Glutamate transport in the brain: Its importance,

regulation, alterations underlying the development of

synaptopathy, and possible peripheral markers

Borisova Tatiana Ph.D. in biochemistry,

Doctor Biol. Sci.

Dept. Neurochemistry

Palladin Institute of Biochemistry NAS of Ukraine

Lithuania, 2012

2007–2013 m. Žmogiškųjų išteklių plėtros veiksmų programos 3 prioriteto „Tyrėjų gebėjimų stiprinimas“

VP1-3.1-ŠMM-05-K priemonės „MTTP tematinių tinklų, asociacijų veiklos stiprinimas“

projektas „Lietuvos Biochemikų draugijos potencialo kurti žinių visuomenę didinimas“

(Nr. VP1-3.1-ŠMM-05-K-01-022)

Lecture Roadmap

Part1 (data of literature)

• CNS disorders and problems

• Ambient glutamate

• Spontaneous exocytosis

• Glutamate receptors and signaling

Part 2 (results)

• Model of cholesterol deficiency

• Glutamate transporters and glutamate uptake

• Extracellular level of glutamate in synaptosomes

• Transporter-mediated glutamate release

• Regulated exocytosis

• Synaptopathy

Part 3 (results)

• Brain-blood glutamate balance

• Blood platelets as potential peripheral models of presynapse

Lithuania, 2012

• Glutamate is the major excitatory neurotransmitter in

the CNS, which is implicated in many aspects of normal

brain functioning, i.e. learning, memory, cognition.

• Abnormal glutamate homeostasis is involved in the

pathogenesis of major neurological and

neurodegenerative disorders and contributes to

neuronal dysfunction in epilepsy, schizophrenia,

amyotrophic lateral sclerosis, Alzheimer’s disease, as

well as hepatic encephalopathy, ischemia, hypoxia,

traumatic brain injury, heavy metals poisoning, brain

cancer, etc.

• Prevention of these disorders and their successful

treatment is restrained by lack of fundamental and

comprehensive knowledge about key processes of

glutamatergic neurotransmission in the CNS.

The human brain

http://ccnmtl.columbia.edu/projects/neuroethics/module4

Lithuania, 2012

Part 1

Discovery timeline highlights major

advances and trends in understanding of

glutamate importance

Bowie, 2008

Lithuania, 2012

Therefore, although L-Glu was originally identified in 1866 by H. Ritthausen, it

took almost another 100 years before its role in the CNS was considered

Bowie 2008

Multiple factors predispose the CNS to disease

Lithuania, 2012

With the advent of reliable

diagnostic criteria, it is now

clear that CNS disorders can

affect any member of any

society at any point in life.

Amongst the many notable

historical personages afflicted

with variousnervous ailments,

both Aristotle and Julius Caesar

were said to have suffered from

epilepsy whereas Mozart may

have been a manic depressive.

Behaviour of the 15 century

French heroine, Joan of Arc was

consistent with schizophrenia

and/or epilepsy.

Although most CNS disorders have unknown aetiology, dysfunction is commonly

attributed to not one but a combination of defects that often include excitatory or

inhibitory neurotransmission, its modulation by other neurotransmitter pathways (e.g.

dopaminergic, cholinergic, serotonergic), environmental or epigenetic factors as well as

genetics.

CNS disorders are treated by targeting

the symptoms

Bowie, 2008

A list of the major CNS

disorders that afflict society and

the drugs commonly used to

treat them

Despite advances in

understanding of the CNS, most

conditions are treated by

attempting

to alleviate symptoms rather

than the roots caused the

disease

Lithuania, 2012

The NMDAR channel blocker, memantine, is currently

used in Europe, the US and Canada in the treatment

of moderate-to-severe Alzheimer’s disease

representing the only therapeutic compound targeted

to iGluRs currently in clinical use.

Glutamatergic neurotransmission

glutamate

transporters

glutamate Na+

synaptic

vesicles

presynaptic

nerve terminal

Presynaptic

nerve terminal

glutamate

receptors

http://www.wormbook.org/chapters Danbolt, 2000 Lithuania, 2012

Tzingounis, 2007

Lithuania, 2012

Ambient extracellular glutamate is the most

important characteristic of glutamatergic

neurotransmission

• Plasma glutamate is 30 -100 µM

• Cerebrospinal fluid glutamate is 3-10 µM

• Synaptic vesicle glutamate transporters in glutamatergic neurons produce a 10,000-fold gradient.

• The concentration of glutamate in the cytoplasm of glutamatergic neurons is ~10,000 µM and synaptic vesicles within these neurons have glutamate concentrations near 100,000 µM

• Excitotoxicity: with swelling and apoptosis predominating at <20 µM glutamate and fast necrosis at >100 µM glutamate

Lithuania, 2012

Ambient extracellular glutamate and

synaptic transmission

In norm:

• Extracellular glutamate controls several important neurological

processes, including neuronal and glial cell differentiation and migration during development.

• Ambient extracellular glutamate also likely plays an important nonpathological role in control of glutamatergic synapse strength. Synapse strength essentially determines information flow in the brain, and activity-dependent changes in glutamatergic synapse strength are now recognized as the basis of learning and memory.

• Ambient extracellular glutamate varies spatially and temporally in healthy brains.

• Ambient extracellular glutamate may be an important unrecognized determinant of neuronal circuit function and plasticity.

Lithuania, 2012

Spatial variation in ambient extracellular

glutamate concentration could differentially

modulate neuronal circuit function

A hypothetical point source (asterisk) for ambient extracellular glutamate is present near the left center of this image. As a

result, ambient extracellular glutamate concentration (green shading) is highest there but drops with distance. Four

hypothetical glutamatergic neural circuits, each possibly leading to a different behavioral output, are represented by

arrows and numbered 1 to 4. All else being equal, circuits 3 and 4 will be weaker than circuits 1 and 2 because of

suppression of synapse strength by ambient extracellular glutamate. This will decrease the probability of behaviors

triggered by circuits 3 and 4, compared to 1 and 2. Temporal variations in glutamate uptake or ambient extracellular

glutamate secretion could alter the relative strength of the circuits at different times. In this way, ambient extracellular

glutamate could serve as a potent modulator of spatially “connected” circuits and regulate behavior.

Featherstone, 2008 Lithuania, 2012

Ambient extracellular glutamate and

synaptic transmission

Under pathological conditions:

• Ambient extracellular glutamate rises substantially during acute

pathological conditions such as seizure, ischemia, or fever, amyotrophic lateral sclerosis (ALS).

• Circadian changes in ambient extracellular glutamate may be caused by circadian rhythms in glial glutamate uptake that are themselves regulated by melatonin.

• Changes in ambient extracellular glutamate and glial activity may also contribute to mood.

• The possibility that ingestion of monosodium glutamate, a common food additive, can alter extracellular glutamate levels in certain regions of the brain has always been controversial and is now generally discounted.

Lithuania, 2012

Regulation of ambient extracellular

glutamate

• Ambient extracellular glutamate is the steady-state balance between glutamate secretion (which will increase ambient extracellular glutamate concentration) and glutamate uptake (which will decrease ambient extracellular glutamate)

• Vesicular release: spontaneous exocytosis

• Nonvesicular release:

• swelling-activated anion channels,

• gap junction hemi-channels,

• purinergic (P2X) receptors,

• cystine-glutamate exchangers,

• diffusion

Lithuania, 2012

Spontaneous exocytosis

Wasser, 2009 Lithuania, 2012

Regulation of synaptic transmission by

ambient extracellular glutamate

• The easiest way to imagine ambient extracellular glutamate affecting any biological process, including synaptic transmission, is via glutamate receptors. A typical initial thought is that ambient extracellular might perpetually activate glutamate receptors.

• Another possibility is that ambient extracellular glutamate might lead to constitutive desensitization of receptors, and therefore, suppression of glutamatergic signaling.

• Which actually occurs depends on the glutamate sensitivity of receptor activation and desensitization relative to the concentration of ambient extracellular glutamate. The exact glutamate sensitivity of activation and desensitization depends on glutamate receptor type.

Lithuania, 2012

Ionotropic glutamate receptors

• There are two main types of glutamate receptor in the nervous system: ionotropic (pore-forming) glutamate receptors and metabotropic (G-protein coupled) glutamate receptors.

• Mammalian ionotropic glutamate receptors are functionally and molecularly differentiated into three subgroups based on agonist pharmacology and subunit composition:

• 1) N-methyl-D-aspartate (NMDA) receptors,

• 2) amino-3-hydroxy-5-methylisoxazole-4-propionic acid

• (AMPA) receptors, and

• 3) kainate receptors.

• As their names imply, NMDA receptors display particular sensitivity to NMDA,AMPA receptors display particular sensitivity to AMPA, and kainate receptors display particular sensitivity to kainate.

• Differences between the three subtypes are attributed to the fact that, although all ionotropic glutamate receptors are thought to be tetrameric, each receptor subtype is assembled from a different set of subunit proteins.

• NMDA receptors are assembled from NR1,NR2,or NR3 subunits,

• AMPA receptors are composed of various combinations of GluR1, GluR2, GluR3, and GluR4 subunits,

• and kainate receptors are composed of GluR5, GluR6,

• GluR7,KA1,and KA2 subunits Lithuania, 2012

Glutamate dependence of activation for various

glutamate receptor subtypes, compared to the

probable concentration of ambient extracellular

glutamate

NMDA receptors (NMDAR) and metabotropic receptors (mGluR) are activated by relatively

low concentrations of glutamate (1 to 20 µM) compared to AMPA receptors (AMPAR) and

kainate receptors (KAR), which are activated only by glutamate concentrations of 100 to

2000 µM. If ambient extracellular glutamate is ~2 µM, then about 40% of NMDARs and 10% of

mGluRs could be constitutively activated in vivo. Featherstone, 2008

Glutamate dependence of desensitization for various

glutamate receptor subtypes, compared to the

probable conсentration of ambient extracellular

glutamate

Compared to activation, steady-state desensitization of ionotropic glutamate receptor occurs

at much lower glutamate concentrations (0.1 to 10 µM). If ambient extracellular glutamate is ~2

µM, then one-half to three-quarters of glutamate receptors might be constitutively

desensitized, and thus functionally silent, in vivo. However, slight changes in ambient

extracellular glutamate concentration or dose-dependence of steady-state desensitization

could have dramatic effects on glutamate receptor availability and synaptic strength. Featherstone, 2008

Structure and domain organization of

glutamate receptors

A, linear representation of the subunit polypeptide chain and schematic illustration of the subunit

topology. Glutamate receptor subunits have a modular structure composed of two large extracellular

domains [the ATD (green) and the LBD (blue)]; a TMD (orange) that forms part of the ion channel pore; and

an intracellular CTD. The LBD is defined by two segments of amino acids termed S1 and S2. The TMD

contains three membrane-spanning helices (M1, M3, and M4) and a membrane re-entrant loop (M2). B,

crystal structure at 3.6 A of the membrane-spanning tetrameric GluA2 AMPA receptor

Traynelis 2010 Lithuania, 2012

Binding sites for the agonists, antagonists,

and modulators shown for the glutamate

receptors

Traynelis, 2010

AMPA and kainate indicates that the ligand selectively targets GluA or GluK receptor

subunits, respectively. The ATDs,LBDs,TMDs,and linkers are shown in purple, orange, green,

andgray, respectively Lithuania, 2012

Conformational changes in the

functioning AMPA receptor

Traynelis, 2010

Lithuania, 2012

Ionotropic glutamate receptors fulfill

distinct roles in the CNS

Table identifying each iGluR subfamily (left column) with the individual subunits that assemble as mature receptors (middle column) and their respective roles within the CNS (right column)

Bowie, 2008 Lithuania, 2012

AMPA receptors and CNS disorders

Bowie, 2008 Lithuania, 2012

Table listing a number of disease states (left column) where defective signaling

through AMPARs has been established. The middle column summarizes key

characteristics associated with each disorder and the right column refers to the

drug classes whose actions may have therapeutic value.

Kainate receptors and CNS disorders

Summary table identifying which KAR subunits are implicated in distinct disorders of

the CNS. As work progress, it is likely that this information will be more complete. (For example, it is commonly assumed that native KARs are heteromers assembled from GluR5–7 subunits with KA1

and/or KA2. In view of this, in disease states where GluR5–7 have been implicated, it is possible that future work will

also implicate KA1 and/or KA2 subunits.) Lithuania, 2012 Bowie, 2008

Metabotropic glutamate receptors

• Mammalian metabotropic glutamate receptors are also divided into three subfamilies based on molecular and pharmacological differences.

• Group I metabotropic glutamate receptors are formed from mGluR1 or mGluR5 subunits and often activate phospholipase C pathways.

• Group II and Group III metabotropic receptors are composed of mGluR2/mGluR3 or mGluR4/mGluR6/mGluR7/mGluR8 subunits, respectively, and generally suppress adenylate cyclase activity.

Lithuania, 2012

Schematic diagram of the mGluR dimer

in different activity states

mGluR dimers contain two large extracellular domains called the Venus flytrap domains (VFDs),

which bind glutamate and other orthosteric ligands. The cysteine-rich domain links the VFDs to

seven transmembrane-spanning domains; the C-terminus faces intracellularly and is often subject

to alternative splicing to generate different C-terminal protein tails. The open-open state (left) is the

inactive state and can be stabilized by antagonists. Either one or two VFDs can then bind

glutamate, resulting in active receptor conformations

Traynelis, 2010 Lithuania, 2012

Glutamatergic neurotransmission

glutamate

transporters

glutamate Na+

synaptic

vesicles

presynaptic

nerve terminal

glutamate

receptors

Danbolt, 2001 universe-review.ca/R10-16-ANS.htm Lithuania, 2012

Part 2

Glutamate transporters

Tzingounis, 2007

Lithuania, 2012

EAAT1

and EAAT2 are

predominantly glial,

whereas EAAT3,

EAAT4 and EAAT5 are

expressed by neurons

throughout

the brain. Notably,

EAAT4 and EAAT5 are

specifically

located in Purkinje

cells (PCs) in the

cerebellum and the

retina, respectively.

DL-threo-b-benzyloxyaspartate L-threo-b-hydroxyaspartate Dihydrokainate

(DL-TBOA) (DL-ТНА) (DHK)

Synaptic vesicles and exocytosis

(Sudhoff, 2004) Lithuania, 2012

Confocal imaging of synaptosomes

labeled with the fluorescent dye R18

Preparations used in the research

Synaptic vesicle

Lithuania, 2012

synaptosomes

www.coloradocollege.edu

Electron microscopy of synaptosomes

brain homogenate crude synaptosomal

fraction

synaptosomes purified

according to Cotman, 1970

Lithuania, 2012

Nervous system cholesterol

homeostasis failure

?

Impairment of

neurotransmission, synaptic

function and plasticity

Impairment of learning and

memory,

neuronal loss

Cholesterol is an

essential component of

mammalian cell

membranes where it is

required to establish

proper membrane

permeability and

fluidity

The central nervous

system, which is equal

to two percent of body

mass, keeps a special

place among other

systems of organism,

since it contains

approximately a

quarter of total

unesterified

cholesterol

Cholesterol

homeostasis breaks are

associated with the

pathogenesis of certain

neurological disorders:

•Niemann-Pick disease C is

due to mutations in either

the NPC1 or NPC2 genes,

resulting in defective

cholesterol transport

•Defective synthesis of

brain cholesterol is the

cause of Smith-Lemli-Optiz

Syndrome.

•A specific down-regulation

of seladin-1, a protein

involved in cholesterol

synthesis, was shown in

Alzheimer's disease

•Low membrane cholesterol

was observed in

hippocampal membranes of

ApoE4 related case of

Alzheimer's disease

•Cholesterol is the

precursor for neurosteroids

which may be modulators of

the pathophysiology of

schizophrenia and bipolar

disorder Lithuania, 2012

1

Confocal imaging of synaptosomes. Dynamics of

cholesterol depletion by MCD (4s). Fluorescent

dye filipin

5мMcontrol 15мM 30мM 60мM 15мM

MCD+

Chol

0

20

40

60

80

100

120

MCD

ch

ole

ste

rol,

% *

*

*

*

Decrease in the level of cholesterol

Two methodological protocols:

-In the presence of the acceptor in the incubation

media (protocol М1).

-After washing of the acceptor (protocol М2).

Cholesterol deficient brain nerve terminals

C М1 М2

Relevance:

MCD as drug

deliverer and as a

component of

nanoparticles

cholesterol

Decreased level of

cholesterol in

neurological disorders

and as a result of

statin treatment

synaptosomes

Lithuania, 2012

Cholesterol acceptor –

methyl-β-cyclodextrin (MCD)

High-affinity Na+ - dependent glutamate

uptake by nerve terminals

L-[14C]glutamate

Na+

K+

EAAT

EAAT3

MCh

Synaptic

vesicles

Presynaptic nerve

terminal

Glutamate uptake activity

depends from:

-Na/K electrochemical gradient

of the plasma membrane;

-cell surface expression of

glutamate transporters

(protein kinase C –dependent

mechanism of regulation);

-the level of membrane

cholesterol;

-acidification of synaptic

vesicles.

Lithuania, 2012

() Changes in

the level

of membrane

cholesterol

()Glutamate

uptake in the

presence of

MCD (М1)

High-affinity Na+-dependent uptake of glutamate under

conditions of cholesterol deficiency

() Synaptic vesicle

acidification

() Glutamate

uptake

(М2)

Tarasenko A., Krisanova N., Sivko R., Himmelreich N., Borisova T. (2010) J. Mol. Neuroscience

Protein

kinase С -

dependent

regulation

• The effects of MCD on synaptic

vesicle acidification. (1) - control

synaptosomes; (2) – M1; (3) -

synaptosomes in the presence of 15

mM MCD, which was added 35 min

before the addition of acridine

orange; (4)-M2.

*

control 5 mM MCD 15mM 15mM MCD+

cholesterol

0

0.5

1

1.5

2

2.5

3

3.5

*

L-[

14C

]glu

tam

ate

,

nm

ol *

min

-1*

mg

of pro

tein

-1

Decreased L-[14C]glutamate uptake under

cholesterol deficiency

М1

0 200 400 600 800 1000 1200 1400 1600

0,6

0,8

1,0

Ft/F

0

Time, s

MCD

1

2

3

М2

control

15 mM MCD

0

1

2

3

4

5

6

0 100 200 300 400 500 600 700

time, s

glu

tam

te, n

mo

l/m

g p

rote

in The reduction of

glutamate uptake

during the removal

of cholesterol (M1)

Lithuania, 2012

The effect of MCD on glutamate uptake by

isolated synaptic vesicles

The initial velocity of L-[14C]glutamate (50mM) uptake by isolated synaptic

vesicles during the application of 15 mM MCD and 15mM MCD complexed

with cholesterol (2.3 mM).

*

**

0

50

100

150

200

250

300

350

400

450

500

control M CD M CD+cholesterol

glu

tam

ate

, p

mo

l/m

in/m

g p

rote

in

Lithuania, 2012

The ambient extracellular glutamate level

Glutamate receptors NMDA

R, AMPA R, mGlu R

Glu

Cys

Transmembrane diffusion

Glu

Glu

Glu

Glu Anion channels

СВ

ЕААТ3

Presynaptic nerve

terminal

Possynaptic membrane

[glu]extracellular=[glu]tonic release–[glu]uptake

Lithuania, 2012

The changes in ambient extracellular

glutamate followed M1 and M2

Confocal imaging of synaptosomes labeled with

a pH sensitive fluorescent dye acridine orange

following the application of MCD. The

measurements were performed by using the

confocal laser scanning microscope LSM 510

META, Carl Zeiss.

3

4

2

1

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0 10 20 30 40

time, min

L[1

4C

]glu

tam

ate

, n

mo

l/m

g p

rote

in

The extracellular level of L-[14C]glutamate in

synaptosomes after the addition of 0, 5, 15,

30 mМ MCD – curves 1; 2; 3; 4, respectively

M1

Lithuania, 2012

0

0,05

0,1

0,15

0,2

0,25

0,3

0,35

L-{

14C

]glu

tam

ate

, n

mo

l/m

g o

f p

rote

in

M2

The extracellular L-[14C]glutamate level

of control (empty bar) and cholesterol-

depleted synaptosomes (shaded bar)

(Р≤0.05, n=8). *, Р≤0.05 as compared to

control.

*

The extracellular level of endogenous

glutamate and glutamine in control

(empty bar) and cholesterol-depleted

(shaded bar) synaptosomes measured

chromatographycaly (Р≤0.05, n=4).

*, Р≤0.05 as compared to control.

*

*

0

2

4

6

8

10

12

14

16

18

20

endogenous glutamate glutamine

nm

ol/m

g p

rote

in

Lithuania, 2012

The possible interrelation between uptake (black arrows), tonic release

(open arrows), transporter-mediated release (grey arrow) and the

intra/extra - cellular concentration of glutamate (indicated by black dots).

Circles inside of synaptosomes showed filled with the neurotransmitter

(black circles) or empty (open circles) synaptic vesicles

In the presence

of MCD (M1)

Cholesterol-deficient

(M2)

Lithuania, 2012

Stimulated by depolarisation of the plasma membrane Са2+-

dependent (exocytosis) and Са2+- independent (transporter-

mediated) glutamate release from nerve terminals

Transporter- mediated release of

glutamate is the main mechanism of

glutamate release during hypoxia,

stoke, insult, seizures, brain trauma exocytosis

EAAT

Glu

SV

МCH

1K+

1 Glu-

n Na+

m H+

in

out

Plasma membrane

http://www.gs.washington.edu Lithuania, 2012

The alterations in exocytotic release of

glutamate followed M1 and M2

Ca2+-dependent (exocytotic) release of glutamate stimulated by high KCl:

(1) - control synaptosomes;

(2) - synaptosomes in the presence of 15 mM MCD (M1), which was added at zero time point just before application of 35 mM KCl;

(3) - synaptosomes in the presence of 15 mM MCD, which was added 35 min before the application of 35 mM KCl;

(4) - synaptosomes were preliminary treated with 15 mM MCD for 35 min, then were washed with 10 volumes of the buffer (M2), then were loaded with L-[14C]glutamate and depolarized with 35 mM KCl

1 2 3 4

0

1

2

3

4

5

6

7

8

9

10

glu

tam

ate

rele

ase, %

*

*** *

Lithuania, 2012

Borisova T., et al. ( 2010) Cell Moll Neurobiol

The opposite effects of M1 and M2 on

transporter-mediated glutamate release

1 2 3 4

0

2

4

6

8

10

12

14

glu

tam

ate

rele

ase, %

**

*

*

Ca2+-independent (transporter-mediated)

release of L-[14C]glutamate

stimulated by high- KCl:

(1) - control synaptosomes;

(2) - synaptosomes in the presence of 15

mM MCD (M1), which was added at

zero time point just before

application of 35 mM KCl;

(3) - synaptosomes in the presence of 15

mM MCD, which was added 35 min

before application of 35 mM KCl;

(4) - synaptosomes were preliminary

treated with 15 mM MCD for 35 min,

then were washed with 10 volumes of

buffer (M2), and then were loaded

with L-[14C]glutamate and depolarized

with 35 mM KCl

Lithuania, 2012

Borisova T., et al. ( 2010) Cell Moll Neurobiol

The possible mechanisms of the action

of cadmium and lead

0 100 200 300 400 500 600 700 800

0,5

0,6

0,7

0,8

0,9

1,0

F/F

0

Time (s)

cadmium

lead

control

control

cadmium

lead

-10

-8

-6

-4

-2

0

2

4

6

0 2 4 6 8 10 12

time,min

extr

acellu

lar

glu

tam

ate

,% o

f to

tal

0 100 200 300 400 500 600

0,2

0,4

0,6

0,8

1,0

Ft/F

o

time,s

Decrease in synaptic

vesicle proton gradient

Decrease in

transporter-

mediated

glutamate uptake

Decrease in

exocytotic

release of

glutamate

Blockage of

potential-

dependent calcium

channels

Binding to SH-

groups of

glutamate

transporters

Synaptic

pathology

Cadmium

Lead

Lithuania, 2012

Borisova et al., 2011, Neurochemistry Int

The effects of 200 mM CdCl2 and 200 mM PbCl2

on the accumulation of acridine orange into

the acidic compartments of synaptosomes

The changes in the extracellular L-

[14C]glutamate level in digitonin-

permeabilised synaptosomes in the control

and in the presence of 200 mM CdCl2 and

PbCl2

-50 0 50 100 150 200 250 300 350 400

0,4

0,6

0,8

1,0

Ft/F

o

time,s

The effect of cadmium on acidification of

isolated synaptic vesicles

The effect of manganese

on acidification of isolated

synaptic vesicles

The possible mechanisms of the action

of cadmium and lead

Nerve terminals

Cd 2+ Pb 2+

Lithuania, 2012

Borisova et al., 2011, Neurochemistry Int

Part 3

Glutamate transport in blood platelets

EAAT

Glu

VGLUT

Secretory

granules

Glutamate receptors

NMDA R, AMPA R

Glu

Blood platelets contains:

-glutamate transporters

ЕААТ 1-3;

-glutamate receptors

NMDA, AMPA, mGlu;

-vesicular glutamate

transporters V GLUT;

-neuronal protein

involved in exocytosis

Lithuania, 2012

Confocal imaging of

blood platelets labeled

with the fluorescent

dye filipin

Blood platelets

Brain-blood glutamate balance

Teichberg, 2009 Lithuania, 2012

Time course of L-

[14C]glutamate uptake by

platelets

А

L-[14C]glutamate uptake by platelets

Km Vmax

Platelets 36 8 mМ 5.2 1.3 pmol х min-1 х mg protein-1

Nerve terminals 10.7 2.5 mМ 12.5 3.2 nmol х min-1 х mg protein-1

Na+-dependent uptake by platelets

1/[glutamate], мicroМ -1

1/V

0, (n

mol/m

in/m

g p

rote

in

Час,с Time,min

n

molm

in/m

g o

f pro

tein

inh

ibit

ion

, %

Influence of DL-ТНА on glutamate uptake by platelets

10 100 mM

Lithuania, 2012

Depolarization of the plasma membrane of

platelets labeled with rodamine 6 G

0 200 400 600 800 1000

0,75

0,80

0,85

0,90

0,95

1,00

Ft /

Fo

Time, s

35 mM KCl

20 mM KCl

контроль

Flow cytometry of platelets in the

presence of KCl

Na+- dependent glutamate uptake by platelets during

depolarization of the plasma membrane

Kasatkina L., Borisova T. ( 2010) Neurochemistry International

Time,s

Secretory granule acidification of

platelets labeled with АО

*

0

0.2

0.4

0.6

0.8

1

1.2

1.4

glu

tam

ate

, p

mo

l/ m

in/

mg

pro

tein

A decrease in the initial velocity of

L-[14C]glutamate uptake by platelets

during the depolarization of the

plasma membrane

In vivo, glutamate uptake in platelets could be targeted under

conditions of hyperkalemia or preudohyperkalemia, i.e.

activation and clotting of platelets, hemolysis, leucocytosis,

acute renal failure, hypofunction of adrenal cortex, lack of

aldosterone, stroke, trauma, etc. In this context, malfunction

of glutamate transporters may be one of the causes for the

development of neurological consequences.

Also, lack function of glutamate transporters has to take place

during depolarization of the platelet plasma membrane

associated with their activation by ADP, thrombin, platelet-

activating factor, etc.

КСl C

0 200 400 600 800 1000

0,75

0,80

0,85

0,90

0,95

1,00

Ft /F

o

час, сTime,s

KCl KCl

Lithuania, 2012

The proton gradient of secretory granules and glutamate

transport in blood platelets during cholesterol depletion of

the plasma membrane by methyl--cyclodextrin

Borisova T., Kasatkina L., ( 2011) Neurochemistry International

Confocal imaging: MCD-

evoked depletion of

membrane cholesterol from

platelets labeled with the

fluorescent dye filipin. The

profiles of the fluorescence

intensity of filipin recorded

along red arrows of the

confocal images.

0 60 120 180 240 300 360 420 480

0,4

0,5

0,6

0,7

0,8

0,9

1,0

15 mM MCD-cholesterol complex (1:0.15)

5 mM MCD

Ft/F

0

Time, s

15 mM MCD

Control

Acidification of secretory granules of platelets

after the application of MCD

0 60 120 180 240 300

4

6

8

10

12

14

5 mM MCD

F

Time, s

1 mM L-GluMCD 15 mM MCD

0 30 60 90 120 150 180 210 240

4

6

8

10

12

14

16

F

Time, s

1 mM L-Glu

thrombin

1 NIH U/ml

Glutamate dehydrogenase assay. Analysis of

the effect of MCD on platelets.

Release of endogenous glutamate from

platelets stimulated by thrombin (1 NIH U/ml). Lithuania, 2012

Related publications 2010-2011:

• T. Borisova, N. Krisanova, R. Sivko, L. Kasatkina, A. Borysov, S. Griffin, M. Wireman Presynaptic malfunction: The neurotoxic effects of cadmium and lead on the proton gradient of synaptic vesicles and glutamate transport. Neurochemistry International - 2011 .- V.59 .- Р. 272-279.

• T. Borisova, L. Kasatkina, L. Ostapchenko The proton gradient of secretory granules and glutamate transport in blood platelets during cholesterol depletion of the plasma membrane by methyl-beta-cyclodextrin. Neurochemistry International-2011.- V.59 .- Р. 965-975.

• T.Borisova, R.Sivko, A.Borysov, N.Krisanova Diverse presynaptic mechanisms underlying methyl-beta-cyclodextrin – mediated changes in glutamate transport. Cellular and Molecular Neurobiology - 2010.- V.30, № 7.- Р. 1013-1023.

• T.Borisova, N.Krisanova, R.Sivko, A.Borysov Cholesterol depletion attenuates tonic release but increases the ambient level of glutamate in rat brain synaptosomes. Neurochemistry International- 2010.- V.56.- Р. 466-478.

• A. S.Tarasenko, R. V. Sivko, N. V.Krisanova, N. H.Himmelreich, T. A. Borisova Cholesterol depletion from the plasma membrane impairs proton and glutamate storage in synaptic vesicles of nerve terminals. Journal of Molecular Neuroscience - 2010.- V.41, № 3.- Р. 358-367.

• L.Kasatkina, T.Borisova Impaired Na+- dependent glutamate uptake in platelets during depolarization of their plasma membrane. Neurochemistry International - 2010.- V.56.- Р. 711-719.

• Krisanova, N.V. Synaptopathy under conditions of altered gravity: Changes in synaptic vesicle fusion and glutamate release / N.V.Krisanova, I.O.Trikash, T.A.Borisova // Neurochemistry International.- 2009.- V.55.- Р. 724-731.

Lithuania, 2012

Thank you for the attention!

Acknowledgements:

Dr. Natalia Krisanova

Roman Sivko

Ludmila Kasatkina

Lithuania, 2012

Acknowledgements:

2007–2013 m. Žmogiškųjų išteklių

plėtros veiksmų programos 3

prioriteto „Tyrėjų gebėjimų

stiprinimas“VP1-3.1-ŠMM-05-K

priemonės „MTTP tematinių tinklų,

asociacijų veiklos stiprinimas

“projektas „Lietuvos Biochemikų

draugijos potencialo kurti žinių

visuomenę didinimas“ (Nr. VP1-3.1-

ŠMM-05-K-01-022)