The activities of our group are focused on the questions how recognition molecules at the cell surface and in the extracellular matrix shape the nervous system during ontogenesis and how these developmental features are recapitulated, at least to some extent, in synaptic plasticity as well as regeneration of the adult peripheral and central nervous systems during acute or chronic injuries. Mouse models for neurodegenerative diseases in humans are used with the aim to ameliorate the deficits resulting from trauma by application of beneficial recognition molecules.
The functions of recognition molecules not only rely on their protein backbones, but also on their glycan moieties which are increasingly recognized as fine-tuners of cell interactions. These two molecular entities, the protein backbone and the attached glycans, can act independently of each other. Also, their expression can be regulated independently of each other in different states of cellular activities.
Neural cell adhesion molecule (NCAM) & polysialic acid (PSA)
Intimately linked to investigations on the functions of glycans is the search for their receptors. For only a few of these glycans the receptors are known. We thus searched for novel receptors and are currently investigating receptors for the unusual glycan polysialic acid which is mainly carried by the neural cell adhesion molecule NCAM. We used an anti-idiotype approach in the search for receptors for PSA and found histone H1 and myristoylated alanine-rich C kinase substrate (MARCKS) as receptors. Histone H1 interacts with polysialic acid extracellularly, while the intracellularly localized MARCKS interacts with PSA within the plane of the plasma membrane from the opposite site. This interaction is noteworthy, because PSA penetrates the surface plasma membrane of live cells from the extracellular space to interact with MARCKS attached to the cytoplasmic side of the surface membrane. This interaction was shown by fluorescence resonance energy transfer (FRET) analysis. Penetration of PSA through the plasma membrane alters the membrane capacity of an artificial lipid bilayer.
NCAM is functionally associated with the dopamine D2 receptor and affects receptor signaling. This interaction increases receptor internalization, resulting in decreased receptor sensitization. Another important functional association of NCAM relates to the direct interaction of NCAM's intracellular domains with the receptor tyrosine kinase TrkB and the inwardly rectifying K+ channel KIR 3.3. Association of NCAM with calmodulin leads to NCAM-mediated neurite outgrowth, while NCAM's interaction with the fibroblast growth factor receptor regulates growth cone functions.
Nervous system recognition molecules and their glycans can be also functional outside the nervous system, for instance in the immune system, and can furthermore mediate interactions of mammalian cells with bacteria and viruses. The PSA tethered to NCAM is targeted by meningococcal infections, often followed by fatal autoimmune reactions, since this glycan is shared in structure with the host's PSA which is crucial for synaptic functions. Blockade of synaptic activity by autoantibodies often leads to paralysis of mental functions. Thus, we have successfully screened for glycomimetic compounds in phage display libraries of peptides and libraries of small organic compounds. These small molecules are presently under investigation for their mimetic potentials in vitro and in vivo.
Neural cell adhesion molecule L1
The L1 recognition molecule is not only important for nervous system development (note: mutants in mice and humans are severely disabled), but for regeneration in acute spinal cord injury model and in mouse models of Parkinson's, Huntington's and Alzheimer's degenerative diseases. Proteolytic cleavage of L1 by cognate proteases is a prerequisite for L1-mediated functions. We identified myelin basic protein (MBP), cathepsin E and the extracellular matrix glycoprotein reelin as proteases that generate distinct L1 fragments with distinct functional roles. MBP and cathepsin E only cleave sumoylated membrane-bound full-length L1 to generate 70 and 30 kDa L1 fragments, respectively, which are transferred from the plasma membrane to the nucleus of L1 expressing neurons and Schwann cells after stimulation with function-triggering L1 antibody. Generation of both fragments promotes neurite outgrowth, neuronal survival, Schwann cell process formation and myelination of dorsal root ganglion axons. L1-induced neurite outgrowth and neuronal survival are reduced in the MBP-deficient shiverer mouse and in wild-type cerebellar neurons treated with MBP antibody or an L1 peptide containing the MBP cleavage site, thus showing a novel function of MBP. Reelin cleaves L1 to generate an 80 kDa transmembrane fragment. Perturbed reelin-L1 interactions not only impair neuronal migration in vivo, but also neurite outgrowth and polarization in vitro.
Proteolysis of L1 is altered in several nervous system diseases. For instance, in Alzheimer's disease patients high levels of soluble L1 fragments can be detected in the cerebrospinal fluid and in a mouse model of Alzheimer´s disease altered levels of the 70 kDa L1 fragment could be observed. For many tumor cell types, not only levels of full-length L1, but also levels of proteolytic fragments are increased when compared to normal tissue. We are thus investigating the mechanisms underlying proteolysis of L1 and the role of proteolytic fragments in cell migration and survival as well as in neurite extension, myelination and formation of synaptic contacts and plasticity and on tumor growth. In addition, function-blocking (tumor metastasis) and function-triggering antibodies (neurite outgrowth, neuronal survival, myelination and synaptic plasticity) reacting with different domains of human L1 have been generated as scFv antibodies for preclinical approaches to therapy. Moreover, L1 peptides coupled to nanoparticles and biodegradable fibers/tubes have been generated and are currently evaluated for their ability to enhance regeneration of the traumatized mouse central and peripheral nervous systems. Since application of L1 via different carriers is beneficial for functional recovery in spinal cord regeneration and in Parkinson's and Huntington's diseases, the influence of L1 on the pathology of Alzheimer's disease was tested in a mouse model of this disease. Application of L1 led to amelioration of plaque load. L1 could be shown to interact with amyloid beta 42 more so than with amyloid beta 40, but not with the amyloid precursor protein.
L1 was also shown to be beneficial in the RETT syndrome of autism spectrum disorders in that neurally-induced reprogrammed human stem cells from a RETT syndrome person which do not express L1 and are mutated in the MeCP2 transcription factor could be induced to express L1 when transfected with wild type MeCP2 and full-length human L1. The RETT syndrome cells which did not show neurite outgrowth in culture could be induced to extend neurites when transfected with wild type MeCP2 and L1. Interestingly, the intracellular domain of L1 binds to MeCP2. The consequences of this interaction are presently under study.
Female mice hemizygous for the X-chromosome linked L1 gene are born with more neurons than their wild type and L1-/y littermates, mitigating the general view that hemizygous animals show an intermediate phenotype between wild type and knockout mice. This phenotype is interesting with regard to autism, since overproduction of neurons has been observed in some persons with autism. The hemizygous L1+/- mice do not show pronounced deficits in behavior, except for a slight abnormality in assessing time intervals. However, they are abnormal in accepted mouse models of autistic behavior.
Close homolog of L1 (CHL1)
The cell adhesion molecule close homolog of L1 (CHL1), which has been linked to mental disorders, binds to a short peptide stretch in the third intracellular loop of the serotonin 2c (5-HT2c) receptor via its intracellular domain. CHL1 deficiency in mice leads to reduced 5-HT2c receptor-related locomotion. This behavioral phenotype is associated with increased levels of serotonin in the striatum, enhanced 5-HT2c receptor levels in striatal plasma membranes as well as altered 5-HT2c receptor induced signaling. In CHL1-deficient brains enhanced binding of phosphatase and tensin homolog (PTEN) and of G-protein-coupled receptor kinase 6 (GRK6) to the 5-HT2c receptor were seen, whereas levels of the phosphorylated 5-HT2c receptor and of receptor-associated ?-arrestin 2 were reduced. These results link CHL1 to 5-HT2c receptor functions and to serotonergic modulation. CHL1 is also associated with the extracellular matrix molecule vitronectin and integrin receptors resulting in CHL1-mediated neurite outgrowth and neuronal migration. Since we showed that CHL1 enhances synaptic vesicle recycling we investigated its association with the SNARE complex and found that CHL1 organizes the presynaptic assembly of this complex.