Cathepsin B-like proteolysis and MARCKS degradation in sub-lethal NMDA-induced collapse of dendritic spines

Sub-lethal excitotoxic injury to dendrites can elicit loss or shrinkage of dendritic spines. Here, we used a cell culture model of sub-lethal NMDA-induced injury to investigate a role for proteolysis in spine collapse. Transient incubation with NMDA-induced spine collapse and spine F-actin loss within 10 min, an effect not mimicked by the actin assembly inhibitor latrunculin A. NMDA-induced spine collapse was significantly attenuated by preincubation with broad-spectrum cysteine protease inhibitors. Results obtained using several class-specific protease inhibitors suggested that this protective effect was due to specific blockade of cathepsin B/L type protease activity, since selective inhibitors of only these proteases significantly attenuated spine loss. Cathepsin B-like immunoreactivity was observed at synaptic sites, but lysosomes were not. Immunoblot analysis showed that MARCKS (myristoylated-alanine-rich C-kinase substrate), a known substrate of cathepsin B, was specifically degraded in response to intense NMDA receptor stimulation. This effect was blocked by preincubation with a cathepsin B-selective inhibitor. Together these data suggest a model in which NMDA-induced spine collapse involves cathepsin B-like proteolysis of MARCKS, and possibly other proteins that regulate the actin-based cytoskeleton.     

家兔脊髓下橄榄投射的突触特点——HRP顺行标记法电镜研究

将HRP注入家兔脊髓腰膨大灰质,在对侧下橄榄背侧副核内显示出大量HRP顺行标记的脊髓纤维终末。并用电子显微镜观察这些标记终末构成的突触。标记终末主要含圆形清亮囊泡,少数含扁形或多形清亮囊泡,清亮囊泡间常夹有散在的大致密芯囊泡。在下橄榄背侧副核内,大部分标记终末形成轴树突触,少数形成轴体突触。有一标记终末同时含圆形及扁形两种清亮囊泡,各与一树突及一胞体构成突触。特别是标记终末还参与构成轴轴突触及突触球。本文还对这些特殊结构的机能意义进行了初步探讨。     

主要躯体感觉皮层突触可塑性的研究进展

感觉经验可以使主要躯体感觉皮层分布发生可塑性改变。无论在幼年还是成年动物,该区均显示出很强的经验依赖型可塑性,但有关以突触为基础的可塑性机制研究较少。最近的研究表明,促成可塑性的机制可能包括兴奋性突触的长时程增强、长时程抑制、抑制性突触数量改变等。     

Increased immunoreactivity of brain spectrin in Alzheimer disease: a marker for synapse loss?

Alzheimer disease (AD) is characterized, among other pathological alterations, by an extensive synapse loss. Brain spectrin is a membrane skeleton protein found in synapses, and its immunoreactivity has been shown to increase in the rat model of denervation. In order to test the hypothesis that there is an increase in brain spectrin immunoreactivity in relation to the synapse pathology in AD, we studied brain sections and homogenates from AD and control cases and found increased anti-brain spectrin immunostaining of neurons, fibers, and plaques, with a relative decrease in the granular pattern of neuropil immunoreactivity. Western blot analysis showed a 25% increase in the 150 kDa bands (degradation products) in the cytosolic fraction and a decrease in the 240 kDa band (intact brain spectrin) in the particulate fraction. Altered immunostaining of brain sections and Western blot was not observed with an antibody against red blood cell spectrin demonstrating the specific change of brain spectrin. These results support the contention that increased brain spectrin immunoreactivity is a marker of synapse or neuronal loss and further supports the concept of synapse pathology in AD.     

Drebrin (developmentally regulated brain protein) is associated with axo-somatic synapses and neuronal gap junctions in rat mesencephalic trigeminal nucleus

Drebrin (developmentally regulated brain protein)-like immunoreactivity was investigated in the adult rat mesencephalic trigeminal nucleus (MTN) using light and electron microscope. Intense immunoreactive puncta were observed on the cytoplasmic membrane and within the cytoplasm. The cytoplasm was also faintly immunopositive for drebrin, and thus MTN somata other than multipolar cells were distinguishable from non-MTN somata. These immunoreactive cell bodies were localized from the level of the superior colliculus to the pons. Electron microscopic observation showed that the post-synaptic cytoplasmic membrane at axo-somatic synapses was immunoreactive for drebrin. Drebrin-like immunoreactivity was also observed on spine-like processes emanating from MTN somata. In addition, the post-synaptic cytoplasmic membrane at axo-somatic synapses was also immunopositive for drebrin. Within the cytoplasm of MTN cell bodies, a part of the rough endoplasmic reticulum and neighboring structures were also immunopositive. Further, both ends of the somato-somatic close appositions that contained neuronal gap junctions harbored immunoreactive structures. We can infer from the results that drebrin is an ideal marker protein for MTN cell bodies. The abundance of drebrin-like immunoreactivity in the MTN neurons suggests that the MTN has highly flexible synaptogenesis.     

Overexpression of wild-type human APP in mice causes cognitive deficits and pathological features unrelated to Aβ levels

Transgenic mice expressing mutant human amyloid precursor protein (APP) develop an age-dependent amyloid pathology and memory deficits, but no overt neuronal loss. Here, in mice overexpressing wild-type human APP (hAPP_wt) we found an early memory impairment, particularly in the water maze and to a lesser extent in the object recognition task, but β-amyloid peptide (Aβ₄₂) was barely detectable in the hippocampus. In these mice, hAPP processing was basically non-amyloidogenic, with high levels of APP carboxy-terminal fragments, C83 and APP intracellular domain. A tau pathology with an early increase in the levels of phosphorylated tau in the hippocampus, a likely consequence of enhanced ERK1/2 activation, was also observed. Furthermore, these mice presented a loss of synapse-associated proteins: PSD95, AMPA and NMDA receptor subunits and phosphorylated CaMKII. Importantly, signs of neurodegeneration were found in the hippocampal CA1 subfield and in the entorhinal cortex that were associated to a marked loss of MAP2 immunoreactivity. Conversely, in mice expressing mutant hAPP, high levels of Aβ₄₂ were found in the hippocampus, but no signs of neurodegeneration were apparent. The results support the notion of Aβ-independent pathogenic pathways in Alzheimer's disease.     

Impaired dendritic development and synaptic formation of postnatal-born dentate gyrus granular neurons in the absence of brain-derived neurotrophic factor signaling

Neurons are continuously added to the hippocampal dentate gyrus throughout life. These neurons must develop dendritic arbors and spines by which they form synapses for making functional connections with existing neurons. The molecular mechanisms that regulate dendritic development and synaptic formation of postnatal-born granular neurons in the dentate gyrus are largely unknown. Hippocampal dentate gyrus (HDG) has been shown to express high level of brain-derived neurotrophic factor (BDNF). Here we reported that when BDNF is conditionally knockout in the postnatal-born granular neurons of the HDG, the mutant neurons exhibit aberrant morphological development with less dendritic branches, shorter dendritic length, and lower density of dendritic spines, while their primary dendrites are not obviously affected. Even though, these BDNF-deficient granular neurons develop immature dendritic spines to initiate synaptic contacts with afferent axons, they fail to develop or maintain mature spine structures. Thus, these postnatal-born neurons have fewer numbers of synapses, particularly mature synaptic spines. These results suggest that BDNF plays an important role during dendritic development, synaptic formation and synaptic maturation in postnatal-born granular neurons of the HDG in vivo.     

Dopamine spillover after quantal release: rethinking dopamine transmission in the nigrostriatal pathway

The predominance of dopamine (DA) receptors at extrasynaptic vs. synaptic sites implies that DA signaling is by diffusion-based volume transmission. In this review, we compare characteristics that regulate extracellular DA behavior in substantia nigra pars compacta (SNc) and striatum, including regional differences in structure (a 40% greater extracellular volume fraction in SNc vs. striatum) and in dynamic DA uptake (a 200-fold greater DA uptake rate in striatum vs. SNc). Furthermore, we test the assumption of diffusion-based volume transmission for SNc and striatum by modeling dynamic DA behavior after quantal release using region-specific parameters for diffusion and uptake at 37 degrees C. Our model shows that DA uptake does not affect peak DA concentration within 1 mum of a release site in either SNc or striatum because of the slow kinetics of DATs vs. diffusion. Rather, diffusion and dilution are the dominant factors governing DA concentration after quantal release. In SNc, limited DAT efficacy is reflected in a lack of influence of uptake on either amplitude or time course of DA transients after quantal release up to 10 mum from a release site. In striatum, the lack of effect of the DAT within 1 mum of a release site means that perisynaptic DATs do not "gate" synaptic spillover. This contrasts with the conventional view of DA synapses, in which DATs efficiently recycle DA by re-uptake into the releasing axon terminal. However, the model also shows that a primary effect of striatal uptake is to curtail DA lifetime after release. In both SNc and striatum, effective DA radius after quantal release is ~2 mum for activation of low-affinity DA receptors and 7-8 mum for high-affinity receptors; the corresponding spheres of influence would encompass tens to thousands of synapses. Thus, the primary mode of intercellular communication by DA, regardless of region, is volume transmission.     

Allelic variants of <Emphasis Type="Italic">SNAP25</Emphasis> in a family-based sample of ADHD

Summary Altered neurotransmission has been suggested to be a crucial factor in the pathophysiology of attention-deficit/hyperactivity disorder ADHD. Subsequently genes encoding for synaptic proteins have been investigated in candidate gene studies. These proteins mediate the release of neurotransmitters into the synaptic cleft in the process of signal transduction by forming a transient complex, enabling the junction of vesicle and synaptic membrane. One of the core proteins of this complex is the synaptosomal-associated protein 25 (SNAP25). It is one of the most validated candidate genes in ADHD according to meta-analyses. However, differing results were observed in previous studies, some of which were not able to observe association with ADHD. In this study we aimed to investigate association of genetic variants of SNAP25 located in the putative promoter region of SNAP25 and a SNP in intron 8, previously reported to associated with ADHD. A family based design was applied to detect preferential transmission of genetic variants. In our German ADHD sample no preferential transmission of either variant could be observed. Further investigation considering sub-sample analysis regarding response to D-amphetamine could enlight the role of SNAP25 in ADHD. Correspondence: Tobias J. Renner, Department of Child and Adolescent Psychiatry and Psychotherapy, University of Würzburg, Füchsleinstr. 15, 97080 Würzburg, Germany     

Electrophysiological identification of a pathway from the septal area to the medial amygdala: sensitivity to estrogen and luteinizing hormone-releasing hormone

Medial amygdala neurons responsive to electrical stimulation of the medial septal area were electrophysiologically identified in ovariectomized, urethane-anesthetized female rats. Peristimulus time histograms were collected and used to define the orthodromic response. The action of iontophoretically applied luteinizing hormone-releasing hormone (LHRH) and an LHRH fragment, Ac-LHRH5-10, on the activity of orthodromically responsive neurons was tested. Of a total of 187 neurons recorded, 119 were identified as orthodromically responsive. Three types of orthodromic responses were observed: excitatory, inhibitory, and complex. Priming the animals with 5 micrograms estradiol benzoate (EB) 48 hr prior to recording had no effect on the overall number of neurons responding to septal area stimulation, but EB priming did significantly reduce the percentage of orthodromically excited neurons. The firing rate of the majority of amygdala neurons responsive to septal area stimulation was not affected by iontophoretically applied LHRH (59 of 76) or LHRH fragment (41 of 65). In some cases, application of LHRH (10 of 76) or Ac-LHRH5-10 (12 of 55) produced a change in neuronal firing that was similar in direction to the orthodromically evoked response. When applied during the collection of peristimulus time histograms, both peptides were also able to modulate the orthodromically evoked response (five of 18 cells tested with LHRH and three of 14 cells tested with Ac-LHRH5-10). The results demonstrate a large projection from the septal area to the amygdala, one component of which is altered by estrogen priming.(ABSTRACT TRUNCATED AT 250 WORDS)