Amiotrofična lateralna skleroza (ALS) je fatalna neurodegenerativna bolest sa
početkom u adultnom dobu koja se karakteriše progresivnim gubitkom gornjih i donjih
motoneurona, što dovodi do atrofije muskulature, mišićne paralize i smrti usled slabosti
respiratorne muskulature. Oko 5-10% ALS slučajeva su sa familijarnom istorijom bolesti
(fALS), dok su preostali slučajevi sporadični (sALS) sa nepoznatim uzrokom bolesti. Cilj
ove disertacije bio je da proceni efekat imunoglobulina G (IgG) izolovanog iz sALS
bolesnika (ALS IgG) na homeostazu kalcijuma i mobilnost endozoma/lizozoma kod
pacovskih kortikalnih astrocita u kulturi. ALS IgG (0.1 mg/ml) poreklom od 7 ALS
pacijenata, za razliku od IgG iz 3 kontrolne osobe, izazvao je prolazne promene
unutarćelijske koncentracije kalcijuma (Ca2+-talas) u astrocitima. Povećana aktivnost
kalcijuma detektovana je kod oko polovine astrocita nakon tretmana sa ALS IgG, bez
obzira na prisustvo vanćelijskog kalcijuma (47,5 ± 12,9 %, 2 mM Ca2+ naspram 48,2 ...± 13,6
%, 0 mM Ca2+). U prisustvu vanćelijskog kalcijuma, maksimalna vrednost promene
dostignuta je ~3x brže (19,7 ± 2,8 s, 2 mM Ca2+ naspram 56,3 ± 6,8 s, 0 mM Ca2+) i ukupno
povećanje kalcijuma (površina pod Ca2+-talasom) bilo je ~1.7x veće (26,0 ± 1,6 F/F0 *s, 2
mM Ca2+ naspram 15,6 ± 1.1 F/F0 *s, 0 mM Ca2+), ali vanćelijski kalcijum nije uticao na
maksimum amplitude kalcijumovog talasa. Primena farmakoloških inhibitora otkrila je da
je aktivacija inozotol 1,4,5-trifosfatnog (IP3), za razliku od rianodinskog receptora
neophodna i dovoljna da inicira povećanu aktivnost, a ulazak kalcijuma kroz TRPC kanale
produžava odgovor. Inhibicija fosfolipaze C (PLC) umanjuje, dok inhibicija
fosfatidilinozitol-3-kinaze (PI3K) u potpunosti sprečava tipičan ALS IgG-izazvan
kalcijumski odgovor. Populacija vezikula obeleženih lizotrekerom koji boji endozome i
lizozome, sastojala se od stacionarnih vezikula (6,1%) sa prosečnom brzinom koja nije
prelazila 67 nm/s i mobilnih vezikula (93.9%) čija je ukupna dužina putanje (TL) pređene
za 15 s iznosila u proseku 3,03±0,01 μm. ALS IgG (0.1 mg/ml) poreklom iz 12 od 13 ALS
bolesnika povećao je TL mobilnih vezikula za ~24% i maksimalni pomeraj (MD) za ~26%
tokom praćenih 4 min, za razliku od kontrolnih IgG (poreklom od 4 osobe) koji nisu uticali
na mobilnost vezikula. ALS IgG-izazvano povećanje mobilnosti bilo je manje izraženo u
rastvoru bez Ca2+, što ukazuje da su promene u homeostazi kalcijuma uključene u proces
kojim ALS IgG povećava mobilnost vezikula. Interesantno, ALS IgG kao i ATP (1 mM)
nisu uticali na oslobađanje sadržaja vezikula, što znači da je brza i kompletna egzocitoza
malo verovatan događaj kod astrocita u kulturi u našim eksperimentalnim uslovima.
Sumarno, ALS IgG utiče na kalcijumovu homeostazu astrocita preko IP3-posredovanog
oslobađanja kalcijuma iz endoplazmatičnog retikuluma i ulaska kalcijuma kroz TRPC
kanale, uz aktivaciju PI3K uzvodno od PLC. ALS IgG povećavaju mobilnost endozoma i
lizozoma, a ovaj efekat delimično zavisi od vanćelijskog kalcijuma. Ova studija je otkrila
molekulske mehanizme kojima astrociti postaju meta humoralnog imunskog odgovora u
ALS, što može da doprinese etiopatogenezi bolesti.
Amyotrophic lateral sclerosis (ALS) is an adult onset fatal neurodegenerative
disease characterized mainly by the progressive loss of upper and lower motor neurons
resulting in wasting, paresis and death from respiratory failure. Approximately 5-10% of
ALS cases are familial (fALS), while the rest are sporadic (sALS) with unknown cause of
disease. The objective of this dissertation was to evaluate the effect of immunoglobulin G
(IgG) isolated from sALS patients (ALS IgG) on calcium homeostasis and mobility of
endosomes/lysosomes in cultured rat cortical astrocytes. ALS IgG (0.1 mg/ml) from 7 ALS
patients, but not IgG from 3 control individuals evoked calcium transients (Ca2+-waves) in
astrocytes. About half of the tested astrocytes responded with elevated calcium activity
regardless of the presence of extracellular calcium (47.5 ± 12.9 %, 2 mM Ca2+ vs. 48.2 ±
13.6 %, 0 mM Ca2+). In presence of extracellular calcium, the peak amplitude developed
~3x faster (19.7 ± 2.8 s, 2 mM Ca2+ vs. 5...6.3 ± 6.8 s, 0 mM Ca2+) and the overall magnitude
of calcium rise (area under the Ca2+-wave) was ~1.7x larger (26.0 ± 1.6 F/F0 *s, 2 mM Ca2+
vs. 15.6 ± 1.1 F/F0 *s, 0 mM Ca2+), while the peak amplitude itself was not affected.
Application of pharmacological inhibitors revealed that activation of inositol 1,4,5-
triphosphate (IP3), but not of ryanodine receptors is necessary and sufficient to initiate
elevated activity, while the influx of extracellular calcium through TRPC channels prolongs
the responses. Inhibition of phospholipase C (PLC) diminishes, while the inhibition of
phosphatidilinositol-3-kinase (PI3K) completely prevents typical ALS IgG evoked calcium
response. The population of vesicles labeled with lysotracker that mainly stains endosomes
and lysosomes, consisted of non-mobile (6.1%) vesicles with the average speed of <67
nm/s and mobile vesicles (93.9%) with total track length (TL) in 15 s averaging at
3.03±0.01 μm. ALS IgG (0.1 mg/ml) from 12 of 13 patients increased the TL of mobile
vesicles by ~24% and the maximal displacement (MD) by ~26% within 4 min, while IgG
from control group (n=4) did not alter the vesicle mobility. The mobility enhancement by
ALS IgG was less pronounced in Ca2+-free extracellular solution, indicating that ALS IgG
vesicle mobility enhancement involves changes in Ca2+ homeostasis. Interestingly, neither
ALS IgG nor ATP (1 mM) triggered the release of vesicular cargo, making rapid and
complete exocytosis unlikely in cultured astrocytes in our experimental conditions. In
conclusion, ALS IgG affect calcium homeostatic system in astrocytes by IP3 mediated
calcium release from the endoplasmic reticulum and entry of extracellular calcium through
TRPC channels, with the activation of PI3K upstream of PLC. ALS IgG enhance the
mobility of endosomes and lysosomes, and this effect is partialy dependent on extracellular
calcium. This study revealed the molecular mechanisms by which astrocytes become
targeted cells for humoral immune response in ALS that could contribute to the
aetiopathogenesis of the disease.
