Efficient generation of lower induced motor neurons by coupling Ngn2 expression with developmental cues.

Human pluripotent stem cells (hPSCs) are a powerful tool for disease modeling of hard-to-access tissues (such as the brain). Current protocols either direct neuronal differentiation with small molecules or use transcription-factor-mediated programming. In this study, we couple overexpression of transcription factor Neurogenin2 (Ngn2) with small molecule patterning to differentiate hPSCs into lower induced motor neurons (liMoNes/liMNs). This approach induces canonical MN markers including MN-specific Hb9/MNX1 in more than 95% of cells. liMNs resemble bona fide hPSC-derived MN, exhibit spontaneous electrical activity, express synaptic markers, and can contact muscle cells in vitro. Pooled, multiplexed single-cell RNA sequencing on 50 hPSC lines reveals reproducible populations of distinct subtypes of cervical and brachial MNs that resemble their in vivo, embryonic counterparts. Combining small molecule patterning with Ngn2 overexpression facilitates high-yield, reproducible production of disease-relevant MN subtypes, which is fundamental in propelling our knowledge of MN biology and its disruption in disease.

Loss of mouse Stmn2 function causes motor neuropathy.

Amyotrophic lateral sclerosis (ALS) is characterized by motor neuron degeneration accompanied by aberrant accumulation and loss of function of the RNA-binding protein TDP43. Thus far, it remains unresolved to what extent TDP43 loss of function directly contributes to motor system dysfunction. Here, we employed gene editing to find whether the mouse ortholog of the TDP43-regulated gene STMN2 has an important function in maintaining the motor system. Both mosaic founders and homozygous loss-of-function Stmn2 mice exhibited neuromuscular junction denervation and fragmentation, resulting in muscle atrophy and impaired motor behavior, accompanied by an imbalance in neuronal microtubule dynamics in the spinal cord. The introduction of human STMN2 through BAC transgenesis was sufficient to rescue the motor phenotypes observed in Stmn2 mutant mice. Collectively, our results demonstrate that disrupting the ortholog of a single TDP43-regulated RNA is sufficient to cause substantial motor dysfunction, indicating that disruption of TDP43 function is likely a contributor to ALS.

Connecting TDP-43 Pathology with Neuropathy.

Transactive response DNA-binding protein 43 kDa (TDP-43), a multifunctional nucleic acid-binding protein, is a primary component of insoluble aggregates associated with several devastating nervous system disorders; mutations in TARDBP, its encoding gene, are a cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here, we review established and emerging roles of TDP-43 and consider how its dysfunction impinges on RNA homeostasis in the nervous system, thereby contributing to neural degeneration. Notably, improper splicing of the axonal growth-associated factor STMN2 has recently been connected to TDP-43 dysfunction, providing a mechanistic link between TDP-43 proteinopathies and neuropathy. This review highlights how a deep understanding of the function of TDP-43 in the brain might be leveraged to develop new targeted therapies for several neurological disorders.

ALS-implicated protein TDP-43 sustains levels of STMN2, a mediator of motor neuron growth and repair.

The findings that amyotrophic lateral sclerosis (ALS) patients almost universally display pathological mislocalization of the RNA-binding protein TDP-43 and that mutations in its gene cause familial ALS have nominated altered RNA metabolism as a disease mechanism. However, the RNAs regulated by TDP-43 in motor neurons and their connection to neuropathy remain to be identified. Here we report transcripts whose abundances in human motor neurons are sensitive to TDP-43 depletion. Notably, expression of STMN2, which encodes a microtubule regulator, declined after TDP-43 knockdown and TDP-43 mislocalization as well as in patient-specific motor neurons and postmortem patient spinal cord. STMN2 loss upon reduced TDP-43 function was due to altered splicing, which is functionally important, as we show STMN2 is necessary for normal axonal outgrowth and regeneration. Notably, post-translational stabilization of STMN2 rescued neurite outgrowth and axon regeneration deficits induced by TDP-43 depletion. We propose that restoring STMN2 expression warrants examination as a therapeutic strategy for ALS.

Regulation of KIF1A-Driven Dense Core Vesicle Transport: Ca2+/CaM Controls DCV Binding and Liprin-α/TANC2 Recruits DCVs to Postsynaptic Sites.

Tight regulation of neuronal transport allows for cargo binding and release at specific cellular locations. The mechanisms by which motor proteins are loaded on vesicles and how cargoes are captured at appropriate sites remain unclear. To better understand how KIF1A-driven dense core vesicle (DCV) transport is regulated, we identified the KIF1A interactome and focused on three binding partners, the calcium binding protein calmodulin (CaM) and two synaptic scaffolding proteins: liprin-α and TANC2. We showed that calcium, acting via CaM, enhances KIF1A binding to DCVs and increases vesicle motility. In contrast, liprin-α and TANC2 are not part of the KIF1A-cargo complex but capture DCVs at dendritic spines. Furthermore, we found that specific TANC2 mutations-reported in patients with different neuropsychiatric disorders-abolish the interaction with KIF1A. We propose a model in which Ca2+/CaM regulates cargo binding and liprin-α and TANC2 recruit KIF1A-transported vesicles.