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Chapter 12
Intermediate Filaments as a Target of Signaling
Mechanisms in Neurotoxicity
Ariane Zamoner and Regina Pessoa-Pureur
Additional information is available at the end of the chapter
http://dx.doi.org/10.5772/66926
Abstract
In this chapter, we deal with the current knowledge and important results on the cyto‐
skeletal proteins and their dierential regulation by kinases/phosphatases and Ca2+‐
mediated mechanisms in developmental rat brain. We focus on the misregulation of the
phosphorylating system associated with intermediate lament proteins of neural cells
and its relevance to cell and tissue dysfunction. Taking into account our ndings, we
propose that intermediate‐lament proteins are dynamic structures whose regulation is
crucial for proper neural cell function. Given their relevance, they must be regulated
in response to extracellular and intracellular signals. The complexity and connection
between signaling pathways regulating intermediate‐lament dynamics remain obscure.
In this chapter, we get light into some kinase/phosphatase cascades downstream of
membrane receptors disrupting the dynamics of intermediate laments and its associa‐
tion with neural dysfunction. However, intermediate laments do not act individually
into the neural cells. Our results evidence the importance of misregulated cytoskeletal
crosstalk in disrupting cytoskeletal dynamics and cell morphology underlying neural
dysfunction in experimental conditions mimicking metabolic diseases and nongenomic
actions of thyroid hormones and as an end point in the neurotoxicity of organic tellurium.
Keywords: intermediate lament, cytoskeleton, cell signaling, calcium, neurotoxicity
1. Introduction
All the cell functions accomplished by the living cell are dependent on a sophisticated net‐
work of protein laments with dierent compositions, distributions and roles into the cell,
forming an integrated meshwork known as the cytoskeleton. However, the most striking fea‐
ture of the cytoskeleton concerns its ability to respond to signals and conditions to which cells
are submied, taking part of adaptive cell response to dierent stimuli. The cytoskeleton is
© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.
an end point of signaling pathways adapting cells to immediate or long‐lasting behaviors in
healthy and sick organisms.
Cytoskeleton of most animal cells is constituted by three interconnected lament subsystems:
microlaments (MFs), microtubules (MTs) and intermediate laments (IFs). Compelling
evidence from the last decades has brought convincing understanding for the highly regu‐
lated and interconnected interactions between the cytoskeletal elements giving support to
sculpting and maintaining cell shape and sustaining all kinds of morphological alterations or
internal organization, as well as their implications for the behavior of animal cells. Figure 1
demonstrates the organization of the cytoskeleton in neurons.
A cohort of accessory proteins and signaling machinery regulates the dynamic turnover of the
cytoskeleton. Although each type of lament has specic cell distribution, molecular constitu‐
ents and equilibrium, the coordinated intertwining among the dierent networks provides
the force for a number of coherent processes in response to all kinds of intra‐ and extracellular
stimuli leading responses so decisive as cell survival or death [1].
This chapter initiates with a brief introduction about the structure and function of IFs, empha‐
sizing those from neural cells. However, the main purposes of the chapter are the experimen‐
tal evidence of our laboratory that the roles of IFs are beyond protection from mechanical and
nonmechanical stress. They might be the end point of misregulated‐signaling mechanisms in
neurotoxic conditions adapting their dynamics, in concert with the other cytoskeletal bers,
to cell survival or death.
Figure 1. Distribution of cytoskeletal constituents into neurons. Neuronal cytoskeleton is composed by microlaments,
microtubules, and intermediate laments. The microtubules are nucleated at the centrosome, then released and delivered
to either the dendrites or the axon. Neurolaments are abundant in axons and the spacing of neurolaments is sensitive
to the level of phosphorylation. The microlaments are dispersed within the cells and they are most abundant near the
plasma membrane.
Cytoskeleton - Structure, Dynamics, Function and Disease234
2. Intermediate laments
2.1. Molecular architecture of intermediate laments
IFs are exible, rod‐shaped bers averaging 10 nm in diameter, a size that is intermediate
between MFs and MTs. They are ubiquitous constituents of the structural scaold of the
eukaryotic cells and considered mechanical integrators of cytomatrix [2]. These cytoskeletal
laments are widespread expressed in practically all animal cell types and are the most diverse
cytoskeletal protein family, encoded by an estimated 70 IF genes in the humans. IFs have
been grouped into six sequence homology classes (SHC) according to the degree of sequence
identity: acidic keratins (SHC group I); basic keratins (SHC group II); desmin, vimentin and
other mesenchymal IF proteins, such as glial brillary acidic protein (GFAP) (SHC group III);
neurolament proteins (SHC group IV); and lamins (SHC group V).
IF building blocks are brous proteins stabilized by multistranded left‐handed coiled coils
giving rise to a rope‐like structure. Their structures are constituted by a long central α‐helical
region, also designed rod domain, with a distinct number of equally sized coiled coils forming
segments anked by non‐α‐helical N‐terminal (the head domain) and C‐terminal domains
(the tail domain). Both head and tail domains are highly varying in size and sequence, thus,
the functional and molecular heterogeneity of IF proteins are a consequence of the highly
variable non‐α‐helical end domains of subunits.
The central rod domain of IF subunits is α‐helical rod highly charged, with a role in the rst
phase of IF assembly. By contrast, the head domain enriched in basic amino acids is essential
for the formation of tetramers (the polymerization units) and complete IF assembly.
The non‐α‐helical tail domain can vary drastically between dierent IF proteins. This domain
is not essential for the assembly of cytoplasmic IFs but plays a signicant role in lament
width control. The functional role of the tail domain is particularly important in the neurola‐
ments, the neuronal‐specic IFs, as discussed below.
Overall, the assembly of subunits giving rise to functional IFs is a complex and multistep
process with individual specicities among the dierent representatives of this molecularly
heterogeneous family. Taking into account the in vitro polymerization of vimentin, lament
assembly starts with the formation of parallel, in‐register dimers. These dimers spontane‐
ously associate laterally into antiparallel, half‐staggered tetramers. Tetramers aggregate into
higher‐order oligomers to form unit length laments (ULFs) that undergo reorganization and
elongation by longitudinal annealing to form immature IFs. The nal step is radial compac‐
tion of the laments from approximately16 nm to a diameter of 10–12 nm [3].
Dierent from the other IFs, NFs comprise three subunits with dierent molecular masses
and distributions into the lament. They are formed by light, medium and heavy molecular
mass NF triplet proteins (NF‐L, NF‐M and NF‐H), respectively. NF‐L can self‐assemble form‐
ing the core of the lament. NF‐M and NF‐H are peripherally disposed on the lament, with
their long and exible tails rich in highly charged domains and multiple phosphorylation
sites, radially projecting out from the lament backbone when NF‐M and NF‐H co‐assemble
Intermediate Filaments as a Target of Signaling Mechanisms in Neurotoxicity
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235
with the short‐tail NF protein NF‐L. Interestingly, NF‐H and NF‐M by their own are not able
to assemble into laments, but by contrast, self‐assembled NF‐L yields normal looking 10‐nm
laments. These side arms of NF‐M and NF‐H contain multiple phosphorylation sites regulat‐
ing the interactions of NFs with each other and with other cytoskeletal structures [4].
2.2. Roles of intermediate laments in neural cell function
Neurons are highly specialized in the transmission and processing of electrical and chemical
signals. A functional nervous system is dependent of a proper axonal array, which in turn
is critically dependent upon the organization of the axonal cytoskeleton. Five main subunit
proteins form the neuronal specic NFs: the group IV NF‐L, NF‐M and NF‐H triplet pro‐
teins, α‐internexin and the group III peripherin. Mature laments are composed of several
combinations of these ve subunits. In most dierentiated neurons, α‐internexin expression
precedes that of the NF triplet and declines somewhat postnatally, while the expression of
the NF triplet sharply rises. Neurolaments found in perikarya, dendrites and axons dier
considerably in their organization and function. Perikarial NFs form a meshwork around
the nucleus. In the axons of mature neurons, a large number of longitudinally oriented and
phosphorylated NFs play a fundamental role increasing the diameter of myelinated axons
and consequently nerve conductivity. Neurolaments present in dendrites are less abundant
and less phosphorylated than those of axons.
Neurolaments are transported from the cell body, where they are synthesized, to be deliv‐
ered along the axon by a mechanism called axonal transport. The motors implicated in the
anterograde transport are kinesins, while the retrograde transport is mediated in association
with dynein, the same motor proteins involved in the fast axonal transport along MTs [4].
The multiple roles of cytoskeletal proteins in the neural cells imply that there is an underlying
cytoskeletal pathology associated with several neurodegenerative processes. The major neu‐
rodegenerative diseases are characterized by the presence of inclusion bodies in implicated
neurons. These inclusion bodies all contain elements of the cytoskeleton. In addition, muta‐
tions and/or accumulations of NFs are frequently observed in several human neurodegenera‐
tive disorders including amyotrophic lateral sclerosis, Alzheimer disease, Parkinson disease,
Charcot‐Marie‐Tooth, giant axonal neuropathy, neuronal intermediate‐lament inclusion
disease and diabetic neuropathy [5]. Multiple factors can potentially induce the accumulation
of NF, including deregulation of NF gene expression, NF mutations, defective axonal trans‐
port, abnormal posranslational modications and proteolysis [4]. Beyond their association
with neural damage in inherited or age‐dependent neurodegenerative diseases, studies from
our laboratory indicated that the disruption of NF homeostasis is a response to toxic agents
and abnormally accumulated metabolites in rat brain.
Astrocytes are important cytoarchitectural elements of the CNS; however, during the past few
years, molecular and functional characterization of astroglial cells indicates that they have a
much broader function than only support the neurons in the brain. Compelling evidence sup‐
ports that astrocytes have specialized functions in inducing and regulating the blood‐brain
barrier (BBB), glutamate uptake, synaptic transmission, plasticity and metabolic homeosta‐
sis of the brain [6]. Astrocytes express 10 dierent isoforms of glial brillary acidic protein
Cytoskeleton - Structure, Dynamics, Function and Disease236
(GFAP), the specic astrocytic IF, together with vimentin, nestin and synemin. However,
GFAP is the main IF protein expressed in mature astrocytes, where it helps maintaining
mechanical strength, as well as cell shape. However, recent evidence has shown that GFAP
plays a role in a variety of additional astrocyte functions, such as cell motility/migration, cell
proliferation, glutamate homeostasis, neurite outgrowth and injury/protection [7].
Astrocytes are also involved in a wide range of CNS pathologies, including trauma, isch‐
emia and neurodegeneration. In such situations, the cells change both their morphology
and their expression of many genes leading to activation of astroglia, or astrogliosis. It is
accepted that the increase of IFs with accompanying cellular hypertrophy and an abnormal
apparent increase in the number of astrocytes characterize astrogliosis. However, upregula‐
tion of IF proteins, in particular GFAP, but also vimentin and nestin, two IF proteins abun‐
dantly expressed in immature astrocytes, is regarded as the hallmark of astrogliosis [7]. In
this regard, the most remarkable evidence of the relevance of GFAP in the physiological roles
of astrocytes in maintaining normal brain function is Alexander disease, a fatal disorder in
which GFAP mutations might compromise the astrocyte stress response [8].
3. Protein phosphorylation in signaling transduction
Phosphorylation is the most widespread type of posranslational modication of the intracel‐
lular signaling proteins. Phosphorylation of proteins occurs within seconds or minutes of a
regulatory signal, typically an extracellular signal.
Phosphorylation is an enzymatic process in which the introduction of a phosphoryl group to
specic amino acid residues of a protein is catalyzed by protein kinases and the removal of
phosphoryl groups is catalyzed by protein phosphatases. For phosphorylation to be useful
in the regulation of a protein activity, it is important to be a reversible process, in which the
phosphorylated form of the protein could restore its original dephosphorylated form when
signal ends, functioning therefore as a molecular switch. The addition of a phosphoryl group
to the side chain of a Ser, Thr, or Tyr residue introduces a bulky, charged group into a polar
region. The oxygen atoms of a phosphoryl group can hydrogen bond with one or several
groups in a protein, commonly the amide groups of the peptide backbone at the α‐helix start
or the charged guanidinium group of an Arg residue inuencing the functionality of the
protein [9].
3.1. Phosphorylation of intermediate‐lament proteins
Phosphorylation, glycosylation and transglutamination take part in the multiple mechanisms
of IF regulation. However, phosphorylation/dephosphorylation is a major regulatory mecha‐
nism orchestrating IF dynamics. Phosphorylation sites of IF subunits are located on their head
and tail domains and phosphorylation plays a major role in regulating the structural organi‐
zation and function of these cytoskeletal proteins in a cell‐ and tissue‐specic manner [10].
Amino‐terminal phosphorylation regulates the assembly/disassembly equilibrium of type
III and IV IFs. Second messenger‐dependent protein kinases add phosphate groups on the
Intermediate Filaments as a Target of Signaling Mechanisms in Neurotoxicity
http://dx.doi.org/10.5772/66926
237
amino‐terminal head domain on GFAP, vimentin and NF‐L. Specic phosphorylating sites
for cAMP‐dependent protein kinase (PKA), Ca2+/calmodulin‐dependent protein kinase II
(PKCaMII) and protein kinase C (PKC) are associated with IF disassembly; however, the
action of the protein phosphatases 1, 2A and 2B (PP1, PP2A and PP2B), respectively, removes
phosphate and restores the IF ability to polymerize [11].
Otherwise, the main phosphorylation sites on NF‐M and NF‐H are located in Lys‐Ser‐Pro
(KSP) repeat regions of the tail domain of these subunits. The KSP repeats are phosphor‐
ylated by proline‐directed kinases such as Cdk5, the mitogen‐activated protein kinases
(MAPK) such as Erk1/2, JNK, p38MAPK as well as glycogen synthase kinase 3 (GSK3).
Phosphorylation of these KSP sites regulates the interactions of NFs with each other and
with other cytoskeletal structures, since the tail domain of NF‐M and NF‐H protrudes later‐
ally from the lament backbone to form “side‐arms” when phosphorylated. These lateral
interactions are central in the formation of a cytoskeletal laice that supports the mature
axon. Moreover, carboxyl‐terminal phosphorylation of NF‐M and NF‐H subunits has long
been considered to regulate their axonal transport rate and in doing so to provide stability
to mature axons [12]. The axonal transport of NFs results from binding to the fast motor
proteins kinesin and dynein intermied with prolonged pauses. It is known that carboxyl‐
terminal phosphorylation of NF‐H progressively restricts the association of NFs with kinesin
and stimulates its interaction with dynein. This event could represent one of the mechanisms
by which aberrant carboxyl‐terminal phosphorylation would slow NF axonal transport. Both
the maintenance of axonal caliber and axonal transport are dependent on the adequately
phosphorylated NF subunits. Consequently, abnormally hyperphosphorylated NF subunits,
commonly found in several neurodegenerative diseases, are intimately associated with neu‐
ral dysfunction and considered a hallmark of neurodegeneration. In addition, demyelinating
diseases might be associated with hypophosphorylated NFs, compromised axonal transport
and decreased axonal diameter, since the phosphorylation of NFs occurs in close proximity
to myelin sheaths, which release signals needed to induce phosphorylation of NFs in mature
axons [13].
In the next sections, we discuss the recent ndings from our laboratory indicating that sig‐
naling mechanisms involved in the regulation of IF phosphorylation/dephosphorylation are
important targets of neurotoxins, metabolites accumulating in neurodegenerative diseases as
well as thyroid hormones, emphasizing the relevance of cytoskeletal homeostasis on the brain
function/dysfunction. To assess the eects of the neurotoxicants on the phosphorylation level
of IF proteins, we developed an approach to measure the in vitro incorporation of radioactive
phosphate (32P‐orthophosphate) into these proteins [14]. In order to shed light onto the sig‐
naling cascades targeted by them, we used pharmacological and immunological approaches,
specic enzyme inhibitors, channel blockers, or glutamate antagonists as well as monoclonal
antibodies directed to signaling cascades or specic phosphorylation sites. We conclude that
misregulated cell signal transduction interferes with the phosphorylation/dephosphorylation
of IFs disrupting the homeostasis of the cytoskeleton of astrocytes and neurons and this is
associated with cell dysfunction and neurodegeneration in experimental models of neurotox‐
icity. Figure 2 corresponds to a schematic representation of the consequences of misregulated
NF phosphorylation for neuronal function.
Cytoskeleton - Structure, Dynamics, Function and Disease238
3.2. Central roles of Ca2+ and glutamate receptors on the regulation of cytoskeletal
dynamics in neural cells
Changes in the cytoplasmic free Ca2+ concentration constitute one of the main pathways by
which information is transferred from extracellular signals received by animal cells to intra‐
cellular sites. However, an augmented Ca2+ inux through the NMDA receptor or voltage‐
dependent calcium channels (VDCCs) can be responsible for the activation of lethal metabolic
pathways in neural cells. Overactivation of glutamate receptors produces neuronal membrane
depolarization. This causes the inux of Ca2+ into the cytoplasm and subsequently triggers cas‐
cade events leading to excitotoxic neuronal death. Excitotoxicity is recognized as a major patho‐
logical process of neuronal death in neurodegenerative diseases involving the CNS. In this
regard, compelling ndings point to the cytoskeleton as an end point of excitotoxic mechanisms.
Dierent toxins and stress conditions are implicated in the misregulation of intracellular
Ca2+‐dependent processes in cells and dierent cell types exhibit a diverse range of transient
responses to their stimuli. Exposure of tissue slices to neurotoxicants or metabolites in toxic
concentrations triggers the activation of ionotropic and metabotropic glutamate receptors
as well as L‐VDCC and the endoplasmic reticulum (ER) Ca2+ channels. These receptors and
channels activate several intracellular‐signaling complexes altering cell behavior in a spatio‐
temporally regulated manner. Metabolism of cyclic nucleotides, membrane phospholipids
as well as endogenous enzymatic regulators are the key biochemical steps coordinating cell
response to an extracellular stimulus [15].
Calcium is a critical regulator of cytoskeletal dynamics. Dysregulation of Ca2+ homeosta‐
sis is an important event in driving the disruption of assembly/disassembly equilibrium as
Figure 2. Schematic representation of disrupted neurolament phosphorylation. The hyperphosphorylation of neurol‐
aments can change the cytoskeleton architecture and lead to neurolament aggregation in perikarya and in axon accounting
for cell damage.
Intermediate Filaments as a Target of Signaling Mechanisms in Neurotoxicity
http://dx.doi.org/10.5772/66926
239
well as the interaction of cytoskeletal proteins with regulatory proteins or cell organelles.
In particular, IF proteins are directly regulated by Ca2+ levels, which crosslink signaling cas‐
cades and connect physiological or pathological extracellular signals with the IF cytoskeleton
inuencing multiple aspects of cell behavior. Consequently, abnormally elicited Ca2+ signals
provoking misregulation of key phosphorylation cascades are able to disrupt cytoskeletal
homeostasis and this is commonly associated with the cell damage.
4. Toxicity of diphenyl ditelluride on the cytoskeleton of neural cells
Many processes in the organic synthesis, vulcanization of rubber and in metal‐oxidizing solu‐
tions to tarnish metals, such as silver, extensively use tellurium. Diphenyl ditelluride (PhTe)2
is the simplest of the aromatic, diorganoyl ditelluride compounds used in organic synthesis.
Indeed, developmental exposure to (PhTe)2 is teratogenic and is associated with long‐term
behavioral and neurochemical changes in rats. Until recently, the general toxicity of (PhTe)2
was considered to be exclusively related to the oxidation of thiol‐containing proteins (for
review, see [16]). However, compelling evidence from our laboratory points to an important
role played by signaling mechanisms involved in regulating IF phosphorylation/dephosphor‐
ylation as target of (PhTe)2 neurotoxicity. In addition, we evidence a remarkable role of Ca2+
mediating these actions secondary to glutamate receptors and L‐VDCC activation.
The neurotoxicity of (PhTe)2 is spatiotemporally regulated, consistent with the window of sus‐
ceptibility of signaling cascades as well as the structural and functional heterogeneity of neu‐
rons in dierent brain regions. In this regard, exposure of cortical slices from 18‐ and 21‐day‐old
rats to (PhTe)2 shows unaltered phosphorylation of IF proteins, while IFs of acute cortical slices
from younger pups (9 and 15 days old) are hypophosphorylated. Activated ionotropic gluta‐
mate receptors, L‐VDCC and ryanodine channels result in PP1‐mediated hypophosphoryla‐
tion of GFAP and NF subunits pointing to the cortical cytoskeleton as a preferential target of
the action of phosphatases in this window of vulnerability. Activation of PP1 is modulated by
dopamine and cyclic AMP‐regulated neuronal phosphoprotein 32 (DARPP‐32), an important
endogenous Ca2+‐mediated inhibitor of PP1 activity. Depending on the site of phosphorylation,
DARPP‐32 is able to produce opposing biochemical eects, that is, inhibition of PP1 activity or
inhibition of protein kinase A (PKA) activity [17]. Decreased cAMP and PKA catalytic subunits
support that (PhTe)2 disrupts the cytoskeletal‐associated phosphorylating/dephosphorylating
system of neurons and astrocytes through PKA‐mediated inactivation of DARPP‐32, promot‐
ing PP1 release and hypophosphorylation of IF proteins of those neural cells [18]. Regarding
neurons, hypophosphorylation of IF proteins could be associated with cell dysfunction since
decreased phosphorylation of KSP repeats in the carboxyl‐terminal domains of NF‐M and
NF‐H correlates with impaired axonal transport and increased NF‐packing density.
In contrast with younger rats, hippocampal slices of 21‐day‐old rats acutely exposed to
(PhTe)2 result in hyperphosphorylated IFs. Hippocampal IF hyperphosphorylation is par‐
tially dependent on L‐VDCC, NMDA and ER Ca2+ channels. The signal evoked by (PhTe)2 is
also transduced through metabotropic glutamate receptors on the plasma membrane, leading
to the activation of phospholipase C (PLC) that produces the intracellular messengers inositol
Cytoskeleton - Structure, Dynamics, Function and Disease240
1,4,5‐trisphosphate (IP3) and diacylglycerol (DAG). IP3 binds to specic receptors on the ER
changing the conformation of IP3 receptors and opening the channel. Released Ca2+ and DAG
directly activate PKCaMII and PCK, resulting in the hyperphosphorylation of some of the
critical amino acid residues in the carboxyl‐terminal tail domain of NF‐L known to interfere
with lament assembly. In addition, the activation of Erk1/2 and p38MAPK results in hyper‐
phosphorylation of KSP repeats of NFM. Interestingly, PKCaMII and PKC are upstream of
MAPK activation implying in a signicant cross‐talk among signaling pathways elicited by
(PhTe)2 that connect the glutamate metabotropic cascade with the activation of Ca2+ channels.
The nal molecular result is the extensive phosphorylation of amino‐ and carboxyl‐terminal
sites on IF proteins and deregulated cytoskeletal homeostasis [19].
4.1. Diphenyl ditelluride disrupts the cytoskeleton and provokes neurodegeneration
in acutely injected young rats
The in vivo exposure to (PhTe)2, in which the neurotoxicant reaches the brain via systemic
circulation, also results in dierent susceptibilities of the IF proteins from neural cells. This
can be evidenced in cerebral cortex and hippocampus of 15‐day‐old rats acutely injected with
a toxic dose of (PhTe)2 (0.3 µmol/kg body weight) [20]. Cortical hyperphosphorylation of neu‐
ronal and glial IF proteins is an early and persistent event up to 6 days after injection, accom‐
panied by increased levels of GFAP and NF‐L. Upregulated gene expression as well as GFAP
and vimentin hyperphosphorylation could be a response to injury and take part in the pro‐
gram of reactive astrogliosis, as further demonstrated in striatum [21] and cerebellum [22] of
(PhTe)2‐injected rats. In addition, hippocampal IFs are not responsive to the insult until wean‐
ing. A strong evidence supports an important role of astrocytes in a more severe cortical than
hippocampal damage following the in vivo (PhTe)2 insult. This supports a direct action of the
neurotoxicant on intracellular signaling pathways and highlights the relevance of the inter‐
play between glial and neuronal cells to adapt the cellular metabolic response to the insult
even when the brain connections are only partially preserved, as shown in acute brain slices.
Of importance, neurodegeneration is part of the deleterious in vivo eects of (PhTe)2 tox‐
icity, as demonstrated in the striatum [23] and cerebellum [22] of (PhTe)2‐injected rats.
Neurodegeneration is associated with alterations in Ca2+ homeostasis and glutamatergic
neurotransmission, upstream of inhibited Akt and activated caspase 3. We therefore propose
that excitotoxicity is a main mechanism of neurodegeneration caused by this compound in
the developing rat brain. On the other hand, most of the actions of (PhTe)2 disrupting the
homeostasis of the cytoskeleton in neural cells are mediated by high Ca2+ levels. Moreover,
a link among disrupted IF homeostasis, activated astrocytes and neuronal apoptosis in
(PhTe)2‐injected rats has been demonstrated by immunohistochemical approaches. In addi‐
tion, MAPK pathway might be a link between altered IF equilibrium and neural cell damage,
since MAPK is implicated in IF hyperphosphorylation and neurodegeneration as well in the
brain structures aained by (PhTe)2 toxicity. Further supporting the cytoskeleton as an end
point of neurotoxicity, hyperphosphorylated NFs can inhibit their proteolytic breakdown by
calpain, a Ca2+‐activated protease. In addition, abnormally phosphorylated NFs accumulate
in the perikarya and the phospho‐NF aggregates can thus become cytotoxic by the enduring
impairment of axonal transport of NFs (see Figure 2). The increased time the NF spent in the
Intermediate Filaments as a Target of Signaling Mechanisms in Neurotoxicity
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241
cell body is thought to result in further aberrant phosphorylation and may prevent them from
entering the axon, resulting in a deleterious feedback loop [24].
In summary, we propose that complex and integrated actions mediate the (PhTe)2 toxicity
directed to the cytoskeleton of neural cells. These molecular mechanisms induce spatiotem‐
poral responses of the cells because of the dierent windows of susceptibility of the develop‐
mental brain. Nonetheless, the Ca2+ ‐initiated events highlight a role for this neurotoxicant as
a disruptor of the cytoskeleton.
5. Cytoskeleton as a target of amino acids and their metabolites
Misregulated cytoskeletal homeostasis is among the molecular mechanisms underlying the
neural cell dysfunction in brain tissue exposed to high levels of amino acids and/or their
metabolites. In humans, several neurological impairments are associated with enzymatic
deciencies or defects in proteins involved in cellular metabolism of neural cells, causing
accumulation of metabolic intermediates associated with neuronal damage. We discuss some
aspects of the molecular mechanisms underlying the disruption of cytoskeletal homeosta‐
sis in response to branched‐chain keto acids (BCKAs) derived from leucine, isoleucine and
valine. We also addressed the eects of homocysteine and quinolinic acid (QUIN), a metabo‐
lite of tryptophan metabolism, directed to the cytoskeleton.
5.1. Branched chain α‐keto acids and the cytoskeleton of neural cells
The branched‐chain ketoacids, α‐ketoisocaproic acid (KIC), α‐keto‐β‐methylvaleric acid
(KMV) and α‐keto‐isovaleric acid (KIV) are produced from the respective branched‐chain
amino acids (BCAAs) leucine, isoleucine and valine, in the reaction catalyzed by the branched‐
chain α‐keto acid dehydrogenase (BCKAD) complex. A deciency of the BCKAD complex is
an inherited metabolic disease known as maple syrup urine disease (MSUD) which lead to the
accumulation of BCAAS and BCKAs in tissues and body uids resulting in dramatic cerebral
symptoms [25].
Curiously, cortical slices of young rats exposed to high levels of the BCAAs individually pre‐
serve the homeostasis of the cytoskeleton. On the other hand, their respective keto acids pro‐
vide an interesting example of the ne‐tune regulation of the cytoskeleton, since KIC [26] and
KMV [27] were dierently deleterious to the homeostasis of the cytoskeleton. KIC and KMV
alter the dynamics of IF proteins of astrocytes and neurons through dierent transduction
mechanisms dependent on excessive intracellular Ca2+ inux, while KIV appears not to be
involved in the disruption of the IF cytoskeleton [28].
The eect of KIC is outlined by hypophosphorylation of GFAP, NF‐M and NF‐L in very young
rats (up to 12 days of age) changing to hyperphosphorylation of the same proteins later in
development (17 days of age). Nonetheless, both responses of the cytoskeletal‐associated
phosphorylating system are regulated by Ca2+ currents through the NMDA and L‐VDCC, as
well as by the intracellular Ca2+storage release from the ER, leading to a dierential activation
of protein phosphatases or kinases [28]. These paradoxical ndings provide an interesting
Cytoskeleton - Structure, Dynamics, Function and Disease242
insight into the dierential susceptibility of cortical IF cytoskeleton to the exposure to
pathological levels of this metabolite. The dierent vulnerabilities of the cytoskeleton of
cortical cells during development might be ascribed to the temporal maturation mediated
by a multitude of developmental processes and signaling pathways. It is conceivable that
they are associated with the pathological role of the developmentally regulated glutamate
receptors in neural cells since the expression paerns of glutamate receptor subunit genes
change during the ontogeny of the brain. Distinct regional and temporal paerns of the
expression of types and subtypes of the glutamate ionotropic receptors during ontogeny
may possibly explain the dierent signaling pathways targeting the cytoskeleton of corti‐
cal neural cells during development.
Interestingly, KMV disturbs the IF‐associated cytoskeletal phosphorylation only in 12‐day‐old
rats without changing the phosphorylation level of these proteins in younger or older animals,
showing a specic window of vulnerability of cytoskeleton to KMV insult in the cerebral cor‐
tex of developing brain. Strikingly, this eect was dependent on intracellular Ca2+ concentra‐
tions; however, in this case ɣ‐amino butyric acid A and B (GABAA and GABAB, respectively)
rather than glutamate receptors were involved in this action. This is in agreement with GABAA
and GABAB receptors mediating the induction and maintenance of Ca2+ levels [27].
Overall, we propose that BCKAs in supra‐physiological concentrations disrupt the cytoskeleton
of rat brain through misregulation of the phosphorylating system associated with the IF cyto‐
skeleton. We evidenced developmentally regulated mechanisms in which Ca2+‐mediated excito‐
toxicity plays a critical role in destabilizing the cytoskeleton that may ultimately disrupt normal
cell function and viability. Although evidence from animal models should be taken with cau‐
tion, we can propose that the disrupted cytoskeleton is part of the physiopathology of MSUD.
5.2. Hyperhomocysteinemia and the cytoskeleton of neural cells
Homocysteine (Hcy) is a sulfur‐containing amino acid generated during methionine metabolism.
Genetic mutations impairing Hcy metabolism cause accumulation of this amino acid aaining
high levels in blood, leading to severe hyperhomocysteinemia and brain damage. Otherwise,
along with genetic factors, mild‐moderate hyperhomocysteinemia is associated with nutritional
imbalance and hormonal factors. Mild hyperhomocysteinemia, which markedly enhance the
vulnerability of neuronal cells to excitotoxicity and oxidative imbalance, is also common in older
people, constituting an independent risk factor for stroke and cognitive impairment [29].
Various existing experimental evidences from our group link hyperhomocysteinemia and cyto‐
skeletal misregulation, supporting that disrupted cytoskeleton could be an end point of neural
dysfunction in this neurometabolic disorder. Experiments with brain slices acutely exposed to
mild Hcy levels (100 µM) showed greater vulnerability of hippocampal cytoskeleton as com‐
pared with cortical one. Moreover, a window of vulnerability of the cytoskeleton of hippocam‐
pal cells is evidenced, since misregulated phosphorylation is detected only at postnatal day
17 [30], reecting an altered activity of the endogenous phosphorylating system associated
with the IFs in this brain structure. As expected, NMDA receptors, L‐VDCC and extracellular
Ca2+inux result in PKC and PKCaMII activation. The prevention of Hcy action through the
inhibition of PKC and MEK, a step that is upstream of MAPK cascade (Raf‐1/MEK/MAPK),
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is consistent with an eect at the level of the monomeric GTPase Raf‐1, supporting a role for
PKC phosphorylating and activating Raf‐1 in the Hcy‐induced modulation of the cytoskeleton.
In contrast with hypophosphorylation found in hippocampal slices, the chemically induced
chronic hyperhomocysteinemia dierently alters the signaling mechanisms directed to the
cytoskeleton, producing PP1‐, PP2A‐ and PP2B‐mediated hypophosphorylation of NF sub‐
units and GFAP in hippocampal slices of 17‐day‐old rats without aecting the cerebral cortex
[31] through glutamate and Ca2+‐mediated mechanisms. Further evidence that homocysteine
targets the cytoskeleton came from cytoskeletal reorganization in primary astrocytes and neu‐
rons exposed to homocysteine [32]. Dramatically altered actin cytoskeleton in primary astro‐
cytes exposed to 100 µM Hcy is consistent with the role of actin as a main determinant of cell
morphology. Concomitant disrupted GFAP meshwork underlies the remodeled actin cyto‐
skeleton and altered cell morphology. These ndings provide further evidence of the cross‐talk
among the dierent cytoskeletal subsystems and the roles played by the toxic levels of Hcy.
Therefore, taking into account our experimental evidence it is conceivable that disturbed cell
signaling is an important determinant of the disrupted homeostasis of the cytoskeleton as a
whole, with widespread consequences on cell function that could be associated with human
hyperhomocysteinemia.
6. Cytoskeleton is a target of quinolinic acid neurotoxicity
Quinolinic acid is a neuroactive metabolite of the kynurenine pathway normally found in
nanomolar concentrations in human brain and cerebrospinal uid (CSF). QUIN is antagonist
of NMDA receptor and it has a high in vivo potency as an excitotoxin supporting involvement
in the pathogenesis of a variety of human neurological diseases. The neurotoxicity of QUIN
results from complex mechanisms including presynaptic receptors, energetic dysfunction,
oxidative stress, transcription factors and behavior [33]. We experimentally demonstrate that
the disruption of the cytoskeleton, in particular, misregulation of the phosphorylation system
associated with the IFs, is a target of QUIN toxicity in injected rat striatum, tissue slices and
primary astrocytes and neurons in culture.
6.1. Eects of intrastriatally injected quinolinic acid on the cytoskeleton of neural cells
Acute intrastriatal injection of QUIN (150 nmol/0.5 µL) in adolescent rats (30 days old) pro‐
vokes NF‐L and GFAP hyperphosphorylation 30 min after infusion, evidencing the suscepti‐
bility of the cytoskeleton of both neurons and astrocytes in the early events of QUIN toxicity.
Hyperphosphorylated NF‐LSer55 destabilizes the NF structure and this might represent an
early step in the pathophysiological cascade of deleterious events exerted by QA in rat striatum.
Experimental insights to get light on the molecular mechanisms underlying this eect point to
NMDA‐mediated Ca2+ events and oxidative stress upstream of activated second messenger‐
dependent protein kinases PKA, PKC and PKCaMII, but not MAPKs after QUIN infusion [34].
A link between misregulation of cell‐signaling mechanisms, disruption of IF phosphory‐
lation and cell damage as part of QUIN toxicity becomes more evident analyzing the
Cytoskeleton - Structure, Dynamics, Function and Disease244
long‐lasting eect of the acute intrastriatal injection of QUIN in adolescent rats on the dynam‐
ics of the phosphorylating system until 21 days after injection [35]. The acutely injected QUIN
alters the homeostasis of IF phosphorylation in a selective manner, progressing from stria‐
tum to cerebral cortex and hippocampus. Twenty‐four hours after QUIN injection, the IFs are
hyperphosphorylated in the striatum. This eect progresses to cerebral cortex causing hypo‐
phosphorylation at day 14 and appears in the hippocampus as hyperphosphorylation at day
21 after QUIN infusion, PKA and PKCaMII mediating this eect. However, MAPKs (Erk1/2,
JNK and p38MAPK) are hyperphosphorylated/activated only in the hippocampus, suggest‐
ing dierent signaling mechanisms in these two brain structures during the rst weeks after
QUIN infusion. Also, PP1 and PP2B‐mediated hypophosphorylation of the IF proteins in the
cerebral cortex 14 days after QUIN injection reinforces the selective signaling mechanisms in
dierent brain structures. Increased GFAP immunocontent in the striatum and cerebral cortex
24 h and 14 days after QUIN injection, respectively, suggests reactive astrocytes in these brain
regions. Yet, we observe biochemical and histopathological alterations in the striatum, cortex
and hippocampus, as well as altered behavioral tests in response to the long‐lasting exposure
to QUIN through glutamate and Ca2+‐mediated mechanisms. Thus, it is tempting to propose
that the long‐lasting deleterious eect of intrastriatal QUIN injection could be due to the fact
that QUIN interferes with the highly regulated signaling mechanisms targeting the cytoskel‐
eton in the immature brain [36].
6.2. Insight into the molecular basis of quinolinic acid action toward the cytoskeleton
Studies in acute brain slices further support the role of glutamatergic signaling and Ca2+ over‐
load disturbing the cytoskeletal equilibrium downstream of QUIN exposure. Moreover, this
experimental approach brings light on the cell‐specic mechanisms targeting the cytoskeleton
in astrocytes and neurons when the cell connections are partially preserved. In astrocytes,
the QUIN action is mainly due to increased Ca2+ inux through NMDA and L‐VDCC. In
neuronal cells, QUIN acts through the activation of metabotropic glutamate receptors and
inux of Ca2+ through NMDA receptors and L‐VDCC, as well as Ca2+ release from intracellu‐
lar stores. These mechanisms then set o a cascade of events including the activation of PKA,
PKCaMII and PKC, which phosphorylate head domain sites on GFAP and NFL. Moreover,
Cdk5 is activated downstream of mGluR5, phosphorylating the KSP repeats on NFM and
NFH. Metabotropic glutamate receptors type 1 (mGluR1) is upstream of PLC, which, in turn,
produce DAG and IP3 promoting hyperphosphorylation of KSP repeats on the tail domain of
NFM and NFH [37].
6.3. The cytoskeleton of astrocytes and neurons responds dierently to quinolinic
acid toxicity
The susceptibility of the cytoskeleton to toxic levels of QUIN is also detectable in isolated astro‐
cytes and neurons growth in primary cultures [38]. In astrocytes, Ca2+‐mediated glutamate mech‐
anisms target the endogenous phosphorylating system, since metabotropic glutamate receptors
and Ca2+ inux through NMDA receptors are upstream of PKA, PKCaMII and PKC activa‐
tion, provoking GFAP hyperphosphorylation. Interestingly, the misregulated phosphorylation
system leads to a reversible and dramatically altered actin cytoskeleton with concomitant change
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of morphology to fusiform and/or aened cells with retracted cytoplasm and disruption of the
GFAP meshwork [39] supporting the dynamic behavior of the cytoskeleton.
Interestingly, neurons show greater vulnerability to QUIN than astrocytes (10×). Neurons
exposed to QUIN presented PKA‐ and PKC‐mediated hyperphosphorylation of NF subunits.
These eects are also downstream of ionotropic and metabotropic glutamate signaling and
Ca2+ inux through NMDA receptors and L‐VDCC. The misregulated signaling pathways dis‐
rupt the neuronal cytoskeleton, evaluated by altered neurite/neuron ratios and neurite out‐
growth. It is important to consider that microtubules play a central role in cell polarity [40].
In particular, microtubules are the main determinants of neuronal polarity and regulation of
microtubule dynamics includes tubulin posranslational modications [40] and phosphoryla‐
tion of microtubule‐associated proteins (MAPs), whose binding to microtubules is essential
for neurite formation [41]. As an example, activated GSK‐3β leads to increased phosphoryla‐
tion of some MAPs, destabilizing microtubules with consequence for neurite stabilization [42].
Therefore, the neurite destabilization could derive from both NFs and microtubules disruption.
Interestingly, we found a protective role of astrocyte‐conditioned medium on the disrupted
neuronal cytoskeleton and morphometric alterations, suggesting that QUIN‐induced trophic
factors secreted by astrocytes are able to modulate signaling mechanisms targeting the neu‐
ronal cytoskeleton. More interestingly, co‐cultured astrocytes and neurons preserve their
cytoskeletal organization and cell morphology together with unaltered activity of the phos‐
phorylating system associated with the cytoskeleton. In other words, co‐cultured astrocytes
and neurons tightly and actively interact with one another reciprocally protecting themselves
against QUIN injury [38]. This evidence raise the question about the role played by the acti‐
vated microglia eliciting signals essential to destabilize the astrocytic and neuronal cytoskel‐
eton but this hypothesis remains to be claried.
All together, we conclude that among the multiple mechanisms through which accumulated
QUIN is able to induce cell damage, our experimental evidence points to Ca2+‐mediated
mechanisms directed to the cytoskeletal disruption as an end point of QUIN toxicity. Both in
vivo and ex vivo approaches clearly demonstrate a wide spectrum of misregulated signaling
mechanisms downstream of QUIN action directly aecting the cytoskeleton and disrupting
cell homeostasis. We also provide evidence that impaired physiological equilibrium of the
signaling cascades directed to the cytoskeleton underlies QUIN cytotoxicity and is associated
with neurodegeneration. The in vitro results showing disorganized cytoskeleton and altered
cell morphology further support the cytoskeleton as a hallmark of stress condition that could
be implicated in the human brain disorders associated with high QUIN levels.
7. Cytoskeleton of neural cells is a target of thyroid hormones
Thyroid hormones are essential for the development and function of central nervous sys‐
tem. In brain, these hormones are essential for myelination [43, 44], neuritogenesis [45],
synaptic plasticity [46–48], IF phosphorylation [49–54], cell dierentiation and maturation
[55]. Considering the role of these hormones on brain development, thyroid diseases might
account for brain injury as well as alteration in mood and cognition [56].
Cytoskeleton - Structure, Dynamics, Function and Disease246
3‐
‐
‐
7.1. Insight into the molecular basis of genomic and nongenomic action of thyroid
hormones toward the cytoskeleton of neural cells
‐
4
nongenomic mechanism [58
34 and reverse
33
43‐
433
59
3‐
‐
51]
3 4
, ‐
34
‐
4
49
‐
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Calcium‐dependent mechanisms play a central role on the thyroid hormone‐induced modula‐
tion of the phosphorylating system associated with IFs. Zamoner and colleagues [49] demon‐
strated that the nongenomic mechanisms underlying the eects T4 targeting the IF‐associated
phosphorylating system in cerebral cortex from 15‐day‐old rats are dependent on extracel‐
lular Ca2+ inux through VDCC, as well as Ca2+ release from ER stores.
Taking into account that in rat the myelination peak is coincident with postnatal day 15 and
that this is a period of intense synaptogenesis, the NF hyperphosphorylation induced by T4 in
cerebral cortex from 15‐day‐old rats appears to be correlated to synaptogenesis and myelination
(for review, see [53]).
In summary, we could suggest that nongenomic actions of T4 targeting the cytoskeleton of
glial cells and neurons might account for neuronal cell migration, myelination, synaptogen‐
esis and synaptic plasticity. Moreover, the modulation of NF phosphorylation by thyroid hor‐
mone may control axonal caliber.
7.2. Hypothyroidism and the cytoskeleton of neural cell
The eects of thyroid hormones in central nervous system during development include the
modulation of the cytoskeleton dynamics. Hypothyroidism in the developing rat brain is asso‐
ciated with oxidative stress and aberrant intraneuronal accumulation of NFs in the perikaryon
of Purkinje neurons (see Figure 2). The authors suggested that the neuron alterations observed
in the developing hypothyroid brain are comparable to those seen in neurodegenerative dis‐
eases [62]. Corroborating these ndings, it has been shown that the eects of hypothyroidism
on neuronal cytoskeleton involve the developmental modulation of specic isoforms of protein
expression, which induce stoichiometric imbalance between the NF triplet [52]. In addition,
thyroid hormone deciency induces a delay and a partial arrest of astrocyte dierentiation,
supported by the decreased expression of GFAP both in cortical [52] and in hippocampal astro‐
cytes [54], which was accompanied by downregulation of the astrocyte glutamate transport‐
ers. These ndings are associated with the extracellular signal‐regulated kinase (ERK)1/2 and
c‐jun terminal kinase (JNK) activation. NF hyperphosphorylation might account for the aber‐
rant intraneuronal accumulation of these cytoskeletal structures previously described [62].
Our research group demonstrated the hyperphosphorylation of tail KSP repeats on NF‐H in
hypothyroid cortical and hippocampal neurons [52, 54]. The carboxyl‐terminal phosphoryla‐
tion of NF‐H progressively restricts association of NFs with kinesin, the axonal anterograde
motor protein and stimulates its interaction with dynein, the axonal retrograde motor protein
[63]. This event could represent one of the mechanisms by which carboxyl‐terminal phos‐
phorylation would slow NF axonal transport.
Taking into account our experimental evidence, we propose that the consequences of congeni‐
tal hypothyroidism to neural cells involve IF hyperphosphorylation, misregulation of gluta‐
mate‐glutamine cycle, oxidative stress and glutamate excitotoxicity. These events suggest a
compromised astroglial defense system that is probably playing a role in the physiopathology
of the neurological dysfunction of hypothyroidism (Figure 3).
Cytoskeleton - Structure, Dynamics, Function and Disease248
Figure 3. Role of glutamate excitotoxicity on intermediate‐lament dynamics and cell damage. Congenital hypothyroid‐
ism leads to glutamate excitotoxicity, calcium overload, and oxidative stress. These events are related to intermediate‐
lament (GFAP and NF) hyperphosphorylation and neural cell damage.
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8. General conclusion
Studies of our group on the endogenous phosphorylating system associated with the IF pro‐
teins of neural cells point to a critical role of disrupted cytoskeleton in response to a variety
of signals both in physiological and in pathological conditions. Our ndings highlight the
IFs as a preferential target of the signal transduction pathways. Importantly, a large body of
evidence shows a link among misregulation of cell‐signaling mechanisms, disruption of IF
phosphorylation and cell damage in response to dierent stress signals. While the exact sig‐
naling pathways regulating NF phosphorylation remains elusive, there is increasing evidence
that known signal transduction cascades are involved. These actions can be initiated by the
activation of NMDA‐, L‐VDCC, or G protein‐coupled receptors and the signal is transduced
downstream of Ca2+ mobilization or monomeric GTPase activation through dierent kinase/
phosphatase pathways, regulating the dynamics of the cytoskeleton. Figure 4 summarizes
Figure 4. Summary of calcium‐associated mechanisms triggered by thyroid hormones, quinolinic acid, diphenyl
ditelluride, branched‐chain keto acids, and homocysteine targeting intermediate‐lament phosphorylation in neural
cells. Calcium inux through the NMDA receptor or voltage‐dependent calcium channels (VDCC) can be responsible for
the activation of lethal metabolic pathways in neural cells. Augmented intracellular Ca2+ levels might be associated with
the modulation of diverse cell‐signaling pathways and exhibit a diverse range of responses to their stimuli.
Cytoskeleton - Structure, Dynamics, Function and Disease250
the calcium‐associated mechanisms triggered by thyroid hormones, quinolinic acid, (PhTe)2,
BCKAs and homocysteine targeting IF phosphorylation in neural cells.
Despite the focus on the misregulation of IF dynamics in response to signaling mechanisms
downstream of metabolites and neurotoxicants, we should consider that cytoskeleton is a
complex meshwork of interconnecting laments [1]. In this regard, the morphological altera‐
tions demonstrated in primary cells in culture mainly reect the reorganization of the mesh‐
work of laments. Taking into account our ndings, we propose that misregulation of kinase/
phosphatase cascades downstream of stressors could disrupt the cytoskeleton as a whole and
this might be an important determinant of neural dysfunction associated with the action of
neurotoxicants and in neurometabolic conditions.
Author details
Ariane Zamoner1 and Regina Pessoa‐Pureur2*
*Address all correspondence to: rpureur@ufrgs.br
1 Department of Biochemistry, Center of Biological Sciences, Federal University of Santa
Catarina, Florianópolis, Brazil
2 Department of Biochemistry, Institute of Basic Sciences of Health, Federal University of Rio
Grande do Sul, Porto Alegre, Brazil
References
[1] Huber F, Boire A, Lopez MP, Koenderink GH. Cytoskeletal crosstalk: when three dier‐
ent personalities team up. Curr Opin Cell Biol. 2015;32:39–47.
[2] Herrmann H, Bar H, Kreplak L, Strelkov SV, Aebi U. Intermediate laments: from cell
architecture to nanomechanics. Nat Rev Mol Cell Biol. 2007;8(7):562–73.
[3] Herrmann H, Aebi U. Intermediate laments: molecular structure, assembly mechanism
and integration into functionally distinct intracellular Scaolds. Annu Rev Biochem.
2004;73:749–89.
[4] Laser‐Azogui A, Kornreich M, Malka‐Gibor E, Beck R. Neurolament assembly and
function during neuronal development. Curr Opin Cell Biol. 2015;32:92–101.
[5] Gentil BJ, Tibshirani M, Durham HD. Neurolament dynamics and involvement in neu‐
rological disorders. Cell Tissue Res. 2015;360(3):609–20.
[6] Seifert G, Schilling K, Steinhauser C. Astrocyte dysfunction in neurological disorders: a
molecular perspective. Nat Rev Neurosci. 2006;7(3):194–206.
[7] Hol EM, Pekny M. Glial brillary acidic protein (GFAP) and the astrocyte interme‐
diate lament system in diseases of the central nervous system. Curr Opin Cell Biol.
2015;32:121–30.
Intermediate Filaments as a Target of Signaling Mechanisms in Neurotoxicity
http://dx.doi.org/10.5772/66926
251
[8] Der Perng M, Su M, Wen SF, Li R, Gibbon T, Presco AR, et al. The Alexander disease‐
causing glial brillary acidic protein mutant, R416W, accumulates into Rosenthal bers
by a pathway that involves lament aggregation and the association of alpha B‐crystal‐
lin and HSP27. Am J Hum Genet. 2006;79(2):197–213.
[9] Ubersax JA, Ferrell JE, Jr. Mechanisms of specicity in protein phosphorylation. Nat Rev
Mol Cell Biol. 2007;8(7):530–41.
[10] Omary MB, Ku NO, Tao GZ, Toivola DM, Liao J. “Heads and tails” of intermediate
lament phosphorylation: multiple sites and functional insights. Trends Biochem Sci.
2006;31(7):383–94.
[11] Sihag RK, Inagaki M, Yamaguchi T, Shea TB, Pant HC. Role of phosphorylation on the
structural dynamics and function of types III and IV intermediate laments. Exp Cell
Res. 2007;313(10):2098–109.
[12] Holmgren A, Bouhy D, Timmerman V. Neurolament phosphorylation and their pro‐
line‐directed kinases in health and disease. J Peripher Nerv Syst. 2012;17(4):365–76.
[13] McLean NA, Popescu BF, Gordon T, Zochodne DW, Verge VM. Delayed nerve stimula‐
tion promotes axon‐protective neurolament phosphorylation, accelerates immune cell
clearance and enhances remyelination in vivo in focally demyelinated nerves. PLoS One.
2014;9(10):e110174.
[14] Funchal C, de Almeida LM, Oliveira Loureiro S, Vivian L, de Lima Pelaez P, Dall Bello
Pessuo F, et al. In vitro phosphorylation of cytoskeletal proteins from cerebral cortex of
rats. Brain Res Brain Res Protoc. 2003;11(2):111–8.
[15] Mehta A, Prabhakar M, Kumar P, Deshmukh R, Sharma PL. Excitotoxicity: bridge to
various triggers in neurodegenerative disorders. Eur J Pharmacol. 2013;698(1–3):6–18.
[16] Pessoa‐Pureur R, Heimfarth L, Rocha JB. Signaling mechanisms and disrupted cyto‐
skeleton in the diphenyl ditelluride neurotoxicity. Oxid Med Cell Longev. 2014;2014:
458601.
[17] Hakansson K, Lindskog M, Pozzi L, Usiello A, Fisone G. DARPP‐32 and modulation
of cAMP signaling: involvement in motor control and levodopa‐induced dyskinesia.
Parkinsonism Relat Disord. 2004;10(5):281–6.
[18] Heimfarth L, Loureiro SO, Reis KP, de Lima BO, Zamboni F, Lacerda S, et al. Diphenyl
ditelluride induces hypophosphorylation of intermediate laments through modula‐
tion of DARPP‐32‐dependent pathways in cerebral cortex of young rats. Arch Toxicol.
2012;86(2):217–30.
[19] Heimfarth L, Loureiro SO, Reis KP, de Lima BO, Zamboni F, Gandol T, et al. Cross‐talk
among intracellular signaling pathways mediates the diphenyl ditelluride actions on the
hippocampal cytoskeleton of young rats. Chem Res Toxicol. 2011;24(10):1754–64.
[20] Heimfarth L, Loureiro SO, Zamoner A, Pelaez PDL, Nogueira CW, Da Rocha JBT, et
al. Eects of in vivo treatment with diphenyl ditelluride on the phosphorylation of
Cytoskeleton - Structure, Dynamics, Function and Disease252
cytoskeletal proteins in cerebral cortex and hippocampus of rats. Neurotoxicology.
2008;29(1):40–7.
[21] Heimfarth L, Reis KP, Loureiro SO, de Lima BO, da Rocha JB, Pessoa‐Pureur R.
Exposure of young rats to diphenyl ditelluride during lactation aects the homeosta‐
sis of the cytoskeleton in neural cells from striatum and cerebellum. Neurotoxicology.
2012;33(5):1106–16.
[22] Heimfarth L, Loureiro SO, Dutra MF, Petenuzzo L, de Lima BO, Fernandes CG, et al.
Disrupted cytoskeletal homeostasis, astrogliosis and apoptotic cell death in the cerebellum
of preweaning rats injected with diphenyl ditelluride. Neurotoxicology. 2013;34:175–88.
[23] Heimfarth L, Loureiro SO, Dutra MF andrade C, Peenuzzo L, Guma FT, et al. In vivo
treatment with diphenyl ditelluride induces neurodegeneration in striatum of young rats:
implications of MAPK and Akt pathways. Toxicol Appl Pharmacol. 2012;264(2):143–52.
[24] Perrot R, Berges R, Bocquet A, Eyer J. Review of the multiple aspects of neurola‐
ment functions and their possible contribution to neurodegeneration. Mol Neurobiol.
2008;38(1):27–65.
[25] Manara R, Del Rizzo M, Burlina AP, Bordugo A, Cion V, Rodriguez‐Pombo P, et al.
Wernicke‐like encephalopathy during classic maple syrup urine disease decompensa‐
tion. J Inherit Metab Dis. 2012;35(3):413–7.
[26] Funchal C, Zamoner A, dos Santos AQ, Loureiro SO, Wajner M, Pessoa‐Pureur R.
Alpha‐ketoisocaproic acid increases phosphorylation of intermediate lament proteins
from rat cerebral cortex by mechanisms involving Ca2+ and cAMP. Neurochem Res.
2005;30(9):1139–46.
[27] Funchal C, Dall Bello Pessuo F, de Almeida LM, de Lima Pelaez P, Loureiro SO, Vivian
L, et al. Alpha‐keto‐beta‐methylvaleric acid increases the in vitro phosphorylation of
intermediate laments in cerebral cortex of young rats through the gabaergic system. J
Neurol Sci. 2004;217(1):17–24.
[28] Funchal C, de Lima Pelaez P, Loureiro SO, Vivian L, Dall Bello Pessuo F, de Almeida
LM, et al. alpha‐Ketoisocaproic acid regulates phosphorylation of intermediate la‐
ments in postnatal rat cortical slices through ionotropic glutamatergic receptors. Brain
Res Dev Brain Res. 2002;139(2):267–76.
[29] Hainsworth AH, Yeo NE, Weekman EM, Wilcock DM. Homocysteine, hyperhomocys‐
teinemia and vascular contributions to cognitive impairment and dementia (VCID).
Biochim Biophys Acta. 2016;1862(5):1008–17.
[30] Loureiro SO, Heimfarth L, Pelaez Pde L, Vanzin CS, Viana L, Wyse AT, et al. Homocysteine
activates calcium‐mediated cell signaling mechanisms targeting the cytoskeleton in rat
hippocampus. Int J Dev Neurosci. 2008;26(5):447–55.
[31] Loureiro SO, Heimfarth L, Pelaez Pde L, Lacerda BA, Vidal LF, Soska A, et al.
Hyperhomocysteinemia selectively alters expression and stoichiometry of intermediate
Intermediate Filaments as a Target of Signaling Mechanisms in Neurotoxicity
http://dx.doi.org/10.5772/66926
253
lament and induces glutamate‐ and calcium‐mediated mechanisms in rat brain during
development. Int J Dev Neurosci. 2010;28(1):21–30.
[32] Loureiro SO, Romao L, Alves T, Fonseca A, Heimfarth L, Moura Neto V, et al.
Homocysteine induces cytoskeletal remodeling and production of reactive oxygen spe‐
cies in cultured cortical astrocytes. Brain Res. 2010;1355:151–64.
[33] Lugo‐Huitron R, Ugalde Muniz P, Pineda B, Pedraza‐Chaverri J, Rios C, Perez‐de la
Cruz V. Quinolinic acid: an endogenous neurotoxin with multiple targets. Oxid Med
Cell Longev. 2013;2013:104024.
[34] Pierozan P, Zamoner A, Soska AK, Silvestrin RB, Loureiro SO, Heimfarth L, et al. Acute
intrastriatal administration of quinolinic acid provokes hyperphosphorylation of cyto‐
skeletal intermediate lament proteins in astrocytes and neurons of rats. Exp Neurol.
2010;224(1):188–96.
[35] Pierozan P, Goncalves Fernandes C, Ferreira F, Pessoa‐Pureur R. Acute intrastriatal
injection of quinolinic acid provokes long‐lasting misregulation of the cytoskeleton in
the striatum, cerebral cortex and hippocampus of young rats. Brain Res. 2014;1577:1–10.
[36] Pierozan P, Fernandes CG, Dutra MF, Pandolfo P, Ferreira F, de Lima BO, et al.
Biochemical, histopathological and behavioral alterations caused by intrastriatal admin‐
istration of quinolic acid to young rats. FEBS J. 2014;281(8):2061–73.
[37] Pierozan P, Zamoner A, Soska AK, de Lima BO, Reis KP, Zamboni F, et al. Signaling
mechanisms downstream of quinolinic acid targeting the cytoskeleton of rat striatal neu‐
rons and astrocytes. Exp Neurol. 2012;233(1):391–9.
[38] Pierozan P, Ferreira F, de Lima BO, Pessoa‐Pureur R. Quinolinic acid induces disrupts
cytoskeletal homeostasis in striatal neurons. Protective role of astrocyte‐neuron interac‐
tion. J Neurosci Res. 2015;93(2):268–84.
[39] Pierozan P, Ferreira F, Ortiz de Lima B, Goncalves Fernandes C, Totarelli Monteforte P,
de Castro Medaglia N, et al. The phosphorylation status and cytoskeletal remodeling of
striatal astrocytes treated with quinolinic acid. Exp Cell Res. 2014;322(2):313–23.
[40] Etienne‐Manneville S. From signaling pathways to microtubule dynamics: the key play‐
ers. Curr Opin Cell Biol. 2010;22(1):104–11.
[41] Caceres A, Kosik KS. Inhibition of neurite polarity by tau antisense oligonucleotides in
primary cerebellar neurons. Nature. 1990;343(6257):461–3.
[42] Wie H, Bradke F. The role of the cytoskeleton during neuronal polarization. Curr Opin
Neurobiol. 2008;18(5):479–87.
[43] Younes‐Rapozo V, Berendonk J, Savignon T, Manhaes AC, Barradas PC. Thyroid hor‐
mone deciency changes the distribution of oligodendrocyte/myelin markers during
oligodendroglial dierentiation in vitro. Int J Dev Neurosci. 2006;24(7):445–53.
[44] Fernandez M, Paradisi M, Del Vecchio G, Giardino L, Calza L. Thyroid hormone induces
glial lineage of primary neurospheres derived from non‐pathological and pathological
Cytoskeleton - Structure, Dynamics, Function and Disease254
rat brain: implications for remyelination‐enhancing therapies. Int J Dev Neurosci.
2009;27(8):769–78.
[45] Martinez R, Gomes FC. Neuritogenesis induced by thyroid hormone‐treated astrocytes
is mediated by epidermal growth factor/mitogen‐activated protein kinase‐phosphati‐
dylinositol 3‐kinase pathways and involves modulation of extracellular matrix proteins.
J Biol Chem. 2002;277(51):49311–8.
[46] Fernandez‐Lamo I, Montero‐Pedrazuela A, Delgado‐Garcia JM, Guadano‐Ferraz A,
Gruart A. Eects of thyroid hormone replacement on associative learning and hippo‐
campal synaptic plasticity in adult hypothyroid rats. Eur J Neurosci. 2009;30(4):679–92.
[47] Vallortigara J, Alfos S, Micheau J, Higueret P, Enderlin V. T3 administration in adult
hypothyroid mice modulates expression of proteins involved in striatal synaptic plastic‐
ity and improves motor behavior. Neurobiol Dis. 2008;31(3):378–85.
[48] Vallortigara J, Chassande O, Higueret P, Enderlin V. Thyroid hormone receptor alpha plays
an essential role in the normalisation of adult‐onset hypothyroidism‐related hypoexpres‐
sion of synaptic plasticity target genes in striatum. J Neuroendocrinol. 2009;21(1):49–56.
[49] Zamoner A, Heimfarth L, Loureiro SO, Royer C, Mena Barreto Silva FR, Pessoa‐Pureur
R. Nongenomic actions of thyroxine modulate intermediate lament phosphorylation in
cerebral cortex of rats. Neuroscience. 2008;156(3):640–52.
[50] Zamoner A, Funchal C, Heimfarth L, Silva F, Pessoa‐Pureur R. Short‐term eects of
thyroid hormones on cytoskeletal proteins are mediated by GABAergic mechanisms in
slices of cerebral cortex from young rats. Cell Mol Neurobiol. 2006;26(2):209–24.
[51] Zamoner A, Funchal C, Jacques‐Silva MC, Gofried C, Mena Barreto Silva FR, Pessoa‐
Pureur R. Thyroid hormones reorganize the cytoskeleton of glial cells through Gfap phos‐
phorylation and Rhoa‐dependent mechanisms. Cell Mol Neurobiol. 2007;27(7):845–65.
[52] Zamoner A, Heimfarth L, Pessoa‐Pureur R. Congenital hypothyroidism is associated
with intermediate lament misregulation, glutamate transporters down‐regulation and
MAPK activation in developing rat brain. Neurotoxicology. 2008;29(6):1092–9.
[53] Zamoner A, Pessoa‐Pureur R. Nongenomic actions of thyroid hormones: every why
has a wherefore. Immunology, Endocrine & Metabolic Agents in Medicinal Chemistry.
2011;11(3):165–78.
[54] Caani D, Goulart PB, de Liz Oliveira Cavalli VL, Winkelmann‐Duarte E, dos Santos
AQ, Pierozan P, et al. Congenital hypothyroidism alters the oxidative status, enzyme
activities and morphological parameters in the hippocampus of developing rats. Mol
Cell Endocrinol. 2013;375(1–2):14–26.
[55] Trentin AG, Alvarez‐Silva M, Moura Neto V. Thyroid hormone induces cerebellar astro‐
cytes and C6 glioma cells to secrete mitogenic growth factors. Am J Physiol Endocrinol
Metab. 2001;281(5):E1088–94.
[56] Samuels MH. Cognitive function in untreated hypothyroidism and hyperthyroidism.
Curr Opin Endocrinol Diabetes Obes. 2008;15(5):429–33.
Intermediate Filaments as a Target of Signaling Mechanisms in Neurotoxicity
http://dx.doi.org/10.5772/66926
255
[57] Davis PJ, Goglia F, Leonard JL. Nongenomic actions of thyroid hormone. Nat Rev
Endocrinol. 2016;12(2):111–21.
[58] Siegrist‐Kaiser CA, Juge‐Aubry C, Tranter MP, Ekenbarger DM, Leonard JL. Thyroxine‐
dependent modulation of actin polymerization in cultured astrocytes. A novel, extra‐
nuclear action of thyroid hormone. J Biol Chem. 1990;265(9):5296–302.
[59] Farwell AP, Dubord‐Tomasei SA, Pietrzykowski AZ, Stachelek SJ, Leonard JL.
Regulation of cerebellar neuronal migration and neurite outgrowth by thyroxine and
3,3’,5’‐triiodothyronine. Brain Res Dev Brain Res. 2005;154(1):121–35.
[60] Trentin AG, Moura Neto V. T3 aects cerebellar astrocyte proliferation, GFAP and bro‐
nectin organization. Neuroreport. 1995;6(2):293–6.
[61] Bergh JJ, Lin HY, Lansing L, Mohamed SN, Davis FB, Mousa S, et al. Integrin alphaV‐
beta3 contains a cell surface receptor site for thyroid hormone that is linked to activa‐
tion of mitogen‐activated protein kinase and induction of angiogenesis. Endocrinology.
2005;146(7):2864–71.
[62] Rahaman SO, Ghosh S, Mohanakumar KP, Das S, Sarkar PK. Hypothyroidism in the
developing rat brain is associated with marked oxidative stress and aberrant intraneu‐
ronal accumulation of neurolaments. Neurosci Res. 2001;40(3):273–9.
[63] Motil J, Chan WK, Dubey M, Chaudhury P, Pimenta A, Chylinski TM, et al. Dynein
mediates retrograde neurolament transport within axons and anterograde delivery
of NFs from perikarya into axons: regulation by multiple phosphorylation events. Cell
Motil Cytoskeleton. 2006;63(5):266–86.
Cytoskeleton - Structure, Dynamics, Function and Disease256
