Austin J Clin Neurol 2015;2(4): 1040.
Scyllo-Inositol, a Therapeutic Agent for Alzheimer’s Disease
Tanaka K, Takenaka S and Yoshida K*
Department of Agrobioscience, Kobe University, Japan
*Corresponding author: Yoshida K, Department of Agrobioscience, Graduate School of Agricultural Science, Kobe University, 1-1 Rokkodai, Nada, Kobe 657 8501, Japan
Received: March 02, 2015; Accepted: March 27, 2015; Published: April 04, 2015
An inositol stereoisomer, scyllo-inositol (SI), has been regarded as a promising therapeutic agent for Alzheimer’s disease (AD), because it is an orally available natural compound that penetrates into the brain and coats the surface of amyloid β-proteins (Aβ) to inhibit their lateral stacking into toxic amyloid fibrils. SI is relatively rare in nature, and we developed a Bacillus subtilis cell factory for the efficient production of SI from abundant myo-inositol (MI).
Keywords: Alzheimer’s disease; Amyloid β-protein; Bacillus subtilis; Scylloinositol
AD: Alzheimer’s disease; Aβ: Amyloid β-protein(s); MI: myo-inositol; SI: scyllo-inositol
Alzheimer’s disease (AD) is the most common and problematic form of dementia. In 2010, 35 million people worldwide suffered from AD, and the number is expected to rise to 115 million in 2050 . Currently, there is no known cure for AD, but only a few medications that address the symptoms of the disease.
On the basis of the amyloid hypothesis, the initial cause of the disease is believed to be abnormal aggregation of amyloid β-protein (Aβ) into fibrillar polymers, which are hallmark lesions in the AD brain . Although it is not yet precisely elucidated how the aggregation of Aβ is initiated [3,4], the excessive Aβ aggregation disrupts the calcium ion homeostasis in neurons, which finally induces apoptosis . Aβ aggregates build up in the mitochondria and inhibit enzymes metabolizing glucose in neurons . Therefore, AD has been regarded as a simple neurodegenerative disease. However, accumulating evidence implies that the development of the disease may involve more intricate events. For instance, Aβ fibrils in AD brain tissues present structural variations that may correlate with phenotypic variations of the disease . In addition, the sustained formation of Aβ aggregates causes chronic activation of the innate immune system and disturbs microglial clearance functions .
On the other hand, another protein, named tau, is also involved in the disease development . The tau proteins form neurofibrillary tangles inside nerve cells  that disturb the cytoskeleton, and thus, the transport system required for biochemical communication among neurons [11,12]. Furthermore, there are a number of hypotheses that attempt to explain the cause of the disease by involving other factors, including herpes simplex virus type 1 , cellular homeostasis of ionic copper, iron, zinc, and aluminum [14,15], extremely low frequency electromagnetic fields , smoking , age-related myelin breakdown [18-20], oxidative stress [21-23], and air pollution . In any case, it is true that all the pathogenic events of the disease are tightly connected to the aggregation of Aβ [25-27]. Therefore, for any therapy for AD to show promise, aggregation of Aβ must be blocked earlier, before neurodegeneration and brain atrophy develop.
Accordingly, various chemical inhibitors targeting Aβ aggregation have been developed, including amyloid-binding dyes , catechols , curcumin , flavonoids , and polyphenols [32,33]. Some of these compounds originate in foods, and can be generally regarded as safe. Such inhibitors, including scyllo-inositol (SI) , (-)-epigallocatechin-3-gallate (EGCG) , and resveratrol  were shown to stabilize nontoxic oligomers of Aβ, and some of them are already in clinical or preclinical trials . However, some of the preemptive clinical trials were not concluded successfully, which indicates that we need to understand the molecular mechanisms of the disease in depth and to devise improved ways of designing trials to accurately evaluate the compounds .
In this mini review, we focus on SI and summarize the current status of the studies on its mechanism of action on Aâ, effectiveness in animal models, ongoing clinical trials, and the efficient production of the compound itself.
What is SI?
Inositol stands for a group of compounds of a six-fold alcohol of cyclohexane. The epimerization of the six hydroxyl groups generates nine stereoisomers. Myo-Inositol (MI) is the most prominent stereoisomer in nature and plays an important role as the structural basis for a number of secondary messengers that are various inositol phosphates. In addition, it serves as an important component of the membrane structural phospholipids, phosphatidylinositol. On the other hand, SI is another stereoisomer that is relatively rare in nature but has been regarded as a possible therapeutic agent for AD, and has received a fast-track designation from the US Food and Drug Administration for the treatment of AD. SI is a naturally occurring molecule that readily crosses the blood–brain barrier. It was shown that the human brain had the highest concentration of inositol in the body, with approximately 5mM MI and 0.5mM SI . The concentration of SI in the brain was elevated in patients with AD . In addition, high cerebral SI was proposed as a new marker of brain metabolism disturbances induced by chronic alcoholism . Furthermore, a higher concentration of SI was found in the normal aging human brain . These facts imply that cerebral SI levels may be controlled in response to functional defects in the brain caused by diseases and aging. Internal SI may be derived from MI through possible inter-conversion between the two inositol stereoisomer’s, which was suggested in a previous study where SI was administered in mice .
How does SI work?
In 2000, SI was first reported to stabilize the non-toxic oligomers of Aβ and to inhibit their toxic aggregation . To elucidate the mechanism by which SI blocks the self-aggregation of Aβ, molecular dynamics simulations of the interaction between SI and simple peptide models were conducted. It was observed that SI was able to bind to the surface of Aβ protofibrils to prevent their aggregation but could not break up the preformed aggregates . In addition, SI preferentially bound to the β-sheet-containing Aβ protofibrils with affinities of 0.2–0.5mM commensurate with its in vitro inhibitory concentrations, and exhibited a higher binding specificity for phenylalanine-lined grooves on the Aβ protofibril surface, indicating that SI obviously coats the surface of Aβ protofibrils and disrupts their stacking into fibrillar aggregates . A series of SI derivatives were synthesized and the effects of these compounds were investigated to reveal that all six hydroxyl groups of SI were involved in the complete inhibition of the fibrillar aggregation of Aβ .
Studies with animal models and clinical trials
SI is an orally available natural product that penetrates into the brain in vivo, and dose-dependently rescues the memory impairment produced by cerebroventricular injection of soluble Aâ in rats . SI blocked the development of aggregation of Aâ in the brain of transgenic AD mice, and was able to reverse defect of memory and alleviated other symptoms [47,48]. There is a patent that claims the use of SI for treating AD , and some clinical investigations of orally-administered SI have been conducted. A phase 2 clinical study was recently conducted on 353 patients with mild to moderate AD for 18 months . The clinical trial helped establish its safety profile, but the higher dose groups (1000 and 2000 mg dosed twice daily) showed greater rates of adverse events, including 9 deaths. Therefore, only the lower dose (250 mg twice daily) will be continued further, although the decision may reduce the ability of the study to establish the potential role of SI in the treatment of serious cases .
Efficient production of SI
As described above, SI is a promising therapeutic agent for AD. However, it is relatively rare in nature, and thus, is not sufficient to satisfy possible demand. At present, SI is produced by an expensive two-step enzymatic conversion  from MI, which (including its derivatives including phytic acid, as described below) is provided by fruits, beans, grains, and nuts . To enable a more efficient production of SI, we devised a bacterial cell factory for the bioconversion of MI into SI.
Bacillus subtilis has the ability to metabolize both MI and SI, and the complete gene set necessary for their utilization has been characterized . The iolABCDEFGHIJ operon encodes enzymes involved in multiple steps of the inositol metabolism, and the transcription of the operon is regulated by the IolR transcriptional repressor . In the first step, MI is converted to scyllo-inosose by the IolG enzyme. B. subtilis possesses two additional inositol dehydrogenases, IolX and IolW, both of which act specifically on SI to convert it to scyllo-inosose . IolX plays a major role in SI catabolism, whereas IolW efficiently reduces scyllo-inosose into SI. Scyllo-Inosose is metabolized sequentially in multiple steps involving the IolE, IolD, IolB, IolC, IolJ, and IolA enzymes to give common intermediates, dihydroxyacetone phosphate and acetyl-CoA . In the B. subtilis chromosome, we deleted all the “useless” genes including iolABCDEFHIJ, iolX, and iolR and overexpressed iolG and iolW under the control of a strong and constitutively active promoter to establish the cell factory with a complete bioconversion of 10 g/L MI into the same amount of SI secreted into the culture medium within 48 h .
Phytic acid (MI-1,2,3,4,5,6-hexaphosphate) is the principal storage form of phosphorus in plants, in particular bran and seeds. Phytases are a class of phosphatases that catalyze the hydrolysis of phytic acid to liberate MI and phosphate [59-61]. Because B. subtilis has a high ability to secrete enzymes [62,63], the cell factory could be modified to secrete phytases, and SI may be produced directly from agricultural waste materials such as rice bran, rich in phytic acid.
MI is synthesized from glucose-6-phosphate in two steps in many organisms . In B. subtilis, glucose-6-phosphate is the starting compound of glycolysis, appearing when glucose is incorporated into the cell via the phosphotransferase system . Glucose-6-phosphate is converted by inositol-3-phosphate synthase to MI 1-phosphate, which is then dephosphorylated by inositol monophosphatase to yield MI. Once we could manipulate the two enzymes function efficiently in B. subtilis, a novel cell factory could be devised to produce SI from glucose.
SI is a promising therapeutic agent for AD because of its ability to inhibit aggregation of Aâ in the brain. SI is relatively rare in nature, and we established the B. subtilis cell factory for production of SI from MI, which allowed 100% conversion of 10 g/L MI into the same amount of SI. By applying our B. subtilis cell factory concept, SI may be produced from raw and cheap materials in the future.
The authors are supported by the Ministry of Education, Culture, Sports, Science and Technology, Japan (Special Coordination Funds for Promoting Science and Technology, Creation of Innovative Centers for Advanced Interdisciplinary Research Areas; the Advanced Low-Carbon Technology Research and Development Program; KAKENHI 26660067; and Grants-in-Aid from the NCCARP project).
- World Alzheimer report. 2010.
- Hardy J, Selkoe DJ. The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics. Science. 2002; 297: 353-356.
- Van Broeck B, Van Broeckhoven C, Kumar-Singh S. Current insights into molecular mechanisms of Alzheimer disease and their implications for therapeutic approaches. Neurodegener Dis. 2007; 4: 349-365.
- Huang Y, Mucke L. Alzheimer mechanisms and therapeutic strategies. Cell. 2012; 148: 1204-1222.
- Yankner BA, Duffy LK, Kirschner DA. Neurotrophic and neurotoxic effects of amyloid beta protein: reversal by tachykinin neuropeptides. Science. 1990; 250: 279-282.
- Chen X, Yan SD. Mitochondrial Abeta: a potential cause of metabolic dysfunction in Alzheimer's disease. IUBMB Life. 2006; 58: 686-694.
- Lu JX, Qiang W, Yau WM, Schwieters CD, Meredith SC, Tycko R. Molecular structure of β-amyloid fibrils in Alzheimer's disease brain tissue. Cell. 2013; 154: 1257-1268.
- Heneka MT, Golenbock DT, Latz E. Innate immunity in Alzheimer's disease. Nat Immunol. 2015; 16: 229-236.
- Mudher A, Lovestone S. Alzheimer's disease-do tauists and baptists finally shake hands? Trends Neurosci. 2002; 25: 22-26.
- Goedert M, Spillantini MG, Crowther RA. Tau proteins and neurofibrillary degeneration. Brain Pathol. 1991; 1: 279-286.
- Iqbal K, Alonso Adel C, Chen S, Chohan MO, El-Akkad E, Gong CX, et al. Tau pathology in Alzheimer disease and other tauopathies. Biochim Biophys Acta. 2005; 1739: 198-210.
- Chun W, Johnson GV. The role of tau phosphorylation and cleavage in neuronal cell death. Front Biosci. 2007; 12: 733-756.
- Itzhaki RF, Wozniak MA. Herpes simplex virus type 1 in Alzheimer's disease: the enemy within. J Alzheimers Dis. 2008; 13: 393-405.
- Xu H, Finkelstein DI, Adlard PA. Interactions of metals and Apolipoprotein E in Alzheimer's disease. Front Aging Neurosci. 2014; 6: 121.
- Shcherbatykh I, Carpenter DO. The role of metals in the etiology of Alzheimer's disease. J Alzheimers Dis. 2007; 11: 191-205.
- Kheifets L, Bowman JD, Checkoway H, Feychting M, Harrington JM, Kavet R, et al. Future needs of occupational epidemiology of extremely low frequency electric and magnetic fields: review and recommendations. Occup Environ Med. 2009; 66: 72-80.
- Cataldo JK, Prochaska JJ, Glantz SA. Cigarette smoking is a risk factor for Alzheimer's Disease: an analysis controlling for tobacco industry affiliation. J Alzheimers Dis. 2010; 19: 465-480.
- Bartzokis G. Alzheimer's disease as homeostatic responses to age-related myelin breakdown. Neurobiol Aging. 2011; 32: 1341-1371.
- Bartzokis G, Lu PH, Mintz J. Quantifying age-related myelin breakdown with MRI: novel therapeutic targets for preventing cognitive decline and Alzheimer's disease. J Alzheimers Dis. 2004; 6: S53-59.
- Bartzokis G, Lu PH, Mintz J. Human brain myelination and amyloid beta deposition in Alzheimer's disease. Alzheimers Dement. 2007; 3: 122-125.
- Su B, Wang X, Nunomura A, Moreira PI, Lee HG, Perry G. Oxidative stress signaling in Alzheimer's disease. Curr Alzheimer Res. 2008; 5: 525-532.
- Kastenholz B, Garfin DE, Horst J, Nagel KA. Plant metal chaperones: a novel perspective in dementia therapy. Amyloid. 2009; 16: 81-83.
- Pohanka M. Alzheimer´s disease and oxidative stress: a review. Curr Med Chem. 2013; 21: 356-364.
- Moulton PV, Yang W. Air pollution, oxidative stress, and Alzheimer's disease. J Environ Public Health. 2012; 2012: 472751.
- Kirkitadze MD, Bitan G, Teplow DB. Paradigm shifts in Alzheimer's disease and other neurodegenerative disorders: the emerging role of oligomeric assemblies. J Neurosci Res. 2002; 69: 567-577.
- Selkoe DJ. Alzheimer's disease is a synaptic failure. Science. 2002; 298: 789-791.
- Selkoe DJ. Soluble oligomers of the amyloid beta-protein impair synaptic plasticity and behavior. Behav Brain Res. 2008; 192: 106-113.
- Necula M, Kayed R, Milton S, Glabe CG. Small molecule inhibitors of aggregation indicate that amyloid beta oligomerization and fibrillization pathways are independent and distinct. J Biol Chem. 2007; 282: 10311-10324.
- Di Giovanni S, Eleuteri S, Paleologou KE, Yin G, Zweckstette, M, Carrupt PA, et al. Entacapone and tolcapone, two catechol O-methyltransferase inhibitors, block fibril formation of a-synuclein and ß-amyloid and protect against amyloid-induced toxicity. J Biol Chem. 2010; 285: 14941-14954.
- Yang F, Lim GP, Begum AN, Ubeda OJ, Simmons MR, Ambegaokar SS, et al. Curcumin inhibits formation of amyloid beta oligomers and fibrils, binds plaques, and reduces amyloid in vivo. J Biol Chem. 2005; 280: 5892-5901.
- Lu JH, Ardah MT, Durairajan SS, Liu LF, Xie LX, Fong WF, et al. Baicalein inhibits formation of a-synuclein oligomers within living cells and prevents Aβ peptide fibrillation and oligomerisation. Chembiochem. 2011; 12: 615-624.
- Ladiwala AR, Lin JC, Bale SS, Marcelino-Cruz AM, Bhattacharya M, Dordick JS, et al. Resveratrol selectively remodels soluble oligomers and fibrils of amyloid Abeta into off-pathway conformers. J Biol Chem. 2010; 285: 24228-24237.
- Ladiwala AR, Dordick JS, Tessier PM. Aromatic small molecules remodel toxic soluble oligomers of amyloid beta through three independent pathways. J Biol Chem. 2011; 286: 3209-3218.
- McLaurin J, Golomb R, Jurewicz A, Antel JP, Fraser PE. Inositol stereoisomers stabilize an oligomeric aggregate of Alzheimer amyloid beta peptide and inhibit abeta -induced toxicity. J Biol Chem. 2000; 275: 18495-18502.
- Rezai-Zadeh K, Shytle D, Sun N, Mori T, Hou H, Jeanniton D, et al. Green tea epigallocatechin-3-gallate (EGCG) modulates amyloid precursor protein cleavage and reduces cerebral amyloidosis in Alzheimer transgenic mice. J Neurosci. 2005; 25: 8807-8814.
- Gravitz L. Drugs: a tangled web of targets. Nature. 2011; 475: S9-11.
- Grill JD, Cummings JL. Current therapeutic targets for the treatment of Alzheimer's disease. Expert Rev Neurother. 2010; 10: 711-728.
- Michaelis T, Helms G, Merboldt KD, Hänicke W, Bruhn H, Frahm J, et al. Identification of Scyllo-inositol in proton NMR spectra of human brain in vivo. NMR Biomed. 1993; 6: 105-109.
- Griffith HR, den Hollander JA, Stewart CC, Evanochko WT, Buchthal SD, Harrell LE, et al. Elevated brain scyllo-inositol concentrations in patients with Alzheimer's disease. NMR Biomed. 2007; 20: 709-716.
- Viola A, Nicoli F, Denis B, Confort-Gouny S, Le Fur Y, Ranjeva JP, et al. High cerebral scyllo-inositol: a new marker of brain metabolism disturbances induced by chronic alcoholism. MAGMA. 2004; 17: 47-61.
- Kaiser LG, Schuff N, Cashdollar N, Weiner MW. Scyllo-inositol in normal aging human brain: 1H magnetic resonance spectroscopy study at 4 Tesla. NMR Biomed. 2005; 18: 51-55.
- Yamashita Y, Yamaoka M, Hasunuma T, Ashida H, Yoshida K. Detection of orally administered inositol stereoisomers in mouse blood plasma and their effects on translocation of glucose transporter 4 in skeletal muscle cells. J Agric Food Chem. 2013; 61: 4850-4854.
- Li G, Rauscher S, Baud S, Pomés R. Binding of inositol stereoisomers to model amyloidogenic peptides. J Phys Chem B. 2012; 116: 1111-1119.
- Li G, Pomès R. Binding mechanism of inositol stereoisomers to monomers and aggregates of Aβ(16-22). J Phys Chem B. 2013; 117: 6603-6613.
- Sun Y, Zhang G, Hawkes CA, Shaw JE, McLaurin J, Nitz M, et al. Synthesis of scyllo-inositol derivatives and their effects on amyloid beta peptide aggregation. Bioorg Med Chem. 2008; 16: 7177-7184.
- Townsend M, Cleary JP, Mehta T, Hofmeister J, Lesne S, O'Hare E, et al. Orally available compound prevents deficits in memory caused by the Alzheimer amyloid-beta oligomers. Ann Neurol. 2006; 60: 668-676.
- McLaurin J, Kierstead ME, Brown ME, Hawkes CA, Lambermon MH, Phinney AL, et al. Cyclohexanehexol inhibitors of Abeta aggregation prevent and reverse Alzheimer phenotype in a mouse model. Nat Med. 2006; 12: 801-808.
- Fenili D, Brown M, Rappaport R, McLaurin J. Properties of scyllo-inositol as a therapeutic treatment of AD-like pathology. J Mol Med (Berl). 2007; 85: 603-611.
- McLaurin J. Methods of preventing, treating and diagnosing disorders of protein aggregation. US patent number 7,521,481.
- Salloway S, Sperling R, Keren R, Porsteinsson AP, van Dyck CH, Tariot PN, et al. A phase 2 randomized trial of ELND005, scyllo-inositol, in mild to moderate Alzheimer disease. Neurology. 2011; 77: 1253-1262.
- Ma K, Thomason LA, McLaurin J. scyllo-Inositol, preclinical, and clinical data for Alzheimer's disease. Adv Pharmacol. 2012; 64: 177-212.
- Yamaguchi M, Kita Y, Mori T, Kanbe K, Tomoda A, Takahashi A, et al. Process for producing scyllo-inositol. Patent number WO2005035774 A1.
- Clements RS Jr, Darnell B. Myo-inositol content of common foods: development of a high-myo-inositol diet. Am J Clin Nutr. 1980; 33: 1954-1967.
- Yamaoka M, Osawa S, Morinaga T, Takenaka S, Yoshida K. A cell factory of Bacillus subtilis engineered for the simple bioconversion of myo-inositol to scyllo-inositol, a potential therapeutic agent for Alzheimer's disease. Microb Cell Fact. 2011; 10: 69.
- Yoshida KI, Aoyama D, Ishio I, Shibayama T, Fujita Y. Organization and transcription of the myo-inositol operon, iol, of Bacillus subtilis. J Bacteriol. 1997; 179: 4591-4598.
- Morinaga T, Ashida H, Yoshida K. Identification of two scyllo-inositol dehydrogenases in Bacillus subtilis. Microbiology. 2010; 156: 1538-1546.
- Yoshida K, Yamaguchi M, Morinaga T, Kinehara M, Ikeuchi M, Ashida H, et al. myo-Inositol catabolism in Bacillus subtilis. J Biol Chem. 2008; 283: 10415-10424.
- Tanaka K, Tajima S, Takenaka S, Yoshida K. An improved Bacillus subtilis cell factory for producing scyllo-inositol, a promising therapeutic agent for Alzheimer's disease. Microb Cell Fact. 2013; 12: 124.
- Oh BC, Choi WC, Park S, Kim YO, Oh TK. Biochemical properties and substrate specificities of alkaline and histidine acid phytases. Appl Microbiol Biotechnol. 2004; 63: 362-372.
- Ragon M, Aumelas A, Chemardin P, Galvez S, Moulin G, Boze H. Complete hydrolysis of myo-inositol hexakisphosphate by a novel phytase from Debaryomyces castellii CBS 2923. Appl Microbiol Biotechnol. 2008; 78: 47-53.
- Escobin-Mopera L, Ohtani M, Sekiguchi S, Sone T, Abe A, Tanaka M, et al. Purification and characterization of phytase from Klebsiella pneumoniae 9-3B. J Biosci Bioeng. 2012; 113: 562-567.
- Antelmann H, Tjalsma H, Voigt B, Ohlmeier S, Bron S, van Dijl JM, et al. A proteomic view on genome-based signal peptide predictions. Genome Res. 2001; 11: 1484-1502.
- van Dijl JM, Braun PG, Robinson C, Quax WJ, Antelmann H, Hecker M, et al. Functional genomic analysis of the Bacillus subtilis Tat pathway for protein secretion. J Biotechnol. 2002; 98: 243-254.
- Majumder AL, Chatterjee A, Ghosh Dastidar K, Majee M. Diversification and evolution of L-myo-inositol 1-phosphate synthase. FEBS Lett. 2003; 553: 3-10.
- Reizer J, Saier MH Jr, Deutscher J, Grenier F, Thompson J, Hengstenberg W. The phosphoenolpyruvate: sugar phosphotransferase system in gram-positive bacteria: properties, mechanism, and regulation. Crit Rev Microbiol. 1988; 15: 297-338.