Motor Neuron Cell Bodies Research Articles

202: Interferon regulator factor (IRF) 8 regulates the microglial response to neuronal injury

The transcription factor interferon regulatory factor (IRF) 8 has a key role in the cellular response to IFN-γ and is involved in myeloid cell differentiation. We have previously identified IRF8 to be a constitutive and IFN-γ-stimulated nuclear factor that regulates the homeostatic properties of microglia. Our study aimed to determine the role of IRF8 in the microglial response to sterile neuronal injury. Facial nerve axotomy (FNA) was performed in wildtype (WT) and IRF8 −/− (KO) mice and the brains removed at different times post-lesion. A subset of mice was injected with bromodeoxyuridine (BrdU) prior to retrieval of the brain. Changes in the facial nucleus (FN) were examined by immunohistochemistry and histochemistry. In brains from IRF8KO mice, nucleoside diphosphatase (NDPase) hisotchemistry revealed gross alterations in the morphology of microglia, which were stunted and hypertrophied. After FNA in WT mice, a progressive increase in microglial activation was observed in the lesioned FN peaking at day 7 and was accompanied by dense staining for Iba1, lectin, NDPase and CD11b. By contrast, in IRF8KO mice, the microglial response was markedly attenuated with little staining for Iba1, while the density of staining for lectin and NDPase was reduced significantly. The attenuated microglial response in IRF8KO mice was paralleled by a significant decrease at day 3 post-lesion in proliferation as monitored by BrdU, phosphohistone 3 and Ki67-positive cells. Furthermore, a decrease in PU.1-positive cells was observed in the FN of IRF8KO mice compared with WT. The wrapping of individual motor neuron cell bodies by microglia involved in synaptic stripping and phagocytosis was incomplete in the absences of IRF8 in the axotomised FN. Quantitative analysis showed that in IRF8KO mice, the degeneration of axotomised motor neurons was significantly increased. These studies extend on our previous finding that IRF8 is a key homeostatic transcription regulator of microglial cell function in the healthy brain, and moreover, has a crucial role in the regulating the response to microglia to neuronal injury.

Adenoviral-mediated mouse model of motor impairment in distal spinal muscular atrophy type V

Distal spinal muscular atrophy type V (dSMA-V) is a hereditary neurodegenerative axonal neuropathy and a glycyl-tRNA synthetase (GARS)-associated neuropathy that is caused by a mutation in GARS. Herein, we report a new GARS-associated neuropathy mouse model using an adenovirus vector system equipped with a neuron-specific promoter. In this model, we showed an increased number of activated microglia around the L129P mutant-expressing motor neuron cell bodies and the increased nerve injury signal in L129P mutant-expressing motor neuron cell bodies. In addition, we found that the mouse model exhibited impaired motor function caused by the L129P mutant. We propose that the neuroinflammation may be associated with motor neuron impairment in GARS-associated neuropathies.

Sensory neurons do not induce motor neuron loss in a human stem cell model of spinal muscular atrophy.

Spinal muscular atrophy (SMA) is an autosomal recessive disorder leading to paralysis and early death due to reduced SMN protein. It is unclear why there is such a profound motor neuron loss, but recent evidence from fly and mouse studies indicate that cells comprising the whole sensory-motor circuit may contribute to motor neuron dysfunction and loss. Here, we used induced pluripotent stem cells derived from SMA patients to test whether sensory neurons directly contribute to motor neuron loss. We generated sensory neurons from SMA induced pluripotent stem cells and found no difference in neuron generation or survival, although there was a reduced calcium response to depolarizing stimuli. Using co-culture of SMA induced pluripotent stem cell derived sensory neurons with control induced pluripotent stem cell derived motor neurons, we found no significant reduction in motor neuron number or glutamate transporter boutons on motor neuron cell bodies or neurites. We conclude that SMA sensory neurons do not overtly contribute to motor neuron loss in this human stem cell system.

Spatiotemporally limited BDNF and GDNF overexpression rescues motoneurons destined to die and induces elongative axon growth

Axonal injury close to cell bodies of motoneurons induces the death of the vast majority of affected cells. Neurotrophic factors, such as brain derived neurotrophic factor (BDNF) and glial cell derived neurotrophic factor (GDNF), delivered close to the damaged motor pool in a non-regulated manner induce good survival of injured motoneurons and sprouting of their axons but fail to induce functional reinnervation.To avoid these drawbacks of high levels of neurotrophic expression, we devised an ex vivo gene therapy system to induce transient expression of BDNF/GDNF in transfected rat adipose tissue-derived stem cells (rASCs) which were grafted around the reimplanted ventral root, embedded in collagen gel.Strong BDNF/GDNF expression was induced in vitro in the first days after transfection with a significant decline in expression 10–14 days following transfection. Numerous axons of injured motoneurons were able to enter the reimplanted root following reimplantation and BDNF or GDNF treatment (192±17 SEM vs 187±12 SEM, respectively) and produce morphological and functional reinnervation. Treatment with a combined cell population (BDNF+GDNF-transfected rASCs) induced slightly improved reinnervation (247±24 SEM). In contrast, only few motoneurons regenerated their axons in control animals (63±4 SEM) which received untransfected cells. The axons of surviving motoneurons showed elongative growth typical of regenerative axons, without aberrant growth or coil formation of sprouting axons.These findings provide evidence that damaged motoneurons require limited and spatially directed amounts of BDNF and GDNF to support their survival and regeneration. Moreover, neurotrophic support appears to be needed only for a critical period of time not longer than for two weeks after injury.

Reconstruction of phrenic neuron identity in embryonic stem cell-derived motor neurons

Air breathing is an essential motor function for vertebrates living on land. The rhythm that drives breathing is generated within the central nervous system and relayed via specialised subsets of spinal motor neurons to muscles that regulate lung volume. In mammals, a key respiratory muscle is the diaphragm, which is innervated by motor neurons in the phrenic nucleus. Remarkably, relatively little is known about how this crucial subtype of motor neuron is generated during embryogenesis. Here, we used direct differentiation of motor neurons from mouse embryonic stem cells as a tool to identify genes that direct phrenic neuron identity. We find that three determinants, Pou3f1, Hoxa5 and Notch, act in combination to promote a phrenic neuron molecular identity. We show that Notch signalling induces Pou3f1 in developing motor neurons in vitro and in vivo. This suggests that the phrenic neuron lineage is established through a local source of Notch ligand at mid-cervical levels. Furthermore, we find that the cadherins Pcdh10, which is regulated by Pou3f1 and Hoxa5, and Cdh10, which is controlled by Pou3f1, are both mediators of like-like clustering of motor neuron cell bodies. This specific Pcdh10/Cdh10 activity might provide the means by which phrenic neurons are assembled into a distinct nucleus. Our study provides a framework for understanding how phrenic neuron identity is conferred and will help to generate this rare and inaccessible yet vital neuronal subtype directly from pluripotent stem cells, thus facilitating subsequent functional investigations.

Altered expression of atypical PKC and Ryk in the spinal cord of a mouse model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive paralysis due to the selective death of motor neurons of unknown causes. Increasing evidence indicates that Wnt signaling is altered in ALS. In this study, we focused on two non-canonical Wnt signaling components, atypical PKC (aPKC) and a Wnt receptor, Ryk, in a mouse model of ALS, SOD1 (G93A). aPKC mediates Wnt signaling to regulate growth cone guidance, axon differentiation and cell survival. Ryk is a Wnt repulsive receptor that regulates axon guidance and inhibits regeneration after spinal cord injury. aPKC expression was increased in motor neurons of the lumbar spinal cord in SOD1 (G93A) mice at both early and late stages. Interestingly, aPKC was co-localized with SOD1 in motor neuron cell bodies and extracellular aggregates, and aPKC-containing extracellular aggregates increased with disease progression. Biochemical fractionation showed that aPKC protein level was increased in the detergent-insoluble protein fraction in SOD1 (G93A) mice at late stage but decreased in the detergent-soluble fraction at symptomatic stage. These results suggest that aPKC may be sequestered in SOD1 aggregates, impairing its ability to protect motor neurons from death. Ryk expression was also increased in the motor neurons and the white matter in the ventral lumbar spinal cord of mutant SOD1 mice with a peak at early stage. These observations indicate that Wnt/aPKC and Wnt/Ryk signaling are altered in SOD1 (G93A) mice, suggesting that changed Wnt signaling may contribute to neurodegeneration in ALS.

Production of RNA for Transcriptomic Analysis from Mouse Spinal Cord Motor Neuron Cell Bodies by Laser Capture Microdissection

Preparation of high-quality RNA from cells of interest is critical to precise and meaningful analysis of transcriptional differences among cell types or between the same cell type in health and disease or following pharmacologic treatments. In the spinal cord, such preparation from motor neurons, the target of interest in many neurologic and neurodegenerative diseases, is complicated by the fact that motor neurons represent <10% of the total cell population. Laser capture microdissection (LMD) has been developed to address this problem. Here, we describe a protocol to quickly recover, freeze, and section mouse spinal cord to avoid RNA damage by endogenous and exogenous RNases, followed by staining with Azure B in 70% ethanol to identify the motor neurons while keeping endogenous RNase inhibited. LMD is then used to capture the stained neurons directly into guanidine thiocyanate lysis buffer, maintaining RNA integrity. Standard techniques are used to recover the total RNA and measure its integrity. This material can then be used for downstream analysis of the transcripts by RNA-seq and qRT-PCR.

Production of RNA for Transcriptomic Analysis from Mouse Spinal Cord Motor Neuron Cell Bodies by Laser Capture Microdissection

Preparation of high-quality RNA from cells of interest is critical to precise and meaningful analysis of transcriptional differences among cell types or between the same cell type in health and disease or following pharmacologic treatments. In the spinal cord, such preparation from motor neurons, the target of interest in many neurologic and neurodegenerative diseases, is complicated by the fact that motor neurons represent <10% of the total cell population. Laser capture microdissection (LMD) has been developed to address this problem. Here, we describe a protocol to quickly recover, freeze, and section mouse spinal cord to avoid RNA damage by endogenous and exogenous RNases, followed by staining with Azure B in 70% ethanol to identify the motor neurons while keeping endogenous RNase inhibited. LMD is then used to capture the stained neurons directly into guanidine thiocyanate lysis buffer, maintaining RNA integrity. Standard techniques are used to recover the total RNA and measure its integrity. This material can then be used for downstream analysis of the transcripts by RNA-seq and qRT-PCR.

Spinal root avulsion: an excellent model for studying motoneuron degeneration and regeneration after severe axonal injury

Spinal root avulsion: an excellent model for studying motoneuron degeneration and regeneration after severe axonal injury

Defects in Synapse Structure and Function in a Fly Model of FUS-Related ALS

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease that leads invariably to fatal paralysis. Although most cases of ALS are sporadic, about 10% are familial. One gene associated with familial ALS encodes the DNA/RNA binding protein Fused in Sarcoma (FUS). There exists a Drosophila model of ALS, in which human FUS with ALS-causing mutations is expressed in motor neurons. These flies exhibit motor neuron degeneration, larval locomotor defects and early death. Similar phenotypes are observed in flies null for the gene Cabeza (Caz), the fly homolog of FUS. We have examined evoked and spontaneous synaptic transmission at the larval neuromuscular junction, larval motor neuron cell body excitability, and presynaptic active zone structure in these fly models of ALS. The amplitude of evoked synaptic currents is decreased by more than 80% in larvae in which human mutant FUS (R521C) is expressed in motor neurons. A similar decrease in evoked synaptic transmission is seen in Caz1 null flies. Furthermore, the frequency of spontaneous miniature synaptic currents is decreased dramatically in FUS-R521C expressing flies. In marked contrast, recordings from motor neuron cell bodies demonstrate that both wild type and mutant FUS expressing neurons can fire normal action potentials, and the voltage-dependent inward and outward currents in the cell bodies are indistinguishable in wild type and mutant FUS motor neurons. Although confocal microscopic analysis of the larval neuromuscular junction does not reveal gross abnormalities, examination of synapses using super-resolution STED microscopy suggests that presynaptic active zones are aberrantly organized in larvae in which FUS-R521C is expressed in the motor neurons. The results are consistent with the idea that defects in synaptic structure and function precede, and may contribute to, the later motor neuron degeneration that is characteristic of ALS.

The singular qualities of motor neurones in health and disease

The singular qualities of motor neurones in health and disease

Defects in Synapse Structure and Function Precede Motor Neuron Degeneration inDrosophilaModels of FUS-Related ALS

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease that leads invariably to fatal paralysis associated with motor neuron degeneration and muscular atrophy. One gene associated with ALS encodes the DNA/RNA-binding protein Fused in Sarcoma (FUS). There now exist two Drosophila models of ALS. In one, human FUS with ALS-causing mutations is expressed in fly motor neurons; in the other, the gene cabeza (caz), the fly homolog of FUS, is ablated. These FUS-ALS flies exhibit larval locomotor defects indicative of neuromuscular dysfunction and early death. The locus and site of initiation of this neuromuscular dysfunction remain unclear. We show here that in FUS-ALS flies, motor neuron cell bodies fire action potentials that propagate along the axon and voltage-dependent inward and outward currents in the cell bodies are indistinguishable in wild-type and FUS-ALS motor neurons. In marked contrast, the amplitude of synaptic currents evoked in the postsynaptic muscle cell is decreased by >80% in FUS-ALS larvae. Furthermore, the frequency but not unitary amplitude of spontaneous miniature synaptic currents is decreased dramatically in FUS-ALS flies, consistent with a change in quantal content but not quantal size. Although standard confocal microscopic analysis of the larval neuromuscular junction reveals no gross abnormalities, superresolution stimulated emission depletion (STED) microscopy demonstrates that the presynaptic active zone protein bruchpilot is aberrantly organized in FUS-ALS larvae. The results are consistent with the idea that defects in presynaptic terminal structure and function precede, and may contribute to, the later motor neuron degeneration that is characteristic of ALS.

Mechanisms for axon maintenance and plasticity in motoneurons: alterations in motoneuron disease

In motoneuron disease and other neurodegenerative disorders, the loss of synapses and axon branches occurs early but is compensated by sprouting of neighboring axon terminals. Defective local axonal signaling for maintenance and dynamics of the axonal microtubule and actin cytoskeleton plays a central role in this context. The molecular mechanisms that lead to defective cytoskeleton architecture in two mouse models of motoneuron disease are summarized and discussed in this manuscript. In the progressive motor neuropathy (pmn) mouse model of motoneuron disease that is caused by a mutation in the tubulin-specific chaperone E gene, death of motoneuron cell bodies appears as a consequence of axonal degeneration. Treatment with bcl-2 overexpression or with glial-derived neurotrophic factor prevents loss of motoneuron cell bodies but does not influence the course of disease. In contrast, treatment with ciliary neurotrophic factor (CNTF) significantly delays disease onset and prolongs survival of pmn mice. This difference is due to the activation of Stat-3 via the CNTF receptor complex in axons of pmn mutant motoneurons. Most of the activated Stat-3 protein is not transported to the nucleus to activate transcription, but interacts locally in axons with stathmin, a protein that destabilizes microtubules. This interaction plays a major role in CNTF signaling for microtubule dynamics in axons. In Smn-deficient mice, a model of spinal muscular atrophy, defects in axonal translocation of β-actin mRNA and possibly other mRNA species have been observed. Moreover, the regulation of local protein synthesis in response to signals from neurotrophic factors and extracellular matrix proteins is altered in motoneurons from this model of motoneuron disease. These findings indicate that local signals are important for maintenance and plasticity of axonal branches and neuromuscular endplates, and that disturbances in these signaling mechanisms could contribute to the pathophysiology of motoneuron diseases.

A novel adenoviral vector-mediated mouse model of Charcot-Marie-Tooth type 2D (CMT2D)

Charcot-Marie-Tooth disease type 2D is a hereditary axonal and glycyl-tRNA synthetase (GARS)-associated neuropathy that is caused by a mutation in GARS. Here, we report a novel GARS-associated mouse neuropathy model using an adenoviral vector system that contains a neuronal-specific promoter. In this model, we found that wild-type GARS is distributed to peripheral axons, dorsal root ganglion (DRG) cell bodies, central axon terminals, and motor neuron cell bodies. In contrast, GARS containing a G240R mutation was localized in DRG and motor neuron cell bodies, but not axonal regions, in vivo. Thus, our data suggest that the disease-causing G240R mutation may result in a distribution defect of GARS in peripheral nerves in vivo. Furthermore, a distributional defect may be associated with axonal degradation in GARS-associated neuropathies.

285: Interferon regulatory factor 8 is essential for regulating the response of microglia to neuronal injury

The transcription factor interferon regulatory factor (IRF) 8 has a key role in the cellular response to IFN-gamma and is involved in myeloid cell differentiation. We have identified IRF8 to be a constitutive and IFN-gamma-stimulated nuclear factor in microglia. The objective of this study was to determine the role of IRF8 in the microglial response to sterile neuronal injury induced by axotomy. Facial nerve axotomy (FNA) was performed in wild type (WT) and IRF8 KO mice and the brain removed at different times post-lesion. A subset of mice was injected with bromodeoxyuridine (BrdU) every 2 h during the 24 h prior to retrieval of the brain. In brains from IRF8 KO mice, NDPase histochemistry revealed gross alterations in the morphology of microglia, which were stunted and hypertrophied. Microglia from IRF8 KO mice also exhibited significant alterations in a number of key myeloid and immune markers. After FNA in WT mice, a progressive increase in microglial activation was observed in the lesioned facial nucleus that peaked at day 7 post-lesion. By contrast, in IRF8 KO mice, the microglial response appeared markedly attenuated and delayed. The attenuated microglial response to FNA in IRF8 KO mice was paralleled by a large decrease in proliferation as monitored by BrdU and phosphohistone 3 positive cells. The wrapping of individual motor neuron cell bodies by microglia involved in synaptic stripping and phagocytosis was incomplete in the absence of IRF8. Quantitative analysis showed that in IRF8 KO mice, the degeneration of axotomized motor neurons was slightly increased. These studies indicate that IRF8: (1) is a key homeostatic transcriptional regulator of microglial cell function in the healthy brain, and (2) has a crucial role in the functional response of microglia to neuronal injury. Supported by NHMRC project Grant APP1007757.

The pre‐synaptic motor nerve terminal as a site for antibody‐mediated neurotoxicity in autoimmune neuropathies and synaptopathies

The pre-synaptic motor nerve terminal is a highly complex and dynamic compartment within the lower motor neuron responsible for converting electrical signals into secreted chemicals. This self-renewing process of synaptic transmission is accomplished by the calcium-triggered fusion of neurotransmitter-containing vesicles with the plasma membrane and the subsequent retrieval and recycling of vesicle components. Besides this conventional physiological role, the highly active process of vesicle fusion and re-uptake into endosomal sorting pathways acts as a conduit for entry of a range of substances into the intracellular compartment of the motor nerve terminal. Whilst this entry portal sub-serves many vital physiological processes, such as those mediated by neurotrophin trafficking, there is also the potential for substantial pathological consequences resulting from uptake of noxious agents, including autoantibodies, viruses and toxins. These may act locally to induce disease within the nerve terminal, or traffic beyond to the motor neuron cell body and central nervous system to exert their pathological effects. This review focuses on the recent evidence that the ganglioside-rich pre-synaptic membrane acts as a binding site for potentially neurotoxic serum autoantibodies that are present in human autoimmune motor neuropathies. Autoantibodies that bind surface antigens induce membrane lytic effects, whereas their uptake attenuates local injury and transfers any potential pathological consequences to the intracellular compartment. Herein the thesis is explored that a balance exists between local injury at the exofacial leaflet of the pre-synaptic membrane and antibody uptake, which dictates the overall level and site of motor nerve injury in this group of disorders.

Transgenic overexpression of the cell adhesion molecule L1 in neurons facilitates recovery after mouse spinal cord injury

It has been shown that the X-chromosome-linked neural cell adhesion molecule L1 plays a beneficial role in regeneration after spinal cord injury (SCI) in young adult rodents when applied in various molecular and cellular forms. In an attempt to further characterize the multiple functions of L1 after severe SCI we analyzed locomotor functions and measured axonal regrowth/sprouting and sparing, glial scarring, and synaptic remodeling at 6weeks after severe spinal cord compression injury at the T7–9 levels of L1-deficient mice (L1−/y) and their wild-type (L1+/y) littermates, as well as mice that overexpress L1 under the control of the neuron-specific Thy-1 promoter (L1tg) and their wild-type littermates (L1+/+). No differences were found in the locomotor scale score and single frame motion analysis between L1−/y and L1+/y mice during 6weeks after SCI, most likely due to the very low expression of L1 in the adult spinal cord of wild-type mice. L1tg mice, however, showed better locomotor recovery than their L1+/+ littermates, being associated with enhanced numbers of catecholaminergic axons in the lumbar spinal cord, but not of cholinergic, GABAergic or glutamatergic terminals around motoneuron cell bodies in the lumbar spinal cord. Additionally, no difference between L1tg and L1+/+ mice was detectable in dieback of corticospinal tract axons. Neuronal L1 overexpression did not influence the size of the glial fibrillary acidic protein-immunoreactive astrocytic scar 6weeks after injury. We conclude that neuronal overexpression of L1 improves functional recovery from SCI by increasing catecholaminergic axonal regrowth/sprouting and/or sparing of severed axons without affecting the glial scar size.

Diagnostic and Therapeutic Potential of Tetanus Toxin-Derivatives in Neurological Diseases

We assessed the ex vivo reactivity of peptidic constructs of Tet1 (analog of tetanus toxin non-virulent C fragment) with sequence homology to the cysteine-active site of thioredoxin (Tet1THO) or tetralysine (Tet1PLYS) with oxidative species or axonopathic sodium cyanate (NaOCN), respectively. We then assessed their neuronal uptake in vivo in laboratory animals. The reactivity of Tet1PLYS with NaOCN (1:2.5 to 1:37.5 molar ratios) or Tet1THO with hydrogen peroxide (1:0.4 to 1:6.2 molar ratios) was assessed by mass spectrometry. Green fluorescence protein (GFP)-tagged Tet1-derivatives (3 mg/ml in artificial cerebrospinal fluid) were administered daily to rats by intramuscular injection in latissimus dorsi at lumborum at the dose of 1 μl/g of body weight, for 3 days. Motor neuron uptake was assessed after double immunolabeling for GFP and choline acetyltransferase. Mass spectrometry analysis successfully demonstrated the ex vivo reactivity of Tet1-derivatives in a concentration-dependent manner. Confocal microscopy revealed the localization of Tet1-derivatives in axons and motor neuron cell bodies. Intramuscular delivery of Tet1-derivatives appears to be a practical approach to circumvent the blood nerve barrier and selectively deliver small molecules to the nervous system, for diagnostic and/or treatment purposes.

The Role of Oxidative Stress in Nervous System Aging

While oxidative stress is implicated in aging, the impact of oxidative stress on aging in the peripheral nervous system is not well understood. To determine a potential mechanism for age-related deficits in the peripheral nervous system, we examined both functional and morphological changes and utilized microarray technology to compare normal aging in wild-type mice to effects in copper/zinc superoxide dismutase-deficient (Sod1−/−) mice, a mouse model of increased oxidative stress. Sod1−/− mice exhibit a peripheral neuropathy phenotype with normal sensory nerve function and deficits in motor nerve function. Our data indicate that a decrease in the synthesis of cholesterol, which is vital to myelin formation, correlates with the structural deficits in axons, myelin, and the cell body of motor neurons in the Sod1+/+ mice at 30 months and the Sod1−/− mice at 20 months compared with mice at 2 months. Collectively, we have demonstrated that the functional and morphological changes within the peripheral nervous system in our model of increased oxidative stress are manifested earlier and resemble the deficits observed during normal aging.

Microfluidic primary culture model of the lower motor neuron–neuromuscular junction circuit

Modelling the complex process of neuromuscular signalling is key to understanding not only normal circuit function but also importantly the mechanisms underpinning a range of degenerative diseases. We describe a novel in vitro model of the lower motor neuron–neuromuscular junction circuit, incorporating primary spinal motor neurons, supporting glia and skeletal muscle. This culture model is designed to spatially mimic the unique anatomical and cellular interactions of this circuit in compartmented microfluidic devices, such that the glial cells are located with motor neuron cell bodies in the cell body chamber and motor neuron axons extend to a distal chamber containing skeletal muscle cells whilst simultaneously allowing targeted intervention. This model is suitable for use in conjunction with a range of downstream experimental approaches and could also be modified to utilise other cellular sources including appropriate immortal cell lines, cells derived from transgenic models of disease and also patient derived stem cells.