Autophagy dysregulation in cell culture and animals models of Spinal Muscular Atrophy

Abnormal autophagy has become a central thread linking neurodegenerative diseases, particularly of the motor neuron. One such disease is spinal muscular atrophy (SMA), a genetic neuromuscular disorder caused by mutations in the SMN1 gene resulting in low levels of Survival Motor Neuron (SMN) protein. Despite knowing the causal protein, the exact intracellular processes that are involved in the selective loss of motor neurons remains unclear. Autophagy induction can be helpful or harmful depending on the situation, and we sought to understand the state of the autophagic response in SMA. We show that cell culture and animal models demonstrate induction of autophagy accompanied by attenuated autophagic flux, resulting in the accumulation of autophagosomes and their associated cargo. Expression of the SMN-binding protein a-COP, a known modulator of autophagic flux, can ameliorate this autophagic traffic jam

Abnormal Golgi morphology and decreased COPI function in cells with low levels of SMN

We report here the finding of abnormal Golgi apparatus morphology in motor neuron like cells depleted of SMN as well as Golgi apparatus morphology in SMA patient fibroblasts. Rescue experiments demonstrate that this abnormality is dependent on SMN, but can also be rescued by expression of the COPI coatomer subunit alpha-COP. A motor neuron-like cell line containing an inducible alpha-COP shRNA was created to generate a parallel system to study knockdown of SMN or alpha-COP. Multiple assays of COPI-dependent intracellular trafficking in cells depleted of SMN demonstrate that alpha-COP function is suboptimal, including failed sequestration of plasma membrane proteins, altered binding of mRNA, and defective targeting and transport of Golgi-resident proteins

Transgenic mice expressing mutant forms VCP/p97 recapitulate the full spectrum of IBMPFD including degeneration in muscle, brain and bone

Inclusion body myopathy associated with Paget's disease of bone and frontotemporal dementia (IBMPFD) is a dominantly inherited degenerative disorder caused by mutations in the valosin-containing protein (VCP) gene. VCP (p97 in mouse, TER94 in Drosophila melanogaster and CDC48 in Saccharomyces cerevisiae) is a highly conserved AAA+-ATPase that regulates a wide array of cellular processes. The mechanism of IBMPFD pathogenesis is unknown. Towards elucidating the pathogenic mechanism we have developed and characterized transgenic mice with ubiquitous expression of wild-type and disease-causing versions of human VCP/p97. Here, we report that mice expressing VCP/p97 harboring the mutations R155H or A232E develop pathology that is limited to muscle, brain and bone, recapitulating the spectrum of disease in humans with IBMPFD. The mice exhibit progressive muscle weakness and pathological examination of muscle shows classic characteristics of inclusion body myopathy including rimmed vacuoles and TDP-43 pathology. The mice exhibit abnormalities in behavioral testing and pathological examination of the brain shows widespread TDP-43 pathology. Furthermore, radiological examination of the skeleton reveals that mutant mice develop severe osteopenia accompanied by focal lytic and sclerotic lesions in vertebrae and femur. In vitro studies indicate that mutant VCP causes inappropriate activation of the NF-κB signaling cascade, which could contribute to the mechanism of pathogenesis in multiple tissues including muscle, bone and brai

Interaction between alpha-COP and SMN ameliorates disease phenotype in a mouse model of spinal muscular atrophy

We report that expression of the α-COP protein rescues disease phenotype in a severe mouse model of Spinal Muscular Atrophy (SMA).. Lentiviral particles expressing α-COP were injected directly into the testes of genetically pure mouse strain of interest resulting in infection of the spermatagonial stem cells. α-COP was stably expressed in brain, skeletal muscle, and spinal cord without altering SMN protein levels. SMA mice transgenic for α-COP live significantly longer than their non-transgenic littermates, and showed increased body mass and normal muscle morphology at postnatal day 15. We previously reported that binding between SMN and α-COP is required for restoration of neurite outgrowth in cells lacking SMN, and we report similar finding here. Lentiviral-mediated transgenic expression of SMN where the dilysine domain in exon 2b was mutated was not able to rescue the SMA phenotype despite robust expression of the mutant SMN protein in brain, muscle and spinal cord. These results demonstrate that α-COP is a validated modifier of SMA disease phenotype in a mammalian, vertebrate model and is a potential target for development of future SMN-independent therapeutic interventions

Identification of a resilient mouse facial motoneuron population following target disconnection by injury or disease

Background: When nerve transection is performed on adult rodents, a substantial population of neurons survives short-term disconnection from target, and the immune system supports this neuronal survival, however long-term survival remains unknown. Understanding the effects of permanent axotomy on cell body survival is important as target disconnection is the first pathological occurrence in fatal motoneuron diseases such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA). Objective: The goal of this study was to determine if facial motoneurons (FMN) could survive permanent target disconnection up to 26 weeks post-operation (wpo) after facial nerve axotomy (FNA). In addition, the potentially additive effects of immunodeficiency and motoneuron disease on post-axotomy FMN survival were examined. Methods: This study included three wild type (WT) mouse strains (C57BL/6J, B6SJL, and FVB/NJ) and three experimental models (RAG-2-/-: immunodeficiency; mSOD1: ALS; Smn-/-/SMN2+/+: SMA). All animals received a unilateral FNA, and FMN survival was quantified at early and extended post-operative timepoints. Results: In the C57BL/6J WT group, FMN survival significantly decreased at 10 wpo (55 ± 6%), and then remained stable out to 26 wpo (47 ± 6%). In the RAG-2-/- and mSOD1 groups, FMN death occurred much earlier at 4 wpo, and survival plateaued at approximately 50% at 10 wpo. The SMA model and other WT strains also exhibited approximately 50% FMN survival after FNA. Conclusion: These results indicate that immunodeficiency and motoneuron disease accelerate axotomy-induced neuron death, but do not increase total neuron death in the context of permanent target disconnection. This consistent finding of a target disconnection-resilient motoneuron population is prevalent in other peripheral nerve injury models and in neurodegenerative disease models as well. Characterization of the distinct populations of vulnerable and resilient motoneurons may reveal new therapeutic approaches for injury and disease

Thrive: Success Strategies for the Modern-Day Faculty Member

The THRIVE collection is intended to help faculty thrive in their roles as educators, scholars, researchers, and clinicians. Each section contains a variety of thought-provoking topics that are designed to be easily digested, guide personal reflection, and put into action. Please use the THRIVE collection to help: Individuals study topics on their own, whenever and wherever they want Peer-mentoring or other learning communities study topics in small groups Leaders and planners strategically insert faculty development into existing meetings Faculty identify campus experts for additional learning, grand rounds, etc. If you have questions or want additional information on a topic, simply contact the article author or email [email protected]://digitalcommons.unmc.edu/facdev_books/1000/thumbnail.jp

Self-oligomerization regulates stability of survival motor neuron protein isoforms by sequestering an SCF^Slmb degron

Spinal muscular atrophy (SMA) is caused by homozygous mutations in human SMN1. Expression of a duplicate gene (SMN2) primarily results in skipping of exon 7 and production of an unstable protein isoform, SMNΔ7. Although SMN2 exon skipping is the principal contributor to SMA severity, mechanisms governing stability of survival motor neuron (SMN) isoforms are poorly understood. We used a Drosophila model system and label-free proteomics to identify the SCFSlmb ubiquitin E3 ligase complex as a novel SMN binding partner. SCFSlmb interacts with a phosphor degron embedded within the human and fruitfly SMN YG-box oligomerization domains. Substitution of a conserved serine (S270A) interferes with SCFSlmb binding and stabilizes SMNΔ7. SMA-causing missense mutations that block multimerization of full-length SMN are also stabilized in the degron mutant background. Overexpression of SMNΔ7S270A, but not wild-type (WT) SMNΔ7, provides a protective effect in SMA model mice and human motor neuron cell culture systems. Our findings support a model wherein the degron is exposed when SMN is monomeric and sequestered when SMN forms higher-order multimers

Dilysine motifs in exon 2b of SMN protein mediate binding to the COPI vesicle protein α-COP and neurite outgrowth in a cell culture model of spinal muscular atrophy

Spinal muscular atrophy (SMA) is a devastating neuromuscular disorder that stems from low levels of survival of motor neuron (SMN) protein. The processes that cause motor neurons and muscle cells to become dysfunctional are incompletely understood. We are interested in neuromuscular homeostasis and the stresses put upon that system by loss of SMN. We recently reported that α-COP, a member of the coatomer complex of coat protein I (COPI) vesicles, is an SMN-binding partner, implicating this protein complex in normal SMN function. To investigate the functional significance of the interaction between α-COP and SMN, we constructed an inducible NSC-34 cell culture system to model the consequences of SMN depletion and find that depletion of SMN protein results in shortened neurites. Heterologous expression of human SMN, and interestingly over-expression of α-COP, restores normal neurite length and morphology. Mutagenesis of the canonical COPI dilysine motifs in exon 2b results in failure to bind to α-COP and abrogates the ability of human SMN to restore neurite outgrowth in SMN-depleted motor neuron-like NSC-34 cells. We conclude that the interaction between SMN and α-COP serves an important function in the growth and maintenance of motor neuron processes and may play a significant role in the pathogenesis of SMA

α-COP binding to the survival motor neuron protein SMN is required for neuronal process outgrowth

Spinal muscular atrophy (SMA), a heritable neurodegenerative disease, results from insufficient levels of the survival motor neuron (SMN) protein. α-COP binds to SMN, linking the COPI vesicular transport pathway to SMA. Reduced levels of α-COP restricted development of neuronal processes in NSC-34 cells and primary cortical neurons. Remarkably, heterologous expression of human α-COP restored normal neurite length and morphology in SMN-depleted NSC-34 cells in vitro and zebrafish motor neurons in vivo. We identified single amino acid mutants of α-COP that selectively abrogate SMN binding, retain COPI-mediated Golgi-ER trafficking functionality, but were unable to support neurite outgrowth in cellular and zebrafish models of SMA. Taken together, these demonstrate the functional role of COPI association with the SMN protein in neuronal development