A tissue’s cells function within the context of an extracellular matrix (ECM) of glycosylated and crosslinked proteins that provide structural support and adhesion, while also modulating intercellular
communication. In the central nervous system (CNS), the ECM is vital for the complex neuronal
migration and neurite projection stages during embryonic development that create the precise
anatomies and morphologies of functioning brain circuitry.
The historical discovery of a plant-derived molecule, Wisteria floribunda agglutinin (WFA),
revolutionised the study of brain ECM because it binds to (and, therefore, is used to stain) one
specific form of ECM called the perineuronal net (PNN). As its name suggests, this creates a ‘cage’
specifically around the soma of parvalbumin-containing interneurons. The critical role of this class of
neurons in brain activity regulation, and known involvement in specific neuropsychiatric pathologies,
ignited much investigation of the PNN. It is now known that its dysfunction is associated with multiple
conditions. In our laboratory, a previous cellular ‘gene trap’ genetic screen identified that the mutation
of protein components of the PNN contributed to the response to lithium, a mood stabiliser treatment
for the psychiatric disorder bipolar disorder.
In this thesis, the further application of gene trap screening to search for genes encoding proteins
that contribute directly to, or regulate, the formation of the perineuronal net on SH-SY5Y
neuroblastoma cells, a commonly used model of neurons is described. The hypothesis was that
identified genes would not only provide greater insight into the PNN structure but might offer new
targets for the treatment of CNS disorders. A ‘library’ of randomly mutated cells was created, and
WFA was used to identify mutant colonies with reduced staining, indicative of PNN dysfunction.
Several PNN-defective cell colonies were isolated, and their mutated genes identified using a
modified polymerase chain reaction (PCR) protocol. Three genes, DCC, FAF1, and GALNTL6 were
among those that were identified and considered the best candidates to take forward for further
analysis. DCC protein is the netrin-1 receptor, which has important roles in CNS development. FAF1,
the FAS associated factor 1 protein, which participates in apoptosis and autophagy processes.
GALNTL6 is a glycosyl transferase enzyme that modifies proteins through O-linked glycosylation –
a very strong candidate in light of the substantial glycosylation that occurs to PNN proteins, and
which is thought to be the target of WFA staining. These three proteins required validation through the generation of independent mutations/inhibition
in cells. CRISPR, siRNA and pharmacological means was used to attempt this. Multiple genetic
ablations failed to produce successful defective gene alleles meaning that full validation of these
three candidate genes was not possible, and their role in PNN function remains unclarified.
However, the still-unknown targets of WFA in a ‘pull-down’ assay of proteins lysed from SH-SY5Y
cells was pursued-associated protein material was assessed by mass spectrometry. Among the top
hits was the protein vimentin which is known to exist within the cytoplasm of the cells but also to be
secreted into the ECM, where it reportedly shows post-translational modification by O-linked
glycosylation.
In summary, despite failure to fully validate screen findings, these studies identified a number of
candidates for further investigation in the context of PNN function and role in associated disease.
The protein vimentin should also be pursued in terms of its potential contribution to PNN function
and as the target of the WFA stain.A tissue’s cells function within the context of an extracellular matrix (ECM) of glycosylated and crosslinked proteins that provide structural support and adhesion, while also modulating intercellular
communication. In the central nervous system (CNS), the ECM is vital for the complex neuronal
migration and neurite projection stages during embryonic development that create the precise
anatomies and morphologies of functioning brain circuitry.
The historical discovery of a plant-derived molecule, Wisteria floribunda agglutinin (WFA),
revolutionised the study of brain ECM because it binds to (and, therefore, is used to stain) one
specific form of ECM called the perineuronal net (PNN). As its name suggests, this creates a ‘cage’
specifically around the soma of parvalbumin-containing interneurons. The critical role of this class of
neurons in brain activity regulation, and known involvement in specific neuropsychiatric pathologies,
ignited much investigation of the PNN. It is now known that its dysfunction is associated with multiple
conditions. In our laboratory, a previous cellular ‘gene trap’ genetic screen identified that the mutation
of protein components of the PNN contributed to the response to lithium, a mood stabiliser treatment
for the psychiatric disorder bipolar disorder.
In this thesis, the further application of gene trap screening to search for genes encoding proteins
that contribute directly to, or regulate, the formation of the perineuronal net on SH-SY5Y
neuroblastoma cells, a commonly used model of neurons is described. The hypothesis was that
identified genes would not only provide greater insight into the PNN structure but might offer new
targets for the treatment of CNS disorders. A ‘library’ of randomly mutated cells was created, and
WFA was used to identify mutant colonies with reduced staining, indicative of PNN dysfunction.
Several PNN-defective cell colonies were isolated, and their mutated genes identified using a
modified polymerase chain reaction (PCR) protocol. Three genes, DCC, FAF1, and GALNTL6 were
among those that were identified and considered the best candidates to take forward for further
analysis. DCC protein is the netrin-1 receptor, which has important roles in CNS development. FAF1,
the FAS associated factor 1 protein, which participates in apoptosis and autophagy processes.
GALNTL6 is a glycosyl transferase enzyme that modifies proteins through O-linked glycosylation –
a very strong candidate in light of the substantial glycosylation that occurs to PNN proteins, and
which is thought to be the target of WFA staining. These three proteins required validation through the generation of independent mutations/inhibition
in cells. CRISPR, siRNA and pharmacological means was used to attempt this. Multiple genetic
ablations failed to produce successful defective gene alleles meaning that full validation of these
three candidate genes was not possible, and their role in PNN function remains unclarified.
However, the still-unknown targets of WFA in a ‘pull-down’ assay of proteins lysed from SH-SY5Y
cells was pursued-associated protein material was assessed by mass spectrometry. Among the top
hits was the protein vimentin which is known to exist within the cytoplasm of the cells but also to be
secreted into the ECM, where it reportedly shows post-translational modification by O-linked
glycosylation.
In summary, despite failure to fully validate screen findings, these studies identified a number of
candidates for further investigation in the context of PNN function and role in associated disease.
The protein vimentin should also be pursued in terms of its potential contribution to PNN function
and as the target of the WFA stain
