Jianning Wei

Demonstration of functional coupling between γ-aminobutyric acid (GABA) synthesis and vesicular GABA transport into synaptic vesicles

l-Glutamic acid decarboxylase (GAD) exists as both membrane-associated and soluble forms in the mammalian brain. Here, we propose that there is a functional and structural coupling between the synthesis of γ-aminobutyric acid (GABA) by membrane-associated GAD and its packaging into synaptic vesicles (SVs) by vesicular GABA transporter (VGAT). This notion is supported by the following observations. First, newly synthesized [3H]GABA from [3H]l-glutamate by membrane-associated GAD is taken up preferentially over preexisting GABA by using immunoaffinity-purified GABAergic SVs. Second, the activity of SV-associated GAD and VGAT seems to be coupled because inhibition of GAD also decreases VGAT activity. Third, VGAT and SV-associated Ca2+/calmodulin-dependent kinase II have been found to form a protein complex with GAD. A model is also proposed to link the neuronal stimulation to enhanced synthesis and packaging of GABA into SVs.

Mode of action of taurine as a neuroprotector

Previously, it has been shown that taurine exerts its protective function against glutamate-induced neuronal excitotoxicity through its action in reducing glutamate-induced elevation of intracellular free calcium, [Ca2+]i. Here, we report the mechanism underlying the effect of taurine in reducing [Ca2+]i. We found that taurine inhibited glutamate-induced calcium influx through L-, P/Q-, N-type voltage-gated calcium channels (VGCCs) and NMDA receptor calcium channel. Surprisingly, taurine had no effect on calcium influx through NMDA receptor calcium channel when cultured neurons were treated with NMDA in Mg2+-free medium. Since taurine was found to prevent glutamate-induced membrane depolarization, we propose that taurine protects neurons against glutamate excitotoxicity by preventing glutamate-induced membrane depolarization, probably through its effect in opening of chloride channels and, therefore, preventing the glutamate-induced increase in calcium influx and other downstream events.

Protective function of taurine in glutamate‐induced apoptosis in cultured neurons

Previously, we showed that taurine protects neurons against glutamate‐induced excitotoxicity by inhibiting the glutamate‐induced increase of [Ca2+]i. In this study, we report that taurine prevents glutamate‐induced chromosomal condensation, indicating that taurine inhibits glutamate‐induced apoptosis. We found that Bcl‐2 was down‐regulated while Bax was up‐regulated by glutamate treatment, and these changes were prevented in the presence of taurine. We have also shown that taurine inhibits glutamate‐induced activation of calpain. Furthermore, calpastatin, a specific calpain inhibitor, also prevented glutamate‐induced cell death. Here we propose the mechanisms underlying glutamate‐induced apoptosis and taurines inhibition of glutamate‐induced apoptosis to be as follows: glutamate stimulation induces [Ca2+]i elevation, which in turn activates calpain; activation of calpain leads to a reduction of Bcl‐2:Bax ratios; with decreased Bcl‐2:Bax ratios Bax homodimers form, Bax homodimerization, and translocation to the mitochondria result in the release of cytochrome c; released cytochrome c in turn activates a downstream caspase cascade leading to apoptosis. The antiapoptotic function of taurine is due to its inhibition of glutamate‐induced membrane depolarization.

Role of glutamate decarboxylase (GAD) isoform, GAD65, in GABA synthesis and transport into synaptic vesicles-Evidence from GAD65-knockout mice studies

In GAD65-knockout mice, lack of GAD65 expression was confirmed. The expression level of vesicular GABA transporter (VGAT) was upregulated, and no change in the synaptic vesicles (SV)-associated GAD67 was found. GAD65(-/-) SV transported cytosolic GABA much more efficiently than that of the wild type, further supporting our model that there is a structural and functional coupling between GABA synthesis and packaging into SV. Both full-length and truncated forms of GAD65 could bind to GABAergic SV, indicating the N-terminus is not required for the anchoring of GAD65 to SV. Although both GAD65(-/-) SV reconstituted with either GAD65 or GAD67 could synthesize GABA from [3H] glutamate and transport this newly synthesized GABA into SV, the combined evidence suggests that GAD65 plays a major role in GABA transmission in normal physiological condition. However, GAD67 could serve this role under some pathological conditions.

Mechanism of Neuroprotective Function of Taurine

Taurine has potent protective function against glutamate-induced neuronal injury presumably through its function in regulation of intracellular free calcium level, [Ca2+]i. In this communication, we report that taurine exerts its protective function through one or more of the following mechanisms: 1. Inhibition of glutamate-induced calcium influx through L-, N- and P/Q-type voltage-gated calcium channels and NMDA receptor calcium channel; 2. Attenuation of glutamate-induced membrane depolarization; 3. Prevention of glutamate-induced apoptosis via preventing glutamate-mediated down-regulation of Bcl-2; 4. Prevention of cleavage of Bcl-2 by calpain. This action of taurine is due to its inhibition on glutamate induced calpain activation. Based on these observations, we propose that taurine protects neurons against glutamate-induced neurotoxicity in part, by preventing glutamate-induced membrane depolarization, elevation of [Ca2+]i, activation of calpain, reduction of Bcl-2 and apoptosis.

BimEL as a possible molecular link between proteasome dysfunction and cell death induced by mutant huntingtin

Huntington’s disease (HD) is a devastating neurodegenerative disorder caused by an expanded polyglutamine repeat within the N‐terminus of the huntingtin protein. It is characterized by a selective loss of medium spiny neurons in the striatum. It has been suggested that impaired proteasome function and endoplasmic reticulum (ER) stress play important roles in mutant huntingtin (mHtt)‐induced cell death. However, the molecular link involved is poorly understood. In the present study, we identified the essential role of the extra long form of Bim (Bcl‐2 interacting mediator of cell death), BimEL, in mHtt‐induced cell death. BimEL protein expression level was significantly increased in cell lines expressing the N‐terminus of mHtt and in a mouse model of HD. Although quantitative RT‐PCR analysis indicated that BimEL mRNA was increased in cells expressing mHtt, we provided evidence showing that, at the post‐translational level, phosphorylation of BimEL played a more important role in regulating BimEL expression. Up‐regulation of BimEL facilitated the translocation of Bax to the mitochondrial membrane, which further led to cytochrome c release and cell death. On the other hand, knocking down BimEL expression prevented mHtt‐induced cell death. Taken together, these findings suggest that BimEL is a key element in regulating mHtt‐induced cell death. A model depicting the role of BimEL in linking mHtt‐induced ER stress and proteasome dysfunction to cell death is proposed.

Role of μ-calpain in proteolytic cleavage of brain l-glutamic acid decarboxylase

Glutamic acid decarboxylase (GAD) is the rate-limiting enzyme for gamma-aminobutyric acid (GABA) biosynthesis. Previously, we reported the presence of truncated forms of GAD in vivo and in vitro. In addition, an unidentified endogenous protease responsible for proteolytic cleavage of full-length GAD (fGAD) to its truncated form (tGAD) was also observed. In this communication, we report that mu-calpain is a good candidate for conversion of fGAD(67) to tGAD(67). This conclusion is based on the following observations: 1. purified recombinant GAD(67) is cleaved by mu-calpain at specific sites; 2. in brain synaptosomal preparation, GAD(67) is cleaved to its truncated form by an endogenous protease which is inhibited by specific calpain inhibitors; 3. in mu-calpain knockout mice, the level of tGAD in the brain is greatly reduced compared with the wild type; 4. when mu-calpain gene is silenced by siRNA, the level of tGAD is also markedly reduced compared to the control group; and 5. mu-calpain is activated by neuronal stimulation and Ca(2+)-influx. The physiological significance of calpain in regulation of GABA synthesis and GABAergic neurotransmission is also discussed.

Post-MPTP Treatment with Granulocyte Colony-Stimulating Factor Improves Nigrostriatal Function in the Mouse Model of Parkinson's Disease

The neuroprotective effects of granulocyte colony-stimulating factor (G-CSF) were reported in several neurological disease models, including Parkinson’s disease (PD). In the present study, we investigated the therapeutic effect of G-CSF after the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD was established. G-CSF was subcutaneously administered into C57BL/6 mice that had undergone systemic MPTP injections. We found that G-CSF treatment markedly increased the number of dopaminergic neurons in the substantia nigra pars compacta (SNpc) of the G-CSF-treated group. Consistent with this finding, we found a significant increase in dopamine release under high K+ stimulation in the striatum of the G-CSF-treated animals compared to the MPTP-exposed mice. Finally, we observed a persistent recovery of locomotor function in the G-CSF-treated animals. These results suggest the potential therapeutic value of G-CSF in treating PD. However, our bromodeoxyuridine labeling experiment failed to identify any newly generated dopaminergic neurons in SNpc. This might indicate an indirect effect of G-CSF on cell proliferation. The underlying mechanism of G-CSF is under further investigation.

Activity-dependent cleavage of brain glutamic acid decarboxylase 65 by calpain

Previously, we reported that l‐glutamic acid decarboxylase isoform 65 (GAD65) could be cleaved in vitro to release a stable truncated form which lacks amino acid 1–69 from the N‐terminus, GAD65(Δ1–69). However, whether such a truncated form is also present under certain physiological conditions remains elusive. In the present study, we showed that, upon sustained neuronal stimulation, GAD65 could be cleaved into a truncated form in a rat synaptosomal preparation. This truncated form had similar electrophoretic mobility to purified recombinant human GAD65(Δ1–69). Furthermore, we demonstrated that this conversion was calcium dependent. Calcium‐chelating reagents such as EDTA and 1,2‐bis‐(o‐aminphenoxy)‐ethane‐N,N,N′,N′‐tetra‐acetic acid tetra‐acetoxy‐methyl ester prevented the cleavage of GAD65. In addition, our data suggested that calpain, a calcium‐dependent cysteine protease, is activated upon neuronal stimulation and could be responsible for the conversion of full‐length GAD65 to truncated GAD65 in the brain. Moreover, calpain inhibitors such as calpain inhibitor I or calpastatin could block the cleavage. Results of our in vitro cleavage assay using purified calpain and immunopurified rat GAD65 also supported the idea that GAD65 could be directly cleaved by calpain.

Palmitoylation and trafficking of GAD65 are impaired in a cellular model of Huntington's disease.

HD (Huntingtons disease) is caused by an expanded polyQ (polyglutamine) repeat in the htt (huntingtin protein). GABAergic medium spiny neurons in the striatum are mostly affected in HD. However, mhtt (mutant huntingtin)-induced molecular changes in these neurons remain largely unknown. The present study focuses on the effect of mhtt on the subcellular localization of GAD (glutamic acid decarboxylase), the enzyme responsible for synthesizing GABA (γ-aminobutyric acid). We report that the subcellular distribution of GAD is significantly altered in two neuronal cell lines that express either the N-terminus of mhtt or full-length mhtt. GAD65 is predominantly associated with the Golgi membrane in cells expressing normal htt; however, it diffuses in the cytosol of cells expressing mhtt. As a result, vesicle-associated GAD65 trafficking is impaired. Since palmitoylation of GAD65 is required for GAD65 trafficking, we then demonstrate that palmitoylation of GAD65 is reduced in the HD model. Furthermore, overexpression of HIP14 (huntingtin-interacting protein 14), the enzyme responsible for palmitoylating GAD65 in vivo, could rescue GAD65 palmitoylation and vesicle-associated GAD65 trafficking. Taken together, our data support the idea that GAD65 palmitoylation is important for the delivery of GAD65 to inhibitory synapses and suggest that impairment of GAD65 palmitoylation by mhtt may lead to altered inhibitory neurotransmission in HD.