Glycogen synthase kinase-3 (GSK-3) plays a critical role in neuronal apoptosis. The two mammalian isoforms of the kinase, GSK-3␣ and GSK-3␤, are inhibited by phosphorylation at Ser-21 and Ser-9, respectively. Depolarization, which is vital for neuronal survival, causes both an increase in Ser-21/9 phosphorylation and an inhibition of GSK-3␣/␤. However, the role of GSK-3 phosphorylation in depolarization-dependent neuron survival and the signaling pathway contributing to GSK-3 phosphorylation during depolarization remain largely unknown. Using several approaches, we showed that both isoforms of GSK-3 are important for mediating neuronal apoptosis. Nonphosphorylatable GSK-3␣/␤ mutants (S21A/S9A) promoted apoptosis, whereas a peptide encompassing Ser-9 of GSK-3␤ protected neurons in a phosphorylation-dependent manner; these results indicate a critical role for Ser-21/9 phosphorylation on depolarization-dependent neuron survival. We found that Ser-21/9 phosphorylation of GSK-3 was mediated by Ca 2؉ / calmodulin-dependent protein kinase II (CaMKII) but not by Akt/PKB, PKA, or p90 RSK . CaMKII associated with and phosphorylated GSK-3␣/␤. Furthermore, the pro-survival effect of CaMKII was mediated by GSK-3 phosphorylation and inactivation. These findings identify a novel Ca 2؉ /calmodulin/CaMKII/ GSK-3 pathway that couples depolarization to neuronal survival.The survival or death of neurons is critical for the establishment of appropriate neural circuitry during brain development (1, 2). Considerable evidence supports that electrical activity plays a crucial role in neuronal survival (3, 4). For example, pharmacological blockade of electrical activity in rat brain induces extensive apoptotic neurodegeneration (5, 6). Deafferentiation of the cerebellar granule layer in adult rats resulted in massive and typical apoptosis of cerebellar granule neurons (CGNs), 3 suggesting the importance of afferent input-related factors for survival of CGNs in vivo (7). In culture, survival of rat CGNs can be maintained by electrical activity, which is effected by depolarizing concentrations of extracellular potassium [KCl] o ϭ 25 mM KCl ((25 K) or potassium depolarization) (8, 9). Lowering [KCl] o to 5 mM KCl ((5 K) or potassium deprivation) triggers typical apoptosis (10). Presumably, this recapitulates the naturally occurring neuronal death that takes place in the newborn rat cerebellum (11). These characteristics, along with an abundant neuronal population and up to 98% homogeneity, make cultured CGNs an excellent and extensively studied model for deciphering the signaling mechanisms that underlie depolarization-dependent neuron survival (4).It has been well documented that depolarizing conditions (such as elevated [KCl] o ) sustain neuronal survival by causing the influx of Ca 2ϩ through L-type Ca 2ϩ channels (8, 12, 13), implicating Ca 2ϩ as a necessary second messenger for survival signaling. When activated by elevated Ca 2ϩ , Ca 2ϩ /calmodulindependent protein kinase II (CaMKII) has been reported to mediate the depolarization-...

RNA interference, which involves the delivery of small interfering RNA (siRNA), has been used to validate target genes, to understand and control cellular metabolic pathways, and to use as a "green" alternative to confer pest tolerance in crops. Conventional siRNA delivery methods such as viruses and Agrobacterium-mediated delivery exhibit plant species range limitations and uncontrolled DNA integration into the plant genome. Here, we synthesize polyethylenimine-functionalized gold nanoclusters (PEI-AuNCs) to mediate siRNA delivery into intact plants and show that these nanoclusters enable efficient gene knockdown. We further demonstrate that PEI-AuNCs protect siRNA from RNase degradation while the complex is small enough to bypass the plant cell wall. Consequently, AuNCs enable gene knockdown with efficiencies of up 76.5 ± 5.9% and 76.1 ± 9.5% for GFP and ROQ1, respectively, with no observable toxicity. Our data suggest that AuNCs can deliver siRNA into intact plant cells for broad applications in plant biotechnology.