Chris I. De Zeeuw

CLASPs Are CLIP-115 and -170 Associating Proteins Involved in the Regional Regulation of Microtubule Dynamics in Motile Fibroblasts

CLIP-170 and CLIP-115 are cytoplasmic linker proteins that associate specifically with the ends of growing microtubules and may act as anti-catastrophe factors. Here, we have isolated two CLIP-associated proteins (CLASPs), which are homologous to the Drosophila Orbit/Mast microtubule-associated protein. CLASPs bind CLIPs and microtubules, colocalize with the CLIPs at microtubule distal ends, and have microtubule-stabilizing effects in transfected cells. After serum induction, CLASPs relocalize to distal segments of microtubules at the leading edge of motile fibroblasts. We provide evidence that this asymmetric CLASP distribution is mediated by PI3-kinase and GSK-3 beta. Antibody injections suggest that CLASP2 is required for the orientation of stabilized microtubules toward the leading edge. We propose that CLASPs are involved in the local regulation of microtubule dynamics in response to positional cues.

Expression of a Protein Kinase C Inhibitor in Purkinje Cells Blocks Cerebellar LTD and Adaptation of the Vestibulo-Ocular Reflex

Cerebellar long-term depression (LTD) is a model system for neuronal information storage that has an absolute requirement for activation of protein kinase C (PKC). It has been claimed to underlie several forms of cerebellar motor learning. Previous studies using various knockout mice (mGluR1, GluRdelta2, glial fibrillary acidic protein) have supported this claim; however, this work has suffered from the limitations that the knockout technique lacks anatomical specificity and that functional compensation can occur via similar gene family members. To overcome these limitations, a transgenic mouse (called L7-PKCI) has been produced in which the pseudosubstrate PKC inhibitor, PKC[19-31], was selectively expressed in Purkinje cells under the control of the pcp-2(L7) gene promoter. Cultured Purkinje cells prepared from heterozygous or homozygous L7-PKCI embryos showed a complete blockade of LTD induction. In addition, the compensatory eye movements of L7-PKCI mice were recorded during vestibular and visual stimulation. Whereas the absolute gain, phase, and latency values of the vestibulo-ocular reflex and optokinetic reflex of the L7-PKCI mice were normal, their ability to adapt their vestibulo-ocular reflex gain during visuo-vestibular training was absent. These data strongly support the hypothesis that activation of PKC in the Purkinje cell is necessary for cerebellar LTD induction, and that cerebellar LTD is required for a particular form of motor learning, adaptation of the vestibulo-ocular reflex.

Microcircuitry and function of the inferior olive

The inferior olive, which provides the climbing fibers to Purkinje cells in the cerebellar cortex, has been implicated in various functions, such as learning and timing of movements, and comparing intended with achieved movements. For example, climbing-fiber activity could transmit error signals during eye-blink conditioning or adaptation of the vestibulo-ocular reflex, or it could carry motor command signals beating on the rhythm of the oscillating and synchronous firing of ensembles of olivary neurons, or both. In this review, we approach the controversial issue of olivocerebellar function from the perspective of the unique organization of the microcircuitry of the olivary neuropil. The characteristic glomeruli are formed by a core of long dendritic or axonal spines, each of which is innervated by both an inhibitory terminal derived from the hindbrain and an excitatory terminal derived from either an ascending or descending input. The dendritic spines, which originate from dendrites with varicosities carrying dendritic lamellar bodies, are coupled by gap junctions. By drawing a comparison with a computational model by Segev and Rall,which might be applicable to the typical olivary spine with its unique morphological features and combined excitatory and inhibitory input, we propose that the microcircuitry of the inferior olive is capable of functioning both in motor learning and motor timing, but does not directly compare intended with achieved movements.

Rescue of behavioral phenotype and neuronal protrusion morphology in Fmr1 KO mice

Lack of fragile X mental retardation protein (FMRP) causes Fragile X Syndrome, the most common form of inherited mental retardation. FMRP is an RNA-binding protein and is a component of messenger ribonucleoprotein complexes, associated with brain polyribosomes, including dendritic polysomes. FMRP is therefore thought to be involved in translational control of specific mRNAs at synaptic sites. In mice lacking FMRP, protein synthesis-dependent synaptic plasticity is altered and structural malformations of dendritic protrusions occur. One hypothesized cause of the disease mechanism is based on exaggerated group I mGluR receptor activation. In this study, we examined the effect of the mGluR5 antagonist MPEP on Fragile X related behavior in Fmr1 KO mice. Our results demonstrate a clear defect in prepulse inhibition of startle in Fmr1 KO mice, that could be rescued by MPEP. Moreover, we show for the first time a structural rescue of Fragile X related protrusion morphology with two independent mGluR5 antagonists.

Bicaudal-D regulates COPI-independent Golgi-ER transport by recruiting the dynein-dynactin motor complex

The small GTPase Rab6a is involved in the regulation of membrane traffic from the Golgi apparatus towards the endoplasmic reticulum (ER) in a coat complex coatomer protein I (COPI)-independent pathway. Here, we used a yeast two-hybrid approach to identify binding partners of Rab6a. In particular, we identified the dynein–dynactin-binding protein Bicaudal-D1 (BICD1), one of the two mammalian homologues of Drosophila Bicaudal-D. BICD1 and BICD2 colocalize with Rab6a on the trans-Golgi network (TGN) and on cytoplasmic vesicles, and associate with Golgi membranes in a Rab6-dependent manner. Overexpression of BICD1 enhances the recruitment of dynein–dynactin to Rab6a-containing vesicles. Conversely, overexpression of the carboxy-terminal domain of BICD, which can interact with Rab6a but not with cytoplasmic dynein, inhibits microtubule minus-end-directed movement of green fluorescent protein (GFP)–Rab6a vesicles and induces an accumulation of Rab6a and COPI-independent ER cargo in peripheral structures. These data suggest that coordinated action between Rab6a, BICD and the dynein–dynactin complex controls COPI-independent Golgi–ER transport.

Distributed synergistic plasticity and cerebellar learning

Studies on synaptic plasticity in the context of learning have been dominated by the view that a single, particular type of plasticity forms the underlying mechanism for a particular type of learning. However, emerging evidence shows that many forms of synaptic and intrinsic plasticity at different sites are induced conjunctively during procedural memory formation in the cerebellum. Here, we review the main forms of long-term plasticity in the cerebellar cortex that underlie motor learning. We propose that the different forms of plasticity in the granular layer and the molecular layer operate synergistically in a temporally and spatially distributed manner, so as to ultimately create optimal output for behaviour.

Spatiotemporal firing patterns in the cerebellum

Neurons are generally considered to communicate information by increasing or decreasing their firing rate. However, in principle, they could in addition convey messages by using specific spatiotemporal patterns of spiking activities and silent intervals. Here, we review expanding lines of evidence that such spatiotemporal coding occurs in the cerebellum, and that the olivocerebellar system is optimally designed to generate and employ precise patterns of complex spikes and simple spikes during the acquisition and consolidation of motor skills. These spatiotemporal patterns may complement rate coding, thus enabling precise control of motor and cognitive processing at a high spatiotemporal resolution by fine-tuning sensorimotor integration and coordination.

Transcription factor GATA-3 alters pathway selection of olivocochlear neurons and affects morphogenesis of the ear.

Patterning the vertebrate ear requires the coordinated expression of genes that are involved in morphogenesis, neurogenesis, and hair cell formation. The zinc finger gene GATA‐3 is expressed both in the inner ear and in afferent and efferent auditory neurons. Specifically, GATA‐3 is expressed in a population of neurons in rhombomere 4 that extend their axons across the floor plate of rhombomere 4 (r4) at embryonic day 10 (E10) and reach the sensory epithelia of the ear by E13.5. The distribution of their cell bodies corresponds to that of the cell bodies of the cochlear and vestibular efferent neurons as revealed by labeling with tracers. Both GATA‐3 heterozygous and GATA‐3 null mutant mice show unusual axonal projections, such as misrouted crossing fibers and fibers in the facial nerve, that are absent in wild‐type littermates. This suggests that GATA‐3 is involved in the pathfinding of efferent neuron axons that navigate to the ear. In the ear, GATA‐3 is expressed inside the otocyst and the surrounding periotic mesenchyme. The latter expression is in areas of branching of the developing ear leading to the formation of semicircular canals. Ears of GATA‐3 null mutants remain cystic, with a single extension of the endolymphatic duct and no formation of semicircular canals or saccular and utricular recesses. Thus, both the distribution of GATA‐3 and the effects of null mutations on the ear suggest involvement of GATA‐3 in morphogenesis of the ear. This study shows for the first time that a zinc finger factor is involved in axonal navigation of the inner ear efferent neurons and, simultaneously, in the morphogenesis of the inner ear. J. Comp. Neurol. 429:615–630, 2001.

A Cre-Dependent GCaMP3 Reporter Mouse for Neuronal Imaging In Vivo

Fluorescent calcium indicator proteins, such as GCaMP3, allow imaging of activity in genetically defined neuronal populations. GCaMP3 can be expressed using various gene delivery methods, such as viral infection or electroporation. However, these methods are invasive and provide inhomogeneous and nonstationary expression. Here, we developed a genetic reporter mouse, Ai38, which expresses GCaMP3 in a Cre-dependent manner from the ROSA26 locus, driven by a strong CAG promoter. Crossing Ai38 with appropriate Cre mice produced robust GCaMP3 expression in defined cell populations in the retina, cortex, and cerebellum. In the primary visual cortex, visually evoked GCaMP3 signals showed normal orientation and direction selectivity. GCaMP3 signals were rapid, compared with virally expressed GCaMP3 and synthetic calcium indicators. In the retina, Ai38 allowed imaging spontaneous calcium waves in starburst amacrine cells during development, and light-evoked responses in ganglion cells in adult tissue. Our results show that the Ai38 reporter mouse provides a flexible method for targeted expression of GCaMP3.

Mammalian Golgi-associated Bicaudal-D2 functions in the dynein–dynactin pathway by interacting with these complexes

Genetic analysis in Drosophila suggests that Bicaudal‐D functions in an essential microtubule‐based transport pathway, together with cytoplasmic dynein and dynactin. However, the molecular mechanism underlying interactions of these proteins has remained elusive. We show here that a mammalian homologue of Bicaudal‐D, BICD2, binds to the dynamitin subunit of dynactin. This interaction is confirmed by mass spectrometry, immunoprecipitation studies and in vitro binding assays. In interphase cells, BICD2 mainly localizes to the Golgi complex and has properties of a peripheral coat protein, yet it also co‐localizes with dynactin at microtubule plus ends. Overexpression studies using green fluorescent protein‐tagged forms of BICD2 verify its intracellular distribution and co‐localization with dynactin, and indicate that the C‐terminus of BICD2 is responsible for Golgi targeting. Overexpression of the N‐terminal domain of BICD2 disrupts minus‐end‐directed organelle distribution and this portion of BICD2 co‐precipitates with cytoplasmic dynein. Nocodazole treatment of cells results in an extensive BICD2–dynactin–dynein co‐localization. Taken together, these data suggest that mammalian BICD2 plays a role in the dynein–dynactin interaction on the surface of membranous organelles, by associating with these complexes.