Fragile X mental retardation protein is translated near synapses in response to neurotransmitter activation

Local translation of proteins in distal dendrites is thought to support synaptic structural plasticity. We have previously shown that metabotropic glutamate receptor (mGluR1) stimulation initiates a phosphorylation cascade, triggering rapid association of some mRNAs with translation machinery near synapses, and leading to protein synthesis. To determine the identity of these mRNAs, a cDNA library produced from distal nerve processes was used to screen synaptic polyribosome-associated mRNA. We identified mRNA for the fragile X mental retardation protein (FMRP) in these processes by use of synaptic subcellular fractions, termed synaptoneurosomes. We found that this mRNA associates with translational complexes in synaptoneurosomes within 1–2 min after mGluR1 stimulation of this preparation, and we observed increased expression of FMRP after mGluR1 stimulation. In addition, we found that FMRP is associated with polyribosomal complexes in these fractions. In vivo, we observed FMRP immunoreactivity in spines, dendrites, and somata of the developing rat brain, but not in nuclei or axons. We suggest that rapid production of FMRP near synapses in response to activation may be important for normal maturation of synaptic connections.

Abnormal dendritic spines in fragile X knockout mice: Maturation and pruning deficits

Fragile X syndrome arises from blocked expression of the fragile X mental retardation protein (FMRP). Golgi-impregnated mature cerebral cortex from fragile X patients exhibits long, thin, tortuous postsynaptic spines resembling spines observed during normal early neocortical development. Here we describe dendritic spines in Golgi-impregnated cerebral cortex of transgenic fragile X gene (Fmr1) knockout mice that lack expression of the protein. Dendritic spines on apical dendrites of layer V pyramidal cells in occipital cortex of fragile X knockout mice were longer than those in wild-type mice and were often thin and tortuous, paralleling the human syndrome and suggesting that FMRP expression is required for normal spine morphological development. Moreover, spine density along the apical dendrite was greater in the knockout mice, which may reflect impaired developmental organizational processes of synapse stabilization and elimination or pruning.

Neurotrophin-3 signals redistribute RNA in neurons

The translocation of specific mRNAs to dendrites and their potential for locally regulated translation are likely to serve as an effector in neuronal plasticity. Whether translation in dendrites is regulated by delivery of the RNA to sites of plasticity or a stationary pool of localized RNA undergoes enhanced translational efficiency is not clear. We show that RNA can translocate into dendrites in response to NT-3. RNA granules were visualized in cultured rat cortical neurons using the dye SYTO 14, which labels poly-ribosome complexes. Long before the morphological effects of NT-3 appeared, there was increased distal translocation of labeled complexes. This effect was blocked by K252a, a potent inhibitor of tyrosine kinase receptors. Therefore, neurons can utilize extracellular signals to alter the distribution of protein synthetic machinery via the active transport of RNA granules.

Interactive cloning with the SH3 domain of N-src identifies a new brain specific ion channel protein, with homology to Eag and cyclic nucleotide-gated channels

We have isolated a novel cDNA, that appears to represent a new class of ion channels, by using the yeast two-hybrid system and the SH3 domain of the neural form of Src (N-src) as a bait. The encoded polypeptide, BCNG-1, is distantly related to cyclic nucleotide-gated channels and the voltage-gated channels, Eag and H-erg. BCNG-1 is expressed exclusively in the brain, as a glycosylated protein of ≈132 kDa. Immunohistochemical analysis indicates that BCNG-1 is preferentially expressed in specific subsets of neurons in the neocortex, hippocampus, and cerebellum, in particular pyramidal neurons and basket cells. Within individual neurons, the BCNG-1 protein is localized to either the dendrites or the axon terminals depending on the cell type. Southern blot analysis shows that several other BCNG-related sequences are present in the mouse genome, indicating the emergence of an entire subfamily of ion channel coding genes. These findings suggest the existence of a new type of ion channel, which is potentially able to modulate membrane excitability in the brain and could respond to regulation by cyclic nucleotides.

Two Heteromeric Kinesin Complexes in Chemosensory Neurons and Sensory Cilia of Caenorhabditis elegans

Chemosensation in the nervous system of the nematode Caenorhabditis elegans depends on sensory cilia, whose assembly and maintenance requires the transport of components such as axonemal proteins and signal transduction machinery to their site of incorporation into ciliary structures. Members of the heteromeric kinesin family of microtubule motors are prime candidates for playing key roles in these transport events. Here we describe the molecular characterization and partial purification of two heteromeric kinesin complexes from C. elegans, heterotrimeric CeKinesin-II and dimeric CeOsm-3. Transgenic worms expressing green fluorescent protein driven by endogenous heteromeric kinesin promoters reveal that both CeKinesin-II and CeOsm-3 are expressed in amphid, inner labial, and phasmid chemosensory neurons. Additionally, immunolocalization experiments on fixed worms show an intense concentration of CeKinesin-II and CeOsm-3 polypeptides in the ciliated endings of these chemosensory neurons and a punctate localization pattern in the corresponding cell bodies and dendrites. These results, together with the phenotypes of known mutants in the pathway of sensory ciliary assembly, suggest that CeKinesin-II and CeOsm-3 drive the transport of ciliary components required for sequential steps in the assembly of chemosensory cilia.

Characterization of the KIF3C Neural Kinesin-like Motor from Mouse

Proteins of the kinesin superfamily define a class of microtubule-dependent motors that play crucial roles in cell division and intracellular transport. To study the molecular mechanism of axonal transport, a cDNA encoding a new kinesin-like protein called KIF3C was cloned from a mouse brain cDNA library. Sequence and secondary structure analysis revealed that KIF3C is a member of the KIF3 family. In contrast to KIF3A and KIF3B, Northern and Western analysis indicated that KIF3C expression is highly enriched in neural tissues such as brain, spinal cord, and retina. When anti-KIF3C antibodies were used to stain the cerebellum, the strongest signal came from the cell bodies and dendrites of Purkinje cells. In retina, anti-KIF3C mainly stains the ganglion cells. Immunolocalization showed that the KIF3C motor in spinal cord and sciatic nerve is mainly localized in cytoplasm. In spinal cord, the KIF3C staining was punctate; double labeling with anti-giantin and anti-KIF3C showed a clear concentration of the motor protein in the Golgi complex. Staining of ligated sciatic nerves demonstrated that the KIF3C motor accumulated at the proximal side of the ligated nerve, which suggests that KIF3C is an anterograde motor. Immunoprecipitation experiments revealed that KIF3C and KIF3A, but not KIF3B, were coprecipitated. These data, combined with previous data from other labs, indicate that KIF3C and KIF3B are “variable” subunits that associate with a common KIF3A subunit, but not with each other. Together these results suggest that KIF3 family members combinatorially associate to power anterograde axonal transport.

KIF3C and KIF3A Form a Novel Neuronal Heteromeric Kinesin That Associates with Membrane Vesicles

We have cloned from rat brain the cDNA encoding an 89,828-Da kinesin-related polypeptide KIF3C that is enriched in brain, retina, and lung. Immunocytochemistry of hippocampal neurons in culture shows that KIF3C is localized to cell bodies, dendrites, and, in lesser amounts, to axons. In subcellular fractionation experiments, KIF3C cofractionates with a distinct population of membrane vesicles. Native KIF3C binds to microtubules in a kinesin-like, nucleotide-dependent manner. KIF3C is most similar to mouse KIF3B and KIF3A, two closely related kinesins that are normally present as a heteromer. In sucrose density gradients, KIF3C sediments at two distinct densities, suggesting that it may be part of two different multimolecular complexes. Immunoprecipitation experiments show that KIF3C is in part associated with KIF3A, but not with KIF3B. Unlike KIF3B, a significant portion of KIF3C is not associated with KIF3A. Consistent with these biochemical properties, the distribution of KIF3C in the CNS has both similarities and differences compared with KIF3A and KIF3B. These results suggest that KIF3C is a vesicle-associated motor that functions both independently and in association with KIF3A.

Microtubule-dependent Recruitment of Staufen-Green Fluorescent Protein into Large RNA-containing Granules and Subsequent Dendritic Transport in Living Hippocampal NeuronsV⃞

Dendritic mRNA transport and local translation at individual potentiated synapses may represent an elegant way to form synaptic memory. Recently, we characterized Staufen, a double-stranded RNA-binding protein, in rat hippocampal neurons and showed its presence in large RNA-containing granules, which colocalize with microtubules in dendrites. In this paper, we transiently transfect hippocampal neurons with human Staufen-green fluorescent protein (GFP) and find fluorescent granules in the somatodendritic domain of these cells. Human Stau-GFP granules show the same cellular distribution and size and also contain RNA, as already shown for the endogenous Stau particles. In time-lapse videomicroscopy, we show the bidirectional movement of these Staufen-GFP–labeled granules from the cell body into dendrites and vice versa. The average speed of these particles was 6.4 μm/min with a maximum velocity of 24.3 μm/min. Moreover, we demonstrate that the observed assembly into granules and their subsequent dendritic movement is microtubule dependent. Taken together, we have characterized a novel, nonvesicular, microtubule-dependent transport pathway involving RNA-containing granules with Staufen as a core component. This is the first demonstration in living neurons of movement of an essential protein constituent of the mRNA transport machinery.

Subcellular Localization of Myosin-V in the B16 Melanoma Cells, a Wild-type Cell Line for the dilute Gene

The discovery that the dilute gene encodes a class V myosin led to the hypothesis that this molecular motor is involved in melanosome transport and/or dendrite outgrowth in mammalian melanocytes. The present studies were undertaken to gain insight into the subcellular distribution of myosin-V in the melanoma cell line B16-F10, which is wild-type for the dilute gene. Immunofluorescence studies showed some degree of superimposed labeling of myosin-V with melanosomes that predominated at the cell periphery. A subcellular fraction highly enriched in melanosomes was also enriched in myosin-V based on Western blot analysis. Immunoelectron microscopy showed myosin-V labeling associated with melanosomes and other organelles. The stimulation of B16 cells with the α-melanocyte-stimulating hormone led to a significant increase in myosin-V expression. This is the first evidence that a cAMP signaling pathway might regulate the dilute gene expression. Immunofluorescence also showed an intense labeling of myosin-V independent of melanosomes that was observed within the dendrites and at the perinuclear region. Although the results presented herein are consistent with the hypothesis that myosin-V might act as a motor for melanosome translocation, they also suggest a broader cytoplasmic function for myosin-V, acting on other types of organelles or in cytoskeletal dynamics.

A universal scaling law between gray matter and white matter of cerebral cortex

Neocortex, a new and rapidly evolving brain structure in mammals, has a similar layered architecture in species over a wide range of brain sizes. Larger brains require longer fibers to communicate between distant cortical areas; the volume of the white matter that contains long axons increases disproportionally faster than the volume of the gray matter that contains cell bodies, dendrites, and axons for local information processing, according to a power law. The theoretical analysis presented here shows how this remarkable anatomical regularity might arise naturally as a consequence of the local uniformity of the cortex and the requirement for compact arrangement of long axonal fibers. The predicted power law with an exponent of 4/3 minus a small correction for the thickness of the cortex accurately accounts for empirical data spanning several orders of magnitude in brain sizes for various mammalian species, including human and nonhuman primates.

Phenotype of arylsulfatase A-deficient mice: Relationship to human metachromatic leukodystrophy

Metachromatic leukodystrophy is a lysosomal sphingolipid storage disorder caused by the deficiency of arylsulfatase A. The disease is characterized by progressive demyelination, causing various neurologic symptoms. Since no naturally occurring animal model of the disease is available, we have generated arylsulfatase A-deficient mice. Deficient animals store the sphingolipid cerebroside-3-sulfate in various neuronal and nonneuronal tissues. The storage pattern is comparable to that of affected humans, but gross defects of white matter were not observed up to the age of 2 years. A reduction of axonal cross-sectional area and an astrogliosis were observed in 1-year-old mice; activation of microglia started at 1 year and was generalized at 2 years. Purkinje cell dendrites show an altered morphology. In the acoustic ganglion numbers of neurons and myelinated fibers are severely decreased, which is accompanied by a loss of brainstem auditory-evoked potentials. Neurologic examination reveals significant impairment of neuromotor coordination.

Dopamine tone regulates D1 receptor trafficking and delivery in striatal neurons in dopamine transporter-deficient mice

In vivo, G protein-coupled receptors (GPCR) for neurotransmitters undergo complex intracellular trafficking that contribute to regulate their abundance at the cell surface. Here, we report a previously undescribed alteration in the subcellular localization of D1 dopamine receptor (D1R) that occurs in vivo in striatal dopaminoceptive neurons in response to chronic and constitutive hyperdopaminergia. Indeed, in mice lacking the dopamine transporter, D1R is in abnormally low abundance at the plasma membrane of cell bodies and dendrites and is largely accumulated in rough endoplasmic reticulum and Golgi apparatus. Decrease of striatal extracellular dopamine concentration with 6-hydroxydopamine (6- OHDA) in heterozygous mice restores delivery of the receptor from the cytoplasm to the plasma membrane in cell bodies. These results demonstrate that, in vivo, in the central nervous system, the storage in cytoplasmic compartments involved in synthesis and the membrane delivery contribute to regulate GPCR availability and abundance at the surface of the neurons under control of the neurotransmitter tone. Such regulation may contribute to modulate receptivity of neurons to their endogenous ligands and related exogenous drugs.

Autoinhibition of serotonin cells: An intrinsic regulatory mechanism sensitive to the pattern of usage of the cells

After periods of high-frequency firing, the normal rhythmically active serotonin (5HT)-containing neurosecretory neurons of the lobster ventral nerve cord display a period of suppressed spike generation and reduced synaptic input that we refer to as “autoinhibition.” The duration of this autoinhibition is directly related to the magnitude and duration of the current injection triggering the high-frequency firing. More interesting, however, is that the autoinhibition is inversely related to the initial firing frequency of these cells within their normal range of firing (0.5–3 Hz). This allows more active 5HT neurons to resume firing after shorter durations of inhibition than cells that initially fired at slower rates. Although superfused 5HT inhibits the spontaneous firing of these cells, the persistence of autoinhibition in saline with no added calcium, in cadmium-containing saline, and in lobsters depleted of serotonin suggests that intrinsic membrane properties account for the autoinhibition. A similar autoinhibition is seen in spontaneously active octopamine neurons but is absent from spontaneously active γ-aminobutyric acid cells. Thus, this might be a characteristic feature of amine-containing neurosecretory neurons. The 5HT cells of vertebrate brain nuclei share similarities in firing frequencies, spike shapes, and inhibition by 5HT with the lobster cells that were the focus of this study. However, the mechanism suggested to underlie autoinhibition in vertebrate neurons is that 5HT released from activated or neighboring cells acts back on inhibitory autoreceptors that are found on the dendrites and cell bodies of these neurons.

Experimental diabetes in rats causes hippocampal dendritic and synaptic reorganization and increased glucocorticoid reactivity to stress

We report that 9 d of uncontrolled experimental diabetes induced by streptozotocin (STZ) in rats is an endogenous chronic stressor that produces retraction and simplification of apical dendrites of hippocampal CA3 pyramidal neurons, an effect also observed in nondiabetic rats after 21 d of repeated restraint stress or chronic corticosterone (Cort) treatment. Diabetes also induces morphological changes in the presynaptic mossy fiber terminals (MFT) that form excitatory synaptic contacts with the proximal CA3 apical dendrites. One effect, synaptic vesicle depletion, occurs in diabetes as well as after repeated stress and Cort treatment. However, diabetes produced other MFT structural changes that differ qualitatively and quantitatively from other treatments. Furthermore, whereas 7 d of repeated stress was insufficient to produce dendritic or synaptic remodeling in nondiabetic rats, it potentiated both dendritic atrophy and MFT synaptic vesicle depletion in STZ rats. These changes occurred in concert with adrenal hypertrophy and elevated basal Cort release as well as hypersensitivity and defective shutoff of Cort secretion after stress. Thus, as an endogenous stressor, STZ diabetes not only accelerates the effects of exogenous stress to alter hippocampal morphology; it also produces structural changes that overlap only partially with those produced by stress and Cort in the nondiabetic state.

Heterogeneity of subcellular localization and electrophoretic mobility of survival motor neuron (SMN) protein in mammalian neural cells and tissues

Spinal muscular atrophy is caused by defects in the survival motor neuron (SMN) gene. To better understand the patterns of expression of SMN in neuronal cells and tissues, we raised a polyclonal antibody (abSMN) against a synthetic oligopeptide from SMN exon 2. AbSMN immunostaining in neuroblastoma cells and mouse and human central nervous system (CNS) showed intense labeling of nuclear “gems,” along with prominent nucleolar immunoreactivity in mouse and human CNS tissues. Strong cytoplasmic labeling was observed in the perikarya and proximal dendrites of human spinal motor neurons but not in their axons. Immunoblot analysis revealed a 34-kDa species in the insoluble protein fractions from human SY5Y neuroblastoma cells, embryonic mouse spinal cord cultures, and human CNS tissue. By contrast, a 38-kDa species was detected in the cytosolic fraction of SY5Y cells. We conclude that SMN protein is expressed prominently in both the cytoplasm and nucleus in multiple types of neurons in brain and spinal cord, a finding consistent with a role for SMN as a determinant of neuronal viability.