sex-stratified-neuronal-cultures-to-study-ischemic-cell-death-pathways

Sex Stratified Neuronal Cultures to Study Ischemic Cell Death Pathways

Sex differences in neuronal susceptibility to ischemic injury and neurodegenerative disease have long been observed, but the signaling mechanisms ...

University of Colorado School of Medicine

Elevated concentrations of intracellular calcium in erythrocytes increase membrane order and susceptibility to secretory phospholipase A2. We hypothesize that calcium aids the formation of domains of ordered lipids within erythrocyte membranes by interacting directly with the inner leaflet of the cell membrane. The interface of these domains with regions of more fluid lipids may create an environment with weakened neighbor-neighbor interactions that would facilitate phospholipid migration into the active site of bound secretory phospholipase A2. This hypothesis was investigated by determining the effects of seven other divalent ions on erythrocyte membrane properties. Changes in membrane order were assessed with steady-state fluorescence spectroscopy and two-photon microscopy with an environment-sensitive probe, laurdan. Each ion increased apparent membrane order in model membranes and in erythrocytes when introduced with an ionophore, suggesting that direct binding to the inner face of the membrane accounts for the effects of calcium on membrane fluidity. Furthermore, the degree to which ions affected membrane properties correlated with the ionic radius and electronegativity of the ions. Lastly, erythrocytes became more susceptible to enzyme hydrolysis in the presence of elevated intracellular levels of nickel and manganese, but not magnesium. These differences appeared related to the ability of the ions to induce a transition in erythrocyte shape.

Divalent cations increase lipid order in erythrocytes and susceptibility to secretory phospholipase A2.

In artificial phospholipid bilayers, dual measurements of laurdan steady-state anisotropy and emission spectra can be used to identify the presence of liquid ordered phases. Human erythrocytes were used as a model to test whether similar measurements could be applied to biological samples. Specifically, laurdan anisotropy and emission spectra were obtained from native erythrocytes before and after treatment with calcium ionophore and from the microvesicles (known to be enriched in liquid ordered domains) shed from the cells during calcium entry. Spectral and anisotropy data were consistent with an increased order and reduced fluidity of erythrocyte membrane lipids upon ionophore treatment. Microvesicle membranes appeared more ordered than native erythrocytes and similar to ionophore-treated cells based on laurdan emission. In contrast, the anisotropy value was lower in microvesicles compared to ionophore-treated cells, suggesting greater probe mobility. Parallel measurements of diphenylhexatriene anisotropy corroborated the laurdan data. These results were consistent with the liquid ordered property of microvesicle membranes based on comparisons to behavior in artificial membranes. Two-photon microscopy was used to examine the distribution of laurdan fluorescence along the surface of erythrocyte membranes before and after ionophore treatment. A dual spatial analysis of laurdan anisotropy, as revealed by the distribution of laurdan emission spectra, and intensity excited by polarized light suggested that the plasma membranes of ionophore-treated erythrocytes may also exhibit elevated numbers of liquid ordered domains.

Use of steady-state laurdan fluorescence to detect changes in liquid ordered phases in human erythrocyte membranes.

Ca(2+)/calmodulin (CaM)-dependent protein kinase II (CaMKII) is a major mediator of cellular Ca(2+) signaling. Several inhibitors are commonly used to study CaMKII function, but these inhibitors all lack specificity. CaM-KIIN is a natural, specific CaMKII inhibitor protein. CN21 (derived from CaM-KIIN amino acids 43-63) showed full specificity and potency of CaMKII inhibition. CNs completely blocked Ca(2+)-stimulated and autonomous substrate phosphorylation by CaMKII and autophosphorylation at T305. However, T286 autophosphorylation (the autophosphorylation generating autonomous activity) was only mildly affected. Two mechanisms can explain this unusual differential inhibitor effect. First, CNs inhibited activity by interacting with the CaMKII T-site (and thereby also interfered with NMDA-type glutamate receptor binding to the T-site). Because of this, the CaMKII region surrounding T286 competed with CNs for T-site interaction, whereas other substrates did not. Second, the intersubunit T286 autophosphorylation requires CaM binding both to the "kinase" and the "substrate" subunit. CNs dramatically decreased CaM dissociation, thus facilitating the ability of CaM to make T286 accessible for phosphorylation. Tat-fusion made CN21 cell penetrating, as demonstrated by a strong inhibition of filopodia motility in neurons and insulin secrection from isolated Langerhans' islets. These results reveal the inhibitory mechanism of CaM-KIIN and establish a powerful new tool for dissecting CaMKII function.

Dual mechanism of a natural CaMKII inhibitor.

CaMKII, a major mediator of synaptic plasticity, forms extra-synaptic clusters under ischemic conditions. This study further supports self-aggregation of CaMKII holoenzymes as the underlying mechanism. Aggregation in vitro was promoted by mimicking ischemic conditions: low pH (6.8 or less), Ca(2+) (and calmodulin), and low ATP and/or high ADP concentration. Mutational analysis showed that high ATP prevented aggregation by a mechanism involving T286 auto-phosphorylation, and indicated requirement for nucleotide binding but not auto-phosphorylation also for extra-synaptic clustering within neurons. These results clarify a previously apparent paradox in the nucleotide and phosphorylation requirement of aggregation, and support a mechanism that involves inter-holoenzyme T286-region/T-site interaction.

Differential regulation by ATP versus ADP further links CaMKII aggregation to ischemic conditions.

Ca(2+)/calmodulin (CaM)-dependent protein kinase II (CaMKII) is a major mediator of physiological glutamate signaling involved in higher brain functions. Here, we show CaMKII involvement in pathological glutamate signaling relevant in stroke. The novel inhibitor tatCN21 was neuroprotective even when added hours after glutamate insults. By contrast, the "traditional" inhibitor KN93 attenuated excitotoxicity only when present during the insult. Both inhibitors efficiently blocked Ca(2+)/CaM-stimulated CaMKII activity, CaMKII interaction with NR2B and aggregation of CaMKII holoenzymes. However, only tatCN21 but not KN93 blocked the Ca(2+)-independent "autonomous" activity generated by Thr-286 autophosphorylation, the hallmark feature of CaMKII regulation. Mutational analysis further validated autonomous CaMKII activity as the drug target crucial for post-insult neuroprotection. Overexpression of CaMKII wild type but not the autonomy-deficient T286A mutant significantly increased glutamate-induced neuronal death. Maybe most importantly, tatCN21 also significantly reduced infarct size in a mouse stroke model (middle cerebral arterial occlusion) when injected (1 mg/kg intravenously) 1 h after onset of arterial occlusion. Together, these data demonstrate that inhibition of autonomous CaMKII activity provides a promising therapeutic avenue for post-insult neuro-protection after stroke.

Effective post-insult neuroprotection by a novel Ca(2+)/ calmodulin-dependent protein kinase II (CaMKII) inhibitor.

Plasminogen activators play an important role in synaptic plasticity associated with the crossed phrenic phenomenon (CPP) and recovery of respiratory function after spinal cord injury. A genetic approach using knockout mice lacking various genes in the plasminogen activator/plasmin system has shown that induction of urokinase plasminogen activator (uPA) is required during the first hour after a C2-hemisection for the acquisition of the CPP response. The uPA knockout mice do not show the structural remodeling of phrenic motor neuron synapses characteristic of the CPP response. As shown here uPA acts in a cell signaling manner via binding to its receptor uPAR rather than as a protease, since uPAR knockout mice or knock-in mice possessing a modified uPA that is unable to bind to uPAR both fail to generate a CPP and recover respiratory function. Microarray data and real-time PCR analysis of mRNAs induced in the phrenic motor nucleus after C2-hemisection in C57Bl/6 mice as compared to uPA knockout mice indicate a potential cell signaling cascade downstream possibly involving β-integrin and Src, and other pathways. Identification of these uPA-mediated signaling pathways may provide the opportunity to pharmacologically upregulate the synaptic plasticity necessary for recovery of phrenic motoneuron activity following cervical spinal cord injury.

Plasminogen activator promotes recovery following spinal cord injury.

Ischemic insults on neurons trigger excessive, pathological glutamate release that causes Ca²⁺ overload resulting in neuronal cell death (excitotoxicity). The Ca²⁺/calmodulin (CaM)-dependent protein kinase II (CaMKII) is a major mediator of physiological excitatory glutamate signals underlying neuronal plasticity and learning. Glutamate stimuli trigger autophosphorylation of CaMKII at T286, a process that makes the kinase "autonomous" (partially active independent from Ca²⁺ stimulation) and that is required for forms of synaptic plasticity. Recent studies suggested autonomous CaMKII activity also as potential drug target for post-insult neuroprotection, both after glutamate insults in neuronal cultures and after focal cerebral ischemia in vivo. However, CaMKII and other members of the CaM kinase family have been implicated in regulation of both neuronal death and survival. Here, we discuss past findings and possible mechanisms of CaM kinase functions in excitotoxicity and cerebral ischemia, with a focus on CaMKII and its regulation.

CaMKII in cerebral ischemia.

Age-related loss of sex steroid hormones is a established risk factor for the development of Alzheimer's disease (AD) in women and men. While the relationships between the sex steroid hormones and AD are not fully understood, findings from both human and experimental paradigms indicate that depletion of estrogens in women and androgens in men increases vulnerability of the aging brain to AD pathogenesis. We review evidence of a wide range of beneficial neural actions of sex steroid hormones that may contribute to their hypothesized protective roles against AD. Both estrogens and androgens exert general neuroprotective actions relevant to a several neurodegenerative conditions, some in a sex-specific manner, including protection from neuron death and promotion of select aspects of neural plasticity. In addition, estrogens and androgens regulate key processes implicated in AD pathogenesis, in particular the accumulation of β-amyloid protein. We discuss evidence of hormone-specific mechanisms related to the regulation of the production and clearance of β-amyloid as critical protective pathways. Continued elucidation of these pathways promises to yield effective hormone-based strategies to delay development of AD.

Rebekah Vest

Department of Anesthesiology

University of Colorado School of Medicine

Sex Stratified Neuronal Cultures to Study Ischemic Cell Death Pathways

Rebekah Vest

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Anesthesiology

University of Colorado School of Medicine

Sex Stratified Neuronal Cultures to Study Ischemic Cell Death Pathways

Department of Anesthesiology