Myelin structure in unfixed, single nerve fibers: Scanning X-ray microdiffraction with a beam size of 200nm.

Previous raster-scanning with a 1µm X-ray beam of individual, myelinated fibers from glutaraldehyde-fixed rat sciatic nerve revealed a spatially-dependent variation in the diffraction patterns from single fibers. Analysis indicated differences in the myelin periodicity, membrane separations, distribution of proteins, and orientation of membrane lamellae. As chemical fixation is known to produce structural artifacts, we sought to determine in the current study whether the structural heterogeneity is intrinsic to unfixed myelin. Using a 200nm-beam that was about five-fold smaller than before, we raster-scanned individual myelinated fibers from both the peripheral (PNS; mouse and rat sciatic nerves) and central (CNS; rat corpus callosum) nervous systems. As expected, the membrane stacking in the internodal region was nearly parallel to the fiber axis and in the paranodal region it was perpendicular to the axis. A myelin lattice was also frequently observed when the incident beam was injected en face to the sheath. Myelin periodicity and diffracted intensity varied with axial position along the fiber, as did the calculated membrane profiles. Raster-scanning with an X-ray beam at sub-micron resolution revealed for the first time that the individual myelin sheaths in unfixed nerve are heterogeneous in both membrane structure and packing.

Arctic ground squirrel hippocampus tolerates oxygen glucose deprivation independent of hibernation season even when not hibernating and after ATP depletion, acidosis, and glutamate efflux.

Cerebral ischemia/reperfusion (I/R) triggers a cascade of uncontrolled cellular processes that perturb cell homeostasis. The arctic ground squirrel (AGS), a seasonal hibernator resists brain damage following cerebral I/R caused by cardiac arrest and resuscitation. However, it remains unclear if tolerance to I/R injury in AGS depends on the hibernation season. Moreover, it is also not clear if events such as depletion of ATP, acidosis, and glutamate efflux that are associated with anoxic depolarization are attenuated in AGS. Here, we employ a novel microperfusion technique to test the hypothesis that tolerance to I/R injury modeled in an acute hippocampal slice preparation in AGS is independent of the hibernation season and persists even after glutamate efflux. Acute hippocampal slices were harvested from summer euthermic AGS, hibernating AGS, and interbout euthermic AGS. Slices were subjected to oxygen glucose deprivation (OGD), an in vitro model of I/R injury to determine cell death marked by lactate dehydrogenase (LDH) release. ATP was assayed using ENLITEN ATP assay. Glutamate and aspartate efflux was measured using capillary electrophoresis. For acidosis, slices were subjected to pH 6.4 or ischemic shift solution (ISS). Acute hippocampal slices from rats were used as a positive control, susceptible to I/R injury. Our results indicate that when tissue temperature is maintained at 36°C, hibernation season has no influence on OGD-induced cell death in AGS hippocampal slices. Our data also show that tolerance to OGD in AGS hippocampal slices occurs despite loss of ATP and glutamate release, and persists during conditions that mimic acidosis and ionic shifts, characteristic of cerebral I/R. Read the Editorial Comment for this article on page 10.

rAAV Gene Therapy in a Canavan's Disease Mouse Model Reveals Immune Impairments and an Extended Pathology Beyond the Central Nervous System.

Aspartoacylase (AspA) gene mutations cause the pediatric lethal neurodegenerative Canavan disease (CD). There is emerging promise of successful gene therapy for CD using recombinant adeno-associated viruses (rAAVs). Here, we report an intracerebroventricularly delivered AspA gene therapy regime using three serotypes of rAAVs at a 20-fold reduced dose than previously described in AspA(-/-) mice, a bona-fide mouse model of CD. Interestingly, central nervous system (CNS)-restricted therapy prolonged survival over systemic therapy in CD mice but failed to sustain motor functions seen in systemically treated mice. Importantly, we reveal through histological and functional examination of untreated CD mice that AspA deficiency in peripheral tissues causes morphological and functional abnormalities in this heretofore CNS-defined disorder. We demonstrate for the first time that AspA deficiency, possibly through excessive N-acetyl aspartic acid accumulation, elicits both a peripheral and CNS immune response in CD mice. Our data establish a role for peripheral tissues in CD pathology and serve to aid the development of more efficacious and sustained gene therapy for this disease.

Claudin-11 Tight Junctions in Myelin Are a Barrier to Diffusion and Lack Strong Adhesive Properties.

The radial component is a network of interlamellar tight junctions (TJs) unique to central nervous system myelin. Ablation of claudin-11, a TJ protein, results in the absence of the radial component and compromises the passive electrical properties of myelin. Although TJs are known to regulate paracellular diffusion, this barrier function has not been directly demonstrated for the radial component, and some evidence suggests that the radial component may also mediate adhesion between myelin membranes. To investigate the physical properties of claudin-11 TJs, we compared fresh, unfixed Claudin 11-null and control nerves using x-ray and neutron diffraction. In Claudin 11-null tissue, we detected no changes in myelin structure, stability, or membrane interactions, which argues against the notion that myelin TJs exhibit significant adhesive properties. Moreover, our osmotic stressing and D2O-H2O exchange experiments demonstrate that myelin lacking claudin-11 is more permeable to water and small osmolytes. Thus, our data indicate that the radial component serves primarily as a diffusion barrier and elucidate the mechanism by which TJs govern myelin function.

Neutron scattering from myelin revisited: bilayer asymmetry and water-exchange kinetics.

Rapid nerve conduction in the central and peripheral nervous systems (CNS and PNS, respectively) of higher vertebrates is brought about by the ensheathment of axons with myelin, a lipid-rich, multilamellar assembly of membranes. The ability of myelin to electrically insulate depends on the regular stacking of these plasma membranes and on the presence of a number of specialized membrane-protein assemblies in the sheath, including the radial component, Schmidt-Lanterman incisures and the axo-glial junctions of the paranodal loops. The disruption of this fine-structure is the basis for many demyelinating neuropathies in the CNS and PNS. Understanding the processes that govern myelin biogenesis, maintenance and destabilization requires knowledge of myelin structure; however, the tight packing of internodal myelin and the complexity of its junctional specializations make myelin a challenging target for comprehensive structural analysis. This paper describes an examination of myelin from the CNS and PNS using neutron diffraction. This investigation revealed the dimensions of the bilayers and aqueous spaces of myelin, asymmetry between the cytoplasmic and extracellular leaflets of the membrane, and the distribution of water and exchangeable hydrogen in internodal multilamellar myelin. It also uncovered differences between CNS and PNS myelin in their water-exchange kinetics.

A single intravenous rAAV injection as late as P20 achieves efficacious and sustained CNS Gene therapy in Canavan mice.

Canavan's disease (CD) is a fatal pediatric leukodystrophy caused by mutations in aspartoacylase (AspA) gene. Currently, there is no effective treatment for CD; however, gene therapy is an attractive approach to ameliorate the disease. Here, we studied progressive neuropathology and gene therapy in short-lived (≤ 1 month) AspA(-/-) mice, a bona-fide animal model for the severest form of CD. Single intravenous (IV) injections of several primate-derived recombinant adeno-associated viruses (rAAVs) as late as postnatal day 20 (P20) completely rescued their early lethality and alleviated the major disease symptoms, extending survival in P0-injected rAAV9 and rAAVrh8 groups to as long as 2 years thus far. We successfully used microRNA (miRNA)-mediated post-transcriptional detargeting for the first time to restrict therapeutic rAAV expression in the central nervous system (CNS) and minimize potentially deleterious effects of transgene overexpression in peripheral tissues. rAAV treatment globally improved CNS myelination, although some abnormalities persisted in the content and distribution of myelin-specific and -enriched lipids. We demonstrate that systemically delivered and CNS-restricted rAAVs can serve as efficacious and sustained gene therapeutics in a model of a severe neurodegenerative disorder even when administered as late as P20.

Curcumin derivatives promote Schwann cell differentiation and improve neuropathy in R98C CMT1B mice.

Charcot-Marie-Tooth disease type 1B is caused by mutations in myelin protein zero. R98C mice, an authentic model of early onset Charcot-Marie-Tooth disease type 1B, develop neuropathy in part because the misfolded mutant myelin protein zero is retained in the endoplasmic reticulum where it activates the unfolded protein response. Because oral curcumin, a component of the spice turmeric, has been shown to relieve endoplasmic reticulum stress and decrease the activation of the unfolded protein response, we treated R98C mutant mice with daily gastric lavage of curcumin or curcumin derivatives starting at 4 days of age and analysed them for clinical disability, electrophysiological parameters and peripheral nerve morphology. Heterozygous R98C mice treated with curcumin dissolved in sesame oil or phosphatidylcholine curcumin performed as well as wild-type littermates on a rotarod test and had increased numbers of large-diameter axons in their sciatic nerves. Treatment with the latter two compounds also increased compound muscle action potential amplitudes and the innervation of neuromuscular junctions in both heterozygous and homozygous R98C animals, but it did not improve nerve conduction velocity, myelin thickness, G-ratios or myelin period. The expression of c-Jun and suppressed cAMP-inducible POU (SCIP)-transcription factors that inhibit myelination when overexpressed-was also decreased by treatment. Consistent with its role in reducing endoplasmic reticulum stress, treatment with curcumin dissolved in sesame oil or phosphatidylcholine curcumin was associated with decreased X-box binding protein (XBP1) splicing. Taken together, these data demonstrate that treatment with curcumin dissolved in sesame oil or phosphatidylcholine curcumin improves the peripheral neuropathy of R98C mice by alleviating endoplasmic reticulum stress, by reducing the activation of unfolded protein response and by promoting Schwann cell differentiation.

SCAP is required for timely and proper myelin membrane synthesis.

Myelination requires a massive increase in glial cell membrane synthesis. Here, we demonstrate that the acute phase of myelin lipid synthesis is regulated by sterol regulatory element-binding protein (SREBP) cleavage activation protein (SCAP), an activator of SREBPs. Deletion of SCAP in Schwann cells led to a loss of SREBP-mediated gene expression involving cholesterol and fatty acid synthesis. Schwann cell SCAP mutant mice show congenital hypomyelination and abnormal gait. Interestingly, aging SCAP mutant mice showed partial regain of function; they exhibited improved gait and produced small amounts of myelin indicating a slow SCAP-independent uptake of external lipids. Accordingly, extracellular lipoproteins partially rescued myelination by SCAP mutant Schwann cells. However, SCAP mutant myelin never reached normal thickness and had biophysical abnormalities concordant with abnormal lipid composition. These data demonstrate that SCAP-mediated regulation of glial lipogenesis is key to the proper synthesis of myelin membrane, and provide insight into abnormal Schwann cell function under conditions affecting lipid metabolism.

Metabolically-incorporated deuterium in myelin localized by neutron diffraction and identified by mass spectrometry.

Myelin is a natural and dynamic multilamellar membrane structure that continues to be of significant biological and neurological interest, especially with respect to its biosynthesis and assembly during its normal formation, maintenance, and pathological breakdown. To explore the usefulness of neutron diffraction in the structural analysis of myelin, we investigated the use of in vivo labeling by metabolically incorporating non-toxic levels of deuterium (2H; D) via drinking water into a pregnant dam (D-dam) and her developing embryos. All of the mice were sacrificed when the pups (D-pups) were 55 days old. Myelinated sciatic nerves were dissected, fixed in glutaraldehyde and examined by neutron diffraction. Parallel samples that were unfixed (trigeminal nerves) were frozen for mass spectrometry (MS). The diffraction patterns of the nerves from deuterium-fed mice (D-mice) vs. the controls (H-mice) had major differences in the intensities of the Bragg peaks but no appreciable differences in myelin periodicity. Neutron scattering density profiles showed an appreciable increase in density at the center of the lipid-rich membrane bilayer. This increase was greater in D-pups than in D-dam, and its localization was consistent with deuteration of lipid hydrocarbon, which predominates over transmembrane protein in myelin. MS analysis of the lipids isolated from the trigeminal nerves demonstrated that in the pups the percentage of lipids that had one or more deuterium atoms was uniformly high across lipid species (97.6% â€‹± â€‹2.0%), whereas in the mother the lipids were substantially less deuterated (60.6% â€‹± â€‹26.4%) with levels varying among lipid species and subspecies. The mass distribution pattern of deuterium-containing isotopologues indicated the fraction (in %) of each lipid (sub-)species having one or more deuteriums incorporated: in the D-pups, the pattern was always bell-shaped, and the average number of D atoms ranged from a low of ∼4 in fatty acid to a high of ∼9 in cerebroside. By contrast, in D-dam most lipids had more complex, overlapping distributions that were weighted toward a lower average number of deuteriums, which ranged from a low of ∼3-4 in fatty acid and in one species of sulfatide to a high of 6-7 in cerebroside and sphingomyelin. The consistently high level of deuteration in D-pups can be attributed to their de novo lipogenesis during gestation and rapid, postnatal myelination. The widely varying levels of deuteration in D-dam, by contrast, likely depends on the relative metabolic stability of the particular lipid species during myelin maintenance. Our current findings demonstrate that stably-incorporated D label can be detected and localized using neutron diffraction in a complex tissue such as myelin; and moreover, that MS can be used to screen a broad range of deuterated lipid species to monitor differential rates of lipid turnover. In addition to helping to develop a comprehensive understanding of the de novo synthesis and turnover of specific lipids in normal and abnormal myelin, our results also suggest application to studies on myelin proteins (which constitute only 20-30% by dry mass of the myelin, vs. 70-80% for lipid), as well as more broadly to the molecular constituents of other biological tissues.

Lack of collagen XV impairs peripheral nerve maturation and, when combined with laminin-411 deficiency, leads to basement membrane abnormalities and sensorimotor dysfunction.

Although the Schwann cell basement membrane (BM) is required for normal Schwann cell terminal differentiation, the role of BM-associated collagens in peripheral nerve maturation is poorly understood. Collagen XV is a BM zone component strongly expressed in peripheral nerves, and we show that its absence in mice leads to loosely packed axons in C-fibers and polyaxonal myelination. The simultaneous lack of collagen XV and another peripheral nerve component affecting myelination, laminin α4, leads to severely impaired radial sorting and myelination, and the maturation of the nerve is permanently compromised, contrasting with the slow repair observed in Lama4-/- single knock-out mice. Moreover, the Col15a1-/-;Lama4-/- double knock-out (DKO) mice initially lack C-fibers and, even over 1 year of age have only a few, abnormal C-fibers. The Lama4-/- knock-out results in motor and tactile sensory impairment, which is exacerbated by a simultaneous Col15a1-/- knock-out, whereas sensitivity to heat-induced pain is increased in the DKO mice. Lack of collagen XV results in slower sensory nerve conduction, whereas the Lama4-/- and DKO mice exhibit increased sensory nerve action potentials and decreased compound muscle action potentials; x-ray diffraction revealed less mature myelin in the sciatic nerves of the latter than in controls. Ultrastructural analyses revealed changes in the Schwann cell BM in all three mutants, ranging from severe (DKO) to nearly normal (Col15a1-/-). Collagen XV thus contributes to peripheral nerve maturation and C-fiber formation, and its simultaneous deletion from neural BM zones with laminin α4 leads to a DKO phenotype distinct from those of both single knock-outs.

P0 (protein zero) mutation S34C underlies instability of internodal myelin in S63C mice.

P0 constitutes 50-60% of protein in peripheral nerve myelin and is essential for its structure and stability. Mutations within the P0 gene (MPZ) underlie a variety of hereditary neuropathies. MpzS63C transgenic mice encode a P0 with a serine to cysteine substitution at position 34 in the extracellular domain of mature P0 (P0S34C), associated with the hypomyelinating Déjérine-Sottas syndrome in human. S63C mice develop a dysmyelinating neuropathy, with packing defects in peripheral myelin. Here, we used x-ray diffraction to examine time-dependent packing defects in unfixed myelin. At ∼7 h post-dissection, WT and S63C(+/+) myelin showed native periods (175 Å) with the latter developing at most a few percent swollen myelin, whereas up to ∼50% of S63C(+/-) (mutant P0 on heterozygous P0 null background) or P0(+/-) myelin swelled to periods of ∼205 Å. In the same time frame, S63C(-/-) myelin was stable, remaining swollen at ∼210 Å. Surprisingly, treatment of whole S63C(-/-) nerves with a reducing agent completely reverted swollen arrays to native spacing and also normalized the swollen arrays that had formed in S63C(+/-) myelin, the genotype most closely related to the human disorder. Western blot revealed P0-positive bands at ∼27 and ∼50 kDa, and MALDI-TOF mass spectrometry showed these bands consisted of Ser(34)-containing peptides or P0 dimers having oxidized Cys(34) residues. We propose that P0S34C forms ectopic disulfide bonds in trans between apposed Cys(34) side chains that retard wrapping during myelin formation causing hypomyelination. Moreover, the new bonds create a packing defect by stabilizing swollen membrane arrays that leads to demyelination.

Evolution of a neuroprotective function of central nervous system myelin.

The central nervous system (CNS) of terrestrial vertebrates underwent a prominent molecular change when a tetraspan membrane protein, myelin proteolipid protein (PLP), replaced the type I integral membrane protein, P0, as the major protein of myelin. To investigate possible reasons for this molecular switch, we genetically engineered mice to express P0 instead of PLP in CNS myelin. In the absence of PLP, the ancestral P0 provided a periodicity to mouse compact CNS myelin that was identical to mouse PNS myelin, where P0 is the major structural protein today. The PLP-P0 shift resulted in reduced myelin internode length, degeneration of myelinated axons, severe neurological disability, and a 50% reduction in lifespan. Mice with equal amounts of P0 and PLP in CNS myelin had a normal lifespan and no axonal degeneration. These data support the hypothesis that the P0-PLP shift during vertebrate evolution provided a vital neuroprotective function to myelin-forming CNS glia.

Differential effects of Phe19 and Phe20 on fibril formation by amyloidogenic peptide A beta 16-22 (Ac-KLVFFAE-NH2).

The sequence KLVFFAE (A beta 16-22) in Alzheimer's beta-amyloid is thought to be a core beta-structure that could act as a template for folding other parts of the polypeptide or molecules into fibrillar assemblies rich in beta-sheet. To elucidate the mechanism of the initial folding process, we undertook combined X-ray fiber/powder diffraction and infrared (IR) spectroscopy to analyze lyophilized A beta 16-22 and solubilized/dried peptide containing nitrile probes at F19 and/or F20. Solubilized/dried wild-type (WT) A beta 16-22 and the peptide containing cyanophenylalanine at F19 (19CN) or at F20 (20CN) gave fiber patterns consistent with slab-like beta-crystallites that were cylindrically averaged around the axis parallel to the polypeptide chain direction. The WT and 19CN assemblies showed 30-A period arrays arising from the stacking of the slabs along the peptide chain direction, whereas the 20CN assemblies lacked any such stacking. The electron density projection along the peptide chain direction indicated similar side-chain dispositions for WT and 20CN, but not for 19CN. These X-ray results and modeling imply that in the assembly of WT A beta 16-22 the F19 side chain is localized within the intersheet space and is involved in hydrophobic contact with amino acids across the intersheet space, whereas the F20 side chain localized near the slab surface is less important for the intersheet interaction, but involved in slab stacking. IR observations for the same peptides in dilute solution showed a greater degree of hydrogen bonding for the nitrile groups in 20CN than in 19CN, supporting this interpretation.

Rapid assessment of internodal myelin integrity in central nervous system tissue.

Monitoring pathology/regeneration in experimental models of de-/remyelination requires an accurate measure not only of functional changes but also of the amount of myelin. We tested whether X-ray diffraction (XRD), which measures periodicity in unfixed myelin, can assess the structural integrity of myelin in fixed tissue. From laboratories involved in spinal cord injury research and in studying the aging primate brain, we solicited "blind" samples and used an electronic detector to record rapidly the diffraction patterns (30 min each pattern) from them. We assessed myelin integrity by measuring its periodicity and relative amount. Fixation of tissue itself introduced +/-10% variation in periodicity and +/-40% variation in relative amount of myelin. For samples having the most native-like periods, the relative amounts of myelin detected allowed distinctions to be made between normal and demyelinating segments, between motor and sensory tracts within the spinal cord, and between aged and young primate CNS. Different periodicities also allowed distinctions to be made between samples from spinal cord and nerve roots and between well-fixed and poorly fixed samples. Our findings suggest that, in addition to evaluating the effectiveness of different fixatives, XRD could also be used as a robust and rapid technique for quantitating the relative amount of myelin among spinal cords and other CNS tissue samples from experimental models of de- and remyelination.

Exploiting Sphingo- and Glycerophospholipid Impairment to Select Effective Drugs and Biomarkers for CMT1A.

In Charcot-Marie-Tooth type 1A (CMT1A), Schwann cells exhibit a preponderant transcriptional deficiency of genes involved in lipid biosynthesis. This perturbed lipid metabolism affects the peripheral nerve physiology and the structure of peripheral myelin. Nevertheless, the identification and functional characterization of the lipid species mainly responsible for CMT1A myelin impairment currently lack. This is critical in the pathogenesis of the neuropathy since lipids are many and complex molecules which play essential roles in the cell, including the structural components of cellular membranes, cell signaling, and membrane trafficking. Moreover, lipids themselves are able to modify gene transcription, thereby affecting the genotype-phenotype correlation of well-defined inherited diseases, including CMT1A. Here we report for the first time a comprehensive lipid profiling in experimental and human CMT1A, demonstrating a previously unknown specific alteration of sphingolipid (SP) and glycerophospholipid (GP) metabolism. Notably, SP, and GP changes even emerge in biological fluids of CMT1A rat and human patients, implying a systemic metabolic dysfunction for these specific lipid classes. Actually, SP and GP are not merely reduced; their expression is instead aberrant, contributing to the ultrastructural abnormalities that we detailed by X-ray diffraction in rat and human internode myelin. The modulation of SP and GP pathways in myelinating dorsal root ganglia cultures clearly sustains this issue. In fact, just selected molecules interacting with these pathways are able to modify the altered geometric parameters of CMT1A myelinated fibers. Overall, we propose to exploit the present SP and GP metabolism impairment to select effective drugs and validate a set of reliable biomarkers, which remain a challenge in CMT1A neuropathy.

Constitutively active Akt induces enhanced myelination in the CNS.

The serine/threonine kinase Akt regulates multiple cellular functions. The current studies identify a new role for Akt in CNS myelination. In earlier studies on cultured oligodendrocytes, we showed that neuregulin signals through phosphatidylinositol-3'-OH kinase and Akt to enhance survival of oligodendrocytes. However, when transgenic animals were generated that overexpressed constitutively active Akt in oligodendrocytes and their progenitor cells, no enhanced survival of oligodendrocytes or progenitors was found. No alteration in the proliferation or death of progenitors was noted. In contrast, the major impact of Akt overexpression in oligodendrocytes was enhanced myelination. Most interestingly, oligodendrocytes in these mice continued actively myelinating throughout life. Thus, expression of constitutively active Akt in oligodendrocytes and their progenitor cells generated no more oligodendrocytes, but dramatically more myelin. The increased myelination continued as these mice aged, resulting in enlarged optic nerves and white matter areas. In older animals with enlarged white matter areas, the density of oligodendrocytes was reduced, but because of the increased area, the total number of oligodendrocytes remained comparable with wild-type controls. Interestingly, in these animals, overexpression of Akt in Schwann cells did not impact myelination. Thus, in vivo, constitutively active Akt enhances CNS myelination but not PNS myelination and has no impact developmentally on oligodendrocyte number. Understanding the unique aspects of Akt signal transduction in oligodendrocytes that lead to myelination rather than uncontrolled proliferation of oligodendrocyte progenitor cells may have important implications for understanding remyelination in the adult nervous system.

Internodal myelination during development quantitated using X-ray diffraction.

Characterizing the formation, accretion, and stability of myelin during development, maturation, and senescence is important for better understanding critical periods in the function of the nervous system in normal growth and following environmental insult or genetic mutation. Although there are numerous studies on the ultrastructural, biochemical, and genetic aspects of myelin development and maturation, few have used X-ray diffraction (XRD), which can rapidly provide unique metrics about internodal myelin based on measurements from whole, unfixed tissue. Besides periodicity (the classic attribute of internodal myelin measured by XRD), other parameters include: relative amount of myelin, membrane dimensions, and packing disorder. To provide a baseline for future experiments on myelin structural integrity, we used XRD to characterize internodal myelin as a function of age (from 5 to 495 days) in the mouse, a species increasingly used for developing transgenic models of human neurological diseases. As expected, the relative amount of myelin increased with age in both PNS and CNS, with the most rapid accumulation occurring in the youngest age group. Changes in rate of myelin accretion yielded three distinct age brackets during which small but significant changes in structural parameters were detected: in PNS, myelin period increased, packing distortion decreased, width of extracellular apposition (EXT) decreased, and widths of cytoplasmic apposition (CYT) and lipid bilayer (LPG) increased; in CNS, myelin period decreased, packing distortion decreased, EXT and CYT decreased, and LPG increased. We propose that the data obtained here can serve as a basis for rapidly detecting abnormal pathologies during myelination.

Simultaneous efflux of endogenous D-ser and L-glu from single acute hippocampus slices during oxygen glucose deprivation.

D-serine and L-glutamate play crucial roles in excitotoxicity through N-methyl-D-aspartate receptor coactivation, but little is known about the temporal profile of efflux during cerebral ischemia. We utilized a newly designed brain slice microperfusion device coupled offline to capillary electrophoresis laser-induced fluorescence to monitor dynamic efflux of endogenous D-ser and L-glu in response to oxygen glucose deprivation (OGD) in single acute hippocampus slices. Efflux profiles with 2-min temporal resolution in response to 24-min OGD show that efflux of D-ser slightly precedes efflux of L-glu by one 2-min sampling interval. Thus both coagonists are available to activate NMDA receptors by the time when glu is released. The magnitude of D-ser efflux relative to baseline values is, however, less than that for L-glu. Peak efflux during OGD, expressed as pre-OGD baseline values, was as follows: D-ser 254% +/- 24%, L-glu 1,675% +/- 259%, L-asp 519% +/- 128%, and L-thr 313% +/- 33%. L-glutamine efflux was shown to decrease significantly in response to OGD. The microperfusion/CE-LIF approach shows several promising attributes for studying endogenous chemical efflux from single, acute brain slices.

Separation and sensitive detection of D-amino acids in biological matrices.

The increase in our understanding of D-amino acid function and distribution in mammals is in many ways a result of the initial development of sensitive enantioselective separation strategies that allow for quantification in real biological samples. This article reviews progress on the development of chiral selective separation and detection of D-amino acids including enzyme-based microbiosensors, GC/MS, HPLC/fluorescence, HPLC/MS-MS, cEKC/fluorescence, and MEKC/fluorescence. Only methods capable of analyzing D-isomers in biological matrices are given here and significant effort is made to highlight approaches that offer speed, resolution, high sensitivity, and versatility.

The Amazon rain forest plant Uncaria tomentosa (cat's claw) and its specific proanthocyanidin constituents are potent inhibitors and reducers of both brain plaques and tangles.

Brain aging and Alzheimer's disease both demonstrate the accumulation of beta-amyloid protein containing "plaques" and tau protein containing "tangles" that contribute to accelerated memory loss and cognitive decline. In the present investigation we identified a specific plant extract and its constituents as a potential alternative natural solution for preventing and reducing both brain "plaques and tangles". PTI-00703 cat's claw (Uncaria tomentosa from a specific Peruvian source), a specific and natural plant extract from the Amazon rain forest, was identified as a potent inhibitor and reducer of both beta-amyloid fibrils (the main component of "plaques") and tau protein paired helical filaments/fibrils (the main component of "tangles"). PTI-00703 cat's claw demonstrated both the ability to prevent formation/aggregation and disaggregate preformed Aß fibrils (1-42 and 1-40) and tau protein tangles/filaments. The disaggregation/dissolution of Aß fibrils occurred nearly instantly when PTI-00703 cat's claw and Aß fibrils were mixed together as shown by a variety of methods including Thioflavin T fluorometry, Congo red staining, Thioflavin S fluorescence and electron microscopy. Sophisticated structural elucidation studies identified the major fractions and specific constituents within PTI-00703 cat's claw responsible for both the observed "plaque" and "tangle" inhibitory and reducing activity. Specific proanthocyanidins (i.e. epicatechin dimers and variants thereof) are newly identified polyphenolic components within Uncaria tomentosa that possess both "plaque and tangle" reducing and inhibitory activity. One major identified specific polyphenol within PTI-00703 cat's claw was epicatechin-4ß-8-epicatechin (i.e. an epicatechin dimer known as proanthocyanidin B2) that markedly reduced brain plaque load and improved short-term memory in younger and older APP "plaque-producing" (TASD-41) transgenic mice (bearing London and Swedish mutations). Proanthocyanidin B2 was also a potent inhibitor of brain inflammation as shown by reduction in astrocytosis and gliosis in TASD-41 transgenic mice. Blood-brain-barrier studies in Sprague-Dawley rats and CD-1 mice indicated that the major components of PTI-00703 cat's claw crossed the blood-brain-barrier and entered the brain parenchyma within 2 minutes of being in the blood. The discovery of a natural plant extract from the Amazon rain forest plant (i.e. Uncaria tomentosa or cat's claw) as both a potent "plaque and tangle" inhibitor and disaggregator is postulated to represent a potential breakthrough for the natural treatment of both normal brain aging and Alzheimer's disease.