Our study demonstrates that impaired retrograde transport as observed in wobbler motor neurons is characterized by the presence of enlarged endosomal vesicles with positive immunostaining for Rab7 and APP, but not for LC3. As such, the type of motor neuron degeneration as observed in WR does not involve autophagosomes. These findings contrast those for motor neurons of SOD1(G93A) mice where extensive autophagosome activation has been demonstrated [13
]. It is unlikely that the vesicular accumulation of APP is instrumental for motor neuron degeneration in the wobbler mouse, as APP transgenic mice crossed into the wobbler mutation did not enhance motor neuron pathology (Schmitt-John et al, unpublished observations).
Characteristic signs of motor neuron death in wobbler are enlarged vesicles blebbing of the Golgi apparatus, increasing in size and number in later stage as well as enlarged vesicles/vacuoles derived from the ER, in agreement with earlier EM studies performed on wobbler motor neurons [11
]. With the exception of this vacuolisation, the affected motor neuron seems to be viable just up to the terminal breakdown. The ultrastructural data indicate that the vesicular shuttle system of ER and Golgi and perhaps of lysosomes (GERL-system) is affected. This would be in agreement with the fact that in wobbler mice the vesicular transport factor VPS54, a component of the GARP complex, is mutated [6
]. In particular, vacuolisation in close proximity to the Golgi apparatus seen on electron micrographs of wobbler motor neurons reflects the function of the GARP complex involved in tethering endosome-derived retrograde vesicles to the Golgi membrane as shown in yeast [7
], but also in mammalian cells [14
Immunofluorescence data indicate an intracellular accumulation of APP in Rab7- but not LC3-positive endosomal compartments in degenerating wobbler motor neurons. Intracellular APP accumulations have earlier been reported for Niemann Pick Disease type 1 (NPC1), where APP was found in Rab5-positive early endosomes rather than in Rab7 positive late endosomes [2
]. Furthermore, Jin et al. [2
] found elevated, proteolytically processed APP (C99 and Aβ42) in NPC1 neurons. The western blotting data suggest that in wobbler spinal cords APP is upregulated but no indications for abnormal APP proteolysis have been found indicating potential amyloid accumulations. Thus, we conclude that the wobbler vesicle trafficking defect leads to an accumulation of native APP variants in abnormally enlarged APP+
The average cross-sectional diameter of these compartments is 3 μm and significantly larger than APP- and Rab7-positive structures in wild-type or SOD1 transgenic mice. The ultrastructural equivalents are vacuoles with low electron density and a size of up to 3 μm. These vacuoles most likely are derived from the Golgi and the ER. Since the occurrence of such structures is found in wobbler, but not in SOD1 and wild type mice these are likely caused by impaired bidirectional vesicle traffic between ER and Golgi. Again, these findings are in agreement with the function of Vps54 and the GARP complex as recently demonstrated [14
endosomes were also found in MDF ("muscle deficient") spinal cord motor neurons. Recently, the mutation underlying the mouse mutant MDF has been revealed as scyl1, a gene that affects Golgi transport and morphology [15
] (see additional file 1
), confirming that vesicular APP/Rab7 staining indeed reflects transport impairment in motor neurons.
Extending our analysis to human MND were able to identify APP+
vesicles in degenerating motor neurons, which could indicate that in a subset of human MND, retrograde vesicle trafficking is affected and could lead to the identification of more MND relevant gene mutations. Thus the correlation between vesicle trafficking defects and ALS is obvious since the affected gene for familial ALS2 Alsin was found to encode a guanine-nucleotide exchange factor for the vesicle traffic associated protein Rab5 [16
] and ALS8 encodes the vesicle associated membrane protein B (VAPB, [18
]). Besides these, several other vesicle trafficking proteins have been associated with neurodegeneration like Dynactin with Neuropathy distal hereditary motor type VIIB (HMN7B, [19
] and Phosphoinositide phosphatase FIG4 in patients with ALS [20
The VPS54 genes of some MND patients, preselected for enlarged APP- and LC3-positive endosomal compartments were sequenced, but so far, mutations or polymorphisms were not detected. A similar sequencing effort, although without pre-selection for APP storage, has recently been published [21
]. In this report a polymorphism in VPS54 has been identified but could not be directly associated with the disease. Thus up to now, mutations or polymorphisms in VPS54 are not defined as a major cause or a risk factor for sporadic forms of MND or ALS.