Similarities between spinocerebellar ataxia type 7 (SCA7) cell models and human brain: proteins recruited in inclusions and activation of caspase-3
C. Zander
1
2
J. Takahashi
0
1
5
K. H. El Hachimi
1
4
H. Fujigasaki
1
2
V. Albanese
1
A. S. Lebre
1
2
G. Stevanin
1
2
C. Duyckaerts
0
1
A. Brice
1
2
3
0
Laboratoire de Neuropathologie
1
Unit, Department of Molecular Medicine, Karolinska Institute
,
Stockholm
,
Sweden
2
INSERM U289, Hopital de la Salpetriere
,
47 boulevard de l'Hopital, 75651 Paris, Cedex 13
,
France
3
Departement de Genetique, Cytogenetique et Embryologie, Hopital de la Salpetriere
,
75651 Paris
4
INSERM U106 and Ecole Pratique des Hautes Etudes
5
Division of Neuropathology, The Jikei University School of Medicine
,
Tokyo
,
Japan
Spinocerebellar ataxia type 7 (SCA7) is an autosomal dominant polyglutamine disorder presenting with progressive cerebellar ataxia and blindness. The molecular mechanisms underlying the selective neuronal death typical of SCA7 are unknown. We have established SCA7 cell culture models in HEK293 and SH-SY5Y cells, in order to analyse the effects of overexpression of the mutant ataxin-7 protein. The cells readily formed anti-ataxin-7 positive, fibrillar inclusions and small, nuclear electron dense structures. We have compared the inclusions in cells expressing mutant ataxin-7 and in human SCA7 brain tissue. There were consistent signs of ongoing abnormal protein folding, including the recruitment of heat-shock proteins and proteasome subunits. Occasionally, sequestered transcription factors were found. Activated caspase-3 was recruited into the inclusions in both the cell models and human SCA7 brain and its expression was upregulated in cortical neurones, suggesting that it may play a role in the disease process. Finally, on the ultrastructural level, there were signs of autophagy and nuclear indentations, indicative of a major stress response in cells expressing mutant ataxin-7.
-
Spinocerebellar ataxia type 7 (SCA7) is one of nine known
polyglutamine disorders (15). The group also includes
Huntingtons disease (HD), spinal and bulbar muscular
atrophy (SBMA), dentatorubral-pallidoluysian atrophy
(DRPLA), SCA1-3, 6 and SCA17/TBP disease. SCA7 is an
autosomal dominant neurodegenerative disorder, characterized
by cerebellar ataxia and visual loss. Pathologically, there is
progressive macular degeneration associated with predominant
neuronal loss in the cerebellar cortex, the brain stem and the
inferior olivary complex. The underlying genetic mutation is an
abnormal expansion of a polymorphic CAG repeat (36300
units in patients, 435 units in controls) in the 5 region of the
SCA7 gene. The propensity of the repeat to expand in SCA7
results in striking anticipation (6).
The SCA7 gene encodes a widely expressed 892 amino acid
protein, ataxin-7, of unknown function (712). The protein has
a functional nuclear localization signal (NLS) (13), but
endogenous ataxin-7 is found both in the cytoplasm and in the
nucleus in neurones. In COS1 cells overexpressed ataxin-7 is
associated with the nuclear matrix and nucleolus (13). The
protein carries a motif related to the phosphate-binding site of
arrestins (14) and several SH3-binding domains through which
it interacts with a Cbl associated protein (15).
Polyglutamine containing proteins have an inherent ability
to aggregate when the length of the polyglutamine tract
exceeds 3540 glutamines, the threshold above which most
of the diseases develop (16). The hallmark of a polyglutamine
disease is the presence of ubiquitinated aggregates, neuronal
intranuclear inclusions (NIIs), which have been found in
human brain, transgenic mice- and cell-models (17). Several
groups of proteins, such as elements of the ubiquitinproteasome
system and transcription factors, are sequestered in the NIIs.
However, whether the inclusions are only a marker of the
disease or part of the disease process is a matter of debate (1820).
Some studies in transfected cell cultures and transgenic mice
have suggested that critical nuclear processes might be
perturbed by the polyglutamine expansion and that altered gene
transcription may be a major cause of cellular dysfunction
(2123). Why this occurs in only selective neuronal
populations, is not known, but it has been hypothesized that the
polyglutamine expansions trigger a toxic gain of function,
eventually through altered interactions with selectively
expressed proteins.
The molecular pathways leading to neuronal dysfunction
and subsequent death in SCA7 are poorly understood. In an
attempt to approach the role of the inclusions and associated
abnormalities in SCA7 pathogenesis, we have characterized
the inclusions and morphological alterations in HEK293 and
SH-SY5Y cells expressing mutant ataxin-7 and the inclusions
in human SCA7 cases. Several heat-shock proteins, subunits of
the proteasome, transcription factors and activated caspase-3
were detected in a subset of inclusions, and overall caspase-3
immunoreactivity was enhanced in post-mortem SCA7 brain
tissue compared to normal brain. There were also
ultrastructural signs of autophagy and nuclear indentations, (...truncated)