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Cytokinesis is the final stage of cell division. Successful cytokinesis requires membrane trafficking pathways regulated by Rabs, molecular switches activated by guanine nucleotide exchange factors (GEFs). Late in cytokinesis, an intercellular cytokinetic bridge (ICB) connecting the two daughter cells undergoes abscission, which requires depolymerization of actin. Rab35 recruits MICAL1 to oxidate and depolymerize actin filaments. We report that DENND2B, a protein previously implicated in cancer, mental retardation and multiple congenital disorders functions as a GEF for Rab35 and recruits and activates the GTPase at the ICB. Unexpectedly, the N terminal region of DENND2B interacts with an active mutant of Rab35, suggesting that DENND2B is both a Rab35 GEF and effector. Knockdown of DENND2B delays abscission resulting in increased multinucleated cells and over-accumulation of F-actin at the ICB. F-actin accumulation leads to formation of a chromatin bridge, a process known to activate the NoCut/abscission checkpoint, and DENND2B knockdown actives Aurora B kinase, a hallmark of checkpoint activation. This study identifies DENND2B as a crucial player in cytokinetic abscission and provides insight into the multisystem disorder associated with DENND2B mutation.

Lewy bodies (LBs), rich in α-synuclein, are a hallmark of Parkinson′s disease (PD). Understanding their biogenesis is likely to provide insight into the pathophysiology of PD, yet a cellular model for LB formation remains elusive. The realization that immune challenge is a trigger for neurodevelopmental disease has been a breakthrough in the understanding of PD. Here, iPSC-derived human dopaminergic (DA) neurons from multiple healthy donors were found to form LB-like inclusions following treatment with α-synuclein preformed fibrils, but only when coupled to an immune challenge (interferon-gamma) or when co-cultured with activated microglia. Human cortical neurons derived from the same iPSC lines did not form LB-like inclusions. Exposure to interferon-gamma impairs autophagy in a lysosomal-specific manner in vitro similar to the disruption of proteostasis pathways that contribute to PD. We find that lysosomal membrane proteins LAMP1 and LAMP2 and transcription factors regulating lysosomal biogenesis and function are downregulated in DA but not cortical neurons. Finally, due to the excellent sample preservation afforded by cells compared to post-mortem PD brain tissue, we conclude that the LB-like inclusions in DA neurons are membrane-bound, suggesting they are not limited to the cytoplasmic compartment.

Epileptic encephalopathies comprise a clinically and genetically heterogenous group of disorders characterized by global developmental delay and ongoing seizure activity. It is generally considered that seizure activity is the primary pathology and leads to altered cognitive function. However, epileptic encephalopathy can also result from primary defects in neurodevelopment that lead to seizures. Here we examine the symptomology of individuals with epileptic encephalopathy resulting from biallelic pathogenic variants in DENND5A, encoding a protein involved in intracellular membrane trafficking. We established a cohort of 15 individuals from 14 families with 21 unique variants in DENND5A and analyzed phenotypic surveys answered by their treating clinicians. We obtained cells derived from several cohort members and examined DENND5A expression and stability, and we examined the role of DENND5A in symmetric cell division capability. Finally, we generated a mouse line expressing a homozygous mutation found in the cohort and analyzed brain morphology in vivo using a 7 Tesla magnetic resonance imaging system. Our cohort study reveals that global developmental delay, gross abnormalities in brain development including ventriculomegaly and corpus callosum dysgenesis, seizures, microcephaly and hypotonia are found in the majority of the cohort. Patient-derived neural precursor cells from induced pluripotent stem cells, as well as patient-derived lymphoblasts, display loss of DENND5A protein and exhibit problems with symmetric cell division, an essential process in early neural development. The introduction of a homozygous human point mutation into the conserved sequence in mouse leads to an animal with reduced brain volume and widespread regional brain dysgenesis and ventriculomegaly, phenotypes shared with the human cohort. Taken together, our multi-dimensional study establishes DENND5A as a critical gene during neurodevelopment and that biallelic loss of function variants, including missense and truncating variants as well as intronic splice site variants, result in a developmental epileptic encephalopathy.

The nervous system spread of alpha-synuclein fibrils leads to Parkinson′s disease (PD) and other synucleinopathies, yet the mechanisms underlying internalization and cell-to-cell transfer are enigmatic. Here we use confocal and superresolution microscopy, subcellular fractionation and electron microscopy of immunogold labelled alpha-synuclein pre-formed fibrils (PFF) to demonstrate that this toxic protein species enters cells using a novel form of ultra-rapid macropinocytosis with transfer to lysosomes in as little as 2 minutes, an unprecedented cell biological kinetic for lysosomal targeting. PFF uptake circumvents classical endosomal pathways and is independent of clathrin. Immunogold-labelled PFF are seen at the highly curved inward edge of membrane ruffles, in newly formed macropinosomes, and in lysosomes. While many of the fibrils remain in lysosomes that continue to take up PFF for hours, a portion are transferred to neighboring naive cells on the external face of vesicles, likely exosomes. These data indicate that PFF uses a novel internalization mechanism as a component of cell-to-cell propagation.