Huntingtin-Associated Protein HAP1 trafficking and orexin neuronal function
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Huntingtin-Associated Protein HAP1 trafficking and orexin neuronal function
Yung-Feng LinPostdoctoral Fellow
Li Lab
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HAP1 and Htt in trafficking
Salinas S et al, Curr Opin Cell Biol 2008
Kinesin light chain (KLC)
Microtubule-dependent transport
14-3-3 Protein trafficking complex assembly
TBP Transcription factor
AHI1 Intracellular trafficking
Androgen receptor (AR)
Membrane receptor
References
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1997, 19981997
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HAP1 expression in hypothalamus
P3 P10 P15
50
75
100
% S
urv
ival
0P1 P2 P9
25
P3 P4 P6 P15
Li et al., J. Neurosci, 2003
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Question:Dose reducing HAP1 expression in hypothalamic neurons in adult mice also lead to neurological phenotypes?
Approach:Use conditional HAP1 knockout mice to examine HAP1 deficiency-related phenotypes
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Orexin neurons are located in the lateral hypothalamic area (LHA) and project to almost all parts of the brain except the cerebellum
My study focuses on orexin neurons because of their importance and availability of orexin-Cre mice
Orexin expression HAP1 expression
Orexin neuronal function:
Coronal sections
LHDMH
VMH
ARC
FeedingLocomotor ActivitySleep/wakefulness
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TKTK NeoNeoE1E1
39513951 69576957
Cre Cre recombinaserecombinase
2 kb2 kb
TKTK
TKTK E1E1
2 kb2 kb
Cre-LoxP system and the Orexin-Hap1 conditional knockout mouse
Hap1Hap1
Hap1-LoxPHap1-LoxP
Hap1-CKOHap1-CKO
3 Kb
1 Kb
5 Kb
WT
KO
M C T Tc Hc
HAP1-loxP
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HAP1 Orexin A Merged
Het
Hom
Homozygous orexin-HAP1 knockout selectively depletes HAP1 in orexin neurons
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Age (day)
0 10 20 30 40 60 80 100
Body weight (g)
0
5
10
15
20
25
30
Orexin-Hap1 +/-Orexin-Hap1 -/-Orexin-Hap1+/+
Age (day)
0 10 20 30 40 50 60 70 80 90 100
% Control body weight
60
70
80
90
100
MaleFemale
Bod
y w
eigh
t (g
)
Bod
y w
eigh
t (%
of
WT
)
Orexin-HAP1 KO mice show decreased body weight
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Orexin-HAP1 KO mice eat less
Male FemaleFood intake (g / day / g body weight)0.0
0.1
0.2
0.3
0.4
+/- -/-
* *
Male FemaleWater intake (g / day / g body weight)
0.0
0.1
0.2
0.3
+/- -/-
Foo
d in
take
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Fed, n=32
Time
8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 1 2 3 4 5 6 7
Movement (beam breaks / hour)0
200
400
600
800
1000
Orexin-Hap1 +/-
Orexin-Hap1 -/-
activ
ity
Fasted, n=32
Time
8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 1 2 3 4 5 6 7
Movement (beam breaks / hour)0
200
400
600
800
1000
Orexin-Hap1 +/-
Orexin-Hap1 -/-
Orexin-HAP1 KO mice also have lower locomotor activity
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WT KO
HAP1 knockout reduces neurite extension of orexin neurons in mouse brain
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Question:
Does lack of HAP1 affect neuronal intracellular trafficking in mouse brain?
Approach:
Sucrose gradient fractionation to examine the distribution of organelle marker proteins in the brains of HAP1 null and WT pups.
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EEA1
Ahi1
Hap1
Syntaxin
SOD2
Inpu
t Sucrose gradient 5-45%
Early endosome
Synaptic vesicle
Mitochondria
Htt (2166)
PSD95Post-synaptic density
GM130Golgi complex
Beclin-1Autophagisome
pHap1A
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5 ~ 45% SucroseInput
WT
KO
WT
KO
WT
KO
Tubulin
Dynactinp150
KinesinLight chain
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5 ~ 45% SucroseInput
WT
KO
WT
KO
LAMP1
BDNF
Lack of HAP1 affects the distribution of lysosomes and BDNF
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Conclusion
HAP1 deficiency
Intracellular trafficking defect
Impaired neurite extension
Orexin neuronal dysfunction
Feeding and locomotor activity changes