Cell Death Of Cholinergic Neurons Research Articles

Microcontact Printing of Cholinergic Neurons in Organotypic Brain Slices.

Alzheimer's disease is a severe neurodegenerative disorder of the brain, characterized by beta-amyloid plaques, tau pathology, and cell death of cholinergic neurons, resulting in loss of memory. The reasons for the damage of the cholinergic neurons are not clear, but the nerve growth factor (NGF) is the most potent trophic factor to support the survival of these neurons. In the present study we aim to microprint NGF onto semipermeable 0.4 μm pore membranes and couple them with organotypic brain slices of the basal nucleus of Meynert and to characterize neuronal survival and axonal growth. The brain slices were prepared from postnatal day 10 wildtype mice (C57BL6), cultured on membranes for 2–6 weeks, stained, and characterized for choline acetyltransferase (ChAT). The NGF was microcontact printed in 28 lines, each with 35 μm width, 35 μm space between them, and with a length of 8 mm. As NGF alone could not be printed on the membranes, NGF was embedded into collagen hydrogels and the brain slices were placed at the center of the microprints and the cholinergic neurons that survived. The ChAT+ processes were found to grow along with the NGF microcontact prints, but cells also migrated. Within the brain slices, some form of re-organization along the NGF microcontact prints occurred, especially the glial fibrillary acidic protein (GFAP)+ astrocytes. In conclusion, we provided a novel innovative microcontact printing technique on semipermeable membranes which can be coupled with brain slices. Collagen was used as a loading substance and allowed the microcontact printing of nearly any protein of interest.

Nerve growth factor released from collagen scaffolds protects axotomized cholinergic neurons of the basal nucleus of Meynert in organotypic brain slices

BackgroundAlzheimeŕs disease is accompanied by cell death of cholinergic neurons, resulting in cognitive impairment and memory loss. Nerve growth factor (NGF) is the most potent protein to support survival of cholinergic neurons. New MethodOrganotypic brain slices of the basal nucleus of Meynert (nBM) are a valuable tool to study cell death of axotomized cholinergic neurons, as well as protective effects of NGF added into the medium. The aim of the present study is to use collagen scaffolds crosslinked with polyethyleneglycole and load with NGF to target delivery of NGF to organotypic nBM brain slices. ResultsCollagen scaffolds (visualized by incorporating AlexaFluor 488 antibodies) slowly degraded when applied onto organotypic brain slices within 2 weeks in culture. GFAP reactive astrocytes and Iba1+ microglia became visible around the collagen scaffolds 7days after incubation, showing reactive gliosis. Cholinergic neurons of the nBM survived (201±21, n=8) when incubated with 100ng/ml NGF in the medium compared to NGF-free medium (69±12, n=7). Collagen scaffolds loaded with NGF (1ng/2μl scaffold) significantly rescued cholinergic cell death in the nBM brain slices (175±12, n=10), which was counteracted by an anti-NGF antibody (77±5, n=5). Comparison with existing MethodsThe combination of coronal brain slices with biomaterial is a novel and potent tool to selectively study neuroprotective effects. ConclusionsCollagen scaffolds loaded with low amounts of a protein/drug of interest can be easily applied directly onto organotypic brain slices, allowing slow targeted release of a protective molecule. Such an approach is highly useful to optimize CollScaff for further in vivo applications.

Cholinergic neurodegeneration in an Alzheimer mouse model overexpressing amyloid-precursor protein with the Swedish-Dutch-Iowa mutations

Alzheimer’s disease (AD) is a chronic neurodegenerative disorder that is mainly characterized by beta-amyloid (Aβ) plaque deposition, Tau pathology and dysfunction of the cholinergic system causing memory impairment. The aim of the present study was to examine (1) anxiety and cognition, (2) Aβ plaque deposition and (3) degeneration of cholinergic neurons in the nucleus basalis of Meynert (nbM) and cortical cholinergic innervation in an Alzheimer mouse model (APP_SweDI; overexpressing amyloid precursor protein (APP) with the Swedish K670N/M671L, Dutch E693Q, and Iowa D694N mutations). Our results show that 12-month-old APP_SweDI mice were more anxious and had more memory impairment. A large number of Aβ plaques were already visible at the age of 6 months and increased with age. A significant decrease in cholinergic neurons was seen in the transgenic mouse model in comparison to the wild-type mice, identified by immunohistochemistry against choline acetyltransferase (ChAT) and p75 neurotrophin receptor as well as by in situ hybridization. Moreover, a significant decrease in cortical cholinergic fiber density was found in the transgenic mice as compared to the wild-type. In the cerebral cortex of APP_SweDI mice, swollen cholinergic varicosities were seen in the vicinity of Aβ plaques. In conclusion, the present study shows that in an AD mouse model (APP_SweDI mice) a high Aβ plaque load in the cortex causes damage to cholinergic axons in the cortex, followed by subsequent retrograde-induced cell death of cholinergic neurons and some forms of compensatory processes. This degeneration was accompanied by enhanced anxiety and impaired cognition.

Muscarinic M1 receptor partially modulates higher sensitivity to cadmium-induced cell death in primary basal forebrain cholinergic neurons: A cholinesterase variants dependent mechanism.

Cadmium is a toxic compound reported to produce cognitive dysfunctions, though the mechanisms involved are unknown. In a previous work we described how cadmium blocks cholinergic transmission and induces greater cell death in primary cholinergic neurons from the basal forebrain. It also induces cell death in SN56 cholinergic neurons from the basal forebrain through M1R blockage, alterations in the expression of AChE variants and GSK-3β, and an increase in Aβ and total and phosphorylated Tau protein levels. It was observed that the silencing or blockage of M1R altered ChAT activity, GSK-3β, AChE splice variants gene expression, and Aβ and Tau protein formation. Furthermore, AChE-S variants were associated with the same actions modulated by M1R. Accordingly, we hypothesized that cholinergic transmission blockage and higher sensitivity to cadmium-induced cell death of primary basal forebrain cholinergic neurons is mediated by M1R blockage, which triggers this effect through alteration of the expression of AChE variants. To prove this hypothesis, we evaluated, in primary culture from the basal forebrain region, whether M1R silencing induces greater cell death in cholinergic neurons than cadmium does, and whether in SN56 cells M1R mediates the mechanisms described so as to play a part in the cadmium induction of cholinergic transmission blockage and cell death in this cell line through alteration of the expression of AChE variants. Our results prove that M1R silencing by cadmium partially mediates the greater cell death observed on basal forebrain cholinergic neurons. Moreover, all previously described mechanisms for blocking cholinergic transmission and inducing cell death on SN56 cells after cadmium exposure are partially mediated by M1R through the alteration of AChE expression. Thus, our results may explain cognitive dysfunctions observed in cadmium toxicity.

Acute and long-term exposure to chlorpyrifos induces cell death of basal forebrain cholinergic neurons through AChE variants alteration.

Chlorpyrifos (CPF) is one of the most widely used organophosphates insecticides that has been reported to induce cognitive disorders both after acute and repeated administration similar to those induced in Alzheimer's disease (AD). However, the mechanisms through which it induces these effects are unknown. On the other hand, the cholinergic system, mainly basal forebrain cholinergic neurons, is involved in learning and memory regulation, and an alteration of cholinergic transmission or/and cholinergic cell loss could induce these effects. In this regard, it has been reported that CPF can affect cholinergic transmission, and alter AChE variants, which have been shown to be related with basal forebrain cholinergic neuronal loss. According to these data, we hypothesized that CPF could induce basal forebrain cholinergic neuronal loss through cholinergic transmission and AChE variants alteration. To prove this hypothesis, we evaluated in septal SN56 basal forebrain cholinergic neurons, the CPF toxic effects after 24h and 14 days exposure on neuronal viability and the cholinergic mechanisms related to it. This study shows that CPF impaired cholinergic transmission, induced AChE inhibition and, only after long-term exposure, increased CHT expression, which suggests that acetylcholine levels alteration could be mediated by these actions. Moreover, CPF induces, after acute and long-term exposure, cell death in cholinergic neurons in the basal forebrain and this effect is independent of AChE inhibition and acetylcholine alteration, but was mediated partially by AChE variants alteration. Our present results provide a new understanding of the mechanisms contributing to the harmful effects of CPF on neuronal function and viability, and the possible relevance of CPF in the pathogenesis of neurodegenerative diseases.

Platelet-derived nerve growth factor supports the survival of cholinergic neurons in organotypic rat brain slices

Platelets play a role in repair of vessels and contain different growth factors, including nerve growth factor (NGF). Since NGF is the most potent growth factor to support survival of cholinergic neurons, we aimed to study the effects of platelet-derived NGF on cholinergic neurons in organotypic brain slices. Brain slices of the nucleus basalis of Meynert (nBM) were cultured with or without NGF (10ng/ml) or platelet extracts (100μg/ml) or fresh platelets (108 platelets/ml). In order to enhance NGF in platelets recombinant NGF (100ng) was loaded into platelets using ultrasound (3h). Our data show that recombinant NGF markedly supports survival of cholinergic neurons. The addition of fresh platelets showed a tendency for enhancing cholinergic neuron numbers, while platelet extracts had no effects. Ultrasound was highly effective to load recombinant NGF into platelets. The addition of NGF-loaded platelets markedly enhanced cholinergic neuron numbers. In conclusion, our data provide evidence that NGF-derived platelets may counteract cell death of cholinergic neurons.

S100b Counteracts Neurodegeneration of Rat Cholinergic Neurons in Brain Slices after Oxygen-Glucose Deprivation

Alzheimer's disease is a severe chronic neurodegenerative disorder characterized by beta-amyloid plaques, tau pathology, cerebrovascular damage, inflammation, reactive gliosis, and cell death of cholinergic neurons. The aim of the present study is to test whether the glia-derived molecule S100b can counteract neurodegeneration of cholinergic neurons after oxygen-glucose deprivation (OGD) in organotypic brain slices of basal nucleus of Meynert. Our data showed that 3 days of OGD induced a marked decrease of cholinergic neurons (60% of control), which could be counteracted by 50 μg/mL recombinant S100b. The effect was dose and time dependent. Application of nerve growth factor or fibroblast growth factor-2 was less protective. C-fos-like immunoreactivity was enhanced 3 hours after OGD indicating metabolic stress. We conclude that S100b is a potent neuroprotective factor for cholinergic neurons during ischemic events.

Rotenone induces cell death of cholinergic neurons in an organotypic co-culture brain slice model.

In Alzheimer and Parkinson's disease cell death of cholinergic and dopaminergic neurons are characteristic hallmarks, respectively. It is well established that rotenone, an inhibitor of complex I of the electron transport chain, induces cell death of dopaminergic neurons, however, not much is known on the effects of rotenone on cholinergic neurons. The aim of the present study was to evaluate the effects of rotenone on cholinergic neurons in an organotypic in vitro brain co-slice model. When co-cultures were treated with 10 μM rotenone for 24 h a significantly decreased number of cholinergic neurons was found in the basal nucleus of Meynert but not in the dorsal striatum. This cell death exhibited apoptotic DAPI-positive malformed nuclei and enhanced TUNEL-positive cells. In summary, inhibition of complex I of the electron transport chain may play a role in neurodegeneration of cholinergic neurons.

Cholinergic neurons degenerate when exposed to conditioned medium of primary rat brain capillary endothelial cells: Counteraction by NGF, MK-801 and inflammation

Alzheimer's disease is characterized by extracellular beta-amyloid plaques, intraneuronal Tau-inclusions and cell death of cholinergic neurons. Recent evidence indicates that the vascular system may play an important role in the development of this progressive neurodegenerative disease. The aim of this study was to observe, if brain capillary endothelial cells (BCEC) may produce and secrete factors which induce cell death of cholinergic neurons, and if this effect is counteracted by (1) NGF, MK-801 or vitamin C, (2) modulated by experimentally-induced inflammation with interleukin-1β and lipopolysaccharide (IL-1β and LPS) or (3) by blocking of different intracellular signalling pathways. Cholinergic neurons were cultivated in organotypic brain slices of the nucleus basalis of Meynert and treated with conditioned medium derived from BCEC, supplemented with different protective factors. BCEC were stimulated with IL-1β and LPS or different intracellular pathway inhibitors before collection of conditioned medium. Cholinergic neurons were detected by immunohistochemistry for choline-acetyltransferase. Possible effects on BCEC viability and proliferation were determined by nuclear staining, BrdU incorporation and release of nitrite and lactate-dehydrogenase. BCEC released factors that can kill cholinergic neurons. This neurotoxic effect was blocked by NGF and MK-801 (a NMDA-antagonist), but not by vitamin C. Pretreatment of BCEC with intracellular pathway inhibitors did not change the neurotoxicity, but pretreatment with IL-1β and LPS abolished the neurotoxic effect. In summary, BCEC produce and secrete molecules which induce excitotoxic cell death of cholinergic neurons. It is concluded that excitotoxic factors secreted by vascular cells may contribute to the development of cholinergic neurodegeneration as it occurs in Alzheimer's disease.

Transplantation of NGF secreting primary monocytes counteracts NMDA-induced cell death of rat cholinergic neurons in vivo

Cholinergic neurons of the basal forebrain degenerate in Alzheimer's disease. Nerve growth factor (NGF) is so far the most potent molecule to counteract this neurodegeneration; however, the delivery of NGF into the brain is very difficult. The aim of the present study was to observe, if transplanted primary monocytes secreting NGF may counteract N-methyl- d-aspartate (NMDA)-induced cell death of cholinergic neurons of the basal nucleus of Meynert (nBM) in vivo. Monocytes were purified by indirect magnetic separation from rat blood. Recombinant NGF was introduced into cells using the novel protein-delivery reagent BioPORTER™ and secretion of NGF was measured by ELISA. Monocytes secreted approximately 4000 pg NGF/day/1 × 10 6 cells. Injection of monocytes onto organotypic brain slices of the nBM in vitro protected cholinergic neurons against cell death. When monocytes were transplanted in vivo into the lateral ventricle, the cells survived for up to 7 days and counteracted the NMDA-induced cell death of cholinergic neurons. In conclusion, primary monocytes secreting recombinant NGF are useful to deliver NGF directly into the brain.

Vascular endothelial growth factor counteracts NMDA-induced cell death of adult cholinergic neurons in rat basal nucleus of Meynert

Vascular endothelial growth factor (VEGF) plays an important role in angiogenesis in the brain and has recently been shown to possess neuroprotective activity. The aim of the present study was to observe if VEGF can counteract the excitotoxic N-methyl- d-aspartate (NMDA)-induced cell death of cholinergic neurons of the basal nucleus of Meynert in vivo in adult rats. Immunohistochemistry was used to detect vascular structures (RECA-1 or laminin staining) and cholinergic neurons (choline-acetyltransferase). To compare the in vivo VEGF effects also in vitro, VEGF was applied to axotomized cholinergic neurons in organotypic brain slices with or without NMDA. Our data provide evidence that VEGF counteracted cell death in vivo in adult rats but did not protect cholinergic neurons in developing brain slices. Nerve growth factor protected cholinergic neurons in vivo as well as in vitro. In conclusion, VEGF exhibits neuroprotective activity on adult cholinergic neurons of the basal nucleus of Meynert.

Tumor necrosis factor-α triggers cell death of sensitized potassium chloride-stimulated cholinergic neurons

Cell death of cholinergic neurons of the basal forebrain plays an important role in neurodegenerative disorders, such as Alzheimer’s disease. Inflammatory cytokines, such as, for example, tumor necrosis factor-α (TNF-α), may be involved in these neurodegenerative processes. The aim of this project was to study the role of TNF-α in the survival and nerve fiber growth of cholinergic neurons of the basal nucleus of Meynert in organotypic brain slices and in adult rats. Cholinergic neurons were visualized by immunohistochemistry for the enzyme choline acetyltransferase and nerve fibers by histochemistry for the enzyme acetylcholinesterase. When co-slices of basal nucleus of Meynert and neocortex were sensitized for 15 min with 30 mM potassium chloride and subsequently incubated for 1 week with 20 ng/ml TNF-α, cholinergic neurons and nerve fibers markedly degenerated. Incubation with different growth factors rescued the loss of cholinergic cell bodies and cholinergic nerve fibers. Injection of 30 mM potassium chloride and 50 ng TNF-α into four defined cortical regions of anesthetized adult rats resulted in predominant cell death of cholinergic neurons on the ipsilateral side. In conclusion, our data show that TNF-α potentiated cell death of cholinergic neurons possibly via retrograde axonal damage in vitro and in vivo. Cortical overactivation combined with an increased expression of pro-inflammatory cytokines may contribute to the cell death observed in Alzheimer’s disease and ageing.

Effect of stimulation of the nucleus basalis of Meynert and somatic afferents on experimentally-induced decreases in cortical blood flow in anesthetized rats

Platelets play a role in repair of vessels and contain different growth factors, including nerve growth factor (NGF). Since NGF is the most potent growth factor to support survival of cholinergic neurons, we aimed to study the effects of platelet-derived NGF on cholinergic neurons in organotypic brain slices. Brain slices of the nucleus basalis of Meynert (nBM) were cultured with or without NGF (10 ng/ml) or platelet extracts (100 μg/ml) or fresh platelets (108 platelets/ml). In order to enhance NGF in platelets recombinant NGF (100 ng) was loaded into platelets using ultrasound (3 h). Our data show that recombinant NGF markedly supports survival of cholinergic neurons. The addition of fresh platelets showed a tendency for enhancing cholinergic neuron numbers, while platelet extracts had no effects. Ultrasound was highly effective to load recombinant NGF into platelets. The addition of NGF-loaded platelets markedly enhanced cholinergic neuron numbers. In conclusion, our data provide evidence that NGF-derived platelets may counteract cell death of cholinergic neurons.