Amyotrophic lateral sclerosis-associated mutant VAPBP56S perturbs calcium homeostasis to disrupt axonal transport of mitochondria.

A proline-to-serine substitution at position 56 in the gene encoding vesicle-associated membrane protein-associated protein B (VAPB; VAPBP56S) causes some dominantly inherited familial forms of motor neuron disease, including amyotrophic lateral sclerosis (ALS) type-8. Here, we show that expression of ALS mutant VAPBP56S but not wild-type VAPB in neurons selectively disrupts anterograde axonal transport of mitochondria. VAPBP56S-induced disruption of mitochondrial transport involved reductions in the frequency, velocity and persistence of anterograde mitochondrial movement. Anterograde axonal transport of mitochondria is mediated by the microtubule-based molecular motor kinesin-1. Attachment of kinesin-1 to mitochondria involves the outer mitochondrial membrane protein mitochondrial Rho GTPase-1 (Miro1) which acts as a sensor for cytosolic calcium levels ([Ca(2+)]c); elevated [Ca(2+)]c disrupts mitochondrial transport via an effect on Miro1. To gain insight into the mechanisms underlying the VAPBP56S effect on mitochondrial transport, we monitored [Ca(2+)]c levels in VAPBP56S-expressing neurons. Expression of VAPBP56S but not VAPB increased resting [Ca(2+)]c and this was associated with a reduction in the amounts of tubulin but not kinesin-1 that were associated with Miro1. Moreover, expression of a Ca(2+) insensitive mutant of Miro1 rescued defective mitochondrial axonal transport and restored the amounts of tubulin associated with the Miro1/kinesin-1 complex to normal in VAPBP56S-expressing cells. Our results suggest that ALS mutant VAPBP56S perturbs anterograde mitochondrial axonal transport by disrupting Ca(2+) homeostasis and effecting the Miro1/kinesin-1 interaction with tubulin.

VAPB interacts with the mitochondrial protein PTPIP51 to regulate calcium homeostasis.

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 (Ca(2+)) and Ca(2+) 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 Ca(2+) by mitochondria following release from ER stores. Finally, we demonstrate that VAPBP56S has altered binding to PTPIP51 and increases Ca(2+) uptake by mitochondria following release from ER stores. Damage to ER, mitochondria and Ca(2+) homeostasis are all seen in ALS and we discuss the implications of our findings in this context.

Dismutase-competent SOD1 mutant accumulation in myelinating Schwann cells is not detrimental to normal or transgenic ALS model mice.

Mutant superoxide dismutase 1 (SOD1) action within non-neuronal cells is implicated in damage to spinal motor neurons in a genetic form of amyotrophic lateral sclerosis (ALS). Central nervous system glial cells such as astrocytes and microglia drive progression in transgenic mutant SOD1 mice, however, the role of myelinating glia remains unclear. Specifically, peripheral myelinating glial cells are likely candidates for mediating degeneration of distal synapses and axons of motor neurons in ALS. Here, we examine the potential contribution of peripheral axon ensheathing Schwann cells to ALS by constructing transgenic mice expressing dismutase active mutant SOD1(G93A) driven by the myelin protein zero (P0) promoter. In this model, mutant SOD1 accumulation in Schwann cells was comparable to levels in mice ubiquitously expressing a SOD1(G93A) transgene that become paralysed. Growth, locomotion and survival of these P0-SOD1(G93A) mice were indistinguishable from normal animals. There was no evidence for spinal motor neuron loss, distal axonal degeneration and p75 neurotrophin receptor (p75(NTR)) upregulation in the periphery of P0-SOD1(G93A) mice, unlike transgenic SOD1(G93A) mice with presymptomatic p75(NTR) induction and death-signalling. Furthermore, Schwann cells were resistant to mutant SOD1 aggregation in vivo and in transfected primary cultures. Increasing mutant SOD1 synthesis in Schwann cells by cross-breeding transgenic P0-SOD1(G93A) and SOD1(G93A) mice did not affect disease onset or survival. We conclude that dismutase-competent mutant SOD1 accumulation within Schwann cells is not pathological to spinal motor neurons or deleterious to disease course in transgenic ALS model mice, in contrast to astrocytes and microglia.

Motor neuron disease occurring in a mutant dynactin mouse model is characterized by defects in vesicular trafficking.

Amyotrophic lateral sclerosis (ALS), a fatal and progressive neurodegenerative disorder characterized by weakness, muscle atrophy, and spasticity, is the most common adult-onset motor neuron disease. Although the majority of ALS cases are sporadic, approximately 5-10% are familial, including those linked to mutations in SOD1 (Cu/Zn superoxide dismutase). Missense mutations in a dynactin gene (DCTN1) encoding the p150(Glued) subunit of dynactin have been linked to both familial and sporadic ALS. To determine the molecular mechanism whereby mutant dynactin p150(Glued) causes selective degeneration of motor neurons, we generated and characterized mice expressing either wild-type or mutant human dynactin p150(Glued). Neuronal expression of mutant, but not wild type, dynactin p150(Glued) causes motor neuron disease in these animals that are characterized by defects in vesicular transport in cell bodies of motor neurons, axonal swelling and axo-terminal degeneration. Importantly, we provide evidence that autophagic cell death is implicated in the pathogenesis of mutant p150(Glued) mice. This novel mouse model will be instrumental for not only clarifying disease mechanisms in ALS, but also for testing therapeutic strategies to ameliorate this devastating disease.

Neurofilament heavy chain side arm phosphorylation regulates axonal transport of neurofilaments.

Neurofilaments possess side arms that comprise the carboxy-terminal domains of neurofilament middle and heavy chains (NFM and NFH); that of NFH is heavily phosphorylated in axons. Here, we demonstrate that phosphorylation of NFH side arms is a mechanism for regulating transport of neurofilaments through axons. Mutants in which known NFH phosphorylation sites were mutated to preclude phosphorylation or mimic permanent phosphorylation display altered rates of transport in a bulk transport assay. Similarly, application of roscovitine, an inhibitor of the NFH side arm kinase Cdk5/p35, accelerates neurofilament transport. Analyses of neurofilament movement in transfected living neurons demonstrated that a mutant mimicking permanent phosphorylation spent a higher proportion of time pausing than one that could not be phosphorylated. Thus, phosphorylation of NFH slows neurofilament transport, and this is due to increased pausing in neurofilament movement.

Identification of a novel, membrane-associated neuronal kinase, cyclin-dependent kinase 5/p35-regulated kinase.

Here we characterize a novel neuronal kinase, cyclin-dependent kinase 5 (cdk5)/p35-regulated kinase (cprk). Cprk is a member of a previously undescribed family of kinases that are predicted to contain two N-terminal membrane-spanning domains and a long C terminus, which harbors a dual-specificity serine/threonine/tyrosine kinase domain. Cprk was isolated in a yeast two-hybrid screen using the neuronal cdk5 activator p35 as "bait." Cprk interacts with p35 in the yeast-two hybrid system, binds to p35 in glutathione S-transferase fusion pull-down assays, and colocalizes with p35 in cultured neurons and transfected cells. In these cells, cprk is present with p35 in the Golgi apparatus. Cprk is expressed in a number of tissues but is enriched in brain and muscle and within the brain is found in a wide range of neuronal populations. Cprk displays catalytic activity in in vitro kinase assays and is itself phosphorylated by cdk5/p35. Cdk5/p35 inhibits cprk activity. Cdk5/p35 may therefore regulate cprk function in the brain.

Role of axonal transport in neurodegenerative diseases.

Many major human neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis (ALS), display axonal pathologies including abnormal accumulations of proteins and organelles. Such pathologies highlight damage to the axon as part of the pathogenic process and, in particular, damage to transport of cargoes through axons. Indeed, we now know that disruption of axonal transport is an early and perhaps causative event in many of these diseases. Here, we review the role of axonal transport in neurodegenerative disease.

TDP-43 mutations in familial and sporadic amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disorder characterized pathologically by ubiquitinated TAR DNA binding protein (TDP-43) inclusions. The function of TDP-43 in the nervous system is uncertain, and a mechanistic role in neurodegeneration remains speculative. We identified neighboring mutations in a highly conserved region of TARDBP in sporadic and familial ALS cases. TARDBPM337V segregated with disease within one kindred and a genome-wide scan confirmed that linkage was restricted to chromosome 1p36, which contains the TARDBP locus. Mutant forms of TDP-43 fragmented in vitro more readily than wild type and, in vivo, caused neural apoptosis and developmental delay in the chick embryo. Our evidence suggests a pathophysiological link between TDP-43 and ALS.

Familial amyotrophic lateral sclerosis-linked SOD1 mutants perturb fast axonal transport to reduce axonal mitochondria content.

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.

A mutation in the small heat-shock protein HSPB1 leading to distal hereditary motor neuronopathy disrupts neurofilament assembly and the axonal transport of specific cellular cargoes.

Distal hereditary motor neuronopathies (dHMNs) are a clinically and genetically heterogeneous group of disorders in which motor neurons selectively undergo age-dependant degeneration. Mutations in the small heat-shock protein HSPB1 (HSP27) are responsible for one form of dHMN. In this study, we have analysed the effect of expressing a form of mutant HSPB1 in primary neuronal cells in culture. Mutant (P182L) but not wild-type HSPB1 led to the formation of insoluble intracellular aggregates and to the sequestration in the cytoplasm of selective cellular components, including neurofilament middle chain subunit (NF-M) and p150 dynactin. These findings suggest a possible pathogenic mechanism for HSPB1 whereby the mutation may lead to preferential motor neuron loss by disrupting selective components essential for axonal structure and transport.

ALS2/Alsin regulates Rac-PAK signaling and neurite outgrowth.

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.

Charcot-Marie-Tooth disease neurofilament mutations disrupt neurofilament assembly and axonal transport.

Charcot-Marie-Tooth disease (CMT) is the most common inherited disorder of the peripheral nervous system, and mutations in neurofilaments have been linked to some forms of CMT. Neurofilaments are the major intermediate filaments of neurones, but the mechanisms by which the CMT mutations induce disease are not known. Here, we demonstrate that CMT mutant neurofilaments disrupt both neurofilament assembly and axonal transport of neurofilaments in cultured mammalian cells and neurones. We also show that CMT mutant neurofilaments perturb the localization of mitochondria in neurones. Accumulations of neurofilaments are a pathological feature of several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Parkinson's disease, dementia with Lewy bodies, and diabetic neuropathy. Our results demonstrate that aberrant neurofilament assembly and transport can induce neurological disease, and further implicate defective neurofilament metabolism in the pathogenesis of human neurodegenerative diseases.

Parkinson's disease alpha-synuclein mutations exhibit defective axonal transport in cultured neurons.

Alpha-synuclein is a major protein constituent of Lewy bodies and mutations in alpha-synuclein cause familial autosomal dominant Parkinson's disease. One explanation for the formation of perikaryal and neuritic aggregates of alpha-synuclein, which is a presynaptic protein, is that the mutations disrupt alpha-synuclein transport and lead to its proximal accumulation. We found that mutant forms of alpha-synuclein, either associated with Parkinson's disease (A30P or A53T) or mimicking defined serine, but not tyrosine, phosphorylation states exhibit reduced axonal transport following transfection into cultured neurons. Furthermore, transfection of A30P, but not wild-type, alpha-synuclein results in accumulation of the protein proximal to the cell body. We propose that the reduced axonal transport exhibited by the Parkinson's disease-associated alpha-synuclein mutants examined in this study might contribute to perikaryal accumulation of alpha-synuclein and hence Lewy body formation and neuritic abnormalities in diseased brain.

p38alpha stress-activated protein kinase phosphorylates neurofilaments and is associated with neurofilament pathology in amyotrophic lateral sclerosis.

Neurofilament middle and heavy chains (NFM and NFH) are heavily phosphorylated on their carboxy-terminal side-arm domains in axons. The mechanisms that regulate this phosphorylation are complex. Here, we demonstrate that p38alpha, a member of the stress-activated protein kinase family, will phosphorylate NFM and NFH on their side-arm domains. Aberrant accumulations of neurofilaments containing phosphorylated NFM and NFH side-arms are a pathological feature of amyotrophic lateral sclerosis (ALS) and we also demonstrate that p38alpha and active forms of p38 family kinases are associated with these accumulations. This is the case for sporadic and familial forms of ALS and also in a transgenic mouse model of ALS caused by expression of mutant superoxide dismutase-1 (SOD1). Thus, p38 kinases may contribute to the aberrant phosphorylation of NFM and NFH side-arms in ALS.