IntroductionL-DOPA-induced dyskinesia (LID) formation requires prominent dopamine oscillations over hours, across years of treatment. Striatal cholinergic interneurons (CIN) have been implicated in both the facilitation and attenuation of LID, but how CIN sense and respond to striatal dopamine levels in this context remains incompletely understood. CIN express both the inhibitory, Gαi-coupled dopamine D2 receptor and the facilitatory, Gαs-coupled dopamine D5 receptor. Although systemic ablation of D5 exacerbates LID, the contribution of CIN-specific D2 expression to LID has not been studied.MethodsWe generated mice with conditional ablation of D2 from choline acetyltransferase-expressing cells (D2ChATKO) and subjected them to unilateral 6-hydroxydopamine lesions followed by chronic L-DOPA dosing. We assessed dyskinetic behaviors across escalating L-DOPA doses and performed postmortem analyses of LID-associated signaling markers in dorsal striatal CIN. Specifically, we quantified p-ERK expression and phosphorylated rpS6240/244, a marker of translational activity, in CIN from mice examined in the L-DOPA ON and OFF states.ResultsD2ChATKO mice exhibited attenuated LID across escalating L-DOPA doses. Postmortem analyses suggested reduced expression of the LID-associated marker p-ERK among CIN of the dorsal striatum. In control mice, L-DOPA increased phosphorylated rpS6240/244 in a subset of CIN located in the dorsolateral striatum, indicating increased translational activity during the L-DOPA ON state. Ablation of D2 from CIN prevented this L-DOPA-associated increase in CIN phosphorylated rpS6240/244.DiscussionTogether, these findings indicate that D2 signaling in CIN promotes LID formation and suggest that CIN D2 is a potential molecular target for mitigating dyskinesias while preserving the therapeutic efficacy of L-DOPA. These results are discussed in the context of recently refined models of how CIN may contribute to aberrant plasticity in the basal ganglia of the Parkinsonian mouse brain.

The striatum is a hub for associative learning where fluctuations in dopamine (DA) and acetylcholine (ACh) regulate behavior. ACh is released by cholinergic interneurons (CIN), which integrate diverse inputs that contextualize DA signals and shape behavior. We previously observed that the GPCR Smoothened (Smo) on CINs suppresses L-DOPA-induced dyskinesias, a motor side effect resulting from medication elevated DA in the Parkinsonian brain. Here, we examine whether Smo signaling modulates ACh dynamics, its coordination with DA, and motor learning in the healthy brain. We find that cholinergic neuron-specific Smo activity bidirectionally modulates ACh inhibition following dopaminergic or cholinergic neuron activity. These effects alter the temporal organization of ACh and its coupling to DA in the dorsolateral striatum. Behaviorally, Smo ablation from cholinergic neurons promotes motor learning and alters adjustments in the effort ortime to obtain reward. These findings identify Smo as a modulator of striatal DA-ACh coordination, striatal learning, and effort-management.

Neuropeptides are key modulators of adult neurocircuits, balancing their sensitivity to both excitation and inhibition, and fine-tuning fast neurotransmitter action under physiological conditions. We found that the mono-carboxypeptidase angiotensin-converting enzyme 2 (ACE2), which can take part in producing and/or processing of several neuroactive peptides in the mammalian brain and is best known for converting the pro-inflammatory peptide angiotensin II (Ang II) to the stress-ameliorating and neuro-protective peptide angiotensin 1–7 (Ang 1–7), is broadly expressed in the striatum. Cholinergic interneurons (CIN) of the striatum, known to express multiple peptide receptors also co-expressed the corresponding and functionally opposing receptors angiotensin type 1 receptor (AT1R) for Ang II and mas receptor (MasR) for Ang 1–7. Accordingly, the conditional, semi acute ablation and/or local pharmacological inhibition of ACE2 increased the frequency of acetylcholine (ACh) bursts, reduced the amplitude of dopamine (DA)-modulated pausing of CIN activity, and disrupted the temporal coordination of extracellular levels of ACh and DA during burst events. Further, ablation of ACE2 in the DLS biased directional movement and impaired motor skill learning. Coinjection of the AT1R inhibitor losartan and the dopamine D2 receptor (D2R) agonist quinpirole reduced steady state level cholinergic activity in an additive manner, and proximity ligation supported close spatial association of AT1R and D2R on CINs. Together our study provides evidence that striatal produced ACE2 impinges on the dynamics of ACh and DA and impacts action selection and motor learning through functional and structural interactions of peptidergic and dopaminergic signaling on CIN. ![Figure][1] Graphical Abstract ### Competing Interest Statement The authors have declared no competing interest. NIH, U54MD017979 [1]: pending:yes

Motor neurons (MNs) and oligodendrocyte precursor cells (OPCs) emerge sequentially from the pMN precursor domain during spinal cord development. MNs diversify into muscle specific subtypes and settle in stereotypic locations in the ventral horns. In contrast, OPCs are mobile and appear to evenly populate the parenchyma. Whether earlier born MNs influence OPC production is controversial. We found that Sonic Hedgehog signaling emanating from nascent MNs of the lateral motor column is critical for maintaining a larger and more yielding pMN domain at limb levels compared to trunk levels during OPC production. Reduced Shh signaling resulted in unrecoverable diminishment of pMN domain based OPC production leaving the spinal cord impoverished of OPC. Our results suggest that production of OPC at limb levels is contingent on completion of MN production.

Background:Striatal Cholinergic Interneurons (CIN) are drivers of L-Dopa induced Dyskinesias (LID). However, what signaling pathways elicit aberrant CIN activity remains unclear. CIN express D2 and D5 receptors suggesting repeated activation of these receptors in response to L-Dopa could promote LID. While the role of D5 in this process has recently been probed, little is known about the role of D2. Method:Mice with CIN-specific D2 ablation (D2 CIN KO) underwent unilateral 6-OHDA lesion and chronic L-Dopa dosing, throughout which LID severity was quantified. The effect of D2 CIN KO on histological markers of LID severity and CIN activity were also quantified postmortem. Results:D2 CIN KO attenuated LID across L-Dopa doses, reduced expression of histological LID marker p-ERK, and prevented L-Dopa-induced increases in CIN activity marker p-rpS6 in the dorsolateral striatum. Conclusion:The activation of D2 specifically on CIN is a key driver of LID.

L-Dopa induced dyskinesia (LID) is a debilitating side effect of dopamine replacement therapy for Parkinson's Disease. The mechanistic underpinnings of LID remain obscure. Here we report that diminished sonic hedgehog (Shh) signaling in the basal ganglia caused by the degeneration of midbrain dopamine neurons facilitates the formation and expression of LID. We find that the pharmacological activation of Smoothened, a downstream effector of Shh, attenuates LID in the neurotoxic 6-OHDA- and genetic aphakia mouse models of Parkinson's Disease. Employing conditional genetic loss-of-function approaches, we show that reducing Shh secretion from dopamine neurons or Smoothened activity in cholinergic interneurons promotes LID. Conversely, the selective expression of constitutively active Smoothened in cholinergic interneurons is sufficient to render the sensitized aphakia model of Parkinson's Disease resistant to LID. Furthermore, acute depletion of Shh from dopamine neurons through prolonged optogenetic stimulation in otherwise intact mice and in the absence of L-Dopa produces LID-like involuntary movements. These findings indicate that augmenting Shh signaling in the L-Dopa treated brain may be a promising therapeutic approach for mitigating the dyskinetic side effects of long-term treatment with L-Dopa.

Oligodendrocyte precursor cells (OPCs) arise sequentially first from a ventral and then from a dorsal precursor domain at the end of neurogenesis during spinal cord development. Whether the sequential production of OPCs is of physiological significance has not been examined. Here we show that ablating Shh signaling from nascent ventricular zone derivatives and partially from the floor plate results in a severe diminishment of ventral derived OPCs but normal numbers of motor neurons in the postnatal spinal cord. In the absence of ventral vOPCs, dorsal dOPCs populate the entire spinal cord resulting in an increased OPC density in the ventral horns. These OPCs take on an altered morphology, do not participate in the removal of excitatory vGlut1 synapses from injured motor neurons, and exhibit morphological features similar to those found in the vicinity of motor neurons in the SOD1 mouse model of Amyotrophic Lateral Sclerosis (ALS). Our data indicates that vOPCs prevent dOPCs from invading ventral spinal cord laminae and suggests that vOPCs have a unique ability to communicate with injured motor neurons.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The dopamine D5 receptor (D5R) is a Gα s -coupled dopamine receptor belonging to the dopamine D1-like receptor family. Together with the dopamine D2 receptor it is highly expressed in striatal cholinergic interneurons and therefore is poised to be a positive regulator of cholinergic activity in response to L-DOPA in the dopamine-depleted parkinsonian brain. Tonically active cholinergic interneurons become dysregulated during chronic L-DOPA administration and participate in the expression of L-DOPA induced dyskinesia. The molecular mechanisms involved in this process have not been elucidated, however a correlation between dyskinesia severity and pERK expression in cholinergic cells has been described. To better understand the function of the D5 receptor and how it affects cholinergic interneurons in L-DOPA induced dyskinesia, we used D5R knockout mice that were rendered parkinsonian by unilateral 6-OHDA injection. In the KO mice, expression of pERK was strongly reduced indicating that activation of these cells is at least in part driven by the D5 receptor. Similarly, pS6, another marker for the activity status of cholinergic interneurons was also reduced. However, mice lacking D5R exhibited slightly worsened locomotor performance in response to L-DOPA and enhanced LID scores. Our findings suggest that D5R can modulate L-DOPA induced dyskinesia and is a critical activator of CINs via pERK and pS6.

L-Dopa induced dyskinesia (LID) is a debilitating side effect of dopamine replacement therapy for Parkinson’s Disease. The mechanistic underpinnings of LID remain obscure. Here we report that diminshed sonic hedgehog (Shh) signaling in the basal ganglia caused by the degeneration of midbrain dopamine neurons (DANs) facilitates the formation and expression of LID. We demonstrate that augmenting Shh signaling with agonists of the Shh effector Smoothened attenuates LID in mouse and macaque models of PD. Employing conditional genetic loss-of-function approaches, we show that reducing Shh secretion from DANs or Smo activity in cholinergic interneurons (CINs) promotes LID. Conversely, the selective expression of constitutively active Smo (SmoM2) in CINs is sufficient to render the sensitized aphakia model of PD resistant to LID. Furthermore, acute depletion of Shh from DANs through prolonged optogenetic stimulation in otherwise intact mice and in the absence of L-Dopa produces LID-like involuntary movements. These findings indicate that augmenting Shh signaling in the L-Dopa treated brain may be a promising and unexpected novel therapeutic approach for mitigating the dyskinetic side effects of long-term treatment with L-Dopa### Competing Interest StatementThe authors have declared no competing interest.

Graded Sonic Hedgehog (Shh) signaling emanating from notochord and floorplate patterns the early neural tube. Soon thereafter, Shh signaling strength within the ventricular zone becomes dis-contiguous and discontinuous along the ventral to dorsal axis suggesting a distribution of Shh that cannot be achieved by diffusion alone. Here we discover that sequential activation of Shh expression by ventricular zone derivatives is critical for counteracting a precocious exhaustion of the Olig2 precursor cell population of the pMN domain at the end of motor neuron genesis and during the subsequent phase of ventral oligodendrocyte precursor production. Selective expression of Shh by motor neurons of the lateral motor column at the beginning of oligodendrogenesis ensures a more yielding pMN domain at limb levels compared to thoracic levels. Thus, patterned expression of Shh by ventricular zone derivatives including earlier born neurons contributes to the scaling of the spinal cord along the anterior – posterior axis by regulating the activity of a select ventricular zone precursor domain at later stages of development.

Sonic hedgehog ( Shh ) is a multifunctional signaling protein governing pattern formation, proliferation and cell survival during embryogenesis. In the adult brain, Shh has neurotrophic function and is implicated in hippocampal neurogenesis but the cellular source of Shh in the hippocampus remains ill defined. Here, we utilize a gene expression tracer allele of Shh ( Shh - nlacZ ) which allowed the identification of a subpopulation of hilar neurons known as mossy cells (MCs) as a prominent and dynamic source of Shh within the dentate gyrus. AAV-Cre mediated ablation of Shh in the adult dentate gyrus led to a marked degeneration of MCs. Conversely, chemical stimulation of hippocampal neurons using the epileptogenic agent kainic acid (KA) increased the number of Shh + MCs indicating that the expression of Shh by MCs confers a survival advantage during the response to excitotoxic insults. In addition, ablation of Shh in the adult dentate gyrus led to increased neural precursor cell proliferation and their migration into the subgranular cell layer demonstrating that MCs-generated Shh is a key modulator of hippocampal neurogenesis.

772–783 (2006). 18. J. Briscoe, EMBO J. 28, 457–465 (2009). 19. T. Mori et al., Glia 54, 21–34 (2006). 20. F. Long, X. M. Zhang, S. Karp, Y. Yang, A. P. McMahon, Development 128, 5099–5108 (2001). 21. B. Djukic, K. B. Casper, B. D. Philpot, L.-S. Chin, K. D. McCarthy, J. Neurosci. 27, 11354–11365 (2007). 22. R. V. Pearse 2nd, K. J. Vogan, C. J. Tabin, Dev. Biol. 239, 15–29 (2001). 23. J. Jeong, J. Mao, T. Tenzen, A. H. Kottmann, A. P. McMahon, Genes Dev. 18, 937–951 (2004). 24. A. D. R. Garcia, R. Petrova, L. Eng, A. L. Joyner, J. Neurosci. 30, 13597–13608 (2010). 25. C. C. Harwell et al., Neuron 73, 1116–1126 (2012). 26. L. E. Gonzalez-Reyes et al., Neuron 75, 306–319 (2012). 27. M. K. Cooper, J. A. Porter, K. E. Young, P. A. Beachy, Science 280, 1603–1607 (1998). 28. A. Rohner et al., Mol. Cancer Ther. 11, 57–65 (2012). 29. A. V. Molofsky et al., Nature 509, 189–194 (2014). 30. X. Tong et al., Nat. Neurosci. 17, 694–703 (2014).

Non cell-autonomous processes are thought to play critical roles in the cellular maintenance of the healthy and diseased brain but mechanistic details remain unclear. We report that the interruption of a non cell-autonomous mode of sonic hedgehog (Shh) signaling originating from dopaminergic neurons causes progressive, adult-onset degeneration of dopaminergic, cholinergic, and fast spiking GABAergic neurons of the mesostriatal circuit, imbalance of cholinergic and dopaminergic neurotransmission, and motor deficits reminiscent of Parkinson's disease. Variable Shh signaling results in graded inhibition of muscarinic autoreceptor- and glial cell line-derived neurotrophic factor (GDNF)-expression in the striatum. Reciprocally, graded signals that emanate from striatal cholinergic neurons and engage the canonical GDNF receptor Ret inhibit Shh expression in dopaminergic neurons. Thus, we discovered a mechanism for neuronal subtype specific and reciprocal communication that is essential for neurochemical and structural homeostasis in the nigrostriatal circuit. These results provide integrative insights into non cell-autonomous processes likely at play in neurodegenerative conditions such as Parkinson's disease.

Thyroid hormone is important for development and plasticity in the immature and adult mammalian brain. Several thyroid hormone-responsive genes are regulated during specific developmental time windows, with relatively few influenced across the lifespan. We provide novel evidence that thyroid hormone regulates expression of the key developmental morphogen sonic hedgehog (Shh), and its coreceptors patched (Ptc) and smoothened (Smo), in the early embryonic and adult forebrain. Maternal hypo- and hyperthyroidism bidirectionally influenced Shh mRNA in embryonic forebrain signaling centers at stages before fetal thyroid hormone synthesis. Further, Smo and Ptc expression were significantly decreased in the forebrain of embryos derived from hypothyroid dams. Adult-onset thyroid hormone perturbations also regulated expression of the Shh pathway bidirectionally, with a significant induction of Shh, Ptc, and Smo after hyperthyroidism and a decline in Smo expression in the hypothyroid brain. Short-term T3 administration resulted in a significant induction of cortical Shh mRNA expression and also enhanced reporter gene expression in Shh+/LacZ mice. Further, acute T3 treatment of cortical neuronal cultures resulted in a rapid and significant increase in Shh mRNA, suggesting direct effects. Chromatin immunoprecipitation assays performed on adult neocortex indicated enhanced histone acetylation at the Shh promoter after acute T3 administration, providing further support that Shh is a thyroid hormone-responsive gene. Our results indicate that maternal and adult-onset perturbations of euthyroid status cause robust and region-specific changes in the Shh pathway in the embryonic and adult forebrain, implicating Shh as a possible mechanistic link for specific neurodevelopmental effects of thyroid hormone.

BACKGROUND & AIMSHedgehog signaling is critical in gastrointestinal patterning. Mice deficient in Hedgehog signaling exhibit abnormalities that mirror deformities seen in the human VACTERL (vertebral, anal, cardiac, tracheal, esophageal, renal, limb) association. However, the direction of Hedgehog signal flow is controversial and the cellular targets of Hedgehog signaling change with time during development. We profiled cellular Hedgehog response patterns from embryonic day 10.5 (E10.5) to adult in murine antrum, pyloric region, small intestine, and colon.METHODSHedgehog signaling was profiled using Hedgehog pathway reporter mice and in situ hybridization. Cellular targets were identified by immunostaining. Ihh-overexpressing transgenic animals were generated and analyzed.RESULTSHedgehog signaling is strictly paracrine from antrum to colon throughout embryonic and adult life. Novel findings include the following: mesothelial cells of the serosa transduce Hedgehog signals in fetal life; the hindgut epithelium expresses Ptch but not Gli1 at E10.5; the 2 layers of the muscularis externa respond differently to Hedgehog signals; organogenesis of the pyloric sphincter is associated with robust Hedgehog signaling; dramatically different Hedgehog responses characterize stomach and intestine at E16; and after birth, the muscularis mucosa and villus smooth muscle consist primarily of Hedgehog-responsive cells and Hh levels actively modulate villus core smooth muscle.CONCLUSIONSThese studies reveal a previously unrecognized association of paracrine Hedgehog signaling with several gastrointestinal patterning events involving the serosa, pylorus, and villus smooth muscle. The results may have implications for several human anomalies and could potentially expand the spectrum of the human VACTERL association.

Activation of macrophages and subsequent “killing” effector functions against infectious pathogens are essential for the establishment of protective immunity. NF-IL6 is a transcription factor downstream of IFN-γ and TNF in the macrophage activation pathway required for bacterial killing. Comparison of microarray expression profiles of Listeria monocytogenes (LM)-infected macrophages from WT and NF-IL6-deficient mice enabled us to identify candidate genes downstream of NF-IL6 involved in the unknown pathways of LM killing independent of reactive oxygen intermediates and reactive nitrogen intermediates. One differentially expressed gene, PKCδ, had higher mRNA levels in the LM-infected NF-IL6-deficient macrophages as compared with WT. To define the role of PKCδ during listeriosis, we infected PKCδ-deficient mice with LM. PKCδ-deficient mice were highly susceptible to LM infection with increased bacterial burden and enhanced histopathology despite enhanced NF-IL6 mRNA expression. Subsequent studies in PKCδ-deficient macrophages demonstrated that, despite elevated levels of proinflammatory cytokines and NO production, increased escape of LM from the phagosome into the cytoplasm and uncontrolled bacterial growth occurred. Taken together these data identified PKCδ as a critical factor for confinement of LM within macrophage phagosomes.