Mechanisms of acute axonal degeneration in the optic nerve in vivo.

Axonal degeneration is an initial key step in traumatic and neurodegenerative CNS disorders. We established a unique in vivo epifluorescence imaging paradigm to characterize very early events in axonal degeneration in the rat optic nerve. Single retinal ganglion cell axons were visualized by AAV-mediated expression of dsRed and this allowed the quantification of postlesional acute axonal degeneration (AAD). EM analysis revealed severe structural alterations of the cytoskeleton, cytoplasmatic vacuolization, and the appearance of autophagosomes within the first hours after lesion. Inhibition of autophagy resulted in an attenuation of acute axonal degeneration. Furthermore, a rapid increase of intraaxonal calcium levels following crush lesion could be visualized using a calcium-sensitive dye. Application of calcium channel inhibitors prevented crush-induced calcium increase and markedly attenuated axonal degeneration, whereas application of a calcium ionophore aggravated the degenerative phenotype. We finally demonstrate that increased postlesional autophagy is calcium dependent and thus mechanistically link autophagy and intraaxonal calcium levels. Both processes are proposed to be major targets for the manipulation of axonal degeneration in future therapeutic settings.

Combined inhibition of Cdk5 and ROCK additively increase cell survival, but not the regenerative response in regenerating retinal ganglion cells.

CNS regeneration is limited by lesion-induced neuronal apoptosis and an environment inhibiting axonal elongation. Inhibition of ROCK has been previously shown to promote regeneration in retinal ganglion cells (RGC) whereas Cdk5 inhibition mainly promoted survival. Therefore, we have evaluated the effects of combined treatment with inhibitors of ROCK and Cdk5. We show that in vitro, the co-application of the Cdk5 inhibitor, Indolinone A, and the ROCK inhibitor, Y-27632, potentiated the survival-promoting effect of either substance alone. However, neurite outgrowth in vitro was promoted only by the presence of Y-27632, not by Indolinone A alone. In the ex vivo explant and the in vivo optic nerve crush model the combination of both inhibitors significantly increased neurite outgrowth at small distances, but this effect leveled off for longer neurites. In summary, the combined treatment with the Cdk5 inhibitor Indolinone A and the ROCK inhibitor Y-27632 results in a strong additive effect on neuronal survival, but is not able to increase the regenerative response beyond the effect of the ROCK inhibitor.

ROCK inhibition and CNTF interact on intrinsic signalling pathways and differentially regulate survival and regeneration in retinal ganglion cells.

Functional regeneration in the CNS is limited by lesion-induced neuronal apoptosis and an environment inhibiting axonal elongation. A principal, yet unresolved question is the interaction between these two major factors. We thus evaluated the role of pharmacological inhibition of rho kinase (ROCK), a key mediator of myelin-derived axonal growth inhibition and CNTF, a potent neurotrophic factor for retinal ganglion cells (RGC), in models of retinal ganglion cell apoptosis and neurite outgrowth/regeneration in vitro and in vivo. Here, we show for the first time that the ROCK inhibitor Y-27632 significantly enhanced survival of RGC in vitro and in vivo. In vitro, the co-application of CNTF and Y-27632 potentiated the effect of either substance alone. ROCK inhibition resulted in the activation of the intrinsic MAPK pathway, and the combination of CNTF and Y-27632 resulted in even more pronounced MAPK activation. While CNTF also induced STAT3 phosphorylation, the additional application of ROCK inhibitor surprisingly diminished the effects of CNTF on STAT3 phosphorylation. ROCK activity was also decreased in an additive manner by both substances. In vivo, both CNTF and Y-27632 enhanced regeneration of RGC into the non-permissive optic nerve crush model and additive effects were observed after combination treatment. Further evaluation using specific inhibitors delineate STAT3 as a negative regulator of neurite growth and positive regulator of cell survival, while MAPK and Akt support neurite growth. These results show that next to neurotrophic factors ROCK inhibition by Y-27632 potently supports survival of lesioned adult CNS neurons. Co-administration of CNTF and Y-27632 results in additive effects on neurite outgrowth and regeneration. The interaction of intracellular signalling pathways may, however, attenuate more pronounced synergy and has to be taken into account for future treatment strategies.

BAG1 promotes axonal outgrowth and regeneration in vivo via Raf-1 and reduction of ROCK activity.

Improved survival of injured neurons and the inhibition of repulsive environmental signalling are prerequisites for functional regeneration. BAG1 (Bcl-2-associated athanogene-1) is an Hsp70/Hsc70-binding protein, which has been shown to suppress apoptosis and enhance neuronal differentiation. We investigated BAG1 as a therapeutic molecule in the lesioned visual system in vivo. Using an adeno-associated viral vector, BAG1 (AAV.BAG1) was expressed in retinal ganglion cells (RGC) and then tested in models of optic nerve axotomy and optic nerve crush. BAG1 significantly increased RGC survival as compared to adeno-associated viral vector enhanced green fluorescent protein (AAV.EGFP) treated controls and this was independently confirmed in transgenic mice over-expressing BAG1 in neurons. The numbers and lengths of regenerating axons after optic nerve crush were also significantly increased in the AAV.BAG1 group. In pRGC cultures, BAG1-over-expression resulted in a approximately 3-fold increase in neurite length and growth cone surface. Interestingly, BAG1 induced an intracellular translocation of Raf-1 and ROCK2 and ROCK activity was decreased in a Raf-1-dependent manner by BAG1-over-expression. In summary, we show that BAG1 acts in a dual role by inhibition of lesion-induced apoptosis and interaction with the inhibitory ROCK signalling cascade. BAG1 is therefore a promising molecule to be further examined as a putative therapeutic tool in neurorestorative strategies.

Imino-tetrahydro-benzothiazole derivatives as p53 inhibitors: discovery of a highly potent in vivo inhibitor and its action mechanism.

Several neurological disorders manifest symptoms that result from the degeneration and death of specific neurons. p53 is an important modulator of cell death, and its inhibition could be a therapeutic approach to several neuropathologies. Here, we report the design, synthesis, and biological evaluation of novel p53 inhibitors based on the imino-tetrahydrobenzothiazole scaffold. By performing studies on their mechanism of action, we find that cyclic analogue 4b and its open precursor 2b are more potent than pifithrin-alpha (PFT-alpha), which is known to block p53 pro-apoptotic activity in vitro and in vivo without acting on other pro-apoptotic pathways. Using spectroscopic methods, we also demonstrate that open form 2b is more stable than 4b in biological media. Compound 2b is converted into its corresponding active cyclic form through an intramolecular dehydration process and was found two log values more active in vivo than PFT-alpha. Thus, 2b can be considered as a new prodrug prototype that prevents in vivo p53-triggered cell death in several neuropathologies and possibly reduces cancer therapy side effects.

JNK isoforms differentially regulate neurite growth and regeneration in dopaminergic neurons in vitro.

Parkinson's disease is characterized by selective and progressive loss of midbrain DAergic neurons (MDN) in the substantia nigra and degeneration of its nigrostriatal projections. Whereas the cellular pathophysiology has been closely linked to an activation of c-Jun N-terminal kinases (JNKs) and c-Jun, the involvement of JNKs in regenerative processes of the nigrostriatal pathway is controversially discussed. In our study, we utilized a mechanical scratch lesion paradigm of midbrain DAergic neurons in vitro and studied regenerative neuritic outgrowth. After a siRNA-mediated knockdown of each of the three JNK isoforms, we found that JNKs differentially regulate neurite regeneration. Knockdown of JNK3 resulted in the most prominent neurite outgrowth impairment. This effect was attenuated again by plasmid overexpression of JNK3. We also evaluated cell survival of the affected neurons at the scratch border. JNK3 was found to be also relevant for survival of MDN which were lesioned by the scratch. Our data suggest that JNK isoforms are involved in differential regulation of cell death and regeneration in MDN depending on their neurite integrity. JNK3 appears to be required for regeneration and survival in the case of an environment permissive for regeneration. Future therapeutic approaches for the DAergic system may thus require isoform specific targeting of these kinases.