Via Luigi Vanvitelli 32
Cellular and molecular mechanisms of motor neuron degeneration in amyotrophic lateral sclerosis (ALS): role of the VAP-B gene in the pathogenesis of ALS
ALS is the most frequent adult-onset Motorneuron Disease whose hallmark is the selective death of motor neurons in the primary motor cortex, brain stem and spinal cord, leading to paralysis of voluntary muscles.
In collaboration with Nica Borgese, the main goal of our laboratory is to investigate the cellular mechanisms of the pathogenicity of mutant VAPB, one of the known gene products that cause familial ALS (FALS). VAPB is an Endoplasmic Reticulum (ER) transmembrane polypeptide that functions as an adaptor, recruiting functionally important proteins, among which lipid exchange proteins, to the ER surface. In particular, VAPB plays a key role in the transport of ceramide, sterols and phosphoinositides from the ER to the Golgi complex. VAPB ligands have been implicated in the generation of membrane contact sites, membrane traffic, ER stress response and organization of the microtubule cytoskeleton.
The mutant form of VAPB, P56S-VAPB, has been linked to a dominantly inherited form of ALS (ALS8). Moreover, sporadic ALS patients have reduced levels of the endogenous protein. In view of the complex variety of VAPB functions, the mechanism of action of the P56S mutation may depend either on the loss of function of a protein with key roles in intracellular traffic and stress response or to a gain of function disease mechanism due to the toxicity of the mutant form of the protein. Aim of our work is to analyze the contribution of mutant VAPB gain or loss of function in the development of ALS.
The ALS-associated P56S mutation dramatically alters VAPB structure, thus preventing the binding to its physiological interactors and causing its aggregation. Although P56S-generated inclusions have been implicated as the cause of motor neuron toxicity, their exact role in ALS pathogenesis has not yet been elucidated. We have shown that the P56S-VAPB inclusions correspond to dramatically restructured ER domains (Fasana et al., 2010), as shown in Fig.1.
Figure 1. Left: Electronmicrograph showing the ultrastructure of ER-derived cytoplasmic inclusions in cells transiently expressing the ALS-associated mutant form of VAPB. The inset shows a higher magnification of rough (as indicated by the presence of ribosomes) N, nucleus. Bar: 200 nm. Right: Tomographic analysis of P56S-VAPB inclusions. The green structure corresponds to paired ER cisternae and shows the convoluted structure of the inclusions.
We have also reported that, at variance with most pathological inclusion bodies, P56S-VAPB inclusions are efficiently degraded by the proteasome, and not by autophagocytosis, in epithelial and motorneuronal cells. Moreover, the presence of these inclusions does not interfere with either general proteostasis or transport of the model secretory protein VSVG (Papiani et al., 2012, Genevini et al., 2014).
While carrying out this work, we developed constructs, antibodies, as well as non neuronal (HeLa) and motorneuronal (NSC34) TetOff cell lines expressing either wild-type (wt)- or P56S-VAPB under a tetracycline repressible promoter (Fig.2).
Figure 2. Screening for NSC34 clones inducibly expressing wild-type (wt, left) or P56S- (right) myc-tagged VAPB.
As the aim of our work is to dissect the different contribution of mutant VAPB gain and loss of function, we have performed VAPB-specific gene silencing experiments and established stable VAPB-downregulated NSC34 cell lines to analyze the effects of VAPB downregulation in a motorneuronal background. Interestingly, in these cells we found that both mild and strong VAPB downregulation cause a delay in neurite extension during NSC34 differentiation (Fig.3).
Figure 3. VAP-B downregulated NSC34 clones show reduced neurite extension.
The results of our studies indicate that P56S-VAPB inclusions are not toxic, while reduced levels of the endogenous VAPB can alter motorneuronal functional viability. These findings suggest that ALS8 pathogenicity is not due to toxic gain of function and that reduced levels of wild-type VAPB (haploinsufficiency) may underlie P56S-VAPB-linked ALS. This finding is relevant not only for ALS8, but also for forms of sporadic ALS, in which VAPB levels are reduced.
Fasana, E., Fossati, M., Ruggiano, A., Brambillasca, S., Hoogenraad, C.C., Navone, F., Francolini, M., and Borgese, N., 2010, A VAPB mutant linked to amyotrophic lateral sclerosis generates a novel form of organized smooth endoplasmic reticulum, The FASEB J 24: 1419-1430.
Papiani, G., Ruggiano, A., Fossati, M., Raimondi, A., Bertoni, G., Francolini, M., Benfante, R., Navone, F., and Borgese, N., 2012, Restructured endoplasmic reticulum generated by mutant amyotrophic lateral sclerosis-linked VAPB is cleared by the proteasome, J Cell Sci 125: 3601-11.
Genevini P., Papiani G., Ruggiano A., Cantoni L., Navone F., Borgese N. Amyotrophic Lateral Sclerosis-Linked Mutant VAPB Inclusions Do Not Interfere with Protein Degradation Pathways or Intracellular Transport in a Cultured Cell Mode PLoS One: 9 e113416.
Navone F., Genevini P., Borgese N. “Autophagy and neurodegeneration: insights from a cultured cell model of ALS” 2015 Cells 4: 354-86. doi:10.3390/cells4030354.