A proline to serine substitution at position 56 in the gene encoding vesicle-associated membrane protein-associated protein B (VAPB) causes some dominantly inherited familial forms of motor neuron disease including amyotrophic lateral sclerosis (ALS) type-8. VAPB is an integral endoplasmic reticulum (ER) protein whose amino-terminus projects into the cytosol. Overexpression of ALS mutant VAPBP56S disrupts ER structure but the mechanisms by which it induces disease are not properly understood. Here we show that VAPB interacts with the outer mitochondrial membrane protein, protein tyrosine phosphatase-interacting protein 51 (PTPIP51). ER and mitochondria are both stores for intracellular calcium (Ca2+) and Ca2+ exchange between these organelles occurs at regions of ER that are closely apposed to mitochondria. These are termed mitochondria-associated membranes (MAM). We demonstrate that VAPB is a MAM protein and that loss of either VAPB or PTPIP51 perturbs uptake of Ca2+ by mitochondria following release from ER stores. Finally, we demonstrate that VAPBP56S has altered binding to PTPIP51 and increases Ca2+ uptake by mitochondria following release from ER stores. Damage to ER, mitochondria and Ca2+ homeostasis are all seen in ALS and we discuss the implications of our findings in this context.

Amyotrophic lateral sclerosis (ALS) is a late-onset neurological disorder characterized by death of motoneurons. Mutations in Cu/Zn superoxide dismutase-1 (SOD1) cause familial ALS but the mechanisms whereby they induce disease are not fully understood. Here, we use time-lapse microscopy to monitor for the first time the effect of mutant SOD1 on fast axonal transport (FAT) of bona fide cargoes in living neurons. We analyzed FAT of mitochondria that are a known target for damage by mutant SOD1 and also of membrane-bound organelles (MBOs) using EGFP-tagged amyloid precursor protein as a marker. We studied FAT in motor neurons derived from SOD1G93A transgenic mice that are a model of ALS and also in cortical neurons transfected with SOD1G93A and three further ALS-associated SOD1 mutants. We find that mutant SOD1 damages transport of both mitochondria and MBOs, and that the precise details of this damage are cargo-specific. Thus, mutant SOD1 reduces transport of MBOs in both anterograde and retrograde directions, whereas mitochondrial transport is selectively reduced in the anterograde direction. Analyses of the characteristics of mitochondrial FAT revealed that reduced anterograde movement involved defects in anterograde motor function. The selective inhibition of anterograde mitochondrial FAT enhanced their net retrograde movement to deplete mitochondria in axons. Mitochondria in mutant SOD1 expressing cells also displayed features of damage. Together, such changes to mitochondrial function and distribution are likely to compromise axonal function. These alterations represent some of the earliest pathological features so far reported in neurons of mutant SOD1 transgenic mice.

Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) are relentlessly progressive neurodegenerative disorders with overlapping clinical, genetic and pathological features. Cytoplasmic inclusions of fused in sarcoma (FUS) are the hallmark of several forms of FTLD and ALS patients with mutations in the FUS gene. FUS is a multifunctional, predominantly nuclear, DNA and RNA binding protein. Here, we report that transgenic mice overexpressing wild-type human FUS develop an aggressive phenotype with an early onset tremor followed by progressive hind limb paralysis and death by 12 weeks in homozygous animals. Large motor neurons were lost from the spinal cord accompanied by neurophysiological evidence of denervation and focal muscle atrophy. Surviving motor neurons in the spinal cord had greatly increased cytoplasmic expression of FUS, with globular and skein-like FUS-positive and ubiquitin-negative inclusions associated with astroglial and microglial reactivity. Cytoplasmic FUS inclusions were also detected in the brain of transgenic mice without apparent neuronal loss and little astroglial or microglial activation. Hemizygous FUS overexpressing mice showed no evidence of a motor phenotype or pathology. These findings recapitulate several pathological features seen in human ALS and FTLD patients, and suggest that overexpression of wild-type FUS in vulnerable neurons may be one of the root causes of disease. Furthermore, these mice will provide a new model to study disease mechanism, and test therapies.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-012-1043-z) contains supplementary material, which is available to authorized users.

FE65 is an adaptor protein that binds to and forms a transcriptionally active complex with the ␥-secretase-derived amyloid precursor protein (APP) intracellular domain. The regulatory mechanisms of FE65-APP-mediated transcription are still not clear. In this report, we demonstrate that Dexras1, a Ras family small G protein, binds to FE65 PTB2 domain and potently suppresses the FE65-APP-mediated transcription. The suppression is not via competition for binding of FE65 between Dexras1 and APP because the two proteins can simultaneously bind to the FE65 PTB2 domain. Phosphorylation of FE65 tyrosine 547 within the PTB2 domain has been shown to enhance FE65-APPmediated transcription but not to influence binding to APP. Here we find that this phosphorylation event reduces the binding between Dexras1 and FE65. We also demonstrate that Dexras1 inhibits the FE65-APP-mediated transcription of glycogen synthase kinase 3␤ (GSK3␤). Moreover, small interfering RNA knockdown of Dexras1 enhances GSK3␤ expression and increases phosphorylation of Tau, a GSK3␤ substrate. Thus, Dexras1 functions as a suppressor of FE65-APP-mediated transcription, and FE65 tyrosine 547 phosphorylation enhances FE65-APP-mediated transcription, at least in part, by modulating the interaction between FE65 and Dexras1. These findings reveal a novel regulatory mechanism for FE65-APP-mediated signaling.FE65 is an adaptor protein with multiple protein-protein interaction domains including a WW domain and two C-terminal PTB domains (1). It is believed that FE65 functions as a "scaffold" protein to recruit various binding partners together to form a functional complex. In fact, FE65 has been shown to interact with a number of proteins. For example, transcription factors CP2 and Tip60 interact with the FE65 PTB1 (2, 3), c-Abl tyrosine kinase and Mena are FE65 WW domain binding partners (4, 5) and the nucleosome assembly factor SET binds FE65 (6). Of great interest, the Alzheimer disease amyloid precursor protein (APP) 2 has been shown to interact with the FE65 PTB2 domain (7-10).APP is a ubiquitously expressed type I integral transmembrane protein with a large ectodomain and a short intracellular domain (11,12). The functions of APP are not properly understood. However, APP is known to be processed first by either ␣-or ␤-secretase and then by ␥-secretase. Cleavage of APP by ␤-and ␥-secretases results in the generation of the 4-kDa ␤-amyloid peptide (A␤). Aggregation of A␤ to form neuritic plaques in brains is a pathological hallmark of Alzheimer disease (for reviews, see Refs. 1 and 13). FE65 has been shown to modulate the production of A␤ (14 -16). In addition to A␤ generation, cleavage of the APP by ␥-secretase releases the APP intracellular domain (AICD). Importantly, AICD has been shown to translocate to the nucleus as a complex with FE65 and the FE65⅐AICD complex strongly stimulates transcription of a GAL4-dependent reporter system (3,(17)(18)(19)(20). However, the control mechanism(s) of FE65-AICD nuclear transcription is currently unclear.Nuclear trans...

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Rac and its downstream effectors p21-activated kinase (PAK) family kinases regulate actin dynamics within growth cones to control neurite outgrowth during development. The activity of Rac is stimulated by guanine nucleotide exchange factors (GEFs) that promote GDP release and GTP binding. ALS2/Alsin is a recently described GEF that contains a central domain that is predicted to regulate the activities of Rac and/or Rho and Cdc42 activities. Mutations in ALS2 cause some recessive familial forms of amyotrophic lateral sclerosis (ALS) but the function of ALS2 is poorly understood. Here we demonstrate that ALS2 is present within growth cones of neurons, in which it co-localizes with Rac. Furthermore, ALS2 stimulates Rac but not Rho or Cdc42 activities, and this induces a corresponding increase in PAK1 activity. Finally, we demonstrate that ALS2 promotes neurite outgrowth. Defects in these functions may therefore contribute to motor neuron demise in ALS.

Primary lateral sclerosis (PLS) is a rare progressive paralytic disorder that results from dysfunction of the upper motoneurons. Although PLS is a sporadic disorder of adult middle age, it has also been described in children as juvenile PLS or JPLS. The causative gene for JPLS was found to be ALS2, which is also responsible for a recessive form of amyotrophic lateral sclerosis, for infantile onset ascending hereditary spastic paralysis (IAHSP) and for a form of complicated hereditary spastic paraplegia (cHSP). ALS2 gene encodes a protein termed alsin, containing multiple guanine nucleotide exchange factor domains, specifically binding to small GTPase Rab5 and acting as a GEF for Rab5. In vitrostudies performed with full-length and truncating forms of alsin protein support its role in endosomal dynamics and trafficking of mitochondria. All ALS2 mutations so far reported generate alsin protein truncation. Here, we describe the first homozygous missense mutation in ALS2, p.G540E. The mutation, which falls within the RCC1 domain, was identified in a 34-year-old patient with typical signs of JPLS such as ascending generalized and severe spasticity involving the limbs and the bulbar region, dysphagia, limb atrophy, preserved cognition and sensation. The father and two proband's sisters were found to be heterozygous carriers of the mutation with no signs of the disease. Studies in the neuronal cell line SK-N-BE indicated that the known subcellular localization of wild-type alsin with the early endosome antigen 1, in enlarged endosomal structures, and transferrin receptor is completely lost by the mutant protein, thus indicating that this mutation leads to protein delocalization. Mutant alsin induced neuronal death itself and also significantly enhanced the apoptogenic effect of NMDA and staurosporine. This effect was associated with decreased Bcl-xL : Bax ratio. In contrast, wild-type alsin was neuroprotective and increased Bcl-xL : Bax ratio. Our results provide the first demonstration that a missense mutation in alsin is cytotoxic. In addition, the identification of Bcl-xL/Bax as target of protection by alsin and of cytotoxicity by the mutant form provides a new signalling event regulated by alsin protein that may be important to define its role in neuronal physiology and neurodegeneration. Finally, the phenotype-genotype correlation in our patient, in view of all other ALS2 mutant cases reported previously, suggests a functional interplay of long and short forms of alsin in relation to disease onset and progression.

Neurofilaments are synthesised in neuronal cell bodies and then transported through axons. Damage to neurofilament transport is seen in amyotrophic lateral sclerosis (ALS). Here, we show that PKN1, a neurofilament head-rod domain kinase is cleaved and activated in SOD1G93A transgenic mice that are a model of ALS. Moreover, we demonstrate that glutamate, a proposed toxic mechanism in ALS leads to caspase cleavage and disruption of PKN1 in neurons. Finally, we demonstrate that a cleaved form of PKN1 but not wild-type PKN1 disrupts neurofilament organisation and axonal transport. Thus, deregulation of PKN1 may contribute to the pathogenic process in ALS.