Mechanisms of Zinc Neurodegenerative Cytotoxicity Present in

Neuronal Cells of Mucolipidosis Type IV (MLIV)

Joshua Silva
California State University, Fullerton











Abstract:
Zinc, a trace metal, has been shown to have significant impacts on cell signaling and
conduction pathways by acting as a second messenger. Zinc is suggested to be vital to
physiological brain processes, and if not regulated properly may cause significant damage to
neuronal cells of the brain. The recent identification of various zinc transporters, ZnTs, within
human cells have allowed for further investigation of these regulation pathways that allow for
proper zinc balance of cells. Calcium channels have even been suggested to play roles in zinc
regulation, such as allowing zinc transfer with calcium transport. One such calcium channel,
TRPML1, has been linked to allowing the transport of zinc due to its role as a non-selective
cation channel. The dysfunction of TRPML1 causes the neurodegenerative disorder,
Mucolipidosis Type IV (MLIV). Dysfunctional TRPML1 displays a toxic increase of zinc within
MLIV patient fibroblasts and murine brain cells, possibly suggesting a mechanism of
neurodegeneration evident within patients. The recent identification and interaction of TRPML1
with ZnT-4 may suggest contributing factors of this cytotoxic zinc increase evident with MLIV
affected cells. SV31, a synaptic vesicle zinc transporter, was recently identified and suggested to
also play an interaction role with the TRPML1 protein. Therefore, this review aims to summarize
various contributing factors of neurodegeneration evident in humans that may correlate with the
MLIV disorder.



Review Hyperlink:
http://www.jneurosci.org/site/misc/ifa_minireviews.xhtml#minirevie
ws
Review Model Hyperlink:
http://www.jneurosci.org/cgi/content/full/26/41/10349






Possible Mechanisms of Zinc Neurodegenerative Cytotoxicity
Present in Neuronal Cells of Mucolipidosis Type IV (MLIV)
Joshua Silva
1
1
California State University, Fullerton Department of Biological Sciences. 800 N State College Blvd, Fullerton, CA 92831

Introduction
Transition metals are well known to
play vital roles in cellular function and
regulation. Although necessary for cell
viability and function, levels of ions that are
unregulated are severely toxic to the
exposed cell. Chelatable, or free, zinc ions
are ions known to affect DNA interaction
proteins as well as enzymatic activity within
the cell (Stefanidou et al., 2006; Maret et al.,
2009). Zinc (Zn
+2
) is more recently regarded
as a second messenger due to its influence
on enzyme activity and controlling
intracellular signaling pathways (Yamasaki
et al., 2007). Within the brain organ, Zn
+2
is
at higher concentrations within synaptic
vesicles at glutamatergic nerve terminals
and is released from synapses with neuronal
excitation affecting the activity of glutamate,
GABA
A
, and glycine receptor proteins
(Sensi et al., 2011). For neuronal brain cells
to properly balance the levels of zinc at rest,
free zinc is often regulated by being
sequestered into cellular organelles as well
bound by metalloproteins, proteins that
regulate trace metal levels within cells.
Zinc is primarily digested and
absorbed through Zip4 proteins of digestive
system enterocytes, intestinal absorptive
cells. Zip4, a member of the Zip family of
Zn
+2
Transporters, transfers zinc from the
digested material to the cytoplasm of the
enterocyte where it will eventually be
transferred to the bloodstream (Eide et al.,
2004; Lichten et al., 2009; Kambe et al.,
2011; Hill et al., 2009). This free zinc within


the blood stream is then transferred from the
blood through cell membranes to be
sequestered and stored within cellular
organelles.
Within neuronal cells, the ability to
regulate free zinc is done so by several zinc
transporters located on membranes of
intracellular organelles. Several well-known
zinc transporters of organelle membranes
consist of ZnT Transporters, Zip
Transporters, and Zn
+2
regulating
metallothioeneins (MTs) (Sensi et al., 2009).
The presence of several of these transporters
on cellular organelles is beneficial due to the
necessity of zinc for reactions to take place
inside the cell (Palmiter et al., 2004). The
association of dysfunctional regulation
between these transporters of zinc within all
cells can lead to cytotoxic conditions and
cell death.
Organellar membrane proteins are
also known to aid trace metal regulation by
interacting with protein partners. Transient
Receptor Potential Mucolipin-1 (TRPML1)
is a lysosomal membrane protein known to
function as a transport channel that is
specific to several ions. TRPML1 has been
characterized as having inward current flow
for ions such as iron, manganese, calcium,
and zinc (Grimm et al., 2007). TRPML1 is
associated with maturation of the lysosome,
regulating lysosomal pH, and lysosomal
degradation. Mucolipidosis type IV (MLIV)
is a genetic disorder with clinical
abnormalities such as neuronal and retinal
cell degeneration, blindness, low gastric acid
production, and anemia (Amir et al., 1987).
MLIV is caused by mutations in the
MCOLN1 gene allowing for a dysfunctional
protein product, TRPML1 (Sun et al., 2000).
In MLIV affected cells, Zn
+2
levels are
significantly higher in the brains of
TRPML1 deficient mice as well as in
fibroblast cells of MLIV affected patients
(Eichelsdoerfer et al., 2010). Although
TRPML1 is suggested to be permeable to
zinc, it is not clear how the disruption of the
TRPML1 protein causes irregular zinc levels
within vesicles of the cell.

Zinc Transport Regulation by ZnT-1

Zinc transporters have recently been
identified within mammalian cells. Most
cases of zinc transporters have had to been
studied through bacterial species and yeast
cells. Current studies suggest that human
CDF, also termed SLC30, is a gene that
codes for 10 Zinc Transporters (ZnTs)
(Cousins et al., 2006). The expression and
cellular distribution of these ZnT proteins
are suggested to be regulated by changes in
zinc levels of the cell (Sekler et al., 2007).
Higher ZnT concentrations have been
associated with higher zinc levels and vice
versa. Studies of these regulation pathways
of zinc within yeast and bacterial
homologues allow for logical approaches to
ZnT function as possible H
+
/Zn
+2

exchangers. These same approaches that
have allowed for investigation of bacterial
and yeast zinc transporter have been used to
correlate the function of human zinc
associated proteins.

ZnT-1, a protein of the SLC30 zinc
transporter family, is typically located on
neuronal cell membranes (Sensi et al.,
2007). Immunostaining methods allowed for
the ZnT-1 proteins to be targeted and stained
within tissue dissections, as shown by Sensi
et al. ZnT-1 was also found to be at higher
concentrations within various regions of the
hippocampus segment of the brain (Sindreu
et al., 2014). Sindrei showed that dissection
and immunostaining of mouse and rat brain
tissue suggests that neuronal cells of these
species displays differential ZnT-1
expression according to exposed zinc
concentrations. The preferential expression
of ZnT-1 proteins at the synaptic membrane
domains of these neuronal cells indicates
that ZnT-1 is a novel post synaptic density
(PSD) protein (Sindreu et al., 2011). This
overall data may suggest the fact that ZnT-1
expression is regulated by zinc
concentrations found at the synapse.
Additionally, ZnT-1 could play a role in
regulating zinc ions within neuronal
synapses at post synaptic membranes.

The overall higher concentrations of
ZnT-1 at PSD regions appear to be
promising. Further investigation is necessary
to determine the exact role of ZnT-1 in zinc
regulation of neuronal cells. Other evidence
has suggested the increased presence of
ZnT-1 proteins as well as its related protein
ZnT-2 during periods of high zinc
consumption (Cousins et al., 2003). This
data from Cousins et al. might even lead to
research into the cooperation of both
proteins in zinc regulation within neuronal
cells. Future studies will include deletion
regions of the ZnT-1 proteins to better
characterize its role in synaptic function and
structure (Sindreu et al., 2011).

Calcium-conducting channels display
activity in zinc ion transport.

Evidence appears to suggest that the
contributions of both calcium (Ca
+2
) and
intracellular accumulation of Zn
+2
ions
contribute to cytotoxic cell injury and death
(Frederickson et al., 2005). The ability of
highly selective calcium channels to allow
the permeation of zinc has been implicated
in several studies. This has been shown with
voltage-gated Ca channels (VGCC) which
are selective for calcium but have been
suggested to transport zinc in the case of
brain cells and cultured cortical neurons,
neurons of the cerebral cortex region of the
brain (Bouron et al., 2013). In this study
done by Bouron et al., it was also shown that
Zn ions entered the neuronal cells even with
exposure to extracellular calcium ions.

A superfamily of mammalian
Transient Receptor Potential (TRP) channels
comprises several members organized into
six subfamilies named TRPA, TRPC,
TRPM, TRPML, TRPP, and TRPV
(Venkatachalam et al., 2007). Initially
TRPM7 was the first TRP protein channel to
be identified as Zn-conducting.
Venkatachalam also addresses the more
recent findings in which investigations have
found that additional proteins within the
subfamily, such as TRPML proteins, shared
high sequence similarity and displayed Zinc
conduction. This is not true for all cases.
Data from studies on TRPM1 indicate that
this calcium channel shares high sequence
similarities to sister proteins that allow for
zinc permeability, but does not allow for
zinc transfer itself (Lambert et al., 2011).
The contribution of calcium channels to
contribute to zinc intake has many
implications for both normal regulation and
diseased states. Therefore it appears that loss
of function of either a zinc transporting
protein or a calcium transporting protein
might lead to cytotoxic states to
dysregulation of zinc, calcium, or both.

Dysfunctional Zinc Regulation Associated
with MLIV Disorder.

TRPML1, of the TRPML family, has
been shown to be permeable to several ions,
which includes the trace metal zinc (Grimm
et al., 2007). With the dysfunction or loss of
presence of TRPML1 on lysosomal
membranes associated with MLIV, an
associated cytotoxic increase in zinc is
evident (Eichelsdoerfer et al., 2010). The
role that TRPML1 itself plays in this zinc
dysregulation is still being investigated.
Eichelsdoerfer also suggests that the loss of
the TRPML1 protein function is associated
with a significant accumulation of chelatable
Zn
+2
within the lysosomes and vacuole
structures of HEK-293 cells. By
incorporating the use of RNA interference
(RNAi) to target ML1 production within
HEK-293 cells, the loss of the TRPML1
protein caused an overall dysfunction within
the lysosome (Eichelsdoerfer et al, 2010). In
these cases, the MLIV disorder is signified
by disrupting proper production of the ML1
protein that ultimately causes dysfunction of
the lysosome. Eichelsdoerfer then shows the
accumulation of zinc within lysosomes by
use of FluoZin-3 fluorescence which binds
free zinc within the cell. These results were
also observed from fibroblast cells of patient
affected by MLIV (Eichelsdoerfer et al.,
2010). Eichelsdoerfer also displayed that
TRPML1 dysfunction led to significant
changes in Zn
+2
ions but not with Iron
(Fe
2+
), suggesting that Zn
+2
may play the key
role in neurodegeneration evident in MLIV.

Cytotoxic Zinc Associated with Loss of
Interaction between TRPML1 and Zinc
Regulating Factors.

It has been shown that the
dysfunction of the TRPML1 protein allows
for a loss of interaction with cooperative
zinc transporters of the lysosome as well as
with proteins that bind and regulate zinc
within the cell (Kukic et al., 2013). Kukic et
al. ultimately showed an overall cytosolic
increase of free zinc with HeLa cells lines.
Kukic aimed with the assumption that if
TRPML1 is directly involved in the
regulation of zinc transport, cellular zinc
trafficking and zinc dependent processes
should change with changes in TRPML1.
By investigating the effects of various zinc
transporters and the inclusion of
dysfunctional TRPML1, it has been
suggested that TRPML1 works with ZnT4, a
zinc transporter, to regulate lysosomal zinc.
Kukic et al. shows that the interaction of
knock down ML1 (TRPML1-KD) HeLa
cells, a cell line specifically targeted to not
produce TRPML1 proteins, displayed a
retention of zinc within the lysosome. This
appears to suggest that TRPML1 has a
functional role in clearing zinc from the
lysosome. When TRPML1 is properly
produced, the cation channel is then able to
allow the secretion of zinc from the
lysosome and into the cytoplasm.

Kukic et al. showed another factor
that plays a role in zinc regulation with
TRPML1. TRPML1 is also suggested to
work with the transcription factor of MTF-1
to regulate free zinc ions. TRPML1 and
ZnT4 protein were shown to interact and
regulate the zinc within the lysosome, while
MTF-1 helps regulate cytosolic zinc within
HeLa cells. Kukic et al. shows that with the
presence of an intracellular zinc increase,
the MTF-1 transcription factor will bind the
extra zinc due to its zinc-finger motifs,
regions of zinc affinity and binding. This
binding to zinc now allows for transfer to
the nucleus, region where DNA is stored,
and induces the activation of protein
products that further assist regulate zinc.
Ultimately, the MTF-1 and zinc complex
will initiate transcription of further zinc
regulators of the cell, including MTs and
some zinc transporters, ZnTs (Guo et al.,
2010). MTs have been shown to function in
binding free zinc within the cell as to aid
regulation of the ion (Sensi et al., 2009). The
additional ZnT proteins allow for removal of
zinc from the cytoplasm to either out of the
cell or into organelles that will store the zinc
for future use. By binding and transporting
zinc, less zinc is free within the cytosol and
is therefore reducing its overall intracellular
activity. This reduction in intracellular
activity is vital to cell viability due to the
toxic effects of prolonged zinc exposure
within the cell.

Chelatable Zinc Responsible for Cell
Pathology of MLIV Disorder.

Further applications of these studies
are necessary to determine the effects of this
Zink Sink, cytotoxic zinc concentrations,
within human neuronal cell lines. If the
presence of zinc levels that are higher than
normal is displayed within neurons, then a
mechanism can be offered to the insight of
neurodegeneration. This is important for the
topic of MLIV due to the unclear
mechanism behind neuronal degeneration of
patients affected with the MLIV disorder.
These consistently observed cytotoxic levels
of zinc explain a method in which neuronal
cell death is applicable. These levels may be
attributed to either the loss of TRPML1, a
known calcium conducting channel that is
now associated with zinc transport, or the
loss of functional relationship between
TRPML1 and zinc transporting proteins.
Overall, these continued discoveries of zinc
transporting/regulating proteins within
mammalian cells are essential to
understanding the entire outcome of the
MLIV disorder pathway.








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