Abstract
Brain insulin resistance is a major factor leading to impaired cognitive function and it is considered as the onset of Alzheimer´s disease. Insulin resistance is intimately linked to inflammatory conditions, many studies have revealed how pro-inflammatory cytokines lead to insulin resistance, by inhibiting IRS1 function. Thus, the dysfunction of insulin signaling is concomitant with inflammatory biomarkers. However, the specific effect of IRS1 impaired function in otherwise healthy brain has not been dissected out. So, we decided in our study, to study the specific role of IRS1 in the hippocampus, in the absence of comorbidities. To that end, shRNA against rat and human IRS1 was designed and tested in cultured HEK cells to evaluate mRNA levels and specificity. The best candidate sequence was encapsulated in an AAV vector (strain DJ8) under the control of the cytomegalovirus promoter and together with the green fluorescent protein gene as a reporter. AAV-CMV-shIRS1-EGFP and control AAV-CMV-EGFP were inoculated into the dorsal hippocampus of female and male Wistar rats. One month later, animals undertook a battery of behavioral paradigms evaluating spatial and social memory and anxiety. Our results suggest that females displayed increased susceptibility to AAV-shIRS1 in the novel recognition object paradigm; whereas both females and males show impaired performance in the T maze when infected with AAV-shIRS1 compared to control. Anxiety parameters were not affected by AAV-shIRS1 infection. We observed specific fluorescence within the hilum of the dentate gyrus, in immuno-characterized parvalbumin and somatostatin neurons. AAV DJ8 did not enter astrocytes. Intense green fibers were found in the fornix, mammillary bodies, and in the medial septum indicating that hippocampal efferent had been efficiently targeted by the AAV DJ8 infection. We observed that AAV-shIRS1 reduced significantly synaptophysin labeling in hippocampal-septal projections compared to controls. These results support that, small alterations in the insulin/IGF1 pathway in specific hippocampal circuitries can underlie alterations in synaptic plasticity and affect behavior, in the absence of inflammatory conditions
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Addgene (2020) AAV Purification. https://www.addgene.org/protocols/aav-purification-iodixanol-gradient-ultracentrifugation/. Accessed 14 Feb 2020
Aggleton JP, Pralus A, Nelson AJD, Hornberger M (2016) Thalamic pathology and memory loss in early Alzheimer’s disease: moving the focus from the medial temporal lobe to Papez circuit. Brain 139:1877–1890. https://doi.org/10.1093/brain/aww083
Aguirre V, Werner ED, Giraud J et al (2002) Phosphorylation of Ser307 in insulin receptor substrate-1 blocks interactions with the insulin receptor and inhibits insulin action. J Biol Chem 277:1531–1537. https://doi.org/10.1074/jbc.M101521200
Al-Delaimy WK, von Muhlen D, Barrett-Connor E (2009) Insulinlike growth factor-1, insulin-like growth factor binding protein-1, and cognitive function in older men and women. J Am Geriatr Soc 57:1441–1446. https://doi.org/10.1111/j.1532-5415.2009.02343.x
Ashrafi G, Wu Z, Farrell RJ, Ryan TA (2017) GLUT4 mobilization supports energetic demands of active synapses. Neuron 93:606-615.e3. https://doi.org/10.1016/j.neuron.2016.12.020
Beam CR, Kaneshiro C, Jang JY et al (2018) Differences between women and men in incidence rates of dementia and Alzheimer’s disease. J Alzheimer’s Dis 64:1077–1083. https://doi.org/10.3233/JAD-180141
Bedse G, Di Domenico F, Serviddio G, Cassano T (2015) Aberrant insulin signaling in Alzheimer’s disease: current knowledge. Front Neurosci 9:1–13. https://doi.org/10.3389/fnins.2015.00204
Burgess N, Maguire EA, O’Keefe J (2002) The human hippocampus and spatial and episodic memory. Neuron 35:625–641
Cao Q, Tan C-C, Xu W et al (2019) The prevalence of dementia: a systematic review and meta-analysis. J Alzheimer’s Dis 73:1–10. https://doi.org/10.3233/jad-191092
Cholerton B, Baker LD, Craft S (2013) Insulin, cognition, and dementia. Eur J Pharmacol 719:170–179. https://doi.org/10.1016/j.ejphar.2013.08.008
Copps KD, White MF (2012) Regulation of insulin sensitivity by serine/threonine phosphorylation of insulin receptor substrate proteins IRS1 and IRS2. Diabetologia 55:2565–2582. https://doi.org/10.1007/s00125-012-2644-8
Costa MM, Violato NM, Taboga SR et al (2012) Reduction of insulin signalling pathway IRS-1/IRS-2/AKT/mTOR and decrease of epithelial cell proliferation in the prostate of glucocorticoid-treated rats. Int J Exp Pathol 93:188–195. https://doi.org/10.1111/j.1365-2613.2012.00817.x
Costello DA, Claret M, Al-Qassab H et al (2012) Brain deletion of insulin receptor substrate 2 disrupts hippocampal synaptic plasticity and metaplasticity. PLoS ONE 7:30–34. https://doi.org/10.1371/journal.pone.0031124
Degroot A, Treit D (2002) Dorsal and ventral hippocampal cholinergic systems modulate anxiety in the plus-maze and shock-probe tests. Brain Res 949:60–70. https://doi.org/10.1016/S0006-8993(02)02965-7
Degroot A, Treit D (2003) Septal gabaergic and hippocampal cholinergic systems interact in the modulation of anxiety. Neuroscience 117:493–501. https://doi.org/10.1016/S0306-4522(02)00651-6
Dineley KT, Jahrling JB, Denner L (2014) Insulin resistance in Alzheimer’s disease. Neurobiol Dis 72:92–103. https://doi.org/10.1016/j.nbd.2014.09.001
Dong X, Park S, Lin X et al (2006) Irs1 and Irs2 signaling is essential for hepatic glucose homeostasis and systemic growth. J Clin Invest 116:101–114. https://doi.org/10.1172/JCI25735
Du G, Yonekubo J, Zeng Y et al (2006) Design of expression vectors for RNA interference based on miRNAs and RNA splicing. FEBS J 273:5421–5427. https://doi.org/10.1111/j.1742-4658.2006.05534.x
Eckstein SS, Weigert C, Lehmann R (2017) Divergent roles of IRS (insulin receptor substrate) 1 and 2 in liver and skeletal muscle. Curr Med Chem 24:1827–1852. https://doi.org/10.2174/0929867324666170426142826
Eichenbaum H (1993) Thinking about brain cell assemblies. Science (80-) 261:993–994
Escobar I, Xu J, Jackson CW, Perez-Pinzon MA (2019) Altered neural networks in the papez circuit: implications for cognitive dysfunction after cerebral ischemia. J Alzheimers Dis 67:425–446. https://doi.org/10.3233/JAD-180875
Folch J, Olloquequi J, Ettcheto M et al (2019) The involvement of peripheral and brain insulin resistance in late-onset Alzheimer’s dementia. Front Aging Neurosci. https://doi.org/10.3389/fnagi.2019.00236
Freude S, Hettich MM, Schumann C et al (2009) Neuronal IGF-1 resistance reduces Aβ accumulation and protects against premature death in a model of Alzheimer’s disease. FASEB J 23:3315–3324. https://doi.org/10.1096/fj.09-132043
Gaffan D, Gaffan EA (1991) Amnesia in man following transection of the fornix: A review. Brain 114:2611–2618. https://doi.org/10.1093/brain/114.6.2611
Gaffan EA, Bannerman DM, Warburton EC, Aggleton JP (2001) Rats’ processing of visual scenes: effects of lesions to fornix, anterior thalamus, mamillary nuclei or the retrohippocampal region. Behav Brain Res 121:103–117. https://doi.org/10.1016/s0166-4328(00)00389-2
Gage FH, Björklund A (1986) Cholinergic septal grafts into the hippocampal formation improve spatial learning and memory in aged rats by an atropine-sensitive mechanism. J Neurosci 6:2837–2847
Golimstok A, Cámpora N, Rojas JI et al (2014) Cardiovascular risk factors and frontotemporal dementia: a case-control study. Transl Neurodegener. https://doi.org/10.1186/2047-9158-3-13
Grillo CA, Piroli GG, Hendry RM, Reagan LP (2009) Insulin-stimulated translocation of GLUT4 to the plasma membrane in rat hippocampus is PI3-kinase dependent. Brain Res 1296:35–45. https://doi.org/10.1016/j.brainres.2009.08.005
Grillo CA, Piroli GG, Lawrence RC et al (2015) Hippocampal insulin resistance impairs spatial learning and synaptic plasticity. Diabetes 64:3927–3936. https://doi.org/10.2337/db15-0596
Guo S, Copps KD, Dong X et al (2009) The Irs1 branch of the insulin signaling cascade plays a dominant role in hepatic nutrient homeostasis. Mol Cell Biol 29:5070–5083. https://doi.org/10.1128/MCB.00138-09
Hanke S, Mann M (2009) The phosphotyrosine interactome of the insulin receptor family and its substrates IRS-1 and IRS-2. Mol Cell Proteom 8:519–534. https://doi.org/10.1074/mcp.M800407-MCP200
Hebert LE, Scherr PA, McCann JJ et al (2001) Is the risk of developing Alzheimer’s disease greater for women than for men? Am J Epidemiol 153:132–136. https://doi.org/10.1093/aje/153.2.132
Hemmings BA, Restuccia DF (2012) PI3K-PKB/Akt pathway. Cold Spring Harb Perspect Biol 4:a011189. https://doi.org/10.1101/cshperspect.a011189
Jeong W, Lee H, Cho S, Seo J (2019) ApoE4-induced cholesterol dysregulation and its brain cell type-specific implications in the pathogenesis of Alzheimer’s disease. Mol Cells. https://doi.org/10.14348/molcells.2019.0200
Jinno S (2009) Structural organization of long-range GABAergic projection system of the hippocampus. Front Neuroanat 3:13. https://doi.org/10.3389/neuro.05.013.2009
Khakpai F, Nasehi M, Haeri-Rohani A et al (2013) Septo-hippocampo-septal loop and memory formation. Basic Clin Neurosci 4:5–23
Kleinridders A, Ferris HA, Cai W, Kahn CR (2014) Insulin action in brain regulates systemic metabolism and brain function. Diabetes 63:2232–2243. https://doi.org/10.2337/db14-0568
Leutgeb S, Leutgeb JK, Barnes CA et al (2005) Neuroscience: Independent codes for spatial and episodic memory in hippocampal neuronal ensembles. Science 309:619–623. https://doi.org/10.1126/science.1114037
Li F, Liu K, Wang A, Harris PWR, Vickers MH, Guan J (2019) Cyclic glycine-proline administration normalizes high-fat diet-induced synaptophysin expression in obese rats. Neuropeptides 76:101935. https://doi.org/10.1016/j.npep.2019.05.006
Long YC, Cheng Z, Copps KD, White MF (2011) Insulin receptor substrates Irs1 and Irs2 coordinate skeletal muscle growth and metabolism via the Akt and AMPK pathways. Mol Cell Biol 31:430–441. https://doi.org/10.1128/MCB.00983-10
Machado-Neto JA, Fenerich BA, Alves APNR et al (2018) Insulin Substrate Receptor (IRS) proteins in normal and malignant hematopoiesis. Clinics. https://doi.org/10.6061/CLINICS/2018/E566S
Marcuzzi A, Loganes C, Valencic E et al (2018) Neuronal dysfunction associated with cholesterol deregulation. Int J Mol Sci. https://doi.org/10.3390/ijms19051523
Milenkovic I, Vasiljevic M, Maurer D et al (2013a) The parvalbumin-positive interneurons in the mouse dentate gyrus express GABAA receptor subunits alpha1, beta2, and delta along their extrasynaptic cell membrane. Neuroscience 254:80–96. https://doi.org/10.1016/j.neuroscience.2013.09.019
Milenkovic I, Vasiljevic M, Maurer D et al (2013b) The parvalbumin-positive interneurons in the mouse dentate gyrus express GABAA receptor subunits α1, β2, and δ along their extrasynaptic cell membrane. Neuroscience 254:80–96. https://doi.org/10.1016/j.neuroscience.2013.09.019
Miyawaki CE (2015) Association of social isolation and health across different racial and ethnic groups of older Americans. Ageing Soc 35:2201–2228. https://doi.org/10.1017/S0144686X14000890
Moloney AM, Griffin RJ, Timmons S et al (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 31:224–243. https://doi.org/10.1016/j.neurobiolaging.2008.04.002
Neves FS, Marques PT, Barros-Aragão F et al (2018) Brain-defective insulin signaling is associated to late cognitive impairment in post-septic mice. Mol Neurobiol 55:435–444. https://doi.org/10.1007/s12035-016-0307-3
Paxinos G, Watson C (2013) The rat brain in stereotaxic coordinates: hard cover. Elsevier Science, Amsterdam
Payami H, Montee KR, Kaye JA et al (1994) Alzheimer’s disease, apolipoprotein E4, and gender. JAMA 271:1316–1317
Pederson TM, Kramer DL, Rondinone CM (2001) Serine/threonine phosphorylation of IRS-1 triggers its degradation: possible regulation by tyrosine phosphorylation. Diabetes 50(1):24–31. https://doi.org/10.2337/diabetes.50.1.24
Ribes-Navarro A, Atef M, Sánchez-Sarasúa S et al (2019) Abscisic acid supplementation rescues high fat diet-induced alterations in hippocampal inflammation and IRSs expression. Mol Neurobiol 56(1):454–464. https://doi.org/10.1007/s12035-018-1091-z
Rondinone CM, Wang LM, Lonnroth P et al (1997) Insulin receptor substrate (IRS) 1 is reduced and IRS-2 is the main docking protein for phosphatidylinositol 3-kinase in adipocytes from subjects with non-insulin-dependent diabetes mellitus. Proc Natl Acad Sci USA 94:4171–4175. https://doi.org/10.1073/pnas.94.8.4171
Sadagurski M, Cheng Z, Rozzo A et al (2011) IRS2 increases mitochondrial dysfunction and oxidative stress in a mouse model of Huntington disease. J Clin Invest 121:4070–4081. https://doi.org/10.1172/JCI46305
Sánchez-Sarasúa S, Moustafa S, García-Avilés Á et al (2016) The effect of abscisic acid chronic treatment on neuroinflammatory markers and memory in a rat model of high-fat diet induced neuroinflammation. Nutr Metab (Lond) 13:73. https://doi.org/10.1186/s12986-016-0137-3
Shirakami A, Toyonaga T, Tsuruzoe K et al (2002) Heterozygous knockout of the IRS-1 gene in mice enhances obesity-linked insulin resistance: a possible model for the development of type 2 diabetes. J Endocrinol 174(2):309–319. https://doi.org/10.1677/joe.0.1740309
Spinelli M, Fusco S, Mainardi M et al (2017) Brain insulin resistance impairs hippocampal synaptic plasticity and memory by increasing GluA1 palmitoylation through. Nat Commun 8(1):2009. https://doi.org/10.1038/s41467-017-02221-9
Spinelli M, Fusco S, Grassi C (2019) Brain insulin resistance and hippocampal plasticity: Mechanisms and biomarkers of cognitive decline. Front Neurosci 13:788. https://doi.org/10.3389/fnins.2019.00788
Talbot K, Wang H-Y, 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 122:1316–1338. https://doi.org/10.1172/JCI59903
Tartaglia N, Du J, Tyler WJ, Neale E, Pozzo-Miller L, Lu B (2001) Protein synthesis-dependent and -independent regulation of hippocampal synapses by brain-derived neurotrophic factor. J Biol Chem 276(40):37585–37593. https://doi.org/10.1074/jbc.M101683200
Uddin MS, Kabir MT, Al Mamun A et al (2019) APOE and Alzheimer’s disease: evidence mounts that targeting apoe4 may combat Alzheimer’s pathogenesis. Mol Neurobiol 56:2450–2465. https://doi.org/10.1007/s12035-018-1237-z
Ujfalussy B, Kiss T, Orbán G et al (2007) Pharmacological and computational analysis of alpha-subunit preferential GABA (A) positive allosteric modulators on the rat septo-hippocampal activity. Neuropharmacology 52:733–743. https://doi.org/10.1016/j.neuropharm.2006.09.022
Ungar L, Altmann A, Greicius MD (2014) Apolipoprotein E, gender, and Alzheimer’s disease: an overlooked, but potent and promising interaction. Brain Imaging Behav 8:262–273. https://doi.org/10.1007/s11682-013-9272-x
Vann SD (2010) Re-evaluating the role of the mammillary bodies in memory. Neuropsychologia 48:2316–2327. https://doi.org/10.1016/j.neuropsychologia.2009.10.019
Vann SD, Nelson AJD (2015) The mammillary bodies and memory: more than a hippocampal relay. Progress in brain research. Elsevier, Amsterdam, pp 163–185
Vann SD, Erichsen JT, O’Mara SM, Aggleton JP (2011) Selective disconnection of the hippocampal formation projections to the mammillary bodies produces only mild deficits on spatial memory tasks: implications for fornix function. Hippocampus 21:945–957. https://doi.org/10.1002/hipo.20796
Whelan SA, Dias WB, Thiruneelakantapillai L et al (2010) Regulation of insulin receptor substrate 1 (IRS-1)/AKT kinase-mediated insulin signaling by O-linked ??-N-acetylglucosamine in 3T3-L1 adipocytes. J Biol Chem 285:5204–5211. https://doi.org/10.1074/jbc.M109.077818
Yamada M, Ohnishi H, Sano SI et al (1997) Insulin receptor substrate (IRS)-1 and IRS-2 are tyrosine-phosphorylated and associated with phosphatidylinositol 3-kinase in response to brain-derived neurotrophic factor in cultured cerebral cortical neurons. J Biol Chem 272(48):30334–30339. https://doi.org/10.1074/jbc.272.48.30334
Yan Y, Dominguez S, Fisher DW, Dong H (2018) Sex differences in chronic stress responses and Alzheimer’s disease. Neurobiol Stress 8:120–126. https://doi.org/10.1016/j.ynstr.2018.03.002
Yankelevitch-Yahav R, Franko M, Huly A, Doron R (2015) The Forced Swim Test as a Model of Depressive-like Behavior. J Vis Exp. https://doi.org/10.3791/52587
Yarchoan M, Toledo JB, Lee EB et al (2014) Abnormal serine phosphorylation of insulin receptor substrate 1 is associated with tau pathology in Alzheimer’s disease and tauopathies. Acta Neuropathol 128:679–689. https://doi.org/10.1007/s00401-014-1328-5
Yuan M, Meyer T, Benkowitz C et al (2017) Somatostatin-positive interneurons in the dentate gyrus of mice provide local- and long-range septal synaptic inhibition. Elife. https://doi.org/10.7554/eLife.21105
Yuki D, Sugiura Y, Zaima N, Akatsu H, Takei S, Yao I, Maesako M, Kinoshita A, Yamamoto T, Kon R, Sugiyama K, Setou M (2014) DHA-PC and PSD-95 decrease after loss of synaptophysin and before neuronal loss in patients with Alzheimer's disease. Sci Rep 4:7130. https://doi.org/10.1038/srep07130
Zarei M, Patenaude B, Damoiseaux J et al (2010) Combining shape and connectivity analysis: an MRI study of thalamic degeneration in Alzheimer’s disease. Neuroimage 49:1–8. https://doi.org/10.1016/j.neuroimage.2009.09.001
Zhang Y, Qiu B, Wang J, Yao Y, Wang C, Liu J (2017) Effects of BDNF-transfected BMSCs on neural functional recovery and synaptophysin expression in rats with cerebral infarction. Mol Neurobiol 54(5):3813–3824. https://doi.org/10.1007/s12035-016-9946-7
Zou D, Chen L, Deng D et al (2016) DREADD in parvalbumin interneurons of the dentate gyrus modulates anxiety, social interaction and memory extinction. Curr Mol Med 16:91–102. https://doi.org/10.2174/1566524016666151222150024
Acknowledgements
The authors want to thank all the people that generously donated to Crowdfunding Precipita (FECYT) that sponsored ARN, and the economic support from the Association of Alzheimer Families, AFA, Castellon.
Funding
This work has been supported by Plan Propi from Universitat Jaume I UJI-B2018-01 and Conselleria de Educació, Cultura I sport AICO/2015/042 to AMSP. Predoctoral fellowship from Conselleria de Innovación, Universidades, Ciencia y Sociedad Digital ACIF/2016/250 to SSS.
Author information
Authors and Affiliations
Contributions
SSS performed the surgeries, conducted the behavioral paradigms, immunofluorescence, and image analysis, and contributed to manuscript writing; ARN generated the shRNA and AAV particles, did the PCR analysis, and contribute to writing of the manuscript; MTB contributed to the immunofluorescence studies; AMSP designed the study and wrote the manuscript.
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that the manuscript has not been submitted elsewhere. Authors report no conflict of interest.
Ethical approval
The procedures followed the directive 86/609/EEC of the European Community on the protection of animals used for scientific purposes. The experiments were approved by the Ethics Committee of the University Jaume I (scientific procedure 2019/VSC/PEA/0143).
Informed consent.
Not applicable.
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Sánchez-Sarasúa, S., Ribes-Navarro, A., Beltrán-Bretones, M.T. et al. AAV delivery of shRNA against IRS1 in GABAergic neurons in rat hippocampus impairs spatial memory in females and male rats. Brain Struct Funct 226, 163–178 (2021). https://doi.org/10.1007/s00429-020-02155-x
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00429-020-02155-x