CYBRAT-AD

Naziv projekta: Cytopathological characterization of the brain in a rat model of sporadic alzheimer’s disease

Trajanje: 15.6.2010. – 15.6.2012.

PROJECT SUMMARY

Background. Sporadic AD is characterized by a slow, clinically asymptomatic onset in humans which is why animal models are needed to reveal the onset and development of neuropathologycal alterations and the time-course of their progression. Currently transgenic mice are widely used animal models for AD investigation and are mostly generated by manipulation of genes involved in amyloid homeostasis due to they can only represent a rare familial form of AD. In contrast to these models, over 20 years the main applicant of the project has been doing a research on characterization of a non-transgenic rat model for predominating sporadic form of AD. The experimental sAD condition in this model is induced by intracerebroventricular injection of streptozotocin (STZ-icv). STZ is selectively toxic to insulin producing/secreting/responding cells and its icv administration leads to development of insulin resistant brain state, recently found in sAD human brain post-mortem as well. STZ-icv rat model also demonstrates cognitive deficits and shares some molecular and structural cerebral features with human sAD. An extensive cytopathological characterization of STZ-icv rat model is a prerequisite for recognizing it as a representative animal sAD model, suitable for usage in translational AD medicine.

Hypothesis. STZ-icv-treated rats exhibit progressive AD-like pathology as time after injection increases.

Research aims. The main objective of the project was to provide the extensive cytopathological characterization of the brain in a specific animal model of sporadic Alzheimer’s disease (sAD). In line with that, project additionally aimed to establish a new approach to AD research and to develop new capacities for AD research in Croatia.

Methods. In line with the specific goals of the project, a number of methods have been used to reveal the specific brain lesions developed over the nine months following the STZ-icv injection, associated with neuronal loss and pathological accumulation of amyloid β and hyperphosphorylated tau protein. These lesions have been then compared with literature reports of lesions found in AD patients post-mortem. Additionally, follow-up of cognitive and cerebral cholinergic deficit in the STZ-icv rat model has been performed, respectively.

Accomplished results. The project has achieved the following results and significant novel findings on STZ-icv rat model:

– STZ-dose- and a bi-phasic time-dependent pattern of changes with acute and slow, progressive chronic memory decline

– STZ-dose-, time-, region- and cholinergic receptor type-dependent alterations in cholinergic neurotransmission, and changes in hippocampal cholinesterase activity which correlate well with cognitive deficits

– STZ-dose- and time-dependent development of structural AD-like changes in the form of neuronal loss and pathological amyloid β and hyperphosphorylated tau protein aggregation, respectively, manifested not earlier than 3 to 6 months after the STZ administration, which further progressed

Conclusions. Bearing in mind the limitations of translating the results from animal studies to humans, project results provided evidence which strongly support the hypothesis that STZ-icv rat model resembles the human sAD condition as much as possible and, to the best of our knowledge, is the only non-transgenic sAD model known until now. Furthermore, the results have revealed that future drug interventional studies in STZ-icv model should be carefully designed taking into consideration the specific time-dependent pattern of cognitive changes when concluding on drug effectiveness. The model offers innovative approach to explore the sAD pathophysiology and novel, more effective therapeutic strategies as well as the opportunity for Croatia to improve its international competitiveness and bring in research funding from both, public and industry.

Project members

Project team and institutions involved:

Project was coordinated by the Project Leader, Melita Šalković-Petrišić, a Professor of Pharmacology at the University of Zagreb, School of Medicine (UZSM), and her team which consisted of three Professors – experts in Cholinergic Pharmacology, Neuroanatomy and Electron Microscopy, two PhD students and one technician. The collaboration with the co-applicant, Dr. Rodrigo Kuljiš, a neurologist, and his institution, the University of Texas, Medical Branch in Galveston, Texas, USA, was active during the preparation of the project proposal and in the first 18 months of the project. The project results published in congress abstracts and those prepared for publication including the cytopathological characterization done in the last 6 project’s months are based on the research carried out completely at two UZSM’s units; Department of Pharmacology and Croatian Institute for Brain Research.

Project leader:

Professor Melita Šalković-Petrišić, MD, PhD (University of Zagreb, School of Medicine, Department of Pharmacology, Šalata 11, Zagreb, Croatia) – melitas@mef.hr

Co-applicant (15.06.2010.-23.12.2011.):

Professor Rodrigo Kuljiš, MD, PhD (University of Texas Medical Branch, Department of Neurology, 301 University Boulevard, Galveston, Texas) – rokuljis@UTMB.EDU

Co-workers:

Professor Vlasta Bradamante, MD, PhD (University of Zagreb, School of Medicine, Department of Pharmacology, Šalata 11, Zagreb, Croatia) – vbradam@mef.hr

Assistant Jelena Osmanović-Barilar, MD (University of Zagreb, School of Medicine, Department of Pharmacology, Šalata 11, Zagreb, Croatia) – josmanov@mef.hr

Research assistant Ana Knezović, dipl.ing.mol.biol.(University of Zagreb, School of Medicine, Department of Pharmacology, Šalata 11, Zagreb, Croatia) – ana.knezovic@mef.hr

Research assistant Marina Knapić, dipl.ing.mol.biol.(University of Zagreb, School of Medicine, Department of Pharmacology, Šalata 11, Zagreb, Croatia) – knapic.marina@gmail.com

Consultancy and expertise:

Professor Goran Šimić, MD, PhD (University of Zagreb, School of Medicine, Croatian Institute for Brain Research, Department of Neuroscience, Šalata 12, Zagreb, Croatia) – gsimic@hiim.hr

Professor Srećko Gajović, MD, PhD (University of Zagreb, School of Medicine and Croatian Institute for Brain Research, Department of Histology and Embriology, Šalata 12, Zagreb, Croatia) – srecko.gajovic@hiim.hr

Financial support

The project was funded by Unity Through Knowledge Fund which involves support of the World bank and Croatian Ministry of Science, Education and Sport(MZOS).

Total amount of 1.027.660,15 HRK has been spent for the project realization.

News

12.10.2012. Award for the best poster: Knezović A, Osmanović-Barilar J, Mandel S, Youdim M, Riederer P, Šalković-Petrišić M. Therapeutic effects o multifunctional iron-chelating agent in streptozotocin-induced rat model of sporadic Alzheimer’s disease. 6th Croatian Congress on Alzheimer’s disease, Primošten, Croatia, October 10-13, 2012.

15.06.2012. Invited lecture (project leader) at the Medical Information Conference Croatia (MICC), Medical School University of Zagreb, Croatia

06.06.-09.06.2012. Participation and poster presentation of the results at the 10th European Congress of Neuropathology, June 6th-9th 2012., Edinburgh, Scotland, UK (link on the programme)

05.06.2012. Submitted article (reviewing in process) “What have we learned from the streptozotocin-induced animal model of sporadic Alzheimer’s disease, about the therapeutic strategies in Alzheimer’s research” in Journal of Neuronal Transmission; Authors: Salkovic-Petrisic M, Knezovic A, Hoyes S, Riederer P.

25.05.2012. Participation and poster presentation of the preliminary results (Ana Knezović PhD thesis-completely based on the UKF project research) at the “PhD Day” of the Doctoral Studies at the Medical School of Zagreb, Zagreb, Croatia

09.05.-12.05.2012. Participation and poster presentation of the results at the 12th International Stockholm/Springfield Symposium on Advances in Alzheimer Therapy, May 9th-12th 2012., Stockholm, Sweden

27.04.-28.04.2012. Project leader participated at the “Interdisciplinary Neuroscience” Symposium in honour of Prof. Dr. Peter Riederer with the lecture „What do cognitive deficits correlate with in a rat model of sporadic Alzheimer’s disease?”, Würzburg, Germany

26.04.2012. The invited lecture held by the project leader Professor Melita Šalković-Petrišić “Modelling of sporadic Alzheimer’s disease” at the 1st WG4 Scientific Meeting: Monoamines and Drugs: New insights into the brain disorders, organized within the COST Action CM1103 “Structure-based drug design for diagnosis and treatment of neurological diseases”, Zagreb, Croatia

21.04.-04.05.2012. Research assistent Ana Knezovic visited Department of Pathology, University of Würzburg, Würzburg, Germany for scientific work

15.04.-20.04.2012. Research assistant Ana Knezović participated FENS-IBRO school: Drugs and the brain: an update in psycho-pharmacology from experimantal to clinic, April 15th-20th 2012., Braga, Portugal

16.03.2012. Poster presentation within the public debate “Early detection of the Alzheimer’s disease” during the Brain awareness week, Zagreb Croatia

12.10.-5.11.2011. Research assistent Ana Knezovic visited University of Texas Medical Branch, Galveston, Texas, USA for scientific work

12.10.-16.10.2011. Project leader prof. Melita Šalković-Petrišić visited UKF project collaborator prof. Rodrigo Kuljis at University of Texas Medical Branch, Galveston, Texas, USA

07.10.2011. PhD Thesis proposal submission of Ana Knezovic at the University of Zagreb, School of Medicine, focused 100% on the research carried out within this UKF project, Zagreb, Croatia

4.10.-5.10.2011. Meeting with prof Harry Steinbuch from University of Maastricht in Maastricht – discussion on the potential FP7 project proposal in 2012

22.-25.9.2011. Participation and poster presentation of the results at the SiNAPSA Neuroscience Conference: Central European FENS Featured Regional Meeting, September 22nd-25th 2011., Ljubljana, Slovenia (link on the programme)

21.9.2011. Project proposal to Alzheimer’s Drug Discovery Foundation: Cerebral glucose transporter-2 as a novel target in age- and sporadic Alzheimer’s disease-related neurodegeneration

24.8.-28.8.2011. Research assistant Ana Knezović participated 10th ISN Advanced School of Neurochemistry: Molecular basis of higher cognitive functions, August 24th-28th 2011., Delphi, Greece

20.7.-26.7.2011. Research assistant Ana Knezović participated FENS-IBRO partially supported school: Metabolic aspects of chronic brain diseases, July 20th-26th 2011., Reisensburg-Günzburg, Germany

19.7.2011. Meeting with UKF project collaborator prof. Rodrigo Kuljis in Paris

16.7.-21.7.2011. Participation and poster presentation of the results at the Alzheimer’s association International Conference, July 16th-21st 2011., Paris, France (link on the programme)

15.-16.06.2011. Research assistant Ana Knezović presented her PhD thesis (based on the UKF project research) at the annual international PhD Symposium of the Medical University of Wien, Wien, Austria

9.3.-13.3.2011. Participation and poster presentation of the results at the 10th International Conference on Alzheimer’s and Parkinson’s Diseases, March 9th-13th 2011., Barcelona, Spain(link on the programme)

09.3.2011. Second meeting with DAAD project collaborators in Barcelona- discussion on the appropriate FP7 call in mid of July 2011 (joined DAAD and UKF collaboration)

18.02.2011. Joint (project leader and coapplicant) publication/review article published in Journal of Alzheimer’s disease, Epub ahead of print

15.01.2011. First meeting with DAAD project collaborators in Würzburg which included discussion on potential FP7 project proposal preparation (joined DAAD and UKF collaboration)

11.11.2010. Project leader participated the lecture „Successful Technology Transfer of University Research Results-ways looking at university research results from a commercial point of view“, held by Dr. Bruno Dalle Carbonare, The Business Development Company, Basel, Swiss, and organized by the Medical Faculty Office for Science and Technology Transfer. After the lecture, the project leader had an individual discussion with Dr. Carbonare regarding the commercial point of this UKF project.

04.-06.10.2010. Meeting of the co-applicant (Professor Kuljis) and the project leader (Professor Salkovic-Petrisic) in Zagreb

2010.-2011. Publications and presentations of coapplicant Professor Rodrigo Kuljis, MD, PhD, before the termination of the collaboration: Three publications/articles in Frontiers in Neurology-Dementia (August 26th 2010., December 2010., April 18th 2011.); Oral presentation at Mitchell Center for Neurodegenerative Diseases Seminar Series, The University of Texas Medical Branch, Galveston, TX, USA (April 8th 2011.); Congress poster presentation at the Alzheimer’s Association International Conference on Alzheimer’s Disease (AAICAD), Paris, France (July 16th-21st 2011.); Panel participation at 5th Annual Bazalides Research Symposium, Alzheimer’s Association, Houston, TX, USA (October 13th 2011.)

29.9.2010. Project leader participated the workshop „Intellectual property and its management in UKF projects“, organized by the Transfer Technology Office of the University of Zagreb and UKF office in Zagreb.

24.9.2010. Project announcement (oral presentation) at the 5th Croatian Congress on Alzheimer’s Disease with International Participation, September 22nd-25th 2010., Zadar, Croatia (link on the programme)

16.9.2010. Project announcement (poster presentation) at the 6th Croatian Congress of Pharmacology with International Participation, September 15th-18th, 2010., Opatija, Croatia (link on the programme)

14.7.2010. Publication (project announcement) in the official journal of the Zagreb Medical School, Zagreb, Croatia

15.6.2010. Official starting date of the project

Publications

Dissemination activities

Project has been publicized in the official Zagreb Medical School’s journal and presented to the Croatian AD-Patient Association, family members and caregivers via oral and poster presentations. The results achieved in the project have already been and will continue to be widely presented on a large number of national and international conferences targeting AD- and pharmacology oriented scientific community, as well as pharmaceutical industry. In addition to 2 already published review articles most closely related to the topic of the UKF project, results on cytopathological, cognitive and cholinergic characterization are in preparation for publication in the respectable journals and for dissemination by 1 PhD Thesis at UZSM.

Congress Abstracts (results):

1. Šalković-Petrišić M, Knezović A, Riederer P. Streptozotocin-induced experimental model of sporadic Alzheimr’s disease as a tool for testing novel therapeutic approaches in this disease. Neurologia Croatica; 6^th Croatian Congress on Alzheimer’s Disease, Primošten, Croatia, October 10-13, 2012

2. Knezović A, Osmanović-Barilar J, Mandel S, Youdim M, Riederer P, Šalković-Petrišić M. Therapeutic effects of multifunctional iron-chelating agent in a streptozotocin-induced rat model of sporadic Alzheimer’s disease. Neurologia Croatica; 6^th Croatian Congress on Alzheimer’s Disease, Primošten, Croatia, October 10-13, 2012

3. Knezović A, Knapić M, Šimić G, Šalković-Petrišić M. The expression of glial fibrillary acidic protein in the brain of streptozotocin rat model of sporadic Alzheimer’s disease. Clinical neuropathology; 31/4; 10th European Congress of Neuropathology organized by the British Neuropahological Society in conjuction with Euro-CNS, Edinburgh, Scotland, June 06-09, 2012

4. Knapić M, Knezović A, Šimić G, Šalković-Petrišić M. Hippocampal and cortical neuronal damage in the streptozotocin rat model of sporadic Alzheimer’s disease. Clinical neuropathology; 31/4; 10th European Congress of Neuropathology organized by the British Neuropahological Society in conjuction with Euro-CNS, Edinburgh, Scotland, June 06-09, 2012

5. Šalković-Petrišić M, Knezović A, Knapić M, Šimić G. Amyloid-beta pathology in the brain of a streptozotocin rat model of sporadic Alzheimer’s disease. Clinical neuropathology; 31/4; 10th European Congress of Neuropathology organized by the British Neuropahological Society in conjuction with Euro-CNS, Edinburgh, Scotland, June 06-09, 2012

6. Knezović A, Šalković-Petrišić M. Alteration of memory functions and cholinergic transmission in the brain of the streptozotocin-induced rat model of sporadic Alzheimer’s disease. Book of abstracts, PhD Day of the School of Medicine University of Zagreb, Zagreb, Croatia, May 25, 2012

7. Šalković-Petrišić M, Knezović A, Knapić M, Mandel S, Youdim M, Riederer P. Neuroprotective-neurorestorative potential of the novel multi-target, iron-chelator, M-30 in non-trangenic rat model of sporadic Alzheimer’s disease. Book of abstracts, 12th International Stockholm/Spriengfield Symposium on Advances in Alzheimer Therapy, May 9-12, 2012

8. Knezović A, Knapić M, Mandel S, Moussa Y, Riederer P, Šalković-Petrišić M. Streptozotocin model of sporadic Alzheimer’s disease in rats: characterization and novel drug testing. Book of abstracts, FENS-IBRO School: Drugs and the brain: an Update in Psycho-pharmacology from experimental to clinic, Brag, Portugal, April 15-20, 2012

9. Knezović A, Kuljis RO, Šalković-Petrišić M. Alterations of cholinergic transmission and memory functions in the non-trangenic model of sporadic Alzheimer’s disease. Book of abstracts, SiNAPSA Neuroscience Conference: Central European FENS Regional Meeting, Ljubljana, Slovenia, September 25-29, 2011

10. Knezović A, Osmanović-Barilar J, Grünblatt E, Riederer P, Šalković-Petrišić M. Fol-degrading enzyme expression in a rat model of sporadic Alzheimer’s disease. 10th ISN Advanced School of Neurochemistry: Molecular basis on higher cognitive functions. Delphi, Greece, August 24-28, 2011

11. Knezović A, Osmanović-Barilar J, Riederer P, Kuljis RO, Šalković-Petrišić M. Metabolic response to central streptozotocin application in a rat model of sporadic Alzheimer’s disease. FENS/IBRO Partially Supported School: Metabolic aspects of chronic brain diseases. Reisensburg/Günzburg, Germany, July 20-26, 2011

12. Kuljis RO. The Fourth Element Hypothesis of Alzheimer’s disease pathogenesis. Alzheimer’s and Dementia. 2011; Alzheimer’s Association International Conference on Alzheimer’s Disease (AAICAD). Paris, France, July 16-21, 2011 (CD version)

13. Knezović A, Šalković-Petrišić M. Early changes of cholinergic receptors in hippocampus and parietotemporal cortex of the rat model of sporadic Alzheimer’s disease. Alzheimer’s and Dementia. 2011; Alzheimer’s Association International Conference on Alzheimer’s Disease (AAICAD). Paris, France, July 16-21, 2011 (CD version)

14. Šalković-Petrišić M, Knezović A, Grünblatt E, Brueckner MK, Arendt T, Hoyer S, Riederer P. Correlation of cognitive deficits and brain cholinesterase activity in streptozotocin-intracerebroventricularly treated rat model of sporadic Alzheimer’s disease. Alzheimer’s and Dementia. 2011; Alzheimer’s Association International Conference on Alzheimer’s Disease (AAICAD). Paris, France, July 16-21, 2011 (CD version)

15. Knezović A, Kuljis RO, Riederer P, Šalković-Petrišić M. Non-trangenic rat model of sporadic Alzheimer’s disease: Is it suitable for testing of novel drug therapies?. Book of abstracts, 7th Young Scientist Association PhD Symposium, Wien, Austria, June 15-16, 2011

16. Knezovć A, Marjanović A,Osmanović-Barilar J, Bradamante V, Riederer P, Šalković-Petrišić M. Early changes of acetylcholnesterase activity and insulin system in the brain of the streptozotocin-intracerebroventricularly treated rat model of sporadic Alzheimer’s disease. Neurodegenerative diseases. 2011; 10^th International Conference on Alzheimer’s & Parkinson’s diseases. Barcelona, Spain, March 09-13, 2011 (CD version)

17. Šalković-Petrišić M, Knezović A,Osmanović-Barilar J, Grünblatt E, Riederer P. Correlation of cognitive deficits and cerebral amyloid angiopathy development in the streptozotocin-rat model of sporadic Alzheimer’s disease. Neurodegenerative diseases. 2011; 10^th International Conference on Alzheimer’s & Parkinson’s diseases. Barcelona, Spain, March 09-13, 2011 (CD version)

Publications:

1. Knezović A, Osmanović Barilar J, Ćurlin M, Hof PR, Šimić G, Riederer P, Šalković-Petrišić M. Staging of cognitive deficits and neuropathological and ultrastructural changes in streptozotocin-induced rat model of Alzheimer’s disease. J Neural Transm 2015; 122(4): 577-592.

2. Šalković-Petrišić M, Knezović A, Hoyer S, Riederer P. 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 2013; 120(1): 233-252

3. Kuljiš RO, Šalković-Petrišić M. Dementia, diabetes, Alzheimer’s disease and insulin resistance in the brain: Progress, dilemmas and new opportunities to tackle intersecting epidemic. J Alzheimers dis. 2011; 25 (1): 29-41.

Project announcement to the scientific community and the Alzheimer’s disease patients and caregivers:

1. Šalković-Petrišić M, Knezović A, Šimić G. Novosti u temeljnim istraživanjima Alzheimerove bolesti: karakterizacija štakorskog modela za testiranje novih lijekova. Brain Awareness week, Zageb, Croatia, March 16th 2012

2. Šalković-Petrišić M, Kuljiš R. Cytopahological characterization of the brain in a rat model of sporadic Alzheimer’s disease. Neurologia Croatica 2010; 59 (Suppl. 2): 19-19.

3. Šalković-Petrišić M, Kuljiš R. A new platform for characterization of the rat model of sporadic Alzheimer’s disease Periodicum Biologorum2010; 112 (Suppl. 1): 44-44.

4. Šalković-Petrišić M. Osobno iskustvo i pogled na translacijska istraživanja (engl. Personal experience and view on translational research). mef.hr 2010; 1: 39-40.

Co-applicants publications which acknowledge UKF project 64/10:

1. Foster PP, Rosenblatt KP, Kuljiš RO. Exercise-induced cognitive plasticity, implications for mild cognitive impairment and Alzheimer’s disease. Front Neurol. 2011; 2: 28.

2. Kuljiš RO. The Fourth Element Targeting hypothesis of Alzheimer’s disease pathogenesis and pathophysiology. Front Neurol. 2010; 1: 144.

3. Kuljiš RO. Integrative understanding of emergent brain properties, Quantum Brain hypothesis and Connectome alterations are key challenges to conquer Alzheimer’s. Front Neurol.2010 Aug 26; 1: 15.

Invited lectures:

1. Knezović A. Cognitive deficits and cholinergic neurotransmission in a rat model of sporadic Alzheimer’s disease. Annual meeting of the Croatian Pharmacological Society. Zagreb, Croatia, November 16, 2012.

2. Šalković-Petrišić M. Personal experience and view of the translational research. Medical Information Conference Croatia (MICC), Zagreb, Croatia, June 15, 2012.

3. Šalković-Petrišić M. What do cognitive deficits correlate with in a rat model of sporadic Alzheimer’s disease? „Interdisciplinary Neuroscience”. Symposium in honour of Professor Peter Riederer. Wuerzburg, Germany, April 27-28, 2012.

4. Šalković-Petrišić M. Modelling of sporadic Alzheimer’s disease. 1st WG4 Scientific Meeting: Monoamines and Drugs: New Insights into the Brain Disorders (part of COST Action CM1103 „Structure-based drug design for diagnosis and treatment of neurological diseases”). Zagreb, Croatia, April 26, 2012.

5. Kuljiš R. Panel participation 5th Annual Bazalides Research Symposium, Alzheimer’s Associatian, Houston & Southeast Texas Chapter, Houston, Texas, USA, October 13, 2012.

6. Kuljiš R. Dementia and diabetes: Biological mechanisms underlying the converging worldwide epidemics of diabetes mellitus and Alzheimer’s disease. Mitchell Center for Neurodegenerative Diseases Seminar Series, The University of Texas Medical Branch at Galveston. Galvestone, Texas, USA, April 8, 2011.

Graduate thesis:

1. Dalia Vađunec “Promjene inzulinskog signalnog puta u olfaktornom bulbusu eksperminetalnog štakorskog modela sporadične Alzheimerove bolesti”

Awards:

Award for the best poster:

Knezović A, Osmanović-Barilar J, Mandel S, Youdim M, Riederer P, Šalković-Petrišić M. Therapeutic effects o multifunctional iron-chelating agent in streptozotocin-induced rat model of sporadic Alzheimer’s disease. 6th Croatian Congress on Alzheimer’s disease, Primošten, Croatia, October 10-13, 2012.