Simone Di Giovanni

Gene profiling in spinal cord injury shows role of cell cycle in neuronal death

Spinal cord injury causes secondary biochemical changes leading to neuronal cell death. To clarify the molecular basis of this delayed injury, we subjected rats to spinal cord injury and identified gene expression patterns by high‐density oligonucleotide arrays (8,800 genes studied) at 30 minutes, 4 hours, 24 hours, or 7 days after injury (total of 26 U34A profiles). Detailed analyses were limited to 4,300 genes consistently expressed above background. Temporal clustering showed rapid expression of immediate early genes (30 minutes), followed by genes associated with inflammation, oxidative stress, DNA damage, and cell cycle (4 and 24 hours). Functional clustering showed a novel pattern of cell cycle mRNAs at 4 and 24 hours after trauma. Quantitative reverse transcription polymerase chain reaction verified mRNA changes in this group, which included gadd45a, c‐myc, cyclin D1 and cdk4, pcna, cyclin G, Rb, and E2F5. Changes in their protein products were quantified by Western blot, and cell‐specific expression was determined by immunocytochemistry. Cell cycle proteins showed an increased expression 24 hours after injury and were, in part, colocalized in neurons showing morphological evidence of apoptosis. These findings suggest that cell cycle–related genes, induced after spinal cord injury, are involved in neuronal damage and subsequent cell death. Ann Neurol 2003

Distinct p53 acetylation cassettes differentially influence gene-expression patterns and cell fate

The activity of the p53 gene product is regulated by a plethora of posttranslational modifications. An open question is whether such posttranslational changes act redundantly or dependently upon one another. We show that a functional interference between specific acetylated and phosphorylated residues of p53 influences cell fate. Acetylation of lysine 320 (K320) prevents phosphorylation of crucial serines in the NH2-terminal region of p53; only allows activation of genes containing high-affinity p53 binding sites, such as p21/WAF; and promotes cell survival after DNA damage. In contrast, acetylation of K373 leads to hyperphosphorylation of p53 NH2-terminal residues and enhances the interaction with promoters for which p53 possesses low DNA binding affinity, such as those contained in proapoptotic genes, leading to cell death. Further, acetylation of each of these two lysine clusters differentially regulates the interaction of p53 with coactivators and corepressors and produces distinct gene-expression profiles. By analogy with the “histone code” hypothesis, we propose that the multiple biological activities of p53 are orchestrated and deciphered by different “p53 cassettes,” each containing combination patterns of posttranslational modifications and protein–protein interactions.

The tumor suppressor protein p53 is required for neurite outgrowth and axon regeneration

Axon regeneration is substantially regulated by gene expression and cytoskeleton remodeling. Here we show that the tumor suppressor protein p53 is required for neurite outgrowth in cultured cells including primary neurons as well as for axonal regeneration in mice. These effects are mediated by two newly identified p53 transcriptional targets, the actin‐binding protein Coronin 1b and the GTPase Rab13, both of which associate with the cytoskeleton and regulate neurite outgrowth. We also demonstrate that acetylation of lysine 320 (K320) of p53 is specifically involved in the promotion of neurite outgrowth and in the regulation of the expression of Coronin 1b and Rab13. Thus, in addition to its recognized role in neuronal apoptosis, surprisingly, p53 is required for neurite outgrowth and axonal regeneration, likely through a different post‐translational pathway. These observations may suggest a novel therapeutic target for promoting regenerative responses following peripheral or central nervous system injuries.

Constitutive activation of MAPK cascade in acute quadriplegic myopathy

Acute quadriplegic myopathy (AQM; also called “critical illness myopathy”) shows acute muscle wasting and weakness and is experienced by some patients with severe systemic illness, often associated with administration of corticosteroids and/or neuroblocking agents. Key aspects of AQM include muscle atrophy and myofilament loss. Although these features are shared with neurogenic atrophy, myogenic atrophy in AQM appears mechanistically distinct from neurogenic atrophy. Using muscle biopsies from AQM, neurogenic atrophy, and normal controls, we show that both myogenic and neurogenic atrophy share induction of myofiber‐specific ubiquitin/proteosome pathways (eg, atrogin‐1). However, AQM patient muscle showed a specific strong induction of transforming growth factor (TGF)–β/MAPK pathways. Atrophic AQM myofibers showed coexpression of TGF‐β receptors, p38 MAPK, c‐jun, and c‐myc, including phosphorylated active forms, and these same fibers showed apoptotic features. Our data suggest a model of AQM pathogenesis in which stress stimuli (sepsis, corticosteroids, pH imbalance, osmotic imbalance) converge on the TGF‐β pathway in myofibers. The acute stimulation of the TGF‐β/MAPK pathway, coupled with the inactivity‐induced atrogin‐1/proteosome pathway, leads to the acute muscle loss seen in AQM patients. Ann Neurol 2004

The non‐apoptotic role of p53 in neuronal biology: enlightening the dark side of the moon

The transcription factor p53 protects neurons from transformation and DNA damage through the induction of cell‐cycle arrest, DNA repair and apoptosis in a range of in vitro and in vivo conditions. Indeed, p53 has a crucial role in eliciting neuronal cell death during development and in adult organisms after exposure to a range of stressors and/or DNA damage. Nevertheless, accumulating evidence challenges this one‐sided view of the role of p53 in the nervous system. Here, we discuss how—unexpectedly—p53 can regulate the proliferation and differentiation of neural progenitor cells independently of its role in apoptosis, and p53 post‐translational modifications might promote neuronal maturation, as well as axon outgrowth and regeneration, following neuronal injury. We hope to encourage a more comprehensive view of the non‐apoptotic functions of p53 during neural development, and to warn against oversimplifications regarding its role in neurons. In addition, we discuss how further insight into the p53‐dependent modulation of these mechanisms is necessary to elucidate the decision‐making processes between neuronal cell death and differentiation during development, and between neuronal degeneration and axonal regeneration after injury.

Role of the cell cycle in the pathobiology of central nervous system trauma

Up-regulation of cell cycle proteins occurs in both mitotic and post-mitotic neural cells after central nervous system (CNS) injury in adult animals. In mitotic cells, such as astroglia and microglia, they induce proliferation, whereas in post-mitotic cells such as neurons they initiate caspase-related apoptosis. We recently reported that early central administration of the cell cycle inhibitor flavopiridol after experimental traumatic brain injury (TBI) significantly reduced lesion volume, scar formation and neuronal cell death, while promoting near complete behavioral recovery. Here we show that in primary neuronal or astrocyte cultures structurally different cell cycle inhibitors (flavopiridol, roscovitine, and olomoucine) significantly reduce up-regulation of cell cycle proteins, attenuate neuronal cell death induced by etoposide, and decrease astrocyte proliferation. Flavopiridol, in a concentration dependent manner, also attenuates proliferation/activation of microglia. In addition, we demonstrate that central administration of flavopiridol improves functional outcome in dose-dependent manner after fluid percussion induced brain injury in rats. Moreover, delayed systemic administration of flavopiridol significantly reduces brain lesion volume and edema development after TBI. These data provide further support for the therapeutic potential of cell cycle inhibitors for the treatment of clinical CNS injury and that protective mechanisms likely include reduction of neuronal cell death, inhibition of glial proliferation and attenuation of microglial activation.

Valproic acid-mediated neuroprotection and regeneration in injured retinal ganglion cells.

PURPOSE Valproic acid (VPA) has been demonstrated to have neuroprotective effects in neurodegenerative conditions. VPA inhibits histone-deacetylases (HDAC) and delays apoptosis in degenerating neurons. The authors investigated whether VPA delays retinal ganglion cell (RGC) death and enhances axonal regeneration after optic nerve crush (ONC). Furthermore, potential molecular targets involved in VPA-mediated protection were analyzed. METHODS ONC was performed on the left eye of rats, which received VPA or Ringers solution subcutaneously (SC; 300 mg/kg twice daily) or intravitreally (single postlesional injection). Densities of fluorogold-labeled RGC were analyzed in retinal flatmounts after 5 or 8 days. Retinal tissue was also harvested and processed to quantify axon growth in retinal explants; evaluate caspase-3 activity; analyze transcription factor cAMP response element binding protein (CREB); and determine acetylated histone 3 and 4, as well as phosphorylated extracellular signal-regulated kinase (pERK) 1/2. RESULTS Five and 8 days after ONC, 93% and 58% RGC survived after subcutaneous VPA treatment in comparison to Ringers solution (62% and 37% viable RGC), respectively (P < 0.001). Likewise, a single intravitreal injection of VPA immediately after injury significantly delayed apoptosis in RGC (P = 0.0016). Injured RGC treated with VPA showed better regeneration of their axons in culture (196 axons/explant) than the crushed controls receiving Ringer (115 axons/explant). RGC axons of the right control eyes regenerated more after VPA treatment. VPA-mediated neuroprotection and neuroregeneration were accompanied by decreased caspase-3 activity, CREB induction, pERK1/2 activation, but not by altered histone-acetylation. CONCLUSIONS VPA provided neuroprotection and axonal regrowth after ONC. Alterations were observed in several pathways; however, the precise mechanism of VPA-mediated protection is not yet fully understood.

The histone acetyltransferase p300 promotes intrinsic axonal regeneration

Axonal regeneration and related functional recovery following axonal injury in the adult central nervous system are extremely limited, due to a lack of neuronal intrinsic competence and the presence of extrinsic inhibitory signals. As opposed to what occurs during nervous system development, a weak proregenerative gene expression programme contributes to the limited intrinsic capacity of adult injured central nervous system axons to regenerate. Here we show, in an optic nerve crush model of axonal injury, that adenoviral (cytomegalovirus promoter) overexpression of the acetyltransferase p300, which is regulated during retinal ganglion cell maturation and repressed in the adult, can promote axonal regeneration of the optic nerve beyond 0.5 mm. p300 acetylates histone H3 and the proregenerative transcription factors p53 and CCAAT-enhancer binding proteins in retinal ganglia cells. In addition, it directly occupies and acetylates the promoters of the growth-associated protein-43, coronin 1 b and Sprr1a and drives the gene expression programme of several regeneration-associated genes. On the contrary, overall increase in cellular acetylation using the histone deacetylase inhibitor trichostatin A, enhances retinal ganglion cell survival but not axonal regeneration after optic nerve crush. Therefore, p300 targets both the epigenome and transcription to unlock a post-injury silent gene expression programme that would support axonal regeneration.

NFAT signaling in neural development and axon growth

The NFAT (nuclear factor of activated T‐cells) family of transcription factors functions as integrators of multiple signaling pathways by binding to chromatin in combination with other transcription factors and coactivators to regulate genes central for many developmental systems. Recent experimental evidence has shown that the calcineurin/NFAT signaling pathway is important in axonal growth and guidance during vertebrate development. In fact, studies with triple NFATc2/c3/c4 mutant mice demonstrate that the extension and organization of sensory axon projection and commissural axon growth are both dependent upon NFAT activity. Neurotrophin and L‐type calcium channel signaling modulate intracellular calcium levels to regulate the nuclear import and transcriptional activity of NFAT by activating the phosphatase calcineurin. The rephosphorylation and subsequent export of NFAT from the nucleus is mediated by several kinases, including GSK‐3β, which contribute to the fine tuning of NFAT transcriptional activity in neurons.

Neuronal plasticity after spinal cord injury: identification of a gene cluster driving neurite outgrowth

Functional recovery after spinal cord injury (SCI) may result in part from axon outgrowth and related plasticity through coordinated changes at the molecular level. We employed microarray analysis to identify a subset of genes the expression patterns of which were temporally coregulated and correlated to functional recovery after SCI. Steady‐state mRNA levels of this synchronously regulated gene cluster were depressed in both ventral and dorsal horn neurons within 24 h after injury, followed by strong re‐induction during the following 2 wk, which paralleled functional recovery. The identified cluster includes neuritin, attractin, microtubule‐ associated protein 1a, and myelin oligodendrocyte protein genes. Transcriptional and protein regulation of this novel gene cluster was also evaluated in spinal cord tissue and in single neurons and was shown to play a role in axonal plasticity. Finally, in vitro transfection experiments in primary dorsal root ganglion cells showed that cluster members act synergistically to drive neurite outgrowth.