, an expanded polyglutamine (polyQ>37) sequence within huntingtin (htt) exon1 leads to enhanced disease risk. It has proved difficult, however, to determine whether the toxic form generated by polyQ expansion is a misfolded or avid-binding monomer, an alpha-helix-rich oligomer, or a beta-sheet-rich amyloid fibril. This study describes an engineered htt exon1 analog featuring a short polyQ sequence that nonetheless quickly forms amyloid fibrils and causes HD-like toxicity in rat neurons and Drosophila. Additional modifications within the polyQ segment produce htt exon1 analogs that populate only spherical oligomers and are non-toxic in cells and flies. Furthermore, in mixture with expanded-polyQ htt exon1, the latter analogs in vitro suppress amyloid formation and promote oligomer formation, and in vivo rescue neurons and flies expressing mhtt exon1 from dysfunction and death. Thus, in these experiments, while htt exon1 toxicity tracks with aggregation propensity, it does so in spite of the toxic construct's possessing polyQ tracts well below those normally considered to be disease-associated. That is, aggregation propensity proves to be a more accurate surrogate for toxicity than is polyQ repeat length itself, strongly supporting a major toxic role for htt exon1 aggregation in HD. In addition, the results suggest that the aggregates that are most toxic in these model systems are amyloid-related. Small molecules with similar amyloid inhibitory properties might be developed into effective therapeutic agents (Drombosky, 2018).
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Donnelly, K. M. and Pearce, M. M. P. (2018). Monitoring cell-to-cell transmission of prion-like protein aggregates in Drosophila melanogaster. J Vis Exp(133). PubMed ID: 29578503
Abstract
Protein aggregation is a central feature of most neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and amyotrophic lateral sclerosis (ALS). Protein aggregates are closely associated with neuropathology in these diseases, although the exact mechanism by which aberrant protein aggregation disrupts normal cellular homeostasis is not known. Emerging data provide strong support for the hypothesis that pathogenic aggregates in AD, PD, HD, and ALS have many similarities to prions, which are protein-only infectious agents responsible for the transmissible spongiform encephalopathies. Prions self-replicate by templating the conversion of natively-folded versions of the same protein, causing spread of the aggregation phenotype. How prions and prion-like proteins in AD, PD, HD, and ALS move from one cell to another is currently an area of intense investigation. A Drosophila melanogaster model was established that permits monitoring of prion-like, cell-to-cell transmission of mutant huntingtin (Htt) aggregates associated with HD is described. This model takes advantage of powerful tools for manipulating transgene expression in many different Drosophila tissues and utilizes a fluorescently-tagged cytoplasmic protein to directly report prion-like transfer of mutant Htt aggregates. Importantly, the approach described in this study can be used to identify novel genes and pathways that mediate spreading of protein aggregates between diverse cell types in vivo. Information gained from these studies will expand the limited understanding of the pathogenic mechanisms that underlie neurodegenerative diseases and reveal new opportunities for therapeutic intervention (Donnelly, 2018).
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Al-Ramahi, I., Lu, B., Di Paola, S., Pang, K., de Haro, M., Peluso, I., Gallego-Flores, T., Malik, N. T., Erikson, K., Bleiberg, B. A., Avalos, M., Fan, G., Rivers, L. E., Laitman, A. M., Diaz-Garcia, J. R., Hild, M., Palacino, J., Liu, Z., Medina, D. L. and Botas, J. .
(2018). High-Throughput Functional Analysis Distinguishes Pathogenic, Nonpathogenic, and Compensatory Transcriptional Changes in Neurodegeneration. Cell Syst. PubMed ID: 29936182
Abstract
Discriminating transcriptional changes that drive disease pathogenesis from nonpathogenic and compensatory responses is a daunting challenge. This is particularly true for neurodegenerative diseases, which affect the expression of thousands of genes in different brain regions at different disease stages. This study integrates functional testing and network approaches to analyze previously reported transcriptional alterations in the brains of Huntington disease (HD) patients. 312 genes were selected whose expression is dysregulated both in HD patients and in HD mice and then replicated and/or antagonized each alteration in a Drosophila HD model. High-throughput behavioral testing in this model and controls revealed that transcriptional changes in synaptic biology and calcium signaling are compensatory, whereas alterations involving the actin cytoskeleton and inflammation drive disease. Knockdown of disease-driving genes in HD patient-derived cells lowered mutant Huntingtin levels and activated macroautophagy, suggesting a mechanism for mitigating pathogenesis. This multilayered approach can thus untangle the wealth of information generated by transcriptomics and identify early therapeutic intervention points (Al-Ramahi, 2018).
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Akbergenova, Y. and Littleton, J. T. (2017). Pathogenic Huntington alters BMP signaling and synaptic growth through local disruptions of endosomal compartments. J Neurosci [Epub ahead of print]. PubMed ID: 28235896
Abstract
Huntington's disease (HD) is a neurodegenerative disorder caused by expansion of a polyglutamine (polyQ) stretch within the Huntingtin (Htt) protein. Pathogenic Htt disrupts multiple neuronal processes, including gene expression, axonal trafficking, proteasome and mitochondrial activity, and intracellular vesicle trafficking. However, the primary pathogenic mechanism and subcellular site of action for mutant Htt are still unclear. Using a Drosophila HD model, this study found that pathogenic Htt expression leads to a profound overgrowth of synaptic connections that directly correlates with the levels of Htt at nerve terminals. Branches of the same nerve containing different levels of Htt show distinct phenotypes, indicating Htt acts locally to disrupt synaptic growth. The effects of pathogenic Htt on synaptic growth arise from defective synaptic endosomal trafficking, leading to expansion of a recycling endosomal signaling compartment containing Sorting Nexin 16, and a reduction in late endosomes containing Rab11. The disruption of endosomal compartments leads to elevated BMP signaling within nerve terminals, driving excessive synaptic growth. Blocking aberrant signaling from endosomes or reducing BMP activity (see Wishful thinking) ameliorates the severity of HD pathology and improves viability. Pathogenic Htt is present largely in a non-aggregated form at synapses, indicating cytosolic forms of the protein are likely to be the toxic species that disrupt endosomal signaling. These data indicate that pathogenic Htt acts locally at nerve terminals to alter trafficking between endosomal compartments, leading to defects in synaptic structure that correlate with pathogenesis and lethality in the Drosophila HD model (Akbergenova, 2017).
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Singh, V., Sharma, R. K., Athilingam, T., Sinha, P., Sinha, N. and Thakur, A. K. (2017). NMR spectroscopy-based metabolomics of Drosophila model of Huntington's disease suggests altered cell energetics. J Proteome Res [Epub ahead of print]. PubMed ID: 28871787
Abstract
Huntington's disease (HD) is a neurodegenerative disorder induced by aggregation of the pathological form of Huntingtin protein that has expanded polyglutamine (polyQ) repeats. In the Drosophila model, for instance, expression of transgenes with polyQ repeats induces HD-like pathologies, progressively correlating with the increasing lengths of these repeats. Previous studies on both animal models and clinical samples have revealed metabolite imbalances during HD progression. To further explore the physiological processes linked to metabolite imbalances during HD, this study has investigated the 1D 1H NMR spectroscopy-based metabolomics profile of Drosophila HD model. Using multivariate analysis (PCA and PLS-DA) of metabolites obtained from methanolic extracts of fly heads displaying retinal deformations due to polyQ overexpression, this study showed that the metabolite imbalance during HD is likely to affect cell energetics. Six out of the 35 metabolites analyzed, namely, nicotinamide adenine dinucleotide (NAD), lactate, pyruvate, succinate, sarcosine, and acetoin, displayed segregation with progressive severity of HD. Specifically, HD progression was seen to be associated with reduction in NAD and increase in lactate-to-pyruvate ratio. Furthermore, comparative analysis of fly HD metabolome with those of mouse HD model and HD human patients revealed comparable metabolite imbalances, suggesting altered cellular energy homeostasis. These findings thus raise the possibility of therapeutic interventions for HD via modulation of cellular energetics (Singh, 2017).
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Raj, K. and Sarkar, S. (2017). Transactivation domain of human c-Myc Is essential to alleviate poly(Q)-mediated neurotoxicity in Drosophila disease models. J Mol Neurosci 62(1):55-66. PubMed ID: 28316031
Abstract
Polyglutamine (poly(Q)) disorders, such as Huntington's disease (HD) and spinocerebellar ataxias, represent a group of neurological disorders which arise due to an atypically expanded poly(Q) tract in the coding region of the affected gene. Pathogenesis of these disorders inside the cells begins with the assembly of these mutant proteins in the form of insoluble inclusion bodies (IBs), which progressively sequester several vital cellular transcription factors and other essential proteins, and finally leads to neuronal dysfunction and apoptosis. Earlier studies have shown that targeted upregulation of Drosophila myc (dmyc) dominantly suppresses the poly(Q) toxicity in Drosophila. The present study examines the ability of the human c-myc proto-oncogene and also identifies the specific c-Myc isoform which drives the mitigation of poly(Q)-mediated neurotoxicity, so that it could be further substantiated as a potential drug target. This study reports that similar to dmyc, tissue-specific induced expression of human c-myc also suppresses poly(Q)-mediated neurotoxicity by an analogous mechanism. Among the three isoforms of c-Myc, the rescue potential was maximally manifested by the full-length c-Myc2 protein, followed by c-Myc1, but not by c-MycS which lacks the transactivation domain. This study suggests that strategies focussing on the transactivation domain of c-Myc could be a very useful approach to design novel drug molecules against poly(Q) disorders (Raj, 2017).
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Dietz, K.N., Di Stefano, L., Maher,
R.C., Zhu, H., Macdonald, M.E., Gusella, J.F. and Walker, J.A.
(2015). The Drosophila Huntington's disease gene
ortholog dhtt influences chromatin regulation during
development. Hum Mol Genet 24: 330-345. PubMed ID: 25168387
Abstract
Huntington's disease is an autosomal dominant neurodegenerative
disorder caused by a CAG expansion mutation in HTT, the gene
encoding huntingtin. Evidence from both human
genotype–phenotype relationships and mouse model systems
suggests that the mutation acts by dysregulating some normal
activity of huntingtin. Recent work in the mouse has revealed a
role for huntingtin in epigenetic regulation during development.
This study examines the role of the Drosophila
huntingtin ortholog (dhtt) in chromatin regulation in the
development of the fly. Although null dhtt mutants
display no overt phenotype, it was found that dhtt acts
as a suppressor of position-effect variegation (PEV), suggesting
that it influences chromatin organization. It was demonstrated
that dhtt affects heterochromatin spreading in a PEV
model by modulating histone H3K9 methylation levels at the
heterochromatin–euchromatin boundary. To gain mechanistic
insights into how dhtt influences chromatin function, a
candidate genetic screen using RNAi lines targeting known PEV
modifier genes was conducted. dhtt was found to modify
phenotypes caused by knockdown of a number of key epigenetic
regulators, including chromatin-associated proteins, histone
demethylases (HDMs) and methyltransferases. Notably, dhtt
strongly modifies phenotypes resulting from loss of the HDM dLsd1,
in both the ovary and wing, and it was demonstrated that dhtt
appears to act as a facilitator of dLsd1 function in
regulating global histone H3K4 methylation levels. These findings
suggest that a fundamental aspect of huntingtin function in
heterochromatin/euchromatin organization is evolutionarily
conserved across phyla (Dietz, 2015).
Highlights
- Huntingtin is well conserved across different Drosophila
species.
- Generation of a new dhtt allele by homologous
recombination.
- Drosophila huntingtin is a suppressor of PEV.
- Drosophila huntingtin affects heterochromatin
spreading in a PEV model.
- Genetic interactions between dhtt and genes encoding
heterochromatin proteins.
- Genetic interactions of dhtt with genes encoding
chromatin-remodeling complex components.
- Loss of dhtt enhances dLsd1 mutant
phenotypes.
- Drosophila huntingtin facilitates histone H3K4
demethylation activity of dLsd1.
Discussion
Previous studies of the inverse relationship between the age at
onset of clinical symptoms and the size of the HTT CAG
repeat mutation have revealed that the repeat confers on the
mutant allele a fully dominant gain-of-function property. However,
it is unknown whether this is the acquisition of enhanced normal
huntingtin function or the acquisition of a novel opportunistic
function, although targeted null and CAG expansion mutations at
the mouse homolog provide support for both possibilities. Despite
earlier studies, details of the normal function of huntingtin
remain relatively elusive, hindering further investigation into
the molecular mechanisms underlying the disease (Dietz, 2015).
This study uses a novel dhtt allele to examine the
normal function of Drosophila huntingtin, focusing on
its potential role in chromatin function during development.
Although dhtt flies are viable and appear grossly
normal, genetic findings indicate that dhtt influences
chromatin regulation: (i) dhtt acts as a suppressor of
PEV, suggesting that it is involved in heterochromatin formation;
(ii) dhtt affects heterochromatin spreading in a PEV
model; (iii) dhtt genetically interacts with a number of
genes encoding proteins known to affect chromatin organization and
function and (iv) dhtt genetically interacts with the
HDM dLsd1 and facilitates its ability in demethylating
histone H3K4 (Dietz, 2015).
PEV is a powerful genetic assay that has been used previously to
identify genes that can regulate chromatin structure. In PEV
models, a chromosomal rearrangement or transposition abnormally
juxtaposes a reporter gene with heterochromatin. A variegated
phenotype is produced since the gene is stochastically silenced in
some of the cells in which it is normally active. The silencing
that occurs in PEV is attributed to the ‘spreading’ of
heterochromatin along the chromosome into a region that would
normally be in a euchromatic form. Thus, since the reporter gene
is on the boundary between these two states, PEV provides a
sensitive system in which to test genetic modifiers of
heterochromatin formation. This study uses two independent PEV
assays [T(2;3)Sbv and In(1)y3P] and
demonstrates that dhtt facilitates heterochromatin
formation, thereby suppressing variegated phenotypes. To date,
approximately 500 dominant Su(var) and E(var)
mutations have been isolated from PEV screens and it is estimated
that these affect about 150 unique genes. Those that have been
molecularly characterized so far have been revealed to generally
encode chromosomal proteins or modifiers of chromosomal proteins
(Dietz, 2015).
Histone post-translational modifications (PTMs) play essential
roles in the transition between active (euchromatin) and inactive
(heterochromatin) chromatin states. In particular, histone
methylation has been widely studied in nearly all model systems
and is generally recognized as an epigenetic marker for
transcriptionally silent heterochromatin. High levels of
methylated histone H3K9me2 are associated with heterochromatin
loci. Using the established PEV model, wm4, this study
demonstrates that the dhtt allele dominantly reduces the
level of histone H3K9me2 at the white locus and the adjacent CG12498
gene at the heterochromatin–euchromatin boundary. This level
of histone H3K9me2 reduction is comparable to that caused by a dLsd1/Su(var)3-3
null allele, an established suppressor of variegation. The loss of
dhtt therefore significantly influences chromatin
structure, thereby shifting the euchromatin–heterochromatin
boundary (Dietz, 2015).
Where in the cell might huntingtin function to affect chromatin
structure and act as a suppressor of variegation? Although the
majority of huntingtin in human and mouse cells has been shown to
reside within the cytoplasm, about 5% is estimated to be nuclear.
A previous report suggests that Drosophila huntingtin is
solely cytoplasmic, but this was based solely on ectopic dhtt
overexpression. Since both fly and mouse loss-of-function
huntingtin models show defects in mitotic spindle orientation in
neuroblast precursors, it is clear that huntingtin does have a
nuclear function. However, it is possible that dhtt
could also influence chromatin structure by acting in the
cytoplasm (Dietz, 2015).
Based on the findings that dhtt dominantly suppresses
PEV and affects chromatin function, it was hypothesized that it
may genetically interact with previously identified PEV modifiers.
The approach to screening for possible dhtt interactors
utilized a collection of RNAi lines targeting known suppressors
and enhancers of PEV. Such a screen has a number of caveats:
first, it relies on RNAi producing a modifiable phenotype in a
relevant tissue. Secondly, due to the nature of the screen, it is
largely limited to looking for interactions in the adult eye and
wing. Interestingly, in some cases, interactions between dhtt
and genes in the wing were found, but not in eye and vice versa.
This may reflect tissue-specific requirements for different genes,
or that dhtt functions only within certain complexes in
certain tissues. Nevertheless, a number of strong genetic
interactors of dhtt were identified, which included
central regulators of chromatin architecture and function, such as
the heterochromatin proteins, HP1a
and HP1b,
brm—the
ATPase subunit of the SWI/SNF (Brm) complex, the transcription
factor dE2F1
and various HDMs and HMTs. Next, the interaction between dhtt
and dLsd1 was evaluated for the following reasons: dLsd1
interacts with both the dhtt allele and dhtt
RNAi and loss of dhtt causes enhancement of dLsd1
phenotypes in both wing and ovary. Additionally, it was found that
dhtt and dLsd1 both affect PEV and
heterochromatin formation to comparable extents (Dietz, 2015).
Although dhtt-deficient flies are fertile and display
no obvious ovarian phenotype, loss of dhtt strongly
enhances the dLsd1 ovary defects. It was hypothesized
that dLsd1 and dhtt collaborat in the
regulation of histone H3K4 methylation at specific loci to control
gene expression critical for oogenesis. Similarly, in contrast to
dLsd1 mutant flies which show elevated levels of histone
H3K4me1 and H3K4me2, any changes in the global levels of these
modifications in dhtt-deficient flies could not be
detected. However, simultaneous knockdown of dLsd1 and dhtt
results in a significant increase in histone H3K4me1 and H3K4me2
levels over that of the dLsd1 knockdown alone. Human
LSD1 is a component of the CoREST/REST (repressor element
silencing transcription factor) complex, which represses the
transcription of neuronal genes in non-neuronal cell lineages.
Within this complex, LSD1 acts to demethylate histone H3K4
residues in nucleosomes at REST target genes, thereby contributing
to their transcriptional repression. Mammalian full-length
huntingtin has been shown to physically interact with this complex
and contribute to its regulation, and it will therefore be
interesting to determine whether dLsd1 and dhtt similarly
associate with each other. Unfortunately due to the lack of
phenotype upon knocking down the Drosophila ortholog of
CoREST, dCoREST, with the available RNAi lines, testing
for a potential interaction with dhtt could not be
performed by this study. The genetic interaction between dhtt
and dLsd1 could potentially account for the strong
effect of dhtt seen on H3K9 methylation at the
heterochromatin/euchromatin boundary in the wm4 PEV
model. dLsd1 has been shown to physically associate with
Su(var)3-9 and to control Su(var)3-9-dependent spreading of
histone H3K9 methylation along euchromatin (Dietz, 2015).
There is considerable evidence suggesting a link between aberrant
acetylation and methylation marks and HD. Mouse Htt has been
implicated in facilitating the trimethylation of histone H3K27 in
developing murine embryoid bodies. The levels of histone H3K4me3
have been shown to change at dysregulated promoters in a mouse HD
model (R6/2) and human HD postmortem brain tissue. The screen in
this study uncovered interactions between dhtt and Drosophila
HDM and HMTs with a variety of different histone H3 specificities
(H3K4, H3K27, H3K9 and H3K79). It is therefore possible that dhtt
has a general role, possibly as a scaffold protein, in
facilitating a number of complexes containing histone-modifying
enzymes with different specificities. Since mammalian full-length
huntingtin has been implicated in the trimethylation of histone
H3K27 by facilitating PRC2 function, it is surprising that the
histone H3K27 methyltransferase, esc,
is the only component of PRC2 found to interact with dhtt.
Although there was no effect of the dhtt null mutation
on the global levels of histone H3K27me levels, it is possible
that dhtt may play a similar role to mouse Htt in
modulating histone H3K27me during development, with histone
H3K27me differences only observed at specific loci (Dietz, 2015).
A number of the dhtt interacting genes found in the
screen encode for important chromatin regulating proteins that
have previously been found to genetically interact with each
other. For example, a strong interaction between dhtt
and brm, the central subunit of the Brm SWI/SNF complex,
was detected. brm is known to interact with E(Pc),
dE2F1, Asx
and Rpd3,
which were also found in the screen. The SIN3
corepressor complex is a class I HDAC complex conserved from Drosophila
to humans and regulates gene transcription through deacetylation
of nucleosomes. Loss of dhtt suppresses both eye and
wing phenotypes caused by Sin3A RNAi. Drosophila
Sin3A has been shown to interact with the HDAC Rpd3 and
the HDM, lid—both
of which were also scored as hits in the screen. Furthermore,
mammalian Sin3A was previously reported as a huntingtin N-terminal
yeast two-hybrid interactor (Dietz, 2015).
Drosophila has proved to be a useful model to
investigate polyglutamine-fragment toxicity. Expression of an
N-terminal fragment with an expanded polyglutamine tract in the
fly has been shown to accumulate in the nucleus. It would
therefore be interesting to evaluate whether the normal chromatin
regulatory functions of dhtt are perturbed in the fly
polyglutamine-fragment models. Although the Drosophila
screen in this study was designed to look initially for phenotypes
in visible external structures of the adult fly (wing and eye),
many of the genes that were found to interact with dhtt
are known to also be expressed and function in the developing
nervous system. It is therefore possible that dhtt may
also exert a role in regulating chromatin function during
neurogenesis and neural function in the fly, leading to subtle
behavioral mutant phenotypes that have been described previously.
This study establishes Drosophila as a system in which
to investigate the normal role of dhtt in chromatin
regulation. It will be particularly useful in examining dhtt
functions that are evolutionarily conserved as these provide
assays with which to determine the impact of the expanded
polyglutamine region on full-length huntingtin function, thereby
deepening our understanding of the mechanism that initiates the HD
disease process (Dietz, 2015).
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Rui,
Y.N., Xu, Z., Patel, B., Chen, Z., Chen, D., Tito, A., David,
G., Sun, Y., Stimming, E.F., Bellen, H.J., Cuervo, A.M. and
Zhang, S. (2015). Huntingtin functions as a scaffold
for selective macroautophagy. Nat Cell Biol 17: 262-275. PubMed
ID: 25686248
Abstract
Selective macroautophagy is an important protective mechanism
against diverse cellular stresses. In contrast to the
well-characterized starvation-induced autophagy, the regulation of
selective autophagy is largely unknown. This study demonstrates
that Huntingtin, the Huntington disease gene product, functions as
a scaffold protein for selective macroautophagy but is dispensable
for non-selective macroautophagy. In Drosophila,
Huntingtin genetically interacts with autophagy pathway
components. In mammalian cells, Huntingtin physically interacts
with the autophagy cargo receptor p62 to facilitate its
association with the integral autophagosome component LC3 and with
Lys-63-linked ubiquitin-modified substrates. Maximal activation of
selective autophagy during stress was attained by the ability of
Huntingtin to bind ULK1, a kinase that initiates autophagy, which
released ULK1 from negative regulation by mTOR. This data uncovers
an important physiological function of Huntingtin and provides a
missing link in the activation of selective macroautophagy in
metazoans (Rui, 2015).
Highlights
- Drosophila huntingtin genetically interacts with
autophagy pathway components.
- Drosophila huntingtin positively regulates autophagy
in vivo.
- Drosophila huntingtin is required for intracellular
quality control.
- Huntingtin’s function is conserved from flies to humans.
- Mammalian Huntingtin is required for selective autophagy.
- Huntingtin physically interacts with p62 and ULK1 proteins.
- Huntingtin facilitates p62-mediated cargo recognition
efficiency.
- Huntingtin–ULK1 and mTOR–ULK1 complexes are
mutually exclusive.
Discussion
Homozygous flies lacking the single htt homologue (dhttko)
are fully viable with only mild phenotypes. In a genetic screen
for the physiological function of Htt, ectopic expression of a
truncated form of the microtubule-binding protein Tau
(Tau-ΔC; truncated after Val 382) induced a prominent
collapse of the thorax in dhttko flies due to severe
muscle loss not observed by Tau expression alone, and accelerated
decline in mobility and lifespan. These phenotypes were fully
rescued by the dhtt genomic rescue transgene (‘dhttRescue’),
suggesting that dhtt protects against Tau-induced
pathogenic effects (Rui, 2015).
Although heterozygous dhttko/+ flies expressing Tau
(ATau; dhttko/+) seem normal, removing a single copy
of the fly LC3 gene, atg8a
(atg8ad4 mutant), in these flies also induces a collapsed
thorax and muscle loss, which can be phenocopied by expressing Tau
in homozygous atg8ad4−/− flies alone. Four
additional components of the early steps of the autophagy pathway,
atg1
(ULK1), atg7
and atg13,
and an adaptor for the selective recognition of autophagic cargo,
also exhibit strong genetic interactions with dhtt.
Consistent with its pivotal role in autophagy initiation, loss of
atg1 induces the strongest defect, and Tau expression can
induce a mild muscle loss phenotype even in heterozygous null atg1Δ3d.
Collectively, these genetic interaction studies suggest a role for
dhtt in autophagy (Rui, 2015).
By using the mCherry–GFP–Atg8a fusion reporter to
directly measure autophagic flux in adult dhttko−/−
brains, this study found similar number of red fluorescent punctae
(acidic autolysosomes originating from autophagosome/lysosome
fusion) in young mutant and control flies, but the number of
punctae were reduced in old dhttko−/− brains
when compared with age-matched controls. As autophagosome
accumulation (co-localized green and red puncta) was not observed,
it was concluded that the absence of dhtt in older
animals was associated with reduced autophagosome formation. The
fact that levels of Ref(2)P
are significantly higher in old dhttko−/−
brains compared with brains from age-matched wild-type controls
suggests a possible preferential compromise in selective autophagy
in these animals (Rui, 2015).
Consistent with the role of basal autophagy in quality control in
non-dividing cells, it was found that brains from 5-week-old
dhttko−/− contained almost double the amount
of ubiquitylated proteins, a marker of quality control failure,
compared with wild-type flies. As genetic interaction analysis and
specific ubiquitin proteasome system (UPS) reporters all failed to
reveal a functional link between dhtt and the UPS
pathway, the study proposes that the defects in autophagic
activity are the main cause of diminished quality control and
increased accumulation of ubiquitylated proteins in dhttko−/−
mutants (Rui, 2015).
Selective autophagy is induced in response to proteotoxic stress.
The truncated Tau-ΔC used in genetic experiments in this
study is preferentially degraded through autophagy in cortical
neurons, serving as a model of proteotoxicity when ectopically
expressed. The lower stability of Tau-ΔC compared with
full-length Tau in wild-type flies and in UPS mutants was
confirmed, but significantly higher levels of Tau-ΔC when
expressed in atg8a and in dhttko−/−
mutant flies were found, suggesting that autophagy is essential
for the clearance of Tau-ΔC also in flies and that dhtt
plays a role in this clearance (Rui, 2015).
In contrast, loss of dhtt does not affect flies’
adaptation to nutrient deprivation, which typically induces robust
‘in bulk’ autophagy. Fat bodies of early third instar
larvae expressing mCherry–Atg8, where starvation-induced
autophagy can be readily detected, fail to reveal any significant
difference between wild-type and dhttko−/−
flies; these flies die at the same rate as wild-type flies when
tested for starvation resistance. Thus, although dhtt is
necessary for selective autophagy of toxic proteins such as
Tau-ΔC, it is dispensable for starvation-induced autophagy
in flies (Rui, 2015).
Expression of human Htt (hHTT) in dhttko−/−
null flies rescues both the mobility and longevity defects of dhttko−/−
mutants and partially rescues the Tau-induced morphological and
behavioural defects of dhttko−/− flies.
hHTT also suppresses almost all of the autophagic defects observed
in dhttko−/−, including decreased levels of
autolysosomes, increased levels of Ref(2)P and of total
ubiquitylated proteins, and accumulation of ectopically expressed
Tau-ΔC, suggesting that the involvement of dhtt
in autophagy is functionally conserved. In fact, confluent mouse
fibroblasts knocked down for Htt (Htt(−)) exhibit
significantly lower basal rates of long-lived proteins’
degradation than control cells, which are no longer evident on
chemical inhibition of lysosomal proteolysis or of macroautophagy,
thus confirming an autophagic origin of the proteolytic defect.
Htt(−) fibroblasts also exhibit higher p62 levels and
accumulate ubiquitin aggregates even in the absence of a
proteotoxic challenge. As in dhttko−/−
flies, Htt knockdown in mammalian cells does not affect
degradation of CL1–GFP (a UPS reporter), β-catenin (a
UPS canonical substrate) or proteasome peptidase activities.
Reduced autophagic degradation in Htt(−) cells is not due
to a primary lysosomal defect, as depletion of Htt does not
reduce lysosomal acidification, endolysosomal number (if anything,
an expansion of this compartment was observed) or other lysosomal
functions such as endocytosis (for example, transferrin
internalization). In fact, analysis of the lysosomal degradation
of LC3-II reveals that autophagic flux and autophagosome formation
are preserved and even enhanced in Htt(−) fibroblasts at
basal conditions (Rui, 2015).
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Pearce, M.M., Spartz, E.J., Hong, W.,
Luo, L. and Kopito, R.R. (2015). Prion-like transmission
of neuronal huntingtin aggregates to phagocytic glia in the Drosophila
brain. Nat Commun 6: 6768. PubMed ID: 25866135
Abstract
The brain has a limited capacity to self-protect against protein
aggregate-associated pathology, and mounting evidence supports a
role for phagocytic glia in this process. This study establishes a
Drosophila model to investigate the role of phagocytic
glia in clearance of neuronal mutant huntingtin (Htt) aggregates
associated with Huntington disease. It was found that glia
regulates steady-state numbers of Htt aggregates expressed in
neurons through a clearance mechanism that requires the glial
scavenger receptor Draper
and downstream phagocytic engulfment machinery. Remarkably, some
of these engulfed neuronal Htt aggregates effect prion-like
conversion of soluble, wild-type Htt in the glial cytoplasm. The
study provided genetic evidence that this conversion depends
strictly on the Draper signalling pathway, unveiling a previously
unanticipated role for phagocytosis in transfer of pathogenic
protein aggregates in an intact brain. These results suggest a
potential mechanism by which phagocytic glia contribute to both
protein aggregate-related neuroprotection and pathogenesis in
neurodegenerative disease (Pearce, 2015).
Highlights
- Clearance of neuronal Htt aggregates by phagocytic glia.
- Prion-like transmission of Htt aggregates from ORNs to glia.
- Antennal injury enhances ORN-to-glial Htt aggregate transfer.
- Glial Htt aggregates associate with cytoplasmic chaperones.
- ORN-to-glia Htt aggregate transfer requires Draper signalling.
Discussion
This study establishes a Drosophila model that
demonstrates an essential role for phagocytic glia in clearance of
Htt aggregates from ORN axons undergoing Wallerian degeneration
after antennal axotomy or from non-severed axons. It was shown
that HttQ91 aggregate clearance is mediated by a pathway that
requires the glial engulfment receptor Draper and downstream
genes, and is genetically indistinguishable from the pathway that
operates in other glial phagocytic processes, including clearance
of axonal or cellular debris following injury and axon pruning
during development. Surprisingly, it was found that HttQ91
aggregates taken up by this Draper-dependent process are able to
access and initiate a prion-like conversion of normally soluble,
cytoplasmic HttQ25 in glia. These two findings have important
implications for the potential role of glia in the suppression
and/or progression of neurodegenerative diseases (Pearce, 2015).
Glial phagocytosis plays an important neuroprotective role in
response to many types of brain injury, including insults
associated with the production of neurodegenerative disease-linked
insoluble protein aggregates. Secretion of pro-inflammatory
cytokines and opsonins by activated glia promotes phagocytic
clearance of damaged neurons, neuronal processes and cellular
debris. In vitro, mouse astrocytes can bind to and degrade
extracellular Aβ aggregates in cell culture and in brain
slices. In vivo, pharmacological activation of microglia promotes
clearance of Aβ deposits in a transgenic mouse model of
Alzheimer disease (AD), and antibodies against Aβ or
α-synuclein promote microglial-mediated phagocytic removal
of the corresponding extracellular aggregates. Compelling evidence
for a neuroprotective role for glial phagocytosis has come from
recent studies linking missense mutations in TREM2, which encodes
a microglial phagocytic receptor, to several neurodegenerative
diseases including AD, Parkinson disease (PD) and amyotrophic
lateral sclerosis (ALS) (Pearce, 2015).
In this study, an essential role for phagocytic glia in clearance
of HttQ91 aggregates from ORN axons was established, but whether
this phagocytosis is initiated as a specific response to the
presence of aggregates or a collateral result of constitutive axon
turnover and remodelling is unclear. This strict dependence on
phagocytosis contrasts with previous work showing that
extracellular aggregates can enter the cytoplasms of many
different types of cultured cells, and cell surface proteins are
only partially responsible for this entry. It is likely that the
discrepancy between these previous studies and the current one at
least in part reflects differences in the extracellular
environment in cell culture, which is homogeneous and effectively
infinite, and in the intact brain, where extracellular space is
severely restricted in volume and is continuously patrolled by
phagocytic glia. This view is supported by observations that, in
the fly brain, aggregate uptake by glia occured only in close
proximity to ORN axons containing HttQ91 aggregates and that
detergent solubilization was required to immunolabel the
aggregates. This study therefore favors a mechanism in which
HttQ91 aggregates are phagocytosed by glia together with
surrounding axonal membrane, analogous to the process by which
supernumerary synapses are eliminated by Draper in Drosophila
development and by the mammalian Draper orthologue, MEGF10, in the
adult mouse brain (Pearce, 2015).
The observation that phagocytosed neuronal HttQ91 aggregates could
nucleate aggregation of cytoplasmic HttQ25 in glia is surprising
because phagocytosis normally leads to encapsulation and
degradation of internalized debris within the membrane-enclosed
phagolysosomal system. However, two independent lines of evidence
were provided to support the conclusion that ORN-derived HttQ91
aggregates encounter HttQ25 in the glial cytoplasm. First, because
HttQ25 is a highly soluble protein that does not aggregate in
cells unless seeded by a pre-existing aggregate, the strict
dependence of HttQ25 puncta formation on HttQ91 expression in ORNs
indicates that these two Htt species must have physically
interacted with one another. In principle, it is possible that,
instead of HttQ91 aggregates being internalized by glia, this
transfer could occur in the opposite direction, namely by the
transfer of soluble HttQ25 into ORNs. However, the finding that
HttQ25 aggregation was blocked by glial-specific knockdown of
Draper and enhanced by antennal injury indicates an absolute
requirement for phagocytic uptake of HttQ91, supporting
ORN-to-glia transfer (Pearce, 2015).
Second, the majority of HttQ91 and HttQ25 aggregates co-localized
with the cytoplasmic chaperones Hsp70/Hsc70 and Hsp90, indicating
that aggregated HttQ25 is in direct contact with the cytoplasm. It
is conceivable that merging of the phagocytic and autophaghic
pathways could provide an opportunity for HttQ91 aggregates and
soluble HttQ25 to encounter one another. However, substantial
(<15%) co-localization of either HttQ91 or HttQ25 puncta with
the autophagy markers, Atg8
and p62,
or with the early endosome marker, Rab5
was not detected. Moreover, the amount of soluble HttQ25 that
could be captured within an autophagosome was miniscule compared
with the total cytoplasmic pool that would be available to be
nucleated by an internalized HttQ91 seed. Altogether, these data
strongly support the conclusion that phagocytosed HttQ91
aggregates are able to access the glial cytoplasm, thereby
affording the opportunity to effect prion-like spreading of
disease pathology (Pearce, 2015).
How do these engulfed HttQ91 aggregates breach the membrane
barrier that separates the phagolysosomal lumen from the
cytoplasm? The dependence of this process on Draper, shark
and the actin-remodelling complex indicates that aggregates must
access the glial cytoplasm at a step during or subsequent to
phagocytic engulfment. It is possible that cytoplasmic entry can
be facilitated by interference of phagocytosed aggregates with
membrane fusion events during phagosome maturation. However, this
‘foot-in-the-door’ mechanism would be favoured by
larger aggregates, which is in opposition to the finding that
smaller HttQ91 aggregates were more strongly associated with
cytoplasmic nucleation of glial HttQ25. It is proposed instead
that slow or inefficient completion of membrane fusion events
could provide a temporary conduit to the cytoplasm for HttQ91
aggregates. Such a mechanism would predict an upper size limit for
cytoplasmic entry that could be exploited by small, newly formed
aggregates. This prediction was supported by the finding that
neither the induced HttQ25 aggregates nor the co-localized
nucleating HttQ91 puncta in glia were labelled with antibodies to
ubiquitin, a marker previously identified with more mature, larger
Htt puncta in both cell culture and transgenic mouse models of HD.
(Pearce, 2015).
The findings described in this study have broader implications
for how potentially toxic protein aggregates are dispersed
throughout the diseased brains. Phagocytic removal of aggregates
is neuroprotective, but it is likely that phagocytes become
impaired in their ability to clear debris as the disease worsens
and that chronic glial activation becomes detrimental to the
health of nearby neurons. The finding that phagocytosed neuronal
Htt aggregates can enter the glial cytoplasm suggests that this
transfer process could generate a reservoir of prion-like species
inside glia, possibly facilitating their spread to other cells. A
growing body of evidence supports the view that glial dysfunction
exacerbates neurodegenerative disease pathogenesis by influencing
the survival of neurons, and determining the mechanism(s) by which
glia contribute to toxicity will be of great value to the
development of therapeutic strategies to combat these devastating
disorders (Pearce, 2015).
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White, J. A., Anderson, E., Zimmerman, K., Zheng, K. H., Rouhani, R. and Gunawardena, S. (2015). Huntingtin differentially regulates the axonal transport of a sub-set of Rab-containing vesicles in vivo. Hum Mol Genet 24(25): 7182-7195. PubMed ID: 26450517
Abstract
Loss of huntingtin (HTT), the Huntington's disease (HD) protein, was previously shown to cause axonal transport defects. Within axons, HTT can associate with kinesin-1 and dynein motors either directly or via accessory proteins for bi-directional movement. However, the composition of the vesicle-motor complex that contains HTT during axonal transport is unknown. This study analyzed the in vivo movement of 16 Rab GTPases within Drosophila larval axons and showed that HTT differentially influences the movement of a particular sub-set of these Rab-containing vesicles. While reduction of HTT perturbed the bi-directional motility of Rab3 and Rab19-containing vesicles, only the retrograde motility of Rab7-containing vesicles was disrupted with reduction of HTT. Interestingly, reduction of HTT stimulated the anterograde motility of Rab2-containing vesicles. Simultaneous dual-view imaging revealed that HTT and Rab2, 7 or 19 move together during axonal transport. Collectively, these findings indicate that HTT likely influences the motility of different Rab-containing vesicles and Rab-mediated functions. These findings have important implications for understanding of the complex role HTT plays within neurons normally, which when disrupted may lead to neuronal death and disease (White, 2015).
This study has identified a novel role for HTT in the regulation of the axonal transport of a particular sub-set of Rab-containing vesicles under physiological conditions. In vivo observations have led to two major findings; (1) HTT differentially regulates the movement of a specific sub-set of Rab-containing vesicles within axons, and (2) HTT is present on these Rab-containing vesicles during axonal transport. At least two possible mechanisms could exist by which HTT exerts a differential control on Rab motility, (1) by associations with specific Rab-containing vesicles, and/or (2) by regulating the motors on moving Rab-containing vesicles, although these two pathways may not be mutually exclusive. Collectively, these findings provide new insight into the normal physiological role of HTT which, when disrupted, may contribute to disease pathology observed in HD (White, 2015).
Different Rab-containing vesicles, even those within the same sub-cellular compartment, move at varying velocities, suggesting that Rab proteins found in the same compartment are differentially regulated. Indeed, different regulatory mechanisms could exist as Rab proteins control trafficking in both the secretory and endocytic pathways. Some Rab proteins are in distinct sub-sets of neurons suggesting that Rabs have roles in diversely regulated mechanisms, and HTT may function to differentially regulate the motility of these neuronal Rab proteins via Rab protein specific associations. Previously, work found that reduction of HTT perturbed the motility of Rab11-containing vesicles but not Rab5-containing vesicles. Since Rab11 is a marker for recycling endosomes and Rab5 is a marker for early endosomes, it is hypothesized that HTT influences the axonal motility of all recycling endosomes. Contrary to this, however, the current systematic in vivo analysis found that HTT does not influence the motility of all Rab proteins found in recycling endosomes, but rather, HTT only affects the movement of particular Rab proteins located in many different compartments. Reduction of HTT function perturbed the bi-directional motility of Rab19, a recycling endosomal Rab, while no effect was seen in the motility of other recycling endosomal Rab proteins. Additionally, reduction of HTT perturbed the bi-directional motility of Rab3, a Rab protein known to be present in synaptic vesicles. Intriguingly, reduction of HTT perturbed the retrograde movement of Rab7 (present on late endosomes), while the anterograde movement of Rab2 (present in ER-Golgi associated compartments) was stimulated by reduction of HTT. Perhaps this differential regulation that is observed with reduction of HTT may be caused by the existence of different HTT-Rab-containing motor complexes. Either several HTT-Rab-containing vesicle complexes may exist or alternatively more than one Rab could be present with a single HTT-Rab-containing vesicle. Perhaps, during long distance transport within axons, HTT-mediated regulation of specific Rab-containing vesicles is required for particular functions at the synapse. Indeed, similar to many Rab proteins, roles for HTT in endocytosis, intracellular trafficking and membrane recycling have also been proposed (White, 2015).
Specific associations between HTT, Rab proteins and linker proteins could perhaps dictate one potential mechanism by which HTT-mediated differential regulation of Rab-containing vesicle motility occurs. It is thought that associations between HTT and motors are facilitated by HTT associated proteins (HAPs). Pal (2006). showed that HTT can mediate the transport of a Rab5-HAP40-HTT-containing early endosome on actin filaments via associations with myosin, the actin motor. HTT can also interact with myosin via optineurin. Optineurin is a binding partner that links both myosin and HTT to the Golgi network via Rab8 for ER-Golgi trafficking in the secretory pathway. HTT can also associate with Rab8 through FIP-2 for regulated cell polarization and morphogenesis. Since reduction of HTT altered the sub-cellular localization of Rab8 the current observations suggest that HTT can play a role in linking Rab8 to vesicles; enabling associations with MT motors during axonal transport. While the involvement of optineurin or FIP-2 in the association of HTT and Rab8 in the context of axonal movement is still unclear, what is clear is that HTT is likely required for the membrane-bound state of Rab8 during axonal transport under physiological conditions (White, 2015).
It has been proposed that a HTT-Rab11-motor complex likely exists during axonal transport. The motility of Rab11-containing vesicles was perturbed with reduction of HTT. Both kinesin-1 and dynein motors were required for MT motility of Rab11. Additionally, membrane binding of Rab11 was decreased in HD knock-in mice, suggesting that similar to Rab8, HTT is also likely required for the membrane-bound state of Rab 11. The Rab11 effector Rip11 regulates the endocytic recycling pathway by forming a complex with Rab11 and kinesin II. Rip11 is also important for the trafficking of Rab11 from apical recycling endosomes to the apical membrane. Perhaps Rip11 may act as a linker that connects Rab11 and HTT similar to optineurin linking Rab8 and HTT. Rabphilin-3A, a Rab3 effector molecule may link Rab3 vesicles to HTT during axonal transport. Studies have shown that Rab3 and Rabphilin-3A are both transported by fast anterograde transport and associate with synaptic vesicles. Thus, although further study is needed, Rab-associated proteins could aid in linking specific Rab-containing vesicles with HTT during axonal transport (White, 2015).
Alternatively, HTT-mediated differential regulation of Rab protein motility could result due to changes in motor protein regulation. Indeed, previous work postulated HTT as a molecular switch that determines the direction of movement during axonal transport. HTT is phosphorylated by Akt (protein kinase B) (a serine-threonine kinase) at serine 421. Constitutively phosphorylated (S421D) HTT can recruit kinesin-1 to the dynactin complex to facilitate anterograde transport while disruption of phosphorylation at S421 (S421A) prevents kinesin association with HTT and the motor complex, enabling retrograde transport. Perhaps HTT's role as a molecular regulator during axonal transport could result in the HTT-mediated motility changes were observe in this study, since reduction of HTT not only perturbed the bi-directional motility of Rab3 and 19, and the retrograde motility of Rab7, but also stimulated the anterograde motility of Rab2, via specific changes to motility parameters; vesicle velocities, pause duration/frequencies and run lengths. While the functional significance of the differential regulation of Rab motility and the mechanistic steps of how HTT controls motors in the context of the different Rab-containing complexes are still unclear, perhaps particular Rab proteins could also exert a regulatory function during vesicle motility by affecting the phosphorylation state of HTT and changing the direction of vesicle movement. Interestingly, several Rabs have been shown to be effectors of kinases. Rab5 and Rab7 are thought to be effectors of PI3K, which is an upstream activator of Akt. Additionally, it has been shown that HTT can act as a scaffold to transport glycolytic machinery down the axon that is required for vesicular motility. Reduction of HTT could decrease glycolysis disrupting the motility of Rab-containing vesicles. Further experiments will be needed to dissect the mechanistic steps involved in the differential regulation of these different HTT-Rab-containing complexes during axonal transport under physiological conditions (White, 2015).
An intriguing result from this analysis was that reduction of HTT influenced the retrograde transport of Rab7, although Rab7-containing vesicles moved bi-directionally. Previous work has implicated Rab7 in neurotrophin receptor trafficking, particularly in the retrograde transport of TrkB/p75NTR-positive signaling endosomes in motor neurons. Consistent with this, CMT2B Rab7 mutants altered trafficking and signaling of retrograde NGF/TrkA trophic signal. Thus, since HTT and Rab7 co-localize on moving vesicles during axonal transport a HTT-Rab7-containing signaling endosome could exist during axonal transport. Alternatively, since Rab7 is a marker for late endosomal and lysosomal compartments, and HTT and dynein were found to be required for the perinuclear positioning of lysosomes, perhaps a HTT-Rab7-containing lysosome could exist during axonal transport. Work has also shown that Rab7 and LC3 (a marker for autophagosomes) are together during the transport of autophagosomes at growth cones and that the retrograde movement of autophagosomes is required for their maturation. Interestingly Rab7 interacting lysosomal protein (RILP) was shown to control lysosomal transport by recruiting dynein-dynactin to Rab7-containing late endosomes/lysosomes. The FYVE (Fab1-YotB-Vac1p-EEA1) and coiled-coil domain-containing 1 protein (FYCO1) was found to function as an adaptor to link autophagosomes to kinesin via Rab7. Additionally, both HTT and HAP1 were identified as regulators of autophagosome transport in neurons. Thus, the current results are consistent with these observations and suggest that perhaps a HTT-Rab7-authophagosome complex and/or a HTT-Rab7-signaling endosomal complex could exist under physiological conditions(White, 2015).
Surprisingly this analysis also revealed that reduction of HTT stimulated the anterograde velocity of Rab2, although Rab2-containing vesicles moved bi-directionally. Rab2 is known to regulate the anterograde and retrograde trafficking of vesicles between the Golgi, the ER-Golgi intermediate compartment and the ER. Rab2 was also one of the Rab proteins that showed the most neuronal sub-cellular localization behaviors: synaptic enrichment with expression of a CA form and loss of synaptic localization with the dominant negative form , suggesting a role for Rab2 at the synapse. While roles for HTT at synapses have been documented, perhaps HTT may function to regulate the anterograde motility of a Rab2-containing complex, although the functional significance for this complex at the synapse is still unknown (White, 2015).
Rab dysfunction has been implicated in many neuronal diseases. For example, a missense mutation in Rab7 was demonstrated in the myelin and axonal disorder Charcot-Marie Tooth disease Type 2B. Altered expression of Rab1, Rab8, and Rab2 was shown to cause Golgi fragmentation in Parkinson's disease. Expansion of a hexanucleotide repeat in C9ORF72, a Rab-associated GEF, was seen in both Amyotrophic Lateral Sclerosis (ALS) and Fronto-Temporal Dementia (FTD), suggesting that this mutant form of the GEF may contribute to the physiology of the disease through Rab dysfunction. Defects in the recycling of Rab7 from lysosomes to early endosomes impaired the transport and degradation of amyloid beta (Aβ) in Alzheimer's disease (AD). Rab6 was shown to modulate the unfolded protein response due to ER stress in AD. Interestingly, defects in Rab11 function were recently observed in HD. Expression of Rab11 was decreased in HD mouse models, and Rab11 activation was impaired by mutant HTTA. Over expression of Rab11 rescued neurodegeneration, dendritic spine loss, synaptic defects and behavioral defects in HD models in both mice and Drosophila. Perhaps defects to HTT-mediated axonal transport of a specific sub-set of Rab-containing vesicles could contribute to neurodegeneration and synaptic defects observed in HD. Thus this work could highlight a potential novel therapeutic pathway for early treatment of HD pathology (White, 2015).
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Maheshwari, M., Bhutani, S., Das, A.,
Mukherjee, R., Sharma, A., Kino, Y., Nukina, N. and Jana, N.R.
(2014). Dexamethasone induces heat shock response and slows down
disease progression in mouse and fly models of Huntington's
disease. Hum Mol Genet 23: 2737-2751. PubMed ID: PubMed
Abstract
Huntington's disease (HD) is an inherited neurodegenerative
disorder caused by abnormal expansion of glutamine repeats in the
protein huntingtin. In HD brain, mutant huntingtin undergoes
proteolytic processing, and its N-terminal fragment containing
poly-glutamine repeats accumulate as insoluble aggregates leading
to the defect in cellular protein quality control system and heat
shock response (HSR). This study demonstrates that the defective
HSR in the brain is due to the down-regulation of heat shock
factor 1 (HSF1) in both mice and fly models of HD. Interestingly,
treatment of dexamethasone (a synthetic glucocorticoid) to HD mice
or flies significantly increases the expression and
transactivation of HSF1 and induction of HSR and these effects are
mediated through the down-regulation of HSP90. Dexamethasone
treatment also significantly decreases the aggregate load and
transient recovery of HD-related behavioural phenotypes in both
disease models. These results suggest that dexamethasone could be
a potential therapeutic molecule for the treatment of HD and
related poly-glutamine disorders (Maheshwari, 2014).
Highlights
- HSF1 is reduced in HD mice brain.
- HSF1 is induced upon dexamethasone treatment.
- Dexamethasone treatment induces transactivation of HSF1 and
up-regulation of HSP70.
- Dexamethasone treatment reduces aggregate load and improves
motor performance of HD mice.
- Effects of dexamethasone in HD are recapitulated in a fly
model.
- Dexamethasone treatment down-regulates HSP90.
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Melkani, G.C., Trujillo, A.S., Ramos,
R., Bodmer, R., Bernstein, S.I. and Ocorr, K. (2013).
Huntington's disease induced cardiac amyloidosis is reversed by
modulating protein folding and oxidative stress pathways in the Drosophila
heart. PLoS Genet 9: e1004024. PubMed ID: 24367279
Abstract
Amyloid-like inclusions have been associated with Huntington's
disease (HD), which is caused by expanded polyglutamine repeats in
the Huntingtin protein. HD patients exhibit a high incidence of
cardiovascular events, presumably as a result of accumulation of
toxic amyloid-like inclusions. This study generated a Drosophila
model of cardiac amyloidosis that exhibits accumulation of PolyQ
aggregates and oxidative stress in myocardial cells upon
heart-specific expression of Huntingtin protein fragments
(Htt-PolyQ) with disease-causing poly-glutamine repeats (PolyQ-46,
PolyQ-72, and PolyQ-102). Cardiac expression of GFP-tagged
Htt-PolyQs results in PolyQ length-dependent functional defects
that include increased incidence of arrhythmias and extreme
cardiac dilation, accompanied by a significant decrease in
contractility. Structural and ultrastructural analysis of the
myocardial cells reveals reduced myofibrillar content,
myofibrillar disorganization, mitochondrial defects and the
presence of PolyQ-GFP positive aggregates. Cardiac-specific
expression of disease causing Poly-Q also shortens lifespan of
flies dramatically. To further confirm the involvement of
oxidative stress or protein unfolding and to understand the
mechanism of PolyQ induced cardiomyopathy, expanded PolyQ-72 was
co-expressed with the antioxidant superoxide
dismutase (SOD) or the myosin chaperone UNC-45.
Co-expression of SOD suppresses PolyQ-72 induced mitochondrial
defects and partially suppresses aggregation as well as
myofibrillar disorganization. However, co-expression of UNC-45
dramatically suppresses PolyQ-72 induced aggregation and partially
suppresses myofibrillar disorganization. Moreover, co-expression
of both UNC-45 and SOD more efficiently suppresses GFP-positive
aggregates, myofibrillar disorganization and physiological cardiac
defects induced by PolyQ-72 than did either treatment alone. The
study demonstrates that mutant-PolyQ induces aggregates, disrupts
the sarcomeric organization of contractile proteins, leads to
mitochondrial dysfunction and increased oxidative stress in
cardiomyocytes leading to abnormal cardiac function. The study
concludes that modulation of both protein unfolding and oxidative
stress pathways in the Drosophila heart model can
ameliorate the detrimental PolyQ effects, thus providing unique
insights into the genetic mechanisms underlying amyloid-induced
cardiac failure in HD patients (Melkani, 2013).
Highlights
- Expression of disease causing PolyQ in the heart
causes cardiac dilation and reduced contractility.
- Cardiac expression of disease-causing PolyQ causes
accumulation of aggregates and myofibrillar defects.
- Ultrastructural analysis revels mitochondrial and myofibrillar
defects upon expression of mutant PolyQ.
- Oxidative stress plays a role in PolyQ-induced cardiac
defects.
- Transgenic over-expression of SODs rescues the Poly-Q induced
cardiac mitochondrial and functional defects.
- Over-expression of the chaperone UNC-45 suppresses Poly-Q
induced cardiomyopathy.
- Combined UNC-45 chaperone and antioxidant treatment is
required for efficient suppression of PolyQ-induced aggregation
and cardiac defects.
Discussion
Huntingtin protein is expressed in many tissues including the
heart and epidemiological studies suggest that HD patients have a
higher susceptibility to cardiac failure compared to age-matched
controls without HD. However, the cellular mechanisms underlying
the cardiac dysfunction in HD have yet to be studied in the heart.
Using the genetically tractable model system Drosophila,
this study shows a direct correlation between the levels of
amyloid accumulation, overall ROS production and the severity of
cardiac dysfunction. Cardiac-specific expression of
disease-causing Htt-PolyQ (PolyQ-46, PolyQ-72 and PolyQ-102) all
elicit cardiac dysfunction compared to hearts expressing the
non-disease-causing PolyQ-25. In addition the qualitative as well
as quantitative defects that were observed in response to PolyQ
expression were dose-dependent. Since mutant Htt-PolyQ protein was
expressed specifically in the heart, it is unlikely that there is
a neuronal contribution to these cardiac defects. It was suggested
that the increased risk of cardiac disease in HD patients is
possibly due to cardiac amyloid accumulation, mitochondrial
defects as well as oxidative stress and that the severity of
disease depended upon the length of the PolyQ repeat (Melkani,
2013).
The likely cause of the observed functional defects was
demonstrated to be severe myofibrillar disorganization and reduced
myosin and actin content in myocardial cells resulting from
cardiac-specific expression of disease causing PolyQ. It has
earlier been shown that the chaperone UNC-45 is required for
preserving myosin accumulation/folding in Drosophila
cardiomyocytes, as its reduction leads to severe disorganization
of myosin-actin containing myofibrils and thus sarcomeres. This
study extended this observation and demonstrated a role for UNC-45
in amyloidosis-induced cardiac defects for the first time. In
support of this hypothesis, it was previously shown that nuclear
or cytoplasmic aggregates (inclusion bodies) of polyglutamine
proteins contained chaperones involved in protein folding.
Furthermore, and consistent with this study's results,
over-expression of the chaperone αB-crystallin reduced
PolyQ-induced aggregation in rat neonatal cardiomyocytes; however,
over-expression of αB-crystallin enhanced amyloid oligomer
formation and toxicity (Melkani, 2013).
Co-over-expression of UNC-45 with disease-causing PolyQ-72
dramatically reduces amyloid aggregate density and ameliorates
cardiac dysfunction by decreasing the incidence of cardiac
arrhythmia, suppressing the mutant-Htt-induced cardiac dilation
and improving cardiac contractility to a dramatic extent.
Importantly, over-expression of UNC-45 in the presence of PolyQ-72
restores myosin-containing myofibrils, suggesting that one effect
of amyloid aggregation is to interfere with proper folding of
muscle myosin in cardiomyocytes (Melkani, 2013).
The fact that UNC-45 over-expression does not completely suppress
the mutant Htt-PolyQ-induced cardiac physiological defects and
lifespan reduction is consistent with the idea that amyloid
accumulation affects additional cellular pathways that result in
cardiac abnormalities. As reported for αB-crystallin,
suppression of aggregates is not sufficient to reduce toxicity and
this possibility may also exist in the case of UNC-45. It is also
possible that the overall high level of oxidative stress produced
by mutant PolyQ is the main determinant for lethality. Expression
of mutant PolyQ leads to mitochondrial defects due to increased
oxidative stress. Several neuronal studies have shown that
expression of mutant polyQ affects SOD expression. Manipulation of
SOD seems to be directly correlated with levels of oxidative
stress in several neurodegenerative diseases. Additionally, SOD
over-expression reduces diabetic cardiomyopathy and some forms of
neurodegeneration by reducing oxidative stress. However, neither
UNC-45 nor SOD has been shown previously to suppress the
PolyQ-induced phenotypes in either neuronal or cardiac animal
disease models (Melkani, 2013).
Data from this study also support a role for oxidative stress
pathways in amyloid-induced cardiac dysfunction and lethality.
Treatment with oxidants aggravates the moderate effects of
PolyQ-46 on heart function, causing an increase in amyloid
aggregate density and more severe cardiac defects. This suggests a
possibly causal relationship between oxidative stress, the
formation of aggregates and cardiac dysfunction. Furthermore,
ultrastructural analysis clearly shows mutant PolyQ-induced
mitochondrial defects, while DHE staining indicates that excess
ROS production occurrs upon expression of mutant PolyQ.
Interestingly, some of the PolyQ aggregates co-localize with
concentrated DHE staining. Significantly, the size and density of
mutant PolyQ-aggregates as well as the severity of the
PolyQ-72-induced cardiac defects are reduced by over-expression of
SOD or by feeding the anti-oxidant resveratrol. This is consistent
with findings that resveratrol provides protection in neuronal
models of Huntington's disease. Interestingly, the anti-oxidant
resveratrol has been shown to affect expression of anti-oxidative
enzymes, including enhanced expression of SOD-1 (Melkani, 2013).
Expression of mutant PolyQ may both induce oxidative stress and
interfere with protein folding pathways. A study using cultured
mouse neurons showed that oxidative stress increases PolyQ
aggregation and that over-expression of SOD1 in conjunction with
the chaperone HSP-70/HSP-40 could suppress Htt-polyQ-induced
aggregation and toxicity. However, simultaneous manipulation of
both of these genetic pathways has not previously been attempted
in vivo. In addition to neurons, expression of the mutated Htt
protein or expression of pre-amyloid oligomers cause cardiac
defects by affecting several pathways including oxidative stress,
mitochondrial abnormalities, presence of protein aggregates and
increased autophagosomal content. However, no attempt has thus far
been made to suppress PolyQ-induced cardiac defects, a crucial
step for understanding the mechanistic basis of disease
progression and amelioration. Indeed, co-expression of UNC-45 and
SOD-1 or expression of UNC-45 in the presence of resveratrol had a
tendency to suppress the PolyQ-72-induced amyloid aggregation and
concomitant cardiac dilation more efficiently than either
treatment alone. Thus, suppression of both protein aggregates and
ROS may be required for the amelioration of PolyQ-induced
cardiomyopathy. As HD is primarily a neurological disease, the
effect of such suppression is worth exploring in neural tissues.
(Melkani, 2013).
In addition to interfering with protein folding pathways,
expression of mutant PolyQ may lead to myofibril loss by directly
interacting with muscle proteins. Previous studies have suggested
that mutant PolyQ may bind directly to contractile proteins and
disturb their function. Integrity of contractile proteins is also
required for maintaining mitochondrial organization and
cardiomyocyte function. Additionally, expression of
aggregation-prone mutant PolyQ may induce oxidative stress due to
mitochondrial damage in the cardiomyocytes, which are heavily
dependent on mitochondrial function and are vulnerable to
oxidative stress. For example, knockdown of SOD results in
mitochondrial defects and severe dilated cardiomyopathy phenotype
in a mouse model. In this study, a dramatic increase in overall
ROS levels in mutant PolyQ expressing hearts was observed.
Moreover, the GFP-positive PolyQ aggregates co-localized with
areas of strong DHE staining and the observation that antioxidant
treatments partially rescued the cardiac defects further support
this hypothesis (Melkani, 2013).
Overall, accumulation of amyloid in the cardiomyocytes could
induce mechanical deficits by affecting the integrity of
contractile proteins as well as mitochondria and lead to
cardiomyocyte death, possibly through activation of autophagy.
Consistent with data from this study, a similar mechanism has been
proposed for cardiomyopathy associated with amyloid producing
mutant αB-crystallin. Both mutant αB-crystallin and
mutant PolyQ caused aggregate formation in cardiomyocytes
suggesting a common mechanism for underlying cardiomyocyte
degeneration. It is unclear at this point whether the presence of
toxic aggregates in cardiomyocytes is directly interfering with
mitochondrial organization leading to cardiac defects or whether
oxidative stress produced by mutant PolyQ leads to mitochondrial
dysfunction that triggers cardiomyocyte dysfunction (Melkani,
2013).
A full understanding of all the molecular details involved in
mutant PolyQ induced cardiomyopathy will require additional study
but some of the key players and interactions in vivo have been
identified. Although dense granular deposits, immunoreactive to an
anti-Huntingtin antibody, have been found in muscle tissue from an
HD patient, no such study has been performed on HD heart biopsy
samples. Thus, this study suggests that it would be useful to look
for accumulation of amyloid protein in the hearts of HD patients,
especially those with heart disease. Delineating how these
aggregates might be toxic to cells will be critical not only for
an understanding of PolyQ-induced cardiomyopathy but also for
gaining insights into aggregation-based neural degeneration. The Drosophila
heart model provides a genetically tractable system whereby these
interactions can be examined in the context of a functioning
organ. Indeed, elucidating the genetics underlying PolyQ-induced
cardiomyopathy should also have an impact on our understanding of
other cardiac diseases associated with oxidative stress,
mitochondrial dysfunction, the unfolded
protein response and proteostasis in general (Melkani,
2013).
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Mason, R.P., Casu, M., Butler, N.,
Breda, C., Campesan, S., Clapp, J., Green, E.W., Dhulkhed, D.,
Kyriacou, C.P. and Giorgini, F. (2013). Glutathione
peroxidase activity is neuroprotective in models of Huntington's
disease. Nat Genet 45: 1249-1254. PubMed ID: 23974869
Abstract
Huntington’s disease (HD) is a fatal neurodegenerative
disorder caused by a CAG repeat expansion encoding a polyglutamine
tract in the huntingtin (htt) protein. This study reports a
genome-wide overexpression suppressor screen which identified 317
open reading frames that ameliorate toxicity of a mutant htt
fragment in yeast, and play roles in diverse cellular processes,
including mitochondrial import and copper metabolism. Two
suppressors encode glutathione peroxidases (GPxs), conserved
antioxidant enzymes that reduce hydrogen peroxide and lipid
hydroperoxides. Using genetic and pharmacological approaches in
yeast, mammalian cells and Drosophila, it was found that
GPx activity robustly ameliorates HD-relevant metrics and is more
protective than other antioxidant approaches tested. Importantly,
it was observed that GPx activity – unlike many antioxidant
treatments – does not inhibit autophagy, an important
mechanism for clearing mutant htt. As previous clinical trials
indicate GPx mimetics are well-tolerated in humans, this study may
have important implications for the treatment of HD (Mason, 2013).
Highlights
- Suppression of Htt103Q toxicity in yeast by ORF
overexpression.
- mGpx1 and ebselen improve HD-relevant phenotypes in PC12
cells.
- mGpx1 and ebselen ameliorate phenotypes in HD flies.
- Ebselen does not inhibit basal or induced autophagy in PC12
cells.
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Bodai, L., Pallos, J., Thompson, L.M.
and Marsh, J.L. (2012). Pcaf modulates polyglutamine
pathology in a Drosophila model of Huntington's disease.
Neurodegener Dis 9: 104-106. PubMed ID: 21912091
Abstract
Huntingtin peptides with elongated polyglutamine domains, the root
causes of Huntington's disease, hinder histone acetylation, which
leads to transcriptional dysregulation. However, the range of
acetyltransferases interacting with mutant Huntingtin has not been
systematically evaluated. This study used genetic interaction
tests in Drosophila to determine whether specific
acetyltransferases belonging to distinct protein families
influence polyglutamine pathology. It was found that flies
expressing a mutant form of the Huntingtin protein (Httex1pQ93)
exhibit reduced viability, which was further decreased by partial
loss of Pcaf or nejire, while the tested MYST
family acetyltransferases do not affect pathology. Reduced levels
of Pcaf also lead to the increased degeneration of
photoreceptor neurons in the retina. Overexpression of Pcaf,
however, was not sufficient to ameliorate these phenotypes, and
the level of soluble Pcaf was unchanged in Httex1pQ93-expressing
flies. These results indicate that while Pcaf has a significant
impact on Huntington's disease pathology, therapeutic strategies
aimed at elevating its levels are likely to be ineffective in
ameliorating Huntington's disease pathology; however, strategies
that aim to increase the specific activity of Pcaf remain to be
tested (Bodai, 2012).
Highlights
- Mutant Huntingtin does not reduce the level of soluble Pcaf.
Discussion
To investigate the consequences of reduced HAT activity on polyQ
pathology, this study used a Drosophila model of HD,
which was previously shown to be sensitive to acetylation levels.
The phenotypes of flies that expressed Httex1pQ93 in the nervous
system were compared with their siblings that, in addition,
carried a HAT mutation as well. It was found that partial loss of
Pcaf (the single fly homolog of human Pcaf and Gcn5) or nejire/dCBP
(nej, homolog of human CBP) by the PcafE333St
or nej3 mutations, respectively, significantly reduces
the viability of Htt-expressing flies, while reducing the MYST
family acetyltransferases enok,
mof
or CG1894
did not have a significant effect on pathology (Bodai, 2012).
Since the role of CBP is well established in HD pathogenesis, the
effect of Pcaf was investigated. First, it was tested
whether Df(3L)iro-2, an independent deletion that
removed the Pcaf gene but did not share the same genetic
background as the PcafE333St null allele, has the same
effect. It was found that Htt-expressing flies heterozygous for Df(3L)iro-2
also exhibit significantly reduced viability. By comparing
Htt-expressing control flies with siblings expressing Htt and also
carrying the PcafE333St allele or the Df(3L)iro-2
deletion, it was found that the average number of rhabdomeres
(light gathering structures of photoreceptor neurons) per
ommatidium are decreased indicating that reduced Pcaf
levels enhance neurodegeneration (Bodai, 2012).
It was also found that partial loss of Pcaf
significantly enhances polyglutamine pathology, but the level of
Pcaf was not reduced by mutant Htt, and HD phenotypes could not be
rescued by the overexpression of Pcaf, indicating that
the interaction of mutant Htt and Pcaf does not involve the
depletion of soluble Pcaf by either degradation or sequestration
to insoluble aggregates. This result, however, does not exclude
the possibility that a soluble toxic form of Htt may inhibit the
function of either Pcaf itself or of Pcaf-containing complexes.
Since Pcaf acts as a catalytic subunit in large multiprotein
complexes in metazoans, it is speculated that its interaction with
a polyQ peptide may cripple entire complexes, which can not be
rescued by overexpression of Pcaf alone (Bodai, 2012).
The study concludes that although Pcaf has a
significant impact on HD pathology, therapeutic strategies aimed
at elevating the levels of Pcaf protein are unlikely to be
effective in ameliorating HD pathology. The question, however,
remains open whether strategies that aim to increase the specific
activity of Pcaf might be useful. Interestingly, of the three HAT
families of proteins tested, only the GNAT and the CBP/p300
families exhibit a strong influence over HD pathology, while the
MYST family members have decidedly less impact (Bodai, 2012).
Go to top
Campesan, S., Green, E.W., Breda, C.,
Sathyasaikumar, K.V., Muchowski, P.J., Schwarcz, R., Kyriacou,
C.P. and Giorgini, F. (2011). The kynurenine pathway
modulates neurodegeneration in a Drosophila model of
Huntington's disease. Curr Biol 21: 961-966. PubMed ID: 21636279
Abstract
Neuroactive metabolites of the kynurenine pathway (KP) of
tryptophan degradation have been implicated in the pathophysiology
of neurodegenerative disorders, including Huntington's disease
(HD). A central hallmark of HD is neurodegeneration caused by a
polyglutamine expansion in the huntingtin (htt) protein. This
study exploits a transgenic Drosophila melanogaster
model of HD to interrogate the therapeutic potential of KP
manipulation. It was observed that genetic and pharmacological
inhibition of kynurenine 3-monooxygenase (KMO) increases levels of
the neuroprotective metabolite kynurenic acid (KYNA) relative to
the neurotoxic metabolite 3-hydroxykynurenine (3-HK) and
ameliorates neurodegeneration. It was also found that genetic
inhibition of tryptophan 2,3-dioxygenase (TDO), the first and
rate-limiting step in the pathway, leads to a similar
neuroprotective shift toward KYNA synthesis. Importantly, it was
demonstrated that the feeding of KYNA and 3-HK to HD model flies
directly modulates neurodegeneration, underscoring the causative
nature of these metabolites. This study provides the first genetic
evidence that inhibition of KMO and TDO activity protects against
neurodegenerative disease in an animal model, indicating that
strategies targeted at two key points within the KP may have
therapeutic relevance in HD, and possibly other neurodegenerative
disorders (Campesan, 2011).
Highlights
- HD model flies exhibit perturbed flux through the KP.
- cinnabar and vermillion mutations are
neuroprotective in HD model flies.
- KMO inhibitors ameliorate neurodegeneration in HD model flies.
- 3-HK and KYNA modulate neurodegeneration.
- Genetic manipulation of the KP modulates neurodegeneration.
Discussion
This study observed that both genetic and chemical inhibition of
KMO ameliorates neurodegeneration in HD model flies, supporting
previous observations that inhibition of KMO is protective in a
yeast model of mutant htt toxicity. It was also found that this
neuroprotection correlates with decreases in 3-HK relative to
KYNA. Importantly, this protection could be abrogated by 3-HK
feeding, showing the causative nature of this metabolite.
Furthermore, a KYNA feeding regime that forced a neuroprotective
ratio of these metabolites also reduced neurodegeneration in HD
flies. Therefore, it was shown for the first time by this study
that KP metabolites directly modulate neurodegeneration in an
animal model of neurodegenerative disease. Finally, genetic
dissection of the KP with vermillion and cardinal
mutants further underscores the critical relationship between
imbalances in the KP and neurodegeneration (Campesan, 2011).
3-HK generates free radicals by auto-oxidation, and this mechanism
is believed to underlie 3-HK-dependent toxicity observed in
neuronal cell lines and in primary neurons. It is therefore likely
that 3-HK was contributing to neurodegeneration by generation of
free radicals in experminents in this study. At endogenous
concentrations in the mammalian brain, KYNA functions as a
competitive inhibitor of the glycine coagonist site of the NMDA
receptor and a potent, noncompetitive inhibitor of the α7
nicotinic acetylcholine receptor. KYNA treatment results in
dose-dependent behavioral changes in rodents, mimicking properties
of NMDA receptor antagonists. Moreover, KYNA is neuroprotective
against excitotoxic lesions and inhibits the release of glutamate
at concentrations in the physiological range. In another study
that used a novel KMO inhibitor, it was found that glutamate
reduction by KYNA is the likely mechanism for neuroprotection in
the central nervous system (CNS) of HD mice. Because flies express
orthologs of both NMDA and α7 nicotinic acetylcholine
receptors, KYNA could be conferring neuroprotection by
antagonizing these receptors and decreasing glutamate-dependent
excitotoxicity, as well as by scavenging free radicals (Campesan,
2011).
Interestingly, it was found that increased 3-HK relative to KYNA
was only neurotoxic in the presence of Htt93Q, suggesting that
general cellular dysfunction due to mutant htt expression confers
a sensitized background for KP modulation. In a related fashion,
it was found that expression of Htt20Q was sufficient to induce
increased 3-HK/KYNA ratios but did not confer
neurodegeneration—providing further support that additional
Htt93Q-dependent cellular defects are required to uncover
KP-dependent toxicity. A similar observation was made in another
study with flies expressing full-length htt constructs where it
was found that whereas expression of either an expanded construct
(128QhttFL) or a nonexpanded construct (16QhttFL) leads to
elevated resting synaptic Ca2+ levels, this perturbation is
associated with pathogenesis only in the case of 128QhttFL flies.
(Campesan, 2011).
The finding in this study that inhibition of TDO is protective in
the fly validates this protein (TDO) as a novel therapeutic target
for HD. TDO and IDO, which are both expressed in the brain, have
distinct structural and biochemical characteristics, and selective
inhibitors are being actively explored as potential therapeutic
compounds. It is now imperative that such compounds be tested in
animal models of HD, and perhaps other neurodegenerative
disorders, to characterize their efficacy and therapeutic
potential (Campesan, 2011).
In summary, results of this study are consistent with the view
that modulation of the KP is central to mutant htt toxicity. The
observations provide genetic and pharmacological support for the
“kynurenine hypothesis,” underscoring the important
role that Drosophila plays in the understanding and
possible development of therapy for human neurodegenerative
disorders. The study favors a model in which increased flux toward
KYNA synthesis is neuroprotective in HD, provides unequivocal
evidence that 3-HK, independent of QUIN, is pathogenic, and shows
that reduction of 3-HK relative to KYNA is therapeutic (Campesan,
2011).
Go to top
Tamura, T., Sone, M., Iwatsubo, T.,
Tagawa, K., Wanker, E.E. and Okazawa, H. (2011). Ku70
alleviates neurodegeneration in Drosophila models of
Huntington's disease. PLoS One 6: e27408. PubMed ID: 22096569
Abstract
DNA damage accumulates in genome DNA during the long life of
neurons, thus DNA damage repair is indispensable to keep normal
functions of neurons. It was previously reported that Ku70, a
critical molecule for DNA double strand break (DSB) repair, is
involved in the pathology of Huntington's disease (HD). Mutant
huntingtin (Htt) impairs Ku70 function via direct interaction, and
Ku70 supplementation recovers phenotypes of a mouse HD model. In
this study, multiple Drosophila HD models were generated
that expressed mutant huntingtin (Htt) in eye or motor neuron by
different drivers and showed various phenotypes. In such fly
models, Ku70 co-expression recovers lifespan, locomotive activity
and eye degeneration. In contrast, Ku70 reduction by heterozygous
null mutation or siRNA-mediated knock down accelerates lifespan
shortening and locomotion disability. These results collectively
support that Ku70 is a critical mediator of the HD pathology and a
candidate therapeutic target in HD (Tamura, 2011).
Highlights
- Ku70 rescues mutant Htt-induced eye degeneration in Drosophila.
- Ku70 elongates lifespan of Drosophila HD model.
- Ku70 reduction affects the lifespan of HD model fly.
- Ku70 reduction accelerates locomotion disability of HD model
fly.
Discussion
This study shows that Ku70 alleviates the HD phenotypes in Drosophila
models and that the functional deficiency of Ku70 accelerates the
lifespan shortening and locomotion disability of HD model flies.
These results are consistent with earlier observation that Ku70
remarkably elongates the lifespan of R6/2 mice, the severest mouse
model of HD, and strongly support the significance of Ku70 as a
disease mediator/modifier gene and a therapeutic target (Tamura,
2011).
Mutant Htt reduces DNA-PK activity and impairs the DNA repair
function of Ku70 in vitro. In the double transgenic mice generated
from R6/2 and Ku70 transgenic mice, Ku70 reduces DNA damage of
striatal neurons. These earlier findings provide the idea that the
functional defect of Ku70 underlies the HD pathology. However, it
is also possible to speculate that Ku70 blocks mutant Htt to
prevent interaction between mutant Htt and other target molecules
and/or to inhibit aggregation of mutant Htt. The two hypotheses
are inseparable and just like two sides of a coin. The two stories
are identical if one focuses on DNA repair. However, if there is
certain pathology other than DNA repair, the blocking effect might
be more important (Tamura, 2011).
With Drosophila, DSBs can not be directly evaluated in a
few motor neurons because there is no fly orthologue of mammalian
H2AX, a direct indicator of DSB, and there is no alternative
method to detect DSB in neurons of Drosophila. However,
results in this study are basically consistent with earlier
observation that impairment of DSB repair is a critical component
in the HD pathology and with previous reports showing DNA damage
signal activation in HD cell models (Tamura, 2011).
The concept of the linkage between DNA damage repair and
neurodegeneration is further supported by multiple autosomal
recessive cerebellar atrophies, in which mutations of DNA repair
genes such as AOA1/EAOH, AOA2, and SCAN1 cause neuronal
dysfunction and cell death. Therefore, impairment of DNA damage
repair can be considered as a common pathological component across
categories of neurodegenerative disorder (Tamura, 2011).
In conclusion, results from this study support that Ku70 is a
critical regulatory factor of Htt toxicity and a candidate for
therapeutic target of HD. This study provides a rationale to
proceed to the next step for translational approaches with Ku70
such as using viral vectors expressing Ku70 or low molecular
weight chemicals against DSB in HD (Tamura, 2011).
Go to top
Besson, M.T., Dupont, P., Fridell, Y.W.
and Liévens, J.C. (2010). Increased energy
metabolism rescues glia-induced pathology in a Drosophila
model of Huntington's disease. Hum Mol Genet 19: 3372-3382. PubMed
ID: 20566711
Abstract
Huntington's disease (HD) is a polyglutamine (polyQ) disease
caused by an expanded CAG tract within the coding region of
Huntingtin protein. Mutant Huntingtin (mHtt) is ubiquitously
expressed, abundantly in neurons but also significantly in glial
cells. Neuron-intrinsic mechanism and alterations in
glia-to-neuron communication both contribute to the neuronal
dysfunction and death in HD pathology. However, the role of glial
cells in HD pathogenesis remains to be determined. In recent
years, development of Drosophila models have facilitated
the dissection of the cellular and molecular events in
polyQ-related diseases. By using genetic approaches in Drosophila,
this study manipulated the expression levels of mitochondrial
uncoupling proteins (UCPs) that regulate production of both ATP
and reactive oxygen species in mitochondria. It was discovered
that enhanced levels of UCPs alleviate the HD phenotype when mHtt
was selectively expressed in glia, including defects in locomotor
behavior and early death of Drosophila. In contrast,
UCPs fail to prevent the HD toxicity in neurons. Increased
oxidative stress defense was found to rescue neuron but not
glia-induced pathology. Evidence is now emerging that UCPs are
fundamental to adapt the energy metabolism in order to meet the
metabolic demand. Thus, the study proposes that UCPs are
glioprotective by rescuing energy-dependent functions in glia that
are challenged by mHtt. In support of this, increasing glucose
entry in glia was found to alleviate glia-induced pathology.
Altogether, data from this study emphasizes the importance of
energy metabolism in the glial alterations in HD and may lead to a
new therapeutic avenue (Besson, 2010).
Highlights
- Drosophila UCP5 rescues glial pathology in HD flies.
- Drosophila UCP5 fails to rescue neuronal pathology in
HD flies.
- Human UCP2 alleviates glial pathology in HD flies.
- Superoxide dismutase and catalase
do not rescue glia-induced pathology in HD flies.
- Drosophila glucose transporter rescues glial
pathology in HD flies.
Discussion
The main outcome of this study is that manipulating energy
metabolism alleviates the glial-induced pathology in a Drosophila
model of HD. Neuron–glia interactions are crucial to ensure
optimal brain function and accumulation of mHtt compromises
crosstalk between the two cell types. Interestingly, whereas both
neuronal and glial expression of mHtt trigger decreased climbing
performance, only flies expressing mHtt in glia suffer from
bang-sensitivity following a mechanical stress. Thus, the
bang-sensitive phenotype likely reflects the disruption of
glial-specific functions by expanded polyQ proteins. The exact
mechanism that triggers the bang-sensitive behavior in Drosophila
is not well understood. So far, bang-sensitivity has been related
to depletion in ATP levels and impairment of the Na+/K+ APTase
pump. Interestingly, two crucial functions of glia are to supply
neurons with energy metabolites and to maintain ionic composition
of the extracellular environment (Besson, 2010).
It was reported that DmUCP5 rescues the bang-sensitive phenotype
and the climbing performance when co-expressed with mHtt in glia.
Moreover, DmUCP5 significantly prolongs the survival of flies
expressing mHtt in glia. Similarly, the exogenous expression of
hUCP2 conferrs protection against mHtt toxicity in glia. In
contrast, DmUCP5 fails to alleviate the deleterious phenotype when
co-expressed with mHtt in neurons. Thus, this study provides the
first evidence that UCPs mediate differential effects on the
glial- and neuronal-specific physiology (Besson, 2010).
Numerous studies have raised the possibility that UCPs can confer
neuroprotection in models of brain injury or neurodegenerative
diseases. Evidence comes mainly from transgenic mice containing a
copy of human UCP2 and UCP3 genes cloned under the regulation of
their endogenous promoters, respectively. Accordingly to the
regional distribution of UCPs, those mice show a moderate
overexpression of UCP2 into the brain. UCP2/UCP3 transgenic mice
exhibit a reduction in cell death occurring after experimental
induction of epileptic seizures or following ischemia and
traumatic brain injury. Acute treatment with
1-methyl-4-phenyl-1,2,5,6-methyl-phenyl-tetrahydropyridine (MPTP)
is currently used to induce dopaminergic neuron degeneration and
reproduce a syndrome clinically similar to Parkinson's disease in
animal models. Whereas UCP2 knock-out mice display an enhanced
sensitivity to MPTP exposure, UCP2/UCP3 transgenic mice show a
lower MPTP-induced loss of dopaminergic neurons in the substantia
nigra (Besson, 2010).
Similarly, selective overexpression of UCP2 in catecholaminergic
neurons by using the tyrosine hydroxylase promoter protects nigral
neurons from acute MPTP toxicity. Stroke, ischemia and acute MPTP
treatment appear to lead to neuronal death by a common pathway:
they all alter mitochondrial metabolism and increase ROS release.
Accordingly, in the aforementioned studies, the neuroprotective
effect of UCP2 is correlated with the reduction of oxidative
stress in these models. Since mHtt is known to interact with
mitochondria and leads to increased ROS production, it is tempting
to propose that UCPs are glioprotective by reducing oxidative
stress in glia. In this study, the potential benefit of increasing
ROS defense was evaluated when mHtt was selectively expressed in Drosophila
glia or neurons. Whereas increasing MnSOD and catalase levels
rescued the early death of flies expressing mHtt in neurons, no
improvement was found when mHtt was present selectively in glia.
Thus, this study further confirms that ROS overproduction is a
crucial pathway by which mHtt leads to neuronal dysfunction and/or
death in HD. However, despite the impact of UCPs on ROS
production, this may not be sufficient to counteract the
mHtt-induced alterations in neurons. The data also reveals that
oxidative stress is likely not the primary cause of HD glial
pathogenesis and the beneficial effects of UCPs on glial cells are
likely not due to the UCP-mediated proton leak and the subsequent
reduction of ROS (Besson, 2010).
In recent years, an increasing amount of data indicate that UCPs
may not only act as uncoupling agents. They may be fundamental for
metabolic sensing and adaptive energetic metabolism in order to
meet the energy demand. For instance, it was reported that UCP2
knock-out fibroblasts display enhanced proliferation associated
with a higher pyruvate oxidation rate and a reduced fatty acid
oxidation in mitochondria. On the contrary, UCP2 overexpression
decreases the glucose-dependent proliferation of CHO cells. This
is consistent with a ‘metabolic hypothesis’ whereby
the role of UCP2 would be to promote oxidation of fatty acids
rather than that of glucose-derived pyruvate. Earlier data also
suggests that UCPs may physically conduct free fatty acid anions
and thereby, actively participate to the fatty acid cycling.
Finally, since UCPs shunt the oxidation of pyruvate in
mitochondria, this may give rise to increased glycolysis followed
by lactate production: a process known as the Warburg effect. In
respect to this, overexpression of UCP3 and UCP4 in muscle and
neuronal cultured cells, respectively, was found to stimulate
glucose transport and shifting the way of ATP production from
mitochondrial production to glycolysis. In the brain, glycolysis
is predominantly an astrocytic metabolic process, whereas
oxidation is primarily neuronal. Therefore, this study proposes
that UCP overexpression in the presence of mHtt would ameliorate
the glial-induced alterations by enhancing glycolysis. In support
of this hypothesis, overexpression of the Drosophila
glucose transporter DmGluT1 ameliorated the locomotor deficiency
and the lifespan of flies expressing mHtt in glia. A role for
glucose metabolism as a modulator for mHtt toxicity was previously
suggested on HD cell models as cell death was significantly
reduced by glucose transporter overexpression. This study provides
the first in vivo evidence that increased entry of glucose is
beneficial against mHtt-induced glial dysfunction. The exact steps
whereby enhanced glucose metabolism rescues glia-induced
alterations in HD flies remain to be clarified. As a possible
mechanism, raised intracellular glucose levels may induce
autophagy via mTOR signaling (Besson, 2010).
In conclusion, this study proposes that defects in energetic
metabolism are involved in mHtt-induced glial alterations and that
increasing glucose metabolism may be beneficial to rescue abnormal
glia-to-neuron communication in HD. Further work is required to
fully understand the importance of energy metabolism in the
abnormal neuron–glia crosstalk in HD pathogenesis (Besson,
2010).
Go to top
Ravikumar, B., Imarisio, S., Sarkar,
S., O'Kane, C.J. and Rubinsztein, D.C. (2008). Rab5
modulates aggregation and toxicity of mutant huntingtin through
macroautophagy in cell and fly models of Huntington disease. J
Cell Sci 121: 1649-1660. PubMed ID: 18430781
Abstract
Huntington disease (HD) is caused by a polyglutamine-expansion
mutation in huntingtin (HTT) that makes the protein toxic and
aggregate-prone. The subcellular localisation of huntingtin and many
of its interactors suggest a role in endocytosis, and it has been
shown that huntingtin interacts indirectly with the early endosomal
protein Rab5 through HAP40. This study shows that Rab5 inhibition
enhances polyglutamine toxicity, whereas Rab5 overexpression
attenuates toxicity in cell and fly models of HD. The study tries to
identify a mechanism for the Rab5 effects in HD model systems, and
it was found that Rab5 acts at an early stage of autophagosome
formation in a macromolecular complex that contains 