GRP78 Overexpression Triggers PINK1-IP3R-Mediated Neuroprotective Mitophagy Part 1

Abstract: 

An experimental model of spinal root avulsion (RA) is useful for studying causal molecular programs that drive retrograde neurodegeneration after neuron-target disconnection. 

Spinal root avulsion is a fatal central nervous system injury that may have a significant impact on the patient's daily life and cognitive ability. Fortunately, however, memory is not necessarily affected, and most patients can improve their memory through some effective cognitive rehabilitation methods.

Several studies have shown that spinal root avulsion does not directly affect a person's memory, but mainly affects the patient's brain cognitive ability, including attention, cognition, and learning. This means that even if affected by spinal root avulsion, many people still have a good memory.

Therefore, for those affected by spinal root avulsion, improving cognitive ability is the key to improving memory. Cognitive rehabilitation is an effective method that can help patients rebuild their brain's cognitive ability and thus improve their memory. These methods include cloze, mathematical operations, etc., which can help patients improve their concentration, sensitivity, and reaction speed.

In addition, a positive attitude is also an important factor in improving memory. Patients with spinal root avulsion, when facing challenges and difficulties, should try to maintain an optimistic attitude and bravely face the various challenges of life to achieve the best results in the rehabilitation process.

In summary, spinal cord root avulsion injury does not directly affect a person's memory. Most people can improve their memory through effective methods such as cognitive rehabilitation. At the same time, an optimistic attitude is also the key to improving memory. During the rehabilitation process, we should firmly believe that we can overcome difficulties and move towards a healthy future step by step. It can be seen that we need to improve our memory. Cistanche can significantly improve memory because Cistanche is a traditional Chinese medicinal material with many unique effects, one of which is to improve memory. The efficacy of Cistanche comes from its various active ingredients, including tannic acid, polysaccharides, flavonoid glycosides, etc. These ingredients can promote brain health in a variety of ways.

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This neurodegenerative process shares common characteristics with neuronal disease-related processes such as the presence of endoplasmic reticulum (ER) stress and autophagy flux blockage. 

We previously found that the overexpression of GRP78 promoted motoneuronal neuroprotection after RA. After that, we aimed to unravel the underlying mechanism by carrying out a comparative unbiased proteomic analysis and pharmacological and genetic interventions. 

Unexpectedly, mitochondrial factors turned out to be most altered when GRP78 was overexpressed, and the abundance of engulfed mitochondria, a hallmark of mitophagy, was also observed by electronic microscopy in RA-injured motoneurons after GRP78 overexpression. 

In addition, GRP78 overexpression increased LC3-mitochondria tagging, promoted PINK1 translocation, and mitophagy induction, and recovered mitochondrial function in ER-stressed cells. 

Lastly, we found that GRP78-promoted pro-survival mitophagy was mediated by PINK1 and IP3R in our in vitro model of motoneuronal death. This data indicates a novel relationship between the GRP78 chaperone and mitophagy, opening novel therapeutical options for drug design to achieve neuroprotection.

Keywords: GRP78/BiP; mitophagy; motoneurons; neurodegeneration; neuroprotection.

1. Introduction

Disruption of functional neuronal connectivity is a common early characteristic of neurodegenerative processes [1]. Axonal degeneration isolates neurons that succumb through a retrograde and progressive dysfunctional process. 

In the face of damage, neurons react, activating endogenous mechanisms of neuroprotection such as the unfolded protein response (UPR), the heat-shock response, the autophagy pathway, the ubiquitin-proteasome system, chaperone expression, the endoplasmic reticulum (ER) associated degradation machinery (ERAD), and the antioxidant defense. 

Although their precise activation is effective in recovering the cell, excessive damage, as well as aging, can result in the defective functioning of one or more of those programs. 

We reasoned that mimicking nature and boosting these endogenous mechanisms may be an efficient strategy for neuroprotection [2,3]. GRP78, also known as BiP or heat shock protein 5a (HSP5a), is a multifunctional protein with critical functions in endogenous mechanisms of neuroprotection [4]. GRP78 orchestrates the UPR, which is activated after ER stress, has ATPase activity and is a Ca2+ binding protein [5,6]. 

It also acts to promote the proper folding of newly synthesized or misfolded proteins and to target disassembled proteins for degradation by the ERAD machinery (reviewed in [7]). 

Evidence also suggests that GRP78 participates in triggering macroautophagy, which removes both soluble and aggregated forms of unfolded proteins and dysfunctional organelles [8–15]. 

Overexpression of GRP78 has been proven to be neuroprotective in several models of neurodegeneration [16–23], and a reduction in GRP78 levels has been observed during aging and throughout the progression of degenerative disorders [24]. 

Some of these studies reported that neuroprotection was mediated by inhibition of apoptosis; however, apoptosis is rarely the main cause of neuronal cell death during neurodegenerative processes [20,25–27]. 

Thus, our main goal is to establish the mechanisms by which GRP78 overexpression leads to neuroprotection using non-transgenic models of neurodegeneration. 

In particular, exploiting the anatomical and technical advantages of several models of spinal motoneuron (MN) axotomy, we previously reported that nerve root avulsion (RA) initiates a retrograde process of motor neurodegeneration (80% of MN loss over a month post-injury) characterized by the presence of ER stress, autophagy flux blockage, vesicle, and protein trafficking arrest, the concurrence of apoptosis/antiapoptosis/anoikis initiation but the absence of an effective apoptosis execution [20,25,28]. 

We discovered that the expression of GRP78 is lost around 5 days after RA within damaged MNs and that its forced overexpression allows their survival after axotomy. As we described previously a correct autophagy resolution increases neuron survival after RA [26], we wonder if it is possible to decipher if selective autophagy may have a protective effect. 

Hence, we used an RA model to clarify the mechanisms that mediate GRP78 neuroprotection using an unbiased proteomic analysis, and we further validated the resulting hypothesis by in vitro depth analysis using tunicamycin treatment that triggers both ER stress and protein trafficking arrest, two main characteristics of the RA model [26].

2. Materials and Methods

2.1. Surgical Procedures

Sprague–Dawley female rats aged 12 weeks were kept under standard conditions of light and temperature and given food and water ad libitum. We performed surgical procedures under anesthesia with a cocktail of ketamine/xylazine (0.1 mL/100 g weight) intraperitoneally, essentially as reported previously [26,27]. 

To perform extra vertebral nerve root avulsion of the L4–L5 roots, we made a midline skin incision to identify each side of the sciatic nerve and applied moderate traction on selected roots away from the intervertebral foramina, obtaining the mixed spinal nerves that contained the motor and sensory roots and dorsal root ganglia. 

The wound was sutured by planes and disinfected with povidone-iodine, and the animals were allowed to recover in a warm environment. Sham-operated animals were used as controls. 

All the procedures that involved animals were performed by Spanish (Real Decreto 53/2013) and European (2010/63/UE), and were approved by the Departament d'Agricultura, Ramaderia, Pesca, Alimentació i Medi Natural of the Catalan Government (Generalitat de Catalunya) legislation.

2.2. Construction, Purification, and Infection with Viral Vectors

cDNA encoding GRP78 (ATCC, LGC Promochem, Teddington, UK) was cloned into the pAC.CMV shuttle vector. Recombinant adenoviruses were constructed by homologous recombination in HEK293 cells. 

The control adenovirus expressing bacterial βgalactosidase (Ad-β-gal) was a kind gift of C.B. Newgard (Duke University, Durham, NC, USA). Viruses were purified using the Vivapure AdenoPackTM 20 kit according to the instructions of the manufacturer (Sartorius, Göttingen, Germany). 

For adeno-associated vector construction, the GRP78 cDNA was cloned into NheI and HindIII sites between the ITR domains of AAV2 under the regulation of a CMV promoter and the woodchuck hepatitis virus responsive element [29]. 

The AAVrh10 vector was generated as previously described [30] by triple transfection of HEK293-AAV cells (Stratagene, Bellingham, WA, USA) with branched polyethyleneimine (Sigma-Aldrich, St. Louis, MO, USA) with the plasmid containing the ITRs of AAV2, the AAV helper plasmid containing Rep2 and Cap for rh10 (kindly provided by J.M. Wilson, University of Pennsylvania, Philadelphia, PA, USA), and the pXX6 plasmid containing helper adenoviral genes [31]. Recombinant vectors were clarified after benzonase treatment (50 U/mL, Novagen, Madison, WI, USA) and polyethylene glycol (PEG 8000, Sigma-Aldrich) precipitation. 

Vectors were purified by iodixanol gradient by the Vector Production Unit at CBATEG-UAB following standard operating procedures. Viral genomes per ml (VG/mL) were quantified using PicoGreen (Invitrogen, Waltham, MA, USA). 

Immediately after RA, the animals were injected with 14 µL of either Ad-GRP78 or Adβ-gal virus (108 pfu/mL) using a 33-gauge needle and a Hamilton syringe into the thecal space at the lumbar site. 

Alternatively, intrathecal administration of 10 µL of 4 × 10−10 viral genomes of AAVrh10 adeno-associated virus to overexpress GFP or GRP78 was slowly injected into the CSF between vertebrae L3 and L4 of isoflurane-anesthetized animals three weeks before RA [32]. 

We used AAVrh10 because it infects specifically 30% of spinal motoneurons [32]. Appropriate access to the intrathecal space was confirmed by the animal tail flick. Needles were held in place at the injection site for 1 min, after which muscle and skin were sutured.

2.3. Sample Preparation and Proteomic Analysis

We anesthetized rats (n = 4–5) at 7 dpi and obtained L4–L5 spinal cord segments (5-mm length) samples, which were snap-frozen in liquid nitrogen. We homogenized the tissue in lysis buffer (20 mM HEPES, pH 7.7, 250 mM sucrose, 1 mM EDTA, 1 mM EGTA, and a cocktail of protease and phosphatase inhibitors) with a Potter homogenizer on ice. 

After centrifugation of lysates at 800× g for 20 min at 4 ◦C, we collected the supernatant as the post-nuclear fraction and quantified protein by BCA assay (Pierce Chemical Co., Dallas, TX, USA). 

For proteomic analysis, we solubilized 75 µg of each sample in 4% SDS, 8 M urea, 0.1 M HEPES, pH 7.7, 0.1 M DTT, added 0.05 M iodoacetamide, and digested with trypsin (1:10 ratio of enzyme: substrate) using the Filter Aided Sample Preparation (FASP) method. 

All samples were treated in parallel. We analyzed samples using an LTQ-OrbitrapVelos mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) coupled to a ProxeonEasyLC (Thermo Fisher Scientific). We loaded the peptide mixtures directly onto the analytical column (2 µL·min−1 ) and separated peptides by reversed-phase chromatography using a 15-cm column with an inner diameter of 100 µm packed with 5-µm C18 particles (NikkyoTechnos Co., Tokyo, Japan). 

Chromatographic gradients started at 97% buffer A (0.1% formic acid (FA) in water), and 3% buffer B (acetonitrile, 0.1% FA) with a flow rate of 500 nl·min−1, and increased to 85% buffer A + 15% buffer B over 4 min, and to 55% buffer A + 45% buffer B over 120 min. 

We operated the instrument in TOP20 Data Dependent Acquisition mode with one full MS scan in the Orbitrap at a resolution of 60,000 and a mass range of m/z 350–2000 followed by MS-MS spectra of the 20 most intense ions. We utilized the ion trap with a collision-induced dissociation method to produce fragment ion spectra and used normalized collision energy at 35%. We acquired all data with Xcalibur software v2.1. 

We used the Proteome Discoverer software suite (v1.3.0.339, Thermo Fisher Scientific) and the Mascot search engine (v2.3.01, Matrix Science50) for peptide identification and quantitation. 

We analyzed the data against the SwissProt Rat database containing the most common contaminants (599 entries). We used a precursor ion mass tolerance of 7 ppm at the MS1 level and allowed up to three miscleavages for trypsin. 

The fragment ion mass tolerance was set to 0.5 Da. Oxidation of methionine and N-terminal protein acetylation were set as variable modifications, and cysteine carbamidomethylation was set as a fixed modification. 

We filtered the peptides based on their false discovery rate (FDR > 5% not considered). For peptide quantification, we considered the chromatographic peak of the peptides calculated by using the Proteome Discoverer and median normalized the areas by log2 transformation using R 3.0.2. We quantified the data using the R package MSstats (v. 2.0.1). 

For each ratio, we calculated the adjusted p-value (p < 0.05 for significance). Finally, we performed Gene Ontology and pathway analysis for regulated proteins with DAVID Annotation Web tools (https: //david.ncifcrf.gov/ accessed on 28 May 2021) and KEGG (https://www.genome.jp/ kegg/pathway.html, accessed on 28 May 2021), as we performed previously (see more details in [25]).

2.4. Immunohistochemistry and Image Analysis

After deep anesthesia with pentobarbital, we transcardially perfused the animals with a saline solution containing 10 U/mL heparin, followed by 4% paraformaldehyde in a 0.1 M phosphate buffer, pH 7.2 for tissue fixation at 7 dpi (n = 4 for each condition), and removed the L4 and L5 segments (5-mm total length) of the spinal cord, which were post-fixed in the same fixative for 4 h and cryopreserved in 30% sucrose overnight. 

Serial transverse sections (20-µm thick) were obtained on gelatinized slides using a cryotome (Leica, Wetzlar, Germany) and stored at −20 ◦C until analysis. For immunohistochemistry, we treated the slides with blocking solution in Tris-buffered saline (TBS) with 0.03% TritonX-100 and 10% bovine serum for 1 h and incubated thereafter with different primary antibodies: rabbit anti-LC3B (ab51520; Abcam; 1:200) or mouse anti-COXIV (A21355; Life Technologies;1:500). 

After several washes with TBS, 0.05% Tween-20, the sections were incubated for 2 h with Cy-2 or Cy-3 conjugated donkey anti-rabbit antibodies (Jackson Immunoresearch, West Grove, PA, USA). 

We counterstained the sections with DAPI (Sigma, St Louis, MO, USA), or NeuroTrace Fluorescent Nissl Stain (Molecular Probes, Leiden, The Netherlands) and mounted the slices with Fluoromount-G mounting medium (Southern Biotech, Birmingham, AL, USA) or hand-made Mowiol. 

Sections to be compared were processed together on the same slide and the same day. 

Images of the spinal cord samples from different treatments and controls were taken under the same exposure time, sensitivity, and resolution for each marker analyzed with the aid of a confocal microscope (Zeiss LSM 700). We analyzed signal intensity with ImageJ software (National Institutes of Health; available at http://rsb.info.nih.gov/ij/, accessed on 28 May 2021).

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