ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
The epilepsies are a common, clinically heterogeneous group of disorders defined by recurrent unprovoked seizures. Here we describe identification of the causative gene in autosomal-dominant partial epilepsy with auditory features (ADPEAF, MIM 600512), a rare form of idiopathic lateral temporal lobe epilepsy characterized by partial seizures with auditory disturbances. We constructed a complete, 4.2-Mb physical map across the genetically implicated disease-gene region, identified 28 putative genes (Fig. 1) and resequenced all or part of 21 genes before identifying presumptive mutations in one copy of the leucine-rich, glioma-inactivated 1 gene (LGI1) in each of five families with ADPEAF. Previous studies have indicated that loss of both copies of LGI1 promotes glial tumor progression. We show that the expression pattern of mouse Lgi1 is predominantly neuronal and is consistent with the anatomic regions involved in temporal lobe epilepsy. Discovery of LGI1 as a cause of ADPEAF suggests new avenues for research on pathogenic mechanisms of idiopathic epilepsies.
Subject(s)
Auditory Diseases, Central/genetics , Epilepsy/genetics , Genes, Dominant , Mutation , Proteins/genetics , Animals , Auditory Diseases, Central/complications , Base Sequence , Chromosome Mapping , Chromosomes, Human, Pair 10 , DNA , Epilepsy/complications , Female , Genotype , Humans , Intracellular Signaling Peptides and Proteins , Male , Pedigree , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
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.
Subject(s)
Dopamine/metabolism , Dyskinesias/prevention & control , Hedgehog Proteins/metabolism , Levodopa/adverse effects , Parkinson Disease/physiopathology , Animals , Disease Models, Animal , Female , Male , MiceABSTRACT
BACKGROUND & AIMS: Hedgehog 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. METHODS: Hedgehog 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. RESULTS: Hedgehog 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. CONCLUSIONS: These 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.
Subject(s)
Body Patterning/genetics , Gastric Mucosa/metabolism , Gastrointestinal Tract/embryology , Hedgehog Proteins/metabolism , Intestine, Small/metabolism , Signal Transduction/genetics , Animals , Body Patterning/physiology , Gastric Mucosa/pathology , Gastrointestinal Tract/growth & development , Gene Expression Regulation, Developmental , Immunohistochemistry , In Situ Hybridization , Intestinal Mucosa/pathology , Intestine, Small/embryology , Intestine, Small/pathology , Mice , Mice, Transgenic , Models, Animal , Stomach/embryology , Stomach/pathologyABSTRACT
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 indicate that vOPCs prevent dOPCs from invading ventral spinal cord laminae and suggest that vOPCs have a unique ability to communicate with injured motor neurons.
Subject(s)
Oligodendrocyte Precursor Cells , Animals , Cell Differentiation , Mice , Motor Neurons , Oligodendroglia , Spinal CordABSTRACT
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.
Subject(s)
Gene Expression , Hedgehog Proteins/genetics , Hippocampus/metabolism , Mossy Fibers, Hippocampal/metabolism , Neurogenesis/genetics , Age Factors , Calbindin 2/genetics , Calbindin 2/metabolism , Cell Proliferation , Cell Survival , GABAergic Neurons/metabolism , Hedgehog Proteins/metabolism , Signal TransductionABSTRACT
Fragile X syndrome, the most common form of inherited mental retardation, is caused by the absence of the FMR-1 gene product FMRP. In addition to the hallmark cognitive defect, other symptoms are also apparent including hyperactivity, seizures and sensory abnormalities including a characteristic increase in sensitivity to auditory, tactile, visual, and olfactory stimuli. Fragile X is a developmental disorder with the first symptoms apparent in the first year of life but little is known about the role of FMRP in developmental processes. The sensory hyperreactivity of fragile X can be reproduced in fmr-1 knockout (KO) mice evident as abnormal audiogenic startle response and increased audiogenic seizure susceptibility. Here, we studied the onset and emergence of the startle deficit in fmr-1 KO mice during development. The startle response was first detectable at the end of the 2nd postnatal week in wild-type mice. The amplitude of startle response showed a substantial increase until the 4th postnatal week followed by a further but moderate increase up to adulthood. Expression of the fmr1 gene was detectable in the startle circuit before the onset and throughout the development of the startle response. Although the onset and amplitude of the startle response were not altered in fmr1 KO mice until the 3rd-4th postnatal week, beyond this age it failed to develop further resulting in an overall response deficit in adult KO mice. This indicates that although Fmrp is dispensable at the initial steps of startle response development, it is necessary for the full development of the response.
Subject(s)
Auditory Pathways/growth & development , Critical Period, Psychological , Fragile X Mental Retardation Protein/physiology , Reflex, Startle/physiology , Acoustic Stimulation/methods , Age Factors , Analysis of Variance , Animals , Auditory Pathways/anatomy & histology , Auditory Pathways/metabolism , Behavior, Animal , Dose-Response Relationship, Radiation , Fragile X Mental Retardation Protein/genetics , In Situ Hybridization/methods , Mice , Mice, Knockout , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reflex, Startle/radiation effects , Reverse Transcriptase Polymerase Chain Reaction/methodsABSTRACT
The Zic genes are a family of zinc finger transcription factors defined by their homology with the Drosophila gene, odd-paired (opa). Zic2 has a critical role in forebrain development, as is evidenced by the fact that, in both mice and humans, diminished expression results in the severe forebrain malformation known as holoprosencephaly. Published information indicates that Zic2 expression is most prominent in the dorsal neural tube/spinal cord and in the hindbrain; however, there is no published description of the pattern of expression of Zic2 in the developing forebrain where the main Zic2 associated phenotype occurs. Using a Zic2-specific antiserum, we present new information about the expression of Zic2 in the developing mouse forebrain. In addition, we show that in sonic hedgehog (Shh) null mice, the expression of Zic2 is expanded ventrally in some structures while absent in others, suggesting that Shh has a role in regulating the expression of Zic2.
Subject(s)
Prosencephalon/metabolism , Transcription Factors/metabolism , Animals , CHO Cells , Cell Nucleus/metabolism , Cricetinae , Fluorescent Antibody Technique , Immunohistochemistry , Mice , Prosencephalon/immunology , Transcription Factors/immunology , TransfectionABSTRACT
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.
Subject(s)
Corpus Striatum/metabolism , Dopaminergic Neurons/metabolism , Hedgehog Proteins/metabolism , Homeostasis/physiology , Nerve Degeneration/metabolism , Parkinson Disease/metabolism , Substantia Nigra/metabolism , Animals , Cholinergic Neurons/metabolism , Cholinergic Neurons/pathology , Corpus Striatum/pathology , Dopaminergic Neurons/pathology , GABAergic Neurons/metabolism , GABAergic Neurons/pathology , Gait/physiology , Glial Cell Line-Derived Neurotrophic Factor/metabolism , Hedgehog Proteins/genetics , Mice , Motor Activity/physiology , Nerve Degeneration/genetics , Nerve Degeneration/pathology , Neural Pathways/metabolism , Neural Pathways/pathology , Parkinson Disease/genetics , Parkinson Disease/pathology , Signal Transduction/physiology , Substantia Nigra/pathologyABSTRACT
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.
Subject(s)
Brain/metabolism , Gene Expression Regulation, Developmental/drug effects , Hedgehog Proteins/genetics , Signal Transduction/drug effects , Thyroid Hormones/pharmacology , Acetylation/drug effects , Animals , Brain/embryology , Brain/growth & development , Cells, Cultured , Female , Fluorescent Antibody Technique , Hedgehog Proteins/metabolism , Hippocampus/embryology , Hippocampus/growth & development , Hippocampus/metabolism , Histones/metabolism , Hypothyroidism/metabolism , In Situ Hybridization , Male , Mice , Mice, Transgenic , Neurons/drug effects , Neurons/metabolism , Patched Receptors , Patched-1 Receptor , Rats , Rats, Sprague-Dawley , Receptors, Cell Surface/genetics , Receptors, Cell Surface/metabolism , Receptors, G-Protein-Coupled/genetics , Receptors, G-Protein-Coupled/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Smoothened Receptor , Thyroid Hormones/metabolism , Triiodothyronine/pharmacologyABSTRACT
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-gamma 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, PKCdelta, had higher mRNA levels in the LM-infected NF-IL6-deficient macrophages as compared with WT. To define the role of PKCdelta during listeriosis, we infected PKCdelta-deficient mice with LM. PKCdelta-deficient mice were highly susceptible to LM infection with increased bacterial burden and enhanced histopathology despite enhanced NF-IL6 mRNA expression. Subsequent studies in PKCdelta-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 PKCdelta as a critical factor for confinement of LM within macrophage phagosomes.
Subject(s)
Listeria monocytogenes/immunology , Macrophages/immunology , Macrophages/microbiology , Phagosomes/immunology , Phagosomes/microbiology , Protein Kinase C-delta/immunology , Animals , Base Sequence , CCAAT-Enhancer-Binding Protein-beta/deficiency , CCAAT-Enhancer-Binding Protein-beta/genetics , CCAAT-Enhancer-Binding Protein-beta/immunology , DNA Primers/genetics , In Vitro Techniques , Interleukin-6/metabolism , Listeria monocytogenes/pathogenicity , Listeriosis/genetics , Listeriosis/immunology , Listeriosis/microbiology , Listeriosis/pathology , Mice , Mice, Knockout , Oligonucleotide Array Sequence Analysis , Protein Kinase C-delta/deficiency , Protein Kinase C-delta/genetics , RNA, Messenger/geneticsABSTRACT
Facial abnormalities in human SHH mutants have implicated the Hedgehog (Hh) pathway in craniofacial development, but early defects in mouse Shh mutants have precluded the experimental analysis of this phenotype. Here, we removed Hh-responsiveness specifically in neural crest cells (NCCs), the multipotent cell type that gives rise to much of the skeleton and connective tissue of the head. In these mutants, many of the NCC-derived skeletal and nonskeletal components are missing, but the NCC-derived neuronal cell types are unaffected. Although the initial formation of branchial arches (BAs) is normal, expression of several Fox genes, specific targets of Hh signaling in cranial NCCs, is lost in the mutant. The spatially restricted expression of Fox genes suggests that they may play an important role in BA patterning. Removing Hh signaling in NCCs also leads to increased apoptosis and decreased cell proliferation in the BAs, which results in facial truncation that is evident by embryonic day 11.5 (E11.5). Together, our results demonstrate that Hh signaling in NCCs is essential for normal patterning and growth of the face. Further, our analysis of Shh-Fox gene regulatory interactions leads us to propose that Fox genes mediate the action of Shh in facial development.