Takeya Kubo

Glyceraldehyde-3-phosphate Dehydrogenase Aggregate Formation Participates in Oxidative Stress-induced Cell Death

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)2 is a classic glycolytic enzyme that also mediates cell death by its nuclear translocation under oxidative stress. Meanwhile, we previously presented that oxidative stress induced disulfide-bonded GAPDH aggregation in vitro. Here, we propose that GAPDH aggregate formation might participate in oxidative stress-induced cell death both in vitro and in vivo. We show that human GAPDH amyloid-like aggregate formation depends on the active site cysteine-152 (Cys-152) in vitro. In SH-SY5Y neuroblastoma, treatment with dopamine decreases the cell viability concentration-dependently (IC50 = 202 μm). Low concentrations of dopamine (50–100 μm) mainly cause nuclear translocation of GAPDH, whereas the levels of GAPDH aggregates correlate with high concentrations of dopamine (200–300 μm)-induced cell death. Doxycycline-inducible overexpression of wild-type GAPDH in SH-SY5Y, but not the Cys-152-substituted mutant (C152A-GAPDH), accelerates cell death accompanying both endogenous and exogenous GAPDH aggregate formation in response to high concentrations of dopamine. Deprenyl, a blocker of GAPDH nuclear translocation, fails to inhibit the aggregation both in vitro and in cells but reduced cell death in SH-SY5Y treated with only a low concentration of dopamine (100 μm). These results suggest that GAPDH participates in oxidative stress-induced cell death via an alternative mechanism in which aggregation but not nuclear translocation of GAPDH plays a role. Moreover, we observe endogenous GAPDH aggregate formation in nigra-striatum dopaminergic neurons after methamphetamine treatment in mice. In transgenic mice overexpressing wild-type GAPDH, increased dopaminergic neuron loss and GAPDH aggregate formation are observed. These data suggest a critical role of GAPDH aggregates in oxidative stress-induced brain damage.

A rapid, targeted, neuron-selective, in vivo knockdown following a single intracerebroventricular injection of a novel chemically modified siRNA in the adult rat brain

There has been a dramatic expansion of the literature on RNA interference and with it, increasing interest in the potential clinical utility of targeted inhibition of gene expression and associated protein knockdown. However, a critical factor limiting the experimental and therapeutic application of RNA interference is the ability to deliver small interfering RNAs (siRNAs), particularly in the central nervous system, without complications such as toxicity and inflammation. Here we show that a single intracerebroventricular injection of Accell siRNA, a new type of naked siRNA that has been modified chemically to allow for delivery in the absence of transfection reagents, even into differentiated cells such mature neurons, leads to neuron-specific protein knockdown in the adult rat brain. Following in vivo delivery, targeted Accell siRNAs were incorporated successfully into various types of mature neurons, but not glia, for 1 week in diverse brain regions (cortex, striatum, hippocampus, midbrain, and cerebellum) with an efficacy of delivery of approximately 97%. Immunohistochemical and Western blotting analyses revealed widespread, targeted inhibition of the expression of two well-known reference proteins, cyclophilin-B (38-68% knockdown) and glyceraldehyde 3-phosphate dehydrogenase (23-34% knockdown). These findings suggest that this novel procedure is likely to be useful in experimental investigations of neuropathophysiological mechanisms.

Glyceraldehyde-3-phosphate Dehydrogenase Aggregates Accelerate Amyloid-β Amyloidogenesis in Alzheimer Disease.

Background: There is currently no strong evidence for a linkage between glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and Alzheimer disease (AD). Results: GAPDH aggregates enhanced amyloid-β peptide (Aβ) amyloidogenesis and augmented Aβ40-induced neurotoxicity, both in vitro and in vivo, concomitant with mitochondrial dysfunction. Conclusion: GAPDH aggregates accelerate Aβ amyloidogenesis. Significance: Aβ amyloidogenesis associated with GAPDH aggregation might underlie AD pathogenesis. Alzheimer disease (AD) is a progressive neurodegenerative disorder characterized by loss of neurons and formation of pathological extracellular deposits induced by amyloid-β peptide (Aβ). Numerous studies have established Aβ amyloidogenesis as a hallmark of AD pathogenesis, particularly with respect to mitochondrial dysfunction. We have previously shown that glycolytic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) forms amyloid-like aggregates upon exposure to oxidative stress and that these aggregates contribute to neuronal cell death. Here, we report that GAPDH aggregates accelerate Aβ amyloidogenesis and subsequent neuronal cell death both in vitro and in vivo. Co-incubation of Aβ40 with small amounts of GAPDH aggregates significantly enhanced Aβ40 amyloidogenesis, as assessed by in vitro thioflavin-T assays. Similarly, structural analyses using Congo red staining, circular dichroism, and atomic force microscopy revealed that GAPDH aggregates induced Aβ40 amyloidogenesis. In PC12 cells, GAPDH aggregates augmented Aβ40-induced cell death, concomitant with disruption of mitochondrial membrane potential. Furthermore, mice injected intracerebroventricularly with Aβ40 co-incubated with GAPDH aggregates exhibited Aβ40-induced pyramidal cell death and gliosis in the hippocampal CA3 region. These observations were accompanied by nuclear translocation of apoptosis-inducing factor and cytosolic release of cytochrome c from mitochondria. Finally, in the 3×Tg-AD mouse model of AD, GAPDH/Aβ co-aggregation and mitochondrial dysfunction were consistently detected in an age-dependent manner, and Aβ aggregate formation was attenuated by GAPDH siRNA treatment. Thus, this study suggests that GAPDH aggregates accelerate Aβ amyloidogenesis, subsequently leading to mitochondrial dysfunction and neuronal cell death in the pathogenesis of AD.

An aggregate-prone mutant of human glyceraldehyde-3-phosphate dehydrogenase augments oxidative stress-induced cell death in SH-SY5Y cells

Glycerladehyde-3-phosphate dehydrogenase (GAPDH), a classic glycolytic enzyme, also has a role in mediating cell death under oxidative stress. Our previous reports suggest that oxidative stress-induced GAPDH aggregate formation is, at least in part, a mechanism to account for the death signaling. Here we show that substitution of cysteine for serine-284 of human GAPDH (S284C-GAPDH) leads to aggregate-prone GAPDH, and that its expression in SH-SY5Y human neuroblastoma results in greater dopamine-induced cell death than expression of wild type-GAPDH. Treatment of purified recombinant S284C-GAPDH in vitro with the nitric oxide donor NOR3 led to greater aggregation than wild type-GAPDH. Several lines of structural analysis revealed that S284C-GAPDH was amyloidogenic. Overexpression of doxycycline-inducible S284C-GAPDH in SH-SY5Y cells accelerated dopamine treatment-induced death and increased formation of GAPDH aggregates, compared to cells expressing wild type-GAPDH. These results suggest that aggregate-prone mutations of GAPDH such as S284C-GAPDH may confer risk of oxidative stress-induced cell death.

Nuclear-translocated Glyceraldehyde-3-phosphate Dehydrogenase Promotes Poly(ADP-ribose) Polymerase-1 Activation during Oxidative/Nitrosative Stress in Stroke

Background: The link between poly(ADP-ribose) polymerase-1 (PARP-1) and nuclear-translocated glyceradehyde-3-phosphate dehydrogenase (GAPDH) in neurons under oxidative/nitrosative stress remains unknown. Results: The N terminus of nuclear GAPDH binds with PARP-1, and this complex promotes PARP-1 overactivation both in vitro and in vivo. Conclusion: Nuclear GAPDH is a key PARP-1 regulator. Significance: GAPDH/PARP-1 signaling underlies oxidative/nitrosative stress-induced brain damage such as stroke. In addition to its role in DNA repair, nuclear poly(ADP-ribose) polymerase-1 (PARP-1) mediates brain damage when it is over-activated by oxidative/nitrosative stress. Nonetheless, it remains unclear how PARP-1 is activated in neuropathological contexts. Here we report that PARP-1 interacts with a pool of glyceradehyde-3-phosphate dehydrogenase (GAPDH) that translocates into the nucleus under oxidative/nitrosative stress both in vitro and in vivo. A well conserved amino acid at the N terminus of GAPDH determines its protein binding with PARP-1. Wild-type (WT) but not mutant GAPDH, that lacks the ability to bind PARP-1, can promote PARP-1 activation. Importantly, disrupting this interaction significantly diminishes PARP-1 overactivation and protects against both brain damage and neurological deficits induced by middle cerebral artery occlusion/reperfusion in a rat stroke model. Together, these findings suggest that nuclear GAPDH is a key regulator of PARP-1 activity, and its signaling underlies the pathology of oxidative/nitrosative stress-induced brain damage including stroke.

Botulinum neurotoxin A subtype 2 reduces pathological behaviors more effectively than subtype 1 in a rat Parkinson’s disease model

Recent reports indicate that interruption of acetylcholine release by intrastriatal injection of botulinum neurotoxin type A (BoNT/A) in a rat Parkinsons disease model reduces pathogenic behavior without adverse side effects such as memory dysfunction. Current knowledge suggests that BoNT/A subtype 1 (BoNT/A1) and BoNT/A subtype 2 (BoNT/A2) exert different effects. In the present study, we compared the effects of BoNT/A1 and BoNT/A2 on rotation behavior and in vivo cleavage of presynaptic protein SNAP-25 in a rat unilateral 6-hydroxydopamine-induced Parkinsons disease model. BoNT/A2 more effectively reduced pathogenic behavior by efficiently cleaving SNAP-25 in the striatum compared with that of BoNT/A1. Our results suggest that BoNT/A2 has greater clinical therapeutic value for treating subjects with Parkinsons disease compared to that of BoNT/A1.

Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH) Aggregation Causes Mitochondrial Dysfunction during Oxidative Stress-induced Cell Death

Glycolytic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional protein that also mediates cell death under oxidative stress. We reported previously that the active-site cysteine (Cys-152) of GAPDH plays an essential role in oxidative stress-induced aggregation of GAPDH associated with cell death, and a C152A-GAPDH mutant rescues nitric oxide (NO)-induced cell death by interfering with the aggregation of wild type (WT)-GAPDH. However, the detailed mechanism underlying GAPDH aggregate-induced cell death remains elusive. Here we report that NO-induced GAPDH aggregation specifically causes mitochondrial dysfunction. First, we observed a correlation between NO-induced GAPDH aggregation and mitochondrial dysfunction, when GAPDH aggregation occurred at mitochondria in SH-SY5Y cells. In isolated mitochondria, aggregates of WT-GAPDH directly induced mitochondrial swelling and depolarization, whereas mixtures containing aggregates of C152A-GAPDH reduced mitochondrial dysfunction. Additionally, treatment with cyclosporin A improved WT-GAPDH aggregate-induced swelling and depolarization. In doxycycline-inducible SH-SY5Y cells, overexpression of WT-GAPDH augmented NO-induced mitochondrial dysfunction and increased mitochondrial GAPDH aggregation, whereas induced overexpression of C152A-GAPDH significantly suppressed mitochondrial impairment. Further, NO-induced cytochrome c release into the cytosol and nuclear translocation of apoptosis-inducing factor from mitochondria were both augmented in cells overexpressing WT-GAPDH but ameliorated in C152A-GAPDH-overexpressing cells. Interestingly, GAPDH aggregates induced necrotic cell death via a permeability transition pore (PTP) opening. The expression of either WT- or C152A-GAPDH did not affect other cell death pathways associated with protein aggregation, such as proteasome inhibition, gene expression induced by endoplasmic reticulum stress, or autophagy. Collectively, these results suggest that NO-induced GAPDH aggregation specifically induces mitochondrial dysfunction via PTP opening, leading to cell death.

Botulinum neurotoxin type A subtype 2 confers greater safety than subtype 1 in a rat Parkinson's disease model.

ABSTRACT Botulinum neurotoxin type A (BoNT/A) cleaves SNAP-25 and interrupts the release of acetylcholine. We previously reported that BoNT/A subtype 2 (BoNT/A2) ameliorates pathologic behavior more effectively than subtype 1 (BoNT/A1) in a rat Parkinson’s disease model. Here, we further show BoNT/A2 has fewer adverse effects than BoNT/A1. We first confirmed that intrastriatal treatments of both BoNT/As had no-effect on dopaminergic terminals in the striatum. SNAP-25 cleaved by BoNT/A2 was strictly localized to the striatum on the injected side; however, SNAP-25 cleaved by BoNT/A1 diffused contralaterally. Furthermore, treatment with BoNT/A1 caused a significant reduction in body weight, while BoNT/A2 treatment did not. These results suggest that BoNT/A2 is more beneficial for clinical application against Parkinson’s disease than BoNT/A1.