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We have previously outlined the main properties of brain metabolic DNA (BMD) and its involvement in circadian oscillations, learning, and post-trial sleep. The presence of BMD in certain subcellular fractions and their behavior in cesium gradients have suggested that BMD originates from cytoplasmic reverse transcription and subsequently acquires a double-stranded configuration. More recently, it has been reported that some DNA sequences of cytoplasmic BMD in learning mice are different from that of the control animals. Furthermore, BMD is located in vicinity of the genes involved in different modifications of synaptic activity, suggesting that BMD may contribute to the brain's response to the changing environment. The present review outlines recent data with a special emphasis on reverse transcription of BMD that may recapitulate the molecular events at the time of the "RNA world" by activating mitochondrial telomerase and generating RNA templates from mitochondrial transcripts. The latter unexpected role of mitochondria is likely to promote a better understanding of mitochondrial contribution to cellular interactions and eukaryotic evolution. An initial step regards the role of human mitochondria in embryonic BMD synthesis, which is exclusively of maternal origin. In addition, mitochondrial transcripts involved in reverse transcription of BMD might possibly reveal unexpected features elucidating mitochondrial involvement in cancer events and neurodegenerative disorders.

While protein synthesis in neurons is largely attributed to cell body and dendrites, the capability of synaptic regions to synthesize new proteins independently of the cell body has been widely demonstrated as an advantageous mechanism subserving synaptic plasticity. Thus, the contribution that local protein synthesis at synapses makes to physiology and pathology of brain plasticity may be more prevalent than initially thought. In this study, we tested if local protein synthesis at synapses is deregulated in the brains of TgCRND8 mice, an animal model for Alzheimer’s disease (AD) overexpressing mutant human amyloid precursor protein (APP). To this end, we used synaptosomes as a model system to study the functionality of the synaptic regions in mouse brains. Our results showed that, while TgCRND8 mice exhibit early signs of brain inflammation and deficits in learning, the electrophoretic profile of newly synthesized proteins in their synaptosomes was subtly different from that of the control mice. Interestingly, APP itself was, in part, locally synthesized in the synaptosomes, underscoring the potential importance of local translation at synapses. More importantly, after the contextual fear conditioning, de novo synthesis of some individual proteins was significantly enhanced in the synaptosomes of control animals, but the TgCRND8 mice failed to display such synaptic modulation by training. Taken together, our results demonstrate that synaptic synthesis of proteins is impaired in the brain of a mouse model for AD, and raise the possibility that this deregulation may contribute to the early progression of the pathology.

In a previous study (Mol Neurobiol 55:7476-7486, 2017), newly synthesized brain metabolic DNA (BMD) from rat subcellular fractions has been shown to behave as a DNA-RNA hybrid when analyzed in cesium gradients at early [3H] thymidine incorporation times but to assume the double-stranded configuration at later times. Conversely, BMD from purified nuclei displayed the dsDNA configuration even at early incorporation times. The results were interpreted to support the BMD origin by reverse transcription in the cytoplasm and its later acquisition of the double-stranded configuration before the partial transfer to the nuclei. This interpretation has now been confirmed by immunofluorescence analyses of newly synthesized BrdU-labeled BMD from the mouse brain that demonstrates its cytoplasmic localization and colocalization with DNA-RNA hybrids. In addition, BrdU-labeled BMD has been shown to colocalize with astroglial anti-GFAP antibodies and with presynaptic anti-synaptophysin antibodies.

Squid giant axon has been an excellent model system for studying fundamental topics in neurobiology such as neuronal signaling. It has been also useful in addressing the questions of local protein synthesis in the axons. Incubation of isolated squid giant axons with [(35)S]methionine followed by immunoprecipitation with a rabbit antibody against all squid neurofilament (NF) proteins demonstrates the local synthesis of a major 180 kDa NF protein and of several NF proteins of lower molecular weights. Their identification as NF proteins is based on their absence in the preimmune precipitates. Immunoprecipitates washed with more stringent buffers confirmed these results. Our data are at variance with a recent study based on the same experimental procedure that failed to visualize the local synthesis of NF proteins by the giant axon and thereby suggested their exclusive derivation from nerve cell bodies (as reported by Gainer et al. in Cell Mol Neurobiol 37:475-486, 2017). By reviewing the pertinent literature, we confute the claims that mRNA translation is absent in mature axons because of a putative translation block and that most proteins of mature axons are synthesized in the surrounding glial cells. Given the intrinsic axonal capacity to synthesize proteins, we stress the glial derivation of axonal and presynaptic RNAs and the related proposal that these neuronal domains are endowed with largely independent gene expression systems (as reported by Giuditta et al. in Physiol Rev 88:515-555, 2008).

The synthesis of brain metabolic DNA (BMD) is modulated by learning and circadian oscillations and is not involved in cell division or DNA repair. Data from rats have highlighted its prevalent association with the mitochondrial fraction and its lack of identity with mtDNA. These features suggested that BMD could be localized in synaptosomes that are the major contaminants of brain mitochondrial fractions. The hypothesis has been examined by immunochemical analyses of the large synaptosomes of squid optic lobes that are readily prepared and identified. Optic lobe slices were incubated with 5-bromo-2-deoxyuridine (BrdU) and the isolated synaptosomal fraction was exposed to the green fluorescent anti-BrdU antibody. This procedure revealed that newly synthesized BrdU-labeled BMD is present in a significant percent of the large synaptosomes derived from the nerve terminals of retinal photoreceptor neurons and in synaptosomal bodies of smaller size. Synaptosomal BMD synthesis was strongly inhibited by actinomycin D. In addition, treatment of the synaptosomal fraction with Hoechst 33258, a blue fluorescent dye specific for dsDNA, indicated that native DNA was present in all synaptosomes. The possible role of synaptic BMD is briefly discussed.

Brain metabolic DNA (BMD) is not involved in cell division or DNA repair but is modulated by memory acquisition, sleep processing, and circadian oscillations. Using routine methods of subcellular fractionation, newly synthesized BMD from male rats is shown to be localized in crude nuclear, mitochondrial, and microsomal fractions and in two fractions of purified nuclei. Sub-fractionation of the mitochondrial fraction indicates the prevalent localization of BMD in free mitochondria and to a lesser degree in synaptosomes and myelin. Cesium density profiles of homogenate, subcellular fractions, and purified nuclei obtained after incorporation periods from 30 min to 4 h indicate that BMD synthesis takes place by reverse transcription in cytoplasmic organelles. Following the acquisition of the double-stranded structure, BMD is transferred to nuclei. Kinetic analyses lasting several weeks highlight the massive BMD turnover in subcellular fractions and purified nuclei and its dependence on age. Data are in agreement with the role of BMD as a temporary information store of cell responses of potential use in comparable forthcoming experiences.