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1.
Elife ; 52016 09 20.
Article in English | MEDLINE | ID: mdl-27648578

ABSTRACT

The regulation of protein degradation is essential for maintaining the appropriate environment to coordinate complex cell signaling events and to promote cellular remodeling. The Autophagy linked FYVE protein (Alfy), previously identified as a molecular scaffold between the ubiquitinated cargo and the autophagic machinery, is highly expressed in the developing central nervous system, indicating that this pathway may have yet unexplored roles in neurodevelopment. To examine this possibility, we used mouse genetics to eliminate Alfy expression. We report that this evolutionarily conserved protein is required for the formation of axonal tracts throughout the brain and spinal cord, including the formation of the major forebrain commissures. Consistent with a phenotype reflecting a failure in axon guidance, the loss of Alfy in mice disrupts localization of glial guidepost cells, and attenuates axon outgrowth in response to Netrin-1. These findings further support the growing indication that macroautophagy plays a key role in the developing CNS.


Subject(s)
Brain/embryology , Neural Pathways/embryology , Neurons/physiology , Vesicular Transport Proteins/metabolism , Adaptor Proteins, Signal Transducing , Animals , Autophagy-Related Proteins , Gene Knockout Techniques , Mice, Inbred C57BL
2.
Neurobiol Dis ; 82: 359-371, 2015 Oct.
Article in English | MEDLINE | ID: mdl-26192625

ABSTRACT

Mutations in the gene for LRRK2 are the most common cause of familial Parkinson's disease (PD) and patients with these mutations manifest clinical features that are indistinguishable from those of the more common sporadic form. Thus, investigations of disease mechanisms based on disease-causing LRRK2 mutations can be expected to shed light on the more common sporadic form as well as the inherited form. We have shown that as human BAC transgenic hLRRK2(R1441G) mice age, they exhibit two abnormalities in the nigrostriatal dopaminergic system: an axonopathy and a diminished number of dendrites in the substantia nigra (SN). To better understand disease mechanisms it is useful to determine where in the affected neural system the pathology first begins. We therefore examined the nigrostriatal dopaminergic system in young mice to determine the initial site of pathology. Brains from hLRRK2(R1441G) and littermate control mice at 2-4months of age were examined by immunohistochemistry, anterograde fluorescent axon labeling and ultrastructural analysis. SN neurons, their projecting axons and the striatal terminal fields were assessed. The first identifiable abnormality in this system is an axonopathy characterized by giant polymorphic axon spheroids, the presence of intra-axonal autophagic vacuoles and intra-axonal myelin invagination. An initial involvement of axons has also been reported for other genetic models of PD. These observations support the concept that axons are involved early in the course of the disease. We suggest that effective neuroprotective approaches will be aimed at preventing axonal degeneration.


Subject(s)
Axons/pathology , Brain/pathology , Parkinsonian Disorders/pathology , Aging/metabolism , Aging/pathology , Animals , Axons/metabolism , Brain/metabolism , Dermoscopy , Disease Progression , Humans , Immunohistochemistry , In Situ Hybridization , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2 , Mice, Inbred C57BL , Mice, Transgenic , Microscopy, Confocal , Microscopy, Electron, Transmission , Neuroanatomical Tract-Tracing Techniques , Parkinsonian Disorders/genetics , Parkinsonian Disorders/metabolism , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , RNA, Messenger/metabolism , Stilbamidines
3.
Neurobiol Dis ; 44(2): 215-22, 2011 Nov.
Article in English | MEDLINE | ID: mdl-21782946

ABSTRACT

The mechanisms underlying the chronic neurodegeneration that occurs in Parkinson's disease (PD) are unknown. One emerging hypothesis is that neural systems deteriorate and eventually degenerate due to a primary failure of either extrinsic neurotrophic support or the intrinsic cellular pathways that mediate such support. One of the cellular pathways that have been often identified in mediating neurotrophic effects is that of PI3K/Akt signaling. In addition, recent observations have suggested a primary failure of PI3K/Akt signaling in animal models and in PD patients. Therefore, to explore the possible role of endogenous Akt signaling in maintaining the viability and functionality of substantia nigra (SN) dopamine neurons, one of the principal systems affected in PD, we have used an adeno-associated viral vector to transduce them with a dominant negative (DN) form of Akt, the pleckstrin homology (PH) domain alone (DN(PH)-Akt). In addition, we have examined the effect of DN(PH)-Akt in murine models of two risk factors for human PD: advanced age and increased expression of α-synuclein. We find that transduction of these neurons in normal adult mice has no effect on any aspect of their morphology at 4 or 7weeks. However, in both aged mice and in transgenic mice with increased expression of human α-synuclein we observe decreased phenotypic expression of the catecholamine synthetic enzyme tyrosine hydroxylase (TH) in dopaminergic axons and terminals in the striatum. In aged transgenic α-synuclein over-expressing mice this reduction was 2-fold as great. We conclude that the two principal risk factors for human PD, advanced age and increased expression of α-synuclein, reveal a dependence of dopaminergic neurons on endogenous Akt signaling for maintenance of axonal phenotype.


Subject(s)
Aging/physiology , Axons/physiology , Dopaminergic Neurons/physiology , Parkinsonian Disorders/physiopathology , Proto-Oncogene Proteins c-akt/physiology , Signal Transduction/physiology , alpha-Synuclein/genetics , Aging/metabolism , Animals , Axons/pathology , Disease Models, Animal , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Parkinsonian Disorders/genetics , Parkinsonian Disorders/metabolism , Proto-Oncogene Proteins c-akt/genetics , alpha-Synuclein/biosynthesis
4.
Ann Neurol ; 70(1): 110-20, 2011 Jul.
Article in English | MEDLINE | ID: mdl-21437936

ABSTRACT

OBJECTIVE: A prevailing concept in neuroscience has been that the adult mammalian central nervous system is incapable of restorative axon regeneration. Recent evidence, however, has suggested that reactivation of intrinsic cellular programs regulated by protein kinase B (Akt)/mammalian target of rapamycin (mTor) signaling may restore this ability. METHODS: To assess this possibility in the brain, we have examined the ability of adenoassociated virus (AAV)-mediated transduction of dopaminergic neurons of the substantia nigra (SN) with constitutively active forms of the kinase Akt and the GTPase Ras homolog enriched in brain (Rheb) to induce regrowth of axons after they have been destroyed by neurotoxin lesion. RESULTS: Both constitutively active myristoylated Akt and hRheb(S16H) induce regrowth of axons from dopaminergic neurons to their target, the striatum. Histological analysis demonstrates that these new axons achieve morphologically accurate reinnervation. In addition, functional reintegration into target circuitry is achieved, as indicated by partial behavioral recovery. INTERPRETATION: We conclude that regrowth of axons within the adult nigrostriatal projection, a system that is prominently affected in Parkinson's disease, can be achieved by activation of Akt/mTor signaling in surviving endogenous mesencephalic dopaminergic neurons by viral vector transduction.


Subject(s)
Axons/physiology , Dopamine/physiology , Monomeric GTP-Binding Proteins/administration & dosage , Nerve Regeneration/physiology , Neuropeptides/administration & dosage , Proto-Oncogene Proteins c-akt/administration & dosage , Signal Transduction , Animals , Axons/metabolism , Dependovirus/genetics , Genetic Vectors/administration & dosage , Genetic Vectors/genetics , Mice , Mice, Inbred C57BL , Mice, Transgenic , Monomeric GTP-Binding Proteins/genetics , Nerve Regeneration/genetics , Neurons/cytology , Neurons/pathology , Neurons/virology , Neuropeptides/genetics , Parkinson Disease/genetics , Parkinson Disease/metabolism , Parkinson Disease/pathology , Proto-Oncogene Proteins c-akt/genetics , Ras Homolog Enriched in Brain Protein , Signal Transduction/genetics , Substantia Nigra/metabolism , Substantia Nigra/pathology , Substantia Nigra/virology
5.
J Neurosci ; 31(6): 2125-35, 2011 Feb 09.
Article in English | MEDLINE | ID: mdl-21307249

ABSTRACT

Axon degeneration is a hallmark of neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease. Such degeneration is not a passive event but rather an active process mediated by mechanisms that are distinct from the canonical pathways of programmed cell death that mediate destruction of the cell soma. Little is known of the diverse mechanisms involved, particularly those of retrograde axon degeneration. We have previously observed in living animal models of degeneration in the nigrostriatal projection that a constitutively active form of the kinase, myristoylated Akt (Myr-Akt), demonstrates an ability to suppress programmed cell death and preserve the soma of dopamine neurons. Here, we show in both neurotoxin and physical injury (axotomy) models that Myr-Akt is also able to preserve dopaminergic axons due to suppression of acute retrograde axon degeneration. This cellular phenotype is associated with increased mammalian target of rapamycin (mTor) activity and can be recapitulated by a constitutively active form of the small GTPase Rheb, an upstream activator of mTor. Axon degeneration in these models is accompanied by the occurrence of macroautophagy, which is suppressed by Myr-Akt. Conditional deletion of the essential autophagy mediator Atg7 in adult mice also achieves striking axon protection in these acute models of retrograde degeneration. The protection afforded by both Myr-Akt and Atg7 deletion is robust and lasting, because it is still observed as protection of both axons and dopaminergic striatal innervation weeks after injury. We conclude that acute retrograde axon degeneration is regulated by Akt/Rheb/mTor signaling pathways.


Subject(s)
Autophagy/physiology , Axons/metabolism , Dopamine/metabolism , Neurons/pathology , Proto-Oncogene Proteins c-akt/metabolism , Retrograde Degeneration/metabolism , Retrograde Degeneration/pathology , Animals , Autophagy/drug effects , Autophagy-Related Protein 7 , Axons/drug effects , Axons/ultrastructure , Dependovirus/genetics , Disease Models, Animal , Gene Expression Regulation/drug effects , Gene Expression Regulation/physiology , Green Fluorescent Proteins/genetics , Medial Forebrain Bundle/pathology , Mice , Mice, Inbred C57BL , Microscopy, Confocal , Microscopy, Electron, Transmission/methods , Microtubule-Associated Proteins/metabolism , Oxidopamine/adverse effects , Proto-Oncogene Proteins c-akt/genetics , Retrograde Degeneration/etiology , Signal Transduction/drug effects , Signal Transduction/genetics , Substantia Nigra/pathology , TOR Serine-Threonine Kinases/metabolism , Tyrosine 3-Monooxygenase/metabolism
6.
Nat Neurosci ; 12(7): 826-8, 2009 Jul.
Article in English | MEDLINE | ID: mdl-19503083

ABSTRACT

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common genetic cause of Parkinson's disease. We created a LRRK2 transgenic mouse model that recapitulates cardinal features of the disease: an age-dependent and levodopa-responsive slowness of movement associated with diminished dopamine release and axonal pathology of nigrostriatal dopaminergic projection. These mice provide a valid model of Parkinson's disease and are a resource for the investigation of pathogenesis and therapeutics.


Subject(s)
Disease Models, Animal , Mice, Transgenic , Parkinson Disease , Protein Serine-Threonine Kinases/genetics , Age Factors , Animals , Antiparkinson Agents/therapeutic use , Apomorphine/therapeutic use , Brain/drug effects , Brain/pathology , Brain/physiopathology , Chromosomes, Artificial, Bacterial , Dopamine/metabolism , Dopamine Uptake Inhibitors/pharmacology , Gene Transfer Techniques , Humans , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2 , Levodopa/therapeutic use , Mice , Movement Disorders/drug therapy , Movement Disorders/genetics , Mutation, Missense , Neurons/pathology , Neurons/physiology , Nomifensine/pharmacology , Parkinson Disease/genetics , Parkinson Disease/pathology , Parkinson Disease/physiopathology , Protein Serine-Threonine Kinases/metabolism
7.
J Neurochem ; 110(1): 23-33, 2009 Jul.
Article in English | MEDLINE | ID: mdl-19490361

ABSTRACT

Following mitosis, specification and migration during embryogenesis, dopamine neurons of the mesencephalon undergo a postnatal naturally occurring cell death event that determines their final adult number, and a period of axonal growth that determines pattern and extent of target contacts. While a number of neurotrophic factors have been suggested to regulate these developmental events, little is known, especially in vivo, of the cell signaling pathways that mediate these effects. We have examined the possible role of Akt/Protein Kinase B by transduction of these neurons in vivo with adeno-associated viral vectors to express either a constitutively active or a dominant negative form of Akt/protein kinase B. We find that Akt regulates multiple features of the postnatal development of these neurons, including the magnitude of the apoptotic developmental cell death event, neuron size, and the extent of target innervation of the striatum. Given the diversity and magnitude of its effects, the regulation of the development of these neurons by Akt may have implications for the many psychiatric and neurologic diseases in which these neurons may play a role.


Subject(s)
Cell Differentiation/genetics , Neurons/enzymology , Proto-Oncogene Proteins c-akt/genetics , Substantia Nigra/enzymology , Substantia Nigra/growth & development , Animals , Animals, Newborn , Apoptosis/genetics , Cell Proliferation , Cell Size , Dopamine/metabolism , Genetic Vectors/genetics , Growth Cones/enzymology , Growth Cones/ultrastructure , Immunohistochemistry , Male , Neural Pathways/cytology , Neural Pathways/enzymology , Neural Pathways/growth & development , Neurogenesis/genetics , Neurons/cytology , Rats , Rats, Sprague-Dawley , Substantia Nigra/cytology , Transduction, Genetic/methods
8.
Mol Cell Neurosci ; 41(4): 440-7, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19409492

ABSTRACT

Brain-derived neurotrophic factor (BDNF) was the first purified molecule identified to directly support the development of mesencephalic dopamine neurons. However, its physiologic role has remained unknown. Based on patterns of expression, it is unlikely to serve as a target-derived neurotrophic factor, but it may instead act locally in the mesencephalon, either released by afferent projections, or in autocrine fashion. To assess a possible local role, we blocked BDNF signaling in the substantia nigra (SN) of postnatal rats by injection of either neutralizing antibodies or a peptide antagonist. These treatments increased the magnitude of developmental cell death in the SN, indicating that endogenous local BDNF does play a regulatory role. However, we also find that elimination of BDNF in brain throughout postnatal development in BDNF(fl/fl):Nestin-Cre mice has no effect on the adult number of SN dopamine neurons. We postulate that other forms of trophic support may compensate for the elimination of BDNF during early development. Although the number of SN dopamine neurons is unchanged, their organization is disrupted. We conclude that BDNF plays a physiologic role in the postnatal development of SN dopamine neurons.


Subject(s)
Brain-Derived Neurotrophic Factor/metabolism , Dopamine/metabolism , Gene Expression Regulation, Developmental/genetics , Neurons/physiology , Substantia Nigra/cytology , Substantia Nigra/growth & development , Age Factors , Analysis of Variance , Animals , Animals, Newborn , Antibodies/pharmacology , Brain-Derived Neurotrophic Factor/antagonists & inhibitors , Brain-Derived Neurotrophic Factor/genetics , Brain-Derived Neurotrophic Factor/immunology , Cell Death/physiology , Female , Gene Expression Regulation, Developmental/drug effects , In Situ Nick-End Labeling/methods , Intermediate Filament Proteins/genetics , Mice , Mice, Inbred C57BL , Mice, Transgenic , Nerve Tissue Proteins/genetics , Nestin , Neurons/drug effects , Peptides/pharmacology , Pregnancy , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Tyrosine 3-Monooxygenase/metabolism
9.
J Neurochem ; 107(6): 1578-88, 2008 Dec.
Article in English | MEDLINE | ID: mdl-19014392

ABSTRACT

Activation of c-jun N-terminal kinase (JNK) by the mitogen-activated protein kinase cascade has been shown to play an important role in the death of dopamine neurons of the substantia nigra, one of the principal neuronal populations affected in Parkinson's disease. However, it has remained unknown whether the JNK2 and JNK3 isoforms, either singly or in combination, are essential for apoptotic death, and, if so, the mechanisms involved. In addition, it has been unclear whether they play a role in axonal degeneration of these neurons in disease models. To address these issues we have examined the effect of single and double jnk2 and jnk3 null mutations on apoptosis in a highly destructive neurotoxin model, that induced by intrastriatal 6-hydroxydopamine. We find that homozygous jnk2/3 double null mutations result in a complete abrogation of apoptosis and a prolonged survival of the entire population of dopamine neurons. In spite of this complete protection at the cell soma level, there was no protection of axons. These studies provide a striking demonstration of the distinctiveness of the mechanisms that mediate cell soma and axon degeneration, and they illustrate the need to identify and target pathways of axon degeneration in the development of neuroprotective therapeutics.


Subject(s)
Apoptosis/physiology , Axons/pathology , Dopamine/metabolism , Mitogen-Activated Protein Kinase 10/metabolism , Mitogen-Activated Protein Kinase 9/metabolism , Neurons/physiology , Retrograde Degeneration/pathology , Substantia Nigra/cytology , Animals , Apoptosis/drug effects , Gene Expression Regulation/drug effects , Gene Expression Regulation/physiology , Mice , Mice, Inbred C57BL , Mice, Knockout , Mitogen-Activated Protein Kinase 10/deficiency , Mitogen-Activated Protein Kinase 9/deficiency , Neurons/drug effects , Oxidopamine/pharmacology , RNA, Messenger/metabolism , Retrograde Degeneration/chemically induced , Retrograde Degeneration/genetics , Silver Staining/methods , Substantia Nigra/drug effects , Sympatholytics/pharmacology , Transcription Factor CHOP/genetics , Transcription Factor CHOP/metabolism , Tyrosine 3-Monooxygenase/metabolism
10.
Exp Neurol ; 175(1): 1-9, 2002 May.
Article in English | MEDLINE | ID: mdl-12009755

ABSTRACT

An emerging theme in programmed cell death (PCD) of neurons is that the mechanisms involved depend on the cellular context and the death-inducing stimulus. One particular class of neurons for which it is important to identify the mechanisms of PCD are the dopamine neurons of the substantia nigra, the neurons which degenerate in Parkinson's disease. PCD has been shown to occur in these neurons during normal development and to be induced in neurotoxin models of parkinsonism. Conventional histologic stains and TUNEL labeling have not revealed morphologic differences in the apoptosis observed in these neurons in any context. We now show that in two models of induced PCD in postmitotic dopamine neurons, one induced by early striatal target injury and another induced by the neurotoxin 6-hydroxydopamine (6OHDA), there are differences in the cellular localization and type of caspase cleavage products. Using two antibodies to caspase cleavage products (fractin and AB127), we show that in the target lesion model immunostaining is localized to the nucleus, whereas in the 6OHDA model intense cytoplasmic as well as nuclear staining is observed. Another antibody, AB246, to a caspase cleavage product of spectrin, immunostains apoptotic profiles only in the 6OHDA model. These findings suggest that the cellular compartment and therefore the role of the caspases may differ in apoptosis induced in pathologic settings, such as that due to neurotoxins, from that observed in models of natural or induced natural cell death. It will be important to recognize these differences in the consideration of caspase inhibitors in the treatment of degenerative neurologic disease.


Subject(s)
Caspases/metabolism , Cell Nucleus/metabolism , Cytoplasm/metabolism , Neurons/metabolism , Peptide Fragments/biosynthesis , Substantia Nigra/metabolism , Actins/metabolism , Animals , Antibody Specificity , Apoptosis/physiology , Cell Compartmentation , Cell Nucleus/pathology , Cytoplasm/pathology , Disease Models, Animal , Dopamine/metabolism , Enzyme Inhibitors/pharmacology , Humans , Immunohistochemistry , Neuroblastoma/chemistry , Neuroblastoma/drug therapy , Neuroblastoma/metabolism , Neurons/pathology , Oxidopamine , Parkinson Disease, Secondary/chemically induced , Parkinson Disease, Secondary/metabolism , Peptide Fragments/analysis , Quinolinic Acid , Rats , Spectrin/metabolism , Substantia Nigra/drug effects , Substantia Nigra/pathology , Tumor Cells, Cultured , Tyrosine 3-Monooxygenase/biosynthesis
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