d-Amino acids: new clinical pathways for brain diseases.

Free d-amino acids (d-AAs) are emerging as a novel and important class of signaling molecules in many organs, including the brain and endocrine systems. There has been considerable progress in our understanding of the fundamental roles of these atypical messengers, with increasingly recognized implications in a wide range of neuropathologies, including schizophrenia (SCZ), epilepsy, Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), substance abuse, and chronic pain, among others. Research has enabled the discovery that d-serine, d-aspartate and more recently d-cysteine are essential for the healthy development and function of the central nervous system (CNS). We discuss recent progress that has profoundly transformed our vision of numerous physiological processes but has also shown how d-AAs are now offering therapeutic promise in clinical settings for several human diseases.

Serine Racemase mediates subventricular zone neurogenesis via fatty acid metabolism.

The adult subventricular zone (SVZ) is a neurogenic niche that continuously produces newborn neurons. Here we show that serine racemase (SR), an enzyme that catalyzes the racemization of L-serine to D-serine and vice versa, affects neurogenesis in the adult SVZ by controlling de novo fatty acid synthesis. Germline and conditional deletion of SR (nestin precursor cells) leads to diminished neurogenesis in the SVZ. Nestin-cre+ mice showed reduced expression of fatty acid synthase and its substrate malonyl-CoA, which are involved in de novo fatty acid synthesis. Global lipidomic analyses revealed significant alterations in different lipid subclasses in nestin-cre+ mice. Decrease in fatty acid synthesis was mediated by phospho Acetyl-CoA Carboxylase that was AMP-activated protein kinase independent. Both L- and D-serine supplementation rescued defects in SVZ neurogenesis, proliferation, and levels of malonyl-CoA in vitro. Our work shows that SR affects adult neurogenesis in the SVZ via lipid metabolism.

Mammalian D-Cysteine: A new addition to the growing family of biologically relevant D-amino acids.

Mammalian D-Cysteine is racemized from L-cysteine by serine racemase, a pyridoxal phosphate (PLP)-dependent enzyme. Endogenous D-Cysteine plays a role in neural development by inhibiting proliferation of neural progenitor cells (NPCs) via protein kinase B (AKT) signaling mediated by the FoxO family of transcription factors. D-Cysteine binds to Myristoylated Alanine Rich C Kinase Substrate (MARCKS) and alters phosphorylation at Ser 159/163 and its translocation from the membrane. By racemizing serine and cysteine, mammalian serine racemase may play important roles in neural development highlighting its importance in psychiatric disorders.

A Novel Stereospecific Bioluminescent Assay for Detection of Endogenous d-Cysteine.

The presence of endogenous d-stereoisomers of amino acids in mammals dispels a long-standing dogma about their existence. d-Serine and d-aspartate function as novel neurotransmitters in mammals. However, the stereoisomer with the fastest, spontaneous in vitro racemization rate, d-cysteine, has not been reported. We utilized a novel, stereospecific, bioluminescent assay to identify endogenous d-cysteine in substantial amounts in the eye, brain, and pancreas of mice. d-Cysteine is enriched in mice embryonic brains at day E9.5 (4.5 mM) and decreases progressively with development (µM levels). d-Cysteine is also present in significantly higher amounts in the human brain white matter compared with gray matter. In the luciferase assay, d-cysteine conjugates with cyano hydroxy benzothiazole in the presence of a base and reducing agent to form d-luciferin. d-Luciferin, subsequently, in the presence of firefly luciferase and ATP, emits bioluminescence proportional to the concentration of d-cysteine. The assay is stereospecific and allows the quantitative estimation of endogenous d-cysteine in tissues in addition to its specificity for d-cysteine. Future efforts aimed at bioluminescent in vivo imaging of d-cysteine may allow a more noninvasive means of its detection, thereby elucidating its function.

Mammalian D-cysteine: A novel regulator of neural progenitor cell proliferation Mammalian D-cysteine: A novel regulator of neural progenitor cell proliferation Endogenous D-cysteine, the stereoisomer with rapid spontaneous in vitro racemization rate, has major neural roles

D-amino acids are being recognized as functionally important molecules in mammals. We recently identified endogenous D-cysteine in mammalian brain. D-cysteine is present in neonatal brain in substantial amounts (mM) and decreases with postnatal development. D-cysteine binds to MARCKS and a host of proteins implicated in cell division and neurodevelopmental disorders. D-cysteine decreases phosphorylation of MARCKS in neural progenitor cells (NPCs) affecting its translocation. D-cysteine controls NPC proliferation by inhibiting AKT signaling. Exogenous D-cysteine inhibits AKT phosphorylation at Thr 308 and Ser 473 in NPCs. D-cysteine treatment of NPCs led to 50% reduction in phosphorylation of Foxo1 at Ser 256 and Foxo3a at Ser 253. We hypothesize that in the developing brain endogenous D-cysteine is as a physiologic regulator of NPC proliferation by inhibiting AKT signaling mediated by Foxo1 and Foxo3a. Endogenous D-cysteine may regulate mammalian neurodevelopment with roles in schizophrenia and Alzheimer's disease (AD).

D-cysteine is an endogenous regulator of neural progenitor cell dynamics in the mammalian brain.

d-amino acids are increasingly recognized as important signaling molecules in the mammalian central nervous system. However, the d-stereoisomer of the amino acid with the fastest spontaneous racemization ratein vitro in vitro, cysteine, has not been examined in mammals. Using chiral high-performance liquid chromatography and a stereospecific luciferase assay, we identify endogenous d-cysteine in the mammalian brain. We identify serine racemase (SR), which generates the N-methyl-d-aspartate (NMDA) glutamate receptor coagonist d-serine, as a candidate biosynthetic enzyme for d-cysteine. d-cysteine is enriched more than 20-fold in the embryonic mouse brain compared with the adult brain. d-cysteine reduces the proliferation of cultured mouse embryonic neural progenitor cells (NPCs) by ∼50%, effects not shared with d-serine or l-cysteine. The antiproliferative effect of d-cysteine is mediated by the transcription factors FoxO1 and FoxO3a. The selective influence of d-cysteine on NPC proliferation is reflected in overgrowth and aberrant lamination of the cerebral cortex in neonatal SR knockout mice. Finally, we perform an unbiased screen for d-cysteine-binding proteins in NPCs by immunoprecipitation with a d-cysteine-specific antibody followed by mass spectrometry. This approach identifies myristoylated alanine-rich C-kinase substrate (MARCKS) as a putative d-cysteine-binding protein. Together, these results establish endogenous mammalian d-cysteine and implicate it as a physiologic regulator of NPC homeostasis in the developing brain.

A Critical Role of Ser26 Hydrogen Bonding in Aß42 Assembly and Toxicity.

Amyloid ß-protein (Aß) assembly is a seminal process in Alzheimer's disease. Elucidating the mechanistic features of this process is thought to be vital for the design and targeting of therapeutic agents. Computational studies of the most pathologic form of Aß, the 42-residue Aß42 peptide, have suggested that hydrogen bonding involving Ser26 may be particularly important in organizing a monomer folding nucleus and in subsequent peptide assembly. To study this question, we experimentally determined structure-activity relationships among Aß42 peptides in which Ser26 was replaced with Gly, Ala, α-aminobutryic acid (Abu), or Cys. We observed that aliphatic substitutions (Ala and Abu) produced substantially increased rates of formation of ß-sheet, hydrophobic surface, and fibrils, and higher levels of cellular toxicity. Replacement of the Ser hydroxyl group with a sulfhydryl moiety (Cys) did not have these effects. Instead, this peptide behaved like native Aß42, even though the hydropathy of Cys was similar to that of Abu and very different from that of Ser. We conclude that H bonding of Ser26 is the factor most important in its contribution to Aß42 conformation, assembly, and subsequent toxicity.

DISC1 in Astrocytes Influences Adult Neurogenesis and Hippocampus-Dependent Behaviors in Mice.

The functional role of genetic variants in glia in the pathogenesis of psychiatric disorders remains poorly studied. Disrupted-In-Schizophrenia 1 (DISC1), a genetic risk factor implicated in major mental disorders, has been implicated in regulation of astrocyte functions. As both astrocytes and DISC1 influence adult neurogenesis in the dentate gyrus (DG) of the hippocampus, we hypothesized that selective expression of dominant-negative C-terminus-truncated human DISC1 (mutant DISC1) in astrocytes would affect adult hippocampal neurogenesis and hippocampus-dependent behaviors. A series of behavioral tests were performed in mice with or without expression of mutant DISC1 in astrocytes during late postnatal development. In conjunction with behavioral tests, we evaluated adult neurogenesis, including neural progenitor proliferation and dendrite development of newborn neurons in the DG. The ameliorative effects of D-serine on mutant DISC1-associated behaviors and abnormal adult neurogenesis were also examined. Expression of mutant DISC1 in astrocytes decreased neural progenitor proliferation and dendrite growth of newborn neurons, and produced elevated anxiety, attenuated social behaviors, and impaired hippocampus-dependent learning and memory. Chronic treatment with D-serine ameliorated the behavioral alterations and rescued abnormal adult neurogenesis in mutant DISC1 mice. Our findings suggest that psychiatric genetic risk factors expressed in astrocytes could affect adult hippocampal neurogenesis and contribute to aspects of psychiatric disease through abnormal production of D-serine.

Reducing synuclein accumulation improves neuronal survival after spinal cord injury.

Spinal cord injury causes neuronal death, limiting subsequent regeneration and recovery. Thus, there is a need to develop strategies for improving neuronal survival after injury. Relative to our understanding of axon regeneration, comparatively little is known about the mechanisms that promote the survival of damaged neurons. To address this, we took advantage of lamprey giant reticulospinal neurons whose large size permits detailed examination of post-injury molecular responses at the level of individual, identified cells. We report here that spinal cord injury caused a select subset of giant reticulospinal neurons to accumulate synuclein, a synaptic vesicle-associated protein best known for its atypical aggregation and causal role in neurodegeneration in Parkinson's and other diseases. Post-injury synuclein accumulation took the form of punctate aggregates throughout the somata and occurred selectively in dying neurons, but not in those that survived. In contrast, another synaptic vesicle protein, synaptotagmin, did not accumulate in response to injury. We further show that the post-injury synuclein accumulation was greatly attenuated after single dose application of either the "molecular tweezer" inhibitor, CLR01, or a translation-blocking synuclein morpholino. Consequently, reduction of synuclein accumulation not only improved neuronal survival, but also increased the number of axons in the spinal cord proximal and distal to the lesion. This study is the first to reveal that reducing synuclein accumulation is a novel strategy for improving neuronal survival after spinal cord injury.

Role of Species-Specific Primary Structure Differences in Aß42 Assembly and Neurotoxicity.

A variety of species express the amyloid ß-protein (Aß (the term "Aß" refers both to Aß40 and Aß42, whereas "Aß40" and "Aß42" refer to each isoform specifically). Those species expressing Aß with primary structure identical to that expressed in humans have been found to develop amyloid deposits and Alzheimer's disease-like neuropathology. In contrast, the Aß sequence in mice and rats contains three amino acid substitutions, Arg5Gly, His13Arg, and Tyr10Phe, which apparently prevent the development of AD-like neuropathology. Interestingly, the brush-tailed rat, Octodon degus, expresses Aß containing only one of these substitutions, His13Arg, and does develop AD-like pathology. We investigate here the biophysical and biological properties of Aß peptides from humans, mice (Mus musculus), and rats (Octodon degus). We find that each peptide displays statistical coil → ß-sheet secondary structure transitions, transitory formation of hydrophobic surfaces, oligomerization, formation of annuli, protofibrils, and fibrils, and an inverse correlation between rate of aggregation and aggregate size (faster aggregation produced smaller aggregates). The rank order of assembly rate was mouse > rat > Aß42. The rank order of neurotoxicity of assemblies formed by each peptide immediately after preparation was Aß42 > mouse ≈ rat. These data do not support long-standing hypotheses that the primary factor controlling development of AD-like neuropathology in rodents is Aß sequence. Instead, the data support a hypothesis that assembly quaternary structure and organismal responses to toxic peptide assemblies mediate neuropathogenetic effects. The implication of this hypothesis is that a valid understanding of disease causation within a given system (organism, tissue, etc.) requires the coevaluation of both biophysical and cell biological properties of that system.

Amyloid ß-Protein Assembly: Differential Effects of the Protective A2T Mutation and Recessive A2V Familial Alzheimer's Disease Mutation.

Oligomeric states of the amyloid ß-protein (Aß) appear to be causally related to Alzheimer's disease (AD). Recently, two familial mutations in the amyloid precursor protein gene have been described, both resulting in amino acid substitutions at Ala2 (A2) within Aß. An A2V mutation causes autosomal recessive early onset AD. Interestingly, heterozygotes enjoy some protection against development of the disease. An A2T substitution protects against AD and age-related cognitive decline in non-AD patients. Here, we use ion mobility-mass spectrometry (IM-MS) to examine the effects of these mutations on Aß assembly. These studies reveal different assembly pathways for early oligomer formation for each peptide. A2T Aß42 formed dimers, tetramers, and hexamers, but dodecamer formation was inhibited. In contrast, no significant effects on Aß40 assembly were observed. A2V Aß42 also formed dimers, tetramers, and hexamers, but it did not form dodecamers. However, A2V Aß42 formed trimers, unlike A2T or wild-type (wt) Aß42. In addition, the A2V substitution caused Aß40 to oligomerize similar to that of wt Aß42, as evidenced by the formation of dimers, tetramers, hexamers, and dodecamers. In contrast, wt Aß40 formed only dimers and tetramers. These results provide a basis for understanding how these two mutations lead to, or protect against, AD. They also suggest that the Aß N-terminus, in addition to the oft discussed central hydrophobic cluster and C-terminus, can play a key role in controlling disease susceptibility.

ADAM9 is a novel product of polymorphonuclear neutrophils: regulation of expression and contributions to extracellular matrix protein degradation during acute lung injury.

A disintegrin and a metalloproteinase domain (ADAM) 9 is known to be expressed by monocytes and macrophages. In this study, we report that ADAM9 is also a product of human and murine polymorphonuclear neutrophils (PMNs). ADAM9 is not synthesized de novo by circulating PMNs. Rather, ADAM9 protein is stored in the gelatinase and specific granules and the secretory vesicles of human PMNs. Unstimulated PMNs express minimal quantities of surface ADAM9, but activation of PMNs with degranulating agonists rapidly (within 15 min) increases PMN surface ADAM9 levels. Human PMNs produce small quantities of soluble forms of ADAM9. Surprisingly, ADAM9 degrades several extracellular matrix (ECM) proteins, including fibronectin, entactin, laminin, and insoluble elastin, as potently as matrix metalloproteinase-9. However, ADAM9 does not degrade types I, III, or IV collagen or denatured collagens in vitro. To determine whether Adam9 regulates PMN recruitment or ECM protein turnover during inflammatory responses, we compared wild-type and Adam9(-/-) mice in bacterial LPS- and bleomycin-mediated acute lung injury (ALI). Adam9 lung levels increase 10-fold during LPS-mediated ALI in wild-type mice (due to increases in leukocyte-derived Adam9), but Adam9 does not regulate lung PMN (or macrophage) counts during ALI. Adam9 increases mortality, promotes lung injury, reduces lung compliance, and increases degradation of lung elastin during LPS- and/or bleomycin-mediated ALI. Adam9 does not regulate collagen accumulation in the bleomycin-treated lung. Thus, ADAM9 is expressed in an inducible fashion on PMN surfaces where it degrades some ECM proteins, and it promotes alveolar-capillary barrier injury during ALI in mice.

Gly25-Ser26 amyloid ß-protein structural isomorphs produce distinct Aß42 conformational dynamics and assembly characteristics.

One of the earliest events in amyloid ß-protein (Aß) self-association is nucleation of Aß monomer folding through formation of a turn at Gly25-Lys28. We report here the effects of structural changes at the center of the turn, Gly25-Ser26, on Aß42 conformational dynamics and assembly. We used "click peptide" chemistry to quasi-synchronously create Aß42 from 26-O-acyliso-Aß42 (iAß42) through a pH jump from 3 to 7.4. We also synthesized Nα-acetyl-Ser26-iAß42 (Ac-iAß42), which cannot undergo O→N acyl chemistry, to study the behavior of this ester form of Aß42 itself at neutral pH. Data from experiments monitoring increases in ß-sheet formation (thioflavin T, CD), hydrodynamic radius (RH), scattering intensity (quasielastic light scattering spectroscopy), and extent of oligomerization (ion mobility spectroscopy-mass spectrometry) were quite consistent. A rank order of Ac-iAß42>iAß42>Aß42 was observed. Photochemically cross-linked iAß42 displayed an oligomer distribution with a prominent dimer band that was not present with Aß42. These dimers also were observed selectively in iAß42 in ion mobility spectrometry experiments. The distinct biophysical behaviors of iAß42 and Aß42 appear to be due to the conversion of iAß42 into "pure" Aß42 monomer, a nascent form of Aß42 that does not comprise the variety of oligomeric and aggregated states present in pre-existent Aß42. These results emphasize the importance of the Gly25-Ser26 dipeptide in organizing Aß42 monomer structure and thus suggest that drugs altering the interactions of this dipeptide with neighboring side-chain atoms or with the peptide backbone could be useful in therapeutic strategies targeting formation of Aß oligomers and higher-order assemblies.

Mechanism of amyloid ß-protein dimerization determined using single-molecule AFM force spectroscopy.

Aß42 and Aß40 are the two primary alloforms of human amyloid ß-protein (Aß). The two additional C-terminal residues of Aß42 result in elevated neurotoxicity compared with Aß40, but the molecular mechanism underlying this effect remains unclear. Here, we used single-molecule force microscopy to characterize interpeptide interactions for Aß42 and Aß40 and corresponding mutants. We discovered a dramatic difference in the interaction patterns of Aß42 and Aß40 monomers within dimers. Although the sequence difference between the two peptides is at the C-termini, the N-terminal segment plays a key role in the peptide interaction in the dimers. This is an unexpected finding as N-terminal was considered as disordered segment with no effect on the Aß peptide aggregation. These novel properties of Aß proteins suggests that the stabilization of N-terminal interactions is a switch in redirecting of amyloids form the neurotoxic aggregation pathway, opening a novel avenue for the disease preventions and treatments.

C-terminal turn stability determines assembly differences between Aß40 and Aß42.

Oligomerization of the amyloid ß-protein (Aß) is a seminal event in Alzheimer's disease. Aß42, which is only two amino acids longer than Aß40, is particularly pathogenic. Why this is so has not been elucidated fully. We report here results of computational and experimental studies revealing a C-terminal turn at Val36-Gly37 in Aß42 that is not present in Aß40. The dihedral angles of residues 36 and 37 in an Ile31-Ala42 peptide were consistent with ß-turns, and a ß-hairpin-like structure was indeed observed that was stabilized by hydrogen bonds and by hydrophobic interactions between residues 31-35 and residues 38-42. In contrast, Aß(31-40) mainly existed as a statistical coil. To study the system experimentally, we chemically synthesized Aß peptides containing amino acid substitutions designed to stabilize or destabilize the hairpin. The triple substitution Gly33Val-Val36Pro-Gly38Val ("VPV") facilitated Aß42 hexamer and nonamer formation, while inhibiting formation of classical amyloid-type fibrils. These assemblies were as toxic as were assemblies from wild-type Aß42. When substituted into Aß40, the VPV substitution caused the peptide to oligomerize similarly to Aß42. The modified Aß40 was significantly more toxic than Aß40. The double substitution d-Pro36-l-Pro37 abolished hexamer and dodecamer formation by Aß42 and produced an oligomer size distribution similar to that of Aß40. Our data suggest that the Val36-Gly37 turn could be the sine qua non of Aß42. If true, this structure would be an exceptionally important therapeutic target.

Structural dynamics of the amyloid ß-protein monomer folding nucleus.

Alzheimer's disease (AD) is linked to the aberrant assembly of the amyloid ß-protein (Aß). The (21)AEDVGSNKGA(30) segment, Aß(21-30), forms a turn that acts as a monomer folding nucleus. Amino acid substitutions within this nucleus cause familial forms of AD. To determine the biophysical characteristics of the folding nucleus, we studied the biologically relevant acetyl-Aß(21-30)-amide peptide using experimental techniques (limited proteolysis, thermal denaturation, urea denaturation followed by pulse proteolysis, and electron microscopy) and computational methods (molecular dynamics). Our results reveal a highly stable foldon and suggest new strategies for therapeutic drug development.

Stability of the allergenic soybean Kunitz trypsin inhibitor.

The soybean Kunitz trypsin inhibitor (SKTI) is a 21.5 kDa allergenic protein that belongs to the family of all antiparallel beta-sheet proteins that are highly resistant to thermal and chemical denaturation. Spectroscopic and biochemical techniques such as circular dichroism (CD), ANS fluorescence and proteolysis were used to study its molecular structure under denaturing conditions such as acid and heat to which these allergens are commonly exposed during food processing. Reduction of native SKTI leads to its complete and rapid proteolysis by pepsin in simulated gastric fluid (SGF). Limited proteolysis with chymotrypsin during renaturation after heating showed that the native structure reforms at around 60 degrees C reversing the denaturation. CD spectra revealed that under acid denaturing conditions, SKTI shows major changes in conformation, indicating the possibility of a molten structure. The existence of this intermediate was established by ANS fluorescence studies at different concentrations of HCl. The remarkable stability of SKTI to both thermal and acid denaturation may be important for its role as a food allergen.

Reversible denaturation of the soybean Kunitz trypsin inhibitor.

The soybean Kunitz trypsin inhibitor (SKTI) is a beta-sheet protein with unusual stability to chemical and thermal denaturation. Different spectroscopic criteria were used to follow the thermal denaturation and renaturation of SKTI. Upon heating to 70 degrees C, changes in UV difference spectra showed increased absorbance at 292 and 297 nm, attributable to perturbation of aromatic residues. Cooling the protein resulted in restoration of the native spectrum unless reduced with dithiothreitol. Far- and near-UV CD spectra also indicate thermal unfolding involving the core tryptophan and tyrosine residues. Both CD and UV-absorbance data suggest a two-state transition with the midpoint at approximately 65 degrees C. CD data along with the increased fluorescence intensity of the reporter fluorophore, 1-anilino-8-naphthalenesulfonate with SKTI, between 60 and 70 degrees C, are consistent with a transition of the native inhibitor to an alternate conformation with a more molten state. Even after heating to 90 degrees C, subsequent cooling of SKTI resulted in >90% of native trypsin inhibition potential. These results indicate that thermal denaturation of SKTI is readily reversible to the native form upon cooling and may provide a useful system for future protein folding studies in the class of disordered beta-sheet proteins.