Basal Forebrain Cholinergic Deficits Reduce Glucose Metabolism and Function of Cholinergic and GABAergic Systems in the Cingulate Cortex

PURPOSE: Reduced brain glucose metabolism and basal forebrain cholinergic neuron degeneration are common features of Alzheimer's disease and have been correlated with memory function. Although regions representing glucose hypometabolism in patients with Alzheimer's disease are targets of cholinergic basal forebrain neurons, the interaction between cholinergic denervation and glucose hypometabolism is still unclear. The aim of the present study was to evaluate glucose metabolism changes caused by cholinergic deficits. MATERIALS AND METHODS: We lesioned basal forebrain cholinergic neurons in rats using 192 immunoglobulin G-saporin. After 3 weeks, lesioned animals underwent water maze testing or were analyzed by 18F-2-fluoro-2-deoxyglucose positron emission tomography. RESULTS: During water maze probe testing, performance of the lesioned group decreased with respect to time spent in the target quadrant and platform zone. Cingulate cortex glucose metabolism in the lesioned group decreased, compared with the normal group. Additionally, acetylcholinesterase activity and glutamate decarboxylase 65/67 expression declined in the cingulate cortex. CONCLUSION: Our results reveal that spatial memory impairment in animals with selective basal forebrain cholinergic neuron damage is associated with a functional decline in the GABAergic and cholinergic system associated with cingulate cortex glucose hypometabolism.

Hipocretinas, péptidos relacionados con la narcolepsia / Hypocretins, peptides associated with narcolepsy

La narcolepsia es un trastorno del sueño caracterizado por excesiva somnolencia diurna, transiciones prematuras de la vigila al sueño de movimientos oculares rápidos, alucinaciones hipnagógicas y cataplexia. Evidencias experimentales indican que la narcolepsia en humanos es una enfermedad neurodegenerativa asociada con la pérdida de neuronas hipocretinérgicas localizadas en el hipotálamo lateral. Además, se sabe que los pacientes narcolépticos presentan reducción significativa en la concentración de hipocretinas (HCRT) en el líquido cefalorraquídeo. Nuestro laboratorio ha generado un nuevo modelo experimental de narcolepsia en rata, que nos permite estudiar la enfermedad desde un enfoque histológico y neuroquímico. Hemos demostrado que la toxina hipocretina 2/saporina destruye selectivamente las neuronas hipocretinérgicas. Además, la pérdida de estas neuronas induce un cuadro conductual similar al observado en otros modelos experimentales o narcolepsia en humanos. En la presente revisión abordamos aspectos generales de la narcolepsia, del sistema hipocretinérgico, así como de los modelos experimentales de narcolepsia y el uso de los transplantes como alternativa para tratar la enfermedad.

Narcolepsy is a chronic disease characterized by excessive somnolence, abrupt transitions from wakefulness to rapid eye movement sleep stage and cataplexy. Experimental evidence show that narcolepsy in humans is a neurodegenerative disease associated with the lost of hypocretin (HCRT) neurons in the lateral hypothalamus. Narcoleptic patients also display a significant diminution in HCRT contents of cerebrospinal fluid. In order to study narcolepsy, several experimental models have been developed. Murine and canine models currently allow us to study this disease. Our laboratory has developed a new experimental rat model of narcolepsy. This model allows us to study the disease from a histological and neurochemical perspective. Elsewhere we have reported that the use of the toxin hypocretin2/saporine (HCRT2/ SAP) selectively destroys hypocretinergic neurons. The loss of these neurons induces a similar behavioural profile as the one observed in other experimental models of narcolepsy. In the present review we describe an overview on narcolepsy, the hypocretinergic system, experimental models in narcolepsy and the use of transplants as an alternative therapeutic tool.

Effect of Basic Fibroblast Growth Factor-Saporin (bFGF-SAP) Conjugate on Bovine Choriocapillary Endothelial Cells

We evaluated the effect of a basic fibroblast growth factor (bFGF) and saporin conjugate (bFGF-SAP) on proliferation, migration and tubule formation in bovine choriocapillary endothelial cells (BCECs). Cell proliferation and MTS assays were done with 0.01, 0.1, 1, 10, and 100 nM bFGF-SAP, and an equimolar concentration of bFGF and saporin. TUNEL assay was performed to confirm apoptosis. Cells were treated with 1, 10, and 100 nM bFGF-SAP and migration assay and tubule formation assay were done. Results were evaluated with image analysis. All experiments were performed in triplicate and repeated three times. Viable cells (ID50 = 0.62) and cell proliferation by MTS assay (ID50 = 0.75 nM) were inhibited. Saporin caused cytotoxicity and inhibition of proliferation at high concentration. DNA fragmentation was identified by TUNEL assay. Migration and tubule formation were also inhibited. All mechanisms responsible for neovascularization were inhibited, and this could be applied in the management of subretinal choroidal neovascularization (SRN).

Transforming growth factor-α conjugated with cytotoxin saporin inhibits specifically proliferating vascular smooth muscle cells / 中国药理学通报

AIM: To testify the special cytotoxicity of TGFa-SAP on proliferating vascular smooth muscle cells and endothelial cells. METHODS: Conjugation of saporin to TGFα was accomplished after derivatization of saporin and TGFα with N-succinimidyl-3 (2-pyridyldithio) proprionate. Cytotoxicity assays were measured by cell count. The studies of influence of TGFα-SAP on values of thymidine and leucine incorporation into SMCs and ECs were measured by 3H-thymidine uptake and 3H-leucine uptake, respectively. RESULTS: Cytotoxicity assays testified TGFα-SAP conjugate could inhibit remarkably proliferation of SMCs in culture. The values of thymidine of TGFα-SAP group (1 × 10-9 mol·L-1 and 1 × 10-7 mol·L-1) in comparison significantly decreased to 60.9% and 56.0% of the control group respectively, suggesting that cellular DNA synthesis obviously decreased as TGFα-SAP was added. But saporin did not affect cellular DNA synthesis at higher level. The rate of 3H-leucine incorporation of TGFα-SAP group significantly decreased to 47.3% of the control group, suggesting that SMCs protein synthesis obviously decreased as TGFα-SAP was added. But TGFαSAP at the same level did not affect DNA synthesis and protein synthesis of ECs compared with the control group. CONCLUSION: TGFα-SAP possesses the more effective cytotoxicity than saporin and the more specific citotoxicity on proliferating vascular smooth muscle cells than on proliferating endothelial cells.

Antiproliferative effect of basic fibroblast growth factor-saporin mitotoxin on keratocytes in culture

We evaluated the effect of the conjugate of basic fibroblast growth factor (FGF2) and saporin (FGF2-SAP) on proliferation of cultured keratocytes. Cultured rabbit and human keratocytes were incubated in medium containing 0.01 to 100 nM of chemical conjugate of EGF2 conjugated by disulfide bond to saporin (CCFS1), FGF2 genetically fused to saporin (rFGF2-SAP), FGF2, or saporin for three hours or four days and cell proliferation was quantified four days after the drug treatment. Proliferation of rabbit and human keratocytes was effectively inhibited by three hour and by four day exposure to CCFS1 and rFGF2-SAP in a dose-dependent manner, whereas it was affected minimally by four day exposure to saporin. Their inhibitory effects were detected at concentrations above 0.1 or 1 nM, and were most prominent in serum-stimulated rabbit keratocytes. These results suggest a potential role for FGF2-SAP in limiting proliferation of keratocytes during corneal wound healing.

Thermo-stability and antitumor activity on colon cancer cell lines of monoclonal anti-CEA antibody-saporin immunotoxin

Eight saporin peaks were obtained from the purification of seed extracts of Saponaria officinalis L. Saporin peak No. 6 (SAP-6) showed the highest activity in the inhibition of protein synthesis (98%) in an in vitro translation study. An immunotoxin (IT) was prepared from SAP-6 conjugated to a monoclonal anti-CEA antibody 26/5/1 (mab B) using N-succinimidyl pyridyl dithiopropionate (SPDP) and 2-iminothiolane as a cross linker. Under thermal stability study by a DSC (differential scanning calorimetry), the IT showed a denature temperature of 75 degrees C. In in vitro translation studies, the purified IT showed the same activity as SAP-6 at 10(-7) M and 10(-9) M protein concentration at 0, 30 and 60-min incubation effects with mab B and SAP-6 not conjugated at 24-hr incubation periods on human promyelocytic cell line HL 60 and on human colon adenocarcinoma cell lines which were SW 403, LoVo and LS 174 T. SAP-6, mab B and IT had no cytotoxic effect on HL-60. The IT showed a higher cytotoxic effect than SAP-6 in CEA-positive cell lines. The IT demonstrated the highest cytotoxic effect of 51% inhibition of control at 10(-7) M on the LS 174 T.

Transforming growth factor-? conjugated with cytotoxin saporin inhibits specifically proliferating vascular smooth muscle cells / 中国药理学通报

AIM To testify the special cytotoxicity of TGF? SAP on proliferating vascular smooth muscle cells and endothelial cells. METHODS Conjugation of saporin to TGF? was accomplished after derivatization of saporin and TGF? with N succinimidyl 3 (2 pyridyldithio) proprionate. Cytotoxicity assays were measured by cell count. The studies of influence of TGF? SAP on values of thymidine and leucine incorporation into SMCs and ECs were measured by 3H thymidine uptake and 3H leucine uptake, respectively. RESULTS Cytotoxicity assays testified TGF? SAP conjugate could inhibit remarkably proliferation of SMCs in culture. The values of thymidine of TGF? SAP group (1?10 -9 mol?L -1 and 1?10 -7 mol?L -1 ) in comparison significantly decreased to 60 9% and 56 0% of the control group respectively, suggesting that cellular DNA synthesis obviously decreased as TGF? SAP was added. But saporin did not affect cellular DNA synthesis at higher level. The rate of 3H leucine incorporation of TGF? SAP group significantly decreased to 47 3% of the control group, suggesting that SMCs protein synthesis obviously decreased as TGF? SAP was added. But TGF? SAP at the same level did not affect DNA synthesis and protein synthesis of ECs compared with the control group. CONCLUSION TGF? SAP possesses the more effective cytotoxicity than saporin and the more specific citotoxicity on proliferating vascular smooth muscle cells than on proliferating endothelial cells.