Proc Natl Acad Sci U S A. 2006 Mar 7;103(10):3887-9.
Neuroprotection by pharmacologic blockade of the GAPDH death cascade.
Hara MR, Thomas B, Cascio MB, Bae BI, Hester LD, Dawson VL, Dawson TM, Sawa A, Snyder SH.
Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
[ Free full text ]

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) participates in a cell death cascade wherein a variety of stimuli activate nitric oxide (NO) synthases with NO nitrosylating GAPDH, conferring on it the ability to bind to Siah, an E3-ubiquitin-ligase, whose nuclear localization signal enables the GAPDH/Siah protein complex to translocate to the nucleus where degradation of Siah targets elicits cell death. R-(-)-Deprenyl (deprenyl) ameliorates the progression of disability in early Parkinson’s disease and also has neuroprotective actions. We show that deprenyl and a related agent, TCH346, in subnanomolar concentrations, prevent S-nitrosylation of GAPDH, the binding of GAPDH to Siah, and nuclear translocation of GAPDH. In mice treated with the dopamine neuronal toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), low doses of deprenyl prevent binding of GAPDH and Siah1 in the dopamine-enriched corpus striatum.

From the full text article:

Efforts to prevent neurodegeneration in disorders such as Alzheimer’s disease, Parkinson’s disease (PD), Huntington’s disease, and stroke have proceeded along two pathways. One involves identifying the fundamental molecular causes of these diseases, although even for Huntington’s disease whose molecular causation is established, curative therapy has not followed directly. Alternatively, elucidating mechanisms of cell death may lead to agents that prevent neurodegeneration without knowing the specific disease etiology. The monoamine oxidase-B (MAO-B) inhibitor, R-(–)-Deprenyl (selegiline, hereafter designated deprenyl) (Fig. 1a) has been used in the therapy of PD with the initial goal of elevating dopamine levels (1–4). Deprenyl can delay the progression of disability in early Parkinson’s disease, and recent studies also indicated that deprenyl reduces neuronal death in a variety of in vivo and in vitro experimental models. These include death of neuronal cultures induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), nitric oxide (NO), or peroxynitrite, as well as in vivo hypoxia models and peripheral or optic nerve crush (5–13). Neuroprotection by deprenyl has been suggested to be independent of MAO-B (14–16). Such a concept has been reinforced by the fact that the deprenyl derivative TCH346 (Fig. 1a) with no inhibitory action for MAO-B, also displays neuroprotective effects in culture models at concentrations as low as 0.1 pM and in intact animals at oral doses as low as 0.3 µg/kg (17–20).

Recently we described a cell death signaling system whereby cell stressors activate inducible or neuronal nitric oxide synthase (NOS) with a specific S-nitrosylation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by NO (21). S-nitrosylation of GAPDH abolishes catalytic activity and confers upon GAPDH the ability to bind to Siah1 (hereafter designated Siah), an E3-ubiquitin-ligase whose nuclear localization signal mediates nuclear translocation of the GAPDH/Siah complex. In the nucleus, GAPDH extends the rapid turnover of Siah, leading to the degradation of selected nuclear targets of Siah and apoptosis. In efforts to identify the target for the neuroprotective actions of TCH346, Waldmeier and colleagues (22) used affinity binding, affinity labeling, and BIAcore technology, and identified GAPDH as its specific target.

We now demonstrate that deprenyl and TCH346 at subnanomolar concentrations prevent the S-nitrosylation of GAPDH, inhibit GAPDH/Siah binding and prevent the nuclear translocation of GAPDH. These actions also occur in intact animals at drug doses as low as 0.01 mg/kg. Thus, the neuroprotective actions of these drugs appear to reflect inhibition of the GAPDH/Siah cell death cascade.

Results and Discussion

In the macrophage cell line RAW264.7, stimulation by LPS and IFN- (LPS–IFN), components of endotoxin, provokes a massive activation of inducible NOS with NO formed mediating the cytotoxic actions of macrophages and also eliciting apoptotic cell death of the macrophages themselves (23). In this system, NO S-nitrosylates GAPDH eliciting GAPDH/Siah binding and nuclear translocation of the protein complex, a process blocked by NOS inhibitors (21). We have replicated these findings revealing S-nitrosylation of GAPDH after induction of inducible NOS. At a 1 nM concentration, deprenyl and TCH346 both prevent GAPDH nitrosylation (Fig. 1 b and c).

We directly demonstrate that the binding of GST-tagged Siah to GAPDH in vitro is prevented by deprenyl with as little as 0.01 nM deprenyl eliciting detectable diminution of binding (Fig. 2a).

We also monitored drug effects upon GAPDH/Siah binding in intact cells. In RAW264.7 cells treatment with LPS-IFN elicits robust binding of GAPDH to Siah (Fig. 2 b and c). At 1 nM, both deprenyl and TCH346 abolish GAPDH/Siah binding.

We wondered whether the prevention of GAPDH S-nitrosylation and binding to Siah by drugs would influence GAPDH nuclear translocation. In RAW264.7 cells 1 nM deprenyl or TCH346 abolish the nuclear translocation of GAPDH after LPS–IFN treatment (Fig. 2d).

To ascertain the influence of deprenyl upon cell death, we used cerebellar granule cells (Fig. 3). Etoposide reduces cell viability by 70% associated with profound augmentation of Siah levels and with nuclear translocation of GAPDH. Treatment with deprenyl abolishes the Siah augmentation and the nuclear translocation of GAPDH and markedly reduces cell death.

In summary, our findings provide compelling evidence that the neuroprotective actions of deprenyl and TCH346 reflect their preventing GAPDH/Siah binding and the nuclear translocation of GAPDH. It appears that the initial action of the drugs is to bind to GAPDH, as Waldmeier and colleagues (22) directly demonstrated binding of TCH346 to GAPDH. Such binding would inhibit S-nitrosylation of GAPDH and its binding to Siah. Recently, Youdim and colleagues (25, 26) observed that rasagiline, a monoamine oxidase inhibitor used in the therapy of PD, is also neuroprotective in multiple animal models and prevents the nuclear translocation of GAPDH.

Although the principal focus for the therapeutic actions of deprenyl has been PD, deprenyl and related drugs might be useful in other neuronal conditions as well as non-nervous system conditions, because the GAPDH/Siah cascade appears fairly universal (21, 27–29). Thus, a wide range of stressors in diverse cell lines elicits nuclear translocation of GAPDH, with antisense to GAPDH preventing nuclear translocation and cell death. The most investigated GAPDH systems involve apoptotic death. Whether GAPDH plays a role in necrosis or in nonapoptotic programmed cell death is unclear.

In addition to the therapeutic relevance of our findings, evidence that cytoprotective actions of these drugs involve blockade of the GAPDH/Siah system provides support for the concept that the GAPDH/Siah signaling cascade is an important component of cell death.

Categories: 2006, Deprenyl, Neuroprotectants, Hypoxia, Sepsis