REEP1 Preserves Motor Function in SOD1G93A Mice by Improving Mitochondrial Function via Interaction with NDUFA4.

A decline in the activities of oxidative phosphorylation (OXPHOS) complexes has been consistently reported in amyotrophic lateral sclerosis (ALS) patients and animal models of ALS, although the underlying molecular mechanisms are still elusive. Here, we report that receptor expression enhancing protein 1 (REEP1) acts as an important regulator of complex IV assembly, which is pivotal to preserving motor neurons in SOD1G93A mice. We found the expression of REEP1 was greatly reduced in transgenic SOD1G93A mice with ALS. Moreover, forced expression of REEP1 in the spinal cord extended the lifespan, decelerated symptom progression, and improved the motor performance of SOD1G93A mice. The neuromuscular synaptic loss, gliosis, and even motor neuron loss in SOD1G93A mice were alleviated by increased REEP1 through augmentation of mitochondrial function. Mechanistically, REEP1 associates with NDUFA4, and plays an important role in preserving the integrity of mitochondrial complex IV. Our findings offer insights into the pathogenic mechanism of REEP1 deficiency in neurodegenerative diseases and suggest a new therapeutic target for ALS.

Cytosolic PINK1 orchestrates protein translation during proteasomal stress by phosphorylating the translation elongation factor eEF1A1.

Mutations in PINK1 (PTEN-induced putative kinase 1) are associated with autosomal recessive early-onset Parkinson's disease. Full-length PINK1 (PINK1-l) has been extensively studied in mitophagy; however, the functions of the short form of PINK1 (PINK1-s) remain poorly understood. Here, we report that PINK1-s is recruited to ribosome fractions after short-term inhibition of proteasomes. The expression of PINK1-s greatly inhibits protein synthesis even without proteasomal stress. Mechanistically, PINK1-s phosphorylates the translation elongation factor eEF1A1 during proteasome inhibition. The expression of the phosphorylation mimic mutation eEF1A1S396E rescues protein synthesis defects and cell viability caused by PINK1 knockout. These findings implicate an important role for PINK1-s in protecting cells against proteasome stress through inhibiting protein synthesis.

Transcriptional factor Nrf2 is essential for aggresome formation during proteasome inhibition.

Aggrephagy, the aggresome-related protein degradation system, represents a protective cellular response to shuttle misfolded proteins into the microtubule-organizing center for degradation through the autophagic pathway during stress conditions, including heat shock, oxidative stress and proteasome inhibition. In response to proteasome failure, many genes are transcriptionally activated to facilitate ubiquitinated proteins to be cleared via the aggrephagy pathway. Although many regulators involved in aggresome formation have been identified, the mechanism how transcriptional activation promotes aggresome formation remains unknown. Here, we have demonstrated that nuclear factor erythroid 2-related factor 2 (Nrf2) accumulated in the nucleus and activated the transcription of sequestosome-1 (p62) during proteasome inhibition in 293 cells. Loss of Nrf2 resulted in failure of aggresome formation and cell death; whereas overexpression of p62 alleviated Nrf2 knockdown-induced aggresome formation defects and promoted cell survival. Notably, blocking Nrf2 activation using a p38/MAPK inhibitor prevented proteasome inhibitor-induced aggresome formation. These findings suggested that Nrf2 may be a critical regulator of aggresome formation, which protects cells from proteasome dysfunction-induced stress.

Cytosolic PINK1 promotes the targeting of ubiquitinated proteins to the aggresome-autophagy pathway during proteasomal stress.

During proteasomal stress, cells can alleviate the accumulation of polyubiquitinated proteins by targeting them to perinuclear aggresomes for autophagic degradation, but the mechanism underlying the activation of this compensatory pathway remains unclear. Here we report that PINK1-s, a short form of Parkinson disease (PD)-related protein kinase PINK1 (PTEN induced putative kinase 1), is a major regulator of aggresome formation. PINK1-s is extremely unstable due to its recognition by the N-end rule pathway, and tends to accumulate in the cytosol during proteasomal stress. Overexpression of PINK1-s induces aggresome formation in cells with normal proteasomal activities, while loss of PINK1-s function leads to a significant decrease in the efficiency of aggresome formation induced by proteasomal inhibition. PINK1-s exerts its effect through phosphorylation of the ubiquitin-binding protein SQSTM1 (sequestosome 1) and increasing its ability to sequester polyubiquitinated proteins into aggresomes. These findings pinpoint PINK1-s as a sensor of proteasomal activities that transduces the proteasomal impairment signal to the aggresome formation machinery.

Parkin-induced ubiquitination of Mff promotes its association with p62/SQSTM1 during mitochondrial depolarization.

The ubiquitin ligase Parkin and autophagic adapter protein p62 are known to function in a common pathway controlling mitochondrial autophagy (mitophagy). However, the evidence supporting that p62 is directly recruited by ubiquitinated proteins remains undetermined. Here, we demonstrate that mitochondrial fission factor (Mff) associates with Parkin and carbonyl cyanide m-chlorophenyl hydrazone treatment significantly increases the affinity of Parkin with Mff. After recruitment to depolarized mitochondria, Parkin mediates poly-ubiquitination of Mff at lysine 251. Replacement of lysine 251 by arginine (K251R) totally abrogates Parkin-stimulated ubiquitination of Mff. Subsequently, the ubiquitinated Mff promotes its association with p62. Mff knockout interferes with p62 translocation to damaged mitochondria. Only re-transfection of Mff WT, but not K251R mutant, rescues this phenotype. Furthermore, loss of Mff results in failure of Parkin translocation and final clearance of damaged mitochondria. Thus, our data reveal functional links among Mff, p62, and the selective autophagy of mitochondria, which are implicated in the pathogenesis of neurodegeneration diseases.

Effects of selenite and selenate application on growth and shoot selenium accumulation of pak choi (Brassica chinensis L.) during successive planting conditions.

Selenate and selenite are two main kinds of inorganic selenium (Se) sources in soil, but these substances can pose threats to the environment. Phytoextraction is an emerging technology to remove Se from polluted soils by using a hyper-accumulator. In this study, a pot experiment was conducted to investigate Se phytoextraction potential of pak choi (Brassica chinensis L.) and to determine the effects of Se on growth and Se accumulation of pak choi under successive planting conditions (four crops). Results showed that Se concentration in pak choi shoots significantly increased as selenate and selenite rates increased. Se concentration increased in successive crops on soil treated with selenite; by contrast, Se concentration decreased in crops on soil treated with selenate. Se concentrations of pak choi on soil treated with selenate were higher than those on soil treated with selenite. The maximum Se accumulations amount in crops on selenite- and selenate-treated soil were 7818 and 8828 µg · pot(-1), respectively. High bioconcentration factor (BCF) values indicated that pak choi could accumulate more Se from Se-contaminated soil. The Se phytoextraction efficiency of pak choi increased under successive planting conditions in selenite and selenate treatments; the maximum Se phytoextraction efficiencies of four successive crops of pak choi on selenite- and selenate-treated soil were 4.91 and 31.90 %, respectively. These differences between selenate and selenite treatments were attributed to the differences in Se forms in soil. Total and available Se contents in soil decreased significantly during repeated planting crops on soil treated with selenate; conversely, total and available Se contents decreased slightly in crops on soil treated with selenite. These results suggested that pak choi could highly tolerate and accumulate Se. Thus, pak choi may remove Se from contaminated soil; indeed, pak choi can be used in the phytoextraction of Se in polluted soil.

[Promotion of Pink1S Auto-phosphorylation with CK2ß].

The aim of this study is to determine the regulatory mechanism of PTEN-induced putative kinase protein 1 short isoform (PINK1S) in cytoplasm. By co-immunoprecipitation (Co-IP) assay, we identified that PINK1S interacted with the beta regulatory subunit of Casein Kinase 2 (CK2ß), but not with the catalytic subunits CK2α1 and CK2α2. Furthermore, cells were transfected with PINK1S and CK2ß, and then PINK1S was purified by immunoprecipitation. After detecting the phosphorylated proteins by Phos-tag Biotin, we found that CK2ß overexpression increased auto-phosphorylation of PINK1S. Finally, we generated CK2ß knockdown cell lines by RNA interference. Purified PINK1S from CK2ß knockdown cells significantly reduced its auto-phosphorylation compared with control cells. These results suggested that CK2ß functions as a regulatory subunit of PINK1S kinase complex promoted its activation by self-phosphorylation.

[CK2beta promotes Pink1/Parkin-mediated MIRO1 degradation].

PTEN-induced putative kinase 1 (PINK1), a Parkinson's disease (PD)-related protein, has two isoforms, the mitochondria-localized full-length isoform PINK1FL and the cytoplasm-localized short isoform PINK1-cyto. Studies have suggested that PINK1FL can selectively accumulate at the surface of damaged mitochondria and cooperate with another Parkinson's Disease-related protein PARKIN to trigger the degradation of MIRO1, a mitochondria trafficking regulator. The functions of PINK1-cyto are, however, not yet clear. To investigate the functions of PINK1-cyto, we expressed different proteins in cultured HEK293 cells by transfecting it with different plasmids, and detected the protein levels by Western blot after expressing for 24 h. We found that in cultured HEK293 cells, PINK1-cyto could also cooperate with PARKIN degrade MIRO1 in the presence of CK23, and the regulatory subunit of Casein Kinase II. Interestingly, this function of CK2P was not dependent on CK2alpha, the catalytic subunit of Casein Kinase II. We also found that CK2P could promote the direct interaction between PINK1-cyto and MIRO1 by immunocoprecipitation analysis. This result suggested that in addition to CK2alpha, CK2beta could also form a kinase complex.