Liliana Mendieta

Neuroprotective effect of alpha-asarone on spatial memory and nitric oxide levels in rats injected with amyloid-β(25-35).

The chemical alpha-asarone is an important active substance of the Acori graminei rhizome (AGR). It has pharmacological effects that include antihyperlipidemic, antiinflammatory, and antioxidant activity. Our aim was to study the effects alpha-asarone on nitric oxide (NO) levels in the hippocampus and temporal cortex of the rat after injection of the fraction 25-35 from amyloid-beta (Abeta((25-35))). In addition we examined the working spatial memory in an eight-arm radial maze. Our results showed a significant increase of nitrites in the hippocampus and temporal cortex of Abeta((25-35))-treated rats. Other evidence of neuronal damage was the expression of a glial-fibrillar-acid protein and a silver staining. There were impairments in the spatial memory evaluated in the eight-arm radial maze. We wanted to determine whether alpha-asarone improves the memory correlated with NO overproduction and neuronal damage caused by the injection of Abeta((25-35)) into rats. Then animals received a 16-day treatment of alpha-asarone before the Abeta((25-35)) injection. Our results show a significant decrease of nitrite levels in the hippocampus and temporal cortex, without astrocytosis and silver-staining cells, which correlates with memory improvement in the alpha-asarone-treated group. Our results suggest that alpha-asarone may protect neurons against Abeta((25-35))-caused neurotoxicity by inhibiting the effects of NO overproduction in the hippocampus and temporal cortex.

Amyloid-β25–35 impairs memory and increases NO in the temporal cortex of rats

beta-Amyloid plays an important role in the neurodegeneration process of Alzheimers disease (AD), but its neurotoxic mechanisms are not clear. It has been associated with the increase of oxidative stress and cognitive impairment because the beta-amyloid peptide 25-35 (Abeta((25-35))) has the critical neurotoxic properties of the full-length Abeta(1-42). Our present study shows the role of Abeta((25-35)) when injected into the temporal cortex on the nitric oxide pathways, 3-nitrotyrosine, neuronal death, and the spatial memory of rats 1 month after the injection. Our data showed that Abeta((25-35)) increases oxidative stress, causes neuronal damage, and decreases spatial memory in rats. Notably, the injection of the fraction Abeta((25-35)) caused an increase of nNOS and iNOS immunoreactivity in the temporal cortex and hippocampus. We demonstrated a significant increase of reactive astrocytosis, which was accompanied by neuronal damage in the temporal cortex and hippocampus of rats injected with Abeta((25-35)). These data suggest that the fraction Abeta((25-35)) injected into the temporal cortex might contribute to understanding the role of nitric oxide on the biological changes related to the neuropathological progression and the memory impairment in AD.

The role of NOS in the impairment of spatial memory and damaged neurons in rats injected with amyloid beta 25-35 into the temporal cortex

The Aβ(25-35) fraction mimics the toxic effects of the complete peptide Aβ(1-42) because this decapeptide is able to cause memory impairment and neurodegenerative events. Recent evidence has shown that the injection of Aβ(25-35) into the temporal cortex (TCx) of the rat increases the nitric oxide (NO) pathways with several consequences, such as neuronal loss in rats. Our aim was to investigate the effects of each NOS isoform by the prior injection of NOS inhibitors before the injection of the Aβ(25-35). One month after the treatment, the animals were tested for their spatial memory in the radial maze. The hippocampus (Hp) and TCx were assessed for NO production, nitration of proteins (3-NT), astrocytosis (GFAP), and neuronal loss. Our findings show a significant impairment in the memory caused by Aβ25-35 injection. In contrast NOS inhibitors plus Aβ25-35 cause a protection yielding a high performance in the memory test and reduction of cell damage in the TCx and the Hp. Particularly, iNOS is the major source of NO and related to the inflammatory response leading to the memory deficits. The inhibition of iNOS is an important target for neuronal protection against the toxicity of the Aβ25-35 over the long term.

The carboxyl-terminal domain of the heavy chain of tetanus toxin prevents dopaminergic degeneration and improves motor behavior in rats with striatal MPP^+-lesions

Recently it has been shown that the C-terminus fragment of the tetanus toxin (Hc-TeTx) is transported retrogradely and had shown neuroprotective effects, preventing neuronal death by apoptosis. This could be a new alternative preventing ongoing cell death and restoring the motor function in Parkinsons disease (PD), which is characterized by dopaminergic neurodegeneration. Our aim was to evaluate the effects of local administration of Hc-TeTx on motor behavior and the dopamine (DA) levels in the striatum of MPP(+)-treated rats. In the rotational behavior task, the Hc-TeTx [2 microM]+MPP(+) group had a decreased number of contralateral rotations and the cylinder test improved for both forelimb-use asymmetry compared to the MPP(+) group. The staircase test showed that the Hc-TeTx+MPP(+) group had an improvement of fine motor skills compared to the same limb performance of the MPP(+) group. The group of animals with Hc-TeTx+MPP(+) had higher DA and metabolite levels compared to the MPP(+) group. Our study clearly shows that Hc-TeTx improves different motor behavior strongly, which favors the hypothesis of the Hc-TeTx fragment enhancing survival pathways that result in amelioration of the dopaminergic system of rats with a dopaminergic lesion.

The amyloid-β25–35 injection into the CA1 region of the neonatal rat hippocampus impairs the long-term memory because of an increase of nitric oxide

Alzheimers disease (AD) is characterized by the amyloid-beta (Abeta) aggregation but it is unclear when this process begins. Previously, we showed that amyloid-beta(25-35) (Abeta(25-35)) increases the nitric oxide (NO) pathways and causes neurodegenerative effects in rats. The excessive increase of NO during brain development can promote a persistent oxidative stress, but the role of the Abeta(25-35) in the neonatal age and its effects over the long term is unclear. Our aim was to evaluate if the Abeta(25-35) injection on postnatal day 7 causes loss in spatial memory by NO pathways in adult rats. Our results showed that neonatal-Abeta(25-35) injection into the hippocampus (Hp) causes significant impairments in the spatial memory after 90 days. The NO levels were found increased and argynophilic in the Hp. Other evidence of neuronal damage was an increase of the immunoreactivity for 3-nitrotyrosine (3-NT) and the glial-fibrilar acid protein (GFAP) in the Hp of the Abeta(25-35) group. In contrast, these effects were blocked by the administration of L-NAME (inhibitor of nitric oxide synthase) before the injection of Abeta(25-35). The L-NAME plus Abeta(25-35) group showed a better performance in the spatial memory compared to the Abeta(25-35) group. In addition in this group we found a decrease of NO, 3-NT and neurodegeneration in the Hp compared to the Abeta(25-35) group. This finding is a novel result about the role of Abeta(25-35) during the neonatal stage that enhances the NO production, which appears to impair the spatial memory in adult rats.

The C-terminal domain of the heavy chain of tetanus toxin given by intramuscular injection causes neuroprotection and improves the motor behavior in rats treated with 6-hydroxydopamine

We have previously shown that the intrastriatal injection of the C-terminal domain of tetanus toxin (Hc-TeTx) protects the nigrostriatal-dopaminergic pathways and improves motor behavior in hemiparkinsonism-rat models caused by MPP(+) (1-methyl-4-phenylpyridinium). Here we have investigated the protective effects of the intramuscular application of the Hc-TeTx on motor asymmetry and neurodegeneration in the striatum of 6-hydroxydopamine (6-OHDA)-treated rats. Adult male rats were intramuscularly injected with the recombinant Hc-TeTx protein (0.1-20μg/kg, daily) 3days before the stereotaxic injection of 6-OHDA into the left striatum. Our results showed that the motor-improvement functions were extended for 4weeks in all Hc-TeTx-treated groups, obtaining the maximum performance with the highest dose of Hc-TeTx (20μg/kg). The improvements found were 97%, 87%, and 70% in the turning behavior, stepping test, and cylinder test, respectively. The striatal levels of dopamine and its metabolites did not vary compared to the control group. Moreover, the peripheral treatment with Hc-TeTx in rats prevents, for 30days, the neurodegeneration in the striatum caused by the toxicity of the 6-OHDA. Our results lead us to believe that the Hc-TeTx could be a potential therapeutic agent in pathologies caused by impairment of dopaminergic innervations such as Parkinsons disease.

Alteration of the sialylation pattern and memory deficits by injection of Aβ(25-35) into the hippocampus of rats.

Sialic acid in glycoconjugates participates in important cellular functions associated with normal development, growth, and communication. Therefore we evaluated the sialylation pattern and memory deficits caused by the injection of Aβ((25-35)) into the hippocampus (Hp) of rats. The eight-arm maze spatial-learning and memory test indicated that the injection of Aβ((25-35)) into subfield CA1 of the Hp impaired both learning and memory. The sialylation pattern was examined using sialic acid-specific lectins. Our results showed that Maackia amurensis agglutinin (MAA, specific for Neu5Acα2,3Gal) showed reactivity in the CA1 and dentate gyrus (DG) subfields of the Hp mainly in the group injected with vehicle, whereas Macrobrachium rosenbergii lectin (MRL, specific for Neu5,9,7Ac) and Sambucus nigra agglutinin (SNA, specific for Neu5Acα2,6Gal-GalNAc) had increased reactivity in the CA1 and DG subfields of the Hp in the Aβ((25-35))-injected group. The staining pattern of the antibody specific for polysialic acid (a linear homopolymer of α-2,8-linked sialic acid) increased in the CA1 and DG subfields of the Hp of the Aβ((25-35)) group compared to the control group. Our results suggest that injection of Aβ((25-35)) causes impairment in spatial memory and alters the sialylation pattern in response to compensatory reorganization and-or sprouting of dendrites and axons of the surviving neurons.

The recombinant C-terminal fragment of tetanus toxin protects against cholinotoxicity by intraseptal injection of β-amyloid peptide (25–35) in rats

The recombinant C-terminal domain of tetanus toxin (Hc-TeTx) is a new non-toxic peptide of the tetanus toxin that exerts a protective action against glutamate excitotoxicity in motoneurons. Moreover, its efficacy as a neuroprotective agent has been demonstrated in several animal models of neurodegeneration. The eleven amino acids in the β amyloid peptide (Aβ25-35) mimic the toxic effects of the full β amyloid peptide (Aβ1-42), causing the impairment of the cholinergic system in the medial septum (MS) which, in turn, alters the septo-hippocampal pathway and leads to learning and memory impairments. The aim of this study was to examine the neuroprotective effects of the Hc-TeTx fragment against cholinotoxicity. The Hc-TeTx fragment (100 ng) was injected into the rats intercranially, with the Aβ(25-35) (2 μg) then injected into their MS. The animals were tested for spatial learning and memory in the eight-arm radial maze. The brains were removed to assess cholinergic markers, such as choline acetyltransferase (ChAT) and acetylcholinesterase (AChE), and to explore neurodegeneration in the MS and hippocampus, using amino-cupric silver and H&E staining. Finally, capase-3, a marker of apoptosis, was examined in the MS. Our results clearly demonstrate that the application of Hc-TeTx prevents the loss of cholinergic markers (ChAT and AChE), the activation of capase-3, and neurodegeneration in the MS and the CA1 and CA3 subfields of the hippocampus. All these improvements were reflected in spatial learning and memory performance, and were significantly higher compared with animals treated with Aβ(25-35). Interestingly, the single administration of Hc-TeTx into the MS modified the ChAT and AChE expression that affect cognitive processes, without inducing neurodegeneration or an increase in capase-3 expression in the MS and hippocampus. In summary, our findings suggest that the recombinant Hc-TeTx fragment offers effective protection for the septo-hippocampal pathway, given that it reduces the neurodegeneration caused by Aβ(25-35) and improves learning and memory processes.

Peripheral Administration of Tetanus Toxin Hc Fragment Prevents MPP + Toxicity In Vivo

Several studies have shown that intrastriatal application of 1-methyl-4-phenylpyridinium (MPP+) produces similar biochemical changes in rat to those seen in Parkinson’s disease (PD), such as dopaminergic terminal degeneration and consequent appearance of motor deficits, making the MPP+ lesion a widely used model of parkinsonism in rodents. Previous results from our group have shown a neuroprotective effect of the carboxyl-terminal domain of the heavy chain of tetanus toxin (Hc-TeTx) under different types of stress. In the present study, pretreatment with the intraperitoneal injection of Hc-TeTx in rats prevents the decrease of tyrosine hydroxylase immunoreactivity in the striatum due to injury with MPP+, when applied stereotaxically in the striatum. Similarly, striatal catecholamine contents are restored, as well as the levels of two other dopaminergic markers, the dopamine transporter (DAT) and the vesicular monoamine transporter-2 (VMAT-2). Additionally, uptake studies of [3H]-dopamine and [3H]-MPP+ reveal that DAT action is not affected by Hc-TeTx, discarding a protective effect due to a reduced entry of MPP+ into nerve terminals. Behavioral assessments show that Hc-TeTx pretreatment improves the motor skills (amphetamine-induced rotation, forelimb use, and adjusting steps) of MPP+-treated rats. Our results lead us to consider Hc-TeTx as a potential therapeutic tool in pathologies caused by impairment of dopaminergic innervation in the striatum, as is the case of PD.