NeuroToxicology 37 (2013) 163–172

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NeuroToxicology

Review

Okadaic acid induced neurotoxicity: An emerging tool to study Alzheimer’s

disease pathology
Pradip K. Kamat a,*, Shivika Rai b, Chandishwar Nath b
a
Division of Physiology and Biophysics, University of Louisville, School of Medicine, KY 40202, United States
b
Division of Pharmacology Central Drug Research Institute (CDRI), P.O. Box 173, Lucknow, U.P. 226001, India

A R T I C L E I N F O A B S T R A C T

Article history: Okadaic acid (OKA) is one of the main polyether toxins produced by marine microalgae which causes
Received 1 January 2013 diarrhetic shellfish poisoning. It is a selective and potent inhibitor of serine/threonine phosphatases 1
Accepted 3 May 2013 and 2A induces hyperphosphorylation of tau in vitro and in vivo. The reduced activity of phosphatases
Available online 17 May 2013
like, protein phosphatase 2A (PP2A) has been implicated in the brain of Alzheimer’s disease (AD)
patients. It is reported that AD is a complex multifactorial neurodegenerative disorder and
Keywords: hyperphosphorylated tau proteins is a major pathological hallmark of AD. The molecular pathogenesis
Okadaic acid
of AD includes an extracellular deposition of beta amyloid (Ab), accumulation of intracellular
Alzheimer’s disease
Neurotoxicity
neurofibrillary tangles (NFT), GSK3b activation, oxidative stress, altered neurotransmitter and
inflammatory cascades. Several lines of evidence suggested that the microinfusion of OKA into the
rat brain causes cognitive deficiency, NFTs-like pathological changes and oxidative stress as seen in AD
pathology via tau hyperphosphorylation caused by inhibition of protein phosphatases. So, communal
data and information inferred that OKA induces neurodegeneration along with tau hyperpho-
sphorylation; GSK3b activation, oxidative stress, neuroinflammation and neurotoxicity which is a
characteristic feature of AD pathology. Through this collected evidence, it is suggested that OKA induced
neurotoxicity may be a novel tool to study Alzheimer’s disease pathology and helpful in development of
new therapeutic approach.
ß 2013 Elsevier Inc. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164
2. Factors responsible for AD pathology and pathological hallmarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164
2.1. Okadaic acid and Alzheimer’s disease pathology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164
2.2. Tau phosphorylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
2.3. Glycogen synthase kinase-3 beta (GSK3b) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166
2.4. Kinases involved in Tau pathology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166
3. Physiopathological conditions and factors involved in AD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166
3.1. Cholinergic function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166
3.2. NMDA receptor function and excitotoxicity: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168
3.3. Role of mitochondria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168
3.4. Oxidative stress. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168
3.5. Neuroinflammation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
3.6. OKA induced neurotoxicity and cell death . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
4. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
5. Future prospectus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170

Abbreviations: OKA, Okadaic acid; PP2A, Protein phosphatase 2A; GSK-3b, Glycogen synthase kinase-3b; PHF, Paired helical filament; NFT, Neurofibrillary tangle; AD,
Alzheimer’s disease; Ab, Beta amyloid; AChE, Acetylcholinestrse activity; ChAT, Choline acetyle transferase activity; ACh, Acetylcholine; NMDA, N-methyl D-aspartate; PKA,
Protein kinase; M1,M2,M3, Muscarinic receptors; nAChR, Nicotinic receptor; ERK, Extracellular receptor kinase.
* Corresponding author at: Division of Physiology and Biophysics, University of Louisville, Louisville, KY 40202, United States. Tel.: +1 52 852 3638.
E-mail address: pradeepkamat2004@gmail.com (P.K. Kamat).

0161-813X/$ – see front matter ß 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.neuro.2013.05.002
164 P.K. Kamat et al. / NeuroToxicology 37 (2013) 163–172
1. Introduction
Okadaic acid (OKA) is one of the major substances among
Diarrheic shellfish poisoning (DSP) toxins worldwide. It was first
isolated from the black sponge Holichondria okadai (Takai et al.,
1987) and later in the dinoflagellate Prorocentrum lima (Murakami
et al., 1994) and in Dinophysis acuta (Garcia et al., 2004). OKA is
known to be a potent inhibitor of Ser/Thr phosphatases and a
specific inhibitor of protein synthesis by hyperphosphorylation of
elongation factor EF2 (Bialojan and Takai, 1988). Previous studies
have shown that OKA is widely distributed in mammals after
consumption (Matias and Creppy, 1996b), the main effects being in
the intestinal tract where it is responsible for DSP (Sueoka et al.,
1997). There is clinical observation that OKA causes memory
impairment in human subjects those who used seafood contami-
nated with dinoflagellate (Helicondria okadai) (http://www.aris-
tatek.com/Newsletter/DEC07/DEC07ts.aspx). Due to its inhibitory
action on protein phosphatases 1 and 2A, OKA used as a tool for
studying the participation of protein phosphorylation in cellular
processes (Arias et al., 1998). Ser/Thr protein phosphatases, PP1
and PP2A, can dephosphorylate tau in vitro. In AD brain, the
activity of PP2A appears to be reduced in both gray and white
matters (Gong et al., 1993; Torrent and Ferrer, 2012) and a down-
regulation of this enzyme are apparently involved in slowing down
the process of tau dephosphorylation (Matsuo et al., 1994).
Alzheimer’s disease (AD) is the most common form of dementia
and characterized by severe neurodegenerative changes, such as
cerebral atrophy, loss of neurons and synapses, and selective
depletion of neurotransmitter systems in cerebral cortex and
certain subcortical region. Brain regions associated with higher
mental functions, particularly the neocortex and hippocampus are
most affected in AD pathology (Hampel et al., 2008). AD pathology
includes the extracellular deposits of b-amyloid (derived from
amyloid precursor protein; APP) in senile plaques (Nunomura
et al., 2010), intracellular formation of NFT (containing an
abnormally phosphorylated form of a microtubule associated
protein, tau) (Binder et al., 2005), and the loss of neuronal synapses
and pyramidal neurons (Capetillo-Zarate et al., 2012). AD is
associated with neurodegenerative changes which compromise
not only cognitive functioning but also lead to a decline in
functional abilities and induce a spectrum of psychological or
behavioral symptoms (Radebaugh et al., 1996). Many efforts are
currently undertaken to investigate AD pathology and develop
appropriate treatment strategies. These strategies focus on long-
term preservation of cognitive and functional abilities or slowing
down disease progression along with reducing behavioral symp-
toms and maintaining the patient’s quality of life (Geldmacher,
2007). These changes result in the development of the typical
symptomology of AD characterized by gross and progressive
impairments of cognitive function and often accompanied by
behavioral disturbances such as aggression and depression. AD
together with other forms of dementia represents a major
challenge for health care systems with aging populations.
Memory impairment induced by intra-hippocampal injection of
OKA has been reported, accompanied by remarkable neuropatho-
logical changes including hippocampal neurodegeneration, a
paired helical filament-like phosphorylation of tau protein, and
formation of b-amyloid containing plaque-like structures (Costa
et al., 2012). Wu et al. (2008) found that the injection of OKA into
rat lateral ventricles constitutes a promising animal model for
investigating selective aspects of AD. Maidana et al. (2006)
investigated OKA affects short- and long-term memory alteration
in rats and caused oxidative damage in hippocampus of rats. Acute
administration of various concentrations of OKA was found to
disrupt two distinct stages of memory formation in day old chick
(Bennett et al., 2003). Different phosphatase enzymes may
contribute to different stages of the enzymatic cascade believed
to underlie memory formation (Bennett et al., 2001). He et al.
(2001) investigated the effects of OKA, on spatial memory and
neuronal survival in rats. It seems that OKA is an extremely useful
tool for studying the cellular processes that are regulated by
reversible phosphorylation of proteins as signal transduction, cell
division and memory (Fernandez et al., 2002).
In this review, we discussed that why OKA induced neurotox-
icity and molecular alteration is better tool to study AD pathology.
There are several factors responsible for AD disease pathology but
tau-hyper phosphorylation is a main pathological hallmark.
Several reports reveal that same pathology is impersonated by
OKA in vivo and in vitro study.
2. Factors responsible for AD pathology and pathological
hallmarks
2.1. Okadaic acid and Alzheimer’s disease pathology
AD is a progressive neurodegenerative disease that causes
cognitive and behavioral deterioration in the aged person. In AD
brain, the activity of PP2A appears to be reduced in both gray and
white matters (Gong et al., 1993) and a down-regulation of this
enzyme are apparently involved in slowing down the process of
tau dephosphorylation (Matsuo et al., 1994). AD is a common
neurodegenerative disease pathologically by histopathological
changes including extracellular deposits of amyloid-beta (Ab)
peptides forming senile plaques (SP) and the intracellular
neurofibrillary tangles (NFT) of hyperphosphorylated tau in the
brain. Natalia et al. (2012) reported that the hyperphosphorylation
of tau protein and formation of intraneuronal neurofibrillary
tangles (NFTs) represents another neuropathological hallmark in
AD brain. Tau is a microtubule-associated protein and localizes
predominantly in the axons of neurons with the primary function
in maintaining microtubules stability. Neurofibrillary tangles
(NFTs) are one of the pathological hallmarks of AD that has been
shown to correlate positively with the severity of dementia in the
neocortex of AD patients. Chopra et al. (2011) also reported that Ab
containing senile plaques and phospho-tau-containing neurofi-
brillary tangles are hallmark lesions of AD pathology. OKA, when
injected into the right lateral dorsal hippocampus area of adult rat
brain depicts an in vivo model of AD tauopathy (Zhang et al., 2010a,
b). Zhou et al. (2008) suggested that the increased demethylation
of PP2A mediated by Ab overproduction may contribute to the
reduced PP2A activity resulting in the compromised dephosphor-
ylation of abnormally hyperphosphorylated tau. Hyperphosphory-
lated tau, which is the major protein of the neurofibrillary tangles
in AD brain, is most probably the result of an imbalance of tau
kinase and phosphatase activities in the affected neurons (Gong
et al., 2000). Selective inhibition of protein phosphatase 2A by OKA
induced an AD-like hyperphosphorylation and accumulation of tau
(Gong et al., 2000). The hyperphosphorylated tau had a reduced
ability to bind to microtubules and to promote microtubule
assembly in vitro (Ballatore et al., 2012). The biochemical evidence
unambiguously demonstrates the hyperphosphorylation and the
accumulation of neurofibril (NF) subunits in AD brain (Fig. 1). The
inhibition of PP-2A/PP-1 activities can lead to the hyperpho-
sphorylation of NF (Wang et al., 2001).
Zhao et al. (1994) and subsequently; our group (Kamat et al.,
2010) also reported that OKA (ICV) causes spatial memory
impairment in rat. Microtubule-associated protein tau is abnor-
mally hyperphosphorylated, glycosylated, and aggregated in
affected neurons in AD and found that the aberrant tau
glycosylation precedes tau hyperphosphorylation in AD brain
(Liu and Wang, 2002). The phosphorylation state of proteins
modulated by protein kinases and protein phosphatases is
 P.K. Kamat et al. / NeuroToxicology 37 (2013) 163–172 165

Fig. 1. Schematic diagram showing the involvement of OKA in the pathogenesis of Alzheimer’s disease. OKA induce APP mismetabolism causing an increase in Ab which
results in neurite plaque formation, microglial activation, oxidative stress and neuroinflammation. OKA induced Ab may also precede formation of neurofibrillary tangles.
Neuritic plaques and neurofibrillary tangles lead to neurotoxicity, neuronal dysfunction and finally to AD like pathology.

important in the regulation of many physiological processes like
the release of neurotransmitters and activation of postsynaptic
receptors (Huganir and Greengard, 1990). Tau phosphorylation
also involve in maintenance of long-term potentiation (Bliss and
Collingridge, 1993), and learning and memory (Zhao et al., 1994).
Immunocytochemical staining revealed hyperphosphorylated tau
accumulation in pyramidal neurons in cornu ammonis and in
neocortical neurons. The topography of these changes recalls the
distribution of NFT in AD brain (van de Nes et al., 2006). The
discovery that rare mutations in the gene encoding for APP always
led to AD in family members carrying the defect resulted in the
proposal of the ‘‘amyloid cascade hypothesis’’ of AD (Pera et al.,
2013). Levine et al. (1995) found that injection of the phosphatase
inhibitor OKA results in modulation of synaptic transmission in the
hippocampus via brain-derived neurotrophic factor (BDNF). BDNF
which is associated with memory rapidly enhanced synaptic
efficacy through increased postsynaptic responsiveness via a
phosphorylation-dependent pathway. It has been reported that
impairment of APP metabolism leading to increased production of
Ab was proposed as the critical event in with other changes,
tangles, neuron loss, synapse loss, and neurotransmission dys-
function (Wang et al., 2012). These neurons therefore seem to have
a central role in the clinical symptoms as well as in the
pathophysiology of the AD disease. Arias et al. (1998) found the
hyperphosphorylation of tau protein due to inhibition of phos-
phatases in vivo induces neuronal stress and subsequent
neurodegeneration. OKA-induced spatial memory impairment is
associated with neuropathological changes like loss of pyramidal
cell in the CAI and apoptotic cell death in the hippocampus, the
brain regions involved in learning memory function (He et al.,
2005). In vitro, OKA treatment in neuroblastoma SKNSH-SY 5Y
cells also produced tau proteins with an increased apparent
molecular weight and a more acidic isoelectric point when
compared to Tau proteins from control cells (Suuronen et al.,
2000). OKA also induces neurotoxicity of in SH-SY5Y cell line and
cultured neuronal cells which developed similar pathological
condition like AD (Zhang et al., 2007).
2.2. Tau phosphorylation
Tau proteins are microtubule-associated proteins in neurons
and under physiological condition, tau plays a key role in
microtubules stabilization, axonal transportation and neurite
outgrowth (Yi and Simpkins, 2008). Tau hyperphosphorylation
and memory deficit are characteristic alterations of AD. Inhibition
of PP2A through hippocampal injection of OKA induces tau
hyperphosphorylation and memory impairment in rats (Yin
et al., 2010). Hyperphosphorylated proteins have been implicated
in the neurodegenerative disorder AD one of the most character-
istics histopathological features of AD is NFT, which contains
distorted microtubule and phosphorylated tau proteins (Reynolds
et al., 2008). Under pathological conditions, deposits of abnormally
hyperphosphorylated tau protein are diagnostic hallmark of AD.
Tau, as the principal component of neurofibrillary tangles, can be
hyperphosphorylated by a reduced activity of PP2A in vitro and by
pharmacological approaches, suggesting a crucial role of PP2A in
tangle formation in AD (Ballatore et al., 2007). Most of the
phosphorylation sites on tau are present in the proline-rich and the
166 P.K. Kamat et al. / NeuroToxicology 37 (2013) 163–172
C-terminal regions flanking the microtubule binding domains
which interact with several kinases (Sergeant et al., 2008). The
mechanisms operative in the onset and progression of neurode-
generative diseases, such as AD likely involve the disruption of
normal phosphorylation events as a result in tau dysfunction
(Abraha et al., 2000). Although the precise reason for the
accumulation of tau in AD is not known, evidence indicates that
it may be related to either an increase in kinase activity or a
decrease in phosphatase activity (Dolan and Johnson, 2010).
2.3. Glycogen synthase kinase-3 beta (GSK3b)
Glycogen synthase kinase-3 beta (GSK3b) is a pivotal molecule
in the development of AD and involved in the formation of paired
helical filament (PHF)-tau. GSK3b is an integral component of the
NFT deposits that disrupt neuronal function, and a marker of
neurodegeneration in AD (Takashima, 2006). The essential
dysfunction appears to be an imbalance in kinase and phosphatase
activities resulting from dysregulation of the phosphorylation/
dephosphorylation system (Gong et al., 2003). GSK3b induces
hyperphosphorylation of tau, NFT formation, neuronal death and
synaptic loss in the AD brain and induces memory deficits
(Takashima, 2006). Alterations in GSK3b and PP2A have been
proposed to be involved in the abnormal tau phosphorylation and
aggregation linked to AD (Martin et al., 2011). GSK3b is recognized
as one of major kinases to phosphorylate tau in AD, thus lots of AD
drug discoveries target GSK3b, however, the inactive form of
GSK3b which is phosphorylated at serine-9 is increased in AD
brains. Lim et al. (2010) addressed this paradoxical condition of AD
in rat neurons treated with OKA which inhibits protein PP2A and
induces tau hyperphosphorylation and cell death. Interestingly,
OKA also induces phosphorylation of GSK3b at serine-9 and other
substrates including tau, beta-catenin and CRMP2 like in AD brains
(Lim et al., 2010). Abnormal hyperphosphorylation of tau appears
to be crucial in neurofibrillary degeneration in AD (Qian et al.,
2010). Inhibition of PP2A with OKA in metabolically active rat
brain slices caused inhibition of GSK3b via an increase in its
phosphorylation (Harris et al., 1993). OKA treatment in human
neuronal and glial cultures severely depleted the content of GSK3b
and down regulated the GSK3b activity by Akt-dependent
inhibition suggest that in primary cultures, Akt selectively
phosphorylates tau at S214 rather than T212 and the possibility
that tau S214 may participate in Akt-mediated anti-apoptotic
signaling. The overall alterations in tau phosphorylation induced
by PP2A inhibition were the result of the combined effects of both
reduced tau dephosphorylation due to PP2A inhibition directly and
reduced phosphorylation by GSK3b due to its inhibition. It is
suggest that GSK3b may still have its kinase activity despite
increase of its phosphorylation in AD brains at least in PP2A-
compromised conditions and that GSK3b inhibitors could be a
valuable drug candidate in AD.
2.4. Kinases involved in Tau pathology
Inactivation of PP1/PP2A via oxidative stress has been shown in
vitro and in vivo to be involved in hyperphosphorylation of tau and
prolonged phosphorylation of ERK 1/2 (Rahman et al., 2005;
Poppek et al., 2006). Thus, it is intriguing to postulate that
oxidative stress mediated PP1 and PP2A inhibition in AD may
account for enhanced ERK1/2 activity and subsequent tau
hyperphosphorylation and neurofibrillary tangle formation. Li
et al. (2004a,b) suggested that decrease in PP-2A activity could
have caused the activation of ERK1/2, MEK1/2, and p70 S6 kinase,
and the abnormal hyperphosphorylation of tau both via an
increase in its phosphorylation and a decrease in its dephosphor-
ylation in AD brain. OKA induced neurotoxicity are mediated by
persistent activation of ERK1/2 and/or protein kinase C and a
resulting oxidative stress (Yi et al., 2009). Cerebral OKA adminis-
tration induces AD like phenotype in rats and phosphorylated tau
as well as cyclin-dependent kinase 5 (cdk5) and its co activator,
p25, were significantly increased in these regions of the brain.
Alterations in glutamate levels associated with hyperactivation of
Cdk5 signaling pathway and tau phosphorylation may participate
in the genesis of this pathological phenotype (Zimmer et al., 2012).
The kinases that phosphorylate tau can be divided into two major
groups, according to motif specificity: proline-directed protein
kinases (PDPK) and non-proline-directed protein kinases (non-
PDPK) (Gendron and Petrucelli, 2009). The PDPK include cyclin-
dependent kinase 5(Cdk5), mitogen-activated protein kinase
(MAPK), and several stress-activated protein kinases (Gendron
and Petrucelli, 2009). Both Cdk5 and GSK3b co-purify with
microtubules and phosphorylate tau within a cellular environment
(Wagner et al., 1996). The phosphorylation of tau by these kinases
inhibits the ability of tau to promote microtubule assembly and
facilitates the polymerization of tau into PHF (Evans et al., 2000).
Among the non-PDPK are cyclic AMP-dependent protein kinase
(PKA), calcium- and calmodulin-dependent protein kinase II
(CaMKII), and microtubule affinity regulating kinase (MARK), the
mammalian homologue of PAR-1 and targets KXGS motifs within
the microtubule binding repeat domains of tau (Paudel, 1997). OKA
caused potassium channel inactivation, which is regulated by
CaMK-II, and may contribute to the electrical memory of the atrial
myocardium (Tessier et al., 2001). Protein phosphatase-mediates
the regulation of protein kinase C during long-term depression in
the adult hippocampus in vivo. The neural substrates of learning
and memory are thought to involve in long-term changes in
synaptic function, including long-term depression (LTD) of
synaptic strength. Thiels et al. (2000) demonstrated that LTD in
the adult hippocampus in vivo involves a decrease in PKC activity.
It is mediated, by dephosphorylation of the catalytic domain of PKC
by protein phosphatases activated after LTD inducing stimulation
which was prevented by OKA. Tau phosphorylation induces its
dissociation from microtubules and prevents its degradation
(Dickey et al., 2007). Unbound tau may then be hyperpho-
sphorylated by other kinases and in fact, the phosphorylation of
tau by MARK/PAR-1 may be a prerequisite for the action of
downstream kinases, including GSK3b and Cdk5 (Nishimura et al.,
1998) (Fig. 2).
3. Physiopathological conditions and factors involved in AD
3.1. Cholinergic function
AD is characterized by neuronal loss and neurofibrillary tangle
formation and primarily affecting pyramidal neurons and their
synapses neurotransmitter specific subcortical nuclei including
the cholinergic nucleus basalis of Meynert and medial septum.
Biochemical investigations of biopsy tissue taken from patients
with AD after the onset of symptoms indicate that selective
neurotransmitter pathology occurs early in the course of the
disease (Francis, 1996). Specifically, presynaptic markers of the
cholinergic system appear uniformly reduced. This is exemplified
by reductions in Acetylcholinestrse activity (AChE), Choline acetyle
transferase (ChAT) activity and Acetylcholine (ACh) synthesis
which are strongly correlated with the degree of cognitive
impairment in patients with AD (Kamat et al., 2012a,b). Moreover,
a few patients with AD do not show large decreases in ChAT
activity, albeit that a small reduction is found in the amygdale
(Palmer et al., 1986) Thus, although diminished ChAT activity is a
necessary correlate of AD, additional factors other than impaired
cholinergic function are likely to participate in the decline in
cognitive function. Other studies have demonstrated a reduction in
 P.K. Kamat et al. / NeuroToxicology 37 (2013) 163–172 167

Fig. 2. Schematic diagram depicting the role of kinases and phosphatases acing on Tau protein in OKA induced AD pathology. Tau contains specific phosphorylation sites that
are apparently critical for hyper-phosphorylation of Tau in AD. Proline-directed protein kinases such as MAPKs, GSK3b and Cdk-5 basically phosphorylate Tau protein, and
the non proline-directed protein kinases such as Cdk5 and MAPK. Several other kinases are also phosphorylate the Tau protein such as GSK3b, ERK1/2, MEK1/2, and p70 S6
kinase, CamkII and p25 and finally involved in development of AD pathology.

the number of nicotinic (Whitehouse and Kellar, 1987) and
muscarinic (M2) ACh receptors in AD brains, most of which are
considered to be located on presynaptic cholinergic terminals, but
a relative preservation of postsynaptic muscarinic (M1, M3
receptors. It is reported that the injection of OKA into the meynert
nucleus basalis, cortex and hippocampus of rat brain induces
decreased acetylcholine level and spatial memory deficit (Tian
et al., 2004). They found that injection of OKA induced hyperpho-
sphorylation of tau and NF and decreased ACh level in the nucleus
basalis of Meynert. These alterations were accompanied by spatial
memory deficit in OKA-injected rats. He et al. (2001) reported that
OKA into the rat brain causes spatial memory deficit and
neurodegeneration in the rat brain. Abnormal hyperphosphoryla-
tion of tau and cholinergic deficit occur in the early stage of AD and
relate to the dementia symptom (Tian et al., 2004). Further, Tian
et al. (2004) also demonstrated that the OKA-induced ACh
reduction may be due to a failure of intraneuronal transport of
ChAT from cell body to the neuronal terminals rather than an
alteration of activity of ChAT or acetylcholinesterase. Insulin
injection into the lateral cerebral ventricle of the rats improve
learning abilities in a OKA injected in CA1 region of the rat
hippocampus and the expressions of nicotinic acetylcholine
receptors were improved (Lin et al., 2012). Cholinergic dysfunction
is a characteristic feature of AD and we found that administration
of OKA in rat brain causes neurodegeneration along with
cholinergic dysfunction (Kamat et al., 2012a,b). It is reported that
muscarinic cholinergic receptor stimulation and activation of
protein kinase C may lead to the inactivation of a protein kinase
GSK3b, which phosphorylates tau, in vitro, in a similar manner to
that found in AD (Lovestone et al., 1994). Neuronal cells culture
transfected with M1 muscarinic receptors show reduced phos-
phorylation of tau after treatment with cholinergic agonists
(Sadot et al., 1996). On the basis of the above evidence, neocortical
cholinergic innervation is probably lost at an early stage of the AD
disease (Dalley et al., 2005). It is reported that both a7 and b2
nAChR agonist can afford neuroprotection against OKA induced
neurotoxicity (Del Barrio et al., 2011). Protection mediated by a7
nAChRs was independent of Ca2+ and involved the intracellular
signaling pathway Janus Kinase-2/Phosphatidylinositol-3-ki-
nase/Akt. However, although the loss of cholinergic function is
correlated with the cognitive impairment in AD (Kamat et al.,
2012a,b) an association between two such indices does not
necessarily indicate a causal relation. Cholinergic neurotransmis-
sion may be a specific target for b-amyloid, as it has been shown to
reduce both choline uptake and ACh release in vitro (Kar et al.,
1998). AD also exhibits extracellular plaques of aggregated
168 P.K. Kamat et al. / NeuroToxicology 37 (2013) 163–172
b-amyloid protein (Ab), intracellular neurofibrillary tangles that
contain hyperphosphorylated tau protein and a profound loss of
basal forebrain cholinergic neurons that innervate the hippocam-
pus and the neocortex (Kar et al., 2004). Pyramidal cells are lost in
the disease, subject to tangle formation, represent a major source
of APP and are regulated by a neurotransmitter ACh, affected early
in the disease (Francis et al., 1999). Observations in cell lines and
primary neuronal cultures it is evident that the activation of
muscarinic, metabotropic glutamate and other phospholipase C-
linked receptors favors the non-amyloidogenic processing of APP
(Nitsch, 1996). Furthermore, b-amyloid neurotoxicity is attenu-
ated by treatment with muscarinic agonists (Emmerling et al.,
1997). No sufficient data are there to report the potential
beneficial effects of cholinomimetic drugs on either increasing
APP or reducing b-amyloid production in patients with AD. There
is some report which shows evidence for reductions in CSF fluid
APP in depressed patients receiving drugs with anticholinergic
side effects (Clarke et al., 1993). Therefore, as a consequence of
reduced cholinergic activity, reduced protein kinase activity may
lead to a higher level of activity of GSK-3 and hence hyperpho-
sphorylation of tau.
3.2. NMDA receptor function and excitotoxicity:
N-methyl-d-aspartate receptors (NMDARs) are glutamate-
gated ion channels that are highly permeable to Ca2+, and Ca2+
influx through NMDARs is essential for synaptogenesis, experi-
ence-dependent synaptic remodeling and long-lasting changes in
synaptic efficacy such as long-term potentiation and long-term
depression (Collingridge et al., 2004). The NMDARs is involved in
cellular mechanism for learning and memory function (Rai et al.,
2012). NMDARs are involved in a wide array of biological
processes which are crucial for brain development and function
(Perez-Otano and Ehlers, 2004). OKA induced dephosphorylation
of one or more proteins other than adenosine kinase as a
consequence of NMDA receptor activation might play an
important role in extracellular adenosine regulation, with
important consequences for the regulation of excitatory synaptic
transmission, plasticity and excitotoxicity (Lu and Rosenberg,
2007). Excitotoxic cell death (ECD) is characteristic of mammalian
brain in several type of neurotoxixity. A key event in ECD is a
massive increase in intracellular Ca2+ by over-stimulation of
NMDARs. Ca2+ and calmodulin play an important role in
decreasing NMDAR activity in neurotoxicity (Shin et al., 2005).
Excitotoxicity is defined as a toxic process characterized by a
sustained stimulation of excitatory amino acids receptors
(Nicholls et al., 2007), mainly involving NMDAR. Different toxic
events derived from excitotoxicity have been characterized in
experimental models, including upregulation of detrimental
signaling pathways, disrupted Ca2+ homeostasis, and recruitment
of reactive oxygen/nitrogen species (ROS/RNS), with further
oxidative/nitrosative stress (Sas et al., 2007), ultimately leading to
cell death. OKA treatment showed elevated Mitochondrial Ca2+ in
rat suggesting OKA might be acting through NMDA system (Kamat
et al., 2011, 2013). Immunohistochemical observation suggest
that OKA induces tau phosphorylation in brain slices to prepare
AD models and causes expressions of P-tau, NR1 and NR2B in brain
slices (Li et al., 2004a,b). NMDA-evoked adenosine accumulation
is mediated by activation of phosphatase 1/2A and associated with
adenosine kinase inhibition. OKA reduces basal synaptic trans-
mission and blocked the induction of synaptic plasticity in the
form of long-term potentiation (LTP). LTP involved in memory
formation and found prolonged inhibition of protein phospha-
tases and suggests reduced post-synaptic signaling as a major
mechanism for basal synaptic transmission and LTP impairments
(Koss et al., 2007).
3.3. Role of mitochondria
Mitochondria play a critical role in regulation of apoptosis, which
is implicated in the aging process age-related mitochondrial
oxidative stress may contribute to apoptosis upon aging (Yamaguchi
and Perkins, 2009). Mitochondria are significantly reduced in
various types of cells obtained from patients with AD (Blass et al.,
1990). Mitochondrial dysfunction is one of the contributory
pathophysiology of AD (Hirai et al., 2001). The most consistent
defect in mitochondrial electron transport enzymes in AD is a
deficiency in cytochrome C oxidase which leads to an increase in ROS
production, a reduction in energy stores and disturbance in energy
metabolism (Cardoso et al., 2004a,b). OKA induces free radical ROS
generation, decreased mitochondrial membrane potential and
mitochondrial activity indicates mitochondrial dysfunction in rat
brain (Kamat et al., 2011). The activation of the permeability
transition pore in mitochondria, which is believed to play a critical
role in cell necrosis and apoptosis, is enhanced in spleen, brain, and
liver of aged mice (Mather and Rottenberg, 2000). Such a
modification of mitochondria may cause the cells more vulnerable
to apoptotic inducers (Zhang and Herman, 2002). Thus, mitochon-
dria appear to influence the aging process via modifying the
regulatory machinery of apoptosis and that mitochondria have a
central role in aging-related neurodegenerative diseases like AD
(Kamat et al., 2011). Mitochondria are critical regulators of cell
death, a key feature of neurodegeneration. In all major examples of
these diseases there is strong evidence that mitochondrial
dysfunction occurs early and acts causally in disease pathogenesis
(Kamat et al., 2011). Targeting basic mitochondrial processes, such
as energy metabolism or free-radical generation, or specific
interactions of disease-related proteins with mitochondria, hold
great promise for therapies (Kamat et al., 2011). Consistently,
oxidative stress-induced autophagy of accumulated amyloid b-
protein in AD causes permeabilization of lysosomal membrane and
leads to neuronal cell death (Zheng et al., 2009). Mitochondria
damaged by significantly increased oxidative stress in pyramidal
neurons of AD are subjected to autophagic degradation, ultimately
leading to neurodegeneration (Moreira et al., 2010). An important
function of mitochondria is to produce ATP and targeting genes
involved in ATP production offers a great opportunity to study the
role of mitochondrial function in aging (Cui et al., 2012).
Mitochondrial dysfunction and increased oxidative damage are
often associated with neurodegenerative disorder like Alzheimer’s
disease (AD), Parkinson disease (PD) and Huntington’s disease (HD)
suggesting that oxidative stress may play an important role in the
pathophysiology of these diseases (Lin and Beal, 2006).
3.4. Oxidative stress
Oxidative stress is one of the most important mechanisms
involved in toxic events observed in neuronal cells in different
neurodegenerative disorders. Besides the clinical study Cohen
(1990) reported in rat brain that OKA selectively inhibits the
serine/threonine phosphatases 1 (PP1) and 2A (PP2A). Arias et al.
(1998) have reported that the protein phosphatase inhibitor OKA
induces heat shock protein expression, neurodegeneration in rat
hippocampus in vivo and also found that protein hyperpho-
sphorylation due to inhibition of phosphatases in vivo induces
neuronal stress and subsequent neurodegeneration. Oxidative
stress was increased with OKA treatment as measured by Free
radical generation and lipid peroxidation (Kamat et al., 2010).
Several data suggest that the bilateral microinfusion of OKA into
the rat brain causes cognitive deficiency, NFTs-like pathological
changes, and oxidative stress as seen in AD pathology via tau
hyperphosphorylation caused by inhibition of protein phospha-
tases (Kamat et al., 2013). AD includes a variety of risk factors,
 P.K. Kamat et al. / NeuroToxicology 37 (2013) 163–172
extracellular deposition of b-amyloid, accumulation of intracellu-
lar neurofibrillary tangles, oxidative neuronal damage and
inflammatory cascades (Chopra et al., 2011). OKA induces
oxidative DNA damage directly and indirectly in SHSY5Y cells,
while it does not induce oxidative DNA damage in HepG2 cells and
showed that OKA induces both cytotoxicity and genotoxicity,
including DNA strand breaks and oxidative DNA damage, in the
cells evaluated (Valdiglesias et al., 2011). Protein phosphatase
inhibitor OKA induced protein phosphorylation is an important
mechanism for the regulation of DNA-PK protein kinase activity
and that the protein phosphatase responsible for reactivation in
vivo is a PP2A-like enzyme (Douglas et al., 2001). However, the
extents of these effects are cell type dependent Reactive nitrogen
species (RNS) and reactive oxygen species (ROS) tirggers neuronal
damage via activation of microglia (Liu et al., 2003). OKA induced
cell damage in neuronal cells and evaluated the effects of OKA as
changes in the quantity of lipid peroxidation products, protein
carbonyl groups, reduced glutathione content (GSH), glutathione
peroxidases (GSH-Px), superoxide dismutase (SDS), catalase, free
radical generation in rat brain and total lactate dehydrogenase
(LDH) (Tunez et al., 2006).
3.5. Neuroinflammation
Neuropathological studies have revealed the presence of a broad
variety of inflammation-related proteins (complement factors,
acute-phase proteins and pro-inflammatory cytokines) in AD brains.
Neuroinflammatory mediators and generation of both reactive
oxygen species (ROS) and reactive nitrogen species (RNS), which
affect synapses and induce neuronal damage (Rai et al., 2012). Glial
cells activated by amyloid-beta deposits and these cells secrete
inflammatory mediators and reactive oxygen species, which can
aggravate the aggregation of amyloid-beta. Some of the products
released by activated glial cells, as well as amyloid-beta itself, can
induce or promote neurodegeneration (Hoozemans et al., 2002).
These constituents of innate immunity are involved in several
crucial pathogenic events of the underlying pathological cascade in
AD, and recent studies have shown that innate immunity is involved
in the etiology of late-onset AD (Eikelenboom et al., 2000).
Neuropathological and experimental studies indicate that fibrillar
amyloid-b (Ab) can activate the innate immunity-related CD14 and
Toll-like receptor signaling pathways of glial cells for pro-
inflammatory cytokine production (Eikelenboom et al., 2010). The
activation of microglia releases proinflammatory cytokines tumor
necrosis factor-alpha (TNF-a) and interleukin-beta (IL1-b) (Tyagi
et al., 2008). Several reports suggested that pro inflammatory
cytokines, oxygen and nitrogen centered free radicals contribute to
the neurodegenerative processes (Tanaka et al., 2006). TNF-a and
IL1-b are suggested as an important mediator in brain pathology of
AD (Tan et al., 2007). OKA administration in rat brain caused elevated
level of cytokine like tumor necrosis factor alpha (TNF-a) and
interleukin-1 b (IL-1b) and their release caused neuroinflammation
(Kamat et al., 2011). Clinical studies suggest that peripheral
inflammation increases the risk of dementia, especially in patients
with preexistent cognitive impairment, and accelerates further
deterioration in demented patients. OKA injection into the CA1
region of the rat hippocampus caused increased in GFAP positive
astrocytes indicates glial activation.
3.6. OKA induced neurotoxicity and cell death
OKA has been shown to be cytotoxic in a variety of cell lines and
Serine/threonine protein phosphatases are important mediators of
general cellular function as well as neurodegenerative processes.
OKA increases phosphorylation of microtubule associated protein
and tau, which are concomitant with early changes in neuronal
169
cytoskeleton that ultimately leads to cell death in primary cortical
neurons and in neuroblastoma cell lines (Arias et al., 1993). In
cerebellar granule cells, OKA induces disintegration of neurites and
swelling of cell bodies (Fernandez et al., 1991). OKA has also been
shown to produce condensation of chromatin, reorganization of
cytoskeleton, and DNA fragmentation characteristic of apoptosis
(Boe et al., 1991; Fernandez-Sanchez et al., 1996). It has been
reported to OKA cause neuronal cell death in vitro (Cagnoli et al.,
1996) and in vivo (He et al., 2001; Yi et al., 2005) by inhibition of
the serine/threonine phosphatases 1 and 2A (Cohen, 1990). OKA
may have rapid metabolic consequences leading to cell death by
altering rates of phosphorylation–dephosphorylation in vivo (Arias
et al., 1998). Loss of cortical pyramidal neurons, synapse loss,
(DeKosky and Scheff, 1990) and reduced glutamate concentration,
together with the formation of neurofibrillary tangles, (Wilcock
et al., 1982) all correlate with the symptoms of dementia. OKA
treated rats showed vacuoles, shrinkage, cell blebbing and
sponginess in cortical and hippocampal areas which indicate
neurodegeneration (Kamat et al., 2011). A neurite network was
generated in a main channel through the neurite outgrowth from
both cell compartments (Kunze et al., 2011). This communication
presents a novel experimental model for Alzheimer studies, where
connected primary neurons were set into subtend, co-pathological
states. Silver staining and immunohistochemistry staining
revealed that OKA treatment induces NFTs-like conformational
changes in both the cortex and hippocampus (Zhang and Simpkins,
2010a,b). Interestingly, mice expressing a dominant negative form
of PP2A in neurons display features of Alzheimer’s pathology (Kins
et al., 2003). Wang et al. (2004) reported that OKA-induced
declines in cell viability and mitochondrial metabolic activity
along with hyperphosphorylation/accumulation of neurofilament-
(NF-) H/M subunits. Li et al. (2006) propose that the absence of
cleavage and degradation of hyperphosphorylated tau (due to
PP2A inhibition) may lead to its accumulation in degenerating
neurons. Axonal swellings filled with vesicles, Cell blebbing,
sponginess and changed morphology of cell were observed in OKA
treated rat brain (Kamat et al., 2011) OKA treatment caused axonal
swellings filled with vesicles microtubule fragments, and transport
molecules such as kinesin and synapsin and immunocytochemis-
try of the APP C-terminus showed that APP accumulated in the
axonal swellings (Yoon et al., 2006). It has reported that inhibition
of protein phosphatases increases tau phosphorylation and
initiates neuronal cell death which, include altered Ca2+ homeo-
stasis and glutamate excitotoxicity (Butterfield and Pocernich,
2003). OKA induced cell death via increased reactive oxygen
species, protein carbonylation, lipid peroxidation, caspase-3
activity and mitochondrial dysfunction in rat brain (Kamat
et al., 2011). It has also reported that OKA offer neuroprotective
effects against MPP(+)-induced apoptosis by blocking ROS
stimulation and ROS-mediated signaling pathways in SH-SY5Y
cells indicated that OKA could provide a therapeutic strategy for
the treatment of neurodegenerative diseases (Ahn et al., 2009).
Glutamate plays an essential role in learning and memory by
triggering NMDA receptors to let a controlled amount of calcium
into a nerve cell. The calcium helps creates the chemical
environment required for information storage. Excess glutamate,
on the other hand, over stimulates NMDA receptors so that they
allow too much calcium into nerve cells that leads to disruption
and death of cells (Kamat et al., 2010).
4. Concluding remarks
In conclusion, Protein phosphorylation having wider implica-
tion in learning and memory, Therefore, OKA may be the suitable
alternative model to understand the mechanism underlying the
process of neurodegeneration linked with memory deficit and AD
170 P.K. Kamat et al. / NeuroToxicology 37 (2013) 163–172
pathology. OKA is known to deteriorate memory in elderly person.
Therefore from translational point of view, OKA induced memory
impairment model may have advantage over the models based on
neurotoxins used in animals or genetic manipulation. There is very
low incidence of genetic based dementia. Such a model may also be
capable to detect the drug acting simultaneously on protein
phosphorylation and memory process. Such a drug will be of
immense important senile dementia of AD type where hyper
phosphorylation of protein are immediately associated with
cognitive dysfunction. OKA leads to phosphorylation of tau and
GSK-3b by inhibition of PP-2A activity. OKA also encourage the
extracellular deposits of b-amyloid in senile plaques, intracellular
formation of NFT (consisting a phosphorylated form of a
microtubule associated protein, tau), and the loss of neuronal
synapses and pyramidal neurons. OKA also persuades oxidative
stress, mitochondrial dysfunction, cholinergic dysfunction altered
NMDARs function, apoptotic cell death, activate inflammatory
cascade and neurotoxicity. All these altered functions are
contributory factors for development of AD like pathology. It is
evident from the shared information that AD like pathology
induced by OKA builds up a similar condition like clinical
neuropathology of AD.
5. Future prospectus
There are many neurotoxins used as a tool to study AD
pathology but none of these are able to target memory deficit
through PP2A inhibition (hyperphosphorylation of tau protein).
Their targets are likes oxidative stress and glucose metabolism by
STZ, MAP protein by Colchicine’s, Ca++ by Kainic acid, and
cholinergic neuron by AF64. In present scenario AChE inhibitors
and NMDA receptor antagonist drugs are the main stay of AD
treatment. Since tau hyperphosphorylation is a main pathological
hallmark of AD pathology. None of the drugs are available, which is
acting through inhibition of tau hyperphosphorylation. So, we
suggest that OKA will be an emerging alternative suitable tool to
search a therapeutic approach for AD pathology.
Conflict of interest
Authors have no conflict of interest.
Acknowledgement
Financial support to Shivika Rai and Pradip Kumar Kamat is
gratefully acknowledged to Council of Scientific and Industrial
Research (CSIR) New Delhi, India.
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