Compared to the previous VAPB transgenic study [24
], our results showed stronger and broader muVAPB aggregates, especially in motor neurons. Our muVAPB transgenic model with wide-spread aggregation formation could provide a useful tool to test the possible gain-of-toxic function due to protein misfolding and aggregation. Yet despite a systematic investigation with multiple analytic assays, we failed to find evidence for neurodegeneration or dysfunction in the transgenic mice. Although a two-fold overexpression of muVAPB may not have reached the toxic threshold, and therefore, may not be sufficient to trigger motor neuron degeneration, the failure of muVAPB to enhance mutant SOD1 toxicity diminishes the possibility of a harmful role of mutant VAPB in motor neuron degeneration. A further support for the innocuous nature of muVAPB is the absence of any pathological hallmarks, including structural changes of ER and mitochondria, evidence of UPR, reactive astrogliosis and microgliosis, redistribution of TDP-43, and activation of cellular protein quality control systems.
Our results contrast with some previous literature reports. Overexpression of muVAPB has been reported to alter ER morphology and modulate UPR in vitro
]. Our data do not support these roles in vivo
. In an analysis of a VAPBP56S transgenic mouse line, Tudor and colleagues reported a redistribution of TDP-43 from nucleus to the cytoplasm and the presence of ubiquitin- and p62-labeled structures in motor neurons [24
]. We have not confirmed their observation in our muVAPB mice. The reason of this discrepancy is not clear. A simple explanation might be that the mutant VAPB was expressed at a higher level in their mice than in the mice used in this study. Consistent in both studies, however, is the observation that overexpression of muVAPB does not cause motor neuron degeneration phenotypes. Therefore, the results from both of these studies do not support the possibility that a gain of toxicity in mutant VAPB causes motor neuron degeneration.
Common in neurodegenerative diseases are cytoplasmic protein aggregates, including mutant or the wild type proteins of SOD1 [37
], TDP-43 [39
], and FUS/TLS [42
]. However, it is not clear what roles these aggregates play in the pathogenesis of the disease. Indeed, it is controversial whether the aggregation is detrimental to cell survival. For example, neurodegeneration is still induced by ALS-related TDP-43 mutations in the absence of cytoplasmic TDP-43 aggregates, suggesting TDP-43 aggregation may not be required for pathogenesis [43
]. Furthermore, inclusion body formation might protect neurons by reducing the levels of toxic soluble forms of mutants, as has been shown in mutant huntingtin [45
]. It is possible that the neurodegeneration is not due to the presence of cytoplasmic aggregates, but by altered function of the mutant protein [46
]. In human MND caused by VAPB mutations, the cellular mechanism leading to motor neuron degeneration and muscle atrophy is not known. Although robust VAPB aggregates were formed in the muVAPB transgenic mice, especially in the motor neurons, our results have revealed that motor neurons could tolerate the VAPB aggregates without degeneration, suggesting that VAPB protein aggregation may not play a significant role in the pathogenesis.
Whether VAPB aggregation has a role in human patients with VAPB mutations is not clear. In fact, it remains to be established that VAPB cytoplasmic aggregates exist in ALS8 patients. Motor neurons derived from iPS cells from ALS8 patients with the known VAPB-P56S mutation have decreased levels of VAPB without VAPB aggregation [48
], which is consistent with the loss-of-function hypothesis. In this study, we detected no decrease in the endogenous VAPB level, nor did we attain evidence for involvement of VAPA or endogenous VAPB in the VAPB aggregates. This result does not support the possibility that the mutant VAPB exerts a dominant negative effect on the normal allele by coaggregation. Furthermore, in the presence of widespread VAPB aggregates, especially in the CNS, no neurodegeneration or motor dysfunction was observed, which does not support the toxic gain-of-function hypothesis. Therefore, future studies to determine whether a loss of function is the underlying pathogenic mechanism for the mutant VAPB is of critical importance.
Previous studies have shown that VAPB levels were reduced in sporadic ALS patients and SOD1G93A mice [12
], suggesting that VAPB dysfunction may contribute to ALS in general. We tested this possibility by overexpression of either the mutant or the wild type VAPB in SOD1G93A mice. Our results demonstrate that neither the mutant nor the wild type VAPB had any impact on the course of the disease in the SOD1G93A mice. These results do not support a general role of VAPB in ALS cases that do not have VAPB mutations.
Since protein aggregation is common in many neurodegenerative diseases, it has been proposed that the protein aggregation weakens the capacity of cellular proteostasis, thus enhancing the cell vulnerability to secondary assault(s) from another misfolding-prone protein [35
]. Our muVAPB/SOD1G93A double transgenic mice enabled us to test whether mutant SOD1 aggregation enhances mutant VAPB aggregation and vice versa in a mammalian system. Our data showed that VAPBP56S and SOD1G93A aggregate independently and do not enhance each other’s aggregation. Therefore, the synergistic protein misfolding and aggregation may not occur in all experimental systems. In addition, since VAPB aggregation did not cause motor neuron degeneration, and in particular, did not enhance mutant SOD1 toxicity, some protein aggregation may be innocuous and all protein aggregation does not inevitably lead to cellular toxicity.