4. DISCUSSION
We show here that ON cells derived from cannabis users suffer
cytoskeleton alterations, decreased attachment, enhanced cell
proliferation and reduced cell death. These behavioural alterations are
accompanied by changes in the expression of proteins that have been
associated with cancer, gastrointestinal disease and neurodevelopmental
pathways (Prescot et al., 2011; Jacobus and Tapert, 2014; Gubatan et
al., 2016; Ghasemiesfe et al., 2019). Thus, the ON cell model provides
relevant information regarding the effects of cannabis on the brain that
may be related to neuropsychiatric disorders.
ON cells are morphologically undifferentiated after 24 h in
vitro , and most of them (>90%) express an antigen
associated with bone marrow stromal cells (CD105), as indicated
previously (Matigian et al., 2010). Furthermore, 60-80% express the
markers of neural progenitors (Nestin) and/or immature neurons
(β-III-Tubulin and GAP43) (Matigian et al., 2010; Galindo et al., 2018)
corroborating their neural lineage. Moreover, around 20% of cells
express GFAP, indicating the presence of astroglial-like cells
previously described as neuroblasts in ON cell populations (Wolozin et
al., 1992; Hahn et al., 2005). Furthermore, multipotent stem cell
marker, Sox2, expressed by ON (Lin et al., 2017) was not detected in our
cell cultures. ON cells from cannabis users also expressed the same
markers as control cells, although there were significantly fewer
CD105+ and GFAP+ cells, and more
Nestin+ cells in these cultures. Nestin and GFAP are
thought to be neural progenitor and stem cell markers, respectively
(Wolozin et al., 1992; Benitez-King et al., 2011), indicating that these
cells are in a different stage of cell growth. Thus, the change we
observed in the number of Nestin+ and
GFAP+ cells could be explained by alterations in cell
growth and differentiation. Likewise, a decrease in CD105 is associated
with the differentiation of mesenchymal stem cells derived from
different tissues (Jin et al., 2009; Alev et al., 2010; Noda et al.,
2019). By contrast, we did not observe a change in the number of
immature β-III-Tubulin+ and GAP43+cells after 24 h in vitro , nor in the number of
NeuN+ cells after 7 days in vitro , indicating
that the changes in the number of neuronal progenitors present in the ON
of cannabis users do not necessarily lead to ON cell differentiation.
We evaluated the proteomic profile of ON cells after 5 days in
vitro , identifying 49 upregulated proteins and 16 downregulated
proteins in the cells from cannabis users as compared to cells from
healthy controls. Several proteins involved in the actin cytoskeleton
signaling pathway, and in the Integrin and ILK signaling pathways, were
significantly affected in ON cells from cannabis users. ILK fulfils an
essential role in connecting the cytoplasmic tail of
integrin
β
subunits to the
actin
cytoskeleton
and in regulating actin polymerization. These proteomic data were in
agreement with changes in cell morphology that was assessed by measuring
the size and shape of β-III-Tubulin stained cells. In control ON cells,
microtubules form an extensive and compact network that appears to
originate from the microtubule organizing centre located close to the
nucleus. There was a robust increase in the size of β-III-Tubulin
stained ON cells from cannabis users, with differences in roundness that
suggests a reorganization of microtubules. Furthermore, changes in
microtubule assembly were accompanied by stronger β-actin expression in
ON cells from cannabis user that was evident in western blots,
suggesting that two cytoskeletal structures may be significantly
affected by cannabis, microtubules and microfilaments. The cytoskeleton
is involved in a number of biological processes, ranging from those that
involve the maintenance of cell shape to those affecting cell
proliferation and other activities (Pedersen et al., 2001; Fletcher and
Mullins, 2010; Kapitein and Hoogenraad, 2015). The mechanisms by which
cannabis alters cytoskeletal architecture are unknown, although THC was
shown to influence the assembly and disassembly of tubulin in
vitro (Tahir and Zimmerman, 1991). Since the cytoskeleton is closely
associated with cell membranes, the interaction of cannabis with the
lipid bilayer or membrane bound enzyme systems may adversely affect the
architecture of the cytoskeleton. In this sense, a recent study using
SR-FTIR spectroscopy found that intracellular lipid chains are
disordered in ON cells from cannabis users, the membrane displaying an
altered lipid composition with a higher rate of membrane lipid renewal
and peroxidation, and more proteins with β-sheet structures
(Saladrigas-Manjon et al., 2020). Thus, these data strongly support the
increased cell size and cytoskeletal alteration in the ON cells of
cannabis users. Interestingly, earlier data showed that ON cells from
bipolar patients were larger and displayed cytoskeletal alterations
(Solis-Chagoyan et al., 2013).
Cannabis may also affect the cytoskeleton indirectly through its effects
on other cell structures or biochemical activities. Indeed, cytoskeletal
abnormalities may be related to other cell functions like cell adhesion
and migration. ACTG1, DOCK1 and RAP1B also belong to FAK signalling and
Paxillin pathways (Figure 3 and 4). Paxillin is a focal
adhesion-associated phosphotyrosine-containing protein. The proteomic
alterations to these pathways suggest there are changes at the cell
surface of ON cells from cannabis users, as demonstrated by the weaker
vinculin staining in ON cells from cannabis users and the decrease in
the total number of attached cells when adhesion was evaluated.
Interestingly, schizophrenic ON cells have half the size and number of
focal adhesion (Fan et al., 2013; Munoz-Estrada et al., 2015; Tee et
al., 2017), and as focal adhesions are related to cell attachment,
detachment and migration (Tee et al., 2017), we evaluated the motility
of these cells. While no changes in cell migration were detected, there
were differences in the expression of proteins involved in migration
(CD9, DOCK1, FERMT2, RAP1B, CAPZB, RAB35, ARPC2, SLC2A1, SYNE1, SYNE2,
MGLL and CALD1). Many of these proteins are not exclusively involved in
migration, which could explain why the alterations in their expression
did not affect migration (e.g. DOCK1 or RAP1B are also involved in actin
cytoskeletal signaling). Hence, the effects of cannabis use on ON cell
migration should be studied further, for example assessing the migration
of these cells in response to different extracellular matrix factors
(Tee et al., 2017).
In terms of cell proliferation, ON cells from cannabis users had a
higher rate of proliferation when evaluated with the Ki67 marker.
However, cell differentiation was not affected, as the number of
NeuN+ neurons and GFAP+ astrocytes
did not change, even though the proteomic analysis suggested alterations
in neural proliferation and axon guidance. In this sense, the STMN2
(stathmin-2) protein is particularly relevant as it is involved in axon
guidance and the Endocannabinoid Developing Neuron pathway, and the
down-regulation seen for in STMN2 was consistent with the low levels
detected in the human brains exposed to THC (Tortoriello et al., 2014).
By reducing STMN2, THC favours ectopic filopodia formation and it alters
axon morphology (Tortoriello et al., 2014). Furthermore, ON cells from
cannabis users underwent less apoptosis, both early (Annexin V+/PI-) and
late apoptosis (Annexin V+/PI+). Indeed, several proteins related to
cell death were differentially expressed in ON cells from cannabis
users. If these alterations were to occur in the foetal or young brain,
they would alter the timing and balance of neuronal birth,
differentiation and death, consequently affecting the timing and success
of synaptic connectivity. Such changes would produce fundamental
differences in brain function, as reported in chronic cannabis users
(Kim et al., 2019; Newman et al., 2019). In fact, chronic cannabis
appeared to directly affect cognitive function, specifically decreasing
attentional performance.
Interestingly, increased cell proliferation and reduced cell adhesion
was evident in olfactory cultures from individuals with schizophrenia,
although no alterations to apoptosis were detected (Feron et al., 1999;
McCurdy et al., 2006). Thus, it is likely that cannabis might predispose
or precipitate alterations leading to other psychiatric diseases.
Accordingly, the proteomic analysis identified protein alterations
common to cannabis and schizophrenic patients, such as those in EWSR1,
RAB32 and STMN2 (Hakak et al., 2001; Bowden et al., 2008). Indeed, EWSR1
was altered in the same direction in post-mortem superior temporal gyrus
tissue and in peripheral blood lymphocytes from individuals with
schizophrenia (Bowden et al., 2008). Monoglyceride lipase (MGLL-1)
metabolizes the endogenous cannabinoid 2-arachidonoylglycerol and ON
cells from cannabis users express this protein this protein weaklier,
while 2-arachidonylglycerol metabolism is enhanced in the prefrontal
cortex of subjects with schizophrenia (Volk and Lewis, 2016). Overall,
these data showing similarities in ON cells derived from cannabis users
and schizophrenia patients, reinforcing the link of cannabis use and a
higher risk of suffering psychoses (Nielsen et al., 2017).
In addition to schizophrenia, the proteomic analysis revealed a
reduction in TBX1 (T-box containing transcription factor)
expression in ON cells from cannabis users. Haplo-insufficiency ofTBX1 is thought to contribute significantly to the
cardiovascular, endocrine and neurogenic phenotypes of DiGeorge Syndrome
(DGS, 22q11.2 deletion syndrome -22q11DS) and Velo-Cardio-Facial
Syndrome (Lindsay et al., 2001; Jonas et al., 2014). Indeed,
approximately 23-30% of late adolescents and young adults with DGS
develop psychotic symptoms. Moreover, the TBX1 gene has been
involved in the pathogenesis of schizophrenia in some patients (Ping et
al., 2016). In animal models, congenic TBX1 heterozygous mice
display autism-related behavioural phenotypes (Hiramoto et al., 2011;
Takahashi et al., 2016). Mutations and haplo-insufficiency of theTBX1 gene are sufficient to cause reduced pre-pulse inhibition, a
behavioural abnormality that is associated with a schizophrenic
endophenotype (Paylor et al., 2006)
ON cells from patients with bipolar disease and from cannabis users
share some characteristics. Indeed, ON cells from bipolar patients are
also larger and have an altered cytoskeleton (Solis-Chagoyan et al.,
2013), similar to the changes found in ON cells from cannabis users.
Moreover, our proteomic analysis detected a down-regulation of SYNE1
(nesprin-1), and polymorphisms of this gene have been associated with
susceptibility to bipolar and unipolar mood disorders (Green et al.,
2013). Apart from mental disorders, proteins differentially expressed in
our samples are also related to other neurological disorders, such as
epilepsy (Katona, 2015), Glut1 syndrome (Brockmann, 2009),
Charcot-Marie-Tooth disease (Alazami et al., 2014), deafness (Morin et
al., 2009), spinocerebellar ataxia (Gros-Louis et al., 2007),
microcephaly/epilepsy/diabetes syndrome (Shalev et al., 2014) and
episodic choreoathetosis/spasticity (Weber and Lerche, 2009).
In summary, our data show that ON cells derived from cannabis users
exhibit changes in cell morphology, weaker adhesion, and alterations to
their cell cycle, cell growth and proliferation, although we did not
observe any effects on differentiation. These changes could be related
to alterations in cytoskeletal proteins and signalling pathways. All
these cellular processes are important for brain development,
maintenance and function. Moreover, some of these alterations have been
described in ON cells of schizophrenia and bipolar patients, providing a
possible link between cannabis consumption and the risk of suffering a
psychiatric disorder.