Aspects of Neuroscience

BCHE4040/LSCI5440
Huntington’s Disease
(HD)
Prof Kwok Fai LAU
School of Life Sciences
2023-24
CUHK 1
Huntington’s Disease (HD)

I wish to draw your attention more particularly to a

form of the disease which exists ……………… almost
exclusively on the east end of Long Island ……………
Let me remark that chorea ……………..
The hereditary chorea is confined to a few families
and has been transmitted to them …………….

• “Chorea” : derived from a Greek word means dance

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Basic facts about HD
• a hereditary syndrome
• Characterized by (1) abnormal and unusual movements
(2) cognitive impairment
(3) disorder in personality
• Mean onset age : 35
• Fatal after 15-20 years after onset

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Huntingtin (Htt)
• It is a soluble protein with 3,144 amino acids
• No sequence homology with other proteins
• It is widely expressed with high level in brain
• A few known sequence motifs are found in Htt Human Molecular Genetics, Volume 15, Issue 9, 1 May
2006, 1513–1523

o HEAT repeat clusters → protein binding

o Polyproline (Pro) stretch → enhance solubility; protein binding
o Polyglutamine (polyQ; encoded by codon CAG) stretch →
expansion of poly Q tract is the cause of HD
Sara IMARISIO et al (2008) Huntington’s disease: from pathology and genetics to potential therapies. Biochem J 412, 191-209 5
Elena Cattaneo etal. (2005) NORMAL HUNTINGTIN FUNCTION: AN ALTERNATIVE APPROACH TO HUNTINGTON’S DISEASE. Nature Reviews Neuroscience 6, 919-930
 Embryonic day 7.5
+/+ -/-

Thickening of embryonic
endoderm (e),
• Homozygous Htt knockout mesoderm (m)
ectoderm (ec)
→ Embryonic lethal
→ Essential for embryonic development
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 Wildtype Hdh conditional KO

• Conditional Hdh KO mice showed progressive degenerative

neuronal phenotype
→ a role in cell viability
→ loss of normal Htt function may contribute to the pathogenesis
of HD
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Neuropathology of HD
• Atrophy of the brain causing enlargement of the lateral ventricles
HD patient brain section with 74 CAG repeat
Anti-ubiquitin Anti-ubiquitin

HD

Anti-Htt Anti-ubiquitin
Normal

• Intra-nuclear inclusions contain Htt and ubiquitin in HD

• The number of inclusion increases with
the number of CAG (Poly glutamine (Q))
repeat
→ Normal < CAG (Q) 35 < HD
→ CAG expansion in mutant Htt
 Comparison of the eyes of adult wild-type flies and flies
containing 108Q

• Expanded CAG repeat itself is toxic

→ Gain of toxic function

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CAG expansion in mutant Htt causes neurodegeneration by
(1) Disrupt the normal Htt physiological functions
(2) Gain of toxic functions

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 Huntingtin interacting proteins

Li SH and Li XJ. Huntingtin-protein interactions and the pathogenesis of Huntington's disease. Trends Genet. 2004 Mar;20(3):146-54.

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• Htt interacts with repressor element-1 silencing transcription (REST)

• REST is a transcription factor that suppresses the expression of

the brain-derived neurotrophic factor (BDNF)

• BDNF is a neurotrophic factor for supporting the survival of

neurons

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 Mechanism that REST suppresses the expression of BDNF

BDNF transcription
REST

REST
X
BDNF transcription

BDNF gene promoter

Neuron restrictive silencer element (NRSE)

BDNF gene

What is the effect of Htt-REST interaction on BDNF expression?

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 CAT
Chloramphenicol (Cm) Acetylated chloramphenicol (Cm-3-ac)

(a) Chloramphenicol
Thin layer chromatography to separate
Cm and Cm-3-ac
BDNF acetyl transferase
promoter (CAT) gene

(b)
NRSE

thymidine
kinase promoter
(tkNRSE)

Conclusion: Htt regulates gene expression via NRSE

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Zuccato C et al. Huntingtin interacts with REST/NRSF to modulate the transcription of NRSE-controlled neuronal genes. Nat Genet. 2003 Sep;35(1):76-83. Epub 2003 Jul 27.
 R6/2 = mouse line HD patient brain
that carries mutant sample
I= input
IP= immunoprecipitation
Htt gene

Conclusion:
• Htt interacts with REST
• Mutant Htt interacts less with REST
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Zuccato C et al. Huntingtin interacts with REST/NRSF to modulate the transcription of NRSE-controlled neuronal genes. Nat Genet. 2003 Sep;35(1):76-83. Epub 2003 Jul 27.
A proposed mechanism that Htt regulates BDNF gene expression

Cattaneo E et al. Normal huntingtin function: an alternative approach to Huntington's disease. Nat Rev Neurosci. 2005 Dec;6(12):919-30

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Htt is detected in nucleus Htt interacts with transcription factors

How could such a big Htt protein enter the nucleus?

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Cleavage of Htt
• N-terminal Htt fragments are found in brain tissue from affected
HD patients but not from controls

Control HD

Htt fragments

T = Total protein
N= Nuclear Extract

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 Expression Full-length Htt Q128

Expression Htt 1-436 Q128

• Small Htt fragments enter nucleus and form intra-nuclear

aggregates
• Nucleus may be an important site for mutant Htt toxicity
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 TUNEL stain for DNA cleavage
Control HD

• DNA cleavage → a sign of apoptosis and the activation of caspases

• Caspases cleave Htt

Htt fragments
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• A small region at the N-terminal of Htt is found to
interact with Translocated Promoter Region (Tpr)
• Tpr is a component of the nuclear pore complex
(NPC), a complex required for the trafficking across
the nuclear membrane

• Expanded CAG reduces Tpr and Htt N-terminal interaction

N-ter Htt 120Q

N-ter Htt Q20Q

• Expanded CAG causes accumulation of mutant Htt in

the nucleus
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 N-terminal Htt mouse line
N-terminal mutant Htt
mouse lines

Figure 6 Reduced lifespan of HD N171-82Q transgenic mice. Mice with N171-18Q had a normal lifespan. Line 77 of N171-82Q showed the shortest
lifespan and died at ∼2.5 months of age; this line could not be maintained (possibly due to slightly higher expression of the transgene protein).
N171-82Q (line 81 and 100) lived to ∼5–6 months, whereas animals from line 6 died at ∼8–11 months of age. The cause of death remains unclear;
there have been no seizures observed in these lines, and the animals appear to take food.

N171-82Q N171-18Q

• N-terminal mutant Htt Stained with an antibody that detect first 17 amino acids of Htt

→ reduces life span of the transgenic mice

→ aggregates to form intranuclear inclusions
→ is sufficient to produce HD-like abnormal clinical syndromes 22
h

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 Dynactin is a protein complex that is required for the
microtubule motor protein dynein

GST-HAP1
Lysates Pull-downs
Q44
Q22

Q44
Q82

Q22

Q82

• Htt interacts with Htt-associated

protein 1 (HAP1)
• HAP1- dynactin p150 -Htt form a trimeric
• The interaction is enhanced by CAG complex
expansion J Neurosci. 1998 Feb 15;18(4):1261-9.
Interaction of huntingtin-associated protein with dynactin P150Glued.
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HAP1
Htt

Int J Dev Neurosci. 2006 Apr-May;24(2-3):103-11.

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Htt modulates vesicle trafficking

• Htt is colocolized with BDNF vesicles in

axons and synaptic terminals

• Htt stimulates BDNF vesicular transport

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http://www.sciencedirect.com/science/article/pii/S0092867404006191#

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HAP1
Htt
X

HAP1 CAG expansion enhances

mHtt HAP1 and mHtt interaction

X
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Mitochondrial dysfunction in HD

reserve of high-energy phosphates

Creatine (Cr) CKm PCr (Store in muscle)

+ +
ATP ADP

• Mitochondrial creatine kinase (CKm) regenerates phosphocreatine (PCr)

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(1) Increased oxidation of biomolecules
• Mitochondria are sites for reactive oxygen species (ROS) production
Oxidation of DNA in HD Oxidation of proteins in HD
patient samples mouse model R6/2

anti-2,4-DNP-hydrazone antibody (specific for protein carbonyls (CO groups))

8-OH-dG (8-hydroxy-2’-deoxyguanosine) is a biomarker for oxidative

Protein Fragmentation
DNA damage
→ impaired protein functions
Polidori MC et al. Oxidative damage to mitochondrial DNA in Huntington's disease parietal cortex. Neurosci Lett. 1999 Sep 3;272(1):53-6.

Zourlidou A et al. Hsp27 overexpression in the R6/2 mouse model of Huntington's disease: chronic neurodegeneration does not induce Hsp27 activation. Hum Mol Genet. 2007 May 1;16(9):1078-90.
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Lipid oxidation is increased in HD mouse model R6/1

striatum

in the mouse brains

Lipid oxidation

Age (weeks)

• Brain mitochondria have a higher concentration of lipids with

polyunsaturated fatty acids, which are more sensitive to
oxidative damage than other lipids
• Damage mitochondrial membrane → increase leakage

Pérez-Severiano F et al. Striatal oxidative damage parallels the expression of a neurological phenotype in mice transgenic for the mutation of
Huntington's disease. Brain Res. 2000 Apr 17;862(1-2):234-7. 31
(2) Mitochondrial Ca2+ homeostasis is
compromised in HD
• Mitochondria are intracellular calcium store

Figure 1. Mitochondrial abnormalities in lymphoblasts from HD patients.

(a) Mitochondrial membrane potential (ψm) was reduced in lymphoblast mitochondria from HD patients (-169 plusminus 4
versus -181 plusminus 2 mV; n = 4 per group; *P < 0.05).
(b) ψm was monitored while aliquots of 7.8 µM Ca2+ (arrows) were added to suspensions of lymphoblast mitochondria
from control subjects or individuals with HD. Mitochondria from individuals with HD depolarized at lower calcium loads
than did mitochondria from controls. In the experiments shown here, the individual with adult-onset HD had 46 repeats,
and the individual with juvenile-onset HD had 65 repeats.
(c) The Ca2+ retention capacity of HD mitochondria was lower than in control mitochondria (63 + 2 versus 146 + 29 nmol/mg
protein; n = 4 per group; *P < 0.05).
(3) Energy metabolism is compromised in HD
• ATP production decreases as Htt CAG repeat number increase

Figure 3.HD CAG size determines [ATP/ADP] in lymphoblastoid cells.

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Impairment of the ubiquitin-proteasome system (UPS)
• Htt inclusions can be stained by anti-unbiquitin
ubiquitin
ubiquitin proteasomes
Unwanted/ ligases
damaged protein
degradation

LMP2 → a proteasome component

GFP- Htt
LMP2

• The proteasome is associated with aggregates of mutant Htt 34

 Control protein

Htt aggregates

Htt aggregates +MG132

(a proteasome inhibitor)

• 26S proteasome activity is inhibited

by htt-aggregates

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 Pathogenetic cellular mechanisms in Huntington
disease.
(1) HTT is translated to produce the full‐length
huntingtin protein as well as an amino‐terminal
HTT exon 1 fragment (the result of aberrant
splicing). The length of the polyglutamine
(polyQ) tract in these proteins depends on the
extent of somatic instability.
(2) Full‐length native huntingtin is cleaved through
proteolysis to generate additional protein
fragments.
(3) Protein fragments enter the nucleus.
(4) Fragments are retained in the nucleus through
self‐association, oligomerization and aggregation
− leading to the formation of inclusions, a
process that causes transcriptional dysregulation
through the sequestration of other proteins and
through other incompletely defined
mechanisms.
(5) Huntingtin fragments oligomerize and aggregate
in the cytoplasm.
(6) The aggregation of huntingtin is exacerbated
through the disease‐related impairment of the
proteostasis network, which also leads to global
cellular impairments.
(7) The aberrant forms of huntingtin result in
additional global cellular impairments, including
synaptic dysfunction, mitochondrial toxicity and
a decreased rate of axonal transport. PRD,
proline‐rich domain; Ub, ubiquitin.

European Journal of Neurology, Volume: 25, Issue: 1, Pages: 24-34, First published: 17
August 2017, DOI: (10.1111/ene.13413)
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Therapy for HD
• No disease-modifying treatments for HD
• Symptomatic treatment
e.g. Xenazine is used to suppress the involuntary movement

• 23 active HD clinical trials are underway (according to

ClinicalTrials.gov 2017)

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Potential mechanism-based strategies for HD

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Imarisio S et al. Huntington's disease: from pathology and genetics to potential therapies. Biochem J. 2008 Jun 1;412(2):191-209.
Huntington’s Disease Therapeutics Research

(1) Reduce Huntingtin Expression

• Small interfering RNA molecules (siRNAs)

• Anti-sense oligonucleotides

• Genome editing techniques such CRISPR/Cas9

• Synthetic zinc finger protein-nuclease complexes

that bind and cleave HTT DNA

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(2) Small Molecule Approaches
e.g. Target the post-translational modifications of mHTT
→ Acetylation of mHTT promotes its clearance by autophagy
→ enhance phosphorylation at certain neuroprotective residues
in Htt.
e.g. Development of small molecule to potentiate neurotrophin BDNF

(3) Stem Cell Therapy

• There have been a number of small trials of foetal striatal (
contains human fetal neural stem cells ) transplantation in HD
patients over the last 15 years in various countries
• Improvement or stabilisation of motor, functional and
neuropsychiatric symptoms has been reported by some but not
all the groups.
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