Synuclein-1 is selectively up-regulated in response to nerve growth factor treatment in PC12 cells

Mutations in the alpha-synuclein gene have recently been identified in families with inherited Parkinson's disease and the protein product of this gene is a component of Lewy bodies, indicating that alpha-synuclein is involved in Parkinson's disease pathogenesis. A role for normal alpha-synuclein in synaptic function, apoptosis or plasticity responses has been suggested. We show here that in rat pheochromocytoma PC12 cells synuclein-1, the rat homolog of human alpha-synuclein, is highly and selectively up-regulated at the mRNA and protein levels after 7 days of nerve growth factor treatment. Synuclein-1 expression appears neither sufficient nor necessary for the neuritic sprouting that occurs within 1-2 days of nerve growth factor treatment. Rather, it likely represents a component of a late neuronal maturational response. Synuclein-1 redistributes diffusely within the cell soma and the neuritic processes in nerve growth factor-treated PC12 cells. Cultured neonatal rat sympathetic neurones express high levels of synuclein-1, with a diffuse intracellular distribution, similar to neuronal PC12 cells. These results suggest that levels of synuclein-1 may be regulated by neurotrophic factors in the nervous system and reinforce a role for alpha-synuclein in plasticity-maturational responses. In contrast, there is no correlation between synuclein expression and apoptotic death following trophic deprivation.     

beta-Amyloid-induced neuronal apoptosis requires c-Jun N-terminal kinase activation

beta-Amyloid (A beta) has been strongly implicated in the pathophysiology of Alzheimer's disease (AD), but the means by which the aggregated form of this molecule induces neuronal death have not been fully defined. Here, we examine the role of the c-Jun N-terminal kinases (JNKs) and of their substrate, c-Jun, in the death of cultured neuronal PC12 cells and sympathetic neurons evoked by exposure to aggregated A beta. The activities of JNK family members increased in neuronal PC12 cells within 2 h of A beta treatment and reached 3--4-fold elevation by 6 h. To test the role of these changes in death caused by A beta, we examined the effects of CEP-1347 (KT7515), an indolocarbazole that selectively blocks JNK activation. Inclusion of CEP-1347 (100--300 nM) in the culture medium effectively blocked the increases in cellular JNK activity caused by A beta and, at similar concentrations, protected both PC12 cells and sympathetic neurons from A beta-evoked-death. Effective protection required addition of CEP-1347 within 2 h of A beta treatment, indicating that the JNK pathway acts relatively proximally and as a trigger in the death mechanism. A dominant-negative c-Jun construct also conferred protection from A beta-evoked death, supporting a model in which JNK activation contributes to death via activation of c-Jun. Finally, CEP-1347 blocked A beta-stimulated activation of caspase-2 and -3, placing these downstream of JNK activation. These observations implicate the JNK pathway as a required element in death evoked by A beta and hence identify it as a potential therapeutic target in AD.     

Central beta-amyloid peptide-induced peripheral interleukin-6 responses in mice

beta-Amyloid peptides (Abetas) share with lipopolysaccharide, a potent pro-inflammatory agent, the property of stimulating glial cells or macrophages to induce various inflammatory mediators. We recently reported that central administration of lipopolysaccharide induces peripheral interleukin-6 responses via both the central and peripheral norepinephrine system. In this study, the effect of intracerebroventricular injection of various synthetic Abetas on plasma interleukin-6 levels was examined in mice. Abeta(1-42) dose-dependently increased plasma interleukin-6 levels: 'aged' Abeta(1-42) was more effective than fresh, whereas Abeta(42-1) had no effect. 'Aged' Abeta(1-42) (205 pmol/mouse i.c.v.)-induced plasma interleukin-6 peaked at 2 h post injection, which is earlier than the peak time of the Abeta(1-42)-induced brain interleukin-6, tumor necrosis factor-alpha and interleukin-1beta levels, which was 4, 4 and 24 h, respectively. Among various peripheral organs, Abeta(1-42) (205 pmol/mouse i.c.v.) significantly increased interleukin-6 mRNA expression in lymph nodes and liver. Abeta(1-42) (205 pmol/mouse i.c.v.) significantly increased norepinephrine turnover in both hypothalamus and spleen. Either central or peripheral norepinephrine depletion effectively inhibited the Abeta(1-42)-induced peripheral interleukin-6 response. Pretreatment with prazosin (alpha(1)-adrenergic antagonist), yohimbine (alpha(2)-adrenergic antagonist), and ICI-118,551 (beta(2)-adrenergic antagonist), but not with betaxolol (beta(1)-adrenergic antagonist), inhibited Abeta(1-42)-induced plasma interleukin-6 levels. These results demonstrate that centrally administered Abeta(1-42) effectively induces the systemic interleukin-6 response which is mediated, in part, by central Abeta(1-42)-induced activation of the central and the peripheral norepinephrine systems.     

肾脏和肾神经在应激、钠盐所致高血压中的作用

本工作采用电生理、生化、放免、电镜等方法,探讨了慢性应激和盐致高血压大鼠交感神经系统和肾脏功能的改变。实验在雄性ＳＤ大鼠上进行。结果表明：（１）高盐大鼠肾血浆流量（ＲＰＦ）和尿钠排泄明显增加,而应激大鼠ＲＰＦ显著下降。（２）电镜显示高盐大鼠近曲和远曲小管上皮细胞及线粒体变大,应激则使细胞萎缩、线粒体变小。（３）高盐大鼠肾皮质ＮａＫＡＴＰ酶活性下降,应激可使其恢复。（４）频谱分析显示应激大鼠低频波动（０２～０９Ｈｚ）明显增加。（５）应激导致大鼠肾素活性（ＰＲＡ）及血管紧张素Ⅱ（ＡＮＧⅡ）水平升高,并能使高盐大鼠低ＰＲＡ和ＡＮＧⅡ水平升高。（６）大鼠去除双侧肾神经后,应激无法造成血压升高、ＲＰＦ下降和ＰＲＡ、ＡＮＧⅡ上升。上述结果提示：肾交感神经系统兴奋性增加介导的肾脏机制,可能在应激和／或盐致高血压发病过程中具有重要作用。     

Induction of SM-20 in PC12 cells leads to increased cytochrome c levels,accumulation of cytochrome c in the cytosol,and caspase-dependent cell death

Sympathetic neurons deprived of nerve growth factor (NGF) release cytochrome c into the cytosol and undergo caspase-dependent cell death through a process that requires de novo gene expression. Expression of the SM-20 gene increases after NGF withdrawal, and ectopic SM-20 expression induces cell death in NGF-maintained neurons. To further evaluate the mechanism by which SM-20 promotes cell death, we developed a PC12-derived cell line in which SM-20 expression can be induced by addition of doxycycline to the culture medium. Induction of SM-20 in either undifferentiated or NGF-differentiated cells resulted in cell death. Cell death was accompanied by an increase in caspase activity and was inhibited by the caspase inhibitor zVAD-FMK. Analysis of cytochrome c in cytosolic and mitochondria-enriched subcellular fractions revealed that induction of SM-20 led to the accumulation of cytochrome c in the cytosol. Surprisingly, SM-20 expression also resulted in a selective increase in the total amount of cytochrome c protein. Thus, induction of SM-20 expression appears to affect both the amount and subcellular localization of cytochrome c in PC12 cells. These results suggest that SM-20 promotes caspase-dependent cell death through a mechanism involving cytochrome c.     

SHP-1 negatively regulates neuronal survival by functioning as a TrkA phosphatase

Nerve growth factor (NGF) mediates the survival and differentiation of neurons by stimulating the tyrosine kinase activity of the TrkA/NGF receptor. Here, we identify SHP-1 as a phosphotyrosine phosphatase that negatively regulates TrkA. SHP-1 formed complexes with TrkA at Y490, and dephosphorylated it at Y674/675. Expression of SHP-1 in sympathetic neurons induced apoptosis and TrkA dephosphorylation. Conversely, inhibition of endogenous SHP-1 with a dominant-inhibitory mutant stimulated basal tyrosine phosphorylation of TrkA, thereby promoting NGF-independent survival and causing sustained and elevated TrkA activation in the presence of NGF. Mice lacking SHP-1 had increased numbers of sympathetic neurons during the period of naturally occurring neuronal cell death, and when cultured, these neurons survived better than wild-type neurons in the absence of NGF. These data indicate that SHP-1 can function as a TrkA phosphatase, controlling both the basal and NGF-regulated level of TrkA activity in neurons, and suggest that SHP-1 regulates neuron number during the developmental cell death period by directly regulating TrkA activity.     

The dorsal compartment locomotory control system in amphioxus larvae

Amphioxus myotomes consist of separate sets of superficial and deep muscle fibers, each with its own innervation, that are thought to be responsible for slow swimming and escape behavior, respectively. Tracings from serial EM sections of the anterior nerve cord in the larva show that the motoneurons and premotor interneurons controlling the superficial fibers (the dorsal compartment, or DC pathway) are linked by specialized junctions of a previously undescribed type, referred to here as juxta-reticular (JR) junctions for the characteristic presence of a cisterna of endoplasmic reticulum on each side. JR junctions link the DC motoneurons with each other, with the largest of the anterior paired neurons (LPN3s) and with one class of ipsilateral projection neurons (IPNs), but occur nowhere else. Because of the paucity of synaptic input to the DC system, larval behavior can only be explained if the JR junctions act as functional links between cells. An analysis of the pattern of cell contacts also suggests that the LPN3s are probably pacemakers for both slow and fast locomotion, but act through junctions in the former case and conventional synapses in the latter. The only major synaptic input to the DC system identified in somites 1 and 2 was from four neurons located in the cerebral vesicle, referred to here as Type 2 preinfundibular projection neurons (PPN2s). They have unusually large varicosities, arranged in series, that make periodic contacts with the DC motoneurons. More caudally, the DC motoneurons receive additional input via similar large varicosities from the receptor cells of the first dorsal ocellus, located in somite 5. The overall circuitry of the locomotory control system suggests that the PPN2s may be instrumental in sustaining slow swimming, whereas mechanical stimulation, especially of the rostrum, preferentially activates the fast mode.     

延髓腹外侧头端区注射肾上腺髓质素对大鼠血压、心率和肾交感神经活动的影响

本研究在 3 4只麻醉Sprague Dawley大鼠观察了延髓腹外侧头端区内微量注射肾上腺髓质素 ( 10μmol/L ,2 0 0nl)对平均动脉压 (MAP)、心率 (HR)和肾交感神经放电 (RSNA)的影响。实验结果如下 :( 1)延髓腹外侧头端区内微量注射肾上腺髓质素可引起MAP、HR、和RSNA明显增加 ,分别由 99 0 9± 3 3 2mmHg ,3 70 78± 7 84bpm和 10 0± 0 %增至 113 5 7± 3 64mmHg (P <0 0 0 1) ,3 83 2 8± 7 3 8bpm (P <0 0 0 1)和 12 3 72±2 74% (P <0 0 0 1) ;( 2 )降钙素基因相关肽受体阻断剂CGRP8 3 7( 10 0 μmol/L ,2 0 0nl)不能阻断肾上腺髓质素的上述效应 ;( 3 )静脉注射NO前体L 精氨酸 ( 10 0mg/kg ,0 2ml)可消除肾上腺髓质素的上述效应。以上结果提示 ,肾上腺髓质素作用于延髓腹外侧头端区可产生显著的心血管作用 ,此作用不是由降钙素基因相关肽受体介导 ,但可被NO所阻断     

Direct measurement of renal sympathetic nervous activity in high-fat diet-related hypertensive rats

The elevation of renal sympathetic nervous activity (SNA) is a possible cause of blood pressure (BP) elevation. Although a high-fat diet (FAT) often induces BP elevation in animals, the effect of FAT on renal SNA in animals is not consistent between studies. Thus, we compared the basal levels of efferent renal SNA and BP in FAT- or high-carbohydrate diet (CHO)-fed rats. Twenty-four male Sprague-Dawley rats were fed FAT (P/F/C=20/45/35% cal) or CHO (20/5/75) from 5 weeks of age. After 20-21 weeks of feeding, a 24-h urine sample was collected to measure sodium excretion. The next day, blood (0.2 ml) was withdrawn from a femoral artery, and basal efferent renal nerve discharges and mean arterial pressure (MAP) were recorded under anesthesia. Immediately after the experiment, abdominal (epididymal, perirenal and mesenteric) adipose tissues were dissected. Total abdominal fat weight was significantly greater in the FAT group than in the CHO group. The plasma level of leptin was significantly higher in the FAT group, but blood glucose and plasma insulin levels did not differ between the two groups. MAP and renal SNA were significantly higher in the FAT group. In addition, the ratio of urinary sodium excretion to dietary sodium intake was significantly lower in the FAT group than in the CHO group. The data suggest that the increased renal SNA may contribute to BP elevation in FAT-fed rats. The present study firstly demonstrated that renal SNA was elevated with FAT-related BP elevation.