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Angela Battaglia Biology 240M 4/30/14

The role of inclusion cells and polyglutamine aggregate
formation in the molecular mechanisms of Huntingtons disease
Introduction
Huntingtons disease is symptomatically characterized by a slow deterioration of mental
functions, particularly in the deep cortical layers and striatum, as well as the deterioration of
involuntary motor system functions, and it is usually fatal within 20 years of the onset of the
disease (Bates et al., 2002) (Bates & Landles, 2004). It is an autosomal dominant disease, and
through studies done in an isolated Venezuelan town where Huntingtons is particularly
common, it has been traced all the way to a single gene (Bates et al., 2002). The wild-type allele
of the gene at this locus codes for a protein called huntingtin, but if the gene is mutated by the
addition of CAG repetitions, it will then produce a disease-causing mutated huntingtin protein
(Bates et al., 2002). The trinucleotide repetitions in the mutated huntingtin protein code for
glutamine and because they are repeated in excess, there are excess amounts of polyglutamine in
the mutated huntingtin protein (Bates et al., 2002). This is the reason that Huntingtons is
considered to be a polyglutamine disease. Other diseases that fall into this category are several
types of spinocerebellar ataxia, Kennedy's disease, and dentatorubro-pallidoluysian atrophy.
Although all of these diseases are different, they all share the underlying factor of excess
polyglutamine (Bates & Landles, 2004). One reason that this is thought to be an issue is because
excess polyglutamine in proteins enables it to more easily form stable aggregates (Bates &
Landles, 2004). These aggregates can then build up to form neural inclusions, also called
inclusion bodies (Bates & Landles, 2004).
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As of currently, it is still highly debated as to whether the formation of this aggregate is
part of the disease-causing mechanism if it is simply a by-product of the true disease-causing
mechanism or if it has neuroprotective qualities (Bates & Landles, 2004). The goal of this paper
is to delve further into the possible mechanisms of Huntingtons disease, specifically into the
role that polyglutamine aggregates play into these mechanisms as well as to further explore the
effect that the formation of these aggregates has been found to have.
How trinucleotide repetition leads to protein aggregates
As mentioned before, the trinucleotide repetition of CAG at the locus that encodes the
huntingtin protein leads to excess amounts of polyglutamine in the mutated disease-causing
huntingtin protein (Bates & Landles, 2004). People without Huntingtons disease have typically
been found to have sequential polyglutamine residues ranging from 10 to 36, while those
diagnosed with the disease generally have more than 41 consecutive residues (Bates & Landles,
2004). The addition of the extra polyglutamine allows these proteins to form stable insoluble
aggregates and inclusion bodies (Bates & Landles, 2004). When studied in an in vitro
morphological study, this process was shown to be aided by a C-terminal 38-residueproline-rich
stretch (C38) and a N-terminal 17-residue amphipathic stretch (N17) which aid in destabilizing
bonds that do not form aggregates and stabilizing the fibrils that help to form aggregates (Crick,
et al., 2013). This means that N17 and C38 both help aid in the overall formation of these
polyglutamine aggregates (Crick, et al., 2013).
In these aggregates, the ubiquitin-proteasome system, heat shock proteins, and molecular
chaperones have been found to change in concentrations as the disease pathology progresses.
The significance of all of these has also been tested with relation to the mechanism of
Huntingtons disease. In order to determine what kind of effect the formation of these aggregates
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has on Huntingtons disease it is important to study the accumulations of each of these
compounds or systems. After this is done, more conclusions can be drawn about the possible
roles that polyglutamine aggregates and inclusion bodies play in the mechanism of Huntingtons
disease.
Investigating polyglutamine and the Ubiquitin-Proteasome System
As the pathogenesis of Huntingtons progresses, the ubiquitin-proteasome system is
impaired (Mitra et al., 2008). The ubiquitin-proteasome system is a main pathway in which
intracellular proteins get degraded. Cell cultures were taken from rat embryo striatas, transfected
in vitro, and then analyzed using live-cell imaging in order to determine the connections between
ubiquitin proteasome system (UPS) and inclusion bodies (Mitra et al., 2008). The results of this
study showed that when looking at cells where inclusion bodies had or were going to form, the
UPS was more impaired before the inclusion body formed, and after inclusion body formation
UPS function was less impaired (Mitra et al., 2008). This implies that the inclusion body cells
have protective qualities when it comes to the UPS, which was one of the original hypotheses
about the effects of polyglutamine aggregation and inclusion bodies (Mitra et al., 2008).
A conflicting theory to these results claims that the ubiquitin-proteasome system is
inhibited by the process of polyglutamine aggregate formation, and that this UPS inhibition leads
to cellular dysfunction (Breuer et al., 2002). This model states that the polyglutamine
intermediates trap certain regulators needed by the UPS and thus inhibit it (Breuer et al., 2002).
Heat shock proteins HSP70 and HSP40 may also play a role in this interaction by preventing the
inhibition of UPS by the polyglutamine intermediates. A summary of this model can be found in
part B of Figure 1. This model contradicts the data found in the previous experiment, although
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Figure 1: Overview of two models by which the aggregation of polyglutamine is
controlled. The first model (A) focuses on the relationship with Heat shock proteins,
where HSP70 and HSP40 inhibit aggregation by inhibiting the formation of
polyglutamine intermediates as well as the recruitment of transcription factors that the
intermediates need. The second model (B) focuses on the connection of both HSP
proteins and the Ubiquitin-proteasome systems with polyglutamine aggregation. Here,
the formation of polyglutamine intermediates inhibits the ubiquitin-proteasome system.
In both of these models, it is thought that polyglutamine aggregate formation leads to
cellular dysfunction (Breuer et al., 2002)
the experiment was done after the model construction. The model was published in a 2002 paper,
while the contradicting research was published in 2008.

Investigating polyglutamine and Heat Shock Proteins
The concentration of heat shock proteins have also been found to change during the
progression of Huntingtons disease. Protein was extracted from the striatal cells of knock-in
mice and analyzed with Western blotting, assays, and immunocytochemistry in order to
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determine the connection between heat shock proteins and polyglutamine in HD mice (Chafekar
& Duennwald, 2012). Through this research, it was found that with an increasing presence of
polyglutamine, there is downregulation of the transcription of two heat shock proteins, HSP1 and
HSP70, thus hindering the heat shock response resulting in a cells reduced capacity to handle
stressful environments (Chafekar & Duennwald, 2012). This allows for more cell damage
because these cells can therefore not properly respond to stressful situations correctly. From this
study, it seems as though the presence of polyglutamine, even before aggregation, plays a
negative role, promoting neurodegeneration and progression of Huntingtons disease by allowing
cell damage.
It has also been found that an opposing relationship also exists between certain heat
shock proteins and polyglutamine aggregation (Breuer et al., 2002). In addition to polyglutamine
inhibiting HSP1 and HSP70 transcription, there is a model that states that HSP70 and HSP40
reduce the formation of polyglutamine aggregates (Breuer et al., 2002). These heat shock
proteins are thought to inhibit the formation of polyglutamine intermediates, as well as the
recruitment of certain transcription factors that are required for the aggregation of these
intermediates (Breuer et al., 2002). In this way, the model claims that heat shock proteins help
decrease cellular dysfunction by decreasing the amount of polyglutamine aggregates that are
formed (Breuer et al., 2002). This model is demonstrated in part A of Figure 1.
Investigating polyglutamine and the molecular chaperone DJ-1
The chaperone DJ -1 has been found to play a role in a few different diseases and its role
in Huntingtons disease was also investigated (Sajjad et al., 2014). In this study conducted by Dr.
Sajjad and colleagues, cells were cultured, and transfected with fluorescent plasmids, protein was
extracted and dot blot and immunoblot analysis was used to determine the results (Sajjad et al.,
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2014). It was found that DJ -1 increased the production of aggregates as well as helped to protect
against neurodegeneration (Sajjad et al., 2014). This research is not about how aggregates
decrease neurodegeneration, but rather how DJ -1 decreases neurodegeneration which presents
the option that it may not be the inclusion cells themselves that affect neurodegeneration. It
instead supports the hypothesis that production of these polyglutamine aggregates is simply a
side product of the production of DJ -1.
Conclusion
Overall, it can be stated that the role of polyglutamine in Huntingtons disease is very
complex, and that more research must be done in order for its full importance to be elucidated.
With respect to the ubiquitin-proteasome system, the aggregates were found to have a positive,
neuroprotective effect in one study (Mitra et al., 2008), although a previously proposed model
states that aggregate formation instead inhibits UPS thus causing cell dysfunction and
degradation (Breuer et al., 2002). In respect to heat shock proteins, the aggregates were found to
have a neurodegenerative effect (Chafekar & Duennwald, 2012), although heat shock proteins
are also thought to attempt to inhibit polyglutamine aggregation in an attempt to stop this
degradation (Breuer et al., 2002). With respect to the molecular chaperone DJ -1, the aggregates
were found to be increased in the presence of DJ -1, but the researchers did not draw any further
conclusions from this increase so here it is thought that the formation of aggregates is simply a
byproduct (Sajjad et al., 2014). As shown by the varied and sometimes contradictory research
presented here, polyglutamine aggregates certainly play a very complex role in the pathogenesis
of Huntingtons disease. Although no further conclusions can be drawn at the moment, more
research should be done to discover more about it because this research could lead to discovery
of a cure for Huntingtons disease.
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References
1. Bates, G., Harper, P., & J ones, A. (2002). Huntington's disease (3rd ed.). Oxford: Oxford
University Press.
2. Chafekar, S. M., Duennwald, M. L., & Borchelt, D. R. (2012). Impaired Heat Shock
Response in Cells Expressing Full-Length Polyglutamine-Expanded Huntingtin. PLoS
ONE, 7(5), e37929.
3. Crick, S. L., Ruff, K. M., Garai, K., Frieden, C., & Pappu, R. V. (2013). Unmasking the roles
of N- and C-terminal flanking sequences from exon 1 of huntingtin as modulators of
polyglutamine aggregation. Proceedings of the National Academy of Sciences, 110(50),
20075-20080.
4. Landles, C., & Bates, G. P. (2004). Huntingtin and the molecular pathogenesis of
Huntington's disease. EMBO Reports, 5(10), 958-963.
5. Mitra, S., Tsvetkov, A. S., & Finkbeiner, S. (2008). Single Neuron Ubiquitin-Proteasome
Dynamics Accompanying Inclusion Body Formation in Huntington Disease. Journal of
Biological Chemistry, 284(7), 4398-4403.
6. Sajjad, M., Green, E., Miller-Fleming, L., Hands, S., Herrera, F., Campesan, S., et al. (2014).
DJ -1 modulates aggregation and pathogenesis in models of Huntingtons disease.
Human Molecular Genetics, 23(3), 755-766.
7. Breuer, P., Sakahira, H., Hayer-Hartl, M., & Hartyl, F. (2002). Molecular chaperones as
modulators of polyglutamine protein aggregation and toxicity. Proceedings of the
National Academy of Sciences, 99(90004), 16412-16418.


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