Cell type-specific dendritic polarity in the absence of spatially organized external cues

Pyramidal neurons of the hippocampus and cortex have polarized dendritic arbors, but little is known about the cellular mechanisms distinguishing apical and basal dendrites. We used morphometric analysis and time lapse imaging of cultured hippocampal neurons to show that glutamatergic neurons develop progressive dendritic asymmetry in the absence of polarized extrinsic cues. Thus, pyramidal neurons have a cellular program for polarized dendrite growth independent of tissue microenvironment.     

Differentiation of Apical and Basal Dendrites in Pyramidal Cells and Granule Cells in Dissociated Hippocampal Cultures

Hippocampal pyramidal cells and dentate granule cells develop morphologically distinct dendritic arbors, yet also share some common features. Both cell types form a long apical dendrite which extends from the apex of the cell soma, while short basal dendrites are developed only in pyramidal cells. Using quantitative morphometric analyses of mouse hippocampal cultures, we evaluated the differences in dendritic arborization patterns between pyramidal and granule cells. Furthermore, we observed and described the final apical dendrite determination during dendritic polarization by time-lapse imaging. Pyramidal and granule cells in culture exhibited similar dendritic patterns with a single principal dendrite and several minor dendrites so that the cell types were not readily distinguished by appearance. While basal dendrites in granule cells are normally degraded by adulthood in vivo, cultured granule cells retained their minor dendrites. Asymmetric growth of a single principal dendrite harboring the Golgi was observed in both cell types soon after the onset of dendritic growth. Time-lapse imaging revealed that up until the second week in culture, final principal dendrite designation was not stabilized, but was frequently replaced by other minor dendrites. Before dendritic polarity was stabilized, the Golgi moved dynamically within the soma and was repeatedly repositioned at newly emerging principal dendrites. Our results suggest that polarized growth of the apical dendrite is regulated by cell intrinsic programs, while regression of basal dendrites requires cue(s) from the extracellular environment in the dentate gyrus. The apical dendrite designation is determined from among multiple growing dendrites of young developing neurons.     

LIMK1 acts downstream of BMP signaling in developing retinal ganglion cell axons but not dendrites

The actin cytoskeleton inside extending axonal and dendritic processes must undergo continuous assembly and disassembly. Some extrinsic factors modulate actin turnover through controlling the activity of LIM kinase 1 (LIMK1), which phosphorylates and inactivates the actin depolymerizing factor cofilin. Here, we for the first time examine the function and regulation of LIMK1 in vivo in the vertebrate nervous system. Upon expression of wildtype or kinase-dead forms of the protein, dendrite growth by Xenopus retinal ganglion cells (RGCs) was unchanged. In contrast, maintaining a low, but significant level, of LIMK1 function in the RGC axon is critical for proper extension. Interestingly, bone morphogenetic protein receptor II (BMPRII) is a major regulator of LIMK1 in extending RGC axons, as expression of a BMPRII lacking the LIMK1 binding region caused a dramatic shortening of the axons. Previously, we found that BMPRIIs stimulate dendrite initiation in vivo. Thus, the fact that manipulation of LIMK1 activity failed to alter dendrite growth suggests that BMPs may activate distinct signalling pathways in axons and dendrites.     

Dendrite remodeling in development and disease

One of the most important features of neuronal function is the capacity to dynamically adapt in response to changes in the environment and neuronal activity. Among cellular elements that show this kind of plasticity are dendrites, the components that receive and process neuronal inputs. Dendrite remodeling occurs during normal development of the nervous system as well as in response to injury or diseases in the adult. In either case, selective stabilization and/or elimination of dendritic branches is likely important to shape dendritic arbors. Here I review examples of the phenomena and consider potential cellular and molecular mechanisms that underlie dendrite remodeling and how they might relate in development and disease.     

The rodent Four-jointed ortholog Fjx1 regulates dendrite extension

The extrinsic and intrinsic factors that regulate the size and complexity of dendritic arborizations are still poorly understood. Here we identify Fjx1, the rodent ortholog of the Drososphila planar cell polarity (PCP) protein Four-jointed (Fj), as a new inhibitory factor that regulates dendrite extension. The Drosophila gene four-jointed (fj) has been suggested to provide directional information in wing discs, but the mechanism how it acts is only poorly understood and the function of its mammalian homolog Fjx1 remains to be investigated. We analyzed the phenotype of a null mutation for mouse Fjx1. Homozygous Fjx1 mutants show an abnormal morphology of dendritic arbors in the hippocampus. In cultured hippocampal neurons from Fjx1 mutant mice, loss of Fjx1 resulted in an increase in dendrite extension and branching. Addition of Fjx1 to cultures of dissociated hippocampal neurons had the opposite effect and reduced the length of dendrites and decreased dendritic branching. Rescue experiments with cultured neurons showed that Fjx1 can act both cell-autonomously and non-autonomously. Our results identify Fjx1 as a new inhibitory factor that regulates dendrite extension.     

In vivo imaging reveals dendritic targeting of laminated afferents by zebrafish retinal ganglion cells

Targeting of axons and dendrites to particular synaptic laminae is an important mechanism by which precise patterns of neuronal connectivity are established. Although axons target specific laminae during development, dendritic lamination has been thought to occur largely by pruning of inappropriately placed arbors. We discovered by in vivo time-lapse imaging that retinal ganglion cell (RGC) dendrites in zebrafish show growth patterns implicating dendritic targeting as a mechanism for contacting appropriate synaptic partners. Populations of RGCs labeled in transgenic animals establish distinct dendritic strata sequentially, predominantly from the inner to outer retina. Imaging individual cells over successive days confirmed that multistratified RGCs generate strata sequentially, each arbor elaborating within a specific lamina. Simultaneous imaging of RGCs and subpopulations of presynaptic amacrine interneurons revealed that RGC dendrites appear to target amacrine plexuses that had already laminated. Dendritic targeting of prepatterned afferents may thus be a novel mechanism for establishing proper synaptic connectivity.     

Ankyrin Repeat‐rich Membrane Spanning/Kidins220 protein regulates dendritic branching and spine stability in vivo

The development of nervous system connectivity depends upon the arborization of dendritic fields and the stabilization of dendritic spine synapses. It is well established that neuronal activity and the neurotrophin BDNF modulate these correlated processes. However, the downstream mechanisms by which these extrinsic signals regulate dendritic development and spine stabilization are less well known. Here we report that a substrate of BDNF signaling, the Ankyrin Repeat‐rich Membrane Spanning (ARMS) protein or Kidins220, plays a critical role in the branching of cortical and hippocampal dendrites and in the turnover of cortical spines. In the barrel somatosensory cortex and the dentate gyrus, regions where ARMS/Kidins220 is highly expressed, no difference in the complexity of dendritic arbors was observed in 1‐month‐old adolescent ARMS/Kidins220^+/? mice compared to wild‐type littermates. However, at 3 months of age, young adult ARMS/Kidins220^+/? mice exhibited decreased dendritic complexity. This suggests that ARMS/Kidins220 does not play a significant role in the initial formation of dendrites but, rather, is involved in the refinement or stabilization of the arbors later in development. In addition, at 1 month of age, the rate of spine elimination was higher in ARMS/Kidins220^+/? mice than in wild‐type mice, suggesting that ARMS/Kidins220^+/? levels regulate spine stability. Taken together, these data suggest that ARMS/Kidins220 is important for the growth of dendritic arbors and spine stability during an activity‐ and BDNF‐dependent period of development. © 2009 Wiley Periodicals, Inc. Develop Neurobiol 2009     

REDD2-mediated inhibition of mTOR promotes dendrite retraction induced by axonal injury

Dendritic defects occur in neurodegenerative diseases accompanied by axonopathy, yet the mechanisms that regulate these pathologic changes are poorly understood. Using Thy1-YFPH mice subjected to optic nerve axotomy, we demonstrate early retraction of retinal ganglion cell (RGC) dendrites and selective loss of mammalian target of rapamycin (mTOR) activity, which precede soma loss. Axonal injury triggered rapid upregulation of the stress-induced protein REDD2 (regulated in development and DNA damage response 2), a potent inhibitor of mTOR. Short interfering RNA-mediated REDD2 knockdown restored mTOR activity and rescued dendritic length, area and branch complexity in a rapamycin-dependent manner. Whole-cell recordings demonstrated that REDD2 depletion leading to mTOR activation in RGCs restored their light response properties. Lastly, we show that REDD2-dependent mTOR activity extended RGC survival following axonal damage. These results indicate that injury-induced stress leads to REDD2 upregulation, mTOR inhibition and dendrite pathology causing neuronal dysfunction and subsequent cell death.During normal neural development there is selective elimination of dendritic and axonal branches without loss of the neuron itself.¹ This developmental pruning refines neuronal processes and ensures precise connectivity. Most of our current knowledge about structural changes in dendrites stems from studies of dendritic remodeling during development.^2,3 In contrast, little is known about how dendritic arbors are affected by trauma or disease in the adult central nervous system (CNS). Defects in dendritic arborization and connectivity are being recognized as one of the first stages of neurodegeneration. Indeed, dendritic abnormalities and loss of synapses have been reported in neuropsychiatric disorders such as schizophrenia and depression, as well as in neurodegenerative conditions including Alzheimer''s disease, stroke and glaucoma.^4,5 Despite the fact that dendritic defects are likely to have devastating consequences on neuronal function and survival, the mechanisms that regulate dendrite degeneration in mature CNS neurons are poorly understood.Recent studies have identified the mammalian target of rapamycin (mTOR) as a critical component of dendritic tree development.^{6, 7, 8, 9} A substantial reduction in the number of dendritic branches and arbor shrinkage were observed in developing hippocampal neurons when mTOR was inhibited.^6,7 In addition, mTOR has been recently implicated in the regulation of dendritic spine morphology, synaptogenesis and synaptic plasticity.^10,11 The emerging developmental role of mTOR in the regulation of dendritic dynamics prompted us to put forward the hypothesis that dysregulation of mTOR function might contribute to dendritic pathology in adult neurons following injury.Many of the signals that impinge upon mTOR activity act through the tuberous sclerosis complex (TSC1/2), a negative regulator of mTOR function. For instance, stress signals such as hypoxia and energy depletion activate TSC1/2 through the REDD (regulated in development and DNA damage response) proteins,^{12, 13, 14} leading to the loss of mTOR activity. REDD2, a member of this family also known as DDIT4L or RTP801L, is an attractive target because in addition to being a potent mTOR inhibitor, it is implicated in stress responses leading to cell death.^15,16 Although REDD2 is enriched in skeletal muscle and has been shown to inhibit mTOR signaling in response to leucine and stretch,¹⁷ its expression and function in the nervous system is currently unknown.We used a model of acute optic nerve lesion in vivo to ask whether axonal damage had a direct effect on retinal ganglion cell (RGC) dendrite morphology and, if so, to identify the molecular mechanisms that regulate this injury-induced response. Our data demonstrate that axonal damage leads to substantial retraction of RGC dendritic arbors before soma loss. Optic nerve lesion led to selective REDD2 upregulation in RGCs, which coincided with the loss of mTOR activity. Short interfering RNA (siRNA)-mediated knockdown of REDD2 restored mTOR function in injured neurons and fully rescued their dendritic arbors, increasing dendritic length, field area and branch complexity. REDD2 depletion also abrogated pathologic RGC hyperexcitability and restored the light response properties of these neurons. Collectively, these data identify the REDD2-mTOR signaling pathway as a critical regulator of dendritic arbor morphology in adult central neurons undergoing axonal damage.     

Different levels of the homeodomain protein cut regulate distinct dendrite branching patterns of Drosophila multidendritic neurons

Functionally similar neurons can share common dendrite morphology, but how different neurons are directed into similar forms is not understood. Here, we show in embryonic and larval development that the level of Cut immunoreactivity in individual dendritic arborization (da) sensory neurons correlates with distinct patterns of terminal dendrites: high Cut in neurons with extensive unbranched terminal protrusions (dendritic spikes), medium levels in neurons with expansive and complex arbors, and low or nondetectable Cut in neurons with simple dendrites. Loss of Cut reduced dendrite growth and class-specific terminal branching, whereas overexpression of Cut or a mammalian homolog in lower-level neurons resulted in transformations toward the branch morphology of high-Cut neurons. Thus, different levels of a homeoprotein can regulate distinct patterns of dendrite branching.     

An autism-associated variant of Epac2 reveals a role for Ras/Epac2 signaling in controlling basal dendrite maintenance in mice

The architecture of dendritic arbors determines circuit connectivity, receptive fields, and computational properties of neurons, and dendritic structure is impaired in several psychiatric disorders. While apical and basal dendritic compartments of pyramidal neurons are functionally specialized and differentially regulated, little is known about mechanisms that selectively maintain basal dendrites. Here we identified a role for the Ras/Epac2 pathway in maintaining basal dendrite complexity of cortical neurons. Epac2 is a guanine nucleotide exchange factor (GEF) for the Ras-like small GTPase Rap, and it is highly enriched in the adult mouse brain. We found that in vivo Epac2 knockdown in layer 2/3 cortical neurons via in utero electroporation reduced basal dendritic architecture, and that Epac2 knockdown in mature cortical neurons in vitro mimicked this effect. Overexpression of an Epac2 rare coding variant, found in human subjects diagnosed with autism, also impaired basal dendritic morphology. This mutation disrupted Epac2's interaction with Ras, and inhibition of Ras selectively interfered with basal dendrite maintenance. Finally, we observed that components of the Ras/Epac2/Rap pathway exhibited differential abundance in the basal versus apical dendritic compartments. These findings define a role for Epac2 in enabling crosstalk between Ras and Rap signaling in maintaining basal dendrite complexity, and exemplify how rare coding variants, in addition to their disease relevance, can provide insight into cellular mechanisms relevant for brain connectivity.     

Principles of branch dynamics governing shape characteristics of cerebellar Purkinje cell dendrites

Neurons develop dendritic arbors in cell type-specific patterns. Using growing Purkinje cells in culture as a model, we performed a long-term time-lapse observation of dendrite branch dynamics to understand the rules that govern the characteristic space-filling dendrites. We found that dendrite architecture was sculpted by a combination of reproducible dynamic processes, including constant tip elongation, stochastic terminal branching, and retraction triggered by contacts between growing dendrites. Inhibition of protein kinase C/protein kinase D signaling prevented branch retraction and significantly altered the characteristic morphology of long proximal segments. A computer simulation of dendrite branch dynamics using simple parameters from experimental measurements reproduced the time-dependent changes in the dendrite configuration in live Purkinje cells. Furthermore, perturbation analysis to parameters in silico validated the important contribution of dendritic retraction in the formation of the characteristic morphology. We present an approach using live imaging and computer simulations to clarify the fundamental mechanisms of dendrite patterning in the developing brain.     

BDNF increases synapse density in dendrites of developing tectal neurons in vivo

Neuronal connections are established through a series of developmental events that involve close communication between pre- and postsynaptic neurons. In the visual system, BDNF modulates the development of neuronal connectivity by influencing presynaptic retinal ganglion cell (RGC) axons. Increasing BDNF levels in the optic tectum of Xenopus tadpoles significantly increases both axon arborization and synapse density per axon terminal within a few hours of treatment. Here, we have further explored the mechanisms by which BDNF shapes synaptic connectivity by imaging tectal neurons, the postsynaptic partners of RGCs. Individual neurons were co-labeled with DsRed2 and a GFP-tagged postsynaptic density protein (PSD95-GFP) to visualize dendritic morphology and postsynaptic specializations simultaneously in vivo. Immunoelectron microscopy confirmed that PSD95-GFP predominantly localized to ultrastructurally identified synapses. Time-lapse confocal microscopy of individual, double-labeled neurons revealed a coincident, activity-dependent mechanism of synaptogenesis and axon and dendritic arbor growth, which is differentially modulated by BDNF. Microinjection of BDNF into the optic tectum significantly increased synapse number in tectal neuron dendritic arbors within 24 hours, without significantly influencing arbor morphology. BDNF function-blocking antibodies had opposite effects. The BDNF-elicited increase in synapse number complements the previously observed increase in presynaptic sites on RGC axons. These results, together with the timescale of the response by tectal neurons, suggest that the effects of BDNF on dendritic synaptic connectivity are secondary to its effects on presynaptic RGCs. Thus, BDNF influences synaptic connectivity in multiple ways: it enhances axon arbor complexity expanding the synaptic territory of the axon, while simultaneously coordinating synapse formation and stabilization with individual postsynaptic cells.     

Developmental mechanisms that regulate retinal ganglion cell dendritic morphology

One of the fundamental features of retinal ganglion cells (RGCs) is that dendrites of individual RGCs are confined to one or a few narrow strata within the inner plexiform layer (IPL), and each RGC synapses only with a small group of presynaptic bipolar and amacrine cells with axons/dendrites ramified in the same strata to process distinct visual features. The underlying mechanisms which control the development of this laminar-restricted distribution pattern of RGC dendrites have been extensively studied, and it is still an open question whether the dendritic pattern of RGCs is determined by molecular cues or by activity-dependent refinement. Accumulating evidence suggests that both molecular cues and activity-dependent refinement might regulate RGC dendrites in a cell subtype-specific manner. However, identification of morphological subtypes of RGCs before they have achieved their mature dendritic pattern is a major challenge in the study of RGC dendritic development. This problem is now being circumvented through the use of molecular markers in genetically engineered mouse lines to identify RGC subsets early during development. Another unanswered fundamental question in the study of activity-dependent refinement of RGC dendrites is how changes in synaptic activity lead to the changes in dendritic morphology. Recent studies have started to shed light on the molecular basis of activity-dependent dendritic refinement of RGCs by showing that some molecular cascades control the cytoskeleton reorganization of RGCs.     

Polarized secretory trafficking directs cargo for asymmetric dendrite growth and morphogenesis

Proper growth of dendrites is critical to the formation of neuronal circuits, but the cellular machinery that directs the addition of membrane components to generate dendritic architecture remains obscure. Here, we demonstrate that post-Golgi membrane trafficking is polarized toward longer dendrites of hippocampal pyramidal neurons in vitro and toward apical dendrites in vivo. Small Golgi outposts partition selectively into longer dendrites and are excluded from axons. In dendrites, Golgi outposts concentrate at branchpoints where they engage in post-Golgi trafficking. Within the cell body, the Golgi apparatus orients toward the longest dendrite, and this Golgi polarity precedes asymmetric dendrite growth. Manipulations that selectively block post-Golgi trafficking halt dendrite growth in developing neurons and cause a shrinkage of dendrites in mature pyramidal neurons. Further, disruption of Golgi polarity produces neurons with symmetric dendritic arbors lacking a single longest principal dendrite. These results define a novel polarized organization of neuronal secretory trafficking and demonstrate a mechanistic link between directed membrane trafficking and asymmetric dendrite growth.     

Development of the mouse retina: emerging morphological diversity of the ganglion cells

The time course and regulatory mechanisms of dendritic development are subjects of intense interest. We approached these problems by investigating dendritic morphology of retinal ganglion cells (RGCs) at four early postnatal stages. The RGCs develop from a diffusely stratified and poorly differentiated group at birth (P0), to 16 distinct, morphologically well-defined subtypes before eye opening (P13). Even before bipolar cells make synaptic contacts with the RGCs (P8), most adultlike RGC subtypes are already present. Similar to previous studies in other mammalian species, our results indicate that the initiation of the RGC morphological maturation is independent of light stimulation and of formation of glutamatergic synapses. This study narrowed down the window of RGCs morphological maturation and highlighted a few early postnatal events as potential factors controlling the developmental process. Because mouse is the most popular mammalian model for genetic manipulation, this study provided a foundation for further exploring regulatory mechanisms of RGC dendritic development.     

The origin recognition core complex regulates dendrite and spine development in postmitotic neurons

The origin recognition complex (ORC) ensures exactly one round of genome replication per cell cycle through acting as a molecular switch that precisely controls the assembly, firing, and inactivation of the replication initiation machinery. Recent data indicate that it may also coordinate the processes of mitosis and cytokinesis and ensure the proper distribution of replicated genome to daughter cells. We have found that the ORC core subunits are highly expressed in the nervous system. They are selectively localized to the neuronal somatodendritic compartment and enriched in the membrane fraction. siRNA knockdown of ORC subunits dramatically reduced dendritic branch formation and severely impeded dendritic spine emergence. Expression of ORC ATPase motif mutants enhanced the branching of dendritic arbors. The ORC core complex thus appears to have a novel role in regulating dendrite and dendritic spine development in postmitotic neurons.