We proposed that group IIA secretory phospholipase A2 (GIIA) participates in neuritogenesis based on our observations that the enzyme migrates to growth cones and neurite tips when PC12 cells are induced to differentiate by nerve growth factor (NGF) (Ferrini et al., Neurochem Res 35:2168–2174, 2010). The involvement of other secretory PLA2 isoforms in neuronal development has been suggested by others but through different mechanisms. In the present study, we compared the subcellular distribution of GIIA and group X sPLA2 (GX) after stimulation of PC12 cells with NGF. We found that GIIA, but not GX, localized at the neuritic tips after treatment with NGF, as demonstrated by immunofluorescence analysis. We also found that NGF stimulated the expression and the activity of GIIA. In addition, NGF induced the expressed myc-tagged GIIA protein to migrate to neurite tips in its active form. We propose that GIIA expression, activity, and subcellular localization is regulated by NGF and that the enzyme may participate in neuritogenesis through intracellular mechanisms, most likely by facilitating the remodelling of glycerophospholipid molecular species by deacylation–reacylation reactions necessary for the incorporation of polyunsaturated fatty acids.

In the present work, the Ca(2+) dependence of mitochondrial H(2) O(2) elimination was investigated. Mitochondria isolated from guinea pig brain were energized by glutamate and malate and incubated with micromolar concentrations of Ca(2+) in the presence of ADP, preventing permeability transition pore formation. After the completion of Ca(2+) uptake, mitochondria were challenged with H(2) O(2) (5 μM), then at various time points residual H(2) O(2) was determined using the Amplex red method and compared with that in mitochondria incubated with H(2) O(2) without Ca(2+) addition. Dose-dependent inhibition of H(2) O(2) elimination by Ca(2+) was detected, which was prevented by the Ca(2+) -uptake inhibitor Ru 360. Stimulation of Ca(2+) release from Ca(2+) -loaded mitochondria by a combined addition of Ru 360 and Na(+) decreased the Ca(2+) -evoked inhibition of H(2) O(2) removal. After Ca(2+) uptake (50 μM), mitochondrial aconitase activity was found to be decreased, which was partially attributable to the impaired elimination of endogenously produced reactive oxygen species. We found that the effects of Ca(2+) and H(2) O(2) on the activity of aconitase were additive. These results confirm that Ca(2+) inhibits elimination of H(2) O(2) in mitochondria and demonstrate that this effect is concentration dependent and reversible. The phenomenon described here can play a role in the modulation of ROS handling under conditions involving excessive cellular Ca(2+) load.

Phospholipases A 2 (PLA 2 s) are involved in neuritogenesis but the identity of the isoforms(s) contributing to this process is still not defined. Several reports have focused on secretory PLA 2 s (sPLA 2 ) as the administration of exogenous sPLA 2 s to PC12 neuronal cells stimulates neurite outgrowth. The present study demonstrates that the endogenous group IIA sPLA 2 (GIIA), constitutively expressed in mammalian neural cells, changes its subcellular localization when PC12 cells are induced to differentiate by NGF treatment. Indeed, confocal analysis showed a time-dependent accumulation of GIIA in growth cones and neurite tips. Under identical conditions the subcellular distribution of another isoform (GV) was unaffected by NGF. Contrary to GX, another sPLA 2 isoform expressed by PC12 cells, the contribution of GIIA to neuritogenesis does not require its release in the extracellular medium.

Several “low molecular weight” or “secretory” phospholipases A2 isoforms may be expressed in mammalian neural cells. Indeed, mRNAs for GIB, GIIA, GIIE, GIII, GV, GX, and GXII were detected in brain tissues despite different levels. However, only the presence of GIB, GIIA, and GV proteins has been clearly demonstrated in neural cells or in the nervous tissue. Although the roles of GIB and GV in the nervous tissue are still elusive, there is evidence to support the involvement of GIIA in physiological and pathological events, including neurotransmission, long-term potentiation, and neuritogenesis. The neurotoxic effects of an increase in GIIA may be envisaged under pathological conditions associated with the activation of astrocytes during inflammation or through activation of neurons and enzymes due to the stimulation of the NMDA glutamate receptor. In the past, elevation of GIIA expression in many acute and chronic neurological diseases is well known. Although each neurodegenerative disease has a separate etiology, many share similar neurochemical common processes, such as excitotoxicity, oxidative stress, and mitochondrial dysfunction, phenomena where GIIA play an important role.

Phospholipases A(2) (PLA(2)) participate in neuronal death signalling pathways because of their ability to release lipid mediators, although the contribution of each isoform and mechanism of neurotoxicity are still elusive. Using a novel fluorogenic method to assess changes in a PLA(2) activity by flow cytometry, here we show that the group IIA secretory phospholipase A(2) isoform (GIIA) was specifically activated in cortical neurons following stimulation of N-methyl-d-aspartate glutamate receptor subtype (NMDAR). For activation, GIIA required Ca(2+) and reactive oxygen/nitrogen species, and inhibition of its activity fully prevented NMDAR-mediated neuronal apoptotic death. Superoxide, nitric oxide or peroxynitrite donors stimulated GIIA activity, which mediated neuronal death. Intriguingly, we also found that GIIA activity induced mitochondrial superoxide production after NMDAR stimulation. These results reveal a novel role for GIIA in excitotoxicity both as target and producer of superoxide in a positive-loop of activation that may contribute to the propagation of neurodegeneration.

Platelet Activating Factor (PAF, 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) was first identi-fied as a lipid mediator of inflammation and immunological response. This compound is present at verylow concentration in normal mammalian brain where it is synthesized by two distinct pathways. The denovo pathway utilizes 1-alkyl-2-acetyl-sn-glycerol and CDP-choline as substrates of a DTT-insensitivephosphocholine transferase (PAF-PCT). The remodeling pathway requires the production of 1-alkyl-2-lyso-sn-glycero-3-phosphocholine (lysoPAF) produced by the hydrolysis of 1-alkyl-2-(long-chain)acyl-sn-glycero-3-phosphocholine (alkylacylGPC) by the action of phospholipases A 2 . Alternatively, lysoPAFcan be produced by transacylation from alkylacyl-GPC to 1-alk-1’-enyl-2-lyso-sn-glycero-phosphoethano-lamine (lysoPlsEtn) produced by a phospholipase A 2 as well. LysoPAF is acetylated to PAF by lysoPAFacetyltransferase(lysoPAF-AcT).TherelativecontributionofthetwopathwaystoPAFsynthesisdependsonseveral factors including the concentration of substrates, energy availability, Ca

The international neurochemistry community has lost an outstanding neurochemist, who was a leader, organizer, and teacher and who nurtured many neurochemists in USA and around the world. Professor Lloyd A. Horrocks, who established collaborations with the worldwide scientific community in the area of lipid neurochemistry, died unexpectedly on 18 August 2007 at the age of 75 in Columbus, Ohio. He was a man of many admirable qualities, known for his keen, yet dry sense of humor. He was a quiet man who used the minimum number of words in his conversation, yet always conveyed a meaningful message to his students, colleagues and friends. Dr Horrocks was a mentor and a friend to most of us and therefore his sudden passing away has deeply saddened his students, colleagues, and friends. Dr Horrocks was born in Cincinnati, Ohio on 13 July, 1932 and into a small family with two sisters. He grew up in Michigan and Illinois and was the son of a YMCA administrator. He obtained his B.A. degree at Ohio Wesleyan University, Delaware, Ohio, 1949–1953 (with Honors in Chemistry). He obtained his Masters degree in 1953 with Professor J. B. Brown at The Ohio State University, Columbus, Ohio. He stayed in the Department of Physiological Chemistry and obtained his PhD degree in 1960 under the guidance of Professor David G. Cornwell. His graduate studies were devoted to the characterization and analysis of fatty acids. In 1960, he chose brain lipids as a topic for research. The focus of his early research work was on the analysis of fatty acids by gas-liquid chromatography and determination of the theoretical basis of calibration factors for the thermal conductivity detector. His interests changed to glycerophospholipid metabolism in the late 1960s when he became interested in composition of myelin and demyelinating diseases. He developed methods for the separation of, and characterization of, phospholipid molecular species from brain tissue. He was well known for his pioneering studies on the metabolism and role of plasmalogen in brain. This included work using fluorometric and spectrophotometric assay methods to determine the presence and activity of plasmalogen-selective-phospholipase A2 and lysoplasmalogenase in the brain. He not only discovered plasmalogen-selective phospholipase A2 in brain but also described the stimulation of cytosolic phospholipase A2 and plasmalogen-selective phospholipase A2 in Alzheimer’s disease. Stimulation of these enzymes is responsible for the decrease in plasmalogen levels and increase in phosphomonoesters and phosphodiesters in brain tissue from AD patients. He was the first to describe the stimulation of phospholipase A2 in ischemic injury and patented the use of CDP-ethanolamine and CDP-choline as a therapy for treating ischemic brain injury. Dr Horrocks also extended his work in neurotrauma to the mechanisms underlying the pathophysiology of spinal cord injury and was the first to note that polyunsaturated fatty acids and eicosanoids are released during and following injury, accompanied by a reduction in plasmalogen and cholesterol levels following spinal cord injury. This was a critical observation in that it formed the biochemical basis for the use of methylprednisolone as a treatment for spinal cord injury. One of his fine attributes was his insistence that his students and post-doctoral investigators from his laboratory participated in ASN and ISN meetings. He wanted his trainees to have an opportunity to interact with other investigators and exchange scientific ideas. He mentored many graduate students and post-doctoral fellows, and was well known for having his trainees be independent and rarely asked them how things were going. He often answered students’ questions with a question, prying the answer from his students and in the end, this resulted in well trained students who were critical thinkers. He was always ready to listen to them and guide them. The students trained by him are now making excellent research/teaching/writing contributions to their respective fields. He was very fond of a quotation by Ralph Waldo Emerson, US essayist & poet (1803–1882): ‘Do not go where the path may lead, go instead where there is no path and leave a trail’. Because of his major contribution and guidance in neurochemistry, Dr Lloyd A. Horrocks will always be remembered by students, colleagues and fellow neurochemists. His work will lead the way for many future neurochemists having an interest in the involvement of neural membrane phospholipids in neurological disorders. Even after his retirement in 1992, Dr Horrocks remained engaged in research and continued directing research and writings. Very recently he contributed review articles on the involvement of phospholipid-derived lipid mediators in neuroinflammation and how these lipid mediators interact with lipid mediators of sphingolipid metabolism and cause neurodegeneration in neurological disorders. Dr Horrocks was also interested in health benefits of docosahexaenoic acid and often compared health benefits of docosahexaenoic acid with statins. Dr Horrocks authored over 300 research papers, reviews, and book chapters, edited 7 books and wrote 2 monographs (“Glycerophospholipids in Brain: Phospholipase A2 in Neurological Disorders” and Neurochemical Aspects of Excitotoxicity published by Springer, New York). Dr Lloyd Horrocks was an active member of several scientific societies: American Society for Biochemistry and Molecular Biology; American Society for Neurochemistry (Local Chairman, 1973 National Meeting; Council Alternate, 1985–1987; Program Chairman, 1988 National Meeting; Program Committee, 1987–1989; Chairman, Standing Rules Committee, 1989–1993); American Oil Chemists’ Society (Board member, Ohio Valley Section, 2000–2005); Biochemical Society; European Society for Neurochemistry; International Society for Neurochemistry (Committee on future of ISN, 1978–1979, Programme Committee, 1979–1981, 1989–1991; Co-organizer, Satellite Symposia, Birmingham, England (1980–1981), Vancouver, British Columbia, Canada (1982–1983), Mantua, Italy (1984–1985) and Puerta La Cruz, Venezuela (1986–1987). Consultant to UNESCO for UN Development Program at the Department of Biochemistry, M.S. University of Baroda, India (1980–1981). He was organizer, International Symposium on Spinal Cord Injury, Florence, Italy (1985). Dr Horrocks received numerous awards and honors, including Omicron Delta Kappa (leadership) 1952; Phi Lambda Upsilon (chemistry) 1952; Sigma Xi (science) 1954; NIH Special Fellowship 1964; Macy Faculty Scholarship 1974; Honorary Member of Tohoku Medical Society (Japan) 1984; NATO Visiting Professorship (National Research Council of Italy) 1986; Foreign Corresponding Member of Royal Academy of Sciences (Spain) 1993; [One of 70 foreign members in 1993; the Academy was founded in the early 1500s]; Neurochemical Research, special issue in his honor, 1997; distinguished Achievement Citation, Ohio Wesleyan Univ., 1998; Hall of Fame, Edgewood High School (Ashtabula, OH, USA), 1998. He served on several editorial boards and study sections: J. Lipid Research, 1976–1983, Editorial Board; J. Neurochem., 1978–1985, Editorial Board; 1976–1977, Advisory Board; International Lecithin Study Group, 1989–1993; Lipids, Associate Editor, 1985–2007; Folia Neuropathologica, 2006–2007; Mol. Chem. Neuropathol. (formerly Neurochem. Pathol., merged into J. Mol. Neurosci. 1999), 1982–1998, Editor-in-Chief for 18 years and Founder of the journal; Neurochem. Res., Associate Editor, 1998–2004; J. Mol. Neurosci., Editorial Board, 1999–2000; Neurological Disorders Program Project Review B Committee (NSB), NIH, 1981–1985; Training Grant and Career Development Review Committee (NST), NINDS, 1990–1993; National Multiple Sclerosis Society, Peer Review Committee B, 1991–1993; Scientific Advisory Board for Jean Carper, nutrition editor for USA Weekend (30 million circulation), 2004–2007; Board of Scientific Counselors, National Institute of Aging, ad hoc, November, 1994 and November, 1998; National Institutes of Health, ad hoc committees; Special Emphasis Panel, ZRG1-BDCN-2, NIH, Feb. 27, 2002, New Jersey Commission on Spinal Cord Injury, (2004-). Dr Lloyd Horrocks is survived by his wife of 51 years, Marjorie, his son, Richard, daughter, Becky and grandchildren, William, Aaron and Valerie Haxe, Ian and Ashley Horrocks, and great-grandson Zachary Haxe-Evans. Dr Horrocks was dedicated to his family, scientific research, and writings. His wife Marjorie was undoubtedly the source of strength and inspiration in his life. She regularly accompanied him to scientific meetings, which he used to enjoy thoroughly. Although a man of few words, he was always known as a respected gentleman who was liked by his family, friends, students and colleagues. We are honored to have worked with him and to have had the opportunity to write this commentary on Dr Lloyd Horrocks and his contributions to neurochemistry. His love for science along with his abilities to organize symposia and write scientific papers, reviews, and monographs are models for young neurochemists. Dr Horrocks’ scientific guidance will be missed in the international neurochemistry community. We can pay him a fitting tribute by fulfilling his dream of studying molecular mechanisms associated with the pathogenesis of neurodegenerative diseases.

Confocal immunofluorescence analysis indicated a relatively high localization of group V secretory phospholipase A(2) (GV) in the nuclei of cultured PC12 and U251 astrocytoma cells. Here, we report the biochemical evidence for the presence of a secretory PLA(2) in the nuclei of neuronal and glial cells from rat brain cortex. Enzymic activity was determined using [(3)H]oleate labelled Escherichia coli membranes in intact nuclei and in their soluble fractions in which the specific activity was significantly more elevated. The treatment of soluble nuclear fractions with inhibitors of cytosolic Ca(2+)-dependent or Ca(2+)-independent phospholipases A(2) was ineffective whereas DTT or Indoxam, a specific inhibitor of all isoforms of sPLA(2), abolished enzyme activity. The enzyme was identified as group V secretory phospholipase A(2) (GV) by Western blot analysis and its nucleoplasmic localization was demonstrated by CLSM.

Purpose: We investigated the effects of BTX-A on visceral afferent nerve transmission by measuring bladder tissue NGF levels in patients with neurogenic detrusor overactivity before and after intravesical treatment with BTX-A. We also compared the bladder tissue NGF content with clinical and urodynamic data.Materials and Methods: A total of 23 patients underwent clinical evaluation and urodynamics with detection of the UDC threshold, maximum pressure and maximum cystometric capacity before, and at the 1 and 3-month followups. Endoscopic bladder Wall biopsies were also obtained at the same time points. NGF levels were measured in tissue homogenate by enzyme-linked immunosorbent assay (Promega, Madison, Wisconsin).Results: At 1 and 3 months mean catheterization and incontinent episodes were significantly decreased (p < 0.05 and < 0.001, respectively). On urodynamics we detected a significant increase in the UDC threshold and maximum cystometric capacity, and a significant decrease in UDC maximum pressure at the 1 and 3-month followups compared to baseline (each p < 0.001). At the same time points we detected a significant decrease in NGF bladder tissue content (each p < 0.02).Conclusions: BTX-A intravesical treatment induces a state of NGF deprivation in bladder tissue that persists at least up to 4 months. As caused by BTX-A, the decrease in acetylcholine release at the presynaptic level may induce a decrease in detrusor contractility and in NGF production by the detrusor muscle. Alternatively BTX-A can decrease the bladder level of neurotransmitters that normally modulate NGF production and release.