Serine/Threonine Protein Phosphatase 2A Regulates the Transport of Axonal Mitochondria.

Microtubule-based transport provides mitochondria to distant regions of neurons and is essential for neuronal health. To identify compounds that increase mitochondrial motility, we screened 1,641 small-molecules in a high-throughput screening platform. Indirubin and cantharidin increased mitochondrial motility in rat cortical neurons. Cantharidin is known to inhibit protein phosphatase 2A (PP2A). We therefore tested two other inhibitors of PP2A: LB-100 and okadaic acid. LB-100 increased mitochondrial motility, but okadaic acid did not. To resolve this discrepancy, we knocked down expression of the catalytic subunit of PP2A (PP2CA). This long-term inhibition of PP2A more than doubled retrograde transport of axonal mitochondria, confirming the importance of PP2A as a regulator of mitochondrial motility and as the likely mediator of cantharidin's effect.

APP and DYRK1A regulate axonal and synaptic vesicle protein networks and mediate Alzheimer's pathology in trisomy 21 neurons.

Trisomy 21 (T21) causes Down syndrome and an early-onset form of Alzheimer's disease (AD). Here, we used human induced pluripotent stem cells (hiPSCs) along with CRISPR-Cas9 gene editing to investigate the contribution of chromosome 21 candidate genes to AD-relevant neuronal phenotypes. We utilized a direct neuronal differentiation protocol to bypass neurodevelopmental cell fate phenotypes caused by T21 followed by unbiased proteomics and western blotting to define the proteins dysregulated in T21 postmitotic neurons. We show that normalization of copy number of APP and DYRK1A each rescue elevated tau phosphorylation in T21 neurons, while reductions of RCAN1 and SYNJ1 do not. To determine the T21 alterations relevant to early-onset AD, we identified common pathways altered in familial Alzheimer's disease neurons and determined which of these were rescued by normalization of APP and DYRK1A copy number in T21 neurons. These studies identified disruptions in T21 neurons in both the axonal cytoskeletal network and presynaptic proteins that play critical roles in axonal transport and synaptic vesicle cycling. These alterations in the proteomic profiles have functional consequences: fAD and T21 neurons exhibit dysregulated axonal trafficking and T21 neurons display enhanced synaptic vesicle release. Taken together, our findings provide insights into the initial molecular alterations within neurons that ultimately lead to synaptic loss and axonal degeneration in Down syndrome and early-onset AD.

A De Novo RAPGEF2 Variant Identified in a Sporadic Amyotrophic Lateral Sclerosis Patient Impairs Microtubule Stability and Axonal Mitochondria Distribution.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that is frequently linked to microtubule abnormalities and mitochondrial trafficking defects. Whole exome sequencing (WES) of patient-parent trios has proven to be an efficient strategy for identifying rare de novo genetic variants responsible for sporadic ALS (sALS). Using a trio-WES approach, we identified a de novo RAPGEF2 variant (c.4069G>A, p.E1357K) in a patient with early-onset sALS. To assess the pathogenic effects of this variant, we have used patient-derived skin fibroblasts and motor neuron-specific overexpression of the RAPGEF2-E1357K mutant protein in Drosophila. Patient fibroblasts display reduced microtubule stability and defective microtubule network morphology. The intracellular distribution, ultrastructure, and function of mitochondria are also impaired in patient cells. Overexpression of the RAPGEF2 mutant in Drosophila motor neurons reduces the stability of axonal microtubules and disrupts the distribution of mitochondria to distal axons and neuromuscular junction (NMJ) synapses. We also show that the recruitment of the pro-apoptotic protein BCL2-associated X (BAX) to mitochondria is significantly increased in patient fibroblasts compared with control cells. Finally, increasing microtubule stability through pharmacological inhibition of histone deacetylase 6 (HDAC6) rescues defects in the intracellular distribution of mitochondria and BAX. Overall, our data suggest that the RAPGEF2 variant identified in this study can drive ALS-related pathogenic effects through microtubule dysregulation.

The Rap activator Gef26 regulates synaptic growth and neuronal survival via inhibition of BMP signaling.

In Drosophila, precise regulation of BMP signaling is essential for normal synaptic growth at the larval neuromuscular junction (NMJ) and neuronal survival in the adult brain. However, the molecular mechanisms underlying fine-tuning of BMP signaling in neurons remain poorly understood. We show that loss of the Drosophila PDZ guanine nucleotide exchange factor Gef26 significantly increases synaptic growth at the NMJ and enhances BMP signaling in motor neurons. We further show that Gef26 functions upstream of Rap1 in motor neurons to restrain synaptic growth. Synaptic overgrowth in gef26 or rap1 mutants requires BMP signaling, indicating that Gef26 and Rap1 regulate synaptic growth via inhibition of BMP signaling. We also show that Gef26 is involved in the endocytic downregulation of surface expression of the BMP receptors thickveins (Tkv) and wishful thinking (Wit). Finally, we demonstrate that loss of Gef26 also induces progressive brain neurodegeneration through Rap1- and BMP signaling-dependent mechanisms. Taken together, these results suggest that the Gef26-Rap1 signaling pathway regulates both synaptic growth and neuronal survival by controlling BMP signaling.