Detection of post-translational modifications by fluorescent staining of two-dimensional gels.

Post-translational modifications (PTMs) are key to the regulation of functional activities of proteins. Quantitative and qualitative information about PTM stages of proteins is crucial in the discovery of biomarkers of disease. Recent commercial availability of fluorescent dyes specifically staining PTMs of proteins such as phosphorylation and glycosylation enables the specific detection of protein regulations taking place with respect to these modifications. Activity and molecular and signalling interactions of many proteins are determined by their extent of phosphorylation. In our search for biomarkers of neurodegenerative diseases such as Multiple Sclerosis (MS), using its animal model, Experimental autoimmune encephalomyelitis (EAE), we have applied the phopshorylation specific fluorescent dye, ProQ Diamond, to study changes taking place in the phosphoproteome. Subsequent Colloidal Coomassie staining of the same gels detects the changes at the whole proteome level. We have detected many changes taking place in the CNS tissue of the EAE animals at the whole proteome as well as at the phosphoproteome level that has given valuable insights into the patho-physiological mechanism of EAE and possibly also MS.

Regulation of GDNF and its receptor components GFR-alpha1, -alpha2 and Ret during development and in the mature retino-collicular pathway.

The development of the retino-tectal projection as part of the central visual pathway is accomplished around postnatal day (P) 10-14 in rodents, and trophic factors are important for topographic refinement of this projection. Emerging data indicate that GDNF may influence synaptic plasticity of this projection. To date, maturation-dependent kinetics of GDNF release and expression and biological function of single GDNF receptors along the retino-collicular pathway are ill-defined. Here, we examined mRNA and protein expression of GDNF and its multicomponent receptor complex in the retina and superior colliculus (SC) during postnatal development of the rat visual system, and after optic nerve (ON) injury by RT-PCR, immunoblotting and immunofluorescence. Stable mRNA transcription of GDNF and its receptors GFR-alpha1, -alpha2 and Ret was found in retina and SC throughout development into adulthood and after ON transection. Expression of GDNF protein increased during retinal development, declined in adulthood and was further reduced in injured retina. In the SC, GDNF peaked at P0, continuously declined with maturation, and was undetectable in the deafferentiated SC. GFR-alpha1 was abundant in retina and SC throughout, while GFR-alpha2 was not expressed. Since Ret was localized primarily to the vascular compartment, the receptor tyrosine kinase may play a minor role in neuronal GDNF signaling. In summary, we provide evidence for GDNF as survival and guidance factor during development of the retino-tectal projection with differential regulation in early and premature retina and SC. Postlesionally, midbrain targets do not induce GDNF, suggesting that retrograde GDNF is not essential for rescue of adult injured retinal ganglion cells (RGCs).

The quest for brain disorder biomarkers.

The identification of disease markers in tissues and body fluids requires an extensive and thorough analysis of its protein constituents. In our efforts to identify biomarkers for affective and neurological disorders we are pursuing several different strategies. On one hand we are using animal models that represent defined phenotypes characteristic for the respective disorder in humans. In addition, we are analyzing human specimens from carefully phenotyped patient groups. Several fractions representing different protein classes from human cerebrospinal fluid obtained by lumbar puncture are used for this purpose. Our biomarker identification efforts range from classical proteomics approaches such as two dimensional gel electrophoresis and mass spectrometry to phage display screens with cerebrospinal fluid antibodies.