Silencing the mitotic checkpoint requires that new MCC formation is prevented and pre-existing MCC is disassembled. This will release the APC/C-Cdc20 from inhibition so it can resume its activity and promote anaphase onset. Very recent studies

have hypothesized that the joint action of the AAA+ ATPase TRIP13 and an adapter protein p31comet can mediate the catalytic disassociation of the mitotic checkpoint protein Mad2 from Cdc20-Mad2 complexes or from the four protein complex BubR1-Bub3-Mad2-Cdc20 known as the mitotic checkpoint complex (MCC). Furthermore, using purified components in vitro, TRIP13 ATPase-dependent activity has been shown to convert closed, “active” Mad2, capable of binding to Cdc20, into its inactive “open” conformer. This is only in conjunction with p31comet, which is proposed to act as an adapter to facilitate recognition of closed Mad2 and deliver it to TRIP13. Viewing all evidence, key mechanistic questions are unresolved, most specifically the role(s) of TRIP13 and p31comet in mitotic checkpoint silencing. An ATP-hydrolysis-deficient mutant of TRIP13 (E253Q), is shown to stably capture its substrates, as its ATP-hydrolysis activity is required for release and remodeling of its substrates (48). By utilizing this mutant we can view TRIP13 substrate binding and elucidate TRIP13 function. Using all purified components, I performed disassembly assays to identify whether TRIP13 and p31comet can disassemble both the initiator of MCC, the Mad1-Mad2 template, and a pre-existing potent inhibitor of the APC/C-Cdc20, the MCC. We can conclude that the joint activity of TRIP13 and p31comet, in the presence of ATP, do not disassemble Mad2 from the Mad1-Mad2 core complex. Additionally, the MCC disassembly assay shows good potential to allow us to discern if there is a role for TRIP13 and p31comet in disassembling the MCC

Stathmin-2 (also known as SCG10) is encoded by one of the most abundantly expressed mRNAs in human motor neurons and is required for axonal regeneration after axotomy in cultured human motor neurons. It has long been associated with growth, maintenance, and homeostasis of neuronal axons. This function is believed to be due to stathmin-2’s ability to bind to two α/β-tubulin heterodimers and correspondingly to affect microtubule dynamics. Moreover, in almost all instances of the fatal, paralytic motor neuron disease ALS, stathmin-2 encoding mRNAs are suppressed in motor neurons as a consequence of missplicing STMN2 pre-mRNA through a mechanism directly dependent on nuclear loss of the RNA binding protein TDP-43. Restoration of stathmin-2 is sufficient to rescue axonal regeneration capacity after axotomy injury in human motor neurons depleted of TDP-43. Stathmin-2 has also been previously correlated with axon regeneration in vivo – multiple investigators have found both mRNA and protein levels increase specifically after injury in the nervous system in instances where regeneration is occurring. Here I show that stathmin-2, despite being a tubulin binding partner in vitro, does not play a major role in microtubule levels or assembly in cultured motor neurons; in fact, I show that the tubulin binding ability of stathmin-2 is not required for axon regeneration of axons in such neurons. I also show that stathmin-2 is transported in fast axonal transport in a tubulin-binding-independent, palmitoylation-dependent manner, and that it can be transported in groups of ~4 round stathmin-2-positive compartments within the axon. It does not co-localize or co-transport with any known canonical axonal cargo marker, but is co-transported with NMNAT2, a protein implicated in the DLK-dependent signaling pathway and Wallerian degeneration (as is stathmin-2). I also demonstrate that stathmin-2 is required for axon regeneration of motor axons in the murine nervous system, with half the normal level of stathmin-2 sufficient for full restoration of regeneration capability. Indeed, absence of stathmin-2 led to an apparent abundant degeneration of axons within the sciatic nerve at sites distal to the injury site, while for wild-type and heterozygous mice those markers were relatively less abundant. Thus, while the molecular events and partners through which stathmin-2 maintains axonal health and innervation capability, my evidence establishes that restoration of stathmin-2 levels represents an attractive therapeutic target not only for ALS and other TDP-43 proteinopathies, but possibly even for acute injuries of the nervous system.

Among all the organs, the brain has the most heterogeneous cellular composition, which mostly remained unclear. With recent advances in omics, the required technologies for unlocking the mysteries of brain heterogeneity are emerging. After reviewing different techniques, we strived to make a periodic table of brain cell types using in situ hybridization techniques using mice as model organisms.

TDP-43 proteinopathies, including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and Alzheimer’s disease (AD), commonly share features of nuclear clearance and cytoplasmic accumulation of the RNA-binding protein TDP-43 within affected neurons. TDP-43 suppression in an adult nervous system changes expression and splicing of RNAs encoded by hundreds of genes, however, the functional consequences and relevance to neurodegeneration of these changes have proven elusive. Our team recently discovered that TDP-43 suppression in human cells drives use of cryptic splice and polyadenylation sites in the pre-mRNA encoded by the STMN2 gene (aka SCG10), leading to loss of the stathmin-2 protein it encodes, preventing axonal recovery after injury. This discovery provides a new gene target in TDP-43 proteinopathy and links dysregulated RNA metabolism to impaired microtubule dynamics, two pathways suggested independently without recognized pathophysiologic commonality. I here demonstrate that concomitant loss of full length stathmin-2 mRNA and accumulation of its cryptically spliced and truncated form is a hallmark of TDP-43 proteinopathy. I show that removal of the proposed TDP-43 binding sites within intron 1 of the stathmin-2 pre-mRNA drives constitutive misprocessing. I determine TDP-43’s role at this site is to sterically block cryptic site utilization, as synthetically targeting other RNA binding proteins or RNA-targeted CRISPR effectors to this locus restores correct stathmin-2 pre-mRNA maturation. By eliminating either cryptic processing site, I determine that cryptic splicing, not cryptic polyadenylation, is the primary driver of stathmin-2 pre-mRNA misprocessing. Next, a therapeutic strategy for TDP-43 proteinopathies is identified using steric binding antisense oligonucleotides (ASOs) to maintain or restore stathmin-2 pre-mRNA maturation and rescue axonal regeneration in human motor neurons with TDP-43 deficiency. I developed AAV-based experimental approaches to modulate stathmin-2 in an adult mammalian central nervous system, including an AAV delivered RNAi against the stathmin-2 pre-mRNA that is used to determine the consequences of chronic reduction of murine stathmin-2, as well as gene therapy cargos to supplement stathmin-2 expression selectively in neurons with dysfunctional TDP-43. Finally, the STMN2 gene is humanized by knock-in of human exon 2a into the corresponding region of murine STMN2, with or without TDP-43 binding sites. I show humanized mice without the STMN2 TDP-43 binding sites constitutively misprocess stathmin-2 pre-mRNA, enabling therapy development in TDP-43 proteinopathies by identification of stathmin-2-restoring ASOs.