ZMBH J.Bericht 2000 - Zentrum für Molekulare Biologie der ...

expression of misfolded PLP is „toxic“ to myelinforming
oligodendrocytes. Jimpy mice can not be
„rescued“ by transgenic complementation with a wildtype
PLP transgene. Moreover, mice which completely
lack expression of PLP or any truncated
polypeptide (a null allele created by gene targeting)
develop normal motor functions and show no signs
of abnormal glial cell death. In fact, CNS myelin is
assembled as a multilamellar and compacted structure
in the absence of PLP. Although the overall stability
of such PLP-deficient myelin appears reduced, these
observations question the view that the major mem-
Figure 1. Mutations of the gene for myelin proteolipid
protein (PLP) cause Pelizaeus-Merzbacher Disease and
severe dysmyelination in PLP-transgenic mice. The molecular
pathomechanism, which is associated with abnormal
protein trafficking, is poorly understood. We are using confocal
microscopy to study the intracellular localisation and
export of myelin proteins into the processes of cultured oligodendrocytes.
The folding-sensitive O10-epitope of PLP
(in green) which is lacking in all mutant PLP isoforms
emerges after the C-terminal epitope (in red) of newly synthezised
PLP (provided by E. Krämer).
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brane protein of CNS myelin is essential to obtain
myelin membrane adhesion and compaction. Recent
evidence suggest that PLP deficiency has profound
effects on axonal integrity which becomes clinically
manifest in aged mice with signs of „neuroaxonal
dystrophy“. The basis of the underlying axon-glia
interaction is currently under investigation.
We have expressed PLP cDNAs from natural mouse
mutants in cultured non-glial cells. These studies show
that fibroblasts are much less sensitive to the overexpression
effect and mutant isoforms of PLP than
oligodendrocytes in vivo. However, also transfected
fibroblastoid cells recognize subtle alterations in the
PLP primary structure, because these translation products
are retained inside the cell and do not reach the
cell surface. A direct demonstration of protein misfolding
is also possible with a conformation-sensitive
monoclonal antibody (O10) which distinguishes
wildtype PLP from the known mutant isoforms.
III. Neuronal members of the proteolipid protein
family: M6A and M6B
L. Dimou, M. Klugmann, H. Werner
PLP is the prototype of a small family of sequencerelated
tetraspan membrane proteins which includes
M6A and M6B proteins, abundantly expressed on
neuronal processes in the central nervous system.
M6B is also detectable in oligodendrocytes. There are
multiple M6B-mRNAs, encoding at least eight protein
isoforms. Smaller, presumably soluble polypeptides
result from alternatively spliced M6B mRNAs
with a stop codon upstream of the exon for the first
transmembrane domain. In cells of the oligodendroglial
lineage, M6B localizes to the cell surface and transiently
also on cellular processes. By in situ-hybrid-
ization, oligodendrocytes express one of two alternative
C-termini, whereas cortical neurons express
both. Regulated expression of the various intracellular
domains of M6B, termed α, β, γ, ψ, and ω (some of
which are structurally conserved in M6A and PLP),
suggests that proteolipids can interact with cytoplasmic
proteins present in a variety of neural cell types.
The N-terminal domains of M6B affect transport features
and the subcellular distribution of this protein
when expressed in fibroblasts. The analysis of M6A
and M6B mouse mutants is ongoing and suggests a
role in neuronal process outgrowth and CNS myelination,
respectively.
IV. NEX and NeuroD regulate hippocampal
granule cell differentition
A. Bartholomä, S. Göbbels, M. Rossner, M.
Schwab
Parts of our laboratory‘s activity was devoted to the
function of basic helix-loop-helix (bHLH) proteins in
the nervous system. The neuronal transcription factors
NEX (neuronal helix-loop-helix protein-1), NeuroD
(neurogenic differentiation factor), and NDRF comprise
a family of Drosophila atonal-related bHLH
proteins with highly overlapping expression in the
developing forebrain. A role for NeuroD and NEX
in terminal neuronal differentiation is demonstrated
by mutations in mice. For example, in the hippocampus,
presumptive granule cells of the dentate gyrus
are generated but fail to mature, lack normal sodium
currents, and show little dendritic arborization. Longterm
hippocampal slice cultures reveal secondary
alterations, such as abnormal projections from the
entorhinal cortex. Ongoing experiments aim at restricting
the mutations of the NeuroD gene to subsets of
cortical neurons, utilizing homologous recombination
of the loxP/Cre system. This will allow to circumvent
perinatal lethality and study the role of bHLH proteins
in adult neuronal functions.
Figure 2. Cre-mediated recombination of genes that are
flanked by loxP sites allows the generation of cell type-specific
somatic mutations in transgenic mice. We have utilized
the spatio-temporal expression pattern of the NEX gene
to drive expression of Cre specifically into CNS neurons. A
„knock in“ strategy was chosen, to insert Cre into exon 2 of
the endogenous NEX gene. To demonstrate the specificity of
this system, a lacZ reporter function was specifically activated
by Cre recombination in neurons of the hippocampus
and neocortex of transgenic mice. This mouse is used to
circumvent the perinatal lethality of a null mutation in the
neuroD gene (provided by S. Goebbels).
External Funding
During the period reported our research was supported
by grants from the Deutsche Forschungsgemeinschaft
(SFB 317 “Molekulare Biologie neuraler Mechanismen
und Interaktionen”, Graduiertenkolleg “Molekulare
und zelluläre Neurobiologie”) from the BMBF
and from the EU.
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