Evidence for Compromised Insulin Signaling and Neuronal Vulnerability in Experimental Model of Sporadic Alzheimer’s Disease

  • Smriti Gupta
  • Kamalendra Yadav
  • Shrikant S. Mantri
  • Nitin K. Singhal
  • Subramaniam Ganesh
  • Rajat Sandhir


Evidence from animal studies categorizes sporadic Alzheimer’s disease (sAD) as a metabolic syndrome with accompanying cognitive deficits. Given that glial cells act as “silent partners” to neurons by providing trophic support and defense, the present study investigated the role of glia in sAD pathology. A streptozotocin (STZ)-induced glial-neuronal co-culture model of sAD was used to study the metabolic status of the two cell types. Real time RT-PCR and Western blotting results indicated that amyloid precursor protein (APP) and β-secretase (BACE1) were highly expressed in co-cultured neurons than in monocultures. Increased amyloidogenesis was accompanied by decreased expression of mediators in insulin signaling pathway that included insulin receptor (IR), insulin receptor substrate 2 (IRS2), insulin-like growth factor 2 (IGF2), insulin-like growth factor 1 receptor (IGF1R), total-glycogen synthase kinase 3β (t-GSK3β), and phosphorylated-GSK3βser9 (p-GSK3βser9), suggesting that neuronal cells are more prone to metabolic variability when cultured in the presence of glial cells. Findings from the sAD model induced by intracerebroventricular (ICV) injection of STZ revealed that increased amyloid beta (Aβ) load in the hippocampus was potentially responsible for the hyperphosphorylation of tau at ser396. Furthermore, impaired cognitive functions and decreased dendritic spine density and axonal thinning in CA1 region of hippocampus were associated with decreased IR and p-GSK3βser9/t-GSK3β expression. Taken together, the present study provides evidence that glia mediated response and insulin signaling defects drive pathological changes in sAD and represent potential targets for delaying sAD progression.


Alzheimer’s disease Amyloid precursor protein Beta amyloid Dendritic spines Diabetes Memory Neurofibriallary tangles 



amyloid precursor protein


a Disintegrin and metalloproteinase domain-containing protein 10

amyloid beta


artificial cerebrospinal fluid


Alzheimer’s disease




blood-brain barrier


Dulbecco’s modified Eagle’s medium


glyceraldehyde-3-phosphate dehydrogenase


glucose oxidase-peroxidise


glucose transporter 1


glucose transporter 3




insulin like growth factor1


insulin like growth factor2


insulin like growth factor 1 receptor


insulin receptor gene




insulin receptor


insulin-resistant brain state


insulin receptor substrate 1


insulin receptor substrate2


3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide


Morris water maze


2-(N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl, amino)-2-deoxyglucose)


neuro-fibrillary tangles


nerve growth factor


phospho-glycogen synthase kinase 3 β


paired helical filament


protein kinase C


Roswell Park Memorial Institute


sporadic Alzheimer’s disease


facilitated glucose transporter member 1


facilitated glucose transporter member 3




type 3 diabetes


total-glycogen synthase kinase3β



This work was supported by UGC-BSR fellowship program (Ref. No. F.4-1/2006 [BSR]/7-209/2009 [BSR] and UGC-SAP DRS phase II program (Ref. No. F.4-7/2015/DRS-II (SAP-II). The authors also acknowledge the finacial support from the DST under PURSE (Phase II) grant. The authors thank Ms. Komal Taneja for her support with Golgi-Cox staining.

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflict of interest.

Supplementary material

12035_2018_985_MOESM1_ESM.pdf (458 kb)
ESM 1 (PDF 458 kb)


  1. 1.
    Ballard C, Gauthier S, Corbett A, Brayne C, Aarsland D, Jones E (2011) Alzheimer’s disease. Lancet 377:1019–1031CrossRefPubMedGoogle Scholar
  2. 2.
    Mancuso M, Calsolaro V, Orsucci D, Carlesi C, Choub A, Piazza S, Siciliano G (2009) Mitochondria, cognitive impairment, and Alzheimer’s disease. Int J Alzheimers Dis 2009:1–8CrossRefGoogle Scholar
  3. 3.
    Holtzman DM, Morris JC, Goate AM (2011) Alzheimer’s disease: the challenge of the second century. Sci Transl Med 3:77Google Scholar
  4. 4.
    Tönnies E, Trushina E (2017) Oxidative stress, synaptic dysfunction, and Alzheimer’s disease. J Alzheimers Dis 57:1105–1121CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Bekris LM, Yu C-E, Bird TD, Tsuang DW (2010) Genetics of Alzheimer disease. J Geriatr Psychiatry Neurol 23:213–227CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Talbot K, Wang H-Y, Kazi H, Han LY, Bakshi KP, Stucky A, Fuino RL, Kawaguchi KR et al (2012) Demonstrated brain insulin resistance in Alzheimer’s disease patients is associated with IGF-1 resistance, IRS-1 dysregulation, and cognitive decline. J Clin Invest 122:1316–1338CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Craft S, Watson GS (2004) Insulin and neurodegenerative disease: shared and specific mechanisms. Lancet Neurol 3:169–178CrossRefPubMedGoogle Scholar
  8. 8.
    Hoyer S (2002) The aging brain. Changes in the neuronal insulin/insulin receptor signal transduction cascade trigger late-onset sporadic Alzheimer disease (SAD). A mini-review. J Neural Transm 109:991–1002CrossRefPubMedGoogle Scholar
  9. 9.
    Sandhir R, Gupta S (2015) Molecular and biochemical trajectories from diabetes to Alzheimer’s disease: a critical appraisal. World J Diabetes 6:1223–1242. CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Pardridge WM (2012) Drug transport across the blood–brain barrier. J Cereb Blood Flow Metab 32:1959–1972CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    De Felice FG, Lourenco MV, Ferreira ST (2014) How does brain insulin resistance develop in Alzheimer’s disease? Alzheimers Dement 10:S26–S32CrossRefPubMedGoogle Scholar
  12. 12.
    Tokutake T, Kasuga K, Yajima R, Sekine Y, Tezuka T, Nishizawa M, Ikeuchi T (2012) Hyperphosphorylation of Tau induced by naturally secreted amyloid-β at nanomolar concentrations is modulated by insulin-dependent Akt-GSK3β signaling pathway. J Biol Chem 287:35222–35233CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    An Y, Varma V, Varma S et al (2017) Evidence for brain glucose dysregulation in Alzheimer’s disease. Alzheimers Dement.
  14. 14.
    Watson GS, Craft S (2003) The role of insulin resistance in the pathogenesis of Alzheimer’s disease: implications for treatment. CNS Drugs 17:27–45CrossRefPubMedGoogle Scholar
  15. 15.
    Rivera E, Goldin A, Fulmer N et al (2005) Insulin and insulin-like growth factor expression and function deteriorate with progression of Alzheimer’s disease: link to brain reductions in acetylcholine. J Alzheimers Dis 8(3):247–268CrossRefPubMedGoogle Scholar
  16. 16.
    Steen E, Terry BM, Rivera EJ et al (2005) Impaired insulin and insulin-like growth factor expression and signaling mechanisms in Alzheimer’s disease--is this type 3 diabetes? J Alzheimers Dis 7(1):63–80CrossRefPubMedGoogle Scholar
  17. 17.
    Hölscher C, Li L (2010) New roles for insulin-like hormones in neuronal signalling and protection: new hopes for novel treatments of Alzheimer’s disease? Neurobiol Aging 31(9):1495–1502CrossRefPubMedGoogle Scholar
  18. 18.
    K1 T, Wang HY, Kazi H et al (2012) Demonstrated brain insulin resistance in Alzheimer’s disease patients is associated with IGF-1 resistance, IRS-1 dysregulation, and cognitive decline. J Clin Invest (4):1316–1338Google Scholar
  19. 19.
    de la Monte SM (2012) Brain insulin resistance and deficiency as therapeutic targets in Alzheimer’s disease. Curr Alzheimer Res 9(1):35–66CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Deng Y, Li B, Liu Y et al (2009) Dysregulation of insulin signaling, glucose transporters, O-GlcNAcylation, and phosphorylation of tau and neurofilaments in the brain implication for alzheimer’s disease. Am J Pathol 175(5):2089–2098CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Shingo AS, Kanabayashi T, Kito S, Murase T (2013) Intracerebroventricular administration of an insulin analogue recovers STZ-induced cognitive decline in rats. Behav Brain Res 241:105–111CrossRefPubMedGoogle Scholar
  22. 22.
    de la Monte, Wands JR (2008) Alzheimer’s disease is type 3 diabetes–evidence reviewed. J Diabetes Sci Technol 26:1101–1113CrossRefGoogle Scholar
  23. 23.
    Salkovic-Petrisic M, Knezovic A et al (2013) What have we learned from the streptozotocin-induced animal model of sporadic Alzheimer’s disease, about the therapeutic strategies in Alzheimer’s research. J Neural Transm 1:233–252CrossRefGoogle Scholar
  24. 24.
    Hoyer S, Müller D, Plaschke K (1994) Desensitization of brain insulin receptor. Effect on glucose/energy and related metabolism. J Neural Transm Suppl 44:259–268PubMedGoogle Scholar
  25. 25.
    Mehla J, Pahuja M, Gupta YK (2013) Streptozotocin-induced sporadic Alzheimer’s disease: selection of appropriate dose. J Alzheimers Dis 33(1):17–21PubMedGoogle Scholar
  26. 26.
    Trinder P (1969) Determination of blood glucose using an oxidase-peroxidase system with a non-carcinogenic chromogen. J Clin Pathol 22:158–161CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Wang X, Michaelis ML, Michaelis EK (2010) Functional genomics of brain aging and Alzheimer’s disease: focus on selective neuronal vulnerability. Curr Genomics 11(8):618–633CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Vorhees CV, Williams MT (2006) Morris water maze: procedures for assessing spatial and related forms of learning and memory. Nat Protoc 1:848–858CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Rajamohamedsait HB, Sigurdsson EM (2012) Histological staining of amyloid and pre-amyloid peptides and proteins in mouse tissue. Methods Mol Biol 849:411–424. CrossRefPubMedGoogle Scholar
  30. 30.
    Guntern R, Bouras C, Hof PR, Vallet PG (1992) An improved thioflavine S method for staining neurofibrillary tangles and senile plaques in Alzheimer’s disease. Experientia 48:8–10CrossRefPubMedGoogle Scholar
  31. 31.
    Fischer AH, Jacobson KA, Rose J, Zeller R (2008) Hematoxylin and eosin staining of tissue and cell sections. Cold Spring Harb Protoc 2008:prot4986Google Scholar
  32. 32.
    Zaqout S, Kaindl AM (2016) Golgi-cox staining step by step. Front Neuroanat 10:38CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Fath T, Eidenmüller J, Brandt R (2002) Tau-mediated cytotoxicity in a pseudohyperphosphorylation model of Alzheimer's disease. J Neurosci 22:9733–9741PubMedGoogle Scholar
  34. 34.
    Wang SS-S, Rymer DL, Good TA (2001) Reduction in cholesterol and sialic acid content protects cells from the toxic effects of β-amyloid peptides. J Biol Chem 276:42027–42034CrossRefPubMedGoogle Scholar
  35. 35.
    Zou C, Wang Y, Shen Z (2005) 2-NBDG as a fluorescent indicator for direct glucose uptake measurement. J Biochem Biophys Methods 64:207–215CrossRefPubMedGoogle Scholar
  36. 36.
    Yuan X, Zhang S, Sun M et al (2013) Putative DHHC-cysteine-rich domain S-acyltransferase in plants. PLoS One 8(10):75985CrossRefGoogle Scholar
  37. 37.
    Bussière T, Bard F, Barbour R, Grajeda H, Guido T, Khan K, Schenk D, Games D et al (2004) Morphological characterization of Thioflavin-S-positive amyloid plaques in transgenic Alzheimer mice and effect of passive Abeta immunotherapy on their clearance. Am J Pathol 165:987–995CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Kluve-Beckerman B, Liepnieks JJ, Wang L, Benson MD (1999) A cell culture system for the study of amyloid pathogenesis. Amyloid formation by peritoneal macrophages cultured with recombinant serum amyloid A. Am J Pathol 155:123–133CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    de la Monte SM, Wands JR (2008) Alzheimer’s disease is type 3 diabetes-evidence reviewed. J Diabetes Sci Technol 2:1101–1113CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Cohen-Pfeffer JL, Gururangan S, Lester T et al (2016) Intracerebroventricular delivery as a safe, long-term route of drug administration. Pediatr Neurol 67:23–35CrossRefPubMedGoogle Scholar
  41. 41.
    de la Monte SM, Longato L, Tong M, Wands JR (2009) Insulin resistance and neurodegeneration: Roles of obesity, type 2 diabetes mellitus and non-alcoholic steatohepatitis. Curr Opin Investig Drugs 10:1049–1060PubMedPubMedCentralGoogle Scholar
  42. 42.
    Velazquez R, Tran A, Ishimwe E, Denner L, Dave N, Oddo S, Dineley KT (2017) Central insulin dysregulation and energy dyshomeostasis in two mouse models of Alzheimer’s disease. Neurobiol Aging 58:1–13CrossRefPubMedGoogle Scholar
  43. 43.
    Leszek J, Trypka E, Tarasov VV et al (2017) Type 3 diabetes mellitus: A novel implication of Alzheimers disease. Curr Top Med Chem 17:1331–1335CrossRefPubMedGoogle Scholar
  44. 44.
    Kleinridders A (2016) Deciphering brain insulin receptor and insulin-like growth factor 1 receptor Signalling. J Neuroendocrinol, 28Google Scholar
  45. 45.
    Knezovic A, Loncar A, Homolak J, Smailovic U, Osmanovic Barilar J, Ganoci L, Bozina N, Riederer P et al (2017) Rat brain glucose transporter-2, insulin receptor and glial expression are acute targets of intracerebroventricular streptozotocin: risk factors for sporadic Alzheimer’s disease? J Neural Transm 124:695–708CrossRefPubMedGoogle Scholar
  46. 46.
    Gasparini L, Netzer WJ, Greengard P, Xu H (2002) Does insulin dysfunction play a role in Alzheimer’s disease? Trends Pharmacol Sci 23:288–293CrossRefPubMedGoogle Scholar
  47. 47.
    Ghasemi R, Haeri A, Dargahi L, Mohamed Z, Ahmadiani A (2013) Insulin in the brain: sources, localization and functions. Mol Neurobiol 47:145–171CrossRefPubMedGoogle Scholar
  48. 48.
    Havrankova J, Schmechel D, Roth J, Brownstein M (1978) Identification of insulin in rat brain. Proc Natl Acad Sci 75:5737–5741CrossRefPubMedPubMedCentralGoogle Scholar
  49. 49.
    Gray SM, Meijer RI, Barrett EJ (2014) Insulin regulates brain function, but how does it get there? Diabetes 63:3992–3997CrossRefPubMedPubMedCentralGoogle Scholar
  50. 50.
    Zhao W, Chen H, Xu H, Moore E, Meiri N, Quon MJ, Alkon DL (1999) Brain insulin receptors and spatial memory. Correlated changes in gene expression, tyrosine phosphorylation, and signaling molecules in the hippocampus of water maze trained rats. J Biol Chem 274:34893–34902CrossRefPubMedGoogle Scholar
  51. 51.
    De Felice FG (2013) Alzheimer’s disease and insulin resistance: translating basic science into clinical applications. J Clin Invest 123:531–539CrossRefPubMedPubMedCentralGoogle Scholar
  52. 52.
    Moloney AM, Griffin RJ, Timmons S, O’Connor R, Ravid R, O’Neill C (2010) Defects in IGF-1 receptor, insulin receptor and IRS 1/2 in Alzheimer’s disease indicate possible resistance to IGF-1 and insulin signalling. Neurobiol Aging 31:224–243CrossRefPubMedGoogle Scholar
  53. 53.
    Jahangir Z, Ahmad W, Shabbiri K (2014) Alternate phosphorylation/O-GlcNAc modification on human insulin IRSs: a road towards impaired insulin signaling in Alzheimer and diabetes. Adv Bioinforma 2014:324753CrossRefGoogle Scholar
  54. 54.
    Malito E, Hulse RE, Tang W-J (2008) Amyloid beta-degrading cryptidases: insulin degrading enzyme, presequence peptidase, and neprilysin. Cell Mol Life Sci 65:2574–2585CrossRefPubMedPubMedCentralGoogle Scholar
  55. 55.
    Bates KA, Verdile G, Li Q-X, Ames D, Hudson P, Masters CL, Martins RN (2009) Clearance mechanisms of Alzheimer’s amyloid-β peptide: implications for therapeutic design and diagnostic tests. Mol Psychiatry 14:469–486CrossRefPubMedGoogle Scholar
  56. 56.
    Rammes G, Mattusch C, Wulff M, Seeser F, Kreuzer M, Zhu K, Deussing JM, Herms J et al (2017) Involvement of GluN2B subunit containing N-methyl-d-aspartate (NMDA) receptors in mediating the acute and chronic synaptotoxic effects of oligomeric amyloid-beta (Aβ) in murine models of Alzheimer’s disease (AD). Neuropharmacology 123:100–115CrossRefPubMedGoogle Scholar
  57. 57.
    Grieb P (2016) Intracerebroventricular streptozotocin injections as a model of Alzheimer’s disease: in search of a relevant mechanism. Mol Neurobiol 53:1741–1752CrossRefPubMedGoogle Scholar
  58. 58.
    Saxena S, Caroni P (2011) Selective neuronal vulnerability in neurodegenerative diseases: from stressor thresholds to degeneration. Neuron 71:35–48CrossRefPubMedGoogle Scholar
  59. 59.
    Bandyopadhyay B, Li G, Yin H, Kuret J (2007) Tau aggregation and toxicity in a cell culture model of tauopathy. J Biol Chem 282:16454–16464CrossRefPubMedGoogle Scholar
  60. 60.
    Evans DB, Rank KB, Bhattacharya K, Thomsen DR, Gurney ME, Sharma SK (2000) Tau phosphorylation at serine 396 and serine 404 by human recombinant tau protein kinase II inhibits Tau’s ability to promote microtubule assembly. J Biol Chem 275:24977–24983CrossRefPubMedGoogle Scholar
  61. 61.
    Umeda T, Tomiyama T, Kitajima E, Idomoto T, Nomura S, Lambert MP, Klein WL, Mori H (2012) Hypercholesterolemia accelerates intraneuronal accumulation of Aβ oligomers resulting in memory impairment in Alzheimer’s disease model mice. Life Sci 91(23–24):1169–1176CrossRefPubMedGoogle Scholar
  62. 62.
    Hooper C, Killick R, Lovestone S (2008) The GSK3 hypothesis of Alzheimer’s disease. J Neurochem 104:1433–1439CrossRefPubMedPubMedCentralGoogle Scholar
  63. 63.
    García-Cáceres C, Quarta C, Varela L, Gao Y, Gruber T, Legutko B, Jastroch M, Johansson P et al (2016) Astrocytic insulin signaling couples brain glucose uptake with nutrient availability. Cell 166:867–880CrossRefPubMedGoogle Scholar
  64. 64.
    Cifelli JL, Dozier L, Chung TS, Patrick GN, Yang J (2016) Benzothiazole amphiphiles promote the formation of dendritic spines in primary hippocampal neurons. J Biol Chem 291:11981–11992CrossRefPubMedPubMedCentralGoogle Scholar
  65. 65.
    Borbély E, Horváth J, Furdan S et al (2014) Simultaneous changes of spatial memory and spine density after intrahippocampal administration of fibrillar aβ1-42 to the rat brain. Biomed Res Int 2014:345305CrossRefPubMedPubMedCentralGoogle Scholar
  66. 66.
    Stockhorst U, de Fries D, Steingrueber H-J, Scherbaum WA (2004) Insulin and the CNS: effects on food intake, memory, and endocrine parameters and the role of intranasal insulin administration in humans. Physiol Behav 83:47–54CrossRefPubMedGoogle Scholar
  67. 67.
    Ruud J, Steculorum SM, Brüninga JC (2017) Neuronal control of peripheral insulin sensitivity and glucose metabolism. Nat Commun 8:15259CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

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Authors and Affiliations

  1. 1.Department of Biochemistry, Basic Medical Science Block II, Sector 25Panjab UniversityChandigarhIndia
  2. 2.National Agri-Food Biotechnology InstituteMohaliIndia
  3. 3.Department of Biological Sciences and BioengineeringIndian Institute of TechnologyKanpurIndia

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