RESUMO
The Rabies virus is a neurotropic virus that manipulates the natural cell death processes of its host to ensure its own survival and replication. Studies have shown that the anti-apoptotic effect of the virus is mediated by one of its protein named, rabies glycoprotein (RVG). Alzheimer's disease (AD) is characterized by the loss of neural cells and memory impairment. We aim to examine whether expression of RVG in the hippocampal cells can shield the detrimental effects induced by Aß. Oligomeric form of Aß (oAß) or vehicle was bilaterally microinjected into the dorsal hippocampus of male Wistar rats. One week later, two µl (108 T.U. /ml) of the lentiviral vector carrying RVG gene was injected into their dorsal hippocampus (post-treatment). In another experiment, the lentiviral vector was microinjected one week before Aß injection (pre-treatment). One week later, the rat's brain was sliced into cross-sections, and the presence of RVG-expressing neuronal cells was confirmed using fluorescent microscopy. Rats were subjected to assessments of spatial learning and memory as well as passive avoidance using the Morris water maze (MWM) and the Shuttle box apparatuses, respectively. Protein expression of AMPA receptor subunit (GluA1) was determined using western blotting technique. In MWM, Aß treated rats showed decelerated acquisition of the task and impairment of reference memory. RVG expression in the hippocampus prevented and restored the deficits in both pre- and post- treatment conditions, respectively. It also improved inhibitory memory in the oAß treated rats. RVG increased the expression level of GluA1 level in the hippocampus. Based on our findings, the expression of RVG in the hippocampus has the potential to enhance both inhibitory and spatial learning abilities, ultimately improving memory performance in an AD rat model. This beneficial effect is likely attributed, at least in part, to the increased expression of GluA1-containing AMPA receptors.
RESUMO
Rabies virus (RABV) is a neurotropic virus exclusively infecting neurons in the central nervous system. RABV encodes five proteins. Among them, the viral glycoprotein (RVG) plays a key role in viral entry into neurons and rabies pathogenesis. It was shown that the nature of the C-terminus of the RABV G protein, which possesses a PDZ-binding motif (PBM), modulates the virulence of the RABV strain. The neuronal protein partners recruited by this PBM may alter host cell function. This study was conducted to investigate the effect of RVG on synaptic function in the hippocampal dentate gyrus (DG) of rat. Two µl (108 T.U./ml) of the lentiviral vector containing RVG gene was injected into the DG of rat hippocampus. After 2 weeks, the rat's brain was cross-sectioned and RVG-expressing cells were detected by fluorescent microscopy. Hippocampal synaptic activity of the infected rats was then examined by recording the local field potentials from DG after stimulation of the perforant pathway. Short-term synaptic plasticity was also assessed by double pulse stimulation. Expression of RVG in DG increased long-term potentiation population spikes (LTP-PS), whereas no facilitation of LTP-PS was found in neurons expressing δRVG (deleted PBM). Furthermore, RVG and δRVG strengthened paired-pulse facilitation. Heterosynaptic long-term depression (LTD) in the DG was significantly blocked in RVG-expressing group compared to the control group. This blockade was dependent to PBM motif as rats expressing δRVG in the DG-expressed LTD comparable to the RVG group. Our data demonstrate that RVG expression facilitates both short- and long-term synaptic plasticity in the DG indicating that it may involve both pre- and postsynaptic mechanisms to alter synaptic function. Further studies are needed to elucidate the underlying mechanisms.
Assuntos
Vírus da Raiva , Animais , Giro Denteado/metabolismo , Estimulação Elétrica , Glicoproteínas/genética , Glicoproteínas/metabolismo , Glicoproteínas/farmacologia , Hipocampo/metabolismo , Potenciação de Longa Duração , Plasticidade Neuronal/fisiologia , Vírus da Raiva/metabolismo , RatosRESUMO
Investigating long-term potentiation (LTP) in disease models provides essential mechanistic insight into synaptic dysfunction and relevant behavioral changes in many neuropsychiatric and neurological diseases. Toxoplasma (T) gondii is an intracellular parasite causing bizarre changes in host's mind including losing inherent fear of life-threatening situations. We examined hippocampal-dependent behavior as well as in vivo short- and long-term synaptic plasticity (STP and LTP) in rats with latent toxoplasmosis. Rats were infected by T. gondii cysts. Existence of REP-529 genomic sequence of the parasite in the brain was detected by RT-qPCR. Four and eight weeks after infection, spatial, and inhibitory memories of rats were assessed by Morris water maze and shuttle box tests, respectively. Eight weeks after infection, STP was assessed in dentate gyrus (DG) and CA1 by double pulse stimulation of perforant pathway and Shaffer collaterals, respectively. High frequency stimulation (HFS) was applied to induce LTP in entorhinal cortex-DG (400 Hz), and CA3-CA1 (200 Hz) synapses. T. gondii infection retarded spatial learning and memory performance at eight weeks post-infection period, whereas inhibitory memory was not changed. Unlike uninfected rats that normally showed paired-pulse depression, the infected rats developed paired-pulse facilitation, indicating an inhibitory synaptic network disruption. T. gondii-infected rats displayed strengthened LTP of both CA1-pyramidal and DG-granule cell population spikes. These data indicate that T. gondii disrupts inhibition/excitation balance and causes bizarre changes to the post-synaptic neuronal excitability, which may ultimately contribute to the abnormal behavior of the infected host.
Assuntos
Via Perfurante , Toxoplasmose , Ratos , Animais , Via Perfurante/fisiologia , Hipocampo/fisiologia , Plasticidade Neuronal/fisiologia , Potenciação de Longa Duração/fisiologia , Sinapses/metabolismo , Giro Denteado/fisiologia , Toxoplasmose/metabolismoRESUMO
Entorhinal cortex (EC) is one of the first cerebral regions affected in the early phase of Alzheimer's disease (AD). Soluble forms of amyloid beta (Aß) impair synaptic transmission in experimental AD models. Protein kinase Mζ (PKMζ) is an atypical persistently active protein kinase C, known to maintain long term synaptic plasticity and memory, but its role in AD has not yet been described. We examined effect of PKMζ overexpression on the late long-term potentiation (L-LTP) in the dentate gyrus (DG) following EC amyloidopathy. Oligomeric Aß 1-42 (oAß) or vehicle was bilaterally microinjected into the EC of the male Wistar rats. After 1 week, 2 µL of lentiviral vector (~108 TU/mL) encoding PKMζ genome was injected into the DG. One week later, synaptic responses and the LTP persistence were assessed in DG of freely moving animals during 90 minutes to 7 days period. Novel object recognition, passive avoidance and spatial memories were also tested. In rats with EC amyloidopathy, LTP was induced with less amplitude compared to the control group, and extinguished after 24 h. PKMζ overexpression in DG augmented synaptic responses (PS-LTP amplitudes) and maintained LTP over 1 week. PKMζ ameliorated recognition and memory deficits in rats with EC amyloidopathy. Microinjection of PKMζ inhibitor, zeta inhibitory peptide, into the DG abolished the boosting effect of PKMζ on synaptic activity and memory performance. PKMζ-dependent pathway could be a potential therapeutic target to combat synaptic failure and memory deficit in the early phase of AD.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Giro Denteado/metabolismo , Córtex Entorrinal/metabolismo , Expressão Gênica , Hipocampo/metabolismo , Potenciação de Longa Duração/genética , Transtornos da Memória/genética , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Sinapses/fisiologia , Transmissão Sináptica/genética , Animais , Células HEK293 , Humanos , Masculino , Memória , Transtornos da Memória/etiologia , Transtornos da Memória/terapia , Terapia de Alvo Molecular , Proteína Quinase C/fisiologia , Ratos WistarRESUMO
Accumulation of amyloid beta (Aß) soluble forms in the cerebral parenchyma is the mainstream concept underlying memory deficit in the early phase of Alzheimer's disease (AD). PKMζ plays a critical role in the maintenance of long-term memory. Yet, the role of this brain-specific enzyme has not been addressed in AD. We examined the impact of hippocampal PKMζ overexpression on AD-related memory impairment in rats. Oligomeric form of Aß (oAß) or vehicle was bilaterally microinjected into the dorsal hippocampus of male Wistar rats under stereotaxic surgery. One week later, 2 µl of lentiviral vector (108 T.U. / ml.) encoding PKMζ genome was microinjected into the dorsal hippocampus. Seven days later, behavioral performance was assessed using shuttle box and Morris water maze. The expression levels of GluA1, GluA2 and KCC2 were determined in the hippocampus using western blot technique. Our data showed that oAß impairs both passive avoidance and spatial learning and memory. However, overexpression of PKMζ in the dorsal hippocampus restored the behavioral performance. This improving effect was blocked by microinjection of ZIP, a PKMζ inhibitor, into the hippocampus. oAß or PKMζ did not significantly change GluA1 level in the hippocampus. Furthermore, PKMζ failed to restore elevated KCC2 level induced by oAß. However, oAß decreased GluA2 level, and overexpression of PKMζ restored its expression toward the control level. In conclusion, hippocampal overexpression of PKMζ restored memory dysfunction induced by amyloidopathy in part, through preserving hippocampal GluA2 containing AMPA receptors. PKMζ's signaling pathway could be considered as a therapeutic target to battle memory deficits in the early phase of AD.
Assuntos
Doença de Alzheimer/enzimologia , Hipocampo/enzimologia , Transtornos da Memória/enzimologia , Proteína Quinase C/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/toxicidade , Animais , Disfunção Cognitiva/enzimologia , Disfunção Cognitiva/etiologia , Hipocampo/patologia , Masculino , Transtornos da Memória/etiologia , Ratos , Ratos Wistar , Receptores de AMPA/metabolismo , Regulação para CimaRESUMO
The hippocampus, a critical cerebral region involved in learning and memory formation, is especially vulnerable to ischemic defect. Here, we developed an injectable electroactive hydrogel based on pluronic-chitosan/aniline-pentamer with proper conductivity around 10-4 S/cm to achieve the functional repair of the hippocampus following the ischemic defect. FTIR, DSC, and TGA measurements were performed to assess the chemical structure and thermal stability of the synthesized hydrogel. Aniline pentamer decreased the swelling capacity, degradation, and drug release rate. Further, contact angle, melting point, and gelation time of hydrogels were enhanced by addition of aniline oligomer. Moreover, it endowed the on-demand electro-responsive drug release. Injectability of hydrogel was evaluated by rheometry, exhibiting proper gelling time at the body temperature. The ionic/electrical conductivity and desired in vitro biocompatibility with PC12 cells were also achieved. Injection of VEGF-loaded electroactive hydrogel in the hippocampal ischemic animal model resulted in decreased infarction volume, improved hippocampal dependent learning, and memory performance. Taken all together, the results confirmed that fabricated injectable hydrogel would be a suitable candidate for ischemic defect treatment and can lead to new horizons to treat neurological disorders.
Assuntos
Quitosana , Hidrogéis , Indutores da Angiogênese , Compostos de Anilina/farmacologia , Animais , Quitosana/análogos & derivados , Hipocampo , Isquemia , RatosRESUMO
Soluble amyloid beta (Aß) oligomers are the most common forms of Aß in the early stage of Alzheimer's disease (AD). They are highly toxic to the neurons but their capability to activate microglia remains controversial. Microglia develop two distinct phenotypes, classic (M1) and alternative (M2). Tuning of microglia to the alternative (anti-inflammatory) state is of major interest in treatment of neuroinflammatory disease. This study aimed to assess tuning the microglia to produce interferon beta (IFN-ß) as an anti-inflammatory cytokine through TLR4 pathway in a rat model of AD. Microglial BV-2 cells were treated with 1 µg/ml lipopolysaccharides (LPS), Monophosphoryl lipid A (MPL), or vehicles for 24 h, and then incubated with Aß oligomer. After 24 h, cell pellets were harvested and TIR-domain-containing adapter-inducing interferon-ß (TRIF), interferon regulatory factor 3 (IRF3), and IFN-ß levels were measured. The ligands/vehicle were microinjected into the right ventricle of male Wistar rats every 3 days. Two weeks later, an osmotic pump filled with oligomeric Aß/vehicle was implanted in the left ventricle. After 2 weeks, TRIF, IRF3, and IFN-ß levels were measured in the hippocampal tissue. TNF-α and IFN-ß levels were assessed in the hippocampus using immunohistochemistry. The oligomeric Aß did not change TRIF, IRF3, and IFN-ß levels in both cell culture and hippocampal tissue. However, pretreatment with LPS or MPL increased the level of these proteins. BV-2 cells morphologically express M1 state in presence of higher dose of Aß oligomer (10 µM). Pretreatment with LPS or MPL decreased the TNF-α and increased the number of IFN-ß positive cells in the hippocampus of Aß-treated rats. In conclusion, pretreatment with low dose TLR4 agonists could induce microglia to produce neuroprotective cytokines including IFN-ß which may be considered as a potential strategy to combat neuronal degeneration in AD.
Assuntos
Doença de Alzheimer/metabolismo , Interferon beta/genética , Microglia/metabolismo , Receptor 4 Toll-Like/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Peptídeos beta-Amiloides/farmacologia , Animais , Linhagem Celular , Células Cultivadas , Hipocampo/metabolismo , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/metabolismo , Interferon beta/metabolismo , Lipídeo A/análogos & derivados , Lipídeo A/farmacologia , Lipopolissacarídeos/farmacologia , Masculino , Camundongos , Microglia/efeitos dos fármacos , Ratos , Ratos Wistar , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Entorhinal cortex (EC) is one of the first cerebral regions affected in Alzheimer's disease (AD). The pathology propagates to neighboring cerebral regions through a prion-like mechanism. In AD, intracellular calcium dyshomeostasis is associated with endoplasmic reticulum (ER) stress. This study was designed to examine hippocampal ER stress following EC amyloidopathy. Aß1-42 was bilaterally microinjected into the EC under stereotaxic surgery. Rats were daily treated with 30 µg of isradipine, nimodipine, or placebo over one week. Passive avoidance and novel object recognition (NOR) tasks were performed using shuttle box and NOR test, respectively. GRP78/BiP and CHOP levels were measured in the hippocampal dentate gyrus (DG) by western blot technique. The glutathione (GSH) level and PDI activity were also assessed in the hippocampus by colorimetric spectrophotometer. Aß treated group developed passive avoidance and novel recognition memory deficit compared to the control group. However, treatment with calcium channel blockers reversed the impairment. BiP and CHOP level increased in the hippocampus following amyloidopathy in the EC. PDI activity and GSH level in the hippocampus decreased in the Aß treated group, but calcium channel blockers restored them toward the control level. In conclusion, memory impairment due to EC amyloidopathy is associated with ER stress related bio-molecular changes in the hippocampus, and treatment with L-type calcium channel blockers may prevent the changes and ultimately improve cognitive performance.