Spontaneous Local Calcium Transients Regulate Oligodendrocyte Development in Culture through Store-Operated Ca2+ Entry and Release.

Oligodendrocytes (OLs) insulate axonal fibers for fast conduction of nerve impulses by wrapping axons of the CNS with compact myelin membranes. Differentiating OLs undergo drastic chances in cell morphology. Bipolar oligodendroglial precursor cells (OPCs) transform into highly ramified multipolar OLs, which then expand myelin membranes that enwrap axons. While significant progress has been made in understanding the molecular and genetic mechanisms underlying CNS myelination and its disruption in diseases, the cellular mechanisms that regulate OL differentiation are not fully understood. Here, we report that developing rat OLs in culture exhibit spontaneous Ca2+ local transients (sCaLTs) in their process arbors in the absence of neurons. Importantly, we find that the frequency of sCaLTs markedly increases as OLs undergo extensive process outgrowth and branching. We further show that sCaLTs are primarily generated through a combination of Ca2+ influx through store-operated Ca2+ entry (SOCE) and Ca2+ release from internal Ca2+ stores. Inhibition of sCaLTs impairs the elaboration and branching of OL processes, as well as substantially reduces the formation of large myelin sheets in culture. Together, our findings identify an important role for spontaneous local Ca2+ signaling in OL development.

Tropomodulin Isoform-Specific Regulation of Dendrite Development and Synapse Formation.

Neurons of the CNS elaborate highly branched dendritic arbors that host numerous dendritic spines, which serve as the postsynaptic platform for most excitatory synapses. The actin cytoskeleton plays an important role in dendrite development and spine formation, but the underlying mechanisms remain incompletely understood. Tropomodulins (Tmods) are a family of actin-binding proteins that cap the slow-growing (pointed) end of actin filaments, thereby regulating the stability, length, and architecture of complex actin networks in diverse cell types. Three members of the Tmod family, Tmod1, Tmod2, and Tmod3 are expressed in the vertebrate CNS, but their function in neuronal development is largely unknown. In this study, we present evidence that Tmod1 and Tmod2 exhibit distinct roles in regulating spine development and dendritic arborization, respectively. Using rat hippocampal tissues from both sexes, we find that Tmod1 and Tmod2 are expressed with distinct developmental profiles: Tmod2 is expressed early during hippocampal development, whereas Tmod1 expression coincides with synaptogenesis. We then show that knockdown of Tmod2, but not Tmod1, severely impairs dendritic branching. Both Tmod1 and Tmod2 are localized to a distinct subspine region where they regulate local F-actin stability. However, the knockdown of Tmod1, but not Tmod2, disrupts spine morphogenesis and impairs synapse formation. Collectively, these findings demonstrate that regulation of the actin cytoskeleton by different members of the Tmod family plays an important role in distinct aspects of dendrite and spine development.SIGNIFICANCE STATEMENT The Tropomodulin family of molecules is best known for controlling the length and stability of actin myofilaments in skeletal muscles. While several Tropomodulin members are expressed in the brain, fundamental knowledge about their role in neuronal function is limited. In this study, we show the unique expression profile and subcellular distribution of Tmod1 and Tmod2 in hippocampal neurons. While both Tmod1 and Tmod2 regulate F-actin stability, we find that they exhibit isoform-specific roles in dendrite development and synapse formation: Tmod2 regulates dendritic arborization, whereas Tmod1 is required for spine development and synapse formation. These findings provide novel insight into the actin regulatory mechanisms underlying neuronal development, thereby shedding light on potential pathways disrupted in a number of neurological disorders.

Phosphoinositide-dependent enrichment of actin monomers in dendritic spines regulates synapse development and plasticity.

Dendritic spines are small postsynaptic compartments of excitatory synapses in the vertebrate brain that are modified during learning, aging, and neurological disorders. The formation and modification of dendritic spines depend on rapid assembly and dynamic remodeling of the actin cytoskeleton in this highly compartmentalized space, but the precise mechanisms remain to be fully elucidated. In this study, we report that spatiotemporal enrichment of actin monomers (G-actin) in dendritic spines regulates spine development and plasticity. We first show that dendritic spines contain a locally enriched pool of G-actin that can be regulated by synaptic activity. We further find that this G-actin pool functions in spine development and its modification during synaptic plasticity. Mechanistically, the relatively immobile G-actin pool in spines depends on the phosphoinositide PI(3,4,5)P3 and involves the actin monomer-binding protein profilin. Together, our results have revealed a novel mechanism by which dynamic enrichment of G-actin in spines regulates the actin remodeling underlying synapse development and plasticity.

p39 Is Responsible for Increasing Cdk5 Activity during Postnatal Neuron Differentiation and Governs Neuronal Network Formation and Epileptic Responses.

Two distinct protein cofactors, p35 and p39, independently activate Cyclin-dependent kinase 5 (Cdk5), which plays diverse roles in normal brain function and the pathogenesis of many neurological diseases. The initial discovery that loss of p35 impairs neuronal migration in the embryonic brain prompted intensive research exploring the function of p35-dependent Cdk5 activity. In contrast, p39 expression is restricted to the postnatal brain and its function remains poorly understood. Despite the robustly increased Cdk5 activity during neuronal differentiation, which activator is responsible for enhancing Cdk5 activation and how the two distinct activators direct Cdk5 signaling to govern neuronal network formation and function still remains elusive. Here we report that p39, but not p35, is selectively upregulated by histone acetylation-mediated transcription, which underlies the robust increase of Cdk5 activity during rat and mouse neuronal differentiation. The loss of p39 attenuates overall Cdk5 activity in neurons and preferentially affects phosphorylation of specific Cdk5 targets, leading to aberrant axonal growth and impaired dendritic spine and synapse formation. In adult mouse brains, p39 deficiency results in dysregulation of p35 and Cdk5 targets in synapses. Moreover, in contrast to the proepileptic phenotype caused by the lack of p35, p39 loss leads to deficits in maintaining seizure activity and induction of immediate early genes that control hippocampal excitability. Together, our studies demonstrate essential roles of p39 in neuronal network development and function. Furthermore, our data support a model in which Cdk5 activators play nonoverlapping and even opposing roles to govern balanced Cdk5 signaling in the postnatal brain. SIGNIFICANCE STATEMENT: Neuronal network development requires tightly regulated activation of Cyclin-dependent kinase 5 (Cdk5) by two distinct cofactors, p35 and p39. Despite the well-known p35-dependent Cdk5 function, why postnatal neurons express abundant p39 in addition to p35 remained unknown for decades. In this study, we discovered that selective upregulation of p39 is the underlying mechanism that accommodates the increased functional requirement of Cdk5 activation during neuronal differentiation. In addition, we demonstrated that p39 selectively directs Cdk5 to phosphorylate protein substrates essential for axonal development, dendritic spine formation, and synaptogenesis. Moreover, our studies suggest opposing roles of p39 and p35 in synaptic Cdk5 function and epileptic responses, arguing that cooperation between Cdk5 activators maintains balanced Cdk5 signing, which is crucial for postnatal brain function.

Amyloid ß oligomers elicit mitochondrial transport defects and fragmentation in a time-dependent and pathway-specific manner.

Small oligomeric forms of amyloid-ß (Aß) are believed to be the culprit for declined brain functions in AD in part through their impairment of neuronal trafficking and synaptic functions. However, the precise cellular actions of Aß oligomers and underlying mechanisms in neurons remain to be fully defined. Previous studies have identified mitochondria as a major target of Aß toxicity contributing to early cognitive decline and memory loss in neurodegenerative diseases including Alzheimer's disease (AD). In this study, we report that Aß oligomers acutely elicit distinct effects on the transport and integrity of mitochondria. We found that acute exposure of hippocampal neurons to Aß oligomers from either synthetic peptides or AD brain homogenates selectively impaired fast transport of mitochondria without affecting the movement of late endosomes and lysosomes. Extended exposure of hipoocampal neurons to Aß oligomers was found to result in mitochondrial fragmentation. While both mitochondrial effects induced by Aß oligomers can be abolished by the inhibition of GSK3ß, they appear to be independent from each other. Aß oligomers impaired mitochondrial transport through HDAC6 activation whereas the fragmentation involved the GTPase Drp-1. These results show that Aß oligomers can acutely disrupt mitochondrial transport and integrity in a time-dependent and pathway-specific manner. These findings thus provide new insights into Aß-induced mitochondrial defects that may contribute to neuronal dysfunction and AD pathogenesis.

Activity-dependent regulation of dendritic growth and maintenance by glycogen synthase kinase 3ß.

Activity-dependent dendritic development represents a crucial step in brain development, but its underlying mechanisms remain to be fully elucidated. Here we report that glycogen synthase kinase 3ß (GSK3ß) regulates dendritic development in an activity-dependent manner. We find that GSK3ß in somatodendritic compartments of hippocampal neurons becomes highly phosphorylated at serine-9 upon synaptogenesis. This phosphorylation-dependent GSK3ß inhibition is mediated by neurotrophin signalling and is required for dendritic growth and arbourization. Elevation of GSK3ß activity leads to marked shrinkage of dendrites, whereas its inhibition enhances dendritic growth. We further show that these effects are mediated by GSK3ß regulation of surface GABAA receptor levels via the scaffold protein gephyrin. GSK3ß activation leads to gephyrin phosphorylation to reduce surface GABAA receptor clusters, resulting in neuronal hyperexcitability that causes dendrite shrinkage. These findings thus identify GSK3ß as a key player in activity-dependent regulation of dendritic development by targeting the excitatory-inhibitory balance of the neuron.

Inhibition of AMPA receptor trafficking at hippocampal synapses by beta-amyloid oligomers: the mitochondrial contribution.

BACKGROUND: Synaptic defects represent a major mechanism underlying altered brain functions of patients suffering Alzheimer's disease (AD) 123. An increasing body of work indicates that the oligomeric forms of beta-amyloid (Abeta) molecules exert profound inhibition on synaptic functions and can cause a significant loss of neurotransmitter receptors from the postsynaptic surface, but the underlying mechanisms remain poorly understood. In this study, we investigated a potential contribution of mitochondria to Abeta inhibition of AMPA receptor (AMPAR) trafficking. RESULTS: We found that a brief exposure of hippocampal neurons to Abeta oligomers not only led to marked removal of AMPARs from postsynaptic surface but also impaired rapid AMPAR insertion during chemically-induced synaptic potentiation. We also found that Abeta oligomers exerted acute impairment of fast mitochondrial transport, as well as mitochondrial translocation into dendritic spines in response to repetitive membrane depolarization. Quantitative analyses at the single spine level showed a positive correlation between spine-mitochondria association and the surface accumulation of AMPARs. In particular, we found that spines associated with mitochondria tended to be more resistant to Abeta inhibition on AMPAR trafficking. Finally, we showed that inhibition of GSK3beta alleviated Abeta impairment of mitochondrial transport, and effectively abolished Abeta-induced AMPAR loss and inhibition of AMPAR insertion at spines during cLTP. CONCLUSIONS: Our findings indicate that mitochondrial association with dendritic spines may play an important role in supporting AMPAR presence on or trafficking to the postsynaptic membrane. Abeta disruption of mitochondrial trafficking could contribute to AMPAR removal and trafficking defects leading to synaptic inhibition.

Sequestration of copper from beta-amyloid promotes selective lysis by cyclen-hybrid cleavage agents.

Decelerated degradation of beta-amyloid (Abeta) and its interaction with synaptic copper may be pathogenic in Alzheimer disease. Recently, Co(III)-cyclen tagged to an aromatic recognition motif was shown to degrade Abeta in vitro. Here, we report that apocyclen attached to selective Abeta recognition motifs (KLVFF or curcumin) can capture copper bound to Abeta and use the Cu(II) in place of Co(III) to become proteolytically active. The resultant complexes interfere with Abeta aggregation, degrade Abeta into fragments, preventing H2O2 formation and toxicity in neuronal cell culture. Because Abeta binds Cu in amyloid plaques, apocyclen-tagged targeting molecules may be a promising approach to the selective degradation of Abeta in Alzheimer disease. The principle of copper capture could generalize to other amyloidoses where copper is implicated.

Astragaloside IV inhibits spontaneous synaptic transmission and synchronized Ca2+ oscillations on hippocampal neurons.

AIM: To investigate the changes in the spontaneous neuronal excitability induced by astragaloside IV (AGS-IV) in the cultured hippocampal network. METHODS: Hippocampal neurons in culture for 9-11 d were used for this study. The spontaneous synaptic activities of these hippocampal neurons were examined by Ca2+ imaging and whole-cell patch-clamp techniques. In total, 40 mg/L AGS-IV dissolved in DMSO and 2 mL/L DMSO were applied to the neurons under a microscope while the experiments were taking place. RESULTS: AGS-IV inhibited the frequencies of synchronized spontaneous Ca2+ oscillations to 59.39%+/- 3.25%(mean+/-SEM), the spontaneous postsynaptic currents to 43.78%+/- 7.72%(mean+/-SEM), and the spontaneous excitatory postsynaptic currents to 49.25%+/- 7.06%(mean+/-SEM) of those of the control periods, respectively, at 16 min after the AGSIV applications. AGS-IV also decreased the peak values of the voltage-gated K+ and Na+ channel currents at that time point. CONCLUSION: These results indicate that AGS-IV suppresses the spontaneous neuronal excitabilities effectively. Such a modulation of neuronal activity could represent new evidence for AGS-IV as a neuroprotector.

Acute impairment of mitochondrial trafficking by beta-amyloid peptides in hippocampal neurons.

Defects in axonal transport are often associated with a wide variety of neurological diseases including Alzheimer's disease (AD). Beta-amyloid (Abeta) is a major component of neuritic plaques associated with pathological conditions of AD brains. Here, we report that a brief exposure of cultured hippocampal neurons to Abeta molecules resulted in rapid and severe impairment of mitochondrial transport without inducing apparent cell death and significant morphological changes. Such acute inhibition of mitochondrial transport was not associated with a disruption of mitochondria potential nor involved aberrant cytoskeletal changes. Abeta also did not elicit significant Ca2+ signaling to affect mitochondrial trafficking. However, stimulation of protein kinase A (PKA) by forskolin, cAMP analogs, or neuropeptides effectively alleviated the impairment. We also show that Abeta inhibited mitochondrial transport by acting through glycogen synthase kinase 3beta (GSK3beta). Given that mitochondria are crucial organelles for many cellular functions and survival, our findings thus identify an important acute action of Abeta molecules on nerve cells that could potentially contribute to various abnormalities of neuronal functions under AD conditions. Manipulation of GSK3beta and PKA activities may represent a key approach for preventing and alleviating Abeta cytotoxicity and AD pathological conditions.

Acute effect of beta amyloid on synchronized spontaneous Ca2+ oscillations in cultured hippocampal networks.

The effects of beta amyloid (Abeta) on cytoplasmic Ca(2+) ([Ca(2+)](c)) have been studied extensively, but the current literature on this aspect is confusing. We reported that 20 microM Abeta(25-35) significantly inhibited the synchronized spontaneous cytoplasmic Ca(2+) transients immediately after application, whereas it had little effect on the baseline [Ca(2+)](c) concentration in neurons. Abeta(1-42) had a similar effect on the Ca(2+) transients as Abeta(25-35), while it increased baseline [Ca(2+)](c) concentration gradually. However, Abeta(1-40) had little effect on either Ca(2+) transients or baseline [Ca(2+)](c). Such differential effects of Abeta on Ca(2+) signals might explain, at least partially, the confusing observations from the previous studies and provide important therapeutic implications for preventing or reversing early neuron damage in Alzheimer's disease.

MEK inhibitor PD98059 acutely inhibits synchronized spontaneous Ca2+ oscillations in cultured hippocampal networks.

AIM: To investigate the changes in synchronized spontaneous Ca2+ oscillations induced by mitogen-activated protein kinase kinase (MEK) inhibitor PD98059 at different concentrations in cultured hippocampal network. METHODS: Hippocampal neurons in culture for 1-2 weeks were used for this study. Spontaneous synaptic activities of these hippocampal neurons were examined by Ca2+ imaging using calcium-sensitive dye. MEK inhibitor PD98059 (10, 30, and 60 micromol/L) and SB202474 (10 and 60 micromol/L), a negative control for mitogen-activated protein kinase (MAPK) cascade study, were applied to the cells under the microscope while imaging was taking place. RESULTS: PD98059 at a lower concentration of 10 micromol/L had little effect on the Ca2+ oscillation. At the higher concentration of 30 micromol/L, 5 min after application of PD98059, the spike frequency was decreased to 25.38% +/-7.40% (mean+/-SEM, n=16, P<0.01 vs medium control) of that of the control period. At an even higher concentration of 60 micromol/L, 5 min after application of PD98059, the spike frequency was decreased to 14.53%+/-5.34% (mean+/-SEM, n=16, P< 0.01 vs medium control) of that of the control period. The spike amplitude underwent a corresponding decrease. However, the negative control SB202474 at concentrations of 10 and 60 micromol/L had little inhibition effect on the Ca2+ oscillation. CONCLUSION: These results indicate that PD98059 inhibits synchronized spontaneous Ca2+ oscillation through inhibition of MEK, which hints that the MAPK cascade is required to maintain synchronized spontaneous Ca2+ oscillation.