Leigh Anne Swayne

Lipidomics era: Accomplishments and challenges

Lipid mediators participate in signal transduction pathways, proliferation, apoptosis, and membrane trafficking in the cell. Lipids are highly complex and diverse owing to the various combinations of polar headgroups, fatty acyl chains, and backbone structures. This structural diversity continues to pose a challenge for lipid analysis. Here we review the current state of the art in lipidomics research and discuss the challenges facing this field. The latest technological developments in mass spectrometry, the role of bioinformatics, and the applications of lipidomics in lipid metabolism and cellular physiology and pathology are also discussed.

Pannexin 2 Is Expressed by Postnatal Hippocampal Neural Progenitors and Modulates Neuronal Commitment

The pannexins (Panx1, -2, and -3) are a mammalian family of putative single membrane channels discovered through homology to invertebrate gap junction-forming proteins, the innexins. Because connexin gap junction proteins are known regulators of neural stem and progenitor cell proliferation, migration, and specification, we asked whether pannexins, specifically Panx2, play a similar role in the postnatal hippocampus. We show that Panx2 protein is differentially expressed by multipotential progenitor cells and mature neurons. Both in vivo and in vitro, Type I and IIa stem-like neural progenitor cells express an S-palmitoylated Panx2 species localizing to Golgi and endoplasmic reticulum membranes. Protein expression is down-regulated during neurogenesis in neuronally committed Type IIb and III progenitor cells and immature neurons. Panx2 is re-expressed by neurons following maturation. Protein expressed by mature neurons is not palmitoylated and localizes to the plasma membrane. To assess the impact of Panx2 on neuronal differentiation, we used short hairpin RNA to suppress Panx2 expression in Neuro2a cells. Knockdown significantly accelerated the rate of neuronal differentiation. Neuritic extension and the expression of antigenic markers of mature neurons occurred earlier in stable lines expressing Panx2 short hairpin RNA than in controls. Together, these findings describe an endogenous post-translational regulation of Panx2, specific to early neural progenitor cells, and demonstrate that this expression plays a role in modulating the timing of their commitment to a neuronal lineage.

Amyloid-β42 signals tau hyperphosphorylation and compromises neuronal viability by disrupting alkylacylglycerophosphocholine metabolism

Perturbation of lipid second messenger networks is associated with the impairment of synaptic function in Alzheimer disease. Underlying molecular mechanisms are unclear. Here, we used an unbiased lipidomic approach to profile alkylacylglycerophosphocholine second messengers in diseased tissue. We found that specific isoforms defined by a palmitic acid (16:0) at the sn-1 position, namely 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (C16:0 PAF) and 1-O-hexadecyl-sn-glycero-3-phosphocholine (C16:0 lyso-PAF), were elevated in the temporal cortex of Alzheimer disease patients, transgenic mice expressing human familial disease-mutant amyloid precursor protein, and human neurons directly exposed to amyloid-β42 oligomers. Acute intraneuronal accumulation of C16:0 PAF but not C16:0 lyso-PAF initiated cyclin-dependent kinase 5-mediated hyperphosphorylation of tau on Alzheimer disease-specific epitopes. Chronic elevation caused a caspase 2 and 3/7-dependent cascade resulting in neuronal death. Pharmacological inhibition of C16:0 PAF signaling, or molecular strategies increasing hydrolysis of C16:0 PAF to C16:0 lyso-PAF, protected human neurons from amyloid-β42 toxicity. Together, these data provide mechanistic insight into how disruptions in lipid metabolism can determine neuronal response to accumulating oligomeric amyloid-β42.

Pannexin 1 regulates postnatal neural stem and progenitor cell proliferation

BackgroundPannexin 1 forms ion and metabolite permeable hexameric channels and is abundantly expressed in the brain. After discovering pannexin 1 expression in postnatal neural stem and progenitor cells we sought to elucidate its functional role in neuronal development.ResultsWe detected pannexin 1 in neural stem and progenitor cells in vitro and in vivo. We manipulated pannexin 1 expression and activity in Neuro2a neuroblastoma cells and primary postnatal neurosphere cultures to demonstrate that pannexin 1 regulates neural stem and progenitor cell proliferation likely through the release of adenosine triphosphate (ATP).ConclusionsPermeable to ATP, a potent autocrine/paracine signaling metabolite, pannexin 1 channels are ideally suited to influence the behavior of neural stem and progenitor cells. Here we demonstrate they play a robust role in the regulation of neural stem and progenitor cell proliferation. Endogenous postnatal neural stem and progenitor cells are crucial for normal brain health, and their numbers decline with age. Furthermore, these special cells are highly responsive to neurological injury and disease, and are gaining attention as putative targets for brain repair. Therefore, understanding the fundamental role of pannexin 1 channels in neural stem and progenitor cells is of critical importance for brain health and disease.

Cysteine String Protein (CSP) Inhibition of N-type Calcium Channels Is Blocked by Mutant Huntingtin

Cysteine string protein (CSP), a 34-kDa molecular chaperone, is expressed on synaptic vesicles in neurons and on secretory vesicles in endocrine, neuroendocrine, and exocrine cells. CSP can be found in a complex with two other chaperones, the heat shock cognate protein Hsc70, and small glutamine-rich tetratricopeptide repeat domain protein (SGT). CSP function is vital in synaptic transmission; however, the precise nature of its role remains controversial. We have previously reported interactions of CSP with both heterotrimeric GTP-binding proteins (G proteins) and N-type calcium channels. These associations give rise to a tonic G protein inhibition of the channels. Here we have examined the effects of huntingtin fragments (exon 1) with (huntingtinexon1/exp) and without (huntingtinexon1/nonexp) expanded polyglutamine (polyQ) tracts on the CSP chaperone system. In vitro huntingtinexon1/exp sequestered CSP and blocked the association of CSP with G proteins. In contrast, huntingtinexon1/nonexp did not interact with CSP and did not alter the CSP/G protein association. Similarly, co-expression of huntingtinexon1/exp with CSP and N-type calcium channels eliminated CSPs tonic G protein inhibition of the channels, while coexpression of huntingtinexon1/nonexp did not alter the robust inhibition promoted by CSP. These results indicate that CSPs modulation of G protein inhibition of calcium channel activity is blocked in the presence of a huntingtin fragment with expanded polyglutamine tracts.

Molecular determinants of cysteine string protein modulation of N-type calcium channels

Cysteine string proteins (CSPs) are secretory vesicle chaperones that are important for neurotransmitter release. We have previously reported an interaction of CSP with both heterotrimeric GTP-binding proteins (G proteins) and N-type calcium channels that results in a tonic G protein inhibition of the channels. In this report we directly demonstrate that two separate regions of CSP associate with G proteins. The N-terminal binding site of CSP, which includes the J domain, binds Gα subunits but not Gαβ subunits whereas the C terminal binding site of CSP associates with either free Gαβ subunits or Gαβ in complex with Gα. The interaction of either binding site of CSP (CSP1-82 or CSP83-198) with G proteins elicits robust tonic inhibition of N-type calcium channel activity. However, CSP1-82 inhibition and CSP83-198 inhibition of calcium channels occur through distinct mechanisms. Calcium channel inhibition by CSP83-198 (but not CSP1-82) is completely blocked by co-expression of the synaptic protein interaction site (synprint) of the N-type channel, indicating that CSP83-198 inhibition is dependent on a physical interaction with the calcium channel. These results suggest that distinct binding sites of CSP can play a role in modulating G protein function and G protein inhibition of calcium channels.

Crosstalk between huntingtin and syntaxin 1A regulates N-type calcium channels

We have identified a novel interaction between huntingtin (htt) and N-type calcium channels, a channel key in coupling calcium influx with synaptic vesicle exocytosis. Htt is a widely expressed 350-kDa cytosolic protein bearing an N-terminal polyglutamine tract. Htt is proteolytically cleaved by calpains and caspases and the resultant htt N-terminal fragments have been proposed to be biologically active; however, the cellular function of htt and/or the htt fragments remains enigmatic. We show that N-terminal fragments of htt (consisting of exon1) and full-length htt associate with the synaptic protein interaction (synprint) region of the N-type calcium channel. Given that synprint has previously been shown to bind syntaxin 1A and that this association elicits inhibition of N-type calcium channels, we tested whether htt(exon1) affects the modulation of these channels. Our data indicate that htt(exon1) enhances calcium influx by blocking syntaxin 1A inhibition of N-type calcium channels and attributes a key role for htt N-terminal fragments in the fine tuning of neurotransmission.

Oilgomerization characteristics of cysteine string protein

CSP function is vital to synaptic transmission, however; the precise nature of its role remains controversial. Conflicting reports support either a role for CSP: (i) in exocytosis or (ii) in the regulation of transmembrane calcium fluxes. Here we have examined the self-association of CSP to form oligomers that are stable upon SDS-PAGE. To understand the structural requirements for CSP self-association a series of CSP deletion mutants were constructed, expressed, and purified. This analysis revealed an interesting pattern of oligomerization. Amino acids between 83 and 136 were observed to be important for self-association. The recombinant CSP oligomers as well as the CSP monomers directly associate with Ni(2+)-NTA agarose. Thus CSP-CSP interactions may be an important consideration for current working models of CSP chaperone activity at the synapse.

Panx1 regulates neural stem and progenitor cell behaviours associated with cytoskeletal dynamics and interacts with multiple cytoskeletal elements

BackgroundPannexins (Panxs) are relatively newly discovered large-pore ion and metabolite permeable channels. Although no proteomics-based interactome has yet been published, Panx1 has been demonstrated to interact with actin in an ectopic expression system. This interaction affects both Panx1 plasma membrane stability as well as cytoskeletal remodelling. The current study builds on our recent discovery of Panx1 expression in ventricular zone (VZ) neural stem and progenitor cells (NSC/NPCs), and on the demonstrated interaction of Panx1 with the cytoskeleton.FindingsHere we demonstrate that Panx1 also plays roles in two additional cell behaviours associated with neurogenesis, including cell migration and neurite extension. Furthermore, we confirm an endogenous interaction between actin and Panx1, and identify a new interaction with actin-related protein 3, an actin cytoskeleton-modulating protein.ConclusionsThis study further establishes the importance of Panx1 in the cell biology of NSC/NPCs and strengthens and expands our knowledge of Panx1 interactions with the cytoskeleton.

Analysis of a pannexin 2-pannexin 1 chimeric protein supports divergent roles for pannexin C-termini in cellular localization

Abstract Pannexins (Panxs) are a three-member family of large pore ion channels permeable to ions and small molecules. Recent elegant work has demonstrated that the Panx1 C-terminus plays an important role in channel trafficking. Panx2, another family member, has a longer and highly dissimilar C-terminus. Interestingly, Panx1 is readily found at the plasma membrane, while Panx2 is mainly present on intracellular membranes. Here we used overlap-extension cloning to create the first chimeric Panx, consisting of Panx2 with the Panx1 C-terminus (Panx2Panx1CT), to determine whether the Panx1 C-terminus influences the trafficking of Panx2. We are the first to observe a high level of co-localization between Panx2 and the endolysosomal enriched mannose-6-phosphate receptor. Interestingly this distinct localization of Panx2 is altered by the presence of the Panx1 C-terminus. These novel observations support previous data indicating the importance of the C-terminus in the control of Panx trafficking, and highlight the complexity of molecular signals involved.