Biology subjects

Pallanck, L. J.

Publications and source records attributed to Pallanck, L. J..

7 recordsLinked to original sources

bioRxiv2026

GBA1 deficiency differentially affects endolysosomal trafficking in neurons versus astrocytes

Mutations in the gene glucosidase, beta acid 1 (GBA1) are the strongest genetic risk factor for Parkinsons disease (PD) and are associated with faster disease progression. GBA1 is expressed in all cell types of the central nervous system, with some evidence supporting higher expression in glial cells than neurons. To elucidate possible specific functions in neurons versus glia, we differentiated human induced pluripotent stem cells (iPSCs) generated from an individual with PD heterozygous for the GBA1 pathogenic null variant IVS2+1 (GBA1IVS/+), homozygous GBA1 IVS2+1 isogenic to GBA1IVS/+ (GBA1IVS/IVS) and a healthy unaffected age-and sex-matched individual (GBA1+/+). GBA1 expression was reduced in GBA1IVS/+ and GBA1IVS/IVS neurons and astrocytes. Endolysosomal trafficking was significantly altered in GBA1-deficient neurons with enlarged early and recycling endosome and lysosome compartments in neurons but not in astrocytes. High molecular weight oligomerization of -synuclein and phosphorylated Ser129 -synuclein were present in GBA1IVS/+ and GBA1IVS/IVS neurons but not in GBA1+/+ neurons, or in GBA1-deficient or GBA1+/+ astrocytes. Transcriptomic analysis of GBA1-deficient neurons and astrocytes revealed cell-type specific profiles. GBA1 deficiency in neurons downregulated immune response and upregulated cholesterol synthesis pathways, while GBA1 deficiency in astrocytes downregulated genes associated with translation and upregulated genes involved in extracellular matrix biogenesis. Transcriptomic analysis also suggests that GBA1 deficiency induces neurotoxic reactivity in astrocytes. Together, these findings indicate that GBA1 deficiency has cell type-specific effects, with increased neuronal vulnerability to endolysosomal trafficking leading to -synucleinopathy while GBA1 deficiency in astrocytes leads to increased neurotoxic reactivity independent of endolysosomal trafficking and -synucleinopathy. HighlightsO_LIiPSC-derived neurons and astrocytes modeled GBA1 deficiency C_LIO_LIEndolysosomal trafficking defects occurred only in GBA1-deficient neurons C_LIO_LI-synuclein oligomers accumulated in GBA1-deficient neurons, not astrocytes C_LIO_LIAstrocyte GBA1 loss drove neurotoxic reactive gene signatures C_LIO_LIGBA1 deficiency causes cell-type specific pathology C_LI

neuroscience↗

bioRxiv2024

A Drosophila model for mechanistic investigation of tau protein spread

Brain protein aggregates are a hallmark of neurodegenerative disease. Previous work indicates that specific protein components of these aggregates are toxic, including tau in Alzheimers disease and related tauopathies. Increasing evidence also indicates that these toxic proteins traffic between cells in a prion-like fashion, thereby spreading pathology from one brain region to another. However, the mechanisms involved in trafficking are poorly understood. We therefore developed a transgenic Drosophila model to facilitate rapid evaluation of candidate tau trafficking modifiers. Our model uses the bipartite Q system to drive co-expression of tau and GFP in the fly eye. We find age-dependent tau spread into the brain, represented by detection of tau, but not GFP in the brain. We also found that tau trafficking was attenuated upon inhibition of the endocytic factor dynamin or the kinase glycogen synthase kinase-3{beta} (GSK-3{beta}). Further work revealed that dynamin promotes tau uptake in recipient tissues, whereas GSK-3{beta} appears to promote tau spread via direct phosphorylation of tau. Our robust and flexible system will promote the identification of tau trafficking components involved in the pathogenesis of neurodegenerative diseases. SUMMARY STATEMENTThe trafficking of toxic proteins in neurodegenerative disease is well-known but poorly understood. Our model will allow rapid and new insight into molecular mechanisms underlying this process.

neuroscience↗

bioRxiv2023

Glucocerebrosidase deficiency leads to neuropathology via cellular immune activation

Mutations in GBA (glucosylceramidase beta), which encodes the lysosomal enzyme glucocerebrosidase (GCase), are the strongest genetic risk factor for the neurodegenerative disorders Parkinsons disease (PD) and Lewy body dementia. Recent work has suggested that neuroinflammation may be an important factor in the risk conferred by GBA mutations. We therefore systematically tested the contributions of immune-related genes to neuropathology in a Drosophila model of GCase deficiency. We identified target immune factors via RNA-Seq and proteomics on heads from GCase-deficient flies, which revealed both increased abundance of humoral factors and increased macrophage activation. We then manipulated the identified immune factors and measured their effect on head protein aggregates, a hallmark of neurodegenerative disease. Genetic ablation of humoral (secreted) immune factors did not suppress the development of protein aggregation. By contrast, re-expressing Gba1b in activated macrophages suppressed head protein aggregation in Gba1b mutants and rescued their lifespan and behavioral deficits. Moreover, reducing the GCase substrate glucosylceramide in activated macrophages also ameliorated Gba1b mutant phenotypes. Taken together, our findings show that glucosylceramide accumulation due to GCase deficiency leads to macrophage activation, which in turn promotes the development of neuropathology. Author SummaryMutations in the gene GBA are the largest risk factor for developing Parkinsons disease and Lewy body dementia, diseases in which important brain cells die. We know that the immune system can be involved in these diseases, and that GBA mutations cause immune changes. We did experiments to learn how the immune system changes could make brain cells more likely to die. Using a fruit fly that was missing the fly version of GBA, we found out that inappropriately activated immune cells, but not secreted immune proteins, were important in the development of brain problems. We also learned that the abnormal activation was triggered by the lack of GBA function in the immune cells, not by signals from the brain or other parts of the body. We would like to find out next whether the immune cells get inside the brain or cause harm from a distance. What we learned matters because it could help us prevent or cure brain diseases associated with GBA mutations. Treating the abnormal activation of immune cells in people with these mutations might help prevent damage to the brain.

genetics↗

bioRxiv2022

Nazo, the Drosophila homolog of the NBIA-mutated protein - c19orf12, is required for triglyceride homeostasis

Lipid dyshomeostasis has been implicated in a variety of diseases ranging from obesity to neurodegenerative disorders such as NBIA. Here, we uncover the physiological role of Nazo, the Drosophila homolog of the NBIA-mutated protein - c19orf12, whose function has been elusive. Ablation of Drosophila c19orf12 homologs leads to dysregulation of multiple lipid metabolism genes. nazo mutants exhibit markedly reduced gut lipid droplet and whole-body triglyceride contents. Consequently, they are sensitive to starvation and oxidative stress. Nazo localizes to ER-lipid droplet contact sites and is required for maintaining normal levels of Perilipin2, an inhibitor of the lipase - Brummer. Concurrent knockdown of Brummer or overexpression of Perilipin2 rescues the nazo phenotype, suggesting that this defect may arise from diminished Perilipin2 on lipid droplets leading to aberrant Brummer-mediated lipolysis. Our findings provide novel insights into the role of c19orf12 as a possible link between lipid dyshomeostasis and neurodegeneration, particularly in the context of NBIA.

cell biology↗

bioRxiv2022

Tissue-Restricted Inhibition of mTOR Using Chemical Genetics

mTOR is a highly conserved eukaryotic protein kinase that coordinates cell growth and metabolism and plays a critical role in cancer, immunity, and aging. It remains unclear how mTOR signaling in individual tissues contributes to whole-organism processes because mTOR inhibitors, like the natural product Rapamycin, are administered systemically and target multiple tissues simultaneously. We developed a chemical-genetic system, termed selecTOR, that restricts the activity of a Rapamycin analog to specific cell populations through targeted expression of a mutant FKBP12 protein. This analog has reduced affinity for its obligate binding partner FKBP12, which reduces its ability to inhibit mTOR in wild-type cells and tissues. Expression of the mutant FKBP12, which contains an expanded binding pocket, rescues the activity of this Rapamycin analog. Using this system, we show that selective mTOR inhibition can be achieved in S. cerevisiae and human cells, and we validate the utility of our system in an intact metazoan model organism by identifying the tissues responsible for a Rapamycin-induced developmental delay in Drosophila. Significance StatementmTOR plays a number of critical organismal roles, including in cell growth, development, immunity and aging, but dissecting the tissue-specific influences of mTOR has proven challenging. This work describes a simple system for identifying the specific tissues and cells responsible for the diverse functions of mTOR, and we show that our system can be used in organisms ranging from yeast to humans.

biochemistry↗

bioRxiv2020

Inactivation of the mitochondrial protease Afg3l2 results in severely diminished respiratory chain activity and widespread defects in mitochondrial gene expression

The m-AAA proteases plays a critical role in the proteostasis of the inner mitochondrial membrane proteins, and mutations in the genes encoding these proteases cause severe incurable neurological diseases. To further explore the biological role of the m-AAA proteases and the pathological consequences of their deficiency, we used a genetic approach in the fruit fly Drosophila melanogaster to inactivate the ATPase family gene 3-like 2 (AFG3L2) gene, which encodes a component of the m-AAA proteases. We found that null alleles of Drosophila AFG3L2 die early in development, but partial inactivation of AFG3L2 using RNAi extended viability to the late pupal and adult stages of development. Flies with partial inactivation of Afg3l2 exhibited marked behavioral defects, neurodegeneration, mitochondrial morphological alterations, and diminished respiratory chain (RC) activity. Further work revealed that reduced RC activity was a consequence of widespread defects in mitochondrial gene expression, including diminished mitochondrial transcription, translation and impaired mitochondrial ribosome biogenesis. These defects were accompanied by the compensatory activation of the mitochondrial unfolded protein response (mito-UPR) and accumulation of unfolded mitochondrial proteins, including proteins involved in transcription. Overexpression of the mito-UPR components partially rescued the Afg3l2-deficient phenotypes, indicating that sequestration of essential components of the mitochondrial gene expression into aggregates partly accounts for these defects. However, Afg3l2 also co-sediments with the mitochondrial ribosome biogenesis machinery, suggesting an additional novel role for Afg3l2 in ribosome biogenesis. Our work suggests that strategies designed to modify mitochondrial stress pathways and mitochondrial gene expression could be therapeutic in the diseases caused by mutations in AFG3L2. Author SummaryMitochondria produce virtually all of the cellular energy through the actions of the respiratory chain (RC) complexes. However, both the assembly of the RC complexes, and their biological functions come at a cost. Biogenesis of the RC complexes depends on the coordinated expression of nuclear and mitochondrially encoded subunits and an imbalance in this process can cause protein aggregation. Moreover, the RC complexes produce highly damaging reactive oxygen species as a side product of their activity. The Mitochondrial AAA+ family of proteases are believed to provide the first line of defense against these insults. The importance of this protease family is best exemplified by the severe neurodegenerative diseases that are caused by mutations in their respective genes. To better understand the biological roles of the AAA+ proteases, and the physiological consequences of their inactivation we used a genetic approach in Drosophila to study the Afg3l2 AAA+ protease. Unexpectedly, we found that Afg3l2 deficiency profoundly impaired mitochondrial gene expression, including transcription, translation and ribosome biogenesis. These phenotypes were accompanied by accumulation of insoluble mitochondrial proteins, and compensatory activation of mito-UPR and autophagy. Our work indicates Afg3l2 plays critical roles in degrading unfolded mitochondrial proteins and regulating mitochondrial gene expression.

neuroscience↗

bioRxiv2020

Glucocerebrosidase reduces the spread of protein aggregation in a Drosophila melanogaster model of neurodegeneration by regulating proteins trafficked by extracellular vesicles

Abnormal protein aggregation within neurons is a key pathologic feature of Parkinsons disease (PD). The spread of protein aggregates in the brain is associated with clinical disease progression, but how this occurs remains unclear. Mutations in the gene glucosidase, beta acid 1 (GBA), which encodes the lysosomal enzyme glucocerebrosidase (GCase), are the most penetrant common genetic risk factor for PD and dementia with Lewy bodies, and also associate with faster disease progression. To explore the mechanism by which mutations in GBA influence pathogenesis of these diseases, we previously created a Drosophila model of GBA deficiency (Gba1b) that manifests neurodegeneration, motor and cognitive deficits, and accelerated protein aggregation. Proteomic analysis of Gba1b mutants revealed dysregulation of proteins involved in extracellular vesicle (EV) biology, and we found altered protein composition of EVs from Gba1b mutants. To further investigate this novel mechanism, we hypothesized that GBA may influence the spread of pathogenic protein aggregates throughout the brain via EVs. We found that protein aggregation is reduced cell-autonomously and non-cell-autonomously by expressing wildtype GCase in specific tissues. In particular, accumulation of insoluble ubiquitinated proteins and Ref(2)P in the brains of Gba1b flies are reduced by ectopic expression of GCase in muscle tissue. Neuronal expression of GCase also cell-autonomously rescued protein aggregation in brain as well as non-cell-autonomously rescued protein aggregation in muscle. Muscle-specific GBA expression rescued the elevated levels of EV-intrinsic proteins and Ref(2)P found in EVs from Gba1b flies. Genetically perturbing EV biogenesis in specific tissues in the absence of GCase revealed differential cell-autonomous effects on protein aggregation but could not replicate the non-cell-autonomous rescue observed with tissue-specific GBA expression. Additionally, we identified ectopically expressed GCase within isolated EVs. Together, our findings suggest that GCase deficiency mediates accelerated spread of protein aggregates between cells and tissues via dysregulated EVs, and EV-mediated trafficking of GCase may partially account for the reduction in aggregate spread. Authors SummaryParkinsons disease (PD) is a common neurodegenerative disease characterized by abnormal clumps of proteins (aggregates) within the brain and other tissues which can lead to cellular dysfunction and death. Mutations in the gene GBA, which encodes glucocerebrosidase (GCase), are the strongest genetic risk factor for PD, and are associated with faster disease progression. GCase-deficient mutant flies display features suggestive of PD including increased protein aggregation in brain and muscle. We found that restoring GCase protein in the muscle of mutant flies reduced protein aggregation in muscle and the brain, suggesting a mechanism involving interaction between tissues. Previous work indicated that GBA influences extracellular vesicles (EVs) - small membrane-bound structures released by cells to communicate and/or transport cargo from cell to cell. Here, we found increased aggregated proteins within EVs of mutant flies, which was reduced by restoring GCase in muscle. In addition, we found GCase within the EVs, possibly explaining how GCase in one tissue such as muscle could reduce protein aggregation in a distant tissue like the brain. Our findings suggest that GCase influences proteins within EVs, affecting the spread of protein aggregation. This may be important to understanding PD progression and could uncover new targets to slow neurodegeneration.

cell biology↗