**5. Effects of membrane lipid rafts on GDNF actions**

Tiny regions in cell membrane exist that differ in composition from other parts of the membrane. In these regions, the extracellular lipids contain rich sphingolipids and cholesterol molecules, which gather and form a mobile platform of fluid double-lipids, i.e. lipid raft. The main components of lipid rafts include GPI-coupled proteins (extramembrane), trans-membrane tyrosine kinases (intermembrane) and two Src family tyrosine kinases (intramembrane) (Simons & Ikonen, 1997). The lipid raft is an essential structure in the membrane for signal transduction, and plays a vital role in cell adherence, axon guidance and synaptic transmission (Paratcha & Ibáñez, 2002).

Substantial studies have focused on GDNF and its receptors, but the studies on the function of lipid rafts and the relationship between GDNF and its receptors are few and far between. To determine whether receptor-mediated GDNF is dependent on the functional lipid rafts, we investigated four transmembrane proteins in the lipid rafts (RET, NCAM140, integrin β1 and N-cadherin) and their involvement in mediating GDNF signal pathways. To this end, the binding of these proteins and GDNF was analyzed, using co-immunoprecipitation, based on the integrity of the membrane lipid rafts. The results demonstrated that all proteins bound to GDNF when the lipid raft was complete, and only RET and N-cadherin bound to GDNF when the lipid raft was not complete. In addition, extraction tests for the membrane lipid rafts have demonstrated that RET and NCAM, but not N-cadherin and integrin β1, may be shifted to the lipid rafts by GDNF. This series of tests indicates the importance of lipid rafts in mediating the interaction of GDNF with its receptors (Figure 4). RET can mediate the protective actions exerted by GDNF upon DA-ergic neurons (Trupp *et al.*, 1996; Durbec *et al.*, 1996). After GDNF stimulation, RET induces multiple signals to mediate the signal pathways of GDNF via two models (intra-lipid raft and extra-lipid raft) (Tansey *et al.*, 2000). Membrane lipid rafts are the basis for the shift and functional activation of RET (Poteryaev *et al.*, 1999). Activated RET influences different downstream targets in lipid rafts. The RET activated extra-raft preferentially binds to SHC, and the RET activated intra-raft binds preferentially to FRS2, thus mediating a series of signal pathways. The membrane lipid rafts provide abundant micro-environments for the binding of RET and kinases/receptor proteins in the raft, and is involved in the GFL signaling pathway. For example, Src family kinases, as proteins in the raft, mediate a series of functions of GDNF signal transduction, after binding to activated RET. In the lipid rafts rich in GFRα1 and Src, GDNF activates Src via a GFRα1 mediated pathway (Tansey *et al.*, 2000). Furthermore, the location of RET on the lipid raft enhances its binding to Src, whose activation is essential for GDNF mediated promotion of survival (Encinas *et al.*, 2001). In a lipid raft, RET cannot be degraded by proteases, and this protective effect may be related to its downstream actions of regulating receptor functions (Pierchala *et al.*, 2006).

total Akt does not change. Finally, through immunoblotting it was discovered that the levels of phosphorylated N-cadherin (Tyr860) and phosphorylated Akt are dose-dependent on GDNF, and that the peak levels of both occur at 50 ng/ml (*in vitro*) and 13 ng/μl (*in vivo*) of GDNF. The levels of phosphorylated N-cadherin (Tyr860) and phosphorylated Akt are also timedependent, and the peak levels of both occur at 15 min (*in vitro*) and 30 min (*in vivo*) after GDNF actions. Statistical analyses show that the two phosphorylations are positively related. Thus, it may be concluded that GDNF activates the PI3K/Akt pathway via N-cadherin to

Tiny regions in cell membrane exist that differ in composition from other parts of the membrane. In these regions, the extracellular lipids contain rich sphingolipids and cholesterol molecules, which gather and form a mobile platform of fluid double-lipids, i.e. lipid raft. The main components of lipid rafts include GPI-coupled proteins (extramembrane), trans-membrane tyrosine kinases (intermembrane) and two Src family tyrosine kinases (intramembrane) (Simons & Ikonen, 1997). The lipid raft is an essential structure in the membrane for signal transduction, and plays a vital role in cell adherence,

Substantial studies have focused on GDNF and its receptors, but the studies on the function of lipid rafts and the relationship between GDNF and its receptors are few and far between. To determine whether receptor-mediated GDNF is dependent on the functional lipid rafts, we investigated four transmembrane proteins in the lipid rafts (RET, NCAM140, integrin β1 and N-cadherin) and their involvement in mediating GDNF signal pathways. To this end, the binding of these proteins and GDNF was analyzed, using co-immunoprecipitation, based on the integrity of the membrane lipid rafts. The results demonstrated that all proteins bound to GDNF when the lipid raft was complete, and only RET and N-cadherin bound to GDNF when the lipid raft was not complete. In addition, extraction tests for the membrane lipid rafts have demonstrated that RET and NCAM, but not N-cadherin and integrin β1, may be shifted to the lipid rafts by GDNF. This series of tests indicates the importance of lipid rafts in mediating the interaction of GDNF with its receptors (Figure 4). RET can mediate the protective actions exerted by GDNF upon DA-ergic neurons (Trupp *et al.*, 1996; Durbec *et al.*, 1996). After GDNF stimulation, RET induces multiple signals to mediate the signal pathways of GDNF via two models (intra-lipid raft and extra-lipid raft) (Tansey *et al.*, 2000). Membrane lipid rafts are the basis for the shift and functional activation of RET (Poteryaev *et al.*, 1999). Activated RET influences different downstream targets in lipid rafts. The RET activated extra-raft preferentially binds to SHC, and the RET activated intra-raft binds preferentially to FRS2, thus mediating a series of signal pathways. The membrane lipid rafts provide abundant micro-environments for the binding of RET and kinases/receptor proteins in the raft, and is involved in the GFL signaling pathway. For example, Src family kinases, as proteins in the raft, mediate a series of functions of GDNF signal transduction, after binding to activated RET. In the lipid rafts rich in GFRα1 and Src, GDNF activates Src via a GFRα1 mediated pathway (Tansey *et al.*, 2000). Furthermore, the location of RET on the lipid raft enhances its binding to Src, whose activation is essential for GDNF mediated promotion of survival (Encinas *et al.*, 2001). In a lipid raft, RET cannot be degraded by proteases, and this protective effect may be related to its downstream actions

protect DA-ergic neurons.

**5. Effects of membrane lipid rafts on GDNF actions** 

axon guidance and synaptic transmission (Paratcha & Ibáñez, 2002).

of regulating receptor functions (Pierchala *et al.*, 2006).

Fig. 4. A membrane lipid raft is an ultrastructure rich in cholesterol and phosphosphingolipids.

NCAM can function as a receptor of GDNF signal transduction (Sjöstrand *et al.*, 2007; Sjöstrand & Ibáñez, 2008). Under the influence of GDNF, NCAM140 binds to Fyn and is recruited to lipid rafts, followed by stimulation of the migration of Schwann cells and promotion of neuronal axonal growth (Paratcha *et al.*, 2003). In membrane lipid rafts, the binding of NCAM and Fyn leads to recruitment of FAK and the activation of the Ras-Raf-ERK1/2 pathway (Beggs *et al.*, 1997; Niethammer *et al.*, 2002).

Integrin β1 is a transmembrane cell adhesion molecule, which is expressed in DA-ergic neurons in the substantia nigra of adult mice (Cao *et al.*, 2008). Inhibitory antibodies can counteract the effects of GDNF on the promotion of survival and growth of DA-ergic

Actions of GDNF on Midbrain Dopaminergic Neurons: The Signaling Pathway 209

neurotoxins and stimulates functional recovery. In the presence of Aβ peptide, GDNF can inhibit Aβ neurotoxin via blocking the activation of gad153, JNK and ERK induced by ER stress, and promote cell survival. In granular cells of the cerebellum, GDNF inhibits the activation of p38-MAPK via PI3K, and prevents the injury induced by TGF-β in the

The summary of research progress on different GDNF supplementation methods is as

Direct injection of GDNF: Some studies have demonstrated that injection of GDNF into the substantia nigra only decreases the apoptosis of DA-ergic neurons, but injection into the striatum corpora repairs the neuronal process of TH-positive neurons and improves the PD symptoms (Bohn *et al.*, 2000). Injection of GDNF into the striatum corpora can decrease the death rate of DA-ergic neurons up to 60%, as well as increase the ispilateral TH-positive immunoreactivity and synaptic growth in the nigrostriatal system. Additionally, injection of GDNF into the striatum corpora can increase the amount, volume and synaptic length of

Injection of GDNF via virus vehicle: This method can transfer the neurotrophic factors into the CNS (Figure 5). In 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) animal models, a lentivirus encoding GDNF was injected into the caudate nucleus, putamen and substantia nigra; a lentivirus encoding for the β-galactosidase enzyme was injected in the control group. Three months later, the PD system scores in the test groups were improved compared to that of the control group (Kordower *et al.*, 2000). Bowenkamp *et al.* used 6- OHDA animal models and obtained similar results (Bowenkamp *et al*., 1996). These findings indicate that GDNF plays a positive role in neuronal reconditioning and synaptic growth in the substantia nigra. However, over-expressed GDNF may also play a negative role in PD treatment. Eslamboli *et al.* reported that high levels of GDNF expression altered bilateral DA transport and synthesis, and produced toxic or adverse effects (Eslamboli *et al.*, 2003; Eslamboli *et al.*, 2005). Thus, low and sustained expression seems to be most prudent. In Huntington's disease, the repeated gene amplification of the huntingtin protein leads to neurological disorders, which can be attenuated by adenovirus-vehicle mediated GDNF

Injection of GDNF via cell mediation: Researchers are searching for other efficacious ways to import GDNF. For example, the BHK-21 cell, transformed by genetic engineering, can persistently express GDNF in neural stem cells (Behrstock & Svendsen, 2004). Here, we introduce a method of delivering GDNF termed encapsulated cell delivery (ECD). This method requires a catheter-like device with a diameter of no more than 1 mm, which is used to inject agents into the striatum corpora. A mobile device, made up of a hollow fiber membrane (< 1.5 cm), is used to control the diffusion of nutrients and injected GDNF. Cell lines of high quality are selected via this fiber membrane. In the process, the membrane is a

GDNF permeable to the blood-brain barrier: Recently, a novel GDNF was developed. It is a fusion protein formed by the combination of GDNF and cell perforin mediated by the transactivator of the HIV virus. Diez *et al.* demonstrated, in the mouse PD model (i.p. injection), that Tat-GDNF could cross the blood-brain barrier and enter into the nigrostriatal system (Dietz *et al.*, 2006). Unfortunately, Tat -GDNF could not protect injured DA-ergic neurons or

protoplasmic membrane (Subramaniam *et al.*, 2008).

DA-ergic neurons as well as the uptake of dopamine.

barrier preventing immunological rejection of allogenic cells.

improve PD symptoms in MPTP models.

follows:

expression.

neurons (Chao *et al.*, 2003). Some reports have demonstrated that the membrane lipid rafts provide a perfect environment for the activation of integrins, mediated by growth factors, in the system of survival for oligodendrocytes. The shift of activated integrin to membrane lipid rafts is an important cue, which provides rationale for the actions of functional T cells and the basis for the study of integrin in other cells (Leitinger & Hogg, 2002).

N-cadherin, a calcium dependent cell adhesion molecule similar to NCAM140 and integrin β1, is a trans-membrane protein that mediates the axonal growth of astrocytes and Schwann cells. As mentioned above, the intracellular domain of N-cadherin is similar to the extradomain of RET. Additionally, N-cadherin can activate the PI3K/Akt and Ras/Raf/MAPK signal pathways (*et al.*,) (73), which provides the rationale for further delving into GDNF and its function.

It is closely related to signal transduction in the membrane and provides a platform for anchoring proteins. A: GFRα1 and integrin β1 integrate into the membrane lipid raft of DAergic neurons, but RET, N-cadherin, and NCAM-140 are outside of the raft. B: A GDNF dimer combines with GFRα1 and forms a GDNF-GFRα1 complex, which binds to RET and gathers in the membrane lipid raft. The combination of the GDNF dimer and NCAM dimer requires another substance. N-cadherin and integrin do not gather in the membrane lipid raft, but N-cadherin can bind to GDNF outside of the raft.
