Neurobiology and cellular pathogenesis of glycolipid storage diseases

Disorders of lysosomal metabolism often involve the accumulation of specific types of glycolipid, particularly gangliosides, because of either degradative failure or other currently unknown mechanisms. Although the precise role of gangliosides in cells remains enigmatic, the presence of specific abnormalities secondary to ganglioside accumulation in lysosomal diseases has suggested important biological functions. Chief among these is the growth of new dendrites on particular classes of mature neurons secondary to an increase in GM2 ganglioside. That GM2 has also been shown to be elevated in normal immature neurons coincident with dendritic sprouting provides a compelling argument that this ganglioside plays a role in dendritic initiation. This discovery has led to the search for other regulators of dendritic differentiation that may in some way be linked to the expression and/or function of GM2 ganglioside. Principal candidates that have emerged include tyrosine kinase receptors, small GTPases and calcium/calmodulin-dependent protein kinase II. Understanding the mechanism underlying ectopic dendritogenesis in lysosomal diseases can be expected to generate significant insight into the control of dendritic plasticity in normal brain. The detrimental aspects of ganglioside accumulation in storage diseases as well as the potential link between gangliosides and dendritogenesis also provide a strong rationale for developing pharmacological means to manipulate ganglioside expression in neurons.     

GM2 ganglioside and pyramidal neuron dendritogenesis

GM2 ganglioside, although scarce in normal adult brain, is the predominant ganglioside accumulating in several types of lysosomal disorders, most notably Tay-Sachs disease. Pyramidal neurons of cerebral cortex in Tay-Sachs, as well as many other types of neuronal storage disorders, are known to exhibit a phenomenon believed unique to storage disorders: growth of ectopic dendrites. Recent studies have shown that a common metabolic abnormality shared by storage diseases with ectopic dendrite growth is the abnormal accumulation of GM2 ganglioside. The correlation between increased levels of GM2 and the presence of ectopic dendrites has been found in both ganglioside and nonganglioside storage disorders, the latter including sphingomyelin-cholesterol lipidosis, mucopolysaccharidosis, and -mannosidosis. Quantitative HPTLC analysis has shown that increases in GM2 occur in proportion to the incidence of ectopic dendrite growth, whereas, other gangliosides, including GM1, lack similar increases. Immunocytochemical studies of all nonganglioside storage diseases which exhibit ectopic dendritogenesis have revealed heightened GM2 ganglioside-immunoreactivity in the cortical pyramidal cell population, whereas neurons in normal adult brain exhibit little or no staining for this ganglioside. Further, studies examining disease development have consistently shown that accumulation of GM2 gangliosideprecedes growth of ectopic dendrites, indicating that it is not simply occurring secondary to new membrane production. These findings have prompted an examination for a similar relationship between GM2 ganglioside and dendritogenesis in cortical neurons of normal developing brain. Results show that GM2 ganglioside-immunoreactivity is consistently elevated in immature neurons during the period when they are undergoing active dendritic initiation, but this staining diminishes dramatically as the dendritic tress of these cells mature. Collectively, these studies on diseased and normal brain offer compelling evidence that GM2 ganglioside plays a pivotal role in the regulation of dendritogenesis in cortical pyramidal neurons.Special issue dedicated to Dr. Leon S. Wolfe.     

MeCP2 mutation results in compartment-specific reductions in dendritic branching and spine density in layer 5 motor cortical neurons of YFP-H mice

Rett Syndrome (RTT) is a neurodevelopmental disorder predominantly caused by mutations in the X-linked gene MECP2. A primary feature of the syndrome is the impaired maturation and maintenance of excitatory synapses in the central nervous system (CNS). Different RTT mouse models have shown that particular Mecp2 mutations have highly variable effects on neuronal architecture. Distinguishing MeCP2 mutant cellular phenotypes therefore demands analysis of specific mutations in well-defined neuronal subpopulations. We examined a transgenically labeled subset of cortical neurons in YFP-H mice crossed with the Mecp2(tm1.1Jae) mutant line. YFP(+) Layer 5 pyramidal neurons in the motor cortex of wildtype and hemizygous mutant male mice were examined for differences in dendrite morphology and spine density. Total basal dendritic length was decreased by 18.6% due to both shorter dendrites and reduced branching proximal to the soma. Tangential dendrite lengths in the apical tuft were reduced by up to 26.6%. Spine density was reduced by 47.4% in the apical tuft and 54.5% in secondary apical dendrites, but remained unaffected in primary apical and proximal basal dendrites. We also found that MeCP2 mutation reduced the number of YFP(+) cells in YFP-H mice by up to 72% in various cortical regions without affecting the intensity of YFP expression in individual cells. Our results support the view that the effects of MeCP2 mutation are highly context-dependent and cannot be generalized across mutation types and cell populations.     

Accelerated dendritic development of rat cortical pyramidal cells and interneurons after biolistic transfection with BDNF and NT4/5

Neurotrophins are candidate molecules for regulating dendritogenesis. We report here on dendritic growth of rat visual cortex pyramidal and interneurons overexpressing 'brain-derived neurotrophic factor' BDNF and 'neurotrophin 4/5' NT4/5. Neurons in organotypic cultures were transfected with plasmids encoding either 'enhanced green fluorescent protein' EGFP, BDNF/EGFP or NT4/5/EGFP either at the day of birth with analysis at 5 days in vitro, or at 5 days in vitro with analysis at 10 days in vitro. In pyramidal neurons, both TrkB ligands increased dendritic length and number of segments without affecting maximum branch order and number of primary dendrites. In the early time window, only infragranular neurons were responsive. Neurons in layers II/III became responsive to NT4/5, but not BDNF, during the later time window. BDNF and NT4/5 transfectants at 10 days in vitro had still significantly shorter dendrites than adult pyramidal neurons, suggesting a massive growth spurt after 10 days in vitro. However, segment numbers were already in the range of adult neurons. Although this suggested a role for BDNF, long-term activity-deprived, and thus BDNF-deprived, pyramidal cells developed a dendritic complexity not different from neurons in active cultures except for higher spine densities on neurons of layers II/III and VI. Neutralization of endogenous NT4/5 causes shorter and less branched dendrites at 10 days in vitro suggesting an essential role for NT4/5. Neutralization of BDNF had no effect. Transfected multipolar interneurons became identifiable during the second time window. Both TrkB ligands significantly increased number of segments and branch order towards the adult state with little effects on dendritic length. The results suggested that early in development BDNF and NT4/5 probably accelerate dendritogenesis in an autocrine fashion. In particular, branch formation was advanced towards the adult pattern in pyramidal cells and interneurons.     

Phosphorylation of Pak1 by the p35/Cdk5 kinase affects neuronal morphology

The small GTPase Rac and its effectors, the Pak1 and p35/Cdk5 kinases, have been assigned important roles in regulating cytoskeletal dynamics in neurons. Our previous work revealed that the neuronal p35/Cdk5 kinase associates with Pak1 in a RacGTP-dependent manner, causing hyperphosphorylation and down-regulation of Pak1 kinase activity. We have now demonstrated direct phosphorylation of Pak1 on threonine 212 by the p35/Cdk5 kinase. In neuronal growth cones, Pak1 phosphorylated on Thr-212 localized to actin and tubulin-rich areas, suggesting a role in regulating growth cone dynamics. The expression of a non-phosphorylatable Pak1 mutant (Pak1A212) induced dramatic neurite disorganization. We also observed a strong association between p35/Cdk5 and the Pak1 C-terminal kinase domain. Overall, our data show that in neurons, membrane-associated, active Pak1 is regulated by the p35/Cdk5 kinase both by association and phosphorylation, which is essential for the proper regulation of the cytoskeleton during neurite outgrowth and remodeling.     

The RhoG/ELMO1/Dock180 signaling module is required for spine morphogenesis in hippocampal neurons

Dendritic spines are actin-rich structures, the formation and plasticity of which are regulated by the Rho GTPases in response to synaptic input. Although several guanine nucleotide exchange factors (GEFs) have been implicated in spine development and plasticity in hippocampal neurons, it is not known how many different Rho GEFs contribute to spine morphogenesis or how they coordinate the initiation, establishment, and maintenance of spines. In this study, we screened 70 rat Rho GEFs in cultured hippocampal neurons by RNA interference and identified a number of candidates that affected spine morphogenesis. Of these, Dock180, which plays a pivotal role in a variety of cellular processes including cell migration and phagocytosis, was further investigated. We show that depletion of Dock180 inhibits spine morphogenesis, whereas overexpression of Dock180 promotes spine morphogenesis. ELMO1, a protein necessary for in vivo functions of Dock180, functions in a complex with Dock180 in spine morphogenesis through activating the Rac GTPase. Moreover, RhoG, which functions upstream of the ELMO1/Dock180 complex, is also important for spine formation. Together, our findings uncover a role for the RhoG/ELMO1/Dock180 signaling module in spine morphogenesis in hippocampal neurons.     

Activity-dependent regulation of h channel distribution in hippocampal CA1 pyramidal neurons

The hyperpolarization-activated cation current, I(h), plays an important role in regulating intrinsic neuronal excitability in the brain. In hippocampal pyramidal neurons, I(h) is mediated by h channels comprised primarily of the hyperpolarization-activated cyclic nucleotide-gated (HCN) channel subunits, HCN1 and HCN2. Pyramidal neuron h channels within hippocampal area CA1 are remarkably enriched in distal apical dendrites, and this unique distribution pattern is critical for regulating dendritic excitability. We utilized biochemical and immunohistochemical approaches in organotypic slice cultures to explore factors that control h channel localization in dendrites. We found that distal dendritic enrichment of HCN1 is first detectable at postnatal day 13, reaching maximal enrichment by the 3rd postnatal week. Interestingly we found that an intact entorhinal cortex, which projects to distal dendrites of CA1 but not area CA3, is critical for the establishment and maintenance of distal dendritic enrichment of HCN1. Moreover blockade of excitatory neurotransmission using tetrodotoxin, 6-cyano-7-nitroquinoxaline-2,3-dione, or 2-aminophosphonovalerate redistributed HCN1 evenly throughout the dendrite without significant changes in protein expression levels. Inhibition of calcium/calmodulin-dependent protein kinase II activity, but not p38 MAPK, also redistributed HCN1 in CA1 pyramidal neurons. We conclude that activation of ionotropic glutamate receptors by excitatory temporoammonic pathway projections from the entorhinal cortex establishes and maintains the distribution pattern of HCN1 in CA1 pyramidal neuron dendrites by activating calcium/calmodulin-dependent protein kinase II-mediated downstream signals.     

The RhoGEF DOCK10 is essential for dendritic spine morphogenesis

By regulating actin cytoskeleton dynamics, Rho GTPases and their activators RhoGEFs are implicated in various aspects of neuronal differentiation, including dendritogenesis and synaptogenesis. Purkinje cells (PCs) of the cerebellum, by developing spectacular dendrites covered with spines, represent an attractive model system in which to decipher the molecular signaling underlying these processes. To identify novel regulators of dendritic spine morphogenesis among members of the poorly characterized DOCK family of RhoGEFs, we performed gene expression profiling of fluorescence-activated cell sorting (FACS)-purified murine PCs at various stages of their postnatal differentiation. We found a strong increase in the expression of the Cdc42-specific GEF DOCK10. Depleting DOCK10 in organotypic cerebellar cultures resulted in dramatic dendritic spine defects in PCs. Accordingly, in mouse hippocampal neurons, depletion of DOCK10 or expression of a DOCK10 GEF-dead mutant led to a strong decrease in spine density and size. Conversely, overexpression of DOCK10 led to increased spine formation. We show that DOCK10 function in spinogenesis is mediated mainly by Cdc42 and its downstream effectors N-WASP and PAK3, although DOCK10 is also able to activate Rac1. Our global approach thus identifies an unprecedented function for DOCK10 as a novel regulator of dendritic spine morphogenesis via a Cdc42-mediated pathway.     

Upregulation of Dendritic Arborization by N-acetyl-D-Glucosamine Kinase Is Not Dependent on Its Kinase Activity

N-acetylglucosamine kinase (GlcNAc kinase or NAGK; EC 2.7.1.59) is highly expressed and plays a critical role in the development of dendrites in brain neurons. In this study, the authors conducted structure-function analysis to verify the previously proposed 3D model structure of GlcNAc/ATP-bound NAGK. Three point NAGK mutants with different substrate binding capacities and reaction velocities were produced. Wild-type (WT) NAGK showed strong substrate preference for GlcNAc. Conversion of Cys143, which does not make direct hydrogen bonds with GlcNAc, to Ser (i.e., C143S) had the least affect on the enzymatic activity of NAGK. Conversion of Asn36, which plays a role in domain closure by making a hydrogen bond with GlcNAc, to Ala (i.e., N36A) mildly reduced NAGK enzyme activity. Conversion of Asp107, which makes hydrogen bonds with GlcNAc and would act as a proton acceptor during nucleophilic attack on the γ-phosphate of ATP, to Ala (i.e., D107A), caused a total loss in enzyme activity. The over-expression of EGFP-tagged WT or any of the mutant NAGKs in rat hippocampal neurons (DIV 5-9) increased dendritic architectural complexity. Finally, the overexpression of the small, but not of the large, domain of NAGK resulted in dendrite degeneration. Our data show the effect of structure on the functional aspects of NAGK, and in particular, that the small domain of NAGK, and not its NAGK kinase activity, plays a critical role in the upregulation of dendritogenesis.     

Signals from the Ras, Rac, and Rho GTPases converge on the Pak protein kinase in Rat-1 fibroblasts

Ras plays a key role in regulating cellular proliferation, differentiation, and transformation. Raf is the major effector of Ras in the Ras > Raf > Mek > extracellular signal-activated kinase (ERK) cascade. A second effector is phosphoinositide 3-OH kinase (PI 3-kinase), which, in turn, activates the small G protein Rac. Rac also has multiple effectors, one of which is the serine threonine kinase Pak (p65(Pak)). Here we show that Ras, but not Raf, activates Pak1 in cotransfection assays of Rat-1 cells but not NIH 3T3 cells. We tested agents that activate or block specific components downstream of Ras and demonstrate a Ras > PI 3-kinase > Rac/Cdc42 > Pak signal. Although these studies suggest that the signal from Ras through PI 3-kinase is sufficient to activate Pak, additional studies suggested that other effectors contribute to Pak activation. RasV12S35 and RasV12G37, two effector mutant proteins which fail to activate PI 3-kinase, did not activate Pak when tested alone but activated Pak when they were cotransfected. Similarly, RacV12H40, an effector mutant that does not bind Pak, and Rho both cooperated with Raf to activate Pak. A dominant negative Rho mutant also inhibited Ras activation of Pak. All combinations of Rac/Raf and Ras/Raf and Rho/Raf effector mutants that transform cells cooperatively stimulated ERK. Cooperation was Pak dependent, since all combinations were inhibited by kinase-deficient Pak mutants in both transformation assays and ERK activation assays. These data suggest that other Ras effectors can collaborate with PI 3-kinase and with each other to activate Pak. Furthermore, the strong correlation between Pak activation and cooperative transformation suggests that Pak activation is necessary, although not sufficient, for cooperative transformation of Rat-1 fibroblasts by Ras, Rac, and Rho.     

A kinase anchor protein 150 (AKAP150)-associated protein kinase A limits dendritic spine density

The A kinase anchor protein AKAP150 recruits the cAMP-dependent protein kinase (PKA) to dendritic spines. Here we show that in AKAP150 (AKAP5) knock-out (KO) mice frequency of miniature excitatory post-synaptic currents (mEPSC) and inhibitory post-synaptic currents (mIPSC) are elevated at 2 weeks and, more modestly, 4 weeks of age in the hippocampal CA1 area versus litter mate WT mice. Linear spine density and ratio of AMPAR to NMDAR EPSC amplitudes were also increased. Amplitude and decay time of mEPSCs, decay time of mIPSCs, and spine size were unaltered. Mice in which the PKA anchoring C-terminal 36 residues of AKAP150 are deleted (D36) showed similar changes. Furthermore, whereas acute stimulation of PKA (2-4 h) increases spine density, prolonged PKA stimulation (48 h) reduces spine density in apical dendrites of CA1 pyramidal neurons in organotypic slice cultures. The data from the AKAP150 mutant mice show that AKAP150-anchored PKA chronically limits the number of spines with functional AMPARs at 2-4 weeks of age. However, synaptic transmission and spine density was normal at 8 weeks in KO and D36 mice. Thus AKAP150-independent mechanisms correct the aberrantly high number of active spines in juvenile AKAP150 KO and D36 mice during development.     

Dapper Antagonist of Catenin-1 Cooperates with Dishevelled-1 during Postsynaptic Development in Mouse Forebrain GABAergic Interneurons

Synaptogenesis has been extensively studied along with dendritic spine development in glutamatergic pyramidal neurons, however synapse development in cortical interneurons, which are largely aspiny, is comparatively less well understood. Dact1, one of 3 paralogous Dact (Dapper/Frodo) family members in mammals, is a scaffold protein implicated in both the Wnt/β-catenin and the Wnt/Planar Cell Polarity pathways. We show here that Dact1 is expressed in immature cortical interneurons. Although Dact1 is first expressed in interneuron precursors during proliferative and migratory stages, constitutive Dact1 mutant mice have no major defects in numbers or migration of these neurons. However, cultured cortical interneurons derived from these mice have reduced numbers of excitatory synapses on their dendrites. We selectively eliminated Dact1 from mouse cortical interneurons using a conditional knock-out strategy with a Dlx-I12b enhancer-Cre allele, and thereby demonstrate a cell-autonomous role for Dact1 during postsynaptic development. Confirming this cell-autonomous role, we show that synapse numbers in Dact1 deficient cortical interneurons are rescued by virally-mediated re-expression of Dact1 specifically targeted to these cells. Synapse numbers in these neurons are also rescued by similarly targeted expression of the Dact1 binding partner Dishevelled-1, and partially rescued by expression of Disrupted in Schizophrenia-1, a synaptic protein genetically implicated in susceptibility to several major mental illnesses. In sum, our results support a novel cell-autonomous postsynaptic role for Dact1, in cooperation with Dishevelled-1 and possibly Disrupted in Schizophrenia-1, in the formation of synapses on cortical interneuron dendrites.     

Pak1 control of E-cadherin endocytosis regulates salivary gland lumen size and shape

Generating and maintaining proper lumen size and shape in tubular organs is essential for organ function. Here, we demonstrate a novel role for p21-activated kinase 1 (Pak1) in defining the size and shape of the Drosophila embryonic salivary gland lumen by regulating the size and elongation of the apical domain of individual cells. Pak1 mediates these effects by decreasing and increasing E-cadherin levels at the adherens junctions and basolateral membrane, respectively, through Rab5- and Dynamin-dependent endocytosis. We also demonstrate that Cdc42 and Merlin act together with Pak1 to control lumen size. A role for Pak1 in E-cadherin endocytosis is supported by our studies of constitutively active Pak1, which induces the formation of multiple intercellular lumens in the salivary gland in a manner dependent on Rab5, Dynamin and Merlin. These studies demonstrate a novel and crucial role for Pak1 and E-cadherin endocytosis in determining lumen size and shape, and also identify a mechanism for multiple lumen formation, a poorly understood process that occurs in normal embryonic development and pathological conditions.     

Changes of Dendritic Spine Density and Morphology in the Superficial Layers of the Medial Entorhinal Cortex Induced by Extremely Low-Frequency Magnetic Field Exposure

In the present study, we investigated the effects of chronic exposure (14 and 28 days) to a 0.5 mT 50 Hz extremely low-frequency magnetic field (ELM) on the dendritic spine density and shape in the superficial layers of the medial entorhinal cortex (MEC). We performed Golgi staining to reveal the dendritic spines of the principal neurons in rats. The results showed that ELM exposure induced a decrease in the spine density in the dendrites of stellate neurons and the basal dendrites of pyramidal neurons at both 14 days and 28 days, which was largely due to the loss of the thin and branched spines. The alteration in the density of mushroom and stubby spines post ELM exposure was cell-type specific. For the stellate neurons, ELM exposure slightly increased the density of stubby spines at 28 days, while it did not affect the density of mushroom spines at the same time. In the basal dendrites of pyramidal neurons, we observed a significant decrease in the mushroom spine density only at the later time point post ELM exposure, while the stubby spine density was reduced at 14 days and partially restored at 28 days post ELM exposure. ELM exposure-induced reduction in the spine density in the apical dendrites of pyramidal neurons was only observed at 28 days, reflecting the distinct vulnerability of spines in the apical and basal dendrites. Considering the changes in spine number and shape are involved in synaptic plasticity and the MEC is a part of neural network that is closely related to learning and memory, these findings may be helpful for explaining the ELM exposure-induced impairment in cognitive functions.     

Activity-dependent synaptogenesis: regulation by a CaM-kinase kinase/CaM-kinase I/betaPIX signaling complex

Neuronal activity augments maturation of mushroom-shaped spines to form excitatory synapses, thereby strengthening synaptic transmission. We have delineated a Ca(2+)-signaling pathway downstream of the NMDA receptor that stimulates calmodulin-dependent kinase kinase (CaMKK) and CaMKI to promote formation of spines and synapses in hippocampal neurons. CaMKK and CaMKI form a multiprotein signaling complex with the guanine nucleotide exchange factor (GEF) betaPIX and GIT1 that is localized in spines. CaMKI-mediated phosphorylation of Ser516 in betaPIX enhances its GEF activity, resulting in activation of Rac1, an established enhancer of spinogenesis. Suppression of CaMKK or CaMKI by pharmacological inhibitors, dominant-negative (dn) constructs and siRNAs, as well as expression of the betaPIX Ser516Ala mutant, decreases spine formation and mEPSC frequency. Constitutively-active Pak1, a downstream effector of Rac1, rescues spine inhibition by dnCaMKI or betaPIX S516A. This activity-dependent signaling pathway can promote synapse formation during neuronal development and in structural plasticity.     

p21-Activated Kinase 2 Regulates Endothelial Development and Function through the Bmk1/Erk5 Pathway

p21-activated kinases (Paks) have been shown to regulate cytoskeleton rearrangements, cell proliferation, attachment, and migration in a variety of cellular contexts, including endothelial cells. However, the role of endothelial Pak in embryo development has not been reported, and currently, there is no consensus on the endothelial function of individual Pak isoforms, in particular p21-activated kinase 2 (Pak2), the main Pak isoform expressed in endothelial cells. In this work, we employ genetic and molecular studies that show that Pak2, but not Pak1, is a critical mediator of development and maintenance of endothelial cell function. Endothelial depletion of Pak2 leads to early embryo lethality due to flawed blood vessel formation in the embryo body and yolk sac. In adult endothelial cells, Pak2 depletion leads to severe apoptosis and acute angiogenesis defects, and in adult mice, endothelial Pak2 deletion leads to increased vascular permeability. Furthermore, ubiquitous Pak2 deletion is lethal in adult mice. We show that many of these defects are mediated through a newly unveiled Pak2/Bmk1 pathway. Our results demonstrate that endothelial Pak2 is essential during embryogenesis and also for adult blood vessel maintenance, and they also pinpoint the Bmk1/Erk5 pathway as a critical mediator of endothelial Pak2 signaling.     

Casein Kinase 2 Phosphorylation of Protein Kinase C and Casein Kinase 2 Substrate in Neurons (PACSIN) 1 Protein Regulates Neuronal Spine Formation

The PACSIN (protein kinase C and casein kinase 2 substrate in neurons) adapter proteins couple components of the clathrin-mediated endocytosis machinery with regulators of actin polymerization and thereby regulate the surface expression of specific receptors. The brain-specific PACSIN 1 is enriched at synapses and has been proposed to affect neuromorphogenesis and the formation and maturation of dendritic spines. In studies of how phosphorylation of PACSIN 1 contributes to neuronal function, we identified serine 358 as a specific site used by casein kinase 2 (CK2) in vitro and in vivo. Phosphorylated PACSIN 1 was found in neuronal cytosol and membrane fractions. This localization could be modulated by trophic factors such as brain-derived neurotrophic factor (BDNF). We further show that expression of a phospho-negative PACSIN 1 mutant, S358A, or inhibition of CK2 drastically reduces spine formation in neurons. We identified a novel protein complex containing the spine regulator Rac1, its GTPase-activating protein neuron-associated developmentally regulated protein (NADRIN), and PACSIN 1. CK2 phosphorylation of PACSIN 1 leads to a dissociation of the complex upon BDNF treatment and induces Rac1-dependent spine formation in dendrites of hippocampal neurons. These findings suggest that upon BDNF signaling PACSIN 1 is phosphorylated by CK2 which is essential for spine formation.     

Dendritic morphology of hippocampal and amygdalar neurons in adolescent mice is resilient to genetic differences in stress reactivity

Many studies have shown that chronic stress or corticosterone over-exposure in rodents leads to extensive dendritic remodeling, particularly of principal neurons in the CA3 hippocampal area and the basolateral amygdala. We here investigated to what extent genetic predisposition of mice to high versus low stress reactivity, achieved through selective breeding of CD-1 mice, is also associated with structural plasticity in Golgi-stained neurons. Earlier, it was shown that the highly stress reactive (HR) compared to the intermediate (IR) and low (LR) stress reactive mice line presents a phenotype, with respect to neuroendocrine parameters, sleep architecture, emotional behavior and cognition, that recapitulates some of the features observed in patients suffering from major depression. In late adolescent males of the HR, IR, and LR mouse lines, we observed no significant differences in total dendritic length, number of branch points and branch tips, summated tip order, number of primary dendrites or dendritic complexity of either CA3 pyramidal neurons (apical as well as basal dendrites) or principal neurons in the basolateral amygdala. Apical dendrites of CA1 pyramidal neurons were also unaffected by the differences in stress reactivity of the animals; marginally higher length and complexity of the basal dendrites were found in LR compared to IR but not HR mice. In the same CA1 pyramidal neurons, spine density of distal apical tertiary dendrites was significantly higher in LR compared to IR or HR animals. We tentatively conclude that the dendritic complexity of principal hippocampal and amygdala neurons is remarkably stable in the light of a genetic predisposition to high versus low stress reactivity, while spine density seems more plastic. The latter possibly contributes to the behavioral phenotype of LR versus HR animals.     

Pak regulates calpain-dependent degradation of E3b1

E3b1, a binding partner of Eps8, plays a critical role in receptor tyrosine kinase (RTK)-mediated Rac activation by facilitating the interaction of Eps8 with Sos-1 and the consequent activation of the Rac-specific guanine nucleotide exchange factor activity of Sos-1. Here we present evidence that E3b1 levels are regulated by the Ca(2+)-activated protease calpain, and also by Pak, a downstream target of Rac signaling. Serum starvation of Rat2 or COS7 cells resulted in rapid loss of E3b1 that was reversed by calpain inhibitors. Loss was also prevented by expressing the constitutively active Pak1 mutant, Pak1(H83,86L). Activation of endogenous Pak by platelet-derived growth factor or the constitutively active Rac1 mutant, Rac1(G12V), also inhibited degradation. In contrast, inhibition of endogenous Pak activity by expressing the Pak auto-inhibitory domain caused degradation of over-expressed E3b1 even in the presence of serum. Taken together, these findings indicate that E3b1 is down-regulated by calpain activation and stabilized by Pak activation. They also suggest that RTK-mediated Rac activation can be modulated by changes in the level of E3b1 in response to signals that affect the activity of calpain or Pak.