The most troublesome symptoms in most patients with acquired neuropathy are those associated with small-fiber dysfunction and include burning pain, numbness and autonomic dysfunction. The central mechanism behind the pathology in neuropathy as well as other neurodegenerative conditions is loss of axonal integrity and function. Whether this is a direct assault on the neuron/axon or an indirect interruption of glial cell axonal support is unclear but it is increasingly apparent that, in the periphery, disrupted Schwann cell metabolism can lead to axonal degeneration. We are therefore pursuing studies of intrinsic axon self-destructive pathways as well as disruption of Schwann cell support functions to unravel underlying mechanisms of neurodegenerative disorders.

Defining the axon degeneration pathway

Defining the axon degeneration pathway.index1 Our laboratory is interested in understanding the program of axon self-destruction that is invoked to eliminate damaged or injured axons. Axon degeneration is an early event in most neurodegenerative conditions (i.e., peripheral neuropathy, Alzheimer’s, Parkinson’s, Multiple sclerosis), making prevention of axonopathy a critical pursuit and prime therapeutic target. In dissecting the molecular mechanism responsible for the delayed axon degeneration observed in wlds mutant mice, we discovered that overexpression of Nmnat enzymes, which synthesize NAD from NMN, prevents axon degeneration in response to injury. This has led to studies aimed at understanding the role of NAD synthetic pathways in axon stability. We are characterizing mice with conditional loss-of-function (and in some cases gain-of-function) mutations in most enzymes of NAD biosynthetic pathways at the phenotypic and molecular levels to understand how NAD biology impacts health and response to stress. This includes studies of Leber congenital amaurosis-associated Nmnat1 mutants in retinal function, where we have established a mouse model of photoreceptor demise similar to that observed in children with blindness due to Nmnat1 mutations. In addition to these studies of NAD related pathways in axon stability, we also conducted genome-scale screens to identify additional components of the axon degeneration pathway. We use DRG neurons, lentivirus siRNA and gRNA libraries, compound libraries, and quantitative image analysis to identify steps that block or enhance axon breakdown. To establish molecular mechanism for the identified candidates, a variety of methods are used including examination of mouse and Drosophila mutants, studies of Cas9-modified iPSC-derived neurons, molecular structure/function analysis, metabolomics, and proteomics to identify interacting proteins. One of the most exciting candidates obtained from the high-throughput axon degeneration screen is the TLR adaptor called Sarm1. Axons degenerate very poorly in Sarm1-deficient neurons and Sarm1 knockout mice are resistant to axonal damage. Sarm1 is also required for axon degeneration in Drosophila, as it was also identified by another group in a similarly aimed Drosophila screen. Sarm1 structure/function analysis indicates that TIR domain dimerization is necessary for its axon destructive role and that the amino terminal domain houses an auto-inhibitory function. Further, Sarm1 is important for cell destruction after mitochondrial inhibition. This is a distinct form of cell destruction that we have termed sarmoptosis. Ongoing studies to dissect the mechanisms of Sarm1-mediated axon destruction and cell death are focused on clarifying the auto-inhibitory role of the N-terminal domain and identifying downstream components that mediate its executioner role, as well as examining its role in peripheral neuropathy and behavioral abnormalities.

Is Axonal Degeneration a Key Early Event in Parkinson’s Disease? Kurowska Z, Kordower JH, Stoessl AJ, Burke RE, Brundin P, Yue Z, Brady ST, Milbrandt J, Trapp BD, Sherer TB, Medicetty S. Journal of Parkinson’s disease. 2016; 6(4):703-707. PubMed [journal] PMID: 27497486

NMNAT1 inhibits axon degeneration via blockade of SARM1-mediated NAD+ depletion. Sasaki Y, Nakagawa T, Mao X, DiAntonio A, Milbrandt J. eLife. 2016; 5. PubMed [journal] PMID: 27735788 PMCID: PMC5063586

 TMEM184b Promotes Axon Degeneration and Neuromuscular Junction Maintenance. Bhattacharya MR, Geisler S, Pittman SK, Doan RA, Weihl CC, Milbrandt J, DiAntonio A. The Journal of neuroscience : the official journal of the Society for Neuroscience. 2016; 36(17):4681-9. PubMed [journal] PMID: 27122027 PMCID: PMC4846669

Axon Self-Destruction: New Links among SARM1, MAPKs, and NAD+ Metabolism. Gerdts J, Summers DW, Milbrandt J, DiAntonio A. Neuron. 2016; 89(3):449-60. NIHMSID: NIHMS746656 PubMed [journal] PMID: 26844829 PMCID:PMC4742785

Neurobiology. SARM1 activation triggers axon degeneration locally via NAD⁺ destruction.  Gerdts J, Brace EJ, Sasaki Y, DiAntonio A, Milbrandt J.  Science (New York, N.Y.). 2015; 348(6233):453.  PubMed [journal] PMID: 25908823

Mitochondrial dysfunction induces Sarm1-dependent cell death in sensory neurons.  Summers DW, DiAntonio A, Milbrandt J.  The Journal of neuroscience : the official journal of the Society for Neuroscience. 2014; 34(28):9338-50.  PubMed [journal]  PMID: 25009267 PMCID: PMC4087211

Sarm1-mediated axon degeneration requires both SAM and TIR interactions.  Gerdts J, Summers DW, Sasaki Y, DiAntonio A, Milbrandt J.  The Journal of neuroscience : the official journal of the Society for Neuroscience. 2013; 33(33):13569-80.  PubMed [journal]  PMID: 23946415  PMCID: PMC3742939

The Phr1 ubiquitin ligase promotes injury-induced axon self-destruction.  Babetto E, Beirowski B, Russler EV, Milbrandt J, DiAntonio A.  Cell reports. 2013; 3(5):1422-9. NIHMSID: NIHMS470714  PubMed [journal]  PMID: 23665224  PMCID: PMC3671584

SCG10 is a JNK target in the axonal degeneration pathway.  Shin JE, Miller BR, Babetto E, Cho Y, Sasaki Y, et al.  Proceedings of the National Academy of Sciences of the United States of America. 2012; 109(52):E3696-705.  PubMed [journal]  PMID: 23188802  PMCID: PMC3535671

Dual leucine zipper kinase is required for retrograde injury signaling and axonal regeneration.  Shin JE, Cho Y, Beirowski B, Milbrandt J, Cavalli V, et al.  Neuron. 2012; 74(6):1015-22. NIHMSID: NIHMS376514  PubMed [journal]  PMID: 22726832  PMCID: PMC3383631

A model of toxic neuropathy in Drosophila reveals a role for MORN4 in promoting axonal degeneration.  Bhattacharya MR, Gerdts J, Naylor SA, Royse EX, Ebstein SY, et al.  The Journal of neuroscience : the official journal of the Society for Neuroscience. 2012; 32(15):5054-61. NIHMSID: NIHMS370502  PubMed [journal]  PMID: 22496551  PMCID: PMC3336743

Mitofusin2 mutations disrupt axonal mitochondrial positioning and promote axon degeneration.  Misko AL, Sasaki Y, Tuck E, Milbrandt J, Baloh RH.  The Journal of neuroscience : the official journal of the Society for Neuroscience. 2012; 32(12):4145-55. NIHMSID: NIHMS365962  PubMed [journal]  PMID: 22442078  PMCID: PMC3319368

Nicotinamide mononucleotide adenylyl transferase 1 protects against acute neurodegeneration in developing CNS by inhibiting excitotoxic-necrotic cell death.  Verghese PB, Sasaki Y, Yang D, Stewart F, Sabar F, et al.  Proceedings of the National Academy of Sciences of the United States of America. 2011; 108(47):19054-9.  PubMed [journal]  PMID: 22058226  PMCID: PMC3223466

Image-based screening identifies novel roles for IkappaB kinase and glycogen synthase kinase 3 in axonal degeneration.  Gerdts J, Sasaki Y, Vohra B, Marasa J, Milbrandt J.  The Journal of biological chemistry. 2011; 286(32):28011-8.  PubMed [journal]  PMID: 21685387  PMCID: PMC3151046

Axonal degeneration is blocked by nicotinamide mononucleotide adenylyltransferase (Nmnat) protein transduction into transected axons.  Sasaki Y, Milbrandt J.  The Journal of biological chemistry. 2010; 285(53):41211-5.  PubMed [journal]  PMID: 21071441  PMCID: PMC3009846

Amyloid precursor protein cleavage-dependent and -independent axonal degeneration programs share a common nicotinamide mononucleotide adenylyltransferase 1-sensitive pathway.  Vohra BP, Sasaki Y, Miller BR, Chang J, DiAntonio A, et al.  The Journal of neuroscience : the official journal of the Society for Neuroscience. 2010; 30(41):13729-38. NIHMSID: NIHMS244770  PubMed [journal]  PMID: 20943913  PMCID: PMC3104322

TMEM184b Promotes Axon Degeneration and Neuromuscular Junction Maintenance. Bhattacharya MR, Geisler S, Pittman SK, Doan RA, Weihl CC, Milbrandt J, DiAntonio A. The Journal of neuroscience : the official journal of the Society for Neuroscience. 2016; 36(17):4681-9. PubMed [journal] PMID: 27122027 PMCID: PMC4846669

Schwann cell metabolism and axon support

milbrandt_web_imageSchwann cell metabolism and axon support. To define crucial aspects of Schwann cell-mediated axon support, we have produced mutant mice with aberrant metabolic homeostasis in Schwann cells. We have disrupted mitochondrial function (Tfam), glucose metabolism (Hk2, Ogt, Oga), sirtuin activity (Sirt2, Nampt), mTOR pathway (Pten, mTOR, Tsc2), and AMPK function (Lkb1, Ampk, Tak1) specifically in Schwann cells. In many of these mice, we observe axon degeneration and loss without myelin abnormalities, indicating that we have disrupted Schwann cell axonal support functions and uncoupled them from myelination. Conversely, other mutants manifest severe hypomyelination with minimal axonal loss, suggesting that Schwann cell axonal support functions can remain intact in the absence of myelination. Overall, the characterization of these mice highlights the role of Schwann cells in promoting axon integrity via glucose and lipid metabolic homeostasis. We have used extensive Schwann cell transcriptomic analysis in nerve injury paradigms as well as in many of the mutant mice above to identify important Schwann cell support pathways. We are continuing to study how abnormal Schwann cell metabolism contributes to axonal demise, with particular focus on the role of Schwann cell glycolytic intermediates in axonal health.

During these studies we found that disruption of Schwann cell metabolism produces devastating effects on the unmyelinated nociceptive and autonomic axons. These unmyelinated fibers are ensheathed and supported by non-myelinating Schwann cells (nmSCs), cells that are reminiscent of the ensheathing glia present in invertebrates. The severe deficits in unmyelinated fibers in these mice prompted us to explore the functions of nmSCs. These oft-neglected cells are poorly characterized due to the lack of tools needed to genetically perturb them in vivo and study them in in vitro. To address this need, we are developing genetic methods to perturb nmSCs specifically using combinations of regulable recombinases driven by Schwann cell-specific promoters. We are utilizing a number of orthogonal expression profiling approaches to identify genes with optimal expression patterns in nmSCs to drive these modified recombinases. Optimized mice will be used to specifically disrupt metabolism in nmSCs so we can study the relationship between nmSC ‘support functions’ and unmyelinated fiber health.

We have also created a high-throughput system for producing myelinating mouse neuron-Schwann cell co-cultures. This required miniaturization of the culture conditions in 96-well plates and microfluidic chambers, as well as new informatics solutions to quantify myelinated axons. We are currently using this system in conjunction with a number of hypomyelinating mouse mutants to identify compounds that enhance myelination as well as examining axonal support by mutant Schwann cells before and after axon injury.

Schwann Cell O-GlcNAc Glycosylation Is Required for Myelin Maintenance and Axon Integrity. Kim S, Maynard JC, Sasaki Y, Strickland A, Sherman DL, Brophy PJ, Burlingame AL, Milbrandt J. The Journal of neuroscience : the official journal of the Society for Neuroscience. 2016; 36(37):9633-46 PubMed [journal] PMID: 27629714 PMCID: PMC5039245

Metabolic regulator LKB1 is crucial for Schwann cell-mediated axon maintenance.  Beirowski B, Babetto E, Golden JP, Chen YJ, Yang K, et al.  Nature neuroscience. 2014; 17(10):1351-61.  PubMed [journal]  PMID: 25195104

Aberrant Schwann cell lipid metabolism linked to mitochondrial deficits leads to axon degeneration and neuropathy.  Viader A, Sasaki Y, Kim S, Strickland A, Workman CS, et al.  Neuron. 2013; 77(5):886-98. NIHMSID: NIHMS437179  PubMed [journal]  PMID: 23473319  PMCID: PMC3594792

An integrated approach to characterize transcription factor and microRNA regulatory networks involved in Schwann cell response to peripheral nerve injury.  Chang LW, Viader A, Varghese N, Payton JE, Milbrandt J, et al.  BMC genomics. 2013; 14:84.  PubMed [journal]  PMID: 23387820  PMCID: PMC3599357

The AMPK β2 subunit is required for energy homeostasis during metabolic stress.  Dasgupta B, Ju JS, Sasaki Y, Liu X, Jung SR, et al.  Molecular and cellular biology. 2012; 32(14):2837-48.  PubMed [journal]  PMID: 22586267  PMCID: PMC3416196

MicroRNAs modulate Schwann cell response to nerve injury by reinforcing transcriptional silencing of dedifferentiation-related genes.  Viader A, Chang LW, Fahrner T, Nagarajan R, Milbrandt J.  The Journal of neuroscience : the official journal of the Society for Neuroscience. 2011; 31(48):17358-69. NIHMSID: NIHMS387250  PubMed [journal]  PMID: 22131398  PMCID: PMC3388739

Sir-two-homolog 2 (Sirt2) modulates peripheral myelination through polarity protein Par-3/atypical protein kinase C (aPKC) signaling.  Beirowski B, Gustin J, Armour SM, Yamamoto H, Viader A, et al.  Proceedings of the National Academy of Sciences of the United States of America. 2011; 108(43):E952-61.  PubMed [journal]  PMID: 21949390  PMCID: PMC3203793

Schwann cell mitochondrial metabolism supports long-term axonal survival and peripheral nerve function.  Viader A, Golden JP, Baloh RH, Schmidt RE, Hunter DA, et al.  The Journal of neuroscience : the official journal of the Society for Neuroscience. 2011; 31(28):10128-40. NIHMSID: NIHMS310348  PubMed [journal]  PMID: 21752989  PMCID: PMC3147283

Congenital hypomyelinating neuropathy with lethal conduction failure in mice carrying the Egr2 I268N mutation.  Baloh RH, Strickland A, Ryu E, Le N, Fahrner T, et al.  The Journal of neuroscience : the official journal of the Society for Neuroscience. 2009; 29(8):2312-21. NIHMSID: NIHMS97310  PubMed [journal]  PMID: 19244508  PMCID: PMC2679588

Genome engineering of neurons and glia using Cas9 methodologies

indexGenome engineering of neurons and glia using Cas9 methodologies. The ability to make precise modifications to the genome has opened up new opportunities to perform functional genomic analyses. The impact of disease-associated variants on cell physiology can be rapidly assessed in the cell types of interest and subsequent dissection of the involved genetic pathways can be pursued in a facile manner in mammalian systems. We are using the Cas9 system to rapidly generate modified iPSC-derived neurons and glia as well as mutant mice. We are using this system to introduce disease-associated mutations, develop fluorescent markers to identify specific classes of iPSC-derived neurons and glia, produce novel tools for in vivo manipulation of metabolism, alter protein post-translational modification, and modify proteins (e.g. BirA) to facilitate their downstream analysis. We have also developed Cas9 transcriptional activators and repressors and are using them to promote the differentiation of patient-derived fibroblasts and/or iPSCs into neural cell types including Schwann cells. In addition, we are using Cas9-mediated genome-scale libraries to select for suppressors and enhancers of molecules involved in axon degeneration and/or Schwann cell-mediated axonal maintenance. New approaches aimed at using Cas9 technology to perform in vivo screens of nervous system function are being pursued using AAV-mediated delivery of gRNAs.

 

 

Motor neuron responses to abnormal Schwann cell function

Motor neuron responses to abnormal Schwann cell function. In ALS, motor neurons become dysfunctional and eventually die. Multiple studies have demonstrated that ALS is caused by abnormalities in the motor neuron environment, including ineffectual glial support. To explore this glial role, we are using Chat-TRAP mice to profile specifically motor neuron translated mRNAs using RNA-seq. We are comparing transcriptomes of normal motor neurons with those of motor neurons surrounded by genetically modified and dysfunctional Schwann cells as well as those from ALS models. Through these studies we hope to identify motor neuron responses to ineffectual Schwann cell support and determine whether these responses contribute to diseases such as ALS.

Unraveling disease mechanisms in cohesinopathies

Unraveling disease mechanisms in cohesinopathies. Autistic spectrum disorders (ASDs) are genetically complex disorders that cause debilitating mental illness in many children. Studies of monogenic syndromes where autistic features are prominent, such as cohesinopathies like Cornelia de Lange syndrome (CdLS) and Roberts’ syndrome, can provide insights into basic mechanisms underlying autism. For example, mutations in the cohesin protein SMC3 and CTCF cause syndromes associated with severe autistic features. These proteins co-localize to hypomethylated DNA and organize chromatin into three-dimensional loops; in their absence chromatin loops become disorganized and transcription is disrupted, perhaps resulting in the abnormal synaptic plasticity that is thought to underlie the neurologic dysfunction in ASDs.

In studies of mutant mice in which CTCF is deleted in postnatal forebrain neurons, we found that they have profound deficits in sociability and spatial memory. Similar studies are ongoing in SMC and Esco1/2 mutant mice. We plan to compare these mice with respect to behavior, brain connectivity, neuron and glia transcriptomes, DNA methylation (DNA methyl-seq) and chromatin (ChIP-seq, ChiA-PET) to associate molecular deficits with the behavioral abnormalities. Overall, we intend to identify plasticity-relevant epigenomic and transcriptomic defects in CTCF and Cohesin-deficient neurons that hinder depolarization-induced epigenetic changes, and which may provide targets for therapeutic intervention.

Nicotinamide adenine dinucleotide (NAD)-regulated DNA methylation alters CCCTC-binding factor (CTCF)/cohesin binding and transcription at the BDNF locus.  Chang J, Zhang B, Heath H, Galjart N, Wang X, et al.  Proceedings of the National Academy of Sciences of the United States of America. 2010; 107(50):21836-41.  PubMed [journal]  PMID: 21106760  PMCID: PMC3003122

Dosage effects of cohesin regulatory factor PDS5 on mammalian development: implications for cohesinopathies.  Zhang B, Chang J, Fu M, Huang J, Kashyap R, et al.  PloS one. 2009; 4(5):e5232.  PubMed [journal]  PMID: 19412548  PMCID: PMC2672303

Mice lacking sister chromatid cohesion protein PDS5B exhibit developmental abnormalities reminiscent of Cornelia de Lange syndrome.  Zhang B, Jain S, Song H, Fu M, Heuckeroth RO, et al.  Development (Cambridge, England). 2007; 134(17):3191-201.  PubMed [journal]  PMID: 17652350