Effect of the sonic hedgehog receptor smoothened on the survival and function of dopaminergic neurons.
To determine the influence of the sonic hedgehog (shh) pathway and its receptor smoothened (smo), on the survival and functionality of dopaminergic (DA) neurons.



During early development, shh induces the differentiation of DA neurons. However, it is unknown whether shh signaling is required in the maturation or maintenance of DA neurons during later development and adulthood due to the lethality of traditional shh knockout models.



We utilized the cre-loxP system to achieve late developmental stage and cell type-specific deletion of the shh receptor, smo, in DA neurons by crossing DATcre (dopamine transporter) mice with Smo(loxP/loxP) mice. We assessed for differences between knockout (ko) and wildtype (wt) mice using combined histochemistry, gene expression analysis, and behavioral evaluation. Number and size of DA neurons in ventral midbrain and the DA neural terminal density in striatum were measured using unbiased stereological quantification. The survival of DA neurons under neurotoxin challenge was examined in the unilateral 6-hydroxydopamine (6-OHDA) Parkinson's disease animal model and the more subtle function under challenge of the dopaminergic system was examined by methamphetamine single- and repeated challenge in wt and ko mice.



Tyrosine hydroxylase (TH) positive neuronal counts and neuronal size in substantia nigra (SN) and ventral tegmental area (VTA) showed no difference between wt and DAT-Smo ko mice in young (5months) or aged (22months) mice. There was also no difference in the striatal DA projections between wt and ko mice in both age groups. In unilateral striatal 6-OHDA lesions modeling Parkinson's disease, using stereotaxic injection of 6-OHDA intrastriatally led to loss of dopaminergic neurons in SN and diminished TH positive projections in striatum. However, there was no differences in survival of DA neurons between wt and ko mice. DAT-Smo ko mice demonstrated hyperactivity compared to wt mice at 5months, but showed no difference in activity at 22months. When injected with a one-time bolus of methamphetamine (METH), despite the higher basal locomotion activity, DAT-Smo ko mice showed a diminished response to a single METH challenge. In METH sensitization testing, ko mice showed decreased sensitization compared to wt mice without evidence of a delayed shift in dynamics of sensitization. Gene expression analysis showed decreased gene expression of smo, Gli 1 (known target gene of smo) and BDNF (brain-derived neurotrophic factor) in the SN. Gene expression was not altered in striatum for the genes examined in this study including dopamine receptor genes, neurotropic genes such as Glial cell line-derived neurotrophic factor (GDNF), and bone morphogenetic protein 7 (BMP7).



Our study showed the smo receptor function is not required for the maturation and survival of DA neurons during late development, aging or under stress challenge. However, smo function has an influence on behavior in young adult mice and in responses of mice to a drug that modulates DA neurochemistry through regulation of gene expression in DA neurons. Since young adult DAT-smo ko mice show hyperactivity and altered response to a psychostimulant drug (METH), this may indicate the involvement of the shh pathway in the development of functional changes that manifest as alterations in DA pathway dynamics.
Department of Neurological Surgery, University Hospitals Case Medical Center, Case Western Reserve University, Cleveland, USA.
Xiaofei
X
Zhou
Department of Neurological Surgery, University Hospitals Case Medical Center, Case Western Reserve University, Cleveland, USA.
Jonathan
J
Pace
Department of Neurological Surgery, University Hospitals Case Medical Center, Case Western Reserve University, Cleveland, USA.
Emily
E
Filichia
Department of Neurological Surgery, University Hospitals Case Medical Center, Case Western Reserve University, Cleveland, USA.
Tao
T
Lv
Department of Neurological Surgery, University Hospitals Case Medical Center, Case Western Reserve University, Cleveland, USA.
Brandon
B
Davis
Department of Neurological Surgery, University Hospitals Case Medical Center, Case Western Reserve University, Cleveland, USA.
Barry
B
Hoffer
Department of Neurological Surgery, University Hospitals Case Medical Center, Case Western Reserve University, Cleveland, USA.
Warren
W
Selman
Department of Neurological Surgery, University Hospitals Case Medical Center, Case Western Reserve University, Cleveland, USA. Electronic address: yxl710@case.edu.
Yu
Y
Luo
D000661:Amphetamine / Q000494:pharmacology; D000818:Animals; D001142:Aromatic-L-Amino-Acid Decarboxylases / Q000378:metabolism; D019208:Brain-Derived Neurotrophic Factor / Q000235:genetics / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics; D000697:Central Nervous System Stimulants / Q000494:pharmacology; D003342:Corpus Striatum / Q000166:cytology; D050483:Dopamine Plasma Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism; D059290:Dopaminergic Neurons / Q000187:drug effects / Q000502:physiology*; D008124:Locomotion / Q000187:drug effects / Q000235:genetics; D051379:Mice; D018345:Mice, Knockout; D057126:Nuclear Receptor Subfamily 4, Group A, Member 2 / Q000235:genetics / Q000378:metabolism; D016627:Oxidopamine / Q000633:toxicity; D000072086:Smoothened Receptor / Q000235:genetics / Q000378:metabolism*; D013378:Substantia Nigra / Q000166:cytology; D013565:Sympatholytics / Q000633:toxicity; D013997:Time Factors; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism; D050493:Vesicular Monoamine Transport Proteins / Q000378:metabolism
D019208:Brain-Derived Neurotrophic Factor; D000697:Central Nervous System Stimulants; D050483:Dopamine Plasma Membrane Transport Proteins; C539883:Nr4a2 protein, mouse; D057126:Nuclear Receptor Subfamily 4, Group A, Member 2; C491022:Smo protein, mouse; D000072086:Smoothened Receptor; D013565:Sympatholytics; D050493:Vesicular Monoamine Transport Proteins; D016627:Oxidopamine; D000661:Amphetamine; D014446:Tyrosine 3-Monooxygenase; D001142:Aromatic-L-Amino-Acid Decarboxylases
6-OHDA; Dopaminergic neurons; Parkinson's disease; Smoothened; Sonic hedgehog