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Huntington's Disease Exam/Essay Summary
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Table of ContentsPart 1: Introduction + Htt Protein..............................................................................................................1
Part 2: Mutant Htt and Pathogenesis........................................................................................................5
Part 3: Toxic gain-of-function....................................................................................................................7
Part 4: Loss-of-function...........................................................................................................................12
Part 5: HD Pathobiology Controversies...................................................................................................16
Part 6: Disease-Modifying Therapeutics..................................................................................................23
Part 7: Biomarkers..................................................................................................................................33
Part 8: Conclusion...................................................................................................................................34
Part 1: Introduction + Htt Protein
Description: rare autosomal dominant progressive neurodegenerative disease
Predominantly affects basal ganglia characterised by affective, cognitive, behavioural, and motor
decline
Neurobiological basis of HD is a CAG repeat expansion to >35 CAG in the gene encoding the
huntintin protein (htt)
Results in an expanded N-terminal polyglutamine tract
Mouse Models of HD used to understand therapeutics (instrumental)
1. BACHD: full-length human mHtt with 97Q under endogenous genomic regulation
a. Conditional Bacterial Artificial Chromosome (BAC) transgenic mouse model of HD (BACHD)
that expressed human full-length mHTT (fl-mHTT) with 97Q
b. Exhibit progressive motor and psychiatric-like behavioural deficits and selective cortical and
striatal atrophy
c. Lack overt striatal neuronal loss – only partially recapitulate HD phenotypes
d. First conditional human genomic transgenic mouse model for HD featuring strategically
placed LoxP sites flanking mHTT-exon1 with the polyQ repeat expression of fl-mHTT can
be genetically reduced in Cre-expressing cell lineages
2. R6/2: mHtt-exon1 transgenic mouse model (Mangiarini et al., 1996)
a. Developed the mouse R6/2 that only expressed exon 1 of the HD gene with an expanded
CAG repeat
b. Causes progressive neurological phenotype in transgenic mice
3. YAC128: slowly progressive mouse w human huntingtin gene + 2 mouse gene
Huntingtin Protein
Soluble 3144 amino acid (348 kDa) protein highest expression in the CNS and testes
Bates et al., 2015
Huntingtin protein with a normal polyQ repeat length of 23 glutamines contains 3144 amino acids
with a molecular weight of 348kDa (Bates, 2015)
o Its expressed throughout the body at varying levels dependening on the cell type
o Normal functions are still being defined
Believed to play role in development of the nervous system, influence brain-derived
neurotrophic factor (BDNF) production and transport, and cell adhesion
Htt protein is encoded by the first exon of the HTT gene, the polyQ domain lies near the N-terminus
of Htt
Huntingtin is linearly organised as series of ordered domains interspersed with intrinsically
disordered segmnets
Folding can occur due to interaction between folded domains
o Known ordered domains are clusters of a-helical HEAT repeats
o HEAT repeats are regions for post-translational modifications like proteolytic cleavage,
phosphorylation and glycosylation
,Htt Protein Structure
Guo et al., 2018: cryo-electron microscopy structure of huntingtin
Used cryo-electron microscopy to determine the structure of full-length human HTT in complex with
HTT-associated protein 40 to an overall resolution of 4A
HTT is largely a-helical and has 3 major domains
o Amino + carboxy domains contain multiple HEAT repeats arranged in solenoid fashion
o Domains are connected by smaller bridge domain containing tandem repeats
Big protein – top 1% in size of proteins in the proteome
Slime mold is the most primitive organism with the huntingtin gene – very well conserved – but has
no glutamine
o Slime mould is one of the first organisms that begins to form multicellular colonies –
associated with development of huntingtin protein – cell-cell communication?
Sea urchins have a huntingtin protein
o One of the most primitive organisms with a functioning nervous system
o Has 2 CAGs in the gene and two glutamine
o Associated with development of nervous system
Saudou and Humbert, 2016: the biology of huntingtin
Multifunctioning scaffolding protein
Abnormal expansion of glutamine tretch (polyQ) in N-terminal sequence = mHTT HD
The mutation causing the polyQ expansion was identified more than 20 years ago
KO of huntingtins is lethal on embryonic day 7.5 showing proof of its early exression
Importance in Understanding HTT functioning
1. Therapeutics
a. Elucidating Htt function is ESSENTIAL in identifying how loss of HTT affects cell physiology
and to understand under which conditions low levels of wt and mHTT can be
toleratedESSENTIAL in emerging therapeutic strategies aimed at lowering HTT levels in HD
patients the goal is to lower levels of mHTT BUT the first clinical trials may not be able to
discriminate between wt and mHTT
2. Pathological mechanisms specific to HD
a. To understand whether pathological mechanisms aare due to mods of HTT function, HTT-
specific gain-of-function or loss-of-function
b. Understand the specificity of HD
3. Pathological steps in disease progression
a. To identify early dysfunctions and lead to identification of makers of disease progression that
can be used to validate early neuroprotective therapies
Function of normal WT Htt protein
1. Protective effects (apoptotic)
Zeitlin et al., 1995: increased apoptosis and early embryonic lethality in mice nullizygous for the
huntingtons disease gene homologue role of Htt protein in antiapoptotic activity protective effects
- Targeted disruption of homologous mouse gene (hdh muring Htt homolog) to examine normal
role of HTT shows that the protien is functionally indispensable
o Nullizygous embryos become developmentally retarded and disorganised
o Die between 8.5 days and 10.5 days of gestation
- Level of regionalised apoptotic cell death in embryonic ectoderm expressing Hdh gene is higher
than normal in null mutants HTT is involved in processes counterbalancing the operation of an
apoptotic pathway
- Shows that HTT is ESSENTIAL for normal functioning any mutations will have significant
detrimental downstream effects on normal functioning
Wild-type huntingtin protects from apoptosis upstream of Caspase-3
WT Htt is neuroprotective indicates that mutations in the Htt gene may confer a loss-of-
function pathway
Rigamonti et al., (2000)
Aim
, Method In vitro ST14A striatal cells were transfected with wt htt
Exposed these cells with pro-apoprtotic conditions
Results During this exposure, cells that showed a decrease in cell viability was correlated
with a reduction in levels of the exogenous htt protein
Overexpression of wt htt increased cell viability demonstrates a neuroprotective
function of wt htt
When cells were transfected with mhtt, there was a faster decline in cell viability
and mitochondrial activity in pro-apoptotic condition when compared to cells
transfected with wt htt
Conclusio Mhtt has a reduced ability to carry out the htt normal neuroprotective functions
n Wt htt directly influences cell survival acts as anti-apoptotic protein in neural
cells
Full-length normal htt modulates the toxicity of the polyQ expansion cells that
express the wt htt are susceptible to fewer death stimuli than cells expressing
truncated mhtt
This functions is partially mediated by preventing caspase-3 activation
Level of protection might take place at the level of caspase-9 activation
Evidence of GOF
Huntingtin inhibits caspase-3 activation
WT huntingtin inhibits caspase-3 activity suggesting a mechanism where caspase-
mediated huntingtin depletion amplification cascade further caspase-3 activation
death
Zhang et al., (2006)
Aim Understand the function of htt
Method Probe the pro-survival function of htt
Modulated levels of wt htt in cellular and invivo models
Results Showed a physical interaction between caspase-3 and wild-type htt in mouse N2a
neuroblastoma cells and found an inverse correlation between wild-type htt expression
and caspase-3 activation.
Htt depletion = caspase-3 activation
Overexpression of htt = caspase-3 inhibition
Htt physically interacts with active caspase-3
Mhtt binds active caspase-3 with lower inhibitor and lower inhibitory effect on
caspase-3 than wt htt
Reduction of htt levels = caspase-3 activiation but cellular response was cell-type
specific
Depletion in htt = overt cell death OR increased vulnerability to cell death
Conclusio Htt inhibits caspase-3 activity
n Caspase-mediated htt depletion = amplification cascade = further caspase-3
activation = cell dysfunction and cell death
Evidence of a LOF mutation htt LOSES its neuroprotective ability
Leavitt et al., 2006: WT Htt protects neurons from excitotoxicity
Aim Test hypothesis that increasing Htt levels protect striatal neurons from NMDAR-mediated
excitotoxicity
Results - Cultured striatal neurons from YAC18 transgenic mice over-expressing full-length
WT Htt were dramatically protected from apoptosis and caspase-3 activation
compared with cultured striatal neurons from non-transgenic FVB/N littermates and
YAC72 mice expressing mHTT
- NMDAR-activation induced by intrastriatal injection of quinolinic acid caused form
of apoptotic neurodegeneration in striatum of mice associated with caspase-3
cleavage of htt in neurons and astrocytes and decreased levels of full length htt
- Over-expression of WT Htt in vivo in YAC18 transgenic mice = protection against
NMDAR-mediated apoptotic neurodegeneration
Conclusio Provided in vitro and in vivo evidence that htt regulates balance between neuronal survival
n and death following acute excitotoxic stress
- Levels of htt may module neuronal sensitivity to excitotoxic neurodegeneration
, Inactivation of Drosophila Huntingtin affects long-term adult functioning and the pathogenesis of
a Huntington’s disease model
Loss of normal dHtt function is a component of toxicity from PolyQ expansions in HD
disrupting normal functioning of WT Htt HD pathogenesis
Zhang et al., 2009
Aim Investigated the function of dHtt in a model of HD
Method - Used reverse transcription-PCR and in situ hybridisation to demonstrate that dhtt is
expressed ubiquitously during all stages of fly development
- Used Gal4-UAS system to express a polyQ-expanded Htt transgene (HD-Q93) in
all fly neurons
Results - Found that dhtt knockout exacerbated HD-Q93 toxicitiy
- HD-Q93; dhtt-ko flies showed normal mobility in beginning of adult life but
declining mobility at 5 days of age that rapidly deteriorated more
uncoordinated, increased frequency of faltering while walking + falling while
climbing
- In standard climbing assay, only 10% of 11-day-old HD-Q93;dhtt-ko mice could
make it to the top (compared to day 3 where they all could and the WT or dhtt-ko
mice of which 94% could)
- Spontaneous locomotion of HD-Q93;dhtt-ko flies showed rapid decline in motility
and were less than half active compared to other groups at day 9
- Life span of HD-Q93;dhtt-ko mice was shortened (half died at day 8 and all at day
14) while only 7% of HD-Q93 mice died at day 8 and half at day 14
- HDD-Q93;dhtt-ko flies developed more severe pathology at 5 days compared to
HD-Q93 flies at same age MB is less organised and were largely
unrecognisable in 95% of HD-Q93;dhtt-ko flies
- HD-Q93;dhtt-ko mice had a 25% increase in total area that is devoid of
neuronal cells compared to HD-Q93 flies increased disorganisation and
increased neuronal loss in brains of HD-Q93;dhtt-ko flies
Conclusio Loss of endogenous dhtt affected the pathogenesis of HD flies renders animals more
n vulnerable to the toxicity associated with PolyQ-expanded Htt (AKA mHtt)
- Dhtt (null mutant in Drosophilia HTT homolog) mutant animals are viable with no
obvious developmental defects
- Dhtt is required for maintaining the mobility and long-term survival of adult
animals, and for modulating axonal terminal complexity in the adult brain
- Removing endogenous dhtt significantly accelerated neurodegenerative phenotype
associated with Drosophilia model of polyQ Htt toxicity (HD-Q93)
- In vivo evidence that disrupting normal function of Htt might contribute to HD
pathogenesis
2. Produces BDNF
Htt is involved in transcription and trafficking of brain-derived neurotrophic factor (BDNF) could
contribute to its prosurvival properties (Gauthier et al., 2004)
- BDNF is a critical factor for cell growth and survival but its not produced in significant quantities by
striatal neurons
o Transfer of BDNF from cortical afferents to striatal cells = striatal survival and activity of
cortico-striatal synapses
- Transfer of BDNF from cortical afferents to striatal cells promotes striatal survival and activity of
cortico-striatal synapses
- BDNF colocalises with Htt
- Large majority of BDNF support in striatum is derived from cortical afferent fibres
- Requires the transcription of BDNF within cortical neurons + successful BDNF trafficking along axon
to presynaptic release sites in the striatum
- Htt promotes BDNF transcription and its transport along microtubules
- Deficits in trafficking of BDNF in HD models probably represents a loss of function feature of
polyQ Htt
Loss of huntingtin-mediated BDNF gene transcription in HD
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