Long-term culture of SH-SY5Y neuroblastoma cells in the absence of neurotrophins: A novel model of neuronal ageing.

BACKGROUND: Studying human ageing is of increasing importance due to the worldwide ageing population. However, it faces the challenge of lengthy experiments to produce an ageing phenotype. Often, to recreate the hallmarks of ageing requires complex empirical conditions that can confound data interpretation. Indeed, many studies use whole organisms with relatively short life spans, which may have little, or limited, relevance to human ageing. There has been extensive use of cell lines to study ageing in human somatic cells, but the modelling of human neuronal ageing is somewhat more complex in vitro. NEW METHOD: We cultured the well-characterised SH-SY5Y human neural cell line to produce high purity cultures of cells differentiated to express a neuronal phenotype, and designed a protocol to maintain these cells in culture until they accumulated biomarkers of cellular ageing. RESULTS: Our data validate a novel and simple technique for the efficient differentiation and long-term maintenance of SH-SY5Y cells, expressing markers of neuronal differentiation and demonstrating electrical activity in culture. Over time in vitro, these cells progressively accumulate markers of ageing such as enhanced production of reactive oxygen species and accumulation of oxidative damage. COMPARISON TO EXISTING METHODS: In comparison to existing techniques to model neuronal ageing our method is cost effective, requiring no specialist equipment or growth factors. CONCLUSIONS: We demonstrate that SH-SY5Y cells, grown under these culture conditions, represent a simple model of neuronal ageing that is amenable to cell biological, biochemical and electrophysiological investigation.

Deep-brain photoreception links luminance detection to motor output in Xenopus frog tadpoles.

Nonvisual photoreceptors are widely distributed in the retina and brain, but their roles in animal behavior remain poorly understood. Here we document a previously unidentified form of deep-brain photoreception in Xenopus laevis frog tadpoles. The isolated nervous system retains sensitivity to light even when devoid of input from classical eye and pineal photoreceptors. These preparations produce regular bouts of rhythmic swimming activity in ambient light but fall silent in the dark. This sensitivity is tuned to short-wavelength UV light; illumination at 400 nm initiates motor activity over a broad range of intensities, whereas longer wavelengths do not cause a response. The photosensitive tissue is located in a small region of caudal diencephalon-this region is necessary to retain responses to illumination, whereas its focal illumination is sufficient to drive them. We present evidence for photoreception via the light-sensitive proteins opsin (OPN)5 and/or cryptochrome 1, because populations of OPN5-positive and cryptochrome-positive cells reside within the caudal diencephalon. This discovery represents a hitherto undescribed vertebrate pathway that links luminance detection to motor output. The pathway provides a simple mechanism for light avoidance and/or may reinforce classical circadian systems.

Leptin prevents hippocampal synaptic disruption and neuronal cell death induced by amyloid ß.

Accumulation of amyloid-ß (Aß) is a key event mediating the cognitive deficits in Alzheimer's disease (AD) as Aß promotes synaptic dysfunction and triggers neuronal death. Recent evidence has linked the hormone leptin to AD as leptin levels are markedly attenuated in AD patients. Leptin is also a potential cognitive enhancer as it facilitates the cellular events underlying hippocampal learning and memory. Here we show that leptin prevents the detrimental effects of Aß(1-42) on hippocampal long-term potentiation. Moreover leptin inhibits Aß(1-42)-driven facilitation of long-term depression and internalization of the 2-amino-3-(5-methyl-3-oxo-1,2- oxazol-4-yl)propanoic acid (AMPA) receptor subunit, GluR1, via activation of PI3-kinase. Leptin also protects cortical neurons from Aß(1-42)-induced cell death by a signal transducer and activator of transcription-3 (STAT-3)-dependent mechanism. Furthermore, leptin inhibits Aß(1-42)-mediated upregulation of endophilin I and phosphorylated tau in vitro, whereas cortical levels of endophilin I and phosphorylated tau are enhanced in leptin-insensitive Zucker fa/fa rats. Thus leptin benefits the functional characteristics and viability of neurons that degenerate in AD. These novel findings establish that the leptin system is an important therapeutic target in neurodegenerative conditions.

Effects of tumour necrosis factor-alpha on developing cerebellar granule and Purkinje neurons in vitro.

Tumour necrosis factor-alpha (TNF-alpha) has been widely implicated in both neurodevelopment and neurodegeneration, yet its effects on individual populations of cerebellar neurons as they develop have not been fully elucidated. Therefore, we established primary neuronal cultures of developing murine cerebellar Purkinje neurons and postnatal cerebellar granule cells to determine the consequences of TNF-alpha exposure for their survival. We discovered that TNF-alpha did not affect the viability of cerebellar granule neurons at any of the ages studied, even though TNF-alpha and its receptors, TNFR1 and TNFR2, are widely expressed in the postnatal cerebellum. In addition, TNF-alpha was neither able to ameliorate, nor enhance, cell death in cerebellar granule cells elicited by a variety of stimuli including homocysteine and alcohol exposure. In contrast, in cultures established at embryonic day 16, TNF-alpha enhanced the number of cerebellar Purkinje neurons in vitro but this effect was not observed in embryonic day 19 cultures. Thus, TNF-alpha has differential and highly specific effects on different populations of cerebellar neurons as they develop.

Neurotrophic effects of leptin on cerebellar Purkinje but not granule neurons in vitro.

As recent evidence has revealed a pro-survival role for the anti-obesity hormone leptin in the nervous system, we investigated the generality of this finding on cerebellar Purkinje and granule neurons in vitro. We found that whilst leptin promoted cerebellar Purkinje neuron survival, it had no affect on cerebellar granule cells. In addition, we discovered that leptin promoted both the outgrowth of neurites from cerebellar Purkinje neurons and increased the complexity of the neurite arbor. Thus, leptin has different effects on two neighbouring populations of neurons within the cerebellum implying specificity of its actions in the central nervous system.

Developmental changes in the response of murine cerebellar granule cells to nitric oxide.

Nitric oxide is a diffusible messenger that plays a multitude of roles within the nervous system including modulation of cell viability. However, its role in regulating neuronal survival during a defined period of neurodevelopment has never been investigated. We discovered that expression of the messenger RNA for both neuronal and endothelial nitric oxide synthase increased in the early postnatal period in the cerebellum in vivo, whilst the expression of inducible nitric oxide synthase remained constant throughout this time in development. Whilst scavenging of nitric oxide was deleterious to the survival of early postnatal cerebellar granule neurons in vitro, this effect was lost in cultures derived at increasing postnatal ages. Conversely, sensitivity to exogenous nitric oxide increased with advancing postnatal age. Thus, we have shown that as postnatal development proceeds, cerebellar granule cells alter their in vitro survival responses to both nitric oxide inhibition and donation, revealing that the nitric oxide's effects on developing neurons vary with the stage of development studied. These findings have important consequences for our understanding of the role of nitric oxide during neuronal development.

Neurotoxic effects of homocysteine on cerebellar Purkinje neurons in vitro.

Whilst a plethora of studies that describe the toxicity of homocysteine to CNS neurons have been published, the effects of homocysteine on the Purkinje neurons of the cerebellum that play a vital role in motor function remain wholly unexplored. We have therefore established cultures of embryonic cerebellar Purkinje neurons and exposed them to a range of concentrations of homocysteine and determined its effects on their survival. The experiments revealed that all concentrations of homocysteine studied, from 50 to 500microM, caused a significant decrease in cerebellar Purkinje neuron number. This loss could be counteracted by the pan-caspase inhibitor z-VAD-fmk in the first 24h following homocysteine exposure, revealing that the initial loss was apoptotic. However, z-VAD-fmk could not prevent homocysteine-mediated loss of cerebellar Purkinje neurons in the longer term, after 6 days in vitro. In addition to its effects on Purkinje neuron survival, homocysteine markedly reduced both the overall magnitude and the complexity of the neurite arbor extended by the cerebellar Purkinje neurons, following 6 days incubation with this agent in vitro. Taken together our data reveal that homocysteine is toxic to cerebellar Purkinje neurons in vitro, inhibiting both their survival and the outgrowth of neurites.

Embryonic cerebellar granule cells are resistant to necrosis induced by homocysteine.

Hyperhomocysteinemia is a risk factor for a range of neurodegenerative conditions, yet its effects in the developing nervous system have been poorly elucidated. We studied the in vitro response of cerebellar granule neurons (CGCs) to homocysteine. We have shown that embryonic CGCs are resistant to homocysteine-induced neurotoxicity, whilst postnatal CGCs are not. This is the first demonstration of a neuronal population undergoing a developmental switch in their response to homocysteine. Greater understanding of this change may have important implications for both neurodegenerative conditions and neurodevelopmental disorders.