A GGGGCC (G4C2) hexanucleotide repeat expansion (HRE) in C9ORF72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Recent studies support an HRE RNA gain-of-function mechanism of neurotoxicity, and we previously identified protein interactors for the G4C2 RNA including RanGAP1. A candidate-based genetic screen in Drosophila expressing 30 G4C2 repeats identified RanGAP (Drosophila ortholog of human RanGAP1), a key regulator of nucleocytoplasmic transport, as a potent suppressor of neurodegeneration. Enhancing nuclear import or suppressing nuclear export of proteins also suppresses neurodegeneration. RanGAP physically interacts with HRE RNA and is mislocalized in HRE-expressing flies, neurons from C9ORF72 ALS patient-derived induced pluripotent stem cells (iPSNs), and in C9ORF72 patient brain tissue. Nuclear import is impaired as a result of HRE expression in the fly model and in C9ORF72 iPSNs, and these deficits are rescued by small molecules and antisense oligonucleotides targeting the HRE G-quadruplexes. Nucleocytoplasmic transport defects may be a fundamental pathway for ALS and FTD amenable to pharmacotherapeutic intervention.
Summary Palmitoylation, a key regulatory mechanism controlling protein targeting, is catalyzed by DHHC-family palmitoyl acyltransferases (PATs). Impaired PAT activity is linked to several neurodevelopmental and neuropsychiatric disorders, suggesting critical roles for palmitoylation in neuronal function. However, few substrates for specific PATs are known, and functional consequences of specific palmitoylation events are frequently uncharacterized. Here, we identify two related PATs, DHHC5 and DHHC8, as specific regulators of the PDZ domain protein GRIP1b. Binding, palmitoylation and dendritic targeting of GRIP1b require a DHHC5/8 PDZ ligand that is absent in all other PATs. Palmitoylated GRIP1b is targeted to trafficking endosomes, and may link endosomes to kinesin motors. Consistent with this trafficking role, GRIP1b's palmitoylation turnover rate approaches the highest of all reported proteins, and palmitoylation increases GRIP1b's ability to accelerate AMPA-R recycling. These findings identify the first neuronal DHHC5/8 substrate, define novel mechanisms controlling palmitoylation specificity, and suggest further links between dysregulated palmitoylation and neurodevelopmental / neuropsychiatric conditions.
Insulin receptor signaling has been postulated to play a role in synaptic plasticity; however, the function of the insulin receptor in CNS is not clear. To test whether insulin receptor signaling affects visual system function, we recorded light-evoked responses in optic tectal neurons in living Xenopus tadpoles. Tectal neurons transfected with dominant-negative insulin receptor (dnIR), which reduces insulin receptor phosphorylation, or morpholino against insulin receptor, which reduces total insulin receptor protein level, have significantly smaller light-evoked responses than controls. dnIR-expressing neurons have reduced synapse density as assessed by EM, decreased AMPA mEPSC frequency, and altered experience-dependent dendritic arbor structural plasticity, although synaptic vesicle release probability, assessed by paired-pulse responses, synapse maturation, assessed by AMPA/NMDA ratio and ultrastructural criteria, are unaffected by dnIR expression. These data indicate that insulin receptor signaling regulates circuit function and plasticity by controlling synapse density.
Accumulating data, including those from large genetic association studies, indicate that alterations in glutamatergic synapse structure and function represent a common underlying pathology in many symptomatically distinct cognitive disorders. In this review, we discuss evidence from human genetic studies and data from animal models supporting a role for aberrant glutamatergic synapse function in the etiology of intellectual disability (ID), autism spectrum disorder (ASD), and schizophrenia (SCZ), neurodevelopmental disorders that comprise a significant proportion of human cognitive disease and exact a substantial financial and social burden. The varied manifestations of impaired perceptual processing, executive function, social interaction, communication, and/or intellectual ability in ID, ASD, and SCZ appear to emerge from altered neural microstructure, function, and/or wiring rather than gross changes in neuron number or morphology. Here, we review evidence that these disorders may share a common underlying neuropathy: altered excitatory synapse function. We focus on the most promising candidate genes affecting glutamatergic synapse function, highlighting the likely disease-relevant functional consequences of each. We first present a brief overview of glutamatergic synapses and then explore the genetic and phenotypic evidence for altered glutamate signaling in ID, ASD, and SCZ.
Sensory experience plays a crucial role in regulating neuronal shape and in developing synaptic contacts during brain formation. These features are required for a neuron to receive, integrate, and transmit signals within the neuronal network so that animals can adapt to the constant changing environment. Insulin receptor signaling, which has been extensively studied in peripheral organ systems such as liver, muscle and adipocyte, has recently been shown to play important roles in the central nervous system. Here we review the current understanding of the underlying mechanisms that regulate structural and functional aspects of circuit development, particularly with respect to the role of insulin receptor signaling in synaptic function and the development of dendritic arbor morphology. The potential link between insulin receptor signaling malfunction and neurological disorders will also be discussed.
Single-cell electroporation allows transfection of plasmid DNA or macromolecules into individual living cells using modified patch electrodes and common electrophysiological equipment. This protocol is optimized for rapid in vivo electroporation of Xenopus laevis tadpole brains with DNA, dextrans, morpholinos and combinations thereof. Experienced users can electroporate roughly 40 tadpoles per hour. The technique can be adapted for use with other charged transfer materials and in other systems and tissues where cells can be targeted with a micropipette. Under visual guidance, an electrode filled with transfer material is placed in a cell body-rich area of the tadpole brain and a train of voltage pulses applied, which electroporates a nearby cell. We show examples of successfully electroporated single cells, instances of common problems and troubleshooting suggestions. Single-cell electroporation is an affordable method to fluorescently label and genetically manipulate individual cells. This powerful technique enables observation of single cells in an otherwise normal environment.
Synaptic dynamics and reorganization are fundamental features of synaptic plasticity both during synaptic circuit development and in the mature CNS underlying learning, memory, and experience-dependent circuit rearrangements. Combining in vivo time-lapse fluorescence imaging and retrospective electron microscopic analysis provides a powerful technique to decipher the rules governing dynamics of neuronal structure and synaptic connections. Here we have generated a membrane-targeted horseradish peroxidase (mHRP) that allows identification of transfected cells without obscuring the intracellular ultrastructure or organelles and in particular allows identification of synaptic sites using electron microscopy. The expression of mHRP does not affect dendritic arbor growth or dynamics of transfected neurons. Co-expression of EGFP and mHRP was used to study neuronal morphology at both the light and electron microscopic levels. mHRP expression greatly facilitates 3D reconstruction based on serial EM sections. We expect this reagent will be valuable for studying the mechanisms that guide construction of neuronal networks.
In mature neurons AMPA receptors cluster at excitatory synapses primarily on dendritic spines, whereas GABA A receptors cluster at inhibitory synapses mainly on the soma and dendritic shafts. The molecular mechanisms underlying the precise sorting of these receptors remain unclear. By directly studying the constitutive exocytic vesicles of AMPA and GABA A receptors in vitro and in vivo, we demonstrate that they are initially sorted into different vesicles in the Golgi apparatus and inserted into distinct domains of the plasma membrane. These insertions are dependent on distinct Rab GTPases and SNARE complexes. The insertion of AMPA receptors requires SNAP25-syntaxin1A/B-VAMP2 complexes, whereas insertion of GABA A receptors relies on SNAP23-syntaxin1A/B-VAMP2 complexes. These SNARE complexes affect surface targeting of AMPA or GABA A receptors and synaptic transmission. Our studies reveal vesicular sorting mechanisms controlling the constitutive exocytosis of AMPA and GABA A receptors, which are critical for the regulation of excitatory and inhibitory responses in neurons.AMPA receptor | GABA A receptor | constitutive exocytosis | TIRFM | SNARE I n the mammalian central nervous system, neurons receive excitatory and inhibitory signals at synapses. Specific receptors at postsynaptic membranes are activated by neurotransmitters released by presynaptic terminals. Most fast excitatory neurotransmission is mediated by AMPA receptors, the majority of which are heterotetramers of GluA1/GluA2 or GluA2/GluA3 subunits in the hippocampus (1). Fast synaptic inhibition is largely mediated by GABA A receptors, which are predominantly heteropentamers of two α subunits, two β subunits, and one γ or δ subunit in the hippocampus (2). Numerous studies have demonstrated AMPA receptors are selectively localized at excitatory synapses on dendritic spines, whereas GABA A receptors cluster at inhibitory synapses localized on dendritic shafts and the soma (3). This segregation of excitatory and inhibitory receptors requires highly precise sorting machinery to target receptors to distinct synapses opposing specific presynaptic terminals. However, it is still not clear whether the receptors are sorted before exocytosis into the plasma membrane or are differentially localized only after exocytosis. For example in a "plasma membrane sorting model," different receptors could be pooled into the same vesicle and inserted along the somatodendritic membrane. The initial sorting would occur on the plasma membrane, where inserted receptors would be segregated by lateral diffusion and stabilization at different postsynaptic zones. Alternatively, in a "vesicle sorting model," different receptors would first be sorted into different vesicles during intracellular trafficking processes and independently inserted to the plasma membrane, where receptors could be further targeted to specific zones and stabilized by synaptic scaffolds. To date there has been no direct evidence to support either model. However, a large body of literature suggests that the e...
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