Progranulin preserves mitochondrial health and turnover in neurons under hyperglycemic stress in a p97-dependent manner

doi:10.21203/rs.3.rs-4510864/v1

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Progranulin preserves mitochondrial health and turnover in neurons under hyperglycemic stress in a p97-dependent manner

https://doi.org/10.21203/rs.3.rs-4510864/v1

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Type II diabetes (T2D) affects over half a billion adults and is a risk factor for neurodegenerative disease. A major component of T2D, hyperglycemia (chronic excess blood glucose) causes mitochondrial damage while impairing their clearance via mitophagy, leading to neuronal cell death. Conversely, restoring mitochondrial turnover to maintain overall mitochondrial health is a means of neuroprotection. We investigated whether progranulin (PGRN), an endogenously-expressed neurotrophic factor that has been shown to be protective against frontotemporal dementia, also protects against hyperglycemic stress at the mitochondrial level. In vitro studies of primary cortex showed that high glucose led to mitochondrial dysfunction that was prevented with PGRN co-treatment. Additionally, we found that the expression and localization of key mitophagy proteins PTEN-INduced Kinase 1 (PINK1), Parkin, and p97 are affected by PGRN. Conversely, pharmacological inhibition of p97 prevented many of PGRN’s protective benefits under high-glucose conditions. These data showcase a novel mechanism of PGRN’s protection and a newly-characterized connection between PGRN and p97. While further studies in vivo are necessary to validate these findings, they provide unique insights into the protective mechanisms of PGRN against diabetic neurodegeneration.

Diabetes

hyperglycemia

progranulin

mitochondria

mitophagy

p97

Type II diabetes (T2D) affects over half a billion adults and is characterized by chronic increased blood glucose (hyperglycemia) due to insulin resistance (Safiri et al. 2022; Chew et al. 2023). This contributes to a host of pathologies including heart disease, cancer, and neurodegeneration, making T2D and downstream cognitive impairment an imminent public health concern. Common to both T2D and neurodegenerative diseases like Alzheimer’s is dysregulation of protein degradation pathways such as autophagy and mitophagy (de Mello et al. 2019; Gómez et al. 2021), which remove protein aggregates and damaged mitochondria, respectively. These pathways are crucial to prevent the onset of neurodegeneration by protein accumulation (Fleming et al. 2022).

The mitochondria are a particular “pain point” in neurodegeneration because neurons have an outsize metabolic demand relative to other cell types (Bélanger, Allaman, and Magistretti 2011; Watts, Pocock, and Claudianos 2018) and are largely post-mitotic, limiting their regenerative capacity in the central nervous system post-stress (Anda et al. 2016; Aranda-Anzaldo 2012). When dysfunctional mitochondria accumulate in diabetes, there is an overall reduced ATP output despite an increased leak of reactive oxygen species (ROS) like superoxide (Rovira-Llopis et al. 2017; Shan et al. 2022). On the other hand, upregulating mitophagy flux under stress preserves the health of the overall mitochondrial population and improves cell viability (Lou et al. 2020; Chen and Chan 2009). This makes mitochondrial restoration through turnover of damaged organelles a promising avenue for neuroprotection under stress conditions.

In the most well-characterized pathway of mitophagy, a diminished mitochondrial membrane potential enables the kinase PTEN-INducible Kinase 1 (PINK1) to accumulate on the outer mitochondrial membrane (OMM) (Eiyama and Okamoto 2015). PINK1 recruits and stabilizes the E3 ubiquitin ligase Parkin to the OMM, where it is phosphorylated by PINK1 and polyubiquitinated, serving as a signal for sequestration and degradation (Palikaras, Lionaki, and Tavernarakis 2018; Campbell, Morris, and Schapira 2018). Important to this is the AAA + ATPase p97 (also known as VCP and Cdc48p) (Xia, Tang, and Ye 2016), a segregase/unfoldase protein that aids in shuttling damaged mitochondria for controlled degradation through PINK1-Parkin signaling (Kim et al. 2013; Tanaka et al. 2010).

Progranulin (PGRN) is a 68 kDa protein that was originally characterized as a pro-peptide involved in lysosomal function (Bateman and Bennett 1998). A body of clinical research over the past three decades has revealed its importance in neuroprotection, with missense mutations to GRN present in familial cases of frontotemporal dementia (Almeida et al. 2016; Gijselinck, Van Broeckhoven, and Cruts 2008; Gass et al. 2006) and GRN knockout replicating frontotemporal dementia in models (C. Zhou et al. 2015; Chang et al. 2017). Conversely, overexpression of and supplementation with PGRN is neuroprotective in vitro and in vivo (Van Kampen and Kay 2017; Minami et al. 2014; Arrant et al. 2017). There is a lack of conclusive evidence on PGRN’s neuroprotective potential under diabetic conditions however, with seemingly disparate findings among different cell populations and conditions (Nicoletto and Canani 2015; B. Zhou et al. 2015; D. Zhou et al. 2019). While there is evidence that PGRN preserves autophagy under neuronal stress (Dedert, Salih, and Xu 2023; Elia et al. 2019), there is scant research addressing whether it mediates mitophagy specifically (D. Zhou et al. 2019; Rodríguez-Periñán et al. 2023) and if under diabetic stress in the brain.

This study provides evidence that PGRN aids in neuroprotection under hyperglycemic stress by preserving mitochondrial health and turnover through p97. Primary cortical cells from C57BL/6 mice cultured under high-glucose conditions showed characteristic neuronal pathologies associated with diabetic stress, which PGRN mitigated. This included mitochondrial morphology, pH, ROS generation, and lysosomal co-localization under high-glucose stress. We also observed a relationship between PGRN expression and p97 levels, and that inhibiting p97 attenuated many of PGRN’s protective effects. These data provide evidence for a novel model of PGRN’s mechanism of neuroprotection under diabetic conditions and serve as a promising foundation for PGRN as a potential protector against diabetic neuronal pathology.

Compounds and reagents

All compounds and reagents used were either produced in-lab (using the components listed in their respective sections) or purchased from commercially-available sources. In the case of the latter, all company and catalog information (Cat#) is listed alongside the relevant reagent.

Organisms used

All procedures involving animals were handled in accordance with the National Institute of Health animal safety guidelines. The protocols used were approved by Saint Louis University’s Institutional Animal Care and Use Committee (IACUC). For in vitro studies, primary cortical neurons were dissected from C57BL/6 mice or mt-Keima mice (a C57BL/6-derived mouse line expressing a fluorescent Keima protein on the mitochondrial COX VIII protein (Sun et al. 2017)). The mt-Keima mice were generously provided by the lab of Dr. Jonathan Brestoff at Washington University in St. Louis. Pups were aged P0-P3 at the time of dissection. For ex vivo studies, C57BL/6 mice were fed either a standard chow diet (Con, containing 10% kCal from fat; Harlan Teklad, Cat# TD.06416) or a high-fat diet (HFD, containing 60% kCal from fat; Harlan Teklad, Cat# TD.06414) from 6 weeks to 22.5 weeks (approximately 4 months) before animals were sacrificed for brain tissue collection.

Cell models used

Cortices from mouse pups were dissected and incubated in an enzymatic digestion solution (Neurobasal medium containing 1.5 mM L-cysteine (Sigma, Cat# 168149-25G), 2 mM CaCl₂ (Sigma, Cat# C4901-100G), 1 mM EDTA (Sigma, Cat# E9884-100G), and 40 units of papain (Worthington Biochemical, Cat# LS003126) at 37°C for 20–25 minutes. The digestion solution was then inactivated by washing tissues thrice with Neurobasal complete medium, containing Neurobasal medium (Gibco, Cat# 35050-061), 1% penicillin/streptomycin (Gibco, Cat# 15140122), 1X GlutaMAX (Gibco, Cat# A1286001), 2% B-27 (Gibco, Cat# 17504-044), and 4% FBS (Avantor, Cat# 97068-086). Cells were triturated using fire-polished glass pipettes and plated on culture dishes coated with 100 µg/ml poly-D lysine (Sigma, Cat# P6407-5MG) and 2 µg/ml laminin (Sigma, Cat# 11243217001). Cells were grown in an incubator maintained at 37°C and 5% CO₂.

Some experiments were performed in the human neuroblastoma cell line SH-SY5Y using wild-type—WT—and homozygous PGRN knockout—GRN-/-) strains. These cell lines were generously provided by the lab of Dr. Andy Nguyen at the Department of Internal Medicine at Saint Louis University School of Medicine. SH-SY5Y cells were incubated in a growth medium consisting of DMEM:F12 (Gibco, Cat# 11320-033) with 10% FBS and 1% pen/strep and passaged once every 2–3 days. At confluence, SH-SY5Y cells were differentiated by washing once with PBS and replacing the growth medium with differentiation medium, which consisted of Neurobasal medium with 1% pen-strep, 2% B27 supplement, and 10 µM all-trans retinoic acid (ATRA; Sigma, Cat# R2625). SH-SY5Y cells were differentiated for 2–3 days before treatment.

Treatment and collection of cell samples

Primary cortical neurons were cultured in Neurobasal complete medium, with the medium changed every 3–4 days and at the start of experimental treatments. At each interval, half of the medium was changed. SH-SY5Y cells were treated in differentiation medium supplemented with glucose (Sigma, Cat# G5767) to achieve a final glucose concentration of 100 mM, with control conditions containing 25 mM glucose, and 200 ng/ml recombinant mouse PGRN (rPGRN; R&D Systems, Cat# 2557-PG-050).

Primary cells were treated with high glucose, i.e. Neurobasal complete medium supplemented with glucose (Sigma, Cat# G5767) to achieve a final glucose concentration of 100 mM, with control conditions containing 25 mM glucose, 200 ng/ml recombinant mouse PGRN (rPGRN; R&D Systems, Cat# 2557-PG-050), and 100 nM of the p97 inhibitor NMS-873 (Selleckchem, Cat# S7285). All inhibitors were used at a 1:1000 dilution, with 0.1% DMSO used as a vehicle control in other groups. Cells were treated for up to 72 hours before live-cell testing, harvest, or fixing for subsequent assays.

Protein lysates from primary cortical cells and SH-SY5Y cells were collected by washing cells thrice with PBS and harvesting using a commercially available lysis buffer (NPER, ThermoFisher, Cat# 87792) containing Halt protease inhibitors (ThermoFisher, Cat# 1860932) and phosphatase inhibitors (ThermoFisher, Cat# 78420).

Calcium imaging

One hour before experimentation, cells were pre-treated with either 200 ng/ml PGRN or 0.1% PBS as a vehicle control. At the end of 72-hour treatment in high or normal glucose, cells were washed twice with HBSS, then incubated in HBSS containing 5 µM of Fura 2-AM (Invitrogen, Cat# F1221) for 30 minutes at 37°C. Cells were then washed thrice with HBSS and imaged using a fluorescence microscope. MetaFluor software was used to collect images at 340 nm and 380 nm fluorescence excitation, and the ratio of 340/380 nm fluorescence intensity was used as the metric of cytosolic calcium levels. Cells were images at baseline for 2–3 minutes, for 2–3 minutes after acute treatment with 50 µM rotenone (with negative control experiments using 0.1% DMSO as a vehicle), and for 1–2 minutes following 10 mM KCl treatment. KCl was added to verify neuronal status of cells studied, as neurons show a characteristic upward spike in cytosolic calcium levels immediately following acute KCl administration.

Mitochondrial viability analyses

Mitochondrial viability was determined by measuring the degree of superoxide production (MitoSOX assay kit; Invitrogen, Cat# M36008). After treatment, cells were washed twice with HBSS, then incubated in HBSS containing 500 nM of MitoSOX at 37°C for 30 minutes. Cells were washed twice with HBSS before imaging using an inverted microscope (IX73, Olympus) with fluorescence light filters (Lambda XL, Sutter Instrument), with pictures taken using a Retiga R1 camera (Q-Imaging).

MitoTracker and LysoTracker imaging and analysis. After treatment, primary cortical cells were washed twice with HBSS, then incubated in 70 nM MitoTracker Red CMXROS (Invitrogen, Cat# M7512) and 100 nM LysoTracker Green DND-26 (Invitrogen, Cat# L7526) for 30 minutes at 37°C. Cells were then immediately imaged under an SP8 LIGHTNING confocal microscope (Leica). The images were uploaded into ImageJ software (NIH), and the green channel (for lysosomes) was thresholded to remove ambiguity, while the red channel (for mitochondria) was unchanged. From there, both the mitochondrial number and the number of mitochondria overlapping with lysosomes were counted by a trained observer.

Immunoblotting

Protein lysates were spun at 14,000 rpm for 10 minutes and the supernatant retained for protein concentration measurement (Pierce BCA protein assay, Invitrogen, Cat# 23227). An equal amount of protein from each sample was treated with 4X LSB (Invitrogen, Cat# NP0007) containing 350 mM dithiothreitol (DTT; Bio-Rad, Cat# 1610611), heated for 5 minutes at 95°C, and run on a NU-PAGE 4–12% bis-tris gel (Invitrogen, Cat# NP0323BOX) using a Novex mini-cell device (Invitrogen, Cat# EI0001). The gel was then transferred to a 0.45 µm nitrocellulose membrane (Bio-Rad, Cat# 1620115) using a Mini Protean Tetra System (Bio-Rad, Cat# 1658004) on ice. For HMC3 analyses, samples were loaded into SDS-PAGE Bio-Rad TGX gels in a Mini Protein Tetra System (Bio-Rad, Cat# 1658004) and transferred using a Bio-Rad Turbo-Blot transfer system.

Membranes were blocked in 5% milk (Bio-Rad, Cat# 1706404) in TBST for one hour. Membranes were incubated in primary antibodies for p97 (Proteintech, Cat# 10736-1-AP) and GAPDH (CST, Cat# 2118S) at a 1:1000 dilution overnight at 4°C. After washing with TBST, membranes incubated in goat anti-rabbit con-jugated with HRP (Invitrogen, Cat# 31460) at a 1:2500 dilution for one hour at room temperature. Membranes were washed with TBST, incubated in Pierce ECL western blotting substrate (ThermoFisher, Cat# 32109), and imaged using an iBright FL1000 (ThermoFisher). Densitometric analysis was performed using ImageJ software (NIH).

Immunofluorescence

Cells were washed twice with PBS, fixed with 4% paraformaldehyde (ThermoFisher, Cat# J19943-K2) for 20 minutes, and permeabilized with 0.3% Triton X-100 (VWR, Cat# 0694-1L) for 5 minutes. Primary cortical cells were blocked in PBS containing 5% goat serum (Gibco, Cat# 16210-064) overnight at 4°C. The following primary antibodies were used for 1 hour at room temperature: MAP2 (Invitrogen, Cat# 13-1500), Parkin (CST, Cat# 4211S), and p97 (Proteintech, Cat# 10736-1-AP) at a 1:200 dilution, and PINK1 (Novus, Cat# NBP2-36488) at a 1:100 dilution.

After washing thrice with PBS, cells were incubated in the following secondary antibodies for 1 hour: goat anti-rabbit conjugated with Alexa Fluor 568 (Invitrogen, Cat# A11036) and goat anti-mouse conjugated with Alexa Fluor 488 (Invitrogen, Cat# A11029) at a 1:500 dilution. Cells were then washed and mounted using coverslips and mounting medium containing DAPI (Fluoroshield, Sigma, Cat# F6507). Slides were imaged using a confocal microscope (SP8 LIGHTNING, Leica) using LASX 1.4.5 software (Leica).

Statistical analyses used

GraphPad PRISM 9.5.0 software was used for statistical analysis of data collected. For comparisons between two groups, Student’s t-test was used. A paired t-test was used when comparing the same samples before and after treatment, and an unpaired t-test when comparing different samples. For comparisons between more than two groups, one-way ANOVA was used. In cases where standard deviations were excessively different (verified by Bartlett’s test), Welch ANOVA was instead used. Fisher’s Least Significant Difference was used for post-hoc (for cases in which Welch ANOVA was used, Student’s t-test with Welch’s correction was used for post-hoc testing instead).

PGRN Protects Against Acute Rotenone-induced Mitochondrial Stress

To validate a more clarion connection between PGRN treatment and protection against acute mitochondrial insults, we pre-treated primary cortical neurons with PGRN for 1 hour, then provided a stressful stimulus through 50 µM rotenone, a known mitochondrial complex I inhibitor and uncoupler (Greenamyre, Betarbet, and Sherer 2003). Acute rotenone stress caused a severe increase in cytosolic calcium (measured by max Δ340/380 nm ratio), indicative of efflux from the mitochondria, a known reservoir of cellular Ca²⁺ (Bravo-Sagua et al. 2017; Finkel et al. 2015). When cells were pre-treated with PGRN, the degree of cytosolic calcium increase due to rotenone decreased from 0.257 ± 0.053 Δ340/380 nm to 0.108 ± 0.034 Δ340/380 nm (p = 0.031) (Fig. 1A). This was also observed in primary cortical neurons, with cytosolic calcium increase diminishing from 0.463 ± 0.051 Δ340/380 nm to 0.332 ± 0.032 Δ340/380 nm (p = 0.028) (Fig. 1B,C). These findings indicate that PGRN has a distinct mitoprotective role under acute stress.

PGRN Preserves Neuronal Mitochondrial Health and Activity Under Hyperglycemic Stress

The maintenance of mitochondrial health is especially crucial in neurons due to their high metabolic requirements and post-mitotic nature, making them vulnerable to excessive ROS generation downstream of mitochondrial activity (Dan Dunn et al. 2015). Given our promising findings of PGRN’s protection against acute mitochondrial stress, we studied mitochondrial health under hyperglycemic stress conditions. This was done by measuring their ROS production using MitoSOX, a dye that fluoresces in response to superoxide production, and by observing their morphology using the fluorescence-based dye MitoTracker Red. After 72 hours of treatment, we observed that, under high-glucose conditions, superoxide production in differentiated SH-SY5Y neurons increased (indicated by greater fluorescence) from 1.000 ± 0.018 AU to 1.123 ± 0.042 AU (p = 0.002) (Fig. 2A). This effect was attenuated by PGRN co-treatment, with fluorescence decreasing from 1.123 ± 0.042 AU to 1.041 ± 0.024 AU (p = 0.032). The morphology of the mitochondria also noticeably changed with high-glucose stress (Fig. 2B). Under control conditions, mitochondrial shape was largely elongated, indicative of healthy organelles (see arrows), while under high-glucose conditions, the mitochondrial population was largely circular, indicating damage and fission (see arrowheads) (Chen and Chan 2009; Liu et al. 2021). However, cells co-treated with high glucose and PGRN had a largely elongated mitochondrial constituency, further suggesting improved overall health due to PGRN.

We also observed altered mitochondrial health in primary cortical neurons from mt-Keima mice, a wild-type mouse model with a pH-sensitive reporter attached to the mitochondrial membrane protein COX VIII (Sun et al. 2017). The amount of red fluorescence intensity (corresponding to mitochondria with a pH of approximately 4.5) increased with high glucose (from 1.000 ± 0.069 AU to 1.290 ± 0.110 AU, p = 0.029), with PGRN attenuating this increase (from 1.290 ± 0.110 AU to 0.981 ± 0.059 AU, p = 0.016) (Fig. 2C,D). These data indicate that PGRN is protective not only against acute stressors (e.g. rotenone) but also chronic stressors such as hyperglycemic conditions. These data showcase a distinct pathological phenotype (increased ROS production and acidity) under our hyperglycemic stress paradigm that was prevented by PGRN administration.

Mitochondrial-lysosomal Interaction Is Modified by PGRN Under Hyperglycemic Stress

When damaged, mitochondria are sequestered into lysosomes for controlled degradation through clearance pathways such as mitophagy (Chen and Chan 2009; Han et al. 2023; Jiang et al. 2022). To capture a snapshot of the degree of mitochondrial-lysosomal localization, we measured the proportion of neuronal mitochondria (stained with MitoTracker Red) that were co-localized with lysosomes (stained with LysoTracker Green). Interestingly, the degree of mitochondrial-lysosomal interaction increased with high glucose (from 9.456 ± 0.649% to 34.914 ± 7.760%, p = 0.032). PGRN treatment significantly increased this percentage further under high glucose (from 34.914 ± 7.760% to 53.035 ± 4.157%, p = 0.013) but not normal glucose (from 9.456 ± 0.649% to 14.985 ± 2.768%, p = 0.389) (Fig. 3A). In no group was the total mitochondrial density affected (F = 0.167, p = 0.916) (Fig. 3B), indicating that the changes observed represent a greater proportion of the mitochondrial population in contact with lysosomes (Fig. 3C, see arrows). This combination of findings suggests that the changes observed are likely not due to excess mitochondrial fusion or fission events and are indicative of overall mitochondrial health.

PGRN Appears to Promote PINK1-Parkin Interaction Under Hyperglycemic Stress

Multiple mechanistic pathways of mitochondrial degradation have been characterized in literature, with the most studied being the PINK1-Parkin mitophagy pathway (Lou et al. 2020; Ashrafi and Schwarz 2013). To assess if PGRN functions to promote mitochondrial turnover through this pathway, we examined the expression of and interaction between key proteins PINK1 and Parkin, which co-localize on the OMM in response to disrupted membrane potential. In primary cortical neurons under high glucose treatment, we observed that PINK1-Parkin co-localization appeared to increase with PGRN treatment, even under high-glucose conditions (Fig. 4A). The number of yellow punctae (representing the co-localization of PINK1 in green and Parkin in red) appeared to increase with high glucose and PGRN co-treatment (Fig. 4B, see arrows), suggesting an increase in PINK1-Parkin interaction. These data indicate that hyperglycemic stress specifically modulates PINK1-Parkin interaction, indicating that this pathway is involved in PGRN’s maintenance of neuronal mitochondria under diabetic conditions.

P97 Expression and Nuclear Localization are Affected by PGRN Treatment and Hyperglycemic Stress

Given our observed effects of PGRN on mitochondrial health and maintenance, we hypothesized that PGRN may exert its mitoprotective effects through expression of p97, an important protein tied to mitochondrial turnover via mitophagy (Tanaka et al. 2010; Fang et al. 2015). We observed an apparent direct relationship between PGRN expression and total p97 levels, with SH-SY5Y cells containing a genetic PGRN knockout (GRN-/-) exhibiting a trend towards a significantly reduced level of p97 (F = 2.917, p = 0.061). Under normal glucose levels, GRN-/- cells showed decreased expression compared to their WT counterparts from 1.000 ± 0.515 AU to 0.445 ± 0.135 AU (p = 0.030), while under high glucose, p97 expression in GRN-/- cells trended towards a decrease compared to WT (from 0.838 ± 0.184 AU to 0.442 ± 0.094 AU, p = 0.095). However, high glucose did not alter p97 expression in either WT or GRN-/- neurons (from 1.000 ± 0.515 AU to 0.838 ± 0.450 AU, p = 0.500) (Fig. 5A). A similar trend was observed in primary cortical neurons supplemented with recombinant PGRN for 72 hours, although this did not reach the threshold for significance (F = 0.949, p = 0.437; Fig. 5B). This lack of change due to hyperglycemia was also present in ex vivo cortical tissues from mice fed a high-fat diet: p97 expression increased with HFD from 1.000 ± 0.342 AU to 1.148 ± 0.467 AU (p = 0.804) (Fig. 5C).

The mean fluorescence intensity of p97 in primary cortical cells showed not only an increase in p97 expression due to high glucose (from 1.000 ± 0.059 AU to 2.075 ± 0.085 AU, p = 0.000), but that PGRN caused a decrease in p97 under high glucose (from 2.075 ± 0.085 AU to 1.495 ± 0.076 AU, p = 0.000) while increasing its expression under normoglycemic conditions (from 1.000 ± 0.059 AU to 1.625 ± 0.078 AU, p = 0.000) (Fig. 5D,E). The increase in mean fluorescence intensity noted lined up with a marked increase in nuclear localization of p97: the percentage of overlap between p97 and the nucleus (stained with DAPI) increased from 7.025 ± 3.565% nuclear overlap under control conditions to 75.677 ± 5.325% under high glucose (p = 0.000) and 35.230 ± 6.875% with high glucose + PGRN co-treatment (p = 0.001 compared to control; p = 0.000 compared to high glucose alone) (Fig. 5F). Of note is that nuclear p97 staining appeared to form puncta adjacent to DAPI-stained puncta (regions indicative of A-T rich heterochromatin (Kapuscinski 1995; Chazotte 2011)) (Fig. 5G; see arrows). These data suggest that PGRN specifically affects p97 protein levels under both basal and stress conditions while high-glucose stress alone does not necessarily do so.

P97 Affects Mitochondrial Structure, Lysosomal Interaction, and pH Due to PGRN Under High Glucose

While p97 is primarily implicated in proteasomal degradation, evidence by Meyer et al. (Meyer, Bug, and Bremer 2012) has demonstrated a role for p97 in lysosomal degradation. Therefore, we considered the potential impact of p97 in lysosomal degradation of mitochondria using a p97-specific AAA + ATPase inhibitor, NMS-873. This allosteric inhibitor binds p97 at its D2 domain with an IC₅₀ of 30 nM while leaving other similar AAA ATPases relatively unaffected (with IC₅₀ values > 10 µM) (Magnaghi et al. 2013). Mitochondrial morphology, assessed by MitoTracker staining, was deleteriously affected by NMS-873 co-treatment under high glucose + PGRN conditions, with a greater proportion of mitochondria appearing to have a circular morphology (Fig. 6A). Of note, the MitoTracker fluorescence intensity of neurons stained also appeared fainter; considering that MitoTracker Red is taken up in proportion to mitochondrial membrane potential, this also suggests decreased mitochondrial health when p97 is inhibited.

From MitoTracker-LysoTracker co-staining, we observed that the percentage of mitochondria co-localized with lysosomes decreased with NMS-873 co-treatment under high glucose + PGRN conditions, from 31.191 ± 5.943% to 18.231 ± 3.120% (p = 0.039) (Fig. 6B). Despite the more fragmented morphology (which would suggest an increased number of mitochondria from excess fission), the concentration of mitochondria/µm² was unchanged due to p97 inhibition (F = 0.143, p = 0.934), indicating that the changes in mitochondrial dynamics represent the overall mitochondrial population (Fig. 6C). We additionally observed that the accumulation of lysosomes changed with NMS-873 treatment: while there was minimal somatic accumulation under basal conditions, NMS-873 treatment caused a massive accumulation of lysosomes in the soma. Under high-glucose conditions—where lysosomal buildup was prevalent in the neurites—lysosome accumulation was largely restricted to the soma with NMS-873 co-treatment (see Supplementary Fig. 1).

In primary cortical neurons from mt-Keima mice, the mean red fluorescence intensity did not significantly change with NMS-873 treatment compared to their respective control or high glucose conditions without PGRN, suggesting no specific change in mitochondrial acidity due to p97 inhibition (Fig. 6D). Red fluorescence intensity only trended towards significance with an increase in neurons treated with PGRN when NMS-873 was added (from 0.727 ± 0.064 AU to 0.901 ± 0.172 AU, p = 0.095), while a trend towards a decrease was observed under high glucose + PGRN conditions with NMS-873 (from 0.981 ± 0.059 AU to 0.820 ± 0.068 AU, p = 0.075) (see Supplementary Fig. 2). These data collectively suggest that p97 inhibition affects mitochondrial health, structure, and lysosomal degradation in cortical neurons, attenuating the protective activity of PGRN under hyperglycemic conditions.

T2D is highly deleterious to cell health, contributing to neurodegeneration in the long-term through impaired mitochondrial dynamics in neurons (Rovira-Llopis et al. 2017). Because the mitochondria are a crucial hub of metabolic activity (Son and Han 2018; Bader and Winklhofer 2020), we explored whether the neuroprotective factor PGRN also protects against diabetic stress on a mitochondrial level. We found that PGRN treatment protects against mitochondrial pathology in terms of structure, health, and activity. Furthermore, we provide evidence that the expression and activity of the AAA + ATPase p97 (also known as VCP and Cdc48p) is affected by PGRN expression and supplementation (Fig. 5) and affects its role in mitochondrial dynamics (Fig. 6), potentially linking these two proteins in the process of mitochondrial turnover.

While PGRN’s exact mechanism of neuroprotection is not conclusive, there is evidence that its lysosomal capacity may play a role, as it is cleaved into granulin protease subunits when trafficked to the lysosome by chaperone proteins like sortilin (X. Zhou et al. 2018) and prosaposin (X. Zhou et al. 2015). Given that neurodegenerative diseases share an underlying inefficacy at protein aggregate clearance through lysosomal degradation pathways such as autophagy (Lim and Yue 2015), it is possible that PGRN protects against neurodegenerative disease at the lysosomal level. Our imaging studies using MitoTracker and LysoTracker indicate that this may be the case, as we observed an increase in mitochondrial-lysosomal interaction with PGRN under high-glucose conditions (Fig. 3A). It is worth noting, however, that PGRN treatment alone (i.e. under normoglycemic conditions) did not elicit a change in lysosomal interaction, suggesting that this reflects a reactive measure to hyperglycemic stress rather than a broad activation of this subcellular trafficking via PGRN.

Mitophagy, a subset of autophagy that selectively clears damaged and defective mitochondria, is of particular importance in neurons (Chen and Chan 2009; Doblado et al. 2021). Because we have seen PGRN’s effects on lysosomal expression in macroautophagy in previous studies (Dedert, Salih, and Xu 2023; Dedert et al. 2022), we explored if PGRN also mediates mitophagy through PINK1 and Parkin expression, two key initiating proteins. Our immunofluorescence data suggest that although hyperglycemic stress seemed to increase Parkin’s interaction with downstream protein PINK1 (Fig. 4). While we also noted an elevated level of mitochondrial-lysosomal interaction (Fig. 3), the fact that mitochondrial pH and ROS generation were akin to control levels with high glucose + PGRN (Fig. 2) suggest that there may be a “bottleneck” under hyperglycemic stress that induces mitochondrial damage and mitophagy initiation. Further testing with PGRN intervention (either through in vivo supplementation or viral overexpression in vitro) is necessary to validate our immunofluorescence findings that indicate that PGRN increases PINK1-Parkin interaction further under hyperglycemic conditions.

A spate of literature characterizing p97’s role in mitophagy has come out in recent years, providing ample evidence that it promotes mitochondrial turnover, such as several key interactions with WIPI2 (Lu et al. 2022), UBXN1 (Mengus et al. 2022), and Parkin (Kim et al. 2013) that promote mitophagy through mitofusin sequestration and degradation (Tanaka et al. 2010; Fang et al. 2015). It has also been shown to interact with UBXD8 to facilitate separation of mitochondria from ER contact sites (Ganji et al. 2023), crucial to enabling PINK1/Parkin- and BNIP3-mediated mitophagy initiation (Li et al. 2021). While these findings are promising, much of the research published to date centers on HeLa cells, with limited neuronal focus (Wani and Weihl 2021; van Swieten and Heutink 2008). This emphasizes the necessity for neuron-specific studies to elucidate the extent of p97 activation in potentially safeguarding against neurodegenerative pathology.

Despite the established connections between PGRN and lysosomal function (X. Zhou et al. 2018; Kao et al. 2017; Paushter et al. 2018), there are limited studies linking PGRN to p97 expression and activity. A recent study of neuron-specific p97 knockout in mice showed a similar profile of differentially expressed genes to GRN heterozygous mice, as well as TDP-43 inclusions (Wani et al. 2021). On the converse, we found that homozygous GRN knockout in SH-SY5Y cells led to a more steep decline in p97 (Fig. 5A) compared to the apparent increase observed from 72 hours of PGRN supplementation in primary cortical cells (Fig. 5B). This may be due in part to the temporal nature of the treatment, and we expect that lentiviral overexpression of GRN over an extended period would more greatly increase endogenous p97 expression. In further support of our proposed connection between PGRN and p97, we found that blocking p97 with the specific pharmacological inhibitor NMS-873 attenuated or negated many of PGRN’s protective effects under high-glucose stress.

Another interesting observation in primary neurons was the increased nuclear localization observed when treated with high glucose or PGRN (Fig. 5D-G). While p97 is best known as a mediator of protein degradation, its segregase activity has been linked to repairing double-stranded DNA breaks (Ramadan et al. 2007; Fujita et al. 2013). Especially intriguing is that p97’s efficacy is tied to buildup and clearance of polyglutamine proteins in the nucleus (e.g. huntingtin, ataxin-3), with failure to clear these proteins impairing p97’s ability to repair double-stranded DNA breaks (Fujita et al. 2013; Boeddrich et al. 2006). However, a study by Kobayashi et al. (Kobayashi et al. 2002) exploring mutant p97 localization in PC12 cells showed no change in subcellular localization due to an inactivating mutation at the D2 domain (the same region inhibited pharmacologically by NMS-873). In addition, p97 segregase activity has been linked to disassociation of PARP1 from chromatin (Alemasova and Lavrik 2019; Krastev et al. 2022); the removal of this early responder to DNA damage74 may explain the close association of p97 and heterochromatin-rich sections of DNA under high-glucose stress (Fig. 5G). That we saw this phenomenon in neurons treated with NMS-873 as well (yet still observed deleterious effects to viability) suggests that either PGRN’s neuroprotection due to p97 is independent of its nuclear localization or that p97’s ability to localize to the nucleus is not affected while another crucial characteristic is (such as ATPase activity (Kobayashi et al. 2002)). Further subcellular fractionation studies are needed to tease out a distinct role for p97 in each cellular compartment.

It is important to point out that despite an increase in mitochondria-lysosome interaction (Fig. 6B), neuronal mitochondrial concentration remained unchanged with high glucose and PGRN (Fig. 6C). P97 inhibition via NMS-873 also did not affect mitochondrial density, except in the case of high glucose treatment alone (Fig. 6B, see Supplementary Fig. 2), suggesting that despite an altered fission-fusion equilibrium under these stress conditions, the overall mitochondrial density was largely unchanged. Based on our data indicating that hyperglycemia reduces the initiating steps of mitophagy (Fig. 4), this is likely not due to a combination of increased mitochondrial fission and clearance. Additional studies using lysosomal (e.g. chloroquine) and proteasomal (e.g. MG132) inhibitors would verify this hypothesis.

Imaging-based studies of mitochondrial pH showed that their acidification decreased with PGRN treatment under high-glucose conditions. However, p97 inhibition caused this to decrease further (although not to the level of statistical significance) despite no changes when inhibiting p97 under high-glucose medium alone (Fig. 6D, see Supplementary Fig. 2). It is possible that the lack of pH decrease may reflect not necessarily a change in mitochondrial turnover but in the lysosomal health itself. As PGRN is an important pro-protease in lysosomal degradation (Elia et al. 2019; X. Zhou et al. 2018; Kao et al. 2017), perhaps p97 plays a role in its efficient trafficking to the lysosome; when blocked, this would impair its effectiveness (i.e. lysosomal acidity), providing an unexpectedly high readout of mitochondrial pH for organelles sequestered in lysosomal vesicles. Verification of this through non-fluorescence-based methods such as electron microscopy would elucidate if these mitochondria simply remain undigested in the lysosome or perhaps untrafficked altogether. A more intricate experimental design, such as acute p97 knock-down via CRISPR- or RNAi alongside PGRN treatment or overexpression, would also help elucidate which effects arising from PGRN are p97-dependent and which are p97-independent.

Although a comprehensive treatment modality remains elusive to treating T2D-derived neurodegeneration, PGRN has emerged as a key protector against neurodegenerative diseases like frontotemporal dementia and Alzheimer’s, conditions that are exacerbated by diabetes. This study provides further evidence of a connection between PGRN and T2D, with the former protecting against the downstream deleterious effects of the latter in the brain. New to this field of research is the role of p97, a regulatory degradative protein, in PGRN’s mechanism of mitochondrial protection—a novel combination of findings that sheds new light on how PGRN protects against neuronal diabetic pathology. While future studies exploring other mechanisms of p97 regulation in vitro and in vivo are necessary, these data provide evidence of a novel connection between these two proteins with regards to neuroprotection and neurodegeneration.

Ethics approval and consent to participate

This animal study was reviewed and approved by the Saint Louis Institute for Animal Care And Use Committee (IACUC).

Consent for publication

Not applicable

Availability of data and materials

The raw data and materials used in this study will be made available by the authors upon request, without undue reservation

Competing interests

The authors declare no competing conflicts of interest.

Funding

This research was funded with the aid of the Saint Louis University start-up fund to FX, the Saint Louis University President’s Research Fund to FX, the Saint Louis University Health Research Fund to FX, the Saint Louis University Graduate-Undergraduate Research Collaboration Fund to CD, the Saint Louis University Knoedler Fund to CD, and the Sigma Xi Grants In Aid of Research award (award #02056) to CD.

Authors’ contributions

CD and FX conceptualized and planned the experimental study. CD performed experiments and performed data analysis. CD and FX prepared and edited the manuscript.

Acknowledgements

The Mt-Keima mice used in this study were a generous gift from the lab of Dr. Jonathan Brestoff at Washington University in St. Louis. The wild-type and GRN-/- SH-SY5Y cells used in this study were generously provided by the lab of Dr. Andy Nguyen at Saint Louis University School of Medicine.

SUPPLEMENTARY MATERIAL

The Supplementary Figures for this article can be found online at: link for supplementary material.

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Supplementary Fig. 1Mitochondrial-lysosomal co-localization in primary cortical neurons treated with high glucose and NMS-873. Regions of overlap were observed in the soma (arrows) and in neurites (arrowheads). Scale bar, 50 μm.
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Supplementary Fig. 2Mt-Keima red fluorescence in primary cortical neurons treated with high glucose, PGRN, and NMS-873. N=17-82 neurons.
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Progranulin preserves mitochondrial health and turnover in neurons under hyperglycemic stress in a p97-dependent manner

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Abstract

Figures

Introduction

Materials and Methods

Compounds and reagents

Organisms used

Cell models used

Treatment and collection of cell samples

Calcium imaging

Mitochondrial viability analyses

Immunoblotting

Immunofluorescence

Statistical analyses used

Results

PGRN Protects Against Acute Rotenone-induced Mitochondrial Stress

PGRN Preserves Neuronal Mitochondrial Health and Activity Under Hyperglycemic Stress

Mitochondrial-lysosomal Interaction Is Modified by PGRN Under Hyperglycemic Stress

PGRN Appears to Promote PINK1-Parkin Interaction Under Hyperglycemic Stress

P97 Expression and Nuclear Localization are Affected by PGRN Treatment and Hyperglycemic Stress

P97 Affects Mitochondrial Structure, Lysosomal Interaction, and pH Due to PGRN Under High Glucose

Discussion

Declarations

References

Supplementary Files

Status:

Version 1