Carolina Cefaliello

Local Gene Expression in Axons and Nerve Endings: The Glia-Neuron Unit

Neurons have complex and often extensively elongated processes. This unique cell morphology raises the problem of how remote neuronal territories are replenished with proteins. For a long time, axonal and presynaptic proteins were thought to be exclusively synthesized in the cell body, which delivered them to peripheral sites by axoplasmic transport. Despite this early belief, protein has been shown to be synthesized in axons and nerve terminals, substantially alleviating the trophic burden of the perikaryon. This observation raised the question of the cellular origin of the peripheral RNAs involved in protein synthesis. The synthesis of these RNAs was initially attributed to the neuron soma almost by default. However, experimental data and theoretical considerations support the alternative view that axonal and presynaptic RNAs are also transcribed in the flanking glial cells and transferred to the axon domain of mature neurons. Altogether, these data suggest that axons and nerve terminals are served by a distinct gene expression system largely independent of the neuron cell body. Such a local system would allow the neuron periphery to respond promptly to environmental stimuli. This view has the theoretical merit of extending to axons and nerve terminals the marginalized concept of a glial supply of RNA (and protein) to the neuron cell body. Most long-term plastic changes requiring de novo gene expression occur in these domains, notably in presynaptic endings, despite their intrinsic lack of transcriptional capacity. This review enlightens novel perspectives on the biology and pathobiology of the neuron by critically reviewing these issues.

Local synthesis of axonal and presynaptic RNA in squid model systems.

The presence of active systems of protein synthesis in axons and nerve endings raises the question of the cellular origin of the corresponding RNAs. Our present experiments demonstrate that, besides a possible derivation from neuronal cell bodies, axoplasmic RNAs originate in periaxonal glial cells and presynaptic RNAs derive from nearby cells, presumably glial cells. Indeed, in perfused squid giant axons, delivery of newly synthesized RNA to the axon perfusate is strongly stimulated by axonal depolarization or agonists of glial glutamate and acetylcholine receptors. Likewise, incubation of squid optic lobe slices with [3H]uridine leads to a marked accumulation of [3H]RNA in the large synaptosomes derived from the nerve terminals of retinal photoreceptor neurons. As the cell bodies of these neurons lie outside the optic lobe, the data demonstrate that presynaptic RNA is locally synthesized, presumably by perisynaptic glial cells. Overall, our results support the view that axons and presynaptic regions are endowed with local systems of gene expression which may prove essential for the maintenance and plasticity of these extrasomatic neuronal domains.

High Fat Diet and Inflammation – Modulation of Haptoglobin Level in Rat Brain

Obesity and dietary fats are well known risk factors for the pathogenesis of neurodegenerative diseases. The analysis of specific markers, whose brain level can be affected by diet, might contribute to unveil the intersection between inflammation/obesity and neurodegeneration. Haptoglobin (Hpt) is an acute phase protein, which acts as antioxidant by binding free haemoglobin (Hb), thus neutralizing its pro-oxidative action. We previously demonstrated that Hpt plays critical functions in brain, modulating cholesterol trafficking in neuroblastoma cell lines, beta-amyloid (Aβ) uptake by astrocyte, and limiting Aβ toxicity on these cells. A major aim of this study was to evaluate whether a long term (12 or 24 weeks) high-fat diet (HFD) influences Hpt and Hb expression in rat hippocampus. We also assessed the development of obesity-induced inflammation by measuring hippocampal level of TNF-alpha, and the extent of protein oxidation by titrating nitro-tyrosine (N-Tyr). Hpt concentration was lower (p < 0.001) in hippocampus of HFD rats than in control animals, both in the 12 and in the 24 weeks fed groups. HFD was also associated in hippocampus with the increase of Hb level (p < 0.01), inflammation and protein oxidative modification, as evidenced by the increase in the concentration of TNF-alpha and nitro-tyrosine. In fact, TNF-alpha concentration was higher in rats receiving HFD for 12 (p < 0.01) or 24 weeks (p < 0.001) compared to those receiving the control diet. N-Tyr concentration was more elevated in hippocampus of HFD than in control rats in both 12 weeks (p = 0.04) and 24 weeks groups (p = 0.01), and a positive correlation between Hb and N-Tyr concentration was found in each group. Finally, we found that the treatment of the human glioblastoma-astrocytoma cell line U-87 MG with cholesterol and fatty acids, such as palmitic and linoleic acid, significantly impairs (p < 0.001) Hpt secretion in the extracellular compartment. We hypothesize that the HFD-dependent decrease of Hpt in hippocampus, as associated with Hb increase, might enhance the oxidative stress induced by free Hb. Altogether our data, identifying Hpt as a molecule modulated in the brain by dietary fats, may represent one of the first steps in the comprehension of the molecular mechanisms underlying the diet-related effects in the nervous system.

Local gene expression in nerve endings

At the Nobel lecture for physiology in 1906, Ramón y Cajal famously stated that “the nerve elements possess reciprocal relationships in contiguity but not in continuity,” summing up the neuron doctrine. Sixty years later, by the time the central dogma of molecular biology formulated the axis of genetic information flow from DNA to mRNA, and then to protein, it became obvious that neurons with extensive ramifications and long axons inevitably incur an innate problem: how can the effect of gene expression be extended from the nucleus to the remote and specific sites of the cell periphery? The most straightforward solution would be to deliver soma‐produced proteins to the target sites. The influential discovery of axoplasmic flow has supported this scheme of protein supply. Alternatively, mRNAs can be dispatched instead of protein, and translated locally at the strategic target sites. Over the past decades, such a local system of protein synthesis has been demonstrated in dendrites, axons, and presynaptic terminals. Moreover, the local protein synthesis in neurons might even involve intercellular trafficking of molecules. The innovative concept of glia‐neuron unit suggests that the local protein synthesis in the axonal and presynaptic domain of mature neurons is sustained by a local supply of RNAs synthesized in the surrounding glial cells and transferred to these domains. Here, we have reviewed some of the evidence indicating the presence of a local system of protein synthesis in axon terminals, and have examined its regulation in various model systems.

Haptoglobin increases with age in rat hippocampus and modulates Apolipoprotein E mediated cholesterol trafficking in neuroblastoma cell lines

Alteration in cholesterol metabolism has been implicated in the pathogenesis of several neurodegenerative disorders. Apolipoprotein E (ApoE) is the major component of brain lipoproteins supporting cholesterol transport. We previously reported that the acute-phase protein Haptoglobin (Hpt) binds ApoE, and influences its function in blood cholesterol homeostasis. Major aim of this study was to investigate whether Hpt influences the mechanisms by which cholesterol is shuttled from astrocytes to neurons. In detail it was studied Hpt effect on ApoE-dependent cholesterol efflux from astrocytes and ApoE-mediated cholesterol incorporation in neurons. We report here that Hpt impairs ApoE-mediated cholesterol uptake in human neuroblastoma cell line SH-SY5Y, and limits the toxicity of a massive concentration of cholesterol for these cells, while it does not affect cholesterol efflux from the human glioblastoma-astrocytoma cell line U-87 MG. As aging is the most important non-genetic risk factor for various neurodegenerative disorders, and our results suggest that Hpt modulates ApoE functions, we evaluated the Hpt and ApoE expression profiles in cerebral cortex and hippocampus of adolescent (2 months), adult (5 and 8 months), and middle-aged (16 months) rats. Hpt mRNA level was higher in hippocampus of 8 and 16 month-old than in 2-month old rats (p < 0.05), and Hpt concentration increased with the age from adolescence to middle-age (p < 0.001). ApoE concentration, in hippocampus, was higher (p < 0.001) in 5 month-old rats compared to 2 month but did not further change with aging. No age-related changes of Hpt (protein and mRNA) were found in the cortex. Our results suggest that aging is associated with changes, particularly in the hippocampus, in the Hpt/ApoE ratio. Age-related changes in the concentration of Hpt were also found in human cerebrospinal fluids. The age-related changes might affect neuronal function and survival in brain, and have important implications in brain pathophysiology.

Synaptosomal protein synthesis is selectively modulated by learning

Synaptosomes from rat brain have long been used to investigate the properties of synaptic protein synthesis. Comparable analyses have now been made in adult male rats trained for a two-way active avoidance task to examine the hypothesis of its direct participation in brain plastic events. Using Ficoll-purified synaptosomes from neocortex, hippocampus and cerebellum, our data indicate that the capacity of synaptosomal protein synthesis and the specific activity of newly synthesized proteins were not different in trained rats in comparison with home-caged control rats. On the other hand, the synthesis of two proteins of 66.5 kDa and 87.6 kDa separated by SDS-PAGE and analyzed by quantitative densitometry was selectively enhanced in trained rats. In addition, the synthesis of the 66.5 kDa protein, but not of the 87.6 kDa protein, correlated with avoidances and escapes and inversely correlated with freezings in the neocortex, while in the cerebellum it correlated with avoidances and escapes. The data demonstrate the participation of synaptic protein synthesis in plastic events of behaving rats, and the selective, region-specific modulation of the synthesis of a synaptic 66.5 kDa protein by the newly acquired avoidance response and by the reprogramming of innate neural circuits subserving escape and freezing responses.

Protein Synthesis in Nerve Terminals and the Glia–Neuron Unit

The progressive philogenetic lengthening of axonal processes and the increase in complexity of terminal axonal arborizations markedly augmented the demands of the neuronal cytoplasmic mass on somatic gene expression. It is proposed that in an adaptive response to this challenge, novel gene expression functions developed in the axon compartment, consisting of axonal and presynaptic translation systems that rely on the delivery of transcripts synthesized in adjacent glial cells. Such intercellular mode of gene expression would allow more rapid plastic changes to occur in spatially restricted neuronal domains, down to the size of individual synapses. The cell body contribution to local gene expression in well-differentiated neurons remains to be defined. The history of this concept and the experimental evidence supporting its validity are critically discussed in this article. The merit of this perspective lies with the recognition that plasticity events represent a major occurrence in the brain, and that they largely occur at synaptic sites, including presynaptic endings.

Training old rats selectively modulates synaptosomal protein synthesis

We have previously shown that the local synthesis of two synaptic proteins of 66.5‐kDa and 87.6‐kDa is selectively enhanced in male adult rats trained for a two‐way active avoidance task. We report here that a comparable but not identical response occurs in 2‐year‐old male rats trained for the same task. In the latter age group, the local synthesis of the 66.5‐kDa protein markedly increases in cerebral cortex, brainstem, and cerebellum, with a somewhat lower increment in synthesis of the 87.6‐kDa protein. On the other hand, the newly synthesized 87.6‐kDa protein correlates with avoidances and escapes and inversely correlates with freezings in cerebral cortex and brainstem, whereas the correlations of the newly synthesized 66.5‐kDa protein remain below significance. These correlative patterns are sharply at variance with those present in trained adult rats. Our data confirm that the local system of synaptic protein synthesis is selectively modulated by training and show that the synaptic response of old rats differs from that of adult rats as reflected in behavioral responses.

Synaptosomal protein synthesis in P2 and Ficoll purified fractions

Cytoplasmic protein synthesis of brain synaptosomes has generally been determined in the Ficoll purified fraction which contains fewer contaminating mitochondria, microsomes and myelin fragments than the parent P2 fraction. Using a highly selective assay of this activity we have compared the total translation activity and the specific activity of the proteins synthesized by either fraction in control rats and in rats trained for a two-way active avoidance task. In control rats the specific activity remained essentially the same in both fractions but in trained rats the value of the Ficoll fraction was markedly lower (38.5%) than in the P2 fraction. Furthermore, the total translation activity of the Ficoll fraction was 30% lower than in the P2 fraction in control rats and 62% lower in trained rats. These decrements indicate that a large proportion of active synaptosomes present in the P2 fraction is not recovered in the Ficoll fraction, notably in rats undergoing plastic brain changes. We conclude that cytoplasmic protein synthesis of brain synaptosomes is better preserved in the P2 fraction.

Synaptic mRNAs are modulated by learning.

We have recently demonstrated that brain plastic events significantly modify synaptic protein synthesis measured by the incorporation of [35S]methionine in brain synaptosomal proteins. Notably, in rats learning a two‐way active avoidance task, the local synthesis of two synaptic proteins was selectively enhanced. Because this effect may be attributed to transcriptional modulation, we used reverse transcriptase–polymerase chain reaction methods to determine the content of discrete synaptosomal mRNAs in rats exposed to the same training protocol. Correlative analyses between behavioral responses and synaptosomal mRNA content showed that GAT‐1 mRNA (a prevalent presynaptic component) correlates with avoidances and escapes in rat cerebellum, while glial fibrillary acid protein mRNA (an astrocytic component) correlates with freezings in cerebellum and cerebral cortex. These observations support the hypothesis that synaptic protein synthesis may be transcriptionally regulated. The cellular origin of synaptic transcripts is briefly discussed, with special regard to those present at large distances from neuron somas.