Molecular Biotechnology

The Molecular Biotechnology Unit develops and establishes cellular and molecular assay systems. The methods comprise cell-based assays, analysis of gene expression, immunological methods, protein biochemistry, complex cell culture models as well as animal experiments. The unit further provides service for cellular compound screening such as efficacy, toxicity and transport. In addition, a main focus is on establishing and validating animal models for the investigation of the enzyme functions.

Pathological role of superantigens derived from expression of human endogenous retroviruses

Multiple Sclerosis (MS) is a progressive and chronic inflammatory disorder of the central nervous system. MS is characterized by a pathological demyelination of axons. Depending on the site of inflammation symptoms vary greatly and include paralysis or numbness of extremities, impaired vision and speech, dizziness, cognitive impairment, and fatigue. An underlying cause for the development of MS has not been identified so far. A widely accepted theory of MS pathology focusses on auto-reactive T-cells acting against axonal myelin. This simplistic view is challenged by a number of clinical observations arguing against a sole autoimmunological process.

First, newly emerging MS lesions are devoid of immune cells and second, MRI imaging suggests alterations in affected areas before the appearance of signs of inflammation. Furthermore, relapses during disease progression do not correlate with the disability patients face in the later stages of MS and standard therapy using immune modulatory and/or immune suppressive drugs has no influence on primary or secondary progredient forms of MS. Consequently, MS consists of 2 arms, 1) an inflammatory part (relapses) and 2) a slowly progressive neurodegenerative part.

The less understood 2nd arm might be correlated with the expression of human endogenous retroviruses (HERVs). HERVs are remnants of an ancient retroviral infection integrating viral DNA into the human genome. A number of HERVs still possess open reading frames, e.g. coding for the former viral envelope protein. These proteins could show superantigenic properties.

Therefore, the project focusses on the immunological properties of HERV-derived viral envelope proteins and the development of humanized monoclonal antibodies as alternative therapy for the treatment of MS.

Novel humanized animal models for studying the pathogenesis of Alzheimer’s disease

Alzheimer’s disease (AD) is characterized by a progressive loss of neurons and is accompanied by an impairment of learning and memory in elderly individuals. Besides major efforts in academic and industrial research, modeling the course of the disease in animals is challenging. A number of established animal models, especially mouse models of AD usually enable investigations only on certain pathological aspects. This is mainly attributed to the overexpression of Alzheimer-related genes in those models frequently including rare mutations devoid in patients with sporadic AD. We have selected 4 key proteins responsible for the development of AD and replaced them in mice by their human counterparts under control of the natural murine promoters. Fully humanization at these AD-specific loci by crossbreeding builds a platform to test new drugs against Alzheimer’s disease.

Rabbit model of in-stent restenosis for drug development

© Photo Fraunhofer IZI

Coronary arteries narrowed by atherosclerotic changes are the underlying cause of angina pectoris and myocardial infarction. To re-open an occluded vessel, percutaneous transluminal angioplasty (PTA) with and without stent application is performed. Frequently, the operated vessel segment is re-occluded by hyperproliferation of smooth muscle cells in combination with invasion of monocytes forming a neointima. The process is called in-stent restenosis (ISR) if occurring after stent application.

The Molecular Biotechnology Unit has an established in vivo model of ISR available. Stent application in atherosclerotic New Zealand white rabbits is a powerful tool to investigate novel drug candidates, biomaterials and medicinal products.

Compound Screening in vitro

Activation of a GPCR by different ligands
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Activation of a GPCR by different ligands

Efficacy

Permanent cell cultures are used for efficacy testing of novel compounds. Assays specific for the investigated target proteins are applied. Aim is the selection of the most potent compounds.

 

Determination of cytotoxicity for 2 compounds (A and B) using a human hepatocyte cell line
© Photo Fraunhofer IZI

Determination of cytotoxicity for 2 compounds (A and B) using a human hepatocyte cell line

Toxicity

Cytotoxic and proliferative effects of novel compounds are screened on mammalian cell lines. > 100 cell lines are available for screening. Due to their pivotal role in drug metabolism, all compounds are tested on permanent human hepatocytes.

Apparent permeability coefficient of selected molecules
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Apparent permeability coefficient of selected molecules

Transport

For the prediction of bioavailability in animal models, compounds are tested on an epithelial cell-derived transport model. The applied mammalian cells are human colorectal adenocarcinoma cell line, CaCo-2. Analysis is performed by LC-MS. The apparent permeability coefficient (Papp) is used to estimate bioavailability.

Cell culture of Iba1-positive primary murine microglia cells
© Photo Fraunhofer IZI

Cell culture of Iba1-positive primary murine microglia cells

Cell culture of GFAP-positive primary murine glia cells
© Photo Fraunhofer IZI

Cell culture of GFAP-positive primary murine glia cells

Primary cell models

The Molecular Biotechnology Unit has a longstanding expertise in experimental research using primary cells of the central nervous system.

For the prediction of bioavailability in animal models, compounds are tested on an epithelial cell-derived transport model. The applied mammalian cells are human colorectal adenocarcinoma cell line, CaCo-2. Analysis is performed by LC-MS. The apparent permeability coefficient (Papp) is used to estimate bioavailability.

Phenotyping transgenic mouse models

8 x PhenoMaster-System (TSE-Systems)
© Photo Fraunhofer IZI

8 x PhenoMaster-System (TSE-Systems)

IntelliCage (NewBehavior) for automated behavior screening in groups
© Photo Fraunhofer IZI

IntelliCage (NewBehavior) for automated behavior screening in groups

Phenotyping of transgenic mouse models follows the SHIRPA protocol. It is divided into primary, secondary and tertiary screen. Comprehensive test batteries for evaluating animal health and specific functions, e.g. motor coordination, cognition and emotion are available. Focus is on automated home cage observation to minimize the contact of humans with the laboratory mice.

 

Histology

Hematoxylin/Oil-Red staining of a plaque in the arterial wall of an atherosclerotic rabbit
© Photo Fraunhofer IZI

Hematoxylin/Oil-Red staining of a plaque in the arterial wall of an atherosclerotic rabbit

Thioflavin S-positive deposits (green) in hippocampus of an Alzheimer mouse model
© Photo Fraunhofer IZI

Thioflavin S-positive deposits (green) in hippocampus of an Alzheimer mouse model

To investigate disease-relevant pathologic alterations, a number of cutting and staining techniques are available.

 

Pharmacology

Concentration of a compound in the circulation after oral exposure
© Photo Fraunhofer IZI

Concentration of a compound in the circulation after oral exposure

The determination of pharmacological parameters, e.g. bioavailability and plasma half-life, is done in-house using catheterized rats. Experiments are performed after extensive testing of compounds in isolated cell cultures and after allowance by the local animal well-fare committee. For measuring compound levels in body fluids, LC-MS-based methods are available.

  • Cynis H, Frost JL, Crehan H , Lemere CA. Immunotherapy targeting pyroglutamate-3 Aβ: prospects and challenges. Mol Neurodegener. 2016 Jun 30;11(1):48. DOI dx.doi.org/10.1186/s13024-016-0115-2
  • Cynis H, Funkelstein L, Toneff T, Mosier C, Ziegler M, Koch B, Demuth HU, Hook V. Pyroglutamate-Amyloid-beta and Glutaminyl Cyclase Are Colocalized with Amyloid-beta in Secretory Vesicles and Undergo Activity-Dependent, Regulated Secretion. Neurodegenerative Diseases. 2014; 14(2):85-97. DOI http://dx.doi.org/10.1159/000358430
  • Höfling C, Indrischek H, Höpcke T, Waniek A, Cynis H, Koch B, Schilling S, Morawski M, Demuth HU, Roßner S, Hartlage-Rübsamen M. Mouse strain and brain region-specific expression of the glutaminyl cyclases QC and isoQC. International Journal of Developmental Neuroscience. 2014 Aug; 36:64-73. DOI http://dx.doi.org/10.1016/j.ijdevneu.2014.05.008
  • Morawski M, Schilling S, Kreuzberger M, Waniek A, Jäger C, Koch B, Cynis H, Kehlen A, Arendt T, Hartlage-Rübsamen M, Demuth HU, Roßner S. Glutaminyl cyclase in human cortex: correlation with (pGlu)-amyloid-beta load and cognitive decline in Alzheimer‘s disease. Journal of Alzheimer‘s Disease. 2014;39(2):385-400. DOI dx.doi.org/10.3233/JAD-131535.
  • Cynis H, Kehlen A, Haegele M, Hoffmann T, Heiser U, Fujii M, Shibazaki Y, Yoneyama H, Schilling S, Demuth HU. Inhibition of Glutaminyl Cyclase alleviates CCL2-mediated inflammation of non-alcoholic fatty liver disease. Int. J. Exp. Pathol. 94 (2013), 3:217-225. DOI dx.doi.org/10.1111/iep.12020
  • Nussbaum J, Schilling S, Cynis H, Silva A, Swanson E, Wangsanut T, Tayler K, Wiltgen B, Hatami A, Rönicke R, Reymann K, Hutter-Paier B, Alexandru A, Jagla W, Graubner S, Glabe CG, Demuth HU, Bloom GS. Prion-like behavior and tau-dependent cytotoxicity of pyroglutamylated amyloid peptides. Nature 485 (2012), 7400:651-655. DOI dx.doi.org/10.1038/nature11060
  • Cynis H, Hoffmann T, Friedrich D, Kehlen A, Gans K, Kleinschmidt M, Rahfeld JU, Wolf R, Wermann M, Stephan A, Haegele M, Sedlmeier R, Graubner S, Jagla W, Müller A, Eichentopf R, Heiser U, Seifert F, Quax PH, de Vries MR, Hesse I, Trautwein D, Wollert U, Berg S, Freyse EJ, Schilling S, Demuth HU. The isoenzyme of Glutaminyl Cyclase is an important regulator of monocycte infiltration under inflammatory conditions. EMBO Mol. Med. 3 (2011), 9:545-558. DOI dx.doi.org/10.1002/emmm.201100158
  • Wirths O, Breyhan H, Cynis H, Schilling S, Demuth HU, Bayer TA. Intraneuronal pyroglutamate-Abeta 3-42 triggers neurodegeneration and lethal neurological deficits in a transgenic mouse model. Acta Neuropathol. 118 (2009), 4:487-496. DOI dx.doi.org/10.1007/s00401-009-0557-5
  • Cynis H, Scheel E, Saido T, Schilling S, Demuth HU. Amyloidogenic processing of amyloid precursor protein: evidence of a pivotal role of Glutaminyl Cyclase in generation of pyroglutamate-modified amyloid-beta. Biochemistry 47 (2008), 28:7405-7413. DOI dx.doi.org/10.1021/bi800250p
  • Schilling S, Zeitschel U, Hoffmann T, Heiser U, Francke M, Kehlen A, Holzer M, Hutter-Paier B, Prokesch M, Windisch M, Jagla W, Schlenzig D, Lindner C, Rudolph T, Reuter G, Cynis H, Montag D, Demuth HU, Rossner S. Glutaminyl Cyclase inhibition attenuates pyroglutamate Abeta and Alzheimer’s disease like pathology. Nat. Med. 14 (2008), 10:1106-1111. DOI dx.doi.org/10.1038/nm.1872
  • Cynis H, Rahfeld JU, Stephan A, Kehlen A, Koch B, Wermann M, Demuth HU, Schilling S. Isolation of an isoenzyme of human Glutaminyl Cyclase: Retention in the Golgi complex suggests involvement in the protein maturation machinery. J. Mol. Biol. 379 (2008), 5:966-980. DOI dx.doi.org/10.1016/j.jmb.2008.03.078
  • Cynis H, Schilling S, Bodnar M, Hoffmann T, Heiser U, Saido TC, Demuth HU. Inhibition of Glutaminyl Cyclase alters pyroglutamate formation in mammalian cells. Biochim. Biophys. Acta 1764 (2006), 10:1618-1625.