05 are labeled with one asterisk (*); with P < 0.01 are labeled with two asterisks (**).
Figure 2
Figure 2
Overexpression of HAP40 leads to mitochondrial fragmentation. Images showing mitochondria in (A) tubular, (B) intermediate, (C) fragmented morphology in STHdhQ7/Q7 striatal cells transfected with mito-eGFP plasmids. (D) STHdhQ7/Q7 striatal cells were co-transfected with the mito-eGFP and the FLAG or FLAG-HAP40 plasmids. Quantification of percentage of the cells with different mitochondrial morphology in STHdhQ7/Q7 striatal cells transfected with the indicated plasmids. Primary cortical neurons were co-transfected with (E) mito-eGFP and mCherry plasmids, or (F) mito-eGFP and HAP40-mCherry plasmids. (G) Average length of mitochondria was quantified in primary cortical neurons with the mCherry or HAP40-mCherry construct. (H) Frequency distribution of mitochondrial length was quantified in primary cortical neurons transfected with the mCherry or HAP40-mCherry construct. Results from three independent experiments were subjected to statistical analysis. Data are represented as mean ± SEM. Data showing significant differences with P < 0.05 are labeled with one asterisk (*); with P < 0.01 are labeled with two asterisks (**); with P < 0.005 are labeled with three asterisks (***); N.S., no significance.
Figure 3
Figure 3
Overexpression of HAP40 increases the level and phosphorylation state of Drp1. (A) Immunoblot detection for Mfn1, Mfn2, OPA1, Drp1, p-Drp1Ser616, Fis1, PHB1, Parkin, PINK1, ADRM1, GAPDH, and tubulin in STHdhQ7/Q7 striatal cells transfected with the FLAG or FLAG-HAP40 plasmid. (B-K) Quantification analyses on protein levels with the indicated protein normalized to the tubulin (B-H) or GAPDH (I-K). Data are from three independent experiments and presented as mean normalized units ± SEM. Data showing significant differences compared with the control (P < 0.05) are labeled with asterisk (*); N.S., no significance.
Figure 4
Figure 4
Reducing ADRM1 increases the phosphorylation state of Drp1. (A) Immunoblot detection for ADRM1, Parkin, p-Drp1Ser616, Drp1, Mfn1, and tubulin in STHdhQ7/Q7 striatal cells transfected with the pLKO.ADRM1 #1, pLKO.ADRM1 #2, or pLKO.Luc. (B-E and G) Quantification analyses on protein levels with the indicated protein normalized to the tubulin. (F) Quantification analyses on phosphorylated Drp1Ser616 normalized to total Drp1 protein in STHdhQ7/Q7 striatal cells transfected with the indicated plasmids. Data are from three independent experiments and presented as mean normalized units ± SEM. Data showing significant differences with P < 0.05 are labeled with one asterisk (*); with P < 0.01 are labeled with two asterisks (**); N.S., no significance.
Figure 5
Figure 5
Depletion of ADRM1 impairs mitochondrial functions and leads to mitochondrial fragmentation. STHdhQ7/Q7 striatal cells were transfected with pLKO.ADRM1 #1, pLKO.ADRM1 #2, or pLKO.Luc shRNA construct. pLKO.Luc that targeted luciferase gene was used as a negative control. (A) Flow cytometric analysis of mitochondrial membrane potential by JC-1 staining in STHdhQ7/Q7 striatal cells depleted for ADRM1. Quantification of the JC-1 associated red/green fluorescent intensity ratio in STHdhQ7/Q7 striatal cells transfected with the indicated plasmids. The ratio of red to green JC-1 signals of cells expressing control pLKO.Luc. was arbitrarily set to be 1. (B) The ATP content of the STHdhQ7/Q7 striatal cells transfected the indicated plasmids. The ATP content of cells expressing control pLKO.Luc. was arbitrarily set to be 1. (C) The total cell number of the STHdhQ7/Q7 striatal cells transfected with the indicated plasmids were measured by the MTT assay. The percentage number of cells expressing pLKO.Luc only was arbitrarily set to be 100%. (D) The normalized ATP content of STHdhQ7/Q7 striatal cells transfected with the pLKO.ADRM1 #1, pLKO.ADRM1 #2, or pLKO.Luc plasmid. (E) Flow cytometric analysis of ROS accumulation in STHdhQ7/Q7 striatal cells depleted for ADRM1. Quantification of percentage of the cells with positive ROS in STHdhQ7/Q7 striatal cells transfected with indicated plasmids. (F) Quantification of percentage of the cells with different mitochondrial morphology in STHdhQ7/Q7 striatal cells transfected with indicated plasmids. Data are from three independent experiments and presented as mean normalized units ± SEM. Data showing significant differences with P < 0.05 are labeled with one asterisk (*); with P < 0.01 are labeled with two asterisks (**); N.S., no significance.
Figure 6
Figure 6
Overexpression of ADRM1 restores mitochondrial functions and reduces HAP40-induced mitochondrial fragmentation. (A) Immunoblot detection for HAP40 and ADRM1 in STHdhQ7/Q7 striatal cells transfected with the FLAG or FLAG-HAP40 plasmid with or without the FLAG-ADRM1 plasmid. Tubulin was used as an internal control for protein loading. (B) Flow cytometric analysis of mitochondrial membrane potential by JC-1 staining in STHdhQ7/Q7 striatal cells in the presence of FLAG or FLAG-ADRM1 plasmids. Quantification of the JC-1 associated red/green fluorescent intensity ratio in STHdhQ7/Q7 striatal cells transfected with indicated plasmids. The ratio of red to green JC-1 signals of cells expressing FLAG only was arbitrarily set to be 1. (C) Flow cytometric analysis of mitochondrial membrane potential by JC-1 in STHdhQ7/Q7 striatal cells in the presence of FLAG-HAP40 and FLAG or FLAG-ADRM1 plasmids. Quantification of the JC-1 associated red/green fluorescent intensity ratio in STHdhQ7/Q7 striatal cells transfected with the FLAG-HAP40 and FLAG or FLAG-ADRM1 plasmids. The ratio of red to green JC-1 signals of cells co-expressing FLAG and FLAG-HAP40 was arbitrarily set to be 1. (D) The cellular ATP content of STHdhQ7/Q7 striatal cells transfected with the indicated plasmids. The ATP content was measured 48 hrs after transfection. The ATP content of cells co-expressing FLAG and HAP40-mCherry was arbitrarily set to be 1. (E) The total cell number of the STHdhQ7/Q7 striatal cells transfected with the indicated plasmids was measured by the MTT assay. Cell number was measured 48 hours after transfection. The percentage number of cells co-expressing FLAG and HAP40-mCherry was arbitrarily set to be 100%. (F) The normalized ATP content of STHdhQ7/Q7 striatal cells transfected with the indicated plasmids. (G) Flow cytometric analysis of ROS accumulation in STHdhQ7/Q7 striatal cells transfected with the indicated plasmids. Quantification of percentage of the cells with positive ROS in STHdhQ7/Q7 striatal cells transfected with indicated plasmids. (H) Quantification of percentage of the cells with different mitochondrial morphology in STHdhQ7/Q7 striatal cells co-transfected with the mito-eGFP and FLAG or FLAG-ADRM1 plasmids. (I) Quantification of percentage of the cells with different mitochondrial morphology in STHdhQ7/Q7 striatal cells co-transfected with the mito-eGFP and FLAG-HAP40 plasmids in the presence or absence of the FLAG or FLAG-ADRM1 plasmid. Data are from three independent experiments and presented as mean normalized units ± SEM. Data showing significant differences with P < 0.05 are labeled with one asterisk (*); N.S., no significance.
Figure 7
Figure 7
Overexpression of ADRM1 reduces the level of phosphorylated Drp1Ser616 in HAP40-expressing cells. (A) Immunoblot detection for p-Drp1Ser616, Drp1, ADRM1, and tubulin in STHdhQ7/Q7 striatal cells co-transfected with the HAP40-mCherry with FLAG or FLAG-ADRM1 plasmid and FLAG or FLAG-ADRM1 plasmid alone. (B-C) Quantification analyses on the indicated protein normalized to the tubulin. (D) Quantification analyses on phosphorylated Drp1Ser616 normalized to total Drp1 protein in STHdhQ7/Q7 striatal cells co-transfected with the indicated plasmids. Data are from three independent experiments and presented as mean normalized units ± SEM. Data showing significant differences compared to the control (P < 0.05) are labeled with asterisk (*); N.S., no significance.
Figure 8
Figure 8
Inhibition of Drp1 alleviates HAP40 overexpression-induced mitochondrial dysfunction. (A) Quantification of percentage of cells with different mitochondrial morphology in STHdhQ7/Q7 striatal cells co-transfected with the mito-eGFP and FLAG-HAP40 plasmids in the presence or absence of Mdivi-1 treatment. (B) Quantification of JC-1 associated red/green fluorescent intensity ratio in STHdhQ7/Q7striatal cells transfected with FLAG or FLAG-HAP40 plasmids with or without Mdivi-1. (C) Flow cytometric analysis of ROS levels in STHdhQ7/Q7 striatal cells transfected with the FLAG or FLAG-HAP40 plasmid in the presence or absence of Mdivi-1 treatment. Quantification of percentage of the cells with positive ROS in STHdhQ7/Q7 striatal cells transfected with the indicated plasmids. Data are from three independent experiments and presented as mean normalized units ± SEM. Data showing significant differences with P < 0.05 are labeled with one asterisk (*); with P < 0.01 are labeled with two asterisks (**); N.S., no significance.
Figure 9
Figure 9
Mdivi-1 treatment relieves mitochondrial dysfunction caused by down-regulation of ADRM1. Quantification of percentage of the cells with different mitochondrial morphology in STHdhQ7/Q7 striatal cells co-transfected with the mito-eGFP and pLKO.ADRM1 #1 (A) or pLKO.ADRM1 #2 (B) shRNA construct in the presence or absence of the Mdivi-1 treatment. (C) Quantification of JC-1 associated red/green fluorescent intensity ratio in STHdhQ7/Q7striatal cells transfected with pLKO.ADRM1 #1, pLKO.ADRM1 #2, or pLKO.Luc shRNA construct with or without Mdivi-1. (D) Flow cytometric analysis of ROS accumulation in STHdhQ7/Q7 striatal cells depleted for ADRM1 with or without Mdivi-1. Quantification of percentage of the cells with positive ROS in STHdhQ7/Q7 striatal cells transfected with indicated plasmids. Data are from three independent experiments and presented as mean normalized units ± SEM. Data showing significant differences with P < 0.05 are labeled with one asterisk (*); N.S., no significance.

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