Kea Joo Lee

Interaction of Reelin with Amyloid Precursor Protein Promotes Neurite Outgrowth

The processing of amyloid precursor protein (APP) to Aβ is an important event in the pathogenesis of Alzheimers disease, but the physiological function of APP is not well understood. Our previous work has shown that APP processing and Aβ production are regulated by the extracellular matrix protein Reelin. In the present study, we examined whether Reelin interacts with APP, and the functional consequences of that interaction in vitro. Using coimmunoprecipitation, we found that Reelin interacted with APP through the central domain of Reelin (repeats 3–6) and the E1 extracellular domain of APP. Reelin increased cell surface levels of APP and decreased endocytosis of APP in hippocampal neurons in vitro. In vivo, Reelin levels were increased in brains of APP knock-out mice and decreased in APP-overexpressing mice. RNA interference knockdown of APP decreased neurite outgrowth in vitro and prevented Reelin from increasing neurite outgrowth. Knock-out of APP or Reelin decreased dendritic arborization in cortical neurons in vivo, and APP overexpression increased dendritic arborization. APP and Reelin have previously been shown to promote neurite outgrowth through interactions with integrins. We confirmed that APP interacted with α3β1 integrin, and α3β1 integrin altered APP trafficking and processing. Addition of an α3β1 integrin antibody prevented APP and Reelin-induced neurite outgrowth. These findings demonstrate that Reelin interacts with APP, potentially having important effects on neurite development.

Effects of aerobic exercise training on cognitive function and cortical vascularity in monkeys.

This study examined whether regular exercise training, at a level that would be recommended for middle-aged people interested in improving fitness could lead to improved cognitive performance and increased blood flow to the brain in another primate species. Adult female cynomolgus monkeys were trained to run on treadmills for 1 h a day, 5 days a week, for a 5 month period (n=16; 1.9+/-0.4 miles/day). A sedentary control group sat daily on immobile treadmills (n=8). Half of the runners had an additional sedentary period for 3 months at the end of the exercise period (n=8). In all groups, half of the monkeys were middle-aged (10-12 years old) and half were more mature (15-17 years old). Starting the fifth week of exercise training, monkeys underwent cognitive testing using the Wisconsin General Testing Apparatus (WGTA). Regardless of age, the exercising group learned to use the WGTA significantly faster (4.6+/-3.4 days) compared to controls (8.3+/-4.8 days; P=0.05). At the end of 5 months of running monkeys showed increased fitness, and the vascular volume fraction in the motor cortex in mature adult running monkeys was increased significantly compared to controls (P=0.029). However, increased vascular volume did not remain apparent after a 3-month sedentary period. These findings indicate that the level of exercise associated with improved fitness in middle-aged humans is sufficient to increase both the rate of learning and blood flow to the cerebral cortex, at least during the period of regular exercise.

BETA AMYLOID-INDEPENDENT ROLE OF AMYLOID PRECURSOR PROTEIN IN GENERATION AND MAINTENANCE OF DENDRITIC SPINES

Synapse loss induced by amyloid beta (Abeta) is thought to be a primary contributor to cognitive decline in Alzheimers disease. Abeta is generated by proteolysis of amyloid precursor protein (APP), a synaptic receptor whose physiological function remains unclear. In the present study, we investigated the role of APP in dendritic spine formation, which is known to be important for learning and memory. We found that overexpression of APP increased spine number, whereas knockdown of APP reduced spine density in cultured hippocampal neurons. This spine-promoting effect of APP required both the extracellular and intracellular domains of APP, and was accompanied by specific upregulation of the GluR2, but not the GluR1, subunit of AMPA receptors. In an in vivo experiment, we found that cortical layers II/III and hippocampal CA1 pyramidal neurons in 1 year-old APP-deficient mice had fewer and shorter dendritic spines than wild-type littermates. In contrast, transgenic mice overexpressing mutant APP exhibited increased spine density compared to control animals, though only at a young age prior to overaccumulation of soluble amyloid. Additionally, increased glutamate synthesis was observed in young APP transgenic brains, whereas glutamate levels were decreased and GABA levels were increased in APP-deficient mice. These results demonstrate that APP is important for promoting spine formation and is required for proper spine development.

Requirement for Plk2 in orchestrated ras and rap signaling, homeostatic structural plasticity, and memory.

Ras and Rap small GTPases are important for synaptic plasticity and memory. However, their roles in homeostatic plasticity are unknown. Here, we report that polo-like kinase 2 (Plk2), a homeostatic suppressor of overexcitation, governs the activity of Ras and Rap via coordination of their regulatory proteins. Plk2 directs elimination of Ras activator RasGRF1 and Rap inhibitor SPAR via phosphorylation-dependent ubiquitin-proteasome degradation. Conversely, Plk2 phosphorylation stimulates Ras inhibitor SynGAP and Rap activator PDZGEF1. These Ras/Rap regulators perform complementary functions to downregulate dendritic spines and AMPA receptors following elevated activity, and their collective regulation by Plk2 profoundly stimulates Rap and suppresses Ras. Furthermore, perturbation of Plk2 disrupts Ras and Rap signaling, prevents homeostatic shrinkage and loss of dendritic spines, and impairs proper memory formation. Our study demonstrates a critical role of Plk2 in the synchronized tuning of Ras and Rap and underscores the functional importance of this regulation in homeostatic synaptic plasticity.

Experience-Dependent Plasticity of Cerebellar Vermis in Basketball Players

The cerebellum is involved in the learning and retention of motor skills. Using animal and human models, a number of studies have shown that long-term motor skill training induces structural and functional plasticity in the cerebellum. The aim of this study was to investigate whether macroscopic alteration in the volume of cerebellum occurs in basketball players who had learned complex motor skills and practiced them intensively for a long time. Three-dimensional magnetic resonance imaging volumetry was performed in basketball players (n = 19) and healthy controls (n = 20), and the volumes of cerebellum and vermian lobules were compared between two groups. Although there was no macroscopic plasticity detected in the cerebellum as a whole, detailed parcellation of cerebellum revealed morphological enlargement in the vermian lobules VI–VII (declive, folium, and tuber) of basketball players (P < 0.0166), which might then be interpreted as evidence for plasticity. This finding suggests that the extensive practice and performance of sports-related motor skills activate structural plasticity of vermian lobules in human cerebellum and suggests that vermian VI–VII plays an important role in motor learning.

Developmental characteristics of dendritic spines in the dentate gyrus of Fmr1 knockout mice

Fragile X Syndrome (FXS) is the most common form of inherited mental retardation. The neuroanatomical phenotype of adult FXS patients, as well as adult Fmr1 knockout (KO) mice, includes elevated dendritic spine density and a spine morphology profile in neocortex that resembles younger individuals. Developmental studies in mouse neocortex have revealed a dynamic phenotype that varies with age, especially during the period of synaptic pruning. Here we investigated the hippocampal dentate gyrus to determine if the FXS spine phenotype is similarly tied to periods of maturation and pruning in this brain region. We used high-voltage electron microscopy to characterize Golgi-stained spines along granule cell dendrites in Fmr1 KO and wildtype (WT) mouse dentate gyrus at postnatal days 15, 21, 30, and 60. In contrast to neocortex, dendritic spine density was higher in Fmr1 KO mice across development. Interestingly, neither genotype showed specific phases of synaptogenesis or pruning, potentially explaining the phenotypic differences from neocortex. Similarly, although the KO mice showed a more immature morphological phenotype overall than WT (higher proportion of thin headed spines, lower proportion of mushroom and stubby spines), both genotypes showed gradual development, rather than impairments during specific phases of maturation. Finally, spine length showed a complex developmental pattern that differs from other brain regions examined, suggesting dynamic regulation by FMRP and other brain region-specific proteins. These findings shed new light on FMRPs role in development and highlight the need for new techniques to further understand the mechanisms by which FMRP affects synaptic maturation.

Morphological changes in dendritic spines of Purkinje cells associated with motor learning.

Experience-dependent changes of spine structure and number may contribute to long-term memory storage. Although several studies demonstrated structural spine plasticity following associative learning, there is limited evidence associating motor learning with alteration of spine morphology. Here, we investigated this issue in the cerebellar Purkinje cells using high voltage electron microscopy (HVEM). Adult rats were trained in an obstacle course, demanding significant motor coordination to complete. Control animals either traversed an obstacle-free runway or remained sedentary. Quantitative analysis of spine morphology showed that the density and length of dendritic spines along the distal dendrites of Purkinje cells were significantly increased in the rats that learned complex motor skills compared to active or inactive controls. Classification of spines into shape categories indicated that the increased spine density and length after motor learning was mainly attributable to an increase in thin spines. These findings suggest that motor learning induces structural spine plasticity in the cerebellar Purkinje neurons, which may play a crucial role in acquiring complex motor skills.

The maintenance of specific aspects of neuronal function and behavior is dependent on programmed cell death of adult‐generated neurons in the dentate gyrus

A considerable number of new neurons are generated daily in the dentate gyrus (DG) of the adult hippocampus, but only a subset of these survive, as many adult‐generated neurons undergo programmed cell death (PCD). However, the significance of PCD in the adult brain for the functionality of DG circuits is not known. Here, we examined the electrophysiological and behavioral characteristics of Bax‐knockout (Bax‐KO) mice in which PCD of post‐mitotic neurons is prevented. The continuous increase in DG cell numbers in Bax‐KO mice resulted in the readjustment of afferent and efferent synaptic connections, represented by age‐dependent reductions in the dendritic arborization of DG neurons and in the synaptic contact ratio of mossy fibers with CA3 dendritic spines. These neuroanatomical changes were associated with reductions in synaptic transmission and reduced performance in a contextual fear memory task in 6‐month‐old Bax‐KO mice. These results suggest that the elimination of excess DG neurons via Bax‐dependent PCD in the adult brain is required for the normal organization and function of the hippocampus.

Specific plasticity of parallel fiber/Purkinje cell spine synapses by motor skill learning.

New synapse formation may underlie learning and memory. To examine specific synaptic plasticity by motor learning, we conducted quantitative analysis of synapses between parallel fibers and Purkinje cell dendritic spines in cerebella of rats trained to complete various obstacle courses. Synapses between parallel fibers and Purkinje cell spines were classified into single synapse boutons, multiple synapse boutons, and multiple synapse spines by their different contact features. Acrobat-trained animals had more single and multiple synaptic boutons, without change of multiple synapse spines, than motor control animals. These results may suggest that motor learning induces specific synaptogenesis and Purkinje cell spines are primary sites in motor learning-dependent cerebellar synaptic plasticity.

Morphological analysis of spine shapes of Purkinje cell dendrites in the rat cerebellum using high-voltage electron microscopy

Morphological changes in spine shapes have been implicated as indications for physiological or pathological status. To investigate the normal distribution ratio of spine shapes of rat Purkinje cells, morphological analysis was conducted using high-voltage electron microscopy following Golgi impregnation. Spines were classified into thin, stubby, mushroom, branched, and unclassified type by their distinct morphological features. In the tertiary branches of Purkinje cell dendrites, proportions of each category were 69.11+/-1.38% (thin), 13.5+/-1.23% (stubby), 10.45+/-0.74% (mushroom), 2.21+/-0.31% (branched), and 4.73+/-0.52% (unclassified). These results suggest that dendritic spines of Purkinje cells may tend to cluster in defined groups by shapes implying that different spine shapes could reflect different functional roles. This classification could be applied for further study of spine plasticity in various conditions.