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The vesicle-associated membrane protein (VAMP) associated protein B (VAPB) is an integral membrane protein localized to the endoplasmic reticulum (ER). The P56S mutation in VAPB has been linked to motor neuron degeneration in amyotrophic lateral sclerosis type 8 (ALS8) and forms ER-like inclusions in various model systems. However, the role of wild-type and mutant VAPB in neurons is poorly understood. Here, we identified Yip1-interacting factor homologue A (YIF1A) as a new VAPB binding partner and important component in the early secretory pathway. YIF1A interacts with VAPB via its transmembrane regions, recycles between the ER and Golgi and is mainly localized to the ER-Golgi intermediate compartments (ERGICs) in rat hippocampal neurons. VAPB strongly affects the distribution of YIF1A and is required for intracellular membrane trafficking into dendrites and normal dendritic morphology. When VAPB-P56S is present, YIF1A is recruited to the VAPB-P56S clusters and loses its ERGIC localization. These data suggest that both VAPB and YIF1A are important for ER-to-Golgi transport and that missorting of YIF1A may contribute to VAPB-associated motor neuron disease.

Mutations in superoxide dismutase (SOD1) cause amyotrophic lateral sclerosis (ALS), an adult-onset progressive paralytic disease characterized by loss of motor neurons, and cause an ALS-like disease when expressed in mice. Recent data have suggested that motor neuron degeneration results from toxic actions of mutant SOD1 operating in both motor neurons and their neighboring glia, raising the question whether mutant SOD1 expression selectively in neurons is sufficient to induce disease. Here we show that neuronal expression of mutant SOD1 is sufficient to cause motor neuron degeneration and paralysis in transgenic mice with cytosolic dendritic ubiquitinated SOD1 aggregates as the dominant pathological feature. In addition, we show that crossing our neuron-specific mutant SOD1 mice with ubiquitously wild-type SOD1-expressing mice leads to dramatic wild-type SOD1 aggregation in oligodendroglia after the onset of neuronal degeneration. Together, our findings support a pathogenic scenario in which mutant SOD1 in neurons triggers neuronal degeneration, which in turn may facilitate aggregate formation in surrounding glial cells.

To obtain insight into the morphological and molecular correlates of motoneuron degeneration in amyotrophic lateral sclerosis (ALS) mice that express G93A mutant superoxide dismutase (SOD)1 (G93A mice), we have mapped and characterized 'sick' motoneurons labelled by the 'stress transcription factors' ATF3 and phospho-c-Jun. Immunocytochemistry and in situ hybridization showed that a subset of motoneurons express ATF3 from a relatively early phase of disease before the onset of active caspase 3 expression and motoneuron loss. The highest number of ATF3-expressing motoneurons occurred at symptom onset. The onset of ATF3 expression correlated with the appearance of ubiquitinated neurites. Confocal double-labelling immunofluorescence showed that all ATF3-positive motoneurons were immunoreactive for phosphorylated c-Jun. Furthermore, the majority of ATF3 and phospho-c-Jun-positive motoneurons were also immunoreactive for CHOP (GADD153) and showed Golgi fragmentation. A subset of ATF3 and phosphorylated c-Jun-immunoreactive motoneurons showed an abnormal appearance characterized by a number of distinctive features, including an eccentric flattened nucleus, perikaryal accumulation of ubiquitin immunoreactivity, juxta-nuclear accumulation of the Golgi apparatus and the endoplasmic reticulum, and intense Hsp70 immunoreactivity. These abnormal cells were not immunoreactive for active caspase 3. We conclude that motoneurons in ALS-SOD1 mice prior to their death and disappearance experience a prolonged sick phase, characterized by the gradual accumulation of ubiquitinated material first in the neurites and subsequently the cell body.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative condition characterized by progressive motor neuron degeneration and muscle paralysis. Genetic evidence from man and mouse has indicated that mutations in the dynein/dynactin motor complex are correlated with motor neuron degeneration. In this study, we have generated transgenic mice with neuron-specific expression of Bicaudal D2 N-terminus (BICD2-N) to chronically impair dynein/dynactin function. Motor neurons expressing BICD2-N showed accumulation of dynein and dynactin in the cell body, Golgi fragmentation and several signs of impaired retrograde trafficking: the appearance of giant neurofilament swellings in the proximal axon, reduced retrograde labelling by tracer injected in the muscle and delayed expression of the injury transcription factor ATF3 after axon transection. Despite these abnormalities, BICD2-N mice did not develop signs of motor neuron degeneration and motor abnormalities. Interestingly, the BICD2-N transgene increased lifespan in 'low copy' SOD1-G93A ALS transgenic mice. Our findings indicate that impaired dynein/dynactin function can explain several pathological features observed in ALS patients, but may be beneficial in some forms of ALS.

The vesicle-associated membrane protein-associated proteins (VAPs) VAPA and VAPB interact with lipid-binding proteins carrying a short motif containing two phenylalanines in an acidic tract (FFAT motif) and targets them to the cytosolic surface of the endoplasmic reticulum (ER). A genetic mutation (P56S) in the conserved major sperm protein homology domain of VAPB has been linked to motor-neuron degeneration in affected amyotrophic lateral sclerosis (ALS) patients. We report that in the CNS, VAPB is abundant in motor neurons and that the P56S substitution causes aggregation of mutant VAPB in immobile tubular ER clusters, perturbs FFAT-motif binding, and traps endogenous VAP in mutant aggregates. Expression of mutant VAPB or reduction of VAP by short hairpin RNA in primary neurons causes Golgi dispersion and cell death. VAPA and VAPB are reduced in human ALS patients and superoxide dismutase 1 (SOD1)-ALS-transgenic mice, suggesting that VAP family proteins may be involved in the pathogenesis of sporadic and SOD1-linked ALS. Our data support a model in which reduced levels of VAP family proteins result in decreased ER anchoring of lipid-binding proteins and cause motor neuron degeneration.