Alpha-synuclein, autophagy-lysosomal pathway, and Lewy bodies: Mutations, propagation, aggregation, and the formation of inclusions.

Research into the pathophysiology of Parkinson's disease (PD) is a fast-paced pursuit, with new findings about PD and other synucleinopathies being made each year. The involvement of various lysosomal proteins, such as TFEB, TMEM175, GBA, and LAMP1/2, marks the rising awareness about the importance of lysosomes in PD and other neurodegenerative disorders. This, along with recent developments regarding the involvement of microglia and the immune system in neurodegenerative diseases, has brought about a new era in neurodegeneration: the role of proinflammatory cytokines on the nervous system, and their downstream effects on mitochondria, lysosomal degradation, and autophagy. More effort is needed to understand the interplay between neuroimmunology and disease mechanisms, as many of the mechanisms remain enigmatic. α-synuclein, a key protein in PD and the main component of Lewy bodies, sits at the nexus between lysosomal degradation, autophagy, cellular stress, neuroimmunology, PD pathophysiology, and disease progression. This review revisits some fundamental knowledge about PD while capturing some of the latest trends in PD research, specifically as it relates to α-synuclein.

SARS-CoV-2 infects cells after viral entry via clathrin-mediated endocytosis.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of COVID-19, so understanding its biology and infection mechanisms is critical to facing this major medical challenge. SARS-CoV-2 is known to use its spike glycoprotein to interact with the cell surface as a first step in the infection process. As for other coronaviruses, it is likely that SARS-CoV-2 next undergoes endocytosis, but whether or not this is required for infectivity and the precise endocytic mechanism used are unknown. Using purified spike glycoprotein and lentivirus pseudotyped with spike glycoprotein, a common model of SARS-CoV-2 infectivity, we now demonstrate that after engagement with the plasma membrane, SARS-CoV-2 undergoes rapid, clathrin-mediated endocytosis. This suggests that transfer of viral RNA to the cell cytosol occurs from the lumen of the endosomal system. Importantly, we further demonstrate that knockdown of clathrin heavy chain, which blocks clathrin-mediated endocytosis, reduces viral infectivity. These discoveries reveal that SARS-CoV-2 uses clathrin-mediated endocytosis to gain access into cells and suggests that this process is a key aspect of virus infectivity.

Modeling Parkinson's disease pathology in human dopaminergic neurons by sequential exposure to α-synuclein fibrils and proinflammatory cytokines.

Lewy bodies (LBs), α-synuclein-enriched intracellular inclusions, are a hallmark of Parkinson's disease (PD) pathology, yet a cellular model for LB formation remains elusive. Recent evidence indicates that immune dysfunction may contribute to the development of PD. In this study, we found that induced pluripotent stem cell (iPSC)-derived human dopaminergic (DA) neurons form LB-like inclusions after treatment with α-synuclein preformed fibrils (PFFs) but only when coupled to a model of immune challenge (interferon-γ or interleukin-1ß treatment) or when co-cultured with activated microglia-like cells. Exposure to interferon-γ impairs lysosome function in DA neurons, contributing to LB formation. The knockdown of LAMP2 or the knockout of GBA in conjunction with PFF administration is sufficient for inclusion formation. Finally, we observed that the LB-like inclusions in iPSC-derived DA neurons are membrane bound, suggesting that they are not limited to the cytoplasmic compartment but may be formed due to dysfunctions in autophagy. Together, these data indicate that immune-triggered lysosomal dysfunction may contribute to the development of PD pathology.

Loss of symmetric cell division of apical neural progenitors drives DENND5A-related developmental and epileptic encephalopathy.

Developmental and epileptic encephalopathies (DEEs) feature altered brain development, developmental delay and seizures, with seizures exacerbating developmental delay. Here we identify a cohort with biallelic variants in DENND5A, encoding a membrane trafficking protein, and develop animal models with phenotypes like the human syndrome. We demonstrate that DENND5A interacts with Pals1/MUPP1, components of the Crumbs apical polarity complex required for symmetrical division of neural progenitor cells. Human induced pluripotent stem cells lacking DENND5A fail to undergo symmetric cell division with an inherent propensity to differentiate into neurons. These phenotypes result from misalignment of the mitotic spindle in apical neural progenitors. Cells lacking DENND5A orient away from the proliferative apical domain surrounding the ventricles, biasing daughter cells towards a more fate-committed state, ultimately shortening the period of neurogenesis. This study provides a mechanism for DENND5A-related DEE that may be generalizable to other developmental conditions and provides variant-specific clinical information for physicians and families.

Loss of symmetric cell division of apical neural progenitors drives DENND5A-related developmental and epileptic encephalopathy.

Developmental and epileptic encephalopathies (DEEs) are a heterogenous group of epilepsies in which altered brain development leads to developmental delay and seizures, with the epileptic activity further negatively impacting neurodevelopment. Identifying the underlying cause of DEEs is essential for progress toward precision therapies. Here we describe a group of individuals with biallelic variants in DENND5A and determine that variant type is correlated with disease severity. We demonstrate that DENND5A interacts with MUPP1 and PALS1, components of the Crumbs apical polarity complex, which is required for both neural progenitor cell identity and the ability of these stem cells to divide symmetrically. Induced pluripotent stem cells lacking DENND5A fail to undergo symmetric cell division during neural induction and have an inherent propensity to differentiate into neurons, and transgenic DENND5A mice, with phenotypes like the human syndrome, have an increased number of neurons in the adult subventricular zone. Disruption of symmetric cell division following loss of DENND5A results from misalignment of the mitotic spindle in apical neural progenitors. A subset of DENND5A is localized to centrosomes, which define the spindle poles during mitosis. Cells lacking DENND5A orient away from the proliferative apical domain surrounding the ventricles, biasing daughter cells towards a more fate-committed state and ultimately shortening the period of neurogenesis. This study provides a mechanism behind DENND5A-related DEE that may be generalizable to other developmental conditions and provides variant-specific clinical information for physicians and families.

Visualization of α-synuclein trafficking via nanogold labeling and electron microscopy.

There is conflicting evidence regarding the mechanisms of α-synuclein internalization, and its trafficking itinerary following cellular entry remains largely unknown. To examine these issues, we describe steps for coupling α-synuclein preformed fibrils (PFFs) to nanogold beads and their subsequent characterization by electron microscopy (EM). Then we describe the uptake of conjugated PFFs by U2OS cells plated on Permanox 8-well chamber slides. This process eliminates the reliance on antibody specificity and the need to employ complex immunoEM staining protocols. For complete details on the use and execution of this protocol, please refer to Bayati et al. (2022).1.

Identification and Utilization of a Chemical Probe to Interrogate the Roles of PIKfyve in the Lifecycle of ß-Coronaviruses.

From a designed library of indolyl pyrimidinamines, we identified a highly potent and cell-active chemical probe (17) that inhibits phosphatidylinositol-3-phosphate 5-kinase (PIKfyve). Comprehensive evaluation of inhibitor selectivity confirmed that this PIKfyve probe demonstrates excellent kinome-wide selectivity. A structurally related indolyl pyrimidinamine (30) was characterized as a negative control that lacks PIKfyve inhibitory activity and exhibits exquisite selectivity when profiled broadly. Chemical probe 17 disrupts multiple phases of the lifecycle of ß-coronaviruses: viral replication and viral entry. The diverse antiviral roles of PIKfyve have not been previously probed comprehensively in a single study or using the same compound set. Our scaffold is a distinct chemotype that lacks the canonical morpholine hinge-binder of classical lipid kinase inhibitors and has a non-overlapping kinase off-target profile with known PIKfyve inhibitors. Our chemical probe set can be used by the community to further characterize the role of PIKfyve in virology.

Rapid macropinocytic transfer of α-synuclein to lysosomes.

The nervous system spread of alpha-synuclein fibrils is thought to cause Parkinson's disease (PD) and other synucleinopathies; however, the mechanisms underlying internalization and cellular spread are enigmatic. Here, we use confocal and superresolution microscopy, subcellular fractionation, and electron microscopy (EM) of immunogold-labeled α-synuclein preformed fibrils (PFFs) to demonstrate that this form of the protein undergoes rapid internalization and is targeted directly to lysosomes in as little as 2 min. Uptake of PFFs is disrupted by macropinocytic inhibitors and circumvents classical endosomal pathways. Immunogold-labeled PFFs are seen at the highly curved inward edge of membrane ruffles, in newly formed macropinosomes, in multivesicular bodies and in lysosomes. While most fibrils remain in lysosomes, a portion is transferred to neighboring naive cells along with markers of exosomes. These data indicate that PFFs use a unique internalization mechanism as a component of cell-to-cell propagation.

Host Kinase CSNK2 is a Target for Inhibition of Pathogenic SARS-like ß-Coronaviruses.

Inhibition of the protein kinase CSNK2 with any of 30 specific and selective inhibitors representing different chemotypes, blocked replication of pathogenic human, bat, and murine ß-coronaviruses. The potency of in-cell CSNK2A target engagement across the set of inhibitors correlated with antiviral activity and genetic knockdown confirmed the essential role of the CSNK2 holoenzyme in ß-coronavirus replication. Spike protein endocytosis was blocked by CSNK2A inhibition, indicating that antiviral activity was due in part to a suppression of viral entry. CSNK2A inhibition may be a viable target for the development of anti-SARS-like ß-coronavirus drugs.

Host kinase CSNK2 is a target for inhibition of pathogenic ß-coronaviruses including SARS-CoV-2.

Inhibition of the protein kinase CSNK2 with any of 30 specific and selective inhibitors representing different chemotypes, blocked replication of pathogenic human and murine ß-coronaviruses. The potency of in-cell CSNK2A target engagement across the set of inhibitors correlated with antiviral activity and genetic knockdown confirmed the essential role of the CSNK2 holoenzyme in ß-coronavirus replication. Spike protein uptake was blocked by CSNK2A inhibition, indicating that antiviral activity was due in part to a suppression of viral entry. CSNK2A inhibition may be a viable target for development of new broad spectrum anti-ß-coronavirus drugs.

Emerging Biofabrication Strategies for Engineering Complex Tissue Constructs.

The demand for organ transplantation and repair, coupled with a shortage of available donors, poses an urgent clinical need for the development of innovative treatment strategies for long-term repair and regeneration of injured or diseased tissues and organs. Bioengineering organs, by growing patient-derived cells in biomaterial scaffolds in the presence of pertinent physicochemical signals, provides a promising solution to meet this demand. However, recapitulating the structural and cytoarchitectural complexities of native tissues in vitro remains a significant challenge to be addressed. Through tremendous efforts over the past decade, several innovative biofabrication strategies have been developed to overcome these challenges. This review highlights recent work on emerging three-dimensional bioprinting and textile techniques, compares the advantages and shortcomings of these approaches, outlines the use of common biomaterials and advanced hybrid scaffolds, and describes several design considerations including the structural, physical, biological, and economical parameters that are crucial for the fabrication of functional, complex, engineered tissues. Finally, the applications of these biofabrication strategies in neural, skin, connective, and muscle tissue engineering are explored.