Jumpei Sasabe

D-Serine is a key determinant of glutamate toxicity in amyotrophic lateral sclerosis

Excitotoxicity has been implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS). More recently, glial involvement has been shown to be essential for ALS‐related motoneuronal death. Here, we identified an N‐methyl‐D‐aspartate (NMDA) receptor co‐agonist, D‐serine (D‐Ser), as a glia‐derived enhancer of glutamate (Glu) toxicity to ALS motoneurons. Cell death assay indicated that primary spinal cord neurons from ALS mice were more vulnerable to NMDA toxicity than those from control mice, in a D‐Ser‐dependent manner. Levels of D‐Ser and its producing enzyme, serine racemase, in spinal cords of ALS mice were progressively elevated, dominantly in glia, with disease progression. In vitro, expression of serine racemase was induced not only by an extracellular pro‐inflammatory factor, but also by transiently expressed G93A‐superoxide dismutase1 in microglial cells. Furthermore, increases of D‐Ser levels were also observed in spinal cords of both familial and sporadic ALS patients. Collectively, Glu toxicity enhanced by D‐Ser overproduced in glia is proposed as a novel mechanism underlying ALS motoneuronal death, and this mechanism may be regarded as a potential therapeutic target for ALS.

Amyloid-beta causes memory impairment by disturbing the JAK2/STAT3 axis in hippocampal neurons.

Elevation of intracranial soluble amyloid-β (Aβ) levels has been implicated in the pathogenesis of Alzheimers disease (AD). Intracellular events in neurons, which lead to memory loss in AD, however, remain elusive. Humanin (HN) is a short neuroprotective peptide abolishing Aβ neurotoxicity. Recently, we found that HN derivatives activate the Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling axis. We here report that an HN derivative named colivelin completely restored cognitive function in an AD model (Tg2576) by activating the JAK2/STAT3 axis. In accordance, immunofluorescence staining using a specific antibody against phospho- (p-) STAT3 revealed that p-STAT3 levels in hippocampal neurons age-dependently decreased in both AD model mice and AD patients. Intracerebroventricular administration of Aβ1–42 downregulated p-STAT3 whereas passive immunization with anti-Aβ antibody conversely restored hippocampal p-STAT3 levels in Tg2576 mice, paralleling the decrease in the brain Aβ burden. Aβ1–42 consistently modulated p-STAT3 levels in primary neurons. Pharmacological inhibition of the JAK2/STAT3 axis not only induced significant loss of spatial working memory by downregulating an acetylcholine-producing enzyme choline acetyltransferase but also desensitized the M1-type muscarinic acetylcholine receptor. Thus, we propose a novel theory accounting for memory impairment related to AD: Aβ-dependent inactivation of the JAK2/STAT3 axis causes memory loss through cholinergic dysfunction. Our findings provide not only a novel pathological hallmark in AD but also a novel target in AD therapy.

d-Amino acid oxidase controls motoneuron degeneration through d-serine

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder involving an extensive loss of motoneurons. Aberrant excitability of motoneurons has been implicated in the pathogenesis of selective motoneuronal death in ALS. d-Serine, an endogenous coagonist of N-methyl-d-aspartate receptors, exacerbates motoneuronal death and is increased both in patients with sporadic/familial ALS and in a G93A-SOD1 mouse model of ALS (mSOD1 mouse). More recently, a unique mutation in the d-amino acid oxidase (DAO) gene, encoding a d-serine degrading enzyme, was reported to be associated with classical familial ALS. However, whether DAO affects the motoneuronal phenotype and d-serine increase in ALS remains uncertain. Here, we show that genetic inactivation of DAO in mice reduces the number and size of lower motoneurons with axonal degeneration, and that suppressed DAO activity in reactive astrocytes in the reticulospinal tract, one of the major inputs to the lower motoneurons, predominantly contributes to the d-serine increase in the mSOD1 mouse. The DAO inactivity resulted from expressional down-regulation, which was reversed by inhibitors of a glutamate receptor and MEK, but not by those of inflammatory stimuli. Our findings provide evidence that DAO has a pivotal role in motoneuron degeneration through d-serine regulation and that inactivity of DAO is a common feature between the mSOD1 ALS mouse model and the mutant DAO-associated familial ALS. The therapeutic benefit of reducing d-serine or controlling DAO activity in ALS should be tested in future studies.

Development of a femtomolar-acting humanin derivative named colivelin by attaching activity-dependent neurotrophic factor to its N terminus: characterization of colivelin-mediated neuroprotection against Alzheimer's disease-relevant insults in vitro and in vivo.

Alzheimers disease (AD) is the most common cause of dementia. Humanin (HN) is a short bioactive peptide abolishing neuronal cell death induced by various familial AD (FAD)-causative genes and amyloid-β (Aβ) in vitro. It has been shown that HN suppresses memory impairment of mice induced by intracerebroventricular administration of Aβ. To potentiate the neuroprotective effect of HN, we synthesized a hybrid peptide named Colivelin composed of activity-dependent neurotrophic factor (ADNF) C-terminally fused to AGA-(C8R)HNG17, a potent HN derivative. Colivelin completely suppresses death induced by overexpressed FAD-causative genes and Aβ1-43 at a concentration of 100 fm, whereas AGA-(C8R)HNG17 does so at a concentration of 10 pm. Colivelin-induced neuroprotection has been confirmed to occur via two neuroprotective pathways: one mediated by Ca2+/calmodulin-dependent protein kinase IV, triggered by ADNF, and one mediated by signal transducer and activator of transcription 3, triggered by HN. In vivo animal studies have further indicated that intracerebroventricular administration of Colivelin not only completely suppresses impairment in spatial working memory induced by repetitive intracerebroventricular injection of Aβ25-35 or Aβ1-42, but also it antagonizes neuronal loss in the CA1 region of hippocampus induced by hippocampal injection of Aβ1-42. In addition, intraperitoneally administered Colivelin suppresses memory impairment caused by a muscarinic acetylcholine receptor antagonist, 3-quinuclidinyl benzilate, indicating that a substantial portion of intraperitoneally administered Colivelin passes through the blood-brain barrier and suppresses functional memory deficit. Thus, Colivelin might serve as a novel drug candidate for treatment of AD.

Implanted cannula-mediated repetitive administration of Aβ25–35 into the mouse cerebral ventricle effectively impairs spatial working memory

Amyloid beta (Abeta) is closely related to the onset of Alzheimers disease (AD). To construct AD animal models, a bolus administration of a large dose of toxic Abeta into the cerebral ventricles of rodents has been performed in earlier studies. In parallel, a continuous infusion system via an osmotic pump into the cerebral ventricle has been developed to make a rat AD model. In this study, we developed a mouse AD model by repetitive administration of Abeta25-35 via a cannula implanted into the cerebral ventricle. Using this administration system, we reproducibly constructed a mouse with impaired spatial working memory. In accordance with the occurrence of the abnormal mouse behavior, we found that the number of choline acetyltransferase (ChAT)-positive neurons was reduced in paraventricular regions of brains of Abeta25-35-administered mice in a dose-dependent manner. Considering that the repetitive administration of a small dose of toxic Abeta via an implanted cannula leads to a brain status more resembling that of the AD patients than a bolus injection of a large dose of Abeta, and therapeutic as well as toxic agents are able to be repeatedly and reliably administered via an implanted cannula, we concluded that the implanted cannula-bearing AD mouse model is useful for development of new AD therapy.

Nasal Colivelin Treatment Ameliorates Memory Impairment Related to Alzheimer's Disease

Humanin (HN) and its derivatives, such as Colivelin (CLN), suppress neuronal death induced by insults related to Alzheimers disease (AD) by activating STAT3 in vitro. They also ameliorate functional memory impairment of mice induced by anticholinergic drugs or soluble toxic amyloid-β (Aβ) in vivo when either is directly administered into the cerebral ventricle or intraperitoneally injected. However, the mechanism underlying the in vivo effect remains uncharacterized. In addition, from the standpoint of clinical application, drug delivery methods that are less invasive and specific to the central nervous system (CNS) should be developed. In this study, we show that intranasally (i.n.) administered CLN can be successfully transferred to CNS via the olfactory bulb. Using several behavioral tests, we have demonstrated that i.n. administered CLN ameliorates memory impairment of AD models in a dose-responsive manner. Attenuation of AD-related memory impairment by HN derivatives such as CLN appears to be correlated with an increase in STAT3 phosphorylation levels in the septohippocampal region, suggesting that anti-AD activities of HN derivatives may be mediated by activation of STAT3 in vivo as they are in vitro. We further demonstrate that CLN treatment inhibits an Aβ induced decrease in the number of choline acetyltransferase (ChAT)-positive neurons in the medial septum. Combined with the finding that HN derivatives upregulate mRNA expression of neuronal ChAT and vesicular acetylcholine transporter (VAChT) in vitro, it is assumed that CLN may ameliorate memory impairment of AD models by supporting cholinergic neurotransmission, which is at least partly mediated by STAT3-mediated transcriptional upregulation of ChAT and VAChT.

Alteration of intrinsic amounts of d-serine in the mice lacking serine racemase and d-amino acid oxidase

For elucidation of the regulation mechanisms of intrinsic amounts of d-serine (d-Ser) which modulates the neuro-transmission of N-methyl-d-aspartate receptors in the brain, mutant animals lacking serine racemase (SRR) and d-amino acid oxidase (DAO) were established, and the amounts of d-Ser in the tissues and physiological fluids were determined. d-Ser amounts in the frontal brain areas were drastically decreased followed by reduced SRR activity. On the other hand, a moderate but significant decrease in d-Ser amounts was observed in the cerebellum and spinal cord of SRR knock-out (SRR−/−) mice compared with those of control mice, although the amounts of d-Ser in these tissues were low. The amounts of d-Ser in the brain and serum were not altered with aging. To clarify the uptake of exogenous d-Ser into the brain tissues, we have determined the d-Ser of SRR−/− mice after oral administration of d-Ser for the first time, and a drastic increase in d-Ser amounts in all the tested tissues was observed. Because both DAO and SRR are present in some brain areas, we have established the double mutant mice lacking SRR and DAO for the first time, and the contribution of both enzymes to the intrinsic d-Ser amounts was investigated. In the frontal brain, most of the intrinsic d-Ser was biosynthesized by SRR. On the other hand, half of the d-Ser present in the hindbrain was derived from the biosynthesis by SRR. These results indicate that the regulation of intrinsic d-Ser amounts is different depending on the tissues and provide useful information for the development of treatments for neuronal diseases.

Activity of D-amino acid oxidase is widespread in the human central nervous system

It has been proposed that D-amino acid oxidase (DAO) plays an essential role in degrading D-serine, an endogenous coagonist of N-methyl-D-aspartate (NMDA) glutamate receptors. DAO shows genetic association with amyotrophic lateral sclerosis (ALS) and schizophrenia, in whose pathophysiology aberrant metabolism of D-serine is implicated. Although the pathology of both essentially involves the forebrain, in rodents, enzymatic activity of DAO is hindbrain-shifted and absent in the region. Here, we show activity-based distribution of DAO in the central nervous system (CNS) of humans compared with that of mice. DAO activity in humans was generally higher than that in mice. In the human forebrain, DAO activity was distributed in the subcortical white matter and the posterior limb of internal capsule, while it was almost undetectable in those areas in mice. In the lower brain centers, DAO activity was detected in the gray and white matters in a coordinated fashion in both humans and mice. In humans, DAO activity was prominent along the corticospinal tract, rubrospinal tract, nigrostriatal system, ponto-/olivo-cerebellar fibers, and in the anterolateral system. In contrast, in mice, the reticulospinal tract and ponto-/olivo-cerebellar fibers were the major pathways showing strong DAO activity. In the human corticospinal tract, activity-based staining of DAO did not merge with a motoneuronal marker, but colocalized mostly with excitatory amino acid transporter 2 and in part with GFAP, suggesting that DAO activity-positive cells are astrocytes seen mainly in the motor pathway. These findings establish the distribution of DAO activity in cerebral white matter and the motor system in humans, providing evidence to support the involvement of DAO in schizophrenia and ALS. Our results raise further questions about the regulation of D-serine in DAO-rich regions as well as the physiological/pathological roles of DAO in white matter astrocytes.

Ischemic Acute Kidney Injury Perturbs Homeostasis of Serine Enantiomers in the Body Fluid in Mice: Early Detection of Renal Dysfunction Using the Ratio of Serine Enantiomers

The imbalance of blood and urine amino acids in renal failure has been studied mostly without chiral separation. Although a few reports have shown the presence of D-serine, an enantiomer of L-serine, in the serum of patients with severe renal failure, it has remained uncertain how serine enantiomers are deranged in the development of renal failure. In the present study, we have monitored serine enantiomers using a two-dimensional HPLC system in the serum and urine of mice after renal ischemia-reperfusion injury (IRI), known as a mouse model of acute kidney injury. In the serum, the level of D-serine gradually increased after renal IRI in parallel with that of creatinine, whereas the L-serine level decreased sharply in the early phase after IRI. The increase of D-serine was suppressed in part by genetic inactivation of a D-serine-degrading enzyme, D-amino acid oxidase (DAO), but not by disruption of its synthetic enzyme, serine racemase, in mice. Renal DAO activity was detected exclusively in proximal tubules, and IRI reduced the number of DAO-positive tubules. On the other hand, in the urine, D-serine was excreted at a rate nearly triple that of L-serine in mice with sham operations, indicating that little D-serine was reabsorbed while most L-serine was reabsorbed in physiological conditions. IRI significantly reduced the ratio of urinary D−/L-serine from 2.82±0.18 to 1.10±0.26 in the early phase and kept the ratio lower than 0.5 thereafter. The urinary D−/L-serine ratio can detect renal ischemia earlier than kidney injury molecule-1 (KIM-1) or neutrophil gelatinase-associated lipocalin (NGAL) in the urine, and more sensitively than creatinine, cystatin C, or the ratio of D−/L-serine in the serum. Our findings provide a novel understanding of the imbalance of amino acids in renal failure and offer a potential new biomarker for an early detection of acute kidney injury.

Glycolytic flux controls d-serine synthesis through glyceraldehyde-3-phosphate dehydrogenase in astrocytes

Significance Neurons require enormous energy to maintain continuous neurotransmission. To meet this requirement, astrocytes support neurons by balancing glycolytic flux with the synaptic level of an excitatory neurotransmitter, glutamate. But to control NMDA-subtype glutamate receptors, regulation of a coagonist, d-serine, as well as of glutamate, is crucial. Here we report that a glycolytic enzyme regulates d-serine synthesis as an indicator of glycolytic activity in astrocytes. This study shows how glutamatergic neurotransmission accommodates to changing energy circumstances through the coagonist. d-Serine is an essential coagonist with glutamate for stimulation of N-methyl-d-aspartate (NMDA) glutamate receptors. Although astrocytic metabolic processes are known to regulate synaptic glutamate levels, mechanisms that control d-serine levels are not well defined. Here we show that d-serine production in astrocytes is modulated by the interaction between the d-serine synthetic enzyme serine racemase (SRR) and a glycolytic enzyme, glyceraldehyde 3-phosphate dehydrogenase (GAPDH). In primary cultured astrocytes, glycolysis activity was negatively correlated with d-serine level. We show that SRR interacts directly with GAPDH, and that activation of glycolysis augments this interaction. Biochemical assays using mutant forms of GAPDH with either reduced activity or reduced affinity to SRR revealed that GAPDH suppresses SRR activity by direct binding to GAPDH and through NADH, a product of GAPDH. NADH allosterically inhibits the activity of SRR by promoting the disassociation of ATP from SRR. Thus, astrocytic production of d-serine is modulated by glycolytic activity via interactions between GAPDH and SRR. We found that SRR is expressed in astrocytes in the subiculum of the human hippocampus, where neurons are known to be particularly vulnerable to loss of energy. Collectively, our findings suggest that astrocytic energy metabolism controls d-serine production, thereby influencing glutamatergic neurotransmission in the hippocampus.