β-Amyloid impairs axonal BDNF retrograde trafficking

The neurotrophin, brain-derived neurotrophic factor (BDNF), is essential for synaptic function, plasticity and neuronal survival. At the axon terminal, when BDNF binds to its receptor, tropomyosin-related kinase B (TrkB), the signal is propagated along the axon to the cell body, via retrograde transport, regulating gene expression and neuronal function. Alzheimer disease (AD) is characterized by early impairments in synaptic function that may result in part from neurotrophin signaling deficits. Growing evidence suggests that soluble β-amyloid (Aβ) assemblies cause synaptic dysfunction by disrupting both neurotransmitter and neurotrophin signaling. Utilizing a novel microfluidic culture chamber, we demonstrate a BDNF retrograde signaling deficit in AD transgenic mouse neurons (Tg2576) that can be reversed by γ-secretase inhibitors. Using BDNF-GFP, we show that BDNF-mediated TrkB retrograde trafficking is impaired in Tg2576 axons. Furthermore, Aβ oligomers alone impair BDNF retrograde transport. Thus, Aβ reduces BDNF signaling by impairing axonal transport and this may underlie the synaptic dysfunction observed in AD.     

Loss of endogenous androgen receptor protein accelerates motor neuron degeneration and accentuates androgen insensitivity in a mouse model of X-linked spinal and bulbar muscular atrophy

X-linked spinal and bulbar muscular atrophy (SBMA; Kennedy's disease) is a polyglutamine (polyQ) disease in which the affected males suffer progressive motor neuron degeneration accompanied by signs of androgen insensitivity, such as gynecomastia and reduced fertility. SBMA is caused by CAG repeat expansions in the androgen receptor (AR) gene resulting in the production of AR protein with an extended glutamine tract. SBMA is one of nine polyQ diseases in which polyQ expansion is believed to impart a toxic gain-of-function effect upon the mutant protein, and initiate a cascade of events that culminate in neurodegeneration. However, whether loss of a disease protein's normal function concomitantly contributes to the neurodegeneration remains unanswered. To address this, we examined the role of normal AR function in SBMA by crossing a highly representative AR YAC transgenic mouse model with 100 glutamines (AR100) and a corresponding control (AR20) onto an AR null (testicular feminization; Tfm) background. Absence of endogenous AR protein in AR100Tfm mice had profound effects upon neuromuscular and endocrine-reproductive features of this SBMA mouse model, as AR100Tfm mice displayed accelerated neurodegeneration and severe androgen insensitivity in comparison to AR100 littermates. Reduction in size and number of androgen-sensitive motor neurons in the spinal cord of AR100Tfm mice underscored the importance of AR action for neuronal health and survival. Promoter-reporter assays confirmed that AR transactivation competence diminishes in a polyQ length-dependent fashion. Our studies indicate that SBMA disease pathogenesis, both in the nervous system and the periphery, involves two simultaneous pathways: gain-of-function misfolded protein toxicity and loss of normal protein function.     

JNK mediates pathogenic effects of polyglutamine-expanded androgen receptor on fast axonal transport

Expansion of the polyglutamine (polyQ) stretch in the androgen receptor (AR) protein leads to spinal and bulbar muscular atrophy (SBMA), a neurodegenerative disease characterized by lower motor neuron degeneration. The pathogenic mechanisms underlying SBMA remain unknown, but recent experiments show that inhibition of fast axonal transport (FAT) by polyQ-expanded proteins, including polyQ-AR, represents a new cytoplasmic pathogenic lesion. Using pharmacological, biochemical and cell biological experiments, we found a new pathogenic pathway that is affected in SBMA and results in compromised FAT. PolyQ-AR inhibits FAT in a human cell line and in squid axoplasm through a pathway that involves activation of cJun N-terminal kinase (JNK) activity. Active JNK phosphorylated kinesin-1 heavy chains and inhibited kinesin-1 microtubule-binding activity. JNK inhibitors prevented polyQ-AR-mediated inhibition of FAT and reversed suppression of neurite formation by polyQ-AR. We propose that JNK represents a promising target for therapeutic interventions in SBMA.     

Neuronal dropout is greater in hindlimb motor nuclei than in forelimb motor nuclei in aged rats

The number of forelimb, ulnar (U) and hindlimb, medial gastrocnemius (MG) motoneurons labeled with retrograde axonal transport of horseradish peroxidase was examined in young and aged rats. No significant difference was found between the mean number of U motoneurons in young and aged rats, whereas the mean number of MG motoneurons was significantly lower in aged rats than in young rats. These results suggest that motoneuronal dropout is greater in hindlimb motor nuclei than in forelimb motor nuclei, which may contribute to differential changes in forelimb and hindlimb muscles with increasing age.     

Spinal and bulbar muscular atrophy: ligand-dependent pathogenesis and therapeutic perspectives

Spinal and bulbar muscular atrophy (SBMA) is a late-onset motor neuron disease characterized by proximal muscle atrophy, weakness, contraction fasciculations, and bulbar involvement. SBMA exclusively affects males, while females are usually asymptomatic. The molecular basis of SBMA is the expansion of a trinucleotide CAG repeat, which encodes the polyglutamine (polyQ) tract in the first exon of the androgen receptor (AR) gene. The histopathological hallmark is the presence of nuclear inclusions containing mutant truncated ARs with expanded polyQ tracts in the residual motor neurons in the brainstem and spinal cord, as well as in some other visceral organs. The AR ligand, testosterone, accelerates AR dissociation from heat shock proteins and thus its nuclear translocation. Ligand-dependent nuclear accumulation of mutant ARs has been implicated in the pathogenesis of SBMA. Transgenic mice carrying the full-length human AR gene with an expanded polyQ tract demonstrate neuromuscular phenotypes, which are profound in males. Their SBMA-like phenotypes are rescued by castration, and aggravated by testosterone administration. Leuprorelin, an LHRH agonist that reduces testosterone release from the testis, inhibits nuclear accumulation of mutant ARs, resulting in the rescue of motor dysfunction in the male transgenic mice. However, flutamide, an androgen antagonist promoting nuclear translocation of the AR, yielded no therapeutic effect. The degradation and cleavage of the AR protein are also influenced by the ligand, contributing to the pathogenesis. Testosterone thus appears to be the key molecule in the pathogenesis of SBMA, as well as main therapeutic target of this disease.     

From the “little brain” gastrointestinal infection to the “big brain” neuroinflammation: A proposed fast axonal transport pathway involved in multiple sclerosis

The human central nervous system (CNS) is targeted by different pathogens which, apart from pathogens’ intranasal inoculation or trafficking into the brain through infected blood cells, may use a distinct pathway to bypass the blood–brain barrier by using the gastrointestinal tract (GIT) retrograde axonal transport through sensory or motor fibres. The recent findings regarding the enteric nervous system (often called the “little brain”) similarities with CNS and GIT axonal transport of infections resulting in CNS neuroinflammation are mainly reviewed in this article. We herein propose that the GIT is the vulnerable area through which pathogens (such as Helicobacter pylori) may influence the brain and induce multiple sclerosis pathologies, mainly via the fast axonal transport by the afferent neurones connecting the GIT to brain.     

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 mouse model of spinal and bulbar muscular atrophy

Spinal and bulbar muscular atrophy (SBMA) is an adult-onset motor neuron disease, caused by the expansion of a trinucleotide repeat (TNR) in exon 1 of the androgen receptor (AR) gene. This disorder is characterized by degeneration of motor and sensory neurons, proximal muscular atrophy, and endocrine abnormalities, such as gynecomastia and reduced fertility. We describe the development of a transgenic model of SBMA expressing a full-length human AR (hAR) cDNA carrying 65 (AR(65)) or 120 CAG repeats (AR(120)), with widespread expression driven by the cytomegalovirus promoter. Mice carrying the AR(120) transgene displayed behavioral and motor dysfunction, while mice carrying 65 CAG repeats showed a mild phenotype. Progressive muscle weakness and atrophy was observed in AR(120) mice and was associated with the loss of alpha-motor neurons in the spinal cord. There was no evidence of neurodegeneration in other brain structures. Motor dysfunction was observed in both male and female animals, showing that in SBMA the polyglutamine repeat expansion causes a dominant gain-of-function mutation in the AR. The male mice displayed a progressive reduction in sperm production consistent with testis defects reported in human patients. These mice represent the first model to reproduce the key features of SBMA, making them a useful resource for characterizing disease progression, and for testing therapeutic strategies for both polyglutamine and motor neuron diseases.     

Lysosomal proliferation and distal degeneration in motor neurons expressing the G59S mutation in the p150Glued subunit of dynactin

An increasing number of neurodegenerative diseases are being linked to mutations in genes encoding proteins required for axonal transport and intracellular trafficking. A mutation in p150(Glued), a component of the cytoplasmic dynein/dynactin microtubule motor complex, results in the human neurodegenerative disease distal spinal and bulbar muscular atrophy (dSBMA). We have developed a transgenic mouse model of dSBMA; these mice exhibit late-onset, slowly progressive muscle weakness but do not have a shortened lifespan, consistent with the human phenotype. Examination of motor neurons from the transgenic model reveals the proliferation of enlarged tertiary lysosomes and lipofuscin granules, indicating significant alterations in the cellular degradative pathway. In addition, we observe deficits in axonal caliber and neuromuscular junction (NMJ) integrity, indicating distal degeneration of motor neurons. However, sciatic nerve ligation studies reveal that inhibition of axonal transport is not evident in this model. Together, these data suggest that mutant p150(Glued) causes neurodegeneration in the absence of significant changes in axonal transport, and therefore other functions of dynein/dynactin, such as trafficking in the degradative pathway and stabilization of the NMJ are likely to be critical in maintaining the health of motor neurons.     

Clearance of the mutant androgen receptor in motoneuronal models of spinal and bulbar muscular atrophy

Spinal and bulbar muscular atrophy (SBMA) is an X-linked motoneuron disease caused by an abnormal expansion of a tandem CAG repeat in exon 1 of the androgen receptor (AR) gene that results in an abnormally long polyglutamine tract (polyQ) in the AR protein. As a result, the mutant AR (ARpolyQ) misfolds, forming cytoplasmic and nuclear aggregates in the affected neurons. Neurotoxicity only appears to be associated with the formation of nuclear aggregates. Thus, improved ARpolyQ cytoplasmic clearance, which indirectly decreases ARpolyQ nuclear accumulation, has beneficial effects on affected motoneurons. In addition, increased ARpolyQ clearance contributes to maintenance of motoneuron proteostasis and viability, preventing the blockage of the proteasome and autophagy pathways that might play a role in the neuropathy in SBMA. The expression of heat shock protein B8 (HspB8), a member of the small heat shock protein family, is highly induced in surviving motoneurons of patients affected by motoneuron diseases, where it seems to participate in the stress response aimed at cell protection. We report here that HspB8 facilitates the autophagic removal of misfolded aggregating species of ARpolyQ. In addition, though HspB8 does not influence p62 and LC3 (two key autophagic molecules) expression, it does prevent p62 bodies formation, and restores the normal autophagic flux in these cells. Interestingly, trehalose, a well-known autophagy stimulator, induces HspB8 expression, suggesting that HspB8 might act as one of the molecular mediators of the proautophagic activity of trehalose. Collectively, these data support the hypothesis that treatments aimed at restoring a normal autophagic flux that result in the more efficient clearance of mutant ARpolyQ might produce beneficial effects in SBMA patients.     

Cleavage, aggregation and toxicity of the expanded androgen receptor in spinal and bulbar muscular atrophy

Spinal and bulbar muscular atrophy (SBMA) is a neurodegenerative disease caused by the expansion of a polyglutamine repeat within the androgen receptor (AR). We have studied the mutant AR in an in vitro system, and find both aggregation and proteolytic processing of the AR protein to occur in a polyglutamine repeat length-dependent manner. In addition, we find the aberrant metabolism of expanded repeat AR to be coupled to cellular toxicity, indicating a likely molecular basis for the toxic gain of AR function that produces neuronal degeneration in SBMA.      

Fast axonal transport of acetylcholinesterase in rat sciatic motoneurons is enhanced following prolonged daily running, but not following swimming

The effects of increases in neuronal activity on fast axonal transport of acetylcholinesterase (AChE) in sciatic motoneurons were studied by subjecting rats to daily running or swimming training (8 weeks). Net accumulation of AChE activity proximal and distal to a ligature served to evaluate orthograde and retrograde transport. Results showed that runners had greater orthograde and retrograde transport of AChE as compared to control animals, while no changes were found in swimmers. These adaptations in the runners were caused by the long-term nature of the training regimen since an acute exercise session had no effect on AChE transport. The observed changes may be attributed to an increase in the mobile fraction of AChE in the motoneurons. Since swimming training had no effect on transport but entails a high level of neuronal activity, it is suggested that increased impulse activity is not the factor mediating the adaptations in axonal transport of AChE which resulted from running training.     

Motoneuronal cell death is not correlated with aggregate formation of androgen receptors containing an elongated polyglutamine tract

Spinal and bulbar muscular atrophy (SBMA) is associated with an abnormal expansion of the (CAG)(n)repeat in the androgen receptor (AR) gene. Similar mutations have been reported in other proteins that cause neurodegenerative disorders. The CAG-coded elongated polyglutamine (polyGln) tracts induce the formation of neuronal intracellular aggregates. We have produced a model to study the effects of potentially 'neurotoxic' aggregates in SBMA using immortalized motoneuronal cells (NSC34) transfected with AR containing polyGln repeats of different sizes [(AR.Q(n = 0, 23 or 46)]. Using chimeras of AR.Q(n) and the green fluorescent protein (GFP), we have shown that aggregate formation occurs when the polyGln tract is elongated and AR is activated by androgens. In NSC34 cells co-expressing the AR with the polyGln of pathological length (AR.Q46) and the GFP we have noted the presence of several dystrophic neurites. Cell viability analyses have shown a reduced growth/survival rate in NSC34 expressing the AR.Q46, whereas testosterone treatment partially counteracted both cell death and the formation of dystrophic neurites. These observations indicate the lack of correlation between aggregate formation and cell survival, and suggest that neuronal degeneration in SBMA might be secondary to axonal/dendritic insults.     

Auditory development in progressive motor neuronopathy mouse mutants

The present study was performed to elucidate the hearing development in the progressive motor neuronopathy (pmn) mouse mutant. This mouse has been used as a model for human motoneuron disease. A missense mutation in the tubulin-specific chaperon E (Tbce) gene on mouse chromosome 13 was localized as the underlying genetic defect. The protein encoded by the Tbce gene is essential for the formation of primary tubulin complexes. Studies on motoneurons show disorganization in microtubules and disturbed axonal transport, followed by retrograde degeneration of the motoneurons. A similar pathomechanism is also possible for hearing disorders where disrupted microtubules could cause functional deficits in spiral ganglion neurons or in cochlear hair cells. Click auditory brainstem response (ABR) audiometry in homozygous pmn mutants showed a normal onset of hearing, but an increasing hearing threshold from postnatal day 26 (P26) on to death, compared to heterozygous mutants and wild-type mice. Histological sections of the cochlea at different ages showed a regular morphology. Additionally, spiral ganglion explants from mutant and wild-type mice were cultured. The neurite length from pmn mutants was shorter than in wild-type mice, and the neurite number/explant was significantly decreased in pmn mutants. We show that the pmn mouse mutant is a model for a progressive rapid hearing loss from P26 on, after initially normal hearing development. Heterozygous mice are not affected by this defect. With the knowledge of the well-known pathomechanism of this defect in motoneurons, a dysfunction of cellular mechanisms regulating tubulin assembling suggests that tubulin assembling plays an essential role in hearing function and maintenance.     

Age-dependent axonal degeneration in an Alzheimer mouse model

Some neurodegenerative diseases including Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS) exhibit prominent defects in axonal transport. These defects can manifest as axonal swellings or spheroids, which correspond to axonal enlargements and aberrant accumulation of axonal cargoes, cytoskeletal proteins and lipids. Recently, a controversial scientific debate focussed on the issue whether Abeta serves as a trigger for aberrant axonal transport in the pathophysiology of AD. Prominent axonopathy has been shown to be induced by overexpression of proteins involved in several neurodegenerative diseases. Neurofilament, apolipoprotein E, Niemann-Pick protein and Tau transgenic mice with axonal trafficking deficits have been reported. Furthermore, motor deficits are frequently observed in patients with AD, which has been attributed to the typical tauopathy in post-mortem brain tissue. In the present report, we analyzed axonal neuropathology in the brain and spinal cord of a transgenic mouse model with abundant intraneuronal Abeta42 production and provide compelling evidence for axonal degeneration. The APP/PS1ki mice showed characteristic axonal swellings, spheroids, axonal demyelination and ovoids, which are myelin remnants of degenerated nerve fibers in an age-dependent manner. Abundant accumulation of intraneuronal N-modified Abeta, Thioflavin S-positive material and ubiquitin was found within the somatodendritic compartment of neurons. We conclude that the intraneuronal accumulation of Abeta-amyloid peptides is followed by axonal degeneration, and thus might be a causative factor for the axonal changes seen in AD.     

Identification of different size motoneurons labeled by the retrograde axonal transport of horseradish peroxidase

Summary Three main groups of motoneurons of different size have been labeled in adult cats by using the method for retrograde axonal transport following injection of horseradish peroxidase in the medial gastrocnemius and soleus muscles. In particular small, medium-size and large neurons which probably correspond respectively to gamma, small alpha and large alpha motoneurons innervating the calf muscles, have been identified and the corresponding area measured.