Experimental approaches to understanding the role of protein phosphorylation in the regulation of neuronal function

Studies by Earl Sutherland and his colleagues on hormonal regulation of the breakdown of glycogen in liver resulted in the discovery that the first step in the action of many hormones is to increase the synthesis of cAMP by activating adenylate cyclase (Raft et al 1957, Sutherland & Rall 1958, Robison et al 1968). It was later established that cAMP exerts its effects by stimulating protein kinases that catalyze the phosphorylation of specific functional proteins and thereby regulate their activity (Walsh et al 1968, Kuo & Greengard 1969, Krebs & Beavo 1979). The discovery that the brain contains a high concentration of cAMP-dependent protein kinase led to the proposal that protein phosphorylation might play an important role in regulation of neuronal properties by neurotransmitters and neurohormones (Miyamoto et al 1969). In particular, it seemed that protein phosphorylation, which usually takes place on a time scale of hundreds of milliseconds or longer, might be a mechanism underlying relatively long-lasting changes in neuronal properties such as "slow" changes in post-synaptic potentials (McAfee & Greengard 1972), changes in the rate of transmitter synthesis (Morgenroth et al 1975), or changes in gene expression (Klein & Berg 1970). The biochemists and neurobiologists who took up the study of brain protein phosphorylation hoped to gain insight into some of the mechanisms underlying changes in neuronal excitability and synaptic efficacy and also, perhaps, into processes that govern the development of various neuronal types during the formation of the nervous system. This line of research was bolstered by the findings that the brain contains not only high concentrations of protein kinases, but also protein phosphatases, adenylate cyclase, and phosphodiesterase (Greengard 1976), and also by the discovery that several neurotransmitters stimulate the synthesis of second messengers such as cyclic AMP and cyclic GMP by binding to specific receptors on the surfaces of neurons (for reviews see Nathanson 1977, Greengard 1981)

Purification and characterization of a calmodulin-dependent protein kinase that is highly concentrated in brain

A calcium and calmodulin-dependent protein kinase has been purified from rat brain. It was monitored during the purification by its ability to phosphorylate the synaptic vesicle-associated protein, synapsin I. A 300-fold purification was sufficient to produce kinase that is 90-95% pure as determined by scans of stained sodium dodecyl sulfate-polyacrylamide gels and has a specific activity of 2.9 mumol of 32P transferred per min/mg of protein. Thus, the kinase is a relatively abundant brain enzyme, perhaps comprising as much as 0.3% of the total brain protein. The Stokes radius (95 A) and sedimentation coefficient (16.4 S) of the kinase indicate a holoenzyme molecular weight of approximately 650,000. The holoenzyme is composed of three subunits as judged by their co-migration with kinase activity during the purification steps and co-precipitation with kinase activity by a specific anti-kinase monoclonal antibody. The three subunits have molecular weights of 50,000, 58,000, and 60,000, and have been termed alpha, beta', and beta, respectively. The alpha- and beta-subunits are distinct peptides, however, beta' may have been generated from beta by proteolysis. All three of these subunits bind calmodulin in the presence of calcium and are autophosphorylated under conditions in which the kinase is active. The subunits are present in a ratio of about 3 alpha-subunits to 1 beta/beta'-subunit. We therefore postulate that the 650,000-Da holoenzyme consists of approximately 9 alpha-subunits and 3 beta/beta'-subunits. The abundance of this calmodulin-dependent protein kinase indicates that its activation is likely to be an important biochemical response to increases in calcium ion concentration in neuronal tissue

Elevated intracellular cAMP exacerbates vulnerability to oxidative stress in optic nerve head astrocytes.

Glaucoma is characterized by a progressive loss of retinal ganglion cells and their axons, but the underlying biological basis for the accompanying neurodegeneration is not known. Accumulating evidence indicates that structural and functional abnormalities of astrocytes within the optic nerve head (ONH) have a role. However, whether the activation of cyclic adenosine 3',5'-monophosphate (cAMP) signaling pathway is associated with astrocyte dysfunction in the ONH remains unknown. We report here that the cAMP/protein kinase A (PKA) pathway is critical to ONH astrocyte dysfunction, leading to caspase-3 activation and cell death via the AKT/Bim/Bax signaling pathway. Furthermore, elevated intracellular cAMP exacerbates vulnerability to oxidative stress in ONH astrocytes, and this may contribute to axonal damage in glaucomatous neurodegeneration. Inhibition of intracellular cAMP/PKA signaling activation protects ONH astrocytes by increasing AKT phosphorylation against oxidative stress. These results strongly indicate that activation of cAMP/PKA pathway has an important role in astrocyte dysfunction, and suggest that modulating cAMP/PKA pathway has therapeutic potential for glaucomatous ONH degeneration

Pericyte-mediated regulation of capillary diameter: a component of neurovascular coupling in health and disease

Because regional blood flow increases in association with the increased metabolic demand generated by localised increases in neural activity, functional imaging researchers often assume that changes in blood flow are an accurate read-out of changes in underlying neural activity. An understanding of the mechanisms that link changes in neural activity to changes in blood flow is crucial for assessing the validity of this assumption, and for understanding the processes that can go wrong during disease states such as ischaemic stroke. Many studies have investigated the mechanisms of neurovascular regulation in arterioles but other evidence suggests that blood flow regulation can also occur in capillaries, because of the presence of contractile cells, pericytes, on the capillary wall. Here we review the evidence that pericytes can modulate capillary diameter in response to neuronal activity and assess the likely importance of neurovascular regulation at the capillary level for functional imaging experiments. We also discuss evidence suggesting that pericytes are particularly sensitive to damage during pathological insults such as ischaemia, Alzheimer’s disease and diabetic retinopathy, and consider the potential impact that pericyte dysfunction might have on the development of therapeutic interventions and on the interpretation of functional imaging data in these disorders

Genomic and proteomic profiling of responses to toxic metals in human lung cells.

Examining global effects of toxic metals on gene expression can be useful for elucidating patterns of biological response, discovering underlying mechanisms of toxicity, and identifying candidate metal-specific genetic markers of exposure and response. Using a 1,200 gene nylon array, we examined changes in gene expression following low-dose, acute exposures of cadmium, chromium, arsenic, nickel, or mitomycin C (MMC) in BEAS-2B human bronchial epithelial cells. Total RNA was isolated from cells exposed to 3 M Cd(II) (as cadmium chloride), 10 M Cr(VI) (as sodium dichromate), 3 g/cm2 Ni(II) (as nickel subsulfide), 5 M or 50 M As(III) (as sodium arsenite), or 1 M MMC for 4 hr. Expression changes were verified at the protein level for several genes. Only a small subset of genes was differentially expressed in response to each agent: Cd, Cr, Ni, As (5 M), As (50 M), and MMC each differentially altered the expression of 25, 44, 31, 110, 65, and 16 individual genes, respectively. Few genes were commonly expressed among the various treatments. Only one gene was altered in response to all four metals (hsp90), and no gene overlapped among all five treatments. We also compared low-dose (5 M, noncytotoxic) and high-dose (50 M, cytotoxic) arsenic treatments, which surprisingly, affected expression of almost completely nonoverlapping subsets of genes, suggesting a threshold switch from a survival-based biological response at low doses to a death response at high doses

Analysis of the Catecholaminergic Phenotype in Human SH-SY5Y and BE(2)-M17 Neuroblastoma Cell Lines upon Differentiation

Human cell lines are often used to investigate cellular pathways relevant for physiological or pathological processes or to evaluate cell toxicity or protection induced by different compounds, including potential drugs. In this study, we analyzed and compared the differentiating activities of three agents (retinoic acid, staurosporine and 12-O-tetradecanoylphorbol-13-acetate) on the human neuroblastoma SH-SY5Y and BE(2)-M17 cell lines; the first cell line is largely used in the field of neuroscience, while the second is still poorly characterized. After evaluating their effects in terms of cell proliferation and morphology, we investigated their catecholaminergic properties by assessing the expression profiles of the major genes involved in catecholamine synthesis and storage and the cellular concentrations of the neurotransmitters dopamine and noradrenaline. Our results demonstrate that the two cell lines possess similar abilities to differentiate and acquire a neuron-like morphology. The most evident effects in SH-SY5Y cells were observed in the presence of staurosporine, while in BE(2)-M17 cells, retinoic acid induced the strongest effects. Undifferentiated SH-SY5Y and BE(2)-M17 cells are characterized by the production of both NA and DA, but their levels are considerably higher in BE(2)-M17 cells. Moreover, the NAergic phenotype appears to be more pronounced in SH-SY5Y cells, while BE(2)-M17 cells have a more prominent DAergic phenotype. Finally, the catecholamine concentration strongly increases upon differentiation induced by staurosporine in both cell lines. In conclusion, in this work the catecholaminergic phenotype of the human BE(2)-M17 cell line upon differentiation was characterized for the first time. Our data suggest that SH-SY5Y and BE(2)-M17 represent two alternative cell models for the neuroscience field