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A pretreatment with m-AIP, a potent inhibitor for CaMKII, abolished the activity and cleavage of caspase-3 mediated by NMDA in the retina (Fig. 1B). Tenneti and Lipton (2000) had demonstrated that an increase in caspase-3 activation in NMDA-treated cerebrocortical neurons was blocked by MK801 as well as memantine, indicating that its activation there was due specifically to NMDA stimulation. As m-AIP inhibited the cleavage of caspase-3 stimulated by intraocular injections of NMDA, one can predict the activation of the protease, caspase-3, to be downstream of CaMKII. While the possibility that m-AIP may have acted through some other unknown mechanism cannot be eliminated, the results strongly support cell death-effector roles for CaMKII activity and caspase-3 in NMDA-mediated cell death in the retina. The prospective roles of transcription vs. post-translational phosphorylation of CaMKII are the subject of additional studies described below.

It has been suggested that the distribution of NMDA receptors in the retina is not correlated with cells that are killed by NMDA (Hof et al., 1998), the receptors having a wider distribution than the vulnerable cells. The neuronal phenotypes for NMDA-sensitive cells may be extended to include those that contain the NMDA receptor together with CaMKII and caspase-3. Future studies are likely to add other proteins to this prospective profile.

BDNF and neuroprotection of RGCs

While the potential for several neurotrophic factors to protect RGCs from stress has been demonstrated (see for example, Chaum, 2003; Chidlow et al., 2007) the prospective role for BDNF has received the most attention. BDNF is a member of the protein family of neurotrophins (NTs) showing widespread expression in the developing and adult mammalian brain (Lessmann et al., 2003). BDNF plays an important role in neuronal survival, differentiation and synaptic plasticity, as well as being important for protection of neurons in various pathological conditions. The effects of BDNF are mediated through the binding

of this factor to its high affinity receptor, TrkB, and a low affinity p75 NTR, respectively. Binding of BDNF to TrkB activates PI3-K/Akt and/or mitogen-activated protein kinase (MAPK) signaling pathways, and thereby mediates numerous cellular functions, including inhibition of apoptosis (Chaum, 2003). The p75 NTR employs distinct signaling pathways to either enhance or suppress TrkB receptor activity, or autonomously activates signaling cascades that result in induction of apoptosis or in the promotion of survival (Roux and Barker, 2002).

In the retina, BDNF has been shown to play critical roles not only in the development and differentiation (Bennett et al., 1999; Bosco and Linden, 1999), but also in survival of retinal neuronal cells of the mature animal both in physiological and pathological conditions (Mey and Thanos, 1993; Unoki and LaVail, 1994; Peinado-Ramon et al., 1996; Kido et al., 2000). The death of RGCs is the hallmark of glaucoma, and the neuroprotective role of BDNF on RGCs has been demonstrated by many studies. For example, administration of exogenous BDNF protects RGCs in various experimental models of glaucoma, including optic nerve axotomy (Mey and Thanos, 1993; Peinado-Ramon et al., 1996), retinal ischemia (Unoki and LaVail, 1994), NMDA-induced neuronal death (Kido et al., 2000), and in eyes with chronic intraocular hypertension (Ko et al., 2000). Transgenic expression of the BDNF gene also prolongs the survival of RGCs in some of the experimental models of glaucoma (Mo et al., 2002; Martin et al., 2003). In the in vitro paradigm, supplements of BDNF in the culture media enhances primary RGC survival (Johnson et al., 1986; Thanos et al., 1989) and has also been shown to rescue transformed RGCs (RGC-5) from cell death following serum deprivation (Krishnamoorthy et al., 2001). Clearly, the mere presence of BDNF in experimental protocols will affect the interpretation of data related to cell death signaling.

There are two sources of BDNF for the RGCs in the retina including that which is retrogradely transported and that which is locally synthesized (Chaum, 2003). The relative contributions of these sources to RGC survival in the in vivo condition

remain to be fully elucidated. The retrogradely transported BDNF has been postulated to be an important trophic factor for RGC survival in glaucoma. Thus, RGCs die by apoptosis in models of glaucoma or retinal ischemia, where retrogradely transported BDNF is interrupted (Pease et al., 2000; Quigley et al., 2000; Lambert et al., 2004). BDNF that is locally synthesized in the retina has also been implicated in RGC protection (Gao et al., 1997; Vecino et al., 1998, 1999; Rudzinski et al., 2004). BDNF is expressed by RGCs, amacrine cells, and other neighboring cells such as Mu¨ller cells in the retina (Vecino et al., 1998, 2002; Garcia et al., 2003). Therefore, both autocrine and paracrine effects on the RGCs need to be examined, and it seems likely that these local sources have multiple roles in the retina. The high affinity receptor for BDNF, TrkB, is present in the RGCs. The local levels of BDNF mRNA and protein in the retina have been shown to be modulated by injury to the optic nerve (Gao et al., 1997), by ocular hypertension (Rudzinski et al., 2004), by injection of NMDA into the eye (Vecino et al., 2002), and by transient retinal ischemia (Vecino et al., 1998; Lonngren et al., 2006). Taken together, these studies suggest an important paracrine/autocrine mechanism for BDNF action within the retina. However, the mechanism through which BDNF is regulated locally to protect RGCs remains unknown.

Expression of BDNF in RGCs/retina: involvement of nuclear CaMKII-ab

Recently, the nuclear isoform of CaMKII-a, CaMKII-aB, has been shown to be involved in a survival response of RGCs (Fan et al., 2007). CaMKII-aB is a splice variant for CaMKII-a that has a nuclear localization signal (Schulman, 2004) (see Diagram 1), which aids the translocation of the CaMKII-a to the nucleus. Its role in the nucleus remains to be clarified. This variant transcript is particularly interesting because of the NMDA-stimulated increase in the CaMKII-aB transcript evident in the in vivo rat model (Laabich et al., 2000). This increase also occurs specifically in pan-purified and cultured RGCs isolated from the Sprague–Dawley (SD) rat retina when

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glutamate is used as the stressor (Fan et al., 2007). Glutamate treatment induced a transient increase in the CaMKII-aB transcript, which was followed by the increase in CaMKII-a protein detected in the nucleus several hours later. This result is of particular interest because it seems that glutamate stimulation induces an alternative splicing of the a gene whose product is targeted to the nucleus at a later stage. While one might conjecture that this delayed nuclear targeting of CaMKII-aB may be relevant to the later appearance of cell death, additional experiments suggest otherwise. Specific knockdown of CaMKII-aB in purified primary RGCs with the aid of RNA interference (RNAi) significantly enhanced gluta- mate-induced cell death, indicating that CaMKII- aB is involved in a cell survival signaling pathway in RGCs (Fan et al., 2007).

The precise mechanisms underlying the role of CaMKII-aB in cell death/survival responses remain unclear. Several reports indicate that CaMKII-a plays a role in Ca2+-mediated transcriptional regulation of genes through phosphorylation of transcription factors such as cAMP response element binding protein (CREB) (Matthews et al., 1994; Sun et al., 1994) activating transcription factor (ATF) (Shimomura et al., 1996; Sun et al., 1996), CCAAT/enhancer-binding protein (C/EBP) (Wegner et al., 1992; Yano et al., 1996), serum response factor (Misra et al., 1994), and NeuroD (Gaudilliere et al., 2004). Thus, it seems likely that the nuclear localized CaMKII-aB detected after NMDA stimulation is evidence of the regulation of gene expression in RGCs. Our studies have revealed that when CaMKII-aB was knocked down, there was a corresponding decrease in the level of BDNF mRNA and protein in primary RGCs. Considering that knockdown of CaMKII-aB also enhanced RGC death, these data may indicate an involvement of CaMKII-aB in regulating BDNF expression and thus cell survival responses. This may be especially relevant to the in vivo condition, where the micro environment of retinal cells is intact and where locally synthesized BDNF may be of significance for the maintenance of cell survival (Murphy and Clarke, 2006). It seems likely that BDNF is not the only survival gene that is regulated by CaMKII-aB. Studies in

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our laboratory also show an increased expression of the anti-apoptotic Bcl-2 gene in RGC-5 cells containing over-expressed CaMKII-aB (unpublished data). Indeed, the target genes whose expressions are regulated by CaMKII-aB are the subjects of further investigation.

It should be noted that among the glutamateresponsive transcription factors, CREB (Jiang et al., 2003) and nuclear factor kB (NFkB) (Jiang et al., 2003; Marini et al., 2004) have also been implicated in BDNF expression. NFkB is a critical regulator of many genes involved in inflammatory processes, cell differentiation, and apoptosis. It has been shown that glutamate-induced NFkB is activated in a Ca2+-dependent manner (Ko et al., 1998; Meffert et al., 2003) and cytoplasmic CaMKII-a plays an important role in mediating NFkB activation in neurons, including RGCs in retina (Lilienbaum and Israel, 2003; Meffert et al., 2003; Fan et al., 2007). These studies indicate that the NFkB machinery is a prospective target for CaMKII-a. As both proand anti-apoptotic properties have been attributed to NFkB in neurons (Mattson et al., 2000; Pizzi et al., 2002; de Erausquin et al., 2003), the balance between cell death and survival in response to external stimuli most likely relies on the activation of distinct NFkB proteins (Pizzi et al., 2002), as well as the expression of genes that are under the control of the NFkB protein(s). For the RGCs this is an active area of research.

Secretion of BDNF: involvement of cytoplasmic CaMKII-a

While BDNF expression is regulated by multiple transcription factors, including CREB, NFkB, and possibly, the nuclear isoform of CaMKII-a, the mechanism by which BDNF is released is not yet completely understood, and yet this is an important consideration for RGC survival. BDNF, like all other neurotrophins, is generated as pre- pro-BDNF, which is further processed in the endoplasmic reticulum, trans-Golgi network, and secretory vesicles, until they are eventually secreted as mature homodimer proteins into the extracellular space (Lessmann et al., 2003). Secretion is observed in other systems in response to

depolarization by K+ or by glutamate stimulation (Lessmann et al., 2003). To investigate the mechanisms underlying the regulation of BDNF secretion, transformed RGCs (RGC-5) have been used. The RGC-5 cells show most of the characteristics of RGCs, respond to glutamate stimulation (Krishnamoorthy et al., 2001) and can be grown in sufficient quantities to measure secretable proteins such as BDNF. RGC-5 cells not only express and secrete BDNF, but also have the BDNF receptor protein, TrkB, thus providing a valuable in vitro model for studying the modulation of BDNF expression and secretion, as well as signaling pathways and modulatory influences.

Fan et al. (2006) demonstrated that glutamate stimulated a transient increase in BDNF mRNA (0.5–2 h) and protein (6–12 h) in RGC-5 cells, and also stimulated an early release (0.5–2 h) of BDNF into the culture media (Fig. 2A). This early release may be triggered by transmitter dependent depolarization. It is noted that at this early stage, although BDNF mRNA is on the rise, the protein translation is not yet underway. Therefore, the early release of BDNF is most likely derived from pre-existing pools within these cells. The released BDNF may exert some protection for glutamate challenged cells, because blocking antibodies against BDNF or its TrkB receptor led to an elevated level of glutamate-stimulated cell death. However, the protection by this small BDNF release was limited and perhaps insufficient to protect all cells because RGC-5 cells did begin to die within 24 h after exposure to glutamate. Although the level of BDNF protein within the RGC-5 cells started to increase at 6–12 h after exposure to glutamate, there was no corresponding increase in its release at these later time points. This could be a critical point with regard to the eventual cell death.

A specific inhibitor for CaMKII, m-AIP, has been shown to be a neuroprotectant for RGCs treated with NMDA in vivo (Laabich and Cooper, 2000) and glutamate in vitro (Fan et al., 2005). The mechanism for the neuroprotective role of m-AIP remains unclear and may be mediated through multiple signaling pathways. Additional studies have revealed that m-AIP enhanced

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glutamate-stimulated BDNF release in RGC-5 cells at a very early stage (30 min–2 h), when glutamate-induced BDNF production is hardly underway, and also promoted the release of BDNF for a prolonged period, perhaps for even longer than the glutamate-stimulated period of BDNF synthesis (Fig. 2B). These results suggest that m-AIP enhances the release of the pre-existing pool of BDNF, and possibly, of newly synthesized BDNF. Obviously, this relatively fast acting and long lasting role for m-AIP in promoting BDNF release is important with respect to neuroprotection, and could be one of the reasons why cell survival both in vivo and in vitro is evidently enhanced in the presence of m-AIP.

The specific mechanisms underlying the regulation of BDNF release are not yet known, but an increase in the activity of cytoplasmic CaMKII induced by glutamate treatment may be involved (Sucher et al., 1997). CaMKII-a is mainly expressed in neurons, and presynaptically, it is associated with synaptic vesicles (Benfenati et al., 1992). CaMKII-a has been shown to serve as a negative activity-dependent regulator of neurotransmitter release at hippocampal synapses (Hinds et al., 2003). This is possibly the case for BDNF release in RGC-5 cells because they do express CaMKII-a (Fan et al., 2005). Inhibition of CaMKII by m-AIP, led to an enhanced glutamatestimulated release of BDNF, and m-AIP alone increased BDNF release by cells that are in control conditions (without glutamate). Therefore, CaMKII can inhibit basal levels BDNF release. However, in contradiction with previous results (Hinds et al., 2003; Fan et al., 2005), a recent study has revealed a strong dependence on Ca2+ influx and activation of CaMKII for an activity-depen- dent postsynaptic BDNF secretion (Kolarow et al., 2007). Further studies are warranted to clarify this important topic. Also, it seems likely that in the in vivo condition, the synthesis and release of BDNF, as well as the expression and activation of TrkB receptors, may be more complex, being regulated and influenced by neighboring cells and other factors that are not present in the in vitro models. Additional studies will be needed to show that results seen in the in vitro model apply to RGCs in the in vivo model.

Nuclear CaMKII-a vs. cytoplasmic CaMKII-a

These recent studies have shown the involvement of CaMKII-a in the regulation of both BDNF expression (nuclear CaMKII-aB isoform) and secretion (cytoplasmic CaMKII-a isoform) in RGCs. Future studies should determine if modulation of BDNF expression and release via CaMKII can protect neurons in glaucomatous animal models. Although application of exogenous BDNF has been shown to be effective, the regulated autocrine/paracrine release of BDNF into the environment of the retina is clearly an important resource for maintaining RGC survival. Thus, the endogenous BDNF and its regulatory machinery should continue to be a target for investigators. Since the application of the CaMKII inhibitor, m-AIP, dramatically enhances BDNF release, this may, in part, explain the prior observations that m-AIP protects neurons in the retina from NMDA-induced cell death in the retina. Thus, modulating CaMKII-a or its nuclear counterpart, CaMKII-aB, to enhance BDNF expression/secretion may be a promising neuroprotective strategy for diseases/disorders such as glaucoma and retinal ischemia where glutamate and excitotoxicity have been implicated.

Patterns of BDNF expression are regulated by the light–dark cycle

Previous studies of BDNF protein in the retina have shown evidence of a diurnal pattern of expression. There is a 1.5-fold higher level of protein at mid-day relative to the mid-night (Pollock et al., 2001). Investigations in our laboratory show a similar trend in mRNA levels with similar mid-day to mid-night ratios of transcribed mRNA, and these observations have been extended to include additional time points. Results of such studies indicate that the peaks of BDNF mRNA expression actually occur shortly after the lights-off condition in the retinas of mice (Fig. 3). This would indicate that the lights-off condition may be a trigger for reducing transcription and the lights-on would be a trigger for ramping up transcription of BDNF. We do know that the levels of CaMKII transcripts and protein