Parkin reverses TDP-43-induced cell death and failure of amino acid homeostasis

Authors

  • Michaeline Hebron,

    1. Department of Neuroscience, Georgetown University Medical Center, Washington, District of Columbia, USA
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  • Wenqiang Chen,

    1. Department of Neuroscience, Georgetown University Medical Center, Washington, District of Columbia, USA
    2. Department of Traditional Chinese Medicine, Xuanwu Hospital, Capital Medical University, Beijing, China
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  • Matthew J. Miessau,

    1. Drug Discovery Program, Lombardi Cancer Center, Georgetown University Medical Center, Washington, District of Columbia, USA
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  • Irina Lonskaya,

    1. Department of Neuroscience, Georgetown University Medical Center, Washington, District of Columbia, USA
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  • Charbel E.-H. Moussa

    Corresponding author
    1. Department of Neuroscience, Georgetown University Medical Center, Washington, District of Columbia, USA
    • Address correspondence and reprint requests to Charbel E.-H. Moussa, Laboratory for Dementia and Parkinsonism, Department of Neuroscience, Georgetown University School of Medicine, 3970 Reservoir Rd, NW, TRB, Room WP09B, Washington DC 20057, USA. E-mail: cem46@georgetown.edu

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Abstract

The E3 ubiquitin ligase Parkin plays a central role in the pathogenesis of many neurodegenerative diseases. Parkin promotes specific ubiquitination and affects the localization of transactivation response DNA-binding protein 43 (TDP-43), which controls the translation of thousands of mRNAs. Here we tested the effects of lentiviral Parkin and TDP-43 expression on amino acid metabolism in the rat motor cortex using high frequency 13C NMR spectroscopy. TDP-43 expression increased glutamate levels, decreased the levels of other amino acids, including glutamine, aspartate, leucine and isoleucine, and impaired mitochondrial tricarboxylic acid cycle. TDP-43 induced lactate accumulation and altered the balance between excitatory (glutamate) and inhibitory (GABA) neurotransmitters. Parkin restored amino acid levels, neurotransmitter balance and tricarboxylic acid cycle metabolism, rescuing neurons from TDP-43-induced apoptotic death. Furthermore, TDP-43 expression led to an increase in 4E-BP levels, perhaps altering translational control and deregulating amino acid synthesis; while Parkin reversed the effects of TDP-43 on the 4E-BP signaling pathway. Taken together, these data suggest that Parkin may affect TDP-43 localization and mitigate its effects on 4E-BP signaling and loss of amino acid homeostasis.

image

TDP-43 increases 4E-BP levels and alters translational control, leading to elevation in glutamate (Glu) and lactate (Lac) and attenuation of glutamine (Gln), aspartate (Asp), leucine (Leu), and isoleucine (IsoLeu). TDP-43 decreases GABA levels and inhibits mitochondrial tri-carboxylic acid cycle (TCA), leading to neuronal loss. Parkin facilitates nuclear TDP-43 translocation to the cytoplasm and decreases oxidative stress, protects TCA cycle and promotes cell survival.

Abbreviations used
ALS

amyotrophic lateral sclerosis

LTP

long-term potentiation

mTOR

mammalian target of Rapamycin

TCA

tricarboxylic acid

Parkin is an E3 ubiquitin ligase involved in protein degradation, while Park2 mutations result in loss of Parkin function, leading to autosomal recessive early onset Parkinson's disease (Kitada et al. 1998; Lucking et al. 2000; Shimura et al. 2000; Cookson and Bandmann 2010). Transactivation response DNA-binding protein 43 (TDP-43) is a 414 amino acid protein with DNA/RNA binding protein properties (Ou et al. 1995). TDP-43 binds to Park2 mRNA and regulates its expression (Polymenidou et al. 2011). TDP-43 increases Parkin expression (Polymenidou et al. 2011; Hebron et al. 2013; Lagier-Tourenne et al. 2012), while TDP-43 depletion down-regulates Parkin mRNA in human stem-cell derived from motor neurons bearing TDP-43 aggregates (Lagier-Tourenne et al. 2012). In healthy neurons, TDP-43 is predominantly nuclear, but in neurodegeneration, including amyotrophic lateral sclerosis (ALS) and Frontotemporal Dementia (FTD-TDP, FTD hereafter), TDP-43 is translocated to the cytosol where it is ubiquitinated and/or phosphorylated and cleaved into smaller fragments (Leigh et al. 1991; Arai et al. 2006; Neumann et al. 2006, 2007a,b; Mackenzie et al. 2007, 2010; Yoshiyama et al. 2007; Hasegawa et al. 2008; Zhang et al. 2009). Lentiviral TDP-43 increases nuclear and cytosolic TDP-43 protein levels, while Parkin co-expression mediates TDP-43 ubiquitination, leading to translocation of nuclear TDP-43 to the cytosol (Hebron et al. 2013). These findings suggest that Parkin may mediate TDP-43 localization and modulate its role in many cellular processes.

Alterations of amino acid metabolism are recognized in neurodegenerative diseases (Young and Penney 1984; Greenamyre et al. 1985; Ellison et al. 1986; Perry et al. 1987; Plaitakis and Caroscio 1987). Glutamate accumulation in cortical neurons increases nuclear TDP-43, which returns to its basal level following glutamate-induced injury (Zheng et al. 2012), and induction of glutamate excito-toxicity does not lead to cytosolic TDP-43 inclusions in cultured organotypic slices (Leggett et al. 2012). However, elevation of TDP-43 in the mouse forebrain reduces the level of glutamate dehydrogenase and γ-amino butyric acid (GABA) (Tsai et al. 2010), suggesting that perturbation of amino acid and neurotransmitter metabolism results from alterations of TDP-43 localization. We previously showed that up-regulation (Herman et al. 2011) or lentiviral expression of human wild type TDP-43 (Herman et al. 2012) can lead to pathological changes, including cleavage, aggregation, and phosphorylation. Parkin affects TDP-43 localization (Hebron et al. 2013) and reverses amyloid-induced changes in mitochondrial tricarboxylic acid (TCA) cycle (Khandelwal et al. 2011; Algarzae et al. 2012). To determine the role of TDP-43 in amino acid metabolism and Parkin effects, we used lentiviral gene delivery that allows examination of the early effects of TDP-43 expression on brain metabolism in vivo. We examined the role of Parkin on TDP-43-mediated changes on amino acid and neurotransmitter metabolism using high frequency 1H-decoupled 13C nuclear magnetic resonance (NMR) spectroscopy.

Materials and methods

Stereotaxic injection

Stereotaxic surgery was performed to inject the lentiviral constructs encoding either LacZ, Parkin and/or TDP-43 into the primary motor cortex of two-month-old male Sprague–Dawley rats weighing between 170–220 g as previously described (Burns et al. 2009). Viral stocks were injected through a microsyringe pump controller (Micro4) using total pump (World Precision Instruments, Inc., Sarasota, FL, USA) delivery of 6 μL at a rate of 0.2 μL/min. The needle (26 gauge) was slightly withdrawn every 2 min to avoid mechanical damage and remained in place at the injection site for an additional minute before slow removal over a period of 2 min. Animals were injected into left side of the motor cortex with 2 × 109 m.o.i (Multiplicity of infection (m.o.i) is the ratio of the number of lentiviral particles to the approximate number of target cells that can be potentially infected with at least one viral particle) Lv-LacZ and into the right side with 1 × 109 m.o.i Lv-Parkin+1 × 109 m.o.i Lv-LacZ; or 1 × 109 m.o.i Lv-TDP-43+1 × 109 m.o.i Lv-LacZ; or 1 × 109 m.o.i Lv-Parkin+1 × 109 m.o.i Lv-TDP-43. All animals were killed 2 weeks post-injection and the left cortex was compared to the right cortex. A total of 8 animals for each treatment were used for western blot and caspase-3 activity, 8 animals of each treatment (32 animals) for IHC, and 5 animals for NMR spectroscopy. This study uses the same models that that we previously published (Hebron et al. 2013) and the same lentiviral titer was used and the experiments were all conducted at the same time. Wild-type C57BL/6 and homozygous Parkin−/− mice generated on C57BL/6 background (Goldberg et al. 2003) were used for western blot. Hemizygous TDP-43 transgenic mice harboring human TDP-43 under the control of mouse Thy1 promoter and generated on C57BL/6 background (Wils et al. 2010) were bred according to Jackson's laboratories' protocol and used for western blot. All studies were approved and conducted according to Georgetown University Animal Care and Use Committee (GUACAC).

Western blot analysis

The motor cortex was dissected out and homogenized in 1xSTEN buffer then centrifuged at 5000 g and the supernatant was collected. Total TDP-43 was probed either with human-specific anti-TDP-43 (1 : 1000) mouse monoclonal (2E2-D3) antibody generated against N-terminal 261 amino acids of the full-length protein (Abnova) or (1 : 1000) rabbit polyclonal (ALS10) antibody (catalog no. 10782-2-AP; ProteinTech, Chicago, IL, USA). Rabbit polyclonal anti-Parkin (PRK8) antibody (Millipore Corporation, Bedford, MA, USA) was used (1 : 1000) for western blot. Rabbit polyclonal antibodies for total 4E-BP1 (1 : 1000), Thr 37/46 phosphorylated 4E-BP1 (1 : 500), Ser 209 phosphorylated eIF-4E (1 : 500) and Thr 389 phospho-p70S6K (1 : 500) were used (Translational control sampler kit, Cell Signaling Technology, Beverly, MA, USA). Rabbit polyclonal antibodies for total mammalian target of Rapamycin (mTOR) (1 :1000) and Ser 2448 phosphorylated mTOR (1 : 1000) were used (Translational control sampler kit, Cell Signaling Technology). A rabbit polyclonal V5 (1 : 1000) epitope (Invitrogen, Carlsbad, CA, USA) and rabbit polyclonal anti-actin (Thermo Scientific, Rockville, MD, USA) were used (1 : 1000). Rabbit polyclonal anti-excitatory amino acid transporter (EAAT)-1 (1 : 1000) antibody (Cell Signaling Technology), and rabbit polyclonal anti-EAAT2 (1 : 1000) antibody (Cell Signaling Technology), and rabbit polyclonal anti-EAAT3 (1 : 1000) antibody (Cell signaling Technology) were used. Western blot were quantified by densitometry using Quantity One 4.6.3 software (Bio-Rad Laboratories, Hercules, CA, USA). Densitometry was obtained as arbitrary numbers measuring band intensity. Data were analyzed as mean ± SEM, using anova with Neuman–Keuls multiple comparison between treatments.

Histology was performed on 20 μm thick brain sections to assess neural disintegrative degeneration in animal models using the FD NeuroSilver™ staining kit II (FD NeuroTechnologies, Inc., Baltimore, MD, USA), which provides high contrast and rapid silver staining for the microscopic detection of neuronal and fiber degeneration in vivo. Total cell counts in cortical subfields were obtained by a blinded investigator using unbiased stereology analysis (Stereologer, Stereology Resource Center, Chester, MD, USA). A minimum of six 20 μm sections were analyzed from 400 μm in each direction from the injection site in the ipsilateral area and an equivalent size area within the same region in the contralateral area. The multilevel sampling design in the Stereologer software, based on the optical fractionator sampling method, was used to estimate positive cell numbers detected by Nissl and Silver (Khandelwal et al. 2011).

Caspase-3 fluorometric activity assay

To measure caspase-3 activity in the animal models, we used EnzChekw caspase-3 assay kit #1 (Invitrogen) on cortical extracts and Z-DEVD-AMC substrate and the absorbance was read according to the manufacturer's protocol.

High frequency 13C/1H NMR Spectroscopy

Animals were fasted overnight with free access to tap water and were intra-peritoneally (i.p.) injected with [1-13C] glucose solution (0.5 mol/L) over 10 s (0.3 mL/25–30 g body weight; 200 mg/kg). 45 min later, animals were killed by cervical dislocation and hemispheres were isolated and immediately homogenized in 6% ice-cold perchloric acid, 50 mM NaH2PO4. After homogenization, the perchloric acid-de-proteinized supernatant were separated by centrifugation at 14 000 g for 30 min, frozen on dry ice and lyophilized overnight. Extracts were then re-suspended in 0.65 mL D2O containing 2 mM sodium [13C]formate as internal intensity and chemical shift reference (δ 171.8). Metabolite pool size was identified on 1H {13C-decoupled} NMR spectra. Peak areas were adjusted for nuclear Overhauser effect, saturation, and natural abundance effects and quantified by reference to [13C] formate. Metabolite pool sizes were determined by integration of resonances in 400 MHz {13C-decoupled} 1H spectra using N-acetylaspartate as internal intensity reference. Incorporation of 13C into isotopomers was measured in reference to [13C]formate. 13C[1H-decoupled] spectra typically 30000-33000 transients, pulse width is 4 s, 83 300 data points were acquired at 9.7 Tesla Varian Spectrometer (Palo Alto, CA, USA) with dual 13C/1H probe. {13C-decoupled}-1H spectra were acquired with 3000 scans, pulse angle 45°, relaxation delay 1 s, line broadening 0.5 Hz, acquired data points 13 132 and transformation size 32 K at 25°C. Spectra were integrated and quantified using MestReNova (Master Lab Research, Bologna, Italy).

Statistical analysis

Data were analyzed using GraphPad PRISM software (San Diego, CA, USA) and statistical analysis was performed using anova, Neuman–Keuls, p < 0.05 or as indicated, n is indicated in the text, and graphs were plotted mean ± SEM.

Results

Parkin reverses TDP-43-induced cell death in rat motor cortex

We previously showed that injection of a lentiviral vector driving wild-type TDP-43 expression in the rat motor cortex (Herman et al. 2012) leads to protein cleavage, aggregation and phosphorylation 2-week post-injection (Herman et al. 2012). We also demonstrated that TDP-43 is expressed in neurons as well as microglia and Parkin translocates TDP-43 from the nucleus to the cytosol (Hebron et al. 2013). These studies were extended to analyze amino acid metabolism via 13C NMR. Lentiviral injection significantly increased (44%, n = 5, p < 0.05) TDP-43 levels in the rat motor cortex (Fig. 1b) compared to LacZ (Fig. 1a) and Parkin (Fig. 1c). However, Parkin co-expression led to TDP-43 localization within the cytoplasm in DAPI (4′,6-Diamidino-2-Phenylindole, Dilactate)-stained cells (Fig. 1c), as we previously showed in these animal models (Hebron et al. 2013). Counter-staining TDP-43-stained cortical sections with 3,3′-Diaminobenzidine showed TDP-43 expression throughout the motor cortex in TDP-43 (Fig. 1f and g) compared to LacZ (Fig. 1e) and Parkin (Fig. 1h). To determine TDP-43 effects on cell survival, Nissl-stained cells were quantified by stereological counting. TDP-43 significantly reduced (31%, n = 8, p < 0.03) the number of Nissl-stained cells in the rat cortex (Fig. 1j and v) compared to LacZ (Fig. 1i and v) and Parkin (Fig. 1l and v); while Parkin co-injection (Fig. 1k and v) reversed Nissl-stained cell loss compared to TDP-43 alone. Co-staining showed endogenous TDP-43 and Parkin expression in LacZ cortex (Fig. 1m), but co-expression of Parkin with TDP-43 (Fig. 1o) resulted in less TDP-43 in DAPI-stained nuclei and more co-localization with cytosolic Parkin compared to TDP-43 (Fig. 1n) or Parkin alone (Fig. 1p, n = 8). Neurodegenerative death was also demonstrated via silver staining, which showed significantly increased staining (56%, n = 8, p < 0.02) in TDP-43 brains (Fig. 1r and v) compared to LacZ (Fig. 1q and v) and Parkin (Fig. 1t and v). High magnification (Fig. 1q) shows silver staining within cell bodies and axonal processes, suggesting that TDP-43 induces neuronal and axonal degeneration. Parkin reduced the number of silver-stained cells back to LacZ level (Fig. 1sand v). Caspase-3 activity was also measured via ELISA and showed that TDP-43 significantly increases caspase-3 levels (Fig. 1u, 94%, n = 8, p < 0.001) compared to LacZ and Parkin; but Parkin co-expression prevents the increase in caspase-3 (Fig. 1v). Taken together, these data indicate that TDP-43 induces cell death in the rat cortex and Parkin reverses TDP-43 effects. To verify equal gene expression via lentiviral delivery, western blot analysis of lentiviral V5 epitope showed equal amount of V5 expression (Fig. 1w, third blot) relative to actin in all injected animals. However, TDP-43 levels were significantly increased (44%, n = 8, p < 0.02) relative to actin (Fig. 1w, second blot) in TDP-43 compared to LacZ or Parkin alone, while Parkin levels were increased (31%, n = 8, < 0.04) relative to actin (Fig. 1w) compared to LacZ.

Figure 1.

Parkin reverses transactivation response DNA-binding protein 43 (TDP-43)-induced apoptotic cell death in rat motor cortex. TDP-43 and DAPI staining of 20 μm thick brain sections injected with lentiviral vector driving expression of (a) LacZ, (b) TDP-43, (c) TDP-43 and Parkin and (d) Parkin. Low magnification shows TDP-43 expression in 3,3′-Diaminobenzidine (DAB) counter-stained 20 μm thick brain sections injected with lentiviral vector driving expression of (e) LacZ, (f) TDP-43, (g) TDP-43 and Parkin, and (h) Parkin. Nissl staining of 20 μm thick brain sections injected with lentiviral vector driving expression of I) LacZ, J) TDP-43, (k) TDP-43 and Parkin, and (l) Parkin alone. TDP-43 and Parkin co-staining of 20 μm thick brain sections injected with lentiviral vector driving expression of (m) LacZ, (n) TDP-43, insert shows silver staining in cell body and axonal processes, (o) TDP-43 and Parkin and (p) Parkin. Silver staining of 20 μm thick brain sections injected with lentiviral vector driving expression of (q) LacZ (r) TDP-43 (s) Parkin and TDP-43 and (t) Parkin alone injected brains. Histograms represent (u) caspase-3 activity and (v) stereological counting of Nissl and silver-positive cells in lentiviral LacZ, TDP-43, Parkin+TDP-43. Western blot analysis on 4–12% SDS NuPAGE gel shows (w) TDP-43 levels relative to actin (first blot) and Parkin levels relative to actin (second blot). V5 epitope level showing equal amount of V5 expression (third blot) relative to actin in LacZ, TDP-43 and Parkin. Asterisk is significantly different to LacZ, mean ± SEM, anova, Neuman–Keuls, p as indicated, n = 8.

Parkin restores TDP-43-induced alteration of amino acid and mitochondrial TCA cycle metabolism

High frequency 13C/1H NMR spectroscopy was performed in cortical extracts to compare the effects of Parkin on TDP-43 in the right hemisphere (ipsilateral) compared to LacZ or Parkin in left hemisphere (contralateral) 2-week post-injection. 1H-decoupled/13C magnetic resonance spectroscopy show that TDP-43 significantly increased (50%, n = 5, < 0.0001) the concentration of 13C label in glutamate (Glu) C4 compared to LacZ and Parkin (Fig. 2a). Parkin co-expression with TDP-43 reversed 13C concentration in Glu C4 back to LacZ level (Fig. 2a). TDP-43 decreased 13C in glutamine (Gln) C4 (34%, n = 5, < 0.002) compared to LacZ and Parkin; while Parkin co-expression with TDP-43 restored 13C back to LacZ level (Fig. 2a). 13C-decoupled 1H magnetic resonance spectroscopy showed that TDP-43 induced a significant increase in total glutamate (21%, < 0.05, n = 5) and a decrease in glutamine (28%, < 0.05, n = 5) compared to LacZ and Parkin, which reversed glutamate and glutamine levels back to control (Fig. 2b). Aspartate levels were also measured because it is an excitatory amino acid that can be transported by EAATs similar to glutamate. TDP-43 significantly decreased 13C concentration in aspartate (Asp) C2 and C3 (47% and 42% respectively, n = 5, < 0.002) compared to LacZ and Parkin (Fig. 2c), but this effect was restored by Parkin (Fig. 2c). TDP-43 significantly decreased (21%, < 0.05, n = 5) aspartate pool size compared to LacZ and Parkin, which reversed aspartate level back to control (Fig. 2d). Measurement of TCA cycle metabolism showed that TDP-43 decreased 13C concentration in succinate (Succ) C2/C3 and citrate (Cit) C3 (53% and 44%, respectively, n = 5, < 0.002) compared to LacZ and Parkin (Fig. 2e) and Parkin co-expression reversed 13C concentration in Succ C2/C3 and Cit C2 back to LacZ level (Fig. 2e). The decrease of 13C flux into TCA cycle intermediates succinate and citrate was echoed by a decrease in metabolite concentration (62% and 51%, respectively, < 0.0001, n = 5), while Parkin reversed mitochondrial TCA metabolism back to control (Fig. 2f). Because leucine and isoleucine are branched-chain amino acids that feed into the TCA cycle (Sears et al. 2009), their metabolism was examined to determine whether they are affected by TCA cycle perturbation. TDP-43 significantly decreased (21% and 18%, respectively, n = 5, < 0.05) 13C concentration in leucine (Leu) C4 and isoleucine (Isoleu) C4 compared to LacZ and Parkin (Fig. 2g). Parkin co-expression with TDP-43 reversed 13C concentration in Leu C4 and Isoleu C4 back to LacZ level (Fig. 2g). TDP-43 also decreased (32% and 22%, respectively, < 0.05, n = 5) the pool sizes of leucine and isoleucine compared to LacZ and Parkin, which returned leucine and isoleucine levels back to control (Fig. 2h).

Figure 2.

High frequency 13C NMR spectroscopy reveals alteration in the levels of amino acid and tricarboxylic acid (TCA) cycle intermediates. Histograms represent (a) 13C concentration derived from glucose in Glu C4 and Gln C4 (b) pool size of total glutamate and glutamine in rat hemispheres injected with lentiviral vector driving the expression of LacZ, transactivation response DNA-binding protein 43 (TDP-43), Parkin, or Parkin+TDP-43. (c) 13C concentration in Asp C2 and C3 and (d) pool size of total of aspartate rat hemispheres injected with vector driving the expression of LacZ, TDP-43, Parkin, or Parkin+TDP-43. (e) 13C concentration in TCA cycle intermediates Succ C2/C3 and Cit C2, and (f) pool size of total succinate and citrate in rat hemispheres injected with lentiviral vector driving the expression of LacZ, TDP-43, Parkin, or Parkin+TDP-43. (g) 13C concentration in Leu C4 and Isoleu C4 and (f) pool size of total leucine and isoleucine in rat hemispheres expressing LacZ, TDP-43, Parkin, or Parkin+TDP-43. Asterisk indicates significantly different, mean ± SEM, anova, Neuman–Keuls, n = 5. p as indicated.

Parkin abrogates TDP-43-induced oxidative stress and loss of GABA neurotransmitter

Lactate metabolism was measured to determine whether TDP-43 alteration of mitochondrial TCA cycle induces oxidative stress and cytosolic lactate accumulation. TDP-43 induced a significant increase (29%, n = 5, < 0.04) in 13C concentration in lactate (Lac) C3 compared to LacZ and Parkin (Fig. 3a). However, Parkin expression with TDP-43 reversed alteration of 13C levels in Lac C3, consistent with Parkin effects on TCA cycle intermediates. In addition, TDP-43 significantly increased lactate level (38%, n = 5, < 0.001) and Parkin restored lactate back to LacZ and Parkin levels (Fig. 3b). Taken together, these data indicate oxidative stress and lactate accumulation as a result of TDP-43-induced mitochondrial TCA cycle changes. GABA levels were also measured to determine whether the changes in excitatory glutamate affected the inhibitory neurotransmitter GABA, which is in part derived from glutamate. TDP-43 significantly decreased 13C concentration in GABA C2 (Fig. 3c, 50%, n = 5, < 0.0001) compared to LacZ and Parkin, but Parkin restored the decrease of 13C in GABA C2. TDP-43 also decreased the total level of GABA (62%, n = 5, < 0.0001) compared to LacZ (Fig. 3d). These data suggest that TDP-43 expression alters neurotransmitter levels and disturbs the balance between inhibitory and excitatory neurotransmitters. However, Parkin reverses GABA level when co-expressed with TDP-43, but GABA remains significantly less than LacZ level (21%, < 0.004).

Figure 3.

Parkin reserves lactate accumulation, loss of GABA levels and restores EAATs function. Histograms represent (a) 13C concentration in Lac C3 and (b) pool size of total lactate in rat hemispheres injected with lentiviral vector driving the expression of LacZ, transactivation response DNA-binding protein 43 (TDP-43), Parkin, or Parkin+TDP-43. (c) 13C concentration in GABAC2 and (d) pool size of GABA in rat hemispheres injected with lentiviral vector driving the expression of LacZ, TDP-43, Parkin, or Parkin+TDP-43. *indicates significantly different, mean ± SEM, anova, Neuman–Keuls, p as indicated, n = 5. (e) western blot analysis on 4–12% SDS NuPAGE gel shows EAAT1 (first blot) and EAAT2 (second blot) and EAAT3 (third blot) relative to actin in LacZ, TDP-43 and Parkin. *indicates significantly different, mean ± SEM, anova, Neuman–Keuls, p as indicated, n = 6.

To ascertain that TDP-43 did not change glutamate and aspartate metabolism via alterations of EAAT levels, which transport glutamate as well as aspartate, western blot was performed and showed no difference in the protein levels of EAAT1, 2, and 3 relative to actin (Fig. 3e), indicating that the changes in glutamate and aspartate were not because of altered levels of transporters.

TDP-43 activates 4E-BP, independent of mTOR, signaling to affect amino acid levels

We examined the effects of TDP-43 expression on markers of translational control, including the mTOR pathway that can affect amino acid metabolism. Inhibition of mTOR inhibits the translation factor eIF-4E that is modulated by eIF-4E-binding proteins (4E-BPs) phosphorylation and cap-dependent translation (Gingras et al. 2001; Grolleau et al. 2002). Kinase signaling via mTOR affects mRNA translation through phosphorylation of downstream kinases such as 4E-BPs and p70S6k (Shima et al. 1998; Gingras et al. 2001; Grolleau et al. 2002; Mamane et al. 2004). No differences were observed in total mTOR (Fig. 4a, first blot) or phosphorylation at Ser 2448 (Fig. 4a, second blot) between TDP-43, Parkin ± TDP-43 and LacZ. The phosphorylation of p70S6k at Thr389 (third blot) was also unaffected between different treatments, suggesting lack of mTOR activation. However, a significant increase in total levels (55%, n = 8, < 0.001) and Thr 37/46 phosphorylated (42%, < 0.01) 4E-BP1 were observed (Fig. 4b and c), indicating increased kinase activity in TDP-43 brains compared to LacZ. A significant decrease (51%, n = 8. < 0.001) in eIF-4E phosphorylation at Ser 209 was observed downstream of 4E-BP1 (Fig. 4b and c), suggesting that TDP-43 increases total 4E-BP1 and may modulate translational control, independent of mTOR activity. Parkin co-expression with TDP-43 significantly reversed total (27%, n = 8, < 0.04) and phosphorylated (39%, < 0.01) 4E-BP1 (Fig. 4d and e), while significantly increasing (19%, n = 8. < 0.05) eIF-4E phosphorylation (Fig. 4d and e), suggesting that Parkin expression modulates TDP-43 effects on 4E-BP signaling.

Figure 4.

Transactivation response DNA-binding protein 43 (TDP-43) activates 4E-BP signaling. Western blot of cortical brain lysates in gene transfer animals on 10% SDS NuPAGE gel showing (a) total mammalian target of Rapamycin (mTOR) levels (first blot) phosphorylated mTOR at Ser 2448 (second blot) and phosphorylated p70S6k levels at Thr 389 (third blot) relative to actin (fourth blot). (b) Western blot of cortical brain lysates on 4–12% SDS NuPAGE gel showing total 4E-BP1 levels (first blot) phosphorylated 4E-BP1 at Thr 37/46 (second blot) and phosphorylated eIF-4E at Ser 209 (third blot) relative to actin (fourth blot). (c) Histograms represent densitometry, western blot on brain lysates on 4–12% SDS NuPAGE gel showing (d) total 4E-BP1 levels (first blot) phosphorylated 4E-BP1 at Thr 37/46 (second blot) and phosphorylated eIF-4E at Ser 209 (third blot) relative to actin (fourth blot). (e) Histograms represent densitometry. (f) Western blot on brain lysates from TDP-43 transgenic and Parkin−/− mice showing total 4E-BP1 levels (first blot) phosphorylated 4E-BP1 at Thr 37/46 (second blot) and phosphorylated eIF-4E at Ser 209 (third blot) relative to actin (fourth blot). (g) Histograms represent densitometry of western blots, *indicates significantly different, Mean ± SEM, anova, Neuman–Keuls, p and n as indicated.

These results were verified in 4–5 months old male control C57BL/6, homozygous Parkin−/− (Goldberg et al. 2003) and hemizygous TDP-43 transgenic mice harboring human TDP-43 (Wils et al. 2010) all generated on C57BL/6 background. Significantly higher TDP-43 levels (190%, n = 3, < 0.0001) were observed in TDP-43 transgenic mice relative to actin compared to control (Fig. 4f and g). However, Parkin−/− mice (first blot) had significantly increased levels of TDP-43 relative to actin (85%, n = 3, < 0.002) compared to control (Fig. 4f and g). A significant increase in the levels of total (165%, n = 3, < 0.002) and phosphorylated (91%, < 0.01) 4E-BP1 (Fig. 4f and g) were observed in TDP-43 transgenic mice, indicating increased kinase activity. Similarly, a significant increase in the levels of both total (70%, n = 3, < 0.02) and phosphorylated (43%, < 0.05) 4E-BP1 (Fig. 4f and g) were observed in Parkin−/− mice. Furthermore, a significant decrease (71%, < 0.001, n = 3) in eIF-4E phosphorylation (Fig. 4f and g) was observed in TDP-43 mice. Phosphorylated eIF-4E was reduced (19%) in Parkin−/− mice. Taken together, these results suggest that Parkin attenuates the effects of TDP-43 on perturbations of translational control.

Discussion

These studies reveal the early effects of TDP-43 on amino acid metabolism. TDP-43 increased glutamate levels and decreased the levels of other amino acids, including its precursor glutamine and the excitatory amino acid aspartate. The increase in glutamate level was associated with apoptotic cell death and increased oxidative stress, including decreased mitochondrial TCA cycle metabolism and cytosolic lactate accumulation. The level of cytotoxic glutamate remained elevated after TDP-43 over-expression despite the decrease in TCA metabolism and glutamine level, suggesting lack of glutamate re-cycling via EAATs. The current data suggest that the effects of TDP-43 on brain metabolism, may be because of neuronal and/or astrocytic changes as lentiviral TDP-43 is expressed in both cell types (Herman et al. 2012). We previously demonstrated that alterations of glutamate transport significantly change 13C flux through the TCA cycle and affect glutamate/glutamine cycling rates in guinea pig brain slices (Moussa Cel et al. 2002; Moussa et al. 2007). Principal component analysis of 13C-labeled metabolome suggested that selective inhibitions of individual EAATs lead to characteristic changes in brain metabolism. This observation indicates that each EAAT may have a very distinctive role in brain function and small perturbations in its expression and/or location could have far reaching - possibly adverse – consequences (Rae et al. 2005, 2006; Moussa et al. 2007).

Parkin reversed TDP-43 effects on TCA cycle and amino acid metabolism, perhaps because of Parkin role in nuclear TDP-43 translocation (Hebron et al. 2013). Nuclear TDP-43 regulates thousands of gene expression and is implicated in many steps of RNA expression and transport (Polymenidou et al. 2011; Tollervey et al. 2011), so its nuclear translocation may affect control of amino acid synthesis. TDP-43 binds to Park2 mRNA and regulates its expression (Polymenidou et al. 2011), and we demonstrated that lentiviral TDP-43 over-expression increases Park2 mRNA and protein levels in vitro and in vivo (Hebron et al. 2013). Inversely, TDP-43 depletion down-regulates Park2 mRNA in human stem-cell derived motor neurons in sporadic ALS (Lagier-Tourenne et al. 2012). Parkin stability is also altered in TDP-43 mice harboring the A315T mutation (Hebron et al. 2013). In post-mortem brains of human ALS and FTD, Parkin level is altered in motor neurons containing TDP-43 aggregates (Lagier-Tourenne et al. 2012). TDP-43 modulation of Parkin levels (Shimura et al. 2000) and detection of ubiquitin-positive TDP-43 inclusions in neurodegeneration (Neumann et al. 2006, 2007a,b; Mackenzie et al. 2007; Yoshiyama et al. 2007; Hasegawa et al. 2008; Zhang et al. 2009), suggest that Parkin may mediate TDP-43 localization via ubiquitination. We showed that co-expression of lentiviral TDP-43 and Parkin promotes K-48 and K-63-linked ubiquitin to TDP-43, leading to translocation of nuclear TDP-43 (Hebron et al. 2013). Together, these data suggest that Parkin mediates TDP-43 localization via ubiquitination.

In healthy neurons, TDP-43 is predominantly nuclear, but in neurodegeneration, including ALS and FTD, TDP-43 is translocated to the cytosol where it is ubiquitinated and/or phosphorylated and cleaved (Leigh et al. 1991; Arai et al. 2006; Neumann et al. 2006, 2007a,b; Mackenzie et al. 2007, 2010; Yoshiyama et al. 2007; Hasegawa et al. 2008; Zhang et al. 2009). It is still unknown whether nuclear or cytosolic (or both) TDP-43 is the culprit in ALS/FTD pathology. Patients with sporadic ALS, as well as familial ALS because of TDP-43 mutations, show abnormal accumulations of TDP-43 in the cytoplasm of spinal cord motor neurons (Neumann et al. 2006; Mackenzie et al. 2007). TDP-43 also accumulates in ubiquitin-positive cytoplasmic or nuclear inclusions in FTD and ALS (Kwiatkowski et al. 2009; Vance et al. 2009). Therefore, TDP-43 localization may be essential for its biological function. The sheer length of motor neurons in the descending spinal motor tracts, which degenerate in ALS (Dickson et al. 2007; Mitchell and Borasio 2007; Geser et al. 2008; McCluskey et al. 2009), may make these neurons more vulnerable to alterations in TDP-43 localization compared to other neurons bearing TDP-43 aggregates. Many RNA binding proteins exhibit dual functions, including nuclear, such as mRNA splicing, and cytoplasmic, such as mRNA transport and silencing (Sau et al. 2011). Deregulation of axodendritic transport is associated with gene mutations causing motor neuron disorders (Sau et al. 2011). Thus, TDP-43 transport from the soma to distant synapses may be required for generation of synaptic proteins. Relevant to the current studies, TDP-43 binds to mRNA of several synaptic proteins and may regulate the level of vesicular glutamate transporters, glutamate receptors, and synaptotagmin, which controls synaptic vesicle dynamics (Polymenidou et al. 2011). Thus, future studies should focus on Parkin-mediated TDP-43 localization to the synapse and maintenance of glutamate metabolism in motor versus non-motor neurons. Recent findings suggest that glutamate increases nuclear TDP-43 (Zheng et al. 2012), while glutamate excito-toxicity does not lead to cytosolic TDP-43 inclusions (Leggett et al. 2012). Defects in glutamate transmission are widely implicated in neurodegeneration (Rahn et al. 2012). The level of some plasma membrane glutamate transporters is altered in ALS, perhaps leading to excito-toxicity and motor neuron degeneration in human ALS patients (Rothstein 1995; Wilson and Shaw 2007; Quinlan 2011). Taken together, these findings and our data suggest that TDP-43 translocation may depend on glutamate levels, which could exacerbate TDP-43 pathology. In addition, Parkin role in reversing mitochondrial TCA cycle metabolism may also be because of its effects on mitochondrial dynamics (Deng et al. 2008; Poole et al. 2010; Tanaka 2010; Ziviani et al. 2010), which promotes mitochondrial fragmentation during mitophagy (Tanaka et al. 2010), thus leading to replenishment of functional mitochondria and increased TCA cycle metabolism.

Parkin plays an important role in synaptic transmission and Parkin deficient animals display defects in synaptic function. Parkin over-expression reverses overgrown neuromuscular synapses and reduces evoked and miniature excitatory junction potentials in motor neurons axons of Parkin mutant larvae (Vincent et al. 2012). Parkin regulates the function and stability of glutamatergic synapses, and Parkin knockdown induces glutamatergic synapse proliferation, leading to excito-toxicity (Helton et al. 2008). Proteomic analysis of Parkin−/− brain lysates show alterations in proteins that are essential for synaptic function (Periquet et al. 2005). Synaptotagmin XI is a member of the synaptotagmin family that is well characterized for vesicle formation and docking (Schiavo et al. 1997; Fukuda et al. 2000). Parkin regulates the function of synaptotagmin that controls storage, docking and release of vesicular glutamate (Huynh et al. 2003). Synaptic stimulation triggers local protein synthesis at the synapse (Jiang and Schuman 2002); while inhibition of protein synthesis disrupts synaptic plasticity, including long-term facilitation, long-term potentiation (LTP), and long-term depression (Kang and Schuman 1996; Martin et al. 1997; Huber et al. 2000). Therefore, local translational control impacts at the level of individual synapses. TDP-43 availability at the synapse may be important to maintain homeostatic level of synaptic proteins. TDP-43 binds to the mRNA of a large number of proteins that are integral to synaptic function and formation (Polymenidou et al. 2011), including synaptotagmin, glutamate transporters, glutamate receptors, and astrocytic glutamate transporters (Polymenidou et al. 2011). One form of LTP, long-lasting late-phase LTP (L-LTP), requires both gene transcription and RNA translation (Klann and Dever 2004). For example, hippocampal long-term depression mediated by metabotropic glutamate receptors requires rapid translation of pre-existing mRNA (Huber et al. 2000). Thus, Parkin may affect TDP-43 localization and increase its bio-availability at synapses to maintain synaptic glutamate metabolism.

The effects of TDP-43 expression on the balance between excitatory and inhibitory neurotransmitters are novel. The decrease in GABA was concurrent with increased glutamate, which is one precursor of GABA synthesis via glutamate dehydrogenase activity. These results are consistent with prior studies indicating that TDP-43 expression reduces the level of glutamate dehydrogenase and GABA levels in the mouse brain (Tsai et al. 2010). In addition, GABA may be derived from succinate, further suggesting that TDP-43 expression targets neurotransmitter synthesis. The simultaneous change in glutamate and GABA are indicative that TDP-43 expression affects neurotransmitter balance, resulting in increased excitatory and decreased inhibitory mechanisms. The change in neurotransmitter levels may affect brain regions that are associated with specific neurodegenerative changes, leading to excito-toxic damage and modulation of neurotransmitter systems in a disease-dependent manner. Furthermore, these studies tested the early effects of TDP-43 expression, independent of the developmental effects of the transgene in animal models expressing TDP-43 or its mutations; therefore, other changes may be associated with neurotransmitter synthesis. TDP-43 may affect translational control via regulation of splicing and transcription of many mRNAs. The effects of TDP-43 expression on 4E-BP activity may affect translational control and amino acid metabolism (Sears et al. 2009), including leucine and isoleucine, which affect the mTOR pathway (Matsumura et al. 2005; Herningtyas et al. 2008). In the current studies, expression of TDP-43 did not change mTOR activity or P70S6k phosphorylation 2-week post-injection. TDP-43 up-regulated the level of total 4E-BP and increased its phosphorylation, leading to decreased phosphorylation of eIF4E, suggesting that TDP-43 affects translation initiation downstream of mTOR. TDP-43 may stimulate the phosphorylation of 4E-BP1 to decrease interaction between eIF4E and 4E-BP, and consequently activate cap-dependent translation (Gingras et al. 2001; Grolleau et al. 2002). Interestingly, TDP-43 over-expression increased glutamate levels, but decreased all other amino acids, including aspartate, leucine, isoleucine and glutamine, suggesting a role for translational control in TDP-43-induced alterations of amino acid and neurotransmitter levels.

Acknowledgements and Conflict of Interest disclosure

This study was supported by NIH grant AG30378 and Georgetown University funding to Charbel E-H Moussa. The authors acknowledge the Drug Discovery Program at the Lombardi Cancer Center, Georgetown University for the NMR studies. The study was conducted in compliance with the ARRIVE guidelines. No conflicts of interests are declared by the authors.

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