A novel antibody targeting sequence 31-35 in amyloid ß protein attenuates Alzheimer's disease-related neuronal damage.

Amyloid ß protein (Aß) plays a critical role in pathogenesis of Alzheimer's disease (AD). Our previous studies indicated that the sequence 31-35 in Aß molecule is an effective active center responsible for Aß neurotoxicity in vivo and in vitro. In the present study, we prepared a novel antibody specifically targeting the sequence 31-35 of amyloid ß protein, and investigated the neuroprotection of the anti-Aß31-35 antibody against Aß1-42 -induced impairments in neuronal viability, spatial memory, and hippocampal synaptic plasticity in rats. The results showed that the anti-Aß31-35 antibody almost equally bound to both Aß31-35 and Aß1-42 , and pretreatment with the antibody dose-dependently prevented Aß1-42 -induced cytotoxicity on cultured primary cortical neurons. In behavioral study, intracerebroventricular (i.c.v.) injection of anti-Aß31-35 antibody efficiently attenuated Aß1-42 -induced impairments in spatial learning and memory of rats. In vivo electrophysiological experiments further indicated that Aß1-42 -induced suppression of hippocampal synaptic plasticity was effectively reversed by the antibody. These results demonstrated that the sequence 31-35 of Aß may be a new therapeutic target, and the anti-Aß31-35 antibody could be a novel immunotheraputic approach for the treatment of AD. © 2016 Wiley Periodicals, Inc.

Humanin protects neurons against apoptosis induced by Abeta31-35 through suppression of intrinsic pathway.

The present study aimed to investigate the effects of humanin (HN) on primary cortical neuronal apoptosis induced by Abeta31-35, and explore the potential mechanisms. Cultured cortical neurons were pretreated with different concentrations of HN (5, 10, 20 micromol/L) for different time period (0, 8 and 16 h) respectively, and then exposed to Abeta31-35 (25 micromol/L) for additional 24 h and the neuronal apoptosis was examined by morphological analysis, flow cytometric assays and TUNEL staining. Caspase activities were measured using a spectrophotometer. Bax expression was measured by Western blot. The results were as follows. (1) Pretreatment with HN (20 micromol/L) for 16 h significantly prevented Abeta31-35-induced apoptosis in cortical neurons; (2) HN significantly decreased Abeta31-35-induced elevation of caspase-3 and -9 activities; (3) HN suppressed Abeta31-35-induced translocation of Bax from the cytosol to mitochondria, but had no effect on overall Bax expression. In conclusions, HN attenuated Abeta31-35-induced cortical neuronal apoptosis by blocking intrinsic caspase-dependent apoptotic pathways.

Protein kinase C mediates amyloid beta-protein fragment 31-35-induced suppression of hippocampal late-phase long-term potentiation in vivo.

Amyloid beta-protein (Abeta) in the brain of Alzheimer's disease (AD) plays a detrimental role in synaptic plasticity and cognitive function. The effects of Abeta on the early-phase long-term potentiation (E-LTP) have been reported widely. However, whether the late-phase long-term potentiation (L-LTP), which differs from E-LTP mechanistically, is also affected by Abeta is still an open question. The present study examined the effects of intracerebraventricular injection of Abeta fragments 25-35 and 31-35 on the L-LTP in the CA1 area of rat hippocampus in vivo, and further investigated its possible underlying mechanism. Our results showed that: (1) Abeta25-35 (6.25-25 nmol) did not affect the baseline field excitatory postsynaptic potentials, but dose-dependently suppressed multiple high-frequency stimuli-induced L-LTP; (2) Abeta31-35, a shorter Abeta fragment than Abeta25-35, also significantly suppressed L-LTP, with the same suppressive effects as Abeta25-35; (3) pretreatment with PMA (6 nmol/5 microl), a membrane permeable PKC agonist, effectively prevented Abeta31-35-induced deficits in the early and the late components of L-LTP; (4) co-application of Abeta31-35 and chelerythrine (12 nmol/5 microl), a PKC antagonist, caused no additive suppression of L-LTP. These results indicate that both Abeta25-35 and Abeta31-35 can impair hippocampal synaptic plasticity in vivo by suppressing the maintenance of L-LTP, and PKC probably mediates the Abeta-induced suppression of hippocampal L-LTP. In addition, the similar efficacy of Abeta31-35 and Abeta25-35 in L-LTP suppression supports the hypothesis we suggested previously that the sequence 31-35 in Abeta might be the shortest active sequence responsible for the neuronal toxicity induced by full length of Abeta molecules.

[Effects of humanin on elevation of intracellular calcium concentration induced by beta-amyloid peptide(31-35) in cultured cortical neurons.].

The disruption of the intracellular Ca(2+) homeostasis has been reported to be one of the mechanisms of beta-amyloid (Abeta) neurotoxicity in Alzheimeros disease (AD). Abeta(31-35), a small active fragment of Abeta, is believed to possess the similar biological activities of full-length Abeta molecule. Humanin (HN) is a recently identified peptide that suppresses neuronal death initiated by AD-related insults. The present study was to investigate the effects of HN on Abeta(31-35)-induced elevation of [Ca(2+)](i) in cultured cortical neurons by real-time fluorescence imaging technique using the Ca(2+)-sensitive dye, Fura-2/AM. The elevation of [Ca(2+)](i) was observed in cultured neurons exposed to Abeta(31-35) (25 mumol/L) (F340/F380: 1 042.56+/- 83.54, compared with control group: 804.73+/- 48.230, P<0.05, n=10). Pretreatment of HN (10 mumol/L) for 10 min significantly decreased the elevation of [Ca(2+)](i) induced by Abeta(31-35) (25 mumol/L) (F340/F380: 918.788+/- 50.73, compared with Abeta(31-35) group, P<0.05, n=10). When neurons were treated with HN and Abeta(31-35) simultaneously, HN (10 mumol/L) could not change the elevation of [Ca(2+)](i) induced by Abeta(31-35) (F340/F380: 1 036.68+/- 88.96, compared with Abeta(31-35) group, P>0.05, n=10), while HN (20 mumol/L) diminished the elevation of [Ca(2+)](i) induced by Abeta(31-35) (25 mumol/L) significantly (F340/F380: 898.56+/- 76.46, compared with Abeta(31-35) group, P<0.05, n=10). The findings imply that: (1) the disruption of the calcium homeostasis induced by Abeta(31-35) is possibly the basis of the neurotoxicity of Abeta(31-35) in cultured cortical neurons; (2) HN suppresses the elevation of [Ca(2+)](i) induced by Abeta(31-35) in a dose- and time-dependent manner.

[Electrophysiological characteristics of central neuronal dendrites and roles of dendritic back-propagating action potentials in modifications of synaptic plasticity].

For expressing the condolences on the passing away of Dr. Hsiang-Tung Chang, one of the distinguished members of the Chinese Academia of Sciences, the pioneer studies on cortical dendritic potentials that Dr. Chang carried out in the 1950s and the prosperous progresses since then, especially, concerning the modifications of synaptic plasticity by the dendritic back-propagating action potentials were briefly reviewed.

[Cerebellum: how does the modular organization in cerebellum fit with its roles in activities of motor learning and classical conditioning?].

Evidence has been accumulating that the cerebellum in vertebrates contributes not only to the well-known postural balance, motor coordination, and the acquisition of skilled movements, but also to the higher cognitive function. However, there exist controversies about how the modular organization, being uniformly distributed throughout the cerebellum, fits with its roles in the motor learning and the classical conditioning, and these have long been hot spots in neuroscience research. Here, some basic advances in these respects were reviewed.

Electroacupuncture attenuates bone cancer pain and inhibits spinal interleukin-1 beta expression in a rat model.

BACKGROUND: Although pain affects the quality of life of cancer patients, current medical treatments are either ineffective or have side effects. In the present study we investigated the effect of electroacupuncture (EA) on cancer-induced hyperalgesia and expression of interleukin-1beta (IL-1beta), upregulation of which is related to the maintenance of persistent pain, in a rat model of bone cancer pain. METHODS: Cancer was induced by injecting AT-3.1 prostate cancer cells into the tibia of male Copenhagen rats. The resulting pain was treated with 10 Hz/2 mA/0.4 ms pulse EA for 30 min daily at the equivalent of the human acupoint GB30 (Huantiao) between Days 14 and 18 after cancer cell inoculation. For sham control, EA needles were inserted into GB30 without stimulation. Thermal hyperalgesia, a decrease in paw withdrawal latency to a noxious thermal stimulus, was measured at baseline and 20 min after EA treatment. IL-1beta and its mRNA were respectively determined by immunohistochemistry and reverse transcription-polymerase chain reaction analysis. RESULTS: Thermal hyperalgesia developed between Days 12 and 18 after cancer cell inoculation. EA significantly (P < 0.05) attenuated this hyperalgesia, increasing paw withdrawal latency from 7.0 +/- 0.3 s to 9.2 +/- 0.4 s, and inhibited the upregulation of IL-1beta and its mRNA compared to the sham control. Intrathecal injection of IL-1 receptor antagonist (IL-1ra, 0.1 mg/rat) also significantly inhibited cancer-induced thermal hyperalgesia. CONCLUSION: The data suggest that EA alleviates bone cancer pain, at least in part by suppressing IL-1beta expression. The results support the clinical use of EA in the treatment of cancer pain.

Effect of lysophosphatidic acid on differentiation of embryonic neural stem cells into neuroglial cells in rats in vitro.

To study the effect of lysophosphatidic acid (LPA) on the differentiation of embryonic neural stem cells (NSCs) into neuroglial cells in rats in vitro, both oligodendrocytes and astrocytes were detected by their marker proteins galactocerebroside (Gal-C) and glial fibrillary acidic protein (GFAP), respectively, using double-labeling immunocytochemistry. RT-PCR assay was also used for analyzing the expression of LPA receptors in NSCs. Our results showed that: (1) LPA at different concentrations (0.01-3.0 mumol/L) was added to culture medium and cell counting was carried out on the 7th day in all groups. Exposure to LPA led to a dose-dependent increase of oligodendrocytes with the response peaked at 1.0 mumol/L, with an increased percentage of 32.6% (P<0.01) of total cells as compared to that of 8.5% in the vehicle group. (2) LPA showed no effect on the differentiation of NSCs into astrocytes. (3) RT-PCR assay showed that LPA(1) and LPA(3) receptors were strongly expressed while LPA(2) receptor expressed weakly in NSCs. These results suggest that LPA at low concentration might act as an extracellular signal through the receptors in NSCs, mainly LPA(1) and LPA(3) receptors, to promote the differentiation of NSCs into oligodendrocytes, while it exhibits little, if any, conceivable effect on the differentiation of NSCs into astrocytes.

Promotive action of lysophosphatidic acid on proliferation of rat embryonic neural stem cells and their differentiation to cholinergic neurons in vitro.

Effects of lysophosphatidic acid (LPA), an extracellular phospholipid signal, on the proliferation of rat embryonic neural stem cells (NSCs) and their differentiation into microtubule-associated protein 2 (MAP2)-positive and choline acetyltransferase (ChAT)-positive, i.e. cholinergic-committed neurons, were observed in vitro by [(3)H]-thymidine incorporation, immunocytochemistry, Western blot and other techniques. The results showed that: (1) Lower concentrations of LPA (0.01~1.0 mumol/L) dose-dependently enhanced the uptake of [(3)H]-thymidine by NSCs cultured in specific serum-free medium, indicating a significant promotive action of LPA on the proliferation of NSCs. (2) After fetal bovine serum which induces and commences the differentiation of NSCs, was used in the medium, the lower concentrations of LPA increased the percentages of both MAP2- and ChAT-immunoreactive neurons, with a peak at 0.1 mumol/L LPA in two cases. (3) The promotive effects of LPA on the differentiation of MAP2- and ChAT-positive neurons were also supported by the up-regulation of the expressions of both MAP2 and ChAT proteins detected by Western blot. (4) At the early phase of differentiation of NSCs, the cell migration and neurite extension were enhanced significantly by lower dosages of LPA under phase-contrast microscope. These results suggest that LPA within certain lower range of concentrations promotes the proliferation of NSCs and their differentiation into unspecific MAP2-positive and specific cholinergic-committed neurons, and also strengthens the migration and neurite extension of the newly-generated neuronal (and also glial as reported elsewhere) progenitors.

[Recent progress in neuroprotection of humanin against Alzheimer's disease-relevant neurotoxicity].

Alzheimer's disease (AD) is the leading cause of dementia for aging people, and far from control due to its obscure mechanism. Humanin, a 24-aa peptide encoded by a newly identified gene cloned from an apparently normal region of AD brain, can specifically attenuate AD-related neurotoxicity. It protects neurons from insults of various AD genes, anti-APP antibodies and Abeta by forming a homodimer outside and interfering directly or indirectly with the activity of Abeta. Humanin seems, however, not to inhibit other toxic insults to neurons, such as Fas or etoposide, an agent against carcinomatous cells in clinical therapy. So Humanin rescues neurons from various AD-related toxicity specifically with efficiency.

Spinal glial activation in a new rat model of bone cancer pain produced by prostate cancer cell inoculation of the tibia.

Studies suggest that astrocytes and microglia in the spinal cord are involved in the development of persistent pain induced by tissue inflammation and nerve injury. However, the role of glial cells in bone cancer pain is not well understood. The present study evaluated the spinal glial activation in a novel rat model of bone cancer pain produced by injecting AT-3.1 prostate cancer cells into the unilateral tibia of male Copenhagen rats. The structural damage to the tibia was monitored by radiological analysis. The thermal hyperalgesia, mechanical hyperalgesia and allodynia, and spontaneous flinch were measured. The results showed that: (1) inoculation of prostate cancer cells, but not the vehicle Hank's solution, induced progressive bone destruction at the proximal epiphysis of the tibia from day 7-20 post inoculation; (2) the inoculation also induced progressive thermal hyperalgesia, mechanical hyperalgesia, mechanical allodynia, and spontaneous flinches; (3) astrocytes and microglia were significantly activated in the spinal cord ipsilateral to the cancer leg, characterized by enhanced immunostaining of both glial fibrillary acidic protein (GFAP, astrocyte marker) and OX-42 (microglial marker); (4) IL-1beta was up-regulated in the ipsilateral spinal cord, evidenced by an increase of IL-1beta immunostained astrocytes. These results demonstrate that injection of AT-3.1 prostate cancer cells into the tibia produces progressive hyperalgesia and allodynia associated with the progression of tibia destruction, indicating the successful establishment of a novel male rat model of bone cancer pain. Further, bone cancer activates spinal glial cells, which may release IL-1beta and other cytokines and contribute to hyperalgesia.

Spinal preprodynorphin mRNA expression in neonatal rats following peripheral inflammation.

Spinal nociceptive neural circuits undergo considerable changes during the postnatal period. This study showed that neonatal rats exhibited earlier upregulation and faster recovery of spinal preprodynorphin (PPD) mRNA than did the adults during complete Freund's adjuvant (CFA)-induced peripheral inflammation. These data suggest that the central nervous systems of neonates and adults respond differently to peripheral noxious inputs, a fact that should be considered when selecting pain treatment strategies for neonate populations.

Mu opioid receptor-containing neurons mediate electroacupuncture-produced anti-hyperalgesia in rats with hind paw inflammation.

Previous studies showed that electroacupuncture (EA) significantly attenuates inflammatory hyperalgesia in a complete Freund's adjuvant (CFA)-induced inflammatory pain rat model. The present study demonstrates that pretreatment with Derm-sap, a selective toxin for neurons that contain mu opioid receptor (MOR), specifically decreases MOR and blocks EA anti-hyperalgesia. These data suggest that spinal MOR-containing neurons are involved in the processes by which EA produces anti-hyperalgesia.

Effects of pertussis toxin on electroacupuncture-produced anti-hyperalgesia in inflamed rats.

Our previous study showed that electroacupuncture (EA) significantly attenuated hyperalgesia in an animal model of persistent inflammatory pain. The present study was designed to show if Gi/o protein is involved in EA-produced anti-hyperalgesia. Spinal Gi/o-protein function was destroyed by intrathecal pretreatment with pertussis toxin (PTX). Seven days after the placement of an intrathecal PE-10 tube, PTX was injected into the intrathecal space of the lumbar spinal cord of rats. Seven days after PTX, complete Freund's adjuvant (CFA) was injected into the plantar surface of one hind paw of the rat to induce hyperalgesia in the injected paw. EA treatment was given at acupoint GB30 immediately post-CFA and then hyperalgesia was assessed by measuring the degree of decreased paw withdrawal latency (PWL) to a noxious thermal stimulus. The results showed that PTX pretreatment prevented EA-produced anti-hyperalgesia in the CFA inflammatory pain model but did not affect either baseline pain threshold or CFA-induced hyperalgesia. The data suggest that EA-produced anti-hyperalgesia is mediated by PTX-sensitive Gi/o proteins and the relevant signaling pathways.

Electroacupuncture suppresses spinal expression of neurokinin-1 receptors induced by persistent inflammation in rats.

It has been demonstrated that electroacupuncture (EA) significantly suppresses behavioral hyperalgesia in a rat model of persistent inflammatory pain and that neurokinin-1 (NK-1)/substance P (SP) receptors play important roles in nociception and hyperalgesia at the spinal cord level. The present study investigated spinal NK-1 receptor involvement in EA-produced suppression of hyperalgesia in a rat model of persistent inflammatory pain. The results showed that hind paw inflammation induced a significant increase of NK-1 receptor expression in the spinal dorsal horn and that this effect was significantly suppressed by EA. This suggests that EA-induced suppression of hyperalgesia is involved, at least partly, in the suppression of the spinal NK-1 receptors induced by sustained peripheral nociceptive input.

Neuroprotective effect of lysophosphatidic acid on AbetaP31-35-induced apoptosis in cultured cortical neurons.

It has been reported that lysophosphatidic acid (LPA) at its lower concentrations prevents apoptosis induced by serum-deprivation in cultured cortical neurons when LPA is added into the cultural medium with serum withdrawal. The present study was designed to investigate whether LPA could also block the apoptosis induced by beta-amyloid peptide fragment 31-35 (AbetaP31-35) in cultured cortical neurons by using techniques of DNA fragmentation electrophoresis, HO33342 staining, and TUNEL examinations. The results showed that pretreatment of LPA suppressed the AbetaP31-35-induced apoptosis only when LPA was applied to the cultured neurons with lower concentrations (1-10 micromol/L) and especially, with a preceding time of 12-24 h before the AbetaP31-35 exposure. These facts imply that LPA also acts as a neuroprotective factor against AbetaP31-35-induced apoptosis, though the mechanism underlying the protective action in this case may be more complex than that involved in the serum deprivation-induced apoptosis.

Dorsal raphe stimulation modulates nociceptive responses in thalamic parafascicular neurons via an ascending pathway: further studies on ascending pain modulation pathways.

A study on the nociceptive responses of single cells within the nucleus parafascicularis (PF) thalami of the rat was undertaken to clarify the reported observations of a pain suppression pathway to this nucleus from the dorsal raphe (DR) nucleus. Two types of nociceptive neuron were identified in the PF which were classified as 'nociceptive-on' and 'nociceptive-off' neurons, respectively. DR stimulation exhibits a simple monophasic 'dose-dependent' relationship between the degree of the inhibition elicited and the stimulation intensity used on the 'nociceptive-off' cells. In contrast, biphasic effects following DR stimulation on the 'nociceptive-on' cells was obtained, with low intensities eliciting suppression while high intensities excited the cells. These effects of low intensity DR stimulation upon the responses of the 'nociceptive-on' cells were diminished but not prevented by transection of the well-known bulbospinal inhibitory fibers descending in the dorsal half of the spinal cord, while the effects of DR stimulation upon the 'nociceptive-off' cells remain unchanged following spinal transection. Thus, our results show that DR stimulation modulates the nociceptive responsiveness of the PF by way of supraspinal pathways in addition to the previously described descending paths.

Spinal antinociceptive effect of agmatine and tentative analysis of involved receptors: study in an electrophysiological model of rats.

The present study was designed to evaluate the antinociceptive effect of agmatine and the receptors involved at the spinal level by using an experimental model in which agmatine was intrathecally (i.t.) administered while the changes of nociceptively-evoked discharges in thalamic parafascicular (PF) neurons were monitored in anesthetized Wistar rats. The results showed that: (1) i.t. agmatine dose-dependently suppressed the nociceptive discharges of PF neurons induced by the tail pinch; (2) i.t. yohimbine did not block the agmatine-induced suppressive effect of nociceptive discharges in these neurons; and (3) the agmatine-induced suppression could be blocked significantly by i.t. idazoxan. The results suggest that agmatine suppresses the transmission of nociceptive inputs at the spinal level mainly through the activation of imidazoline receptors other than alpha(2)-adrenoceptors.

Effects of aging on hyperalgesia and spinal dynorphin expression in rats with peripheral inflammation.

The aging process is associated with various morphological and biochemical changes in the nervous system that may affect the processing of noxious inputs. This study showed greater hyperalgesia and up-regulation of spinal dynorphin (DYN) expression in aging than in young adult rats during CFA-induced peripheral inflammation. These data indicate that nociception is regulated differently in aging individuals, a fact that should be considered when selecting treatment strategies for aging populations with persistent pain.

Strain differences in pain sensitivity and expression of preprodynorphin mRNA in rats following peripheral inflammation.

The experience of pain is highly variable among individuals, which may be due in part to the effects of genetic factors on the central transmission and modulation of noxious inputs. This study examined behavioral responses and the expression of preprodynorphin (PPD) mRNA at the spinal level during complete Freund's adjuvant-induced inflammation of the unilateral hind paw in male Fischer 344 (F344), Sprague-Dawley (SD), and Lewis (LEW) rats. Experiments showed that F344 rats exhibited stronger hind paw hyperalgesia and greater spinal PPD mRNA induction than SD or LEW rats. These results indicate that genetic factors that determine the spinal PPD mRNA and dynorphin production underlie strain-dependent differences in pain perception.