Biology subjects

Bellen, H. J.

Publications and source records attributed to Bellen, H. J..

26 records · Page 2Linked to original sources

bioRxiv2021

Neuronal ROS-Induced Glial Lipid Droplet Formation is Altered by Loss of Alzheimers Disease-associated Genes

A growing list of Alzheimers disease (AD) genetic risk factors is being identified, but the contribution of these genetic mutations to disease remains largely unknown. Accumulating data support a role of lipid dysregulation and excessive ROS in the etiology of AD. Here, we identified cell-specific roles for eight AD risk-associated genes in ROS-induced glial lipid droplet (LD) formation. We demonstrate that ROS-induced glial LD formation requires two ABCA transporters (ABCA1 and ABCA7) in neurons, the APOE receptor (LRP1), endocytic genes (PICALM, CD2AP, and AP2A2) in glia, and retromer genes (VPS26 and VPS35) in both neurons and glia. Moreover, ROS strongly enhances A{beta}42-toxicity in flies and A{beta}42-plaque formation in mice. Finally, an ABCA1-activating peptide restores glial LD formation in the APOE4-associated loss of LD. This study places AD risk factors in a neuron-to-glia lipid transfer pathway with a critical role in protecting neurons from ROS-induced toxicity.

genetics↗

bioRxiv2021

Low doses of the organic insecticide spinosad trigger lysosomal defects, ROS driven lipid dysregulation and neurodegeneration in flies

The plight of insect populations around the world and the threats it poses to agriculture and ecosystems has thrown insecticide use into the spotlight. Spinosad is an organic insecticide, considered less harmful to beneficial insects than synthetic insecticides, but its mode of action remains unclear. Using Drosophila, we show that low doses of spinosad reduce cholinergic response in neurons by antagonizing D6 nAChRs. D6 nAChRs are transported to lysosomes that become enlarged and accumulate upon spinosad treatment. Oxidative stress is initiated in the central nervous system, and spreads to midgut and disturbs lipid storage in metabolic tissues in a D6-dependent manner. Spinosad toxicity was ameliorated with the antioxidant N-Acetylcysteine amide (NACA). Chronic exposures lead to mitochondrial defects, severe neurodegeneration and blindness in adult animals. The many deleterious effects of low doses of this insecticide reported here point to an urgent need for rigorous investigation of its impacts on beneficial insects.

neuroscience↗

bioRxiv2020

Two neuronal peptides encoded from a single transcript regulate mitochondrial function in Drosophila

Naturally produced peptides (<100 amino acids) are important regulators of physiology, development, and metabolism. Recent studies have predicted that thousands of peptides may be translated from transcripts containing small open reading frames (smORFs). Here, we describe two peptides in Drosophila encoded by conserved smORFs, Sloth1 and Sloth2. These peptides are translated from the same bicistronic transcript and share sequence similarities, suggesting that they encode paralogs. Yet, Sloth1 and Sloth2 are not functionally redundant, and loss of either peptide causes animal lethality, reduced neuronal function, impaired mitochondrial function, and neurodegeneration. We provide evidence that Sloth1/2 are highly expressed in neurons, imported to mitochondria, and regulate mitochondrial complex III assembly. These results suggest that phenotypic analysis of smORF genes in Drosophila can provide a wealth of information on the biological functions of this poorly characterized class of genes.

genomics↗

bioRxiv2020

TFEB/Mitf links impaired nuclear import to autophagolysosomal dysfunction in C9-ALS

Disrupted nucleocytoplasmic transport (NCT) has been implicated in neurodegenerative disease pathogenesis; however, the mechanisms by which impaired NCT causes neurodegeneration remain unclear. In a Drosophila screen, we identified Ref(2)p/p62, a key regulator of autophagy, as a potent suppressor of neurodegeneration caused by the GGGGCC hexanucleotide repeat expansion (G4C2 HRE) in C9orf72 that causes amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). We found that p62 is increased and forms ubiquitinated aggregates due to decreased autophagic cargo degradation. Immunofluorescence and electron microscopy of Drosophila tissues demonstrate an accumulation of lysosome-like organelles that precedes neurodegeneration. These phenotypes are partially caused by cytoplasmic mislocalization of Mitf/TFEB, a key transcriptional regulator of autophagolysosomal function. Additionally, TFEB is mislocalized and downregulated in human cells expressing GGGGCC repeats and in C9-ALS patient motor cortex. Our data suggest that the C9orf72-HRE impairs Mitf/TFEB nuclear import, thereby disrupting autophagy and exacerbating proteostasis defects in C9-ALS/FTD.

cell biology↗

bioRxiv2019

Retromer subunit, VPS29, regulates synaptic transmission and is required for endolysosomal function in the aging brain

Retromer, including Vps35, Vps26, and Vps29, is a protein complex responsible for recycling proteins within the endolysosomal pathway. Although implicated in both Parkinsons and Alzheimers disease, our understanding of retromer function in the adult brain remains limited, in part because Vps35 and Vps26 are essential for development. In Drosophila, we find that Vps29 is dispensable for embryogenesis but required for retromer function in aging adults, including for synaptic transmission, survival, and locomotion. Unexpectedly, in Vps29 mutants, Vps35 and Vps26 proteins are normally expressed and associated, but retromer is mislocalized from neuropil to soma with the Rab7 GTPase. Further, Vps29 phenotypes are suppressed by reducing Rab7 or overexpressing the GTPase activating protein, TBC1D5. With aging, retromer insufficiency triggers progressive endolysosomal dysfunction, with ultrastructural evidence of impaired substrate clearance and lysosomal stress. Our results reveal the role of Vps29 in retromer localization and function, highlighting requirements for brain homeostasis in aging.\n\nImpact StatementVps29 promotes retromer localization in the adult Drosophila brain, engaging Rab7 and TBC1D5, and its loss triggers age-dependent neuronal impairments in endolysosomal trafficking and synaptic transmission.

cell biology↗

bioRxiv2019

De novo EIF2AK1 and EIF2AK2 variants are associated with developmental delay, leukoencephalopathy, and neurologic decompensation

EIF2AK1 and EIF2AK2 encode members of the Eukaryotic Translation Initiation Factor 2 Alpha Kinase (EIF2AK) family that inhibits protein synthesis in response to physiologic stress conditions. EIF2AK2 is also involved in innate immune response and the regulation of signal transduction, apoptosis, cell proliferation, and differentiation. Despite these findings, human disorders associated with deleterious variants in EIF2AK1 and EIF2AK2 have not been reported. Here, we describe the identification of eight unrelated individuals with heterozygous de novo missense variants in EIF2AK1 (1/8) or EIF2AK2 (7/8). Features seen in these eight individuals include white matter alterations (8/8), developmental delay (8/8), impaired language (8/8), cognitive impairment (7/8), ataxia (6/8), dysarthria in probands with verbal ability (6/6), hypotonia (6/8), hypertonia (5/8), and involuntary movements (3/8). Individuals with EIF2AK2 variants also exhibit neurological regression in the setting of febrile illness or infection. We use mammalian cell lines and patient-derived fibroblasts to further confirm the pathogenicity of variants in these genes and found reduced kinase activity. EIF2AKs phosphorylate Eukaryotic Translation Initiation Factor 2 Subunit 1, (EIF2S1, also known as EIF2), which then inhibits EIF2B activity. Deleterious variants in genes encoding EIF2B proteins cause childhood ataxia with central nervous system hypomyelination/vanishing white matter disease (CACH/VWM), a leukoencephalopathy characterized by neurologic regression in the setting of febrile illness and other stressors. Our findings indicate that EIF2AK2 missense variants cause a neurodevelopmental syndrome that may share phenotypic and pathogenic mechanisms with CACH/VWM.

genetics↗

bioRxiv2019

An efficient CRISPR-based strategy to insert small and large fragments of DNA using short homology arms

We previously reported a CRISPR-mediated knock-in strategy into introns of Drosophila genes, generating an attP-FRT-SA-T2A-GAL4-polyA-3XP3-EGFP-FRT-attP transgenic library for multiple uses (Lee et al., 2018b). The method relied on double stranded DNA (dsDNA) homology donors with [~]1 kb homology arms. Here, we describe three new simpler ways to edit genes in flies. We create single stranded DNA (ssDNA) donors using PCR and add 100 nt of homology on each side of an integration cassette, followed by enzymatic removal of one strand. Using this method, we generated GFP-tagged proteins that mark organelles in S2 cells. We then describe two dsDNA methods using cheap synthesized donors flanked by 100 nt homology arms and gRNA target sites cloned into a plasmid. Upon injection, donor DNA (1 to 5 kb) is released from the plasmid by Cas9. The cassette integrates efficiently and precisely in vivo. The approach is fast, cheap, and scalable.

genomics↗

bioRxiv2019

ANKLE2, a target of Zika virus, controls asymmetric cell division of neuroblasts and uncovers a novel microcephaly pathway

Neuroblasts in flies divide asymmetrically by establishing polarity, distributing cell fate determinants asymmetrically, and positioning their spindle for cell division. The apical complex contains aPKC, Bazooka (Par3), and Par6, and its activity depends on L(2)gl. We show that Ankle2 interacts with L(2)gl and affects aPKC. Reducing Ankle2 levels disrupts ER and nuclear envelope morphology, releasing the kinase Ballchen/VRK1 into the cytosol. These defects are associated with reduced phosphorylation of aPKC, disruption of Par complex localization, and spindle alignment defects. Importantly, removal of one copy of ballchen/VRK1 or l(2)gl suppresses the loss of Ankle2 and restores viability and brain size. The Zika virus NS4A protein interacts with Drosophila Ankle2 and VRK1 in dividing neuroblasts. Human mutational studies implicate this neural cell division pathway in microcephaly and motor neuron disease. In summary, NS4A, ANKLE2, VRK1 and LLGL1 define a novel pathway that impinges on asymmetric determinants of neural stem cell division.

genetics↗