The role of transmembrane agrin in reorganizing the cytoskeleton in neurons and non-neuronal cells

The brain belongs to the most fascinating organs that developed in evolution. Its
complexity is responsible for recording and organizing impressions from the environment,
for our thoughts and feelings, for our personality. Knowledge of the mechanisms involved
in the development of the brain, in thinking and transmission of neural signals is likely to
also help our understanding of disease mechanisms underlying Alzheimer’s and
Parkinson’s disease or neuromuscular diseases including muscular dystrophies.
The brain is a complex network of billions of neurons and supporting cells. Ramòn Y Cajal
showed in the 19th
century with the Golgi-technique that each neuron is a unit that
communicates with other neurons by special contacts called synapses (Cajal, 1928). Most
of our current knowledge of how synapses work and of how they develop derives from our
profound understanding of the neuromuscular junction (NMJ), which is relatively simply
structured and organized. The key regulator for the development and maintenance of the
NMJ is the highly glycosylated heparansulfate proteoglycan (HSPG) agrin. Owing to
alternative first exon usage, agrin can be expressed in a secreted form (SS-NtA-Agrin) or a
transmembrane form (TM-Agrin). The amino-terminus of SS-NtA-Agrin binds to the
extracellular matrix (ECM) via laminin. That of TM-Agrin consists of a short intracellular
and a transmembrane region. TM-Agrin is preferentially expressed in the CNS, particularly
by neurons of the brain (Neumann et al., 2001).
This thesis examines the function of TM-Agrin in non-neuronal and neuronal cells. Using
transfection of cDNAs encoding full-length TM-Agrin, chimeric constructs and mutants
thereof, I show that TM-Agrin has a strong effect on cell morphology. In particular, during
my research, cells expressing TM-Agrin formed long and numerous actin-containing
microprocesses. In the chimeric constructs I replaced the intracellular part, the extracellular
part or the TM-domain of TM-Agrin with a corresponding part of another TM-protein. In
the mutant the glycosaminoglycan (GAG)-attachment site between the 7th and 8th
follistatin-like (FS) domain was mutated so that sugar chain could not attach. By this
means I managed to elucidate that the described effect is dependent on the close
association of the extracellular part of TM-Agrin with the membrane and, additionally, on
the presence of the GAG-chain localized between the 7th and 8th FS domain.
To evaluate whether similar effects of TM-Agrin can also be observed in neuronal cells,
we also transfected primary hippocampal mouse neurons. Indeed, transfected neurons
showed a curvy growth and developed microspikes on axons and dendrites indicating that
TM-Agrin also affects neuritogenesis. To test whether these effects could be based on
overexpression-induced self-dimerization of TM-Agrin, and whether TM-Agrin could
directly activate a signalling cascade, we also used antibody-induced dimerization, a
method that has been shown to allow activation of single transmembrane domain receptors
(Heldin, 1995; Weiss and Schlessinger, 1998). Indeed anti-agrin antibodies induced doseand
time-dependent formation of microspikes on primary mouse hippocampal neurons,
suggesting that TM-Agrin may have a function in inducing the reorganization of the actincytoskeleton
and also in development of neurites and their outgrowth.
In the last part of the work we created a transgenic mouse in which the expression of a
miniaturized version of mouse neural agrin could be temporally controlled. In Duchenne
muscular dystrophy (DMD), dystrophin has mutated, which leads to fragility of muscle
membranes to cause muscle wasting. It had been shown that overexpression of utrophin, an
autosomal homologue of dystrophin can functionally compensate for the loss of
dystrophin. With this mouse model we tested whether overexpression of agrin also causes
upregulation of utrophin in vivo. This could be an appropriate way to ameliorate and
eventually also cure DMD.