ABSTRACT
Sensorimotor information processing underlies normal cognitive and behavioral traits and has classically been evaluated through prepulse inhibition (PPI) of a startle reflex. PPI is a behavioral dimension deregulated in several neurological and psychiatric disorders, yet the mechanisms underlying the cross-diagnostic nature of PPI deficits across these conditions remain to be understood. To identify circuitry mechanisms for PPI, we performed circuitry recording over the prefrontal cortex and striatum, two brain regions previously implicated in PPI, using wild-type (WT) mice compared to Disc1-locus-impairment (LI) mice, a model representing neuropsychiatric conditions. We demonstrated that the corticostriatal projection regulates neurophysiological responses during the PPI testing in WT, whereas these circuitry responses were disrupted in Disc1-LI mice. Because our biochemical analyses revealed attenuated brain-derived neurotrophic factor (Bdnf) transport along the corticostriatal circuit in Disc1-LI mice, we investigated the potential role of Bdnf in this circuitry for regulation of PPI. Virus-mediated delivery of Bdnf into the striatum rescued PPI deficits in Disc1-LI mice. Pharmacologically augmenting Bdnf transport by chronic lithium administration, partly via phosphorylation of Huntingtin (Htt) serine-421 and its integration into the motor machinery, restored striatal Bdnf levels and rescued PPI deficits in Disc1-LI mice. Furthermore, reducing the cortical Bdnf expression negated this rescuing effect of lithium, confirming the key role of Bdnf in lithium-mediated PPI rescuing. Collectively, the data suggest that striatal Bdnf supply, collaboratively regulated by Htt and Disc1 along the corticostriatal circuit, is involved in sensorimotor gating, highlighting the utility of dimensional approach in investigating pathophysiological mechanisms across neuropsychiatric disorders.
Subject(s)
Brain-Derived Neurotrophic Factor , Corpus Striatum , Nerve Tissue Proteins , Prefrontal Cortex , Prepulse Inhibition , Animals , Brain-Derived Neurotrophic Factor/metabolism , Corpus Striatum/metabolism , Humans , Mice , Nerve Tissue Proteins/metabolism , Prefrontal Cortex/metabolism , Prepulse Inhibition/physiology , Reflex, Startle/physiology , Sensory Gating/physiologyABSTRACT
Cleavage of mutant huntingtin (HTT) is an essential process in Huntington's disease (HD), an inherited neurodegenerative disorder. Cleavage generates N-ter fragments that contain the polyQ stretch and whose nuclear toxicity is well established. However, the functional defects induced by cleavage of full-length HTT remain elusive. Moreover, the contribution of non-polyQ C-terminal fragments is unknown. Using time- and site-specific control of full-length HTT proteolysis, we show that specific cleavages are required to disrupt intramolecular interactions within HTT and to cause toxicity in cells and flies. Surprisingly, in addition to the canonical pathogenic N-ter fragments, the C-ter fragments generated, that do not contain the polyQ stretch, induced toxicity via dilation of the endoplasmic reticulum (ER) and increased ER stress. C-ter HTT bound to dynamin 1 and subsequently impaired its activity at ER membranes. Our findings support a role for HTT on dynamin 1 function and ER homoeostasis. Proteolysis-induced alteration of this function may be relevant to disease.
Subject(s)
Dynamin I/metabolism , Huntington Disease/metabolism , Microtubule-Associated Proteins/metabolism , Nerve Tissue Proteins/metabolism , Peptides/metabolism , Proteolysis , Serotonin Plasma Membrane Transport Proteins/metabolism , Animals , Drosophila Proteins , Drosophila melanogaster , Dynamin I/genetics , Endoplasmic Reticulum/genetics , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum Stress/genetics , Humans , Huntingtin Protein , Huntington Disease/genetics , Mice , Microtubule-Associated Proteins/genetics , Nerve Tissue Proteins/genetics , Peptides/genetics , Serotonin Plasma Membrane Transport Proteins/geneticsABSTRACT
Cell pathology in lysosomal storage diseases is characterized by the formation of distended vacuoles with characteristics of lysosomes. Our previous studies in mucopolysaccharidosis type IIIB (MPSIIIB), a disease in which a genetic defect induces the accumulation of undigested heparan sulfate (HS) fragments, led to the hypothesis that abnormal lysosome formation was related to events occurring at the Golgi level. We reproduced the enzyme defect of MPSIIIB in HeLa cells using tetracycline-inducible expression of shRNAs directed against α-N-acetylglucosaminidase (NAGLU) and addressed this hypothesis. HeLa cells deprived of NAGLU accumulated abnormal lysosomes. The Golgi matrix protein GM130 was over-expressed. The cis- and medial-Golgi compartments were distended, elongated and formed circularized ribbons. The Golgi microtubule network was enlarged with increased amounts of AKAP450, a partner of GM130 controlling this network. GM130 down-regulation prevented pathology in HeLa cells deprived of NAGLU, whereas GM130 over-expression in control HeLa cells mimicked the pathology of deprived cells. We concluded that abnormal lysosomes forming in cells accumulating HS fragments were the consequence of GM130 gain-of-function and subsequent alterations of the Golgi ribbon architecture. These results indicate that GM130 functions are modulated by HS glycosaminoglycans and therefore possibly controlled by extracellular cues.
Subject(s)
Autoantigens/metabolism , Membrane Proteins/metabolism , Mucopolysaccharidosis III/pathology , Acetylglucosaminidase/antagonists & inhibitors , Golgi Apparatus/ultrastructure , HeLa Cells , Humans , Lysosomes/pathology , Microtubules/ultrastructure , Models, Biological , Vacuoles/ultrastructureABSTRACT
Neurotransmitters are released at synapses by synaptic vesicles (SVs), which originate from SV precursors (SVPs) that have traveled along the axon. Because each synapse maintains a pool of SVs, only a small fraction of which are released, it has been thought that axonal transport of SVPs does not affect synaptic function. Here, studying the corticostriatal network both in microfluidic devices and in mice, we find that phosphorylation of the Huntingtin protein (HTT) increases axonal transport of SVPs and synaptic glutamate release by recruiting the kinesin motor KIF1A. In mice, constitutive HTT phosphorylation causes SV over-accumulation at synapses, increases the probability of SV release, and impairs motor skill learning on the rotating rod. Silencing KIF1A in these mice restored SV transport and motor skill learning to wild-type levels. Axonal SVP transport within the corticostriatal network thus influences synaptic plasticity and motor skill learning.
ABSTRACT
The accumulation of intracellular storage vesicles is a hallmark of lysosomal storage diseases. Neither the identity nor origin of these implicated storage vesicles have yet been established. The vesicles are often considered as lysosomes, endosomes, and/or autophagosomes that are engorged with undigested materials. Our studies in the mouse model of mucopolysaccharidosis type IIIB, a lysosomal storage disease that induces neurodegeneration, showed that large storage vesicles in cortical neurons did not receive material from either the endocytic or autophagy pathway, which functioned normally. Storage vesicles expressed GM130, a Golgi matrix protein, which mediates vesicle tethering in both pre- and cis-Golgi compartments. However, other components of the tethering/fusion complex were not associated with GM130 on storage vesicles, likely accounting for both the resistance of the vesicles to brefeldin A and the alteration of Golgi ribbon architecture, which comprised distended cisterna connected to LAMP1-positive storage vesicles. We propose that alteration in the GM130-mediated control of vesicle trafficking in pre-Golgi and Golgi compartments affects Golgi biogenesis and gives rise to a dead-end storage compartment. Vesicle accumulation, Golgi disorganization, and alterations of other GM130 functions may account for neuron dysfunction and death.
Subject(s)
Cytoplasmic Vesicles/pathology , Golgi Apparatus/pathology , Neurons/ultrastructure , Animals , Autophagy/physiology , Biological Transport/physiology , Cells, Cultured , Cytoplasmic Vesicles/metabolism , Disease Models, Animal , Golgi Apparatus/metabolism , Lysosomes/metabolism , Lysosomes/pathology , Membrane Proteins/metabolism , Metabolic Networks and Pathways/physiology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Mucopolysaccharidosis III/complications , Mucopolysaccharidosis III/metabolism , Mucopolysaccharidosis III/pathology , Neurons/metabolism , Neurons/pathology , Neurons/physiologyABSTRACT
Behavioral manifestations mark the onset of disease expression in children with mucopolysaccharidosis type III (MPSIII, Sanfilippo syndrome), a genetic disorder resulting from interruption of the lysosomal degradation of heparan sulfate. In the mouse model of MPSIII type B (MPSIIIB), cortical neuron pathology and dysfunction occur several months before neuronal loss and are primarily cell autonomous. The gene coding for GAP43, a neurite growth potentiator, is overexpressed in the MPSIIIB mouse cortex, and neurite dystrophy was reported in other types of lysosomal storage diseases. We therefore examined the development of the neuritic trees in pure populations of MPSIIIB mouse embryo cortical neurons grown for up to 12 days in primary culture. Dynamic observation of living neurons and quantification of neurite growth parameters indicated more frequent neurite elongation and branching and less frequent neurite retraction, resulting in a relative overgrowth of MPSIIIB neuron neuritic trees, involving both dendrites and axons, compared with normal controls. Neurite overgrowth was concomitant with more than twofold increased expression of GAP43 mRNAs and proteins. Correction of the genetic defect leads to expression of the missing lysosomal enzyme, normal GAP43 mRNA expression, and reduced neurite outgrowth. These results indicate that heparan sulfate oligosaccharide storage modifies GAP43 expression in MPSIIIB cortical neurons with potential consequences for neurite development and neuronal functions that may be relevant to clinical manifestations.
Subject(s)
Cerebral Cortex/metabolism , GAP-43 Protein/metabolism , Mucopolysaccharidosis III/metabolism , Neurites/metabolism , Animals , Blotting, Western , Cell Shape/physiology , Cells, Cultured , Cerebral Cortex/cytology , Disease Models, Animal , Fluorescent Antibody Technique , GAP-43 Protein/genetics , Gene Expression , Genetic Vectors/metabolism , Lentivirus/metabolism , Mice , Mucopolysaccharidosis III/genetics , Neuronal Plasticity/physiology , Neurons/cytology , Neurons/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Statistics, Nonparametric , Time FactorsABSTRACT
Biochemical disorders in lysosomal storage diseases consist of the interruption of metabolic pathways involved in the recycling of the degradation products of one or several types of macromolecules. The progressive accumulation of these primary storage products is the direct consequence of the genetic defect and represents the initial pathogenic event. Downstream consequences for the affected cells include the accumulation of secondary storage products and the formation of histological storage lesions, which appear as intracellular vacuoles that represent the pathological hallmark of lysosomal storage diseases. Relationships between storage products and storage lesions are not simple and are still largely not understood. Primary storage products induce malfunction of the organelles where they accumulate, these being primarily, but not only, lysosomes. Consequences for cell metabolism and intracellular trafficking combine the effects of primary storage product toxicity and the compensatory mechanisms activated to protect the cell. Induced disorders extend far beyond the primarily interrupted metabolic pathway.
Subject(s)
Lysosomal Storage Diseases/metabolism , Lysosomes/metabolism , Metabolic Networks and Pathways , Animals , Autoantigens/metabolism , Biological Transport , Brain/metabolism , Brain/pathology , Golgi Apparatus/pathology , Humans , Lysosomal Storage Diseases/pathology , Lysosomes/pathology , Membrane Proteins/metabolismABSTRACT
Mutations in the X-linked MECP2 gene are responsible for Rett syndrome (RTT), a severe neurological disorder for which there is no treatment. Several studies have linked the loss of MeCP2 function to alterations of brain-derived neurotrophic factor (BDNF) levels, but non-specific overexpression of BDNF only partially improves the phenotype of Mecp2-deficient mice. We and others have previously shown that huntingtin (HTT) scaffolds molecular motor complexes, transports BDNF-containing vesicles, and is under-expressed in Mecp2 knockout brains. Here, we demonstrate that promoting HTT phosphorylation at Ser421, either by a phospho-mimetic mutation or inhibition of the phosphatase calcineurin, restores endogenous BDNF axonal transport in vitro in the corticostriatal pathway, increases striatal BDNF availability and synaptic connectivity in vivo, and improves the phenotype and the survival of Mecp2 knockout mice-even though treatments were initiated only after the mice had already developed symptoms. Stimulation of endogenous cellular pathways may thus be a promising approach for the treatment of RTT patients.
Subject(s)
Brain-Derived Neurotrophic Factor , Huntingtin Protein/chemistry , Methyl-CpG-Binding Protein 2 , Rett Syndrome/genetics , Animals , Brain-Derived Neurotrophic Factor/genetics , Disease Models, Animal , Female , Homeostasis , Male , Methyl-CpG-Binding Protein 2/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Phenotype , PhosphorylationABSTRACT
Studies have suggested that amyloid precursor protein (APP) regulates synaptic homeostasis, but the evidence has not been consistent. In particular, signaling pathways controlling APP transport to the synapse in axons and dendrites remain to be identified. Having previously shown that Huntingtin (HTT), the scaffolding protein involved in Huntington's disease, regulates neuritic transport of APP, we used a microfluidic corticocortical neuronal network-on-a-chip to examine APP transport and localization to the pre- and post-synaptic compartments. We found that HTT, upon phosphorylation by the Ser/Thr kinase Akt, regulates APP transport in axons but not dendrites. Expression of an unphosphorylatable HTT decreased axonal anterograde transport of APP, reduced presynaptic APP levels, and increased synaptic density. Ablating in vivo HTT phosphorylation in APPPS1 mice, which overexpress APP, reduced presynaptic APP levels, restored synapse number and improved learning and memory. The Akt-HTT pathway and axonal transport of APP thus regulate APP presynaptic levels and synapse homeostasis.
Subject(s)
Alzheimer Disease/metabolism , Amyloid beta-Protein Precursor/metabolism , Huntingtin Protein/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Synapses/metabolism , Animals , Axonal Transport , Brain/diagnostic imaging , Disease Models, Animal , Homeostasis , Magnetic Resonance Imaging , Male , Memory , Mice, Transgenic , Microfluidic Analytical Techniques , Morris Water Maze Test , PhosphorylationABSTRACT
Huntington's disease (HD), a devastating neurodegenerative disorder, strongly affects the corticostriatal network, but the contribution of pre- and postsynaptic neurons in the first phases of disease is unclear due to difficulties performing early subcellular investigations in vivo. Here, we have developed an on-a-chip approach to reconstitute an HD corticostriatal network in vitro, using microfluidic devices compatible with subcellular resolution. We observed major defects in the different compartments of the corticostriatal circuit, from presynaptic dynamics to synaptic structure and transmission and to postsynaptic traffic and signaling, that correlate with altered global synchrony of the network. Importantly, the genetic status of the presynaptic compartment was necessary and sufficient to alter or restore the circuit. This highlights an important weight for the presynaptic compartment in HD that has to be considered for future therapies. This disease-on-a-chip microfluidic platform is thus a physiologically relevant in vitro system for investigating pathogenic mechanisms and for identifying drugs.
Subject(s)
Corpus Striatum , Huntington Disease , Lab-On-A-Chip Devices , Nerve Net , Presynaptic Terminals , Synaptic Transmission , Animals , Corpus Striatum/metabolism , Corpus Striatum/pathology , Huntington Disease/genetics , Huntington Disease/metabolism , Huntington Disease/pathology , Mice , Mice, Transgenic , Nerve Net/metabolism , Nerve Net/pathology , Presynaptic Terminals/metabolism , Presynaptic Terminals/pathologyABSTRACT
Mucopolysaccharidoses type III (MPSIII, Sanfilippo syndrome) are genetic diseases due to deficient heparan sulfate (HS) saccharide digestion by lysosomal exoglycanases. Progressive accumulation of undigested saccharides causes early-onset behavioural and cognitive symptoms. The precise role of these saccharides in the pathophysiological cascade is still unclear. We showed that exposure of wild-type neural cells to exogenous soluble HS fragments of at least eight saccharides activated integrin-based focal adhesions (FAs), which attach cells to the extracellular matrix. FAs were constitutively activated in MPSIII type B astrocytes or neural stem cells unless undigested saccharides were cleared by exogenous supply of the missing exoglycanase. Defective cell polarisation and oriented migration in response to focal extracellular stimuli in affected cells suggest improper sensing of the environment. We consistently observed abnormal organisation of the rostral migratory stream in the brain of adult mice with MPSIII type B. These results suggest that cell polarisation and oriented migration defects participate to the neurological disorders associated with Sanfilippo syndrome.