The present experiments reveal the alterations of the hippocampal neuronal populations in chronic epilepsy. The mice were injected with a single dose of pilocarpine. They had status epilepticus and spontaneously recurrent motor seizures. Three months after pilocarpine treatment, the animals were investigated with the Barnes maze to determine their learning and memory capabilities. Their hippocampi were analyzed 2 weeks later (at 3.5 months) with standard immunohistochemical methods and cell counting. Every animal displayed hippocampal sclerosis. The neuronal loss was evaluated with neuronal-N immunostaining, and the activation of the microglia was measured with Iba1 immunohistochemistry. The neuropeptide Y, parvalbumin, and calretinin immunoreactive structures were qualitatively and quantitatively analyzed in the hippocampal formation. The results were compared statistically to the results of the control mice. We detected neuronal loss and strongly activated microglia populations. Neuropeptide Y was significantly upregulated in the sprouting axons. The number of parvalbumin- and calretinin-containing interneurons decreased significantly in the Ammon’s horn and dentate gyrus. The epileptic animals displayed significantly worse learning and memory functions. We concluded that degeneration of the principal neurons, a numerical decrease of PV-containing GABAergic neurons, and strong peptidergic axonal sprouting were responsible for the loss of the hippocampal learning and memory functions.

Neurogenesis plays a crucial role in cognitive processes. During aging and in Alzheimer's disease (AD), altered neurogenesis and neuroinflammation are evident both in C57BL/6J, APPSwe/PS1dE9 (Tg) mice and humans. AD pathology may slow down upon drug treatment, for example, in a previous study of our group P33, a putative neuroprotective agent was found to exert advantageous effects on the elevated levels of APP, Aβ, and neuroinflammation. In the present study, we aimed to examine longitudinal alterations in neurogenesis, neuroinflammation and AD pathology in a transgenic (Tg) mouse model, and assessed the putative beneficial effects of long-term P33 treatment on AD-specific neurological alterations. Hippocampal cell proliferation and differentiation were significantly reduced between 8 and 12 months of age. Regarding neuroinflammation, significantly elevated astrogliosis and microglial activation were observed in 6- to 7-month-old Tg animals. The amounts of the molecules involved in the amyloidogenic pathway were altered from 4 months of age in Tg animals. P33-treatment led to significantly increased neurogenesis in 9-month-old animals. Our data support the hypothesis that altered neurogenesis may be a consequence of AD pathology. Based on our findings in the transgenic animal model, early pharmacological treatment before the manifestation of AD symptoms might ameliorate neurological decline.

Alzheimer’s disease (AD) is the most common form of dementia characterized by cognitive dysfunctions. Pharmacological interventions to slow the progression of AD are intensively studied. A potential direction targets neuronal sigma-1 receptors (S1Rs). S1R ligands are recognized as promising therapeutic agents that may alleviate symptom severity of AD, possibly via preventing amyloid-β-(Aβ-) induced neurotoxicity on the endoplasmic reticulum stress-associated pathways. Furthermore, S1Rs may also modulate adult neurogenesis, and the impairment of this process is reported to be associated with AD. We aimed to investigate the effects of two S1R agonists, dimethyltryptamine (DMT) and PRE084, in an Aβ-induced in vivo mouse model characterizing neurogenic and anti-neuroinflammatory symptoms of AD, and the modulatory effects of S1R agonists were analyzed by immunohistochemical methods and western blotting. DMT, binding moderately to S1R but with high affinity to 5-HT receptors, negatively influenced neurogenesis, possibly as a result of activating both receptors differently. In contrast, the highly selective S1R agonist PRE084 stimulated hippocampal cell proliferation and differentiation. Regarding neuroinflammation, DMT and PRE084 significantly reduced Aβ1–42-induced astrogliosis, but neither had remarkable effects on microglial activation. In summary, the highly selective S1R agonist PRE084 may be a promising therapeutic agent for AD. Further studies are required to clarify the multifaceted neurogenic and anti-neuroinflammatory roles of these agonists.

Background Heat-shock protein B1 (HSPB1) is among the most well-known and versatile member of the evolutionarily conserved family of small heat-shock proteins. It has been implicated to serve a neuroprotective role against various neurological disorders via its modulatory activity on inflammation, yet its exact role in neuroinflammation is poorly understood. In order to shed light on the exact mechanism of inflammation modulation by HSPB1, we investigated the effect of HSPB1 on neuroinflammatory processes in an in vivo and in vitro model of acute brain injury. Methods In this study, we used a transgenic mouse strain overexpressing the human HSPB1 protein. In the in vivo experiments, 7-day-old transgenic and wild-type mice were treated with ethanol. Apoptotic cells were detected using TUNEL assay. The mRNA and protein levels of cytokines and glial cell markers were examined using RT-PCR and immunohistochemistry in the brain. We also established primary neuronal, astrocyte, and microglial cultures which were subjected to cytokine and ethanol treatments. TNFα and hHSPB1 levels were measured from the supernates by ELISA, and intracellular hHSPB1 expression was analyzed using fluorescent immunohistochemistry. Results Following ethanol treatment, the brains of hHSPB1-overexpressing mice showed a significantly higher mRNA level of pro-inflammatory cytokines ( Tnf , Il1b ), microglia ( Cd68 , Arg1 ), and astrocyte ( Gfap ) markers compared to wild-type brains. Microglial activation, and 1 week later, reactive astrogliosis was higher in certain brain areas of ethanol-treated transgenic mice compared to those of wild-types. Despite the remarkably high expression of pro-apoptotic Tnf , hHSPB1-overexpressing mice did not exhibit higher level of apoptosis. Our data suggest that intracellular hHSPB1, showing the highest level in primary astrocytes, was responsible for the inflammation-regulating effects. Microglia cells were the main source of TNFα in our model. Microglia isolated from hHSPB1-overexpressing mice showed a significantly higher release of TNFα compared to wild-type cells under inflammatory conditions. Conclusions Our work provides novel in vivo evidence that hHSPB1 overexpression has a regulating effect on acute neuroinflammation by intensifying the expression of pro-inflammatory cytokines and enhancing glial cell activation, but not increasing neuronal apoptosis. These results suggest that hHSPB1 may play a complex role in the modulation of the ethanol-induced neuroinflammatory response.

Double staining protocols using the most popular immunoperoxidase techniques may raise difficulties. The two ordinary detection systems may cross-talk, when the primary antibodies are derived from phylogenetically closely related animals. A color shift of the 3,3'-diaminobenzidine (DAB) polymer may occur during the second development, resulting in poor distinction between the two kinds of deposits. A post-DAB technique, sulfide-silver-gold intensification, was fine tuned to eliminate these difficulties, which may be especially suitable for colocalization of cell nuclei and perikarya of the same cells. The revised method was probed in combination with a subsequent other immunoperoxidase step or fluorochrome-tagged reagents. The nuclear antigens (BrdU, c-Fos, and Prox-1) were first visualized with DAB polymer, which were then treated with SSGI, turning the deposit black. Thereafter, cytoplasmic antigens (doublecortin, neuronal nuclei, and calbindin) were detected with either another immunoperoxidase using DAB again or immunofluorescence labeling. In both approaches, the immunopositive nuclei and cytoplasmic sites could be easily distinguished even at low magnifications. Different shielding or eluting posttreatments were compared for consecutive acetylcholinesterase histochemistry terminated with DAB development and immunohistochemistry in the same sections. In conclusion, we recommend post-DAB treatments that abolish interactions between detection systems and allow clear distinction between the two signals under various conditions.

Regulated intramembrane proteolysis (RIP) of the amyloid precursor protein (APP) leads to the formation of fragments, among which the intracellular domain of APP (AICD) was also identified to be a causative of early pathological events. AICD-counteracting proteins, such as Fe65, may serve as alternative therapeutic targets of Alzheimer’s disease (AD). The detection of elevated levels of Fe65 in the brains of both human patients and APP transgenic mice may further strengthen the hypothesis that influencing the interaction between Fe65 and APP may have a beneficial effect on the course of AD. Based on a PXP motif, proven to bind to the WW domain of Fe65, a new pentapeptide was designed and tested. The impedimental effect of P33 on the production of beta amyloid (Aβ) (soluble fraction and aggregated plaques) and on the typical features of the AD pathology (decreased dendritic spine density, synaptic markers, elevated inflammatory reactions) was also demonstrated. Significant enhancements of both learning ability and memory function were observed in a Morris water maze paradigm. The results led us to formulate the theory that P33 acts by altering the conformation of Fe65 via binding to its WW domain, consequently hindering any interactions between Fe65 and key members involved in APP processing.

Misfolding, oligomerization and aggregation of the amyloid-beta (Aβ) peptide is widely recognized as a central event in the pathogenesis of Alzheimer’s disease (AD). Recent studies have identified soluble Aβ oligomers as the main pathogenic agents and provided evidence that such oligomeric Aβ aggregates are neurotoxic, disrupt synaptic plasticity and inhibit long-term potentiation (LTP). A promising therapeutic strategy in the battle against AD is the application of short synthetic peptides which are designed to bind to specific Aβ-regions thereby neutralizing or interfering with the devastating properties of oligomeric Aβ species. In the present study we investigated the neuroprotective properties of the amyloid sequence derived pentapeptide Leu-Pro-Tyr-Phe-Asp-NH2 (LPYFDa) in vitro as well as its memory preserving capacity against Aβ42-induced learning deficits in vivo. In vitro we could show that neurons in culture treated with LPYFDa are protected against Aβ42-induced cell death. Moreover, in vivo LPYFDa prevented memory impairment tested in a contextual fear conditioning paradigm in mice after bilateral intrahippocampal Aβ42 injections. We thus showed for the first time that an anti-amyloid peptide like LPYFDa can preserve memory by reverting Aβ42 oligomer induced learning deficits. Journal of Alzheimer’s Disease (2010), 19:991-1005.

Several animal models of Alzheimer’s disease have been used in laboratory experiments. Intrahippocampal injection of fibrillar amyloid-beta (fAβ) peptide represents one of the most frequently used models, mimicking Aβ deposits in the brain. In our experiment synthetic fAβ1–42 peptide was administered to rat hippocampus. The effect of the Aβ peptide on spatial memory and dendritic spine density was studied. The fAβ1–42-treated rats showed decreased spatial learning ability measured in Morris water maze (MWM). Simultaneously, fAβ1–42 caused a significant reduction of the dendritic spine density in the rat hippocampus CA1 region. The decrease of learning ability and the loss of spine density were in good correlation. Our results prove that both methods (MWM and dendritic spine density measurement) are suitable for studying Aβ-triggered neurodegeneration processes.

Hsp27 belongs to the small heat shock protein family, which are ATP-independent chaperones. The most important function of Hsp27 is based on its ability to bind non-native proteins and inhibit the aggregation of incorrectly folded proteins maintaining them in a refolding-competent state. Additionally, it has anti-apoptotic and antioxidant activities. To study the effect of Hsp27 on memory and synaptic functions, amyloid-β (Aβ) accumulation, and neurodegeneration, we generated transgenic mice overexpressing human Hsp27 protein and crossed with APPswe/PS1dE9 mouse strain, a mouse model of Alzheimer's disease (AD). Using different behavioral tests, we found that spatial learning was impaired in AD model mice and was rescued by Hsp27 overexpression. Electrophysiological recordings have revealed that excitability of neurons was significantly increased, and long-term potentiation (LTP) was impaired in AD model mice, whereas they were normalized in Hsp27 overexpressing AD model mice. Using anti-amyloid antibody, we counted significantly less amyloid plaques in the brain of APPswe/PS1dE9/Hsp27 animals compared to AD model mice. These results suggest that overexpression of Hsp27 protein might ameliorate certain symptoms of AD.

Uj, standardizalhato modszert dolgoztunk ki toxikus �beta-amiloid (Abeta) 1-42 peptid oligomerek előallitasara, a preparalt oligomereket fiziko-kemiai modszerekkel jellemeztuk. Ket uj neuroprotektiv peptidmimetikum vegyuletcsaladot talaltunk, ezek az anyagok megvedik a neuronokat az Alzheimer-kor (AK) allatmodelljeben az Abeta neurotoxikus hatasatol. Mindket vegyuletcsoportot szabadalmilag vedjuk, mint az AK potencialis gyogyszerjelolt vegyuleteit. Uj ex vivo modszert dolgoztunk ki az Abeta peptidek toxicitasanak meresere (patkany hippocampus szelet, MTT-teszt), a modszer alkalmas az uj neuroprotektiv vegyuleteink aktivitasmeresere is. Az ex vivo hippocampus szeleteket sikerrel alkalmaztuk a neuronalis plaszticitas (LTP) meresere, az Abeta-toxicitas meghatarozasara, multielektrod array (MEA) technikaval. In vivo, egysejt-elvezeteses elektrofiziologiai meresekkel bizonyitottuk az uj peptidmimetikumaink neuroprotektiv hatasat. Proteomikai modszerekkel azonositottuk az Abeta peptidekkel kolcsonhatasba lepő feherjeket, ezek elsősorban plazmamembran, ill. intraneuronalis feherjek (mitokondrium, endoplazmas reticulum, mikrotubularis rendszer). Az intraneuronalis feherjek es az Abeta peptidek kolcsonhatasai kulcsszerepet jatszhatnak az AK patogeneziseben. Igazoltuk, hogy a Zn2+ ionok toxikus Abeta-aggregatumok kepződeset indukaljak. Az AK transzgen allatmodelljen bizonyitottuk, hogy a Zn-kelatorok (pl. Perindopril) neuroprotektiv hatasuak. Uj AK-allatmodellt dolgoztunk ki az Abeta oligomerek icv bevitelevel. | A new method was introduced for the preparation of toxic beta-amyloid (Abeta) 1-42 oligomers, these assemblies were characterized with physicochemical methods. Two families of novel neuroprotective peptidomimetics were found, these substances protect neurons against the toxic effect of Abeta in tg mouse models of Alzheimer’s disease (AD). Both groups of the novel substances will be patented as putative drug candidates for AD treatment. A new ex vivo method was introduced for toxicity measurement of Abeta peptides (rat hippocampal slices, MTT-assay); this method proved to be suitable for activity measurement of the novel neuroprotective substances. Hippocampal slices were successfully used for measurement of neuronal plasticity (LTP) for demonstrating neurotoxicity of Abeta aggregates, applying multielectrode array (MEA) technique. The neuroprotective effect of our novel peptidomimetics was demonstrated also in vivo, using one-cell electrophysiology. Proteomic methods were used for identification of proteins interacting with Abeta peptides; these are mainly plasma membrane and intraneuronal (mitochondrial, endoplasmatic reticular and microtubular) proteins. Interaction of intracellular proteins with Abeta may play key role in AD pathogenesis. The role of Zn2+ ions in formation of toxic Abeta-aggregates was demonstrated. Zn2+-chelators (e.g. Perindopril) were neuroprotective in a tg-mouse model of AD. A new AD rat model was introduced using icv administration of synthetic Abeta oligomers.

Misfolding, oligomerization, and aggregation of the amyloid-beta (Abeta) peptide is widely recognized as a central event in the pathogenesis of Alzheimer's disease (AD). Recent studies have identified soluble Abeta oligomers as the main pathogenic agents and provided evidence that such oligomeric Abeta aggregates are neurotoxic, disrupt synaptic plasticity, and inhibit long-term potentiation. A promising therapeutic strategy in the battle against AD is the application of short synthetic peptides which are designed to bind to specific Abeta-regions thereby neutralizing or interfering with the devastating properties of oligomeric Abeta species. In the present study, we investigated the neuroprotective properties of the amyloid sequence derived pentapeptide LPYFDa in vitro as well as its memory preserving capacity against Abeta(42)-induced learning deficits in vivo. In vitro we showed that neurons in culture treated with LPYFDa are protected against Abeta (42) -induced cell death. Moreover, in vivo LPYFDa prevented memory impairment tested in a contextual fear conditioning paradigm in mice after bilateral intrahippocampal Abeta (42) injections. We thus showed for the first time that an anti-amyloid peptide like LPYFDa can preserve memory by reverting Abeta (42) oligomer-induced learning deficits.

Activity-dependent adaptive changes in the nervous system involve structural and functional changes in the cortical circuitry. In this work the cortical function was studied by repeated recording of the somatosensory and motor potentials evoked by whisker deflections after altered sensory-motor experience in adult mice. The latencies of motor and somatosensory evoked potentials were found to shorten, while their amplitudes decreased, after a behavioural challenge involving the vibrissal apparatus. Sensory deprivation achieved by whisker trimming resulted in a partial reversal of the changes observed after increased activity. The derived parameters imply that cortical information processing speeds up as a result of experience, while decreased activity has the opposite effect. The methods used throughout the experiment were minimally invasive, and thus proved to be sufficient for the long-term follow-up of cortical functions.