Lectin-based Isolation and Culture of Mouse Embryonic Motoneurons

Spinal motoneurons develop towards postmitotic stages through early embryonic nervous system development and subsequently grow out dendrites and axons. Neuroepithelial cells of the neural tube that express Nkx6.1 are the unique precursor cells for spinal motoneurons1. Though postmitotic motoneurons move towards their final position and organize themselves into columns along the spinal tract2,3. More than 90% of all these differentiated and positioned motoneurons express the transcription factors Islet 1/2. They innervate the muscles of the limbs as well as those of the body and the inner organs. Among others, motoneurons typically express the high affinity receptors for brain derived neurotrophic factor (BDNF) and Neurotrophin-3 (NT-3), the tropomyosin-related kinase B and C (TrkB, TrkC). They do not express the tropomyosin-related kinase A (TrkA)4. Beside the two high affinity receptors, motoneurons do express the low affinity neurotrophin receptor p75NTR. The p75NTR can bind all neurotrophins with similar but lower affinity to all neurotrophins than the high affinity receptors would bind the mature neurotrophins. Within the embryonic spinal cord, the p75NTR is exclusively expressed by the spinal motoneurons5. This has been used to develop motoneuron isolation techniques to purify the cells from the vast majority of surrounding cells6. Isolating motoneurons with the help of specific antibodies (panning) against the extracellular domains of p75NTR has turned out to be an expensive method as the amount of antibody used for a single experiment is high due to the size of the plate used for panning. A much more economical alternative is the use of lectin. Lectin has been shown to specifically bind to p75NTR as well7. The following method describes an alternative technique using wheat germ agglutinin for a preplating procedure instead of the p75NTR antibody. The lectin is an extremely inexpensive alternative to the p75NTR antibody and the purification grades using lectin are comparable to that of the p75NTR antibody. Motoneurons from the embryonic spinal cord can be isolated by this method, survive and grow out neurites

Missense mutation in the tubulin-specific chaperone E (Tbce) gene in the mouse mutant progressive motor neuronopathy, a model of human motoneuron disease

Progressive motor neuronopathy (pmn) mutant mice have been widely used as a model for human motoneuron disease. Mice that are homozygous for the pmn gene defect appear healthy at birth but develop progressive motoneuron disease, resulting in severe skeletal muscle weakness and respiratory failure by postnatal week 3. The disease starts at the motor endplates, and then leads to axonal loss and finally to apoptosis of the corresponding cell bodies. We localized the genetic defect in pmn mice to a missense mutation in the tubulin-specific chaperone E (Tbce) gene on mouse chromosome 13. The human orthologue maps to chromosome 1q42.3. The Tbce gene encodes a protein (cofactor E) that is essential for the formation of primary α-tubulin and β-tubulin heterodimeric complexes. Isolated motoneurons from pmn mutant mice exhibit shorter axons and axonal swelling with irregularly structured β-tubulin and tau immunoreactivity. Thus, the pmn gene mutation provides the first genetic evidence that alterations in tubulin assembly lead to retrograde degeneration of motor axons, ultimately resulting in motoneuron cell death

Smn, the spinal muscular atrophy–determining gene product, modulates axon growth and localization of β-actin mRNA in growth cones of motoneurons

Spinal muscular atrophy (SMA), a common autosomal recessive form of motoneuron disease in infants and young adults, is caused by mutations in the survival motoneuron 1 (SMN1) gene. The corresponding gene product is part of a multiprotein complex involved in the assembly of spliceosomal small nuclear ribonucleoprotein complexes. It is still not understood why reduced levels of the ubiquitously expressed SMN protein specifically cause motoneuron degeneration. Here, we show that motoneurons isolated from an SMA mouse model exhibit normal survival, but reduced axon growth. Overexpression of Smn or its binding partner, heterogeneous nuclear ribonucleoprotein (hnRNP) R, promotes neurite growth in differentiating PC12 cells. Reduced axon growth in Smn-deficient motoneurons correlates with reduced β-actin protein and mRNA staining in distal axons and growth cones. We also show that hnRNP R associates with the 3′ UTR of β-actin mRNA. Together, these data suggest that a complex of Smn with its binding partner hnRNP R interacts with β-actin mRNA and translocates to axons and growth cones of motoneurons

A transgene carrying an A2G missense mutation in the SMN gene modulates phenotypic severity in mice with severe (type I) spinal muscular atrophy

5q spinal muscular atrophy (SMA) is a common autosomal recessive disorder in humans and the leading genetic cause of infantile death. Patients lack a functional survival of motor neurons (SMN1) gene, but carry one or more copies of the highly homologous SMN2 gene. A homozygous knockout of the single murine Smn gene is embryonic lethal. Here we report that in the absence of the SMN2 gene, a mutant SMN A2G transgene is unable to rescue the embryonic lethality. In its presence, the A2G transgene delays the onset of motor neuron loss, resulting in mice with mild SMA. We suggest that only in the presence of low levels of full-length SMN is the A2G transgene able to form partially functional higher order SMN complexes essential for its functions. Mild SMA mice exhibit motor neuron degeneration, muscle atrophy, and abnormal EMGs. Animals homozygous for the mutant transgene are less severely affected than heterozygotes. This demonstrates the importance of SMN levels in SMA even if the protein is expressed from a mutant allele. Our mild SMA mice will be useful in (a) determining the effect of missense mutations in vivo and in motor neurons and (b) testing potential therapies in SMA

CDNF rescues motor neurons in models of amyotrophic lateral sclerosis by targeting endoplasmic reticulum stress.

Amyotrophic lateral sclerosis is a progressive neurodegenerative disease that affects motor neurons (MNs) in the spinal cord, brainstem, and motor cortex, leading to paralysis and eventually to death within 3 to 5 years of symptom onset. To date, no cure or effective therapy is available. The role of chronic endoplasmic reticulum (ER) stress in the pathophysiology of amyotrophic lateral sclerosis, as well as a potential drug target, has received increasing attention. Here, we investigated the mode of action and therapeutic effect of the ER-resident protein cerebral dopamine neurotrophic factor (CDNF) in three preclinical models of amyotrophic lateral sclerosis, exhibiting different disease development and etiology: (i) the conditional choline acetyltransferase (ChAT)-tTA/TRE-hTDP43-M337V rat model previously described, (ii) the widely used SOD1-G93A mouse model, and (iii) a novel slow-progressive TDP43-M337V mouse model. To specifically analyse the ER stress response in MNs, we used three main methods: (i) primary culture of MNs derived from E13 days embryos, (ii) immunohistochemical analyses of spinal cord sections with ChAT as spinal MNs marker, and (iii) qPCR analyses of lumbar MNs isolated via laser microdissection. We show that intracerebroventricular administration of CDNF significantly halts the progression of the disease and improves motor behavior in TDP43-M337V and SOD1-G93A rodent models of amyotrophic lateral sclerosis. CDNF rescues motor neurons in vitro and in vivo from ER stress-associated cell death and its beneficial effect is independent of genetic disease etiology. Notably, CDNF regulates the unfolded protein response (UPR) initiated by transducers IRE1α, PERK, and ATF6, thereby enhancing MN survival. Thus, CDNF holds great promise for the design of new rational treatments for amyotrophic lateral sclerosis

Differentiation defects in primary motoneurons from a SMARD1 mouse model that are insensitive to treatment with low dose PEGylated IGF1

Muscle atrophy and diaphragmatic palsy are the clinical characteristics of spinal muscular atrophy with respiratory distress type 1 (SMARD1), and are well represented in the neuromuscular degeneration $(Nmd^{2J})$ mouse, modeling the juvenile form of SMARD1. Both in humans and mice mutations in the IGHMBP2 gene lead to motoneuron degeneration. We could previously demonstrate that treatment with a polyethylene glycol-coupled variant of IGF1 (PEG-IGF1) improves motor functions accompanied by reduced fiber degeneration in the gastrocnemius muscle and the diaphragm, but has no beneficial effect on motoneuron survival. These data raised the question which cell autonomous disease mechanisms contribute to dysfunction and loss of Ighmbp2-deficient motoneurons. An analysis of primary Ighmbp2-deficient motoneurons exhibited differentiation deficits such as reduced spontaneous $Ca^{2+}$ transients and altered axon elongation, which was not compensated by PEG-IGF1. This points to an IGF1 independent mechanism of motoneuron degeneration that deserves treatment approaches in addition to IGF1

Cell-autonomous axon growth of young motoneurons is triggered by a voltage-gated sodium channel

Spontaneous electrical activity preceding synapse formation contributes to the precise regulation of neuronal development. Examining the origins of spontaneous activity revealed roles for neurotransmitters that depolarize neurons and activate ion channels. Recently, we identified a new molecular mechanism underlying fluctuations in spontaneous neuronal excitability. We found that embryonic motoneurons with a genetic loss of the low-threshold sodium channel Na$_V$1.9 show fewer fluctuations in intracellular calcium in axonal compartments and growth cones than wild-type littermates. As a consequence, axon growth of Na$_V$1.9-deficient motoneurons in cell culture is drastically reduced while dendritic growth and cell survival are not affected. Interestingly, Na$_V$1.9 function is observed under conditions that would hardly allow a ligand- or neurotransmitter-dependent depolarization. Thus, Na$_V$1.9 may serve as a cell-autonomous trigger for neuronal excitation. In this addendum, we discuss a model for the interplay between cell-autonomous local neuronal activity and local cytoskeleton dynamics in growth cone function

The SMA Modifier Plastin 3 Targets Cell Membrane-Associated Proteins in Motoneurons

Loss of the Survival Motor Neuron (SMN) gene inevitably leads to spinal muscular atrophy (SMA), one of the most common fatal neuromuscular diseases in children with FDA and EMA approved therapies. However, the cellular mechanisms leading to neuromuscular junction (NMJ) dysfunction due to impaired Ca 2+ homeostasis in the presynaptic compartment remain largely unexplained. In the last decade, the so-called SMA modifiers have gained attention. The F-actin bundler Plastin 3 (PLS3) is one of them and counteracts neurotransmission defects, including altered vesicle endocytosis, in Smn-deficient NMJs. Properly bundled F-actin is the basis for the translocation and arrangement of transmembrane proteins at the cell surface. Our recently published data by Hennlein et al., J Cell Biol. (2023) clearly showed that Smn deficiency impairs the F-actin dependent translocation of the high-affinity BDNF receptor TrkB to the cell surface resulting in reduced BDNF-mediated TrkB activation in motor axon terminals. Strikingly, the overexpression of PLS3 restores TrkB availability, and significantly improves the clustering of the active zone-associated voltage-gated calcium channel Ca v 2.2 in growth cones of Smn-deficient motoneurons. These observations raise the question of how PLS3 mediates the proper cell surface localization and cluster-like formation of Ca v 2.2 in motor axon terminals

Axonal and dendritic localization of mRNAs for glycogen-metabolizing enzymes in cultured rodent neurons

Background: Localization of mRNAs encoding cytoskeletal or signaling proteins to neuronal processes is known to contribute to axon growth, synaptic differentiation and plasticity. In addition, a still increasing spectrum of mRNAs has been demonstrated to be localized under different conditions and developing stages thus reflecting a highly regulated mechanism and a role of mRNA localization in a broad range of cellular processes. Results: Applying fluorescence in-situ-hybridization with specific riboprobes on cultured neurons and nervous tissue sections, we investigated whether the mRNAs for two metabolic enzymes, namely glycogen synthase (GS) and glycogen phosphorylase (GP), the key enzymes of glycogen metabolism, may also be targeted to neuronal processes. If it were so, this might contribute to clarify the so far enigmatic role of neuronal glycogen. We found that the mRNAs for both enzymes are localized to axonal and dendritic processes in cultured lumbar spinal motoneurons, but not in cultured trigeminal neurons. In cultured cortical neurons which do not store glycogen but nevertheless express glycogen synthase, the GS mRNA is also subject to axonal and dendritic localization. In spinal motoneurons and trigeminal neurons in situ, however, the mRNAs could only be demonstrated in the neuronal somata but not in the nerves. Conclusions: We could demonstrate that the mRNAs for major enzymes of neural energy metabolism can be localized to neuronal processes. The heterogeneous pattern of mRNA localization in different culture types and developmental stages stresses that mRNA localization is a versatile mechanism for the fine-tuning of cellular events. Our findings suggest that mRNA localization for enzymes of glycogen metabolism could allow adaptation to spatial and temporal energy demands in neuronal events like growth, repair and synaptic transmission