Huntingtin-Associated Protein HAP1 trafficking and orexin neuronal function

Yung-Feng LinPostdoctoral Fellow

Li Lab

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

1995

1997, 19981997

2002

2004

2003

2003

2006

2006

2007

20072008

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

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

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

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

HAP1 Orexin A Merged

Het

Hom

Homozygous orexin-HAP1 knockout selectively depletes HAP1 in orexin neurons

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

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

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

WT KO

HAP1 knockout reduces neurite extension of orexin neurons in mouse brain

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.

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

5 ~ 45% SucroseInput

WT

KO

WT

KO

WT

KO

Tubulin

Dynactinp150

KinesinLight chain

5 ~ 45% SucroseInput

WT

KO

WT

KO

LAMP1

BDNF

Lack of HAP1 affects the distribution of lysosomes and BDNF

Conclusion

HAP1 deficiency

Intracellular trafficking defect

Impaired neurite extension

Orexin neuronal dysfunction

Feeding and locomotor activity changes