Biology subjects

Brown, H. J.

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

10 recordsLinked to original sources

bioRxiv2025

DRP1 induces neuroinflammation via transcriptional regulation of NF-ĸB

Neuroinflammation is a major pathogenic mechanism in neurodegenerative diseases. Understanding the regulation of neuroinflammation is critical to therapeutic development. We report here that dynamin related protein 1 (DRP1), well-recognized for its role in mitochondrial fission, is a transcription factor that regulates neuroinflammation. Using multiple inflammatory models, we provide evidence demonstrating that DRP1, when challenged with pro-inflammatory lipopolysaccharides, translocates from the cytosol to the nucleus, then binds to the promoter region of Rela (encoding NF-{kappa}B) to activate its gene products and other downstream inflammatory cytokines. Our data also demonstrate the significant role of the proinflammatory lipocalin 2 in the brain. In combination, this study highlights a previously unidentified function of DRP1 in mediating neuroinflammation via the NF-{kappa}B-lipocalin 2 axis. Through such mechanisms of DRP1, this study also provides potential therapeutic targets for neurodegenerative diseases and other conditions linked to inflammation.

neuroscience↗

bioRxiv2025

Imbalanced mitochondrial dynamics in human and mouse PD brains

Mitochondrial dysfunction is a major pathogenic mechanism in Parkinsons disease (PD). Emerging studies have shown that dysregulation in mitochondrial dynamics (fission/fusion/movement) has a major negative impact on mitochondria - both morphologically and functionally. Partial genetic deletion and pharmacological inhibition of the mitochondrial fission dynamin-related protein 1 (Drp1) have been demonstrated to be beneficial in experimental models of PD. However, the expression of DRP1 (and other fission and fusion genes/proteins) has not been investigated in the brains of Parkinsons patients. Without these data, the question remains whether targeting DRP1 is a valid therapeutic target for PD. To address this gap of knowledge, first, we used post-mortem substantia nigra specimens of Parkinsons patients and controls. Significant increases in the levels of both DNM1L, which encodes DRP1, as well as the DRP1 protein were detected in Parkinsons patients. Immunostaining revealed increased DRP1 expression in dopamine (DA) neurons, astrocytes, and microglia. In addition to DRP1, the levels of other fission and fusion genes/proteins were also altered in Parkinsons patients. To complement these human studies and given the significant role of -synuclein in PD pathogenesis, we performed time-course studies (3-, 6- and 12-month) using transgenic mice overexpressing human wild-type SNCA under the mouseThy-1 promoter. As early as 6 months old, we detected an upregulation of Dnm1l and Drp1 in the nigral DA neurons of the SNCA mice as compared to their WT littermates. Furthermore, these mutant animals exhibited more Drp1 phosphorylation at serine 616, which promotes its translocation to mitochondria to induce fragmentation. Together, this study shows an upregulation of DRP1/Drp1 and alterations in other fission/fusion proteins in both human and mouse PD brains, leading to a pro-fission phenotype, providing additional evidence that blocking mitochondrial fission or promoting fusion is a potential therapeutic strategy for PD.

neuroscience↗

bioRxiv2024

Tubulin-like proteins CetZ1 and CetZ2 coordinate cellular morphogenesis in the archaeon Haloferax volcanii

CetZs are archaeal tubulin superfamily cytoskeletal proteins implicated in the control of cell shape and motility. In the pleomorphic archaeon Haloferax volcanii, CetZ1 is required for the transformation of a discoid, or plate-like, cell morphology to the rod shape during early-mid log phase cultures and for the development of swimming motility. In this study, we found that the paralog CetZ2 is not required for rod development or reversion to plates in log phase but was strongly upregulated later in stationary phase, where it promotes the maintenance of plate cell shape. CetZ2 cytoskeletal structures visualized through the production of a functional fluorescent tagged CetZ2 were most dynamic specifically in mid-stationary phase, where they showed directional movement around the cell edge and other complex cytomotive-like behaviours. Furthermore, in mid-stationary phase, the dynamics of CetZ1 and CetZ2 cytoskeletal structures were specifically dependent on the presence of one another and their GTPase activities that control the polymerization-depolymerization cycle. Together, the results suggest that CetZ2 counteracts the CetZ1-based rod development pathway to maintain plate shape, and they imply that additional stationary-phase factors are also involved. CetZ1 and CetZ2 are co-conserved in many haloarchaea, suggesting that their coordinated functions at different stages of the growth cycle are widely utilized to control cell shapeshifting. More generally, our findings show that CetZ paralogues can have distinct and non-redundant functions, indicating that diversification and specialisation within the tubulin superfamily has occurred multiple times in archaeal evolution. SignificanceThis study describes the first defined function of a CetZ2 protein, which represents a distinct haloarchaeal protein family within the near-universal tubulin superfamily of cytoskeletal proteins. Using the model archaeal organism Haloferax volcanii, we found that CetZ2 maintains the plate or disk-like cell morphology specifically in the stationary phase of the growth cycle, by counteracting CetZ1-based rod development. We also showed for the first time that CetZ2 can form dynamic cytoskeletal filaments that show directional movement around the cell in mid-stationary phase. The CetZ1-CetZ2 interplay we detected specifically at this stage may represent a paradigm for understanding the evolution of antagonistic cytoskeletal functions, such as those that led to a reliance on active and inactive subunits during the early evolution of microtubules in eukaryotes.

microbiology↗

bioRxiv2024

MinD proteins regulate CetZ1 localisation in Haloferax volcanii

CetZ proteins are archaea-specific homologues of the cytoskeletal proteins FtsZ and tubulin. In the pleomorphic archaeon Haloferax volcanii, CetZ1 contributes to the development of rod shape and motility, and has been implicated in the proper assembly and positioning of the archaellum and chemotaxis motility proteins. CetZ1 shows complex subcellular localization, including irregular midcell structures and filaments along the long axis of developing rods and patches at the cell poles of the motile rod cell type. The polar localizations of archaellum and chemotaxis proteins are also influenced by MinD4, the only previously characterized archaeal member of the MinD family of ATPases, which are better known for their roles in the positioning of the division ring in bacteria. Using minD mutant strains and CetZ1 subcellular localization studies, we show here that a second minD homolog, minD2, has a strong influence on motility and the localization of CetZ1. Knockout of the minD2 gene altered the distribution of a fluorescent CetZ1-mTq2 fusion protein in a broad midcell zone and along the edges of rod cells, and inhibited the localization of CetZ1-mTq2 at the cell poles. MinD4 had a similar but weaker influence on motility and CetZ1-mTq2 localization. The MinD2/4 mutant strains formed rod cell shapes like the wildtype at an early log stage of growth. Our results are consistent with distinct roles for CetZ1 in rod shape formation and at the poles of mature rods, that are positioned through the action of the MinD proteins and contribute to the development of swimming motility in multiple ways. They represent the first report of MinD proteins controlling the positioning of tubulin superfamily proteins in archaea.

microbiology↗

bioRxiv2024

CetZ1-dependent assembly and positioning of the motility machinery in haloarchaea

The archaeal tubulin-like cytoskeletal protein CetZ1 is required for rod-cell morphogenesis during the development of motility in Haloferax volcanii. This is expected to improve swimming speed and directionality. Here, we found that deletion of cetZ1 or expression of a GTPase-defective mutant caused a substantial defect in the assembly of the motility machinery, including the archaellum base marker protein ArlD1, the chemotaxis sensory array adapter CheW1, and signal transducer CheY. Furthermore, overexpression of cetZ1 reduced the assembly and polar placement of the motility machinery without detectably affecting the rod shape of motile cells. In contrast, deletion of the conserved paralog cetZ2 caused no defects in swimming or rod shape, although expression of the cetZ2 GTPase-defective mutant reduced motility whereas cetZ2 overexpression caused mild hyper-motility; these effects were dependent on the presence of cetZ1. A functional CetZ1-mTq2 fusion strongly localized at the poles of mature motile cells, where it partially co-localized with the motility machinery markers. These results suggest that CetZ1 has another role in the organisation or structure of the cell poles that promotes the assembly of the motility machinery. The multiple roles and locations of CetZ1 during motile cell development are reminiscent of the multiple functions of eukaryotic cytoskeletal proteins.

microbiology↗

bioRxiv2024

Dynamic self-association of archaeal tubulin-like protein CetZ1 drives Haloferax volcanii morphogenesis

Tubulin superfamily (TSF) proteins include the well-known eukaryotic tubulin and bacterial FtsZ families, and lesser-known archaeal CetZ family. In eukaryotes and bacteria, GTP-dependent polymerization and self-association of tubulin and FtsZ protofilaments are integral to the formation of cytoskeletal structures with essential roles in cell division, growth, and morphology. Archaeal CetZs are implicated in the control of cell shape and motility through unknown mechanisms. Here, we reveal a sequence of subcellular localization patterns of CetZ1, the prototypical member of the CetZ family, during stages of Haloferax volcanii rod cell development, in which it plays an essential role. Like tubulin and FtsZ, we found that CetZ1 formed GTP-dependent polymers in vitro, which appear to associate laterally as irregular polymer bundles. Mutations targeting regions predicted to mediate self-association and dynamic turnover of CetZ1, including the longitudinal (GTPase T7 and T4 loops) and lateral assembly interfaces, perturbed or altered rod shape development and subcellular assembly and dynamics, and caused corresponding effects on polymerization in vitro. Remarkably, a conspicuous amphipathic protrusion in the large microtubule (M-) loop, a characteristic of the CetZ1 subfamily, also strongly influenced function and assembly. Our findings reveal the importance of dynamic CetZ1 self-association in cellular morphogenesis involving multiple regions of the TSF fold, including tubulin- and FtsZ-like structural characteristics and CetZ1-specific features. Furthermore, they support a mechanism involving CetZ1 dynamic guidance of cell envelope-associated structures that reshape the cell during morphogenesis.

molecular biology↗

bioRxiv2024

A vector system for single and tandem expression of cloned genes and multi-colour fluorescent tagging in Haloferax volcanii

Archaeal cell biology is an emerging field expected to identify fundamental cellular processes, help resolve the deep evolutionary history of cellular life, and contribute new components and functions in biotechnology and synthetic biology. To facilitate these, we have developed plasmid vectors that allow convenient cloning and production of proteins and fusion proteins with flexible, rigid, or semi-rigid linkers in the model archaeon Haloferax volcanii. For protein subcellular localization studies using fluorescent protein (FP) tags, we created vectors incorporating a range of codon-optimized fluorescent proteins for N- or C-terminal tagging, including GFP, mNeonGreen, mCherry, YPet, mTurquoise2 and mScarlet-I. Obtaining functional fusion proteins can be challenging with proteins involved in multiple interactions, mainly due to steric interference. We demonstrated the use of the new vector system to screen for improved function in cytoskeletal protein FP fusions, and identified FtsZ1-FPs that are functional in cell division and CetZ1-FPs that are functional in motility and rod cell development. Both the type of linker and the type of FP influenced the functionality of the resulting fusions. The vector design also facilitates convenient cloning and tandem expression of two genes or fusion genes, controlled by a modified tryptophan-inducible promoter, and we demonstrated its use for dual-colour imaging of tagged proteins in H. volcanii cells. These tools should promote further development and applications of archaeal molecular and cellular biology and biotechnology.

microbiology↗

bioRxiv2020

Tau pathology spreads between anatomically-connected regions of the brain and is modulated by a LRRK2 mutation

Tau pathology is a diagnostic feature of Alzheimers disease (AD) but is also a prominent feature of Parkinsons disease (PD), including genetic forms of PD with mutations in leucine-rich repeat kinase 2 (LRRK2). In both diseases, tau pathology is progressive and correlates with cognitive decline. Neuropathological staging studies in humans and mouse models have suggested that tau spreads through the brain, but it is unclear how neuroanatomical connections, spatial proximity, and regional vulnerability contribute to pathology spread. Further, it is unknown how mutations in the LRRK2 gene may modulate susceptibility to tau pathologys initiation or spread. In this study, we used seed-based models of tauopathy to capture spatiotemporal patterns of pathology in mice. Following the injection of AD brain-derived tau into the brains of non-transgenic mice, tau pathology spreads progressively through the brain in a spatiotemporal pattern that is well-explained by anatomical connectivity. We validated and compared network models based on diffusion along anatomical connections to predict tau spread, estimate regional vulnerability to tau pathology, and investigate gene expression patterns related to regional vulnerability. We further investigated tau pathology spread in mice harboring a mutation in LRRK2 and found that while tau pathology spread is still constrained by anatomical connectivity, it spreads preferentially in a retrograde direction to regions that are otherwise resilient in wildtype mice. This study provides a quantitative demonstration that tau pathology spreads along anatomical connections, explores the kinetics of this spread, and provides a platform for investigating the effect of genetic risk factors and treatments on the progression of tauopathies.

neuroscience↗

bioRxiv2020

Improved growth and morphological plasticity of Haloferax volcanii

Some microbes display pleomorphism, showing variable cell shapes in a single culture, whereas others differentiate to adapt to changed environmental conditions. The pleomorphic archaeon Haloferax volcanii commonly forms discoid-shaped ( plate) cells in culture, but may also be present as rods, and can develop into motile rods in soft agar, or longer filaments in certain biofilms. Here we report improvement of H. volcanii growth in both semi-defined and complex media by supplementing with eight trace-element micronutrients. With these supplemented media, transient development of plate cells into uniformly-shaped rods was clearly observed during the early log phase of growth; cells then reverted to plates for the late log and stationary phases. In media prepared with high-purity water and reagents, without supplemental trace elements, rods and other complex elongated morphologies ( pleomorphic rods) were observed at all growth stages of the culture; the highly-elongated cells sometimes displayed a substantial tubule at one or less frequently both poles, as well as unusual tapered and highly-curved forms. Polar tubules were observed forming by initial mid-cell narrowing or tubulation, causing a dumbbell-like shape, followed by cell division towards one end. Formation of the uniform early-log rods, as well as the pleomorphic rods and tubules were dependent on the function of the tubulin-like cytoskeletal protein, CetZ1. Our results have revealed the remarkable morphological plasticity of H. volcanii cells in response to multiple culture conditions, and should facilitate the use of this species in further studies of archaeal biology.

microbiology↗

bioRxiv2020

Snca-GFP knock-in mice reflect patterns of endogenous expression and pathological seeding

Alpha-synuclein (aSyn) participates in synaptic vesicle trafficking and synaptic transmission, but its misfolding is also strongly implicated in Parkinsons disease (PD) and other neurodegenerative disorders known as synucleinopathies where misfolded aSyn accumulates in different regions of the central and peripheral nervous systems. Although increased aSyn expression levels or altered aggregation propensities likely underlie familial PD with SNCA amplification or mutations, the majority of synucleinopathies arise sporadically, indicating that disease can develop under normal levels of wildtype aSyn. We report here the development and characterization of a mouse line expressing an aSyn-GFP fusion protein under the control of native Snca regulatory elements. Regional and subcellular localization of the aSyn-GFP fusion protein in brains and peripheral tissues of knock-in (KI) mice are indistinguishable from that of wildtype littermates. Importantly, similar to wildtype aSyn, aSyn-GFP disperses from synaptic vesicles upon membrane depolarization, indicating that the tag does not alter normal aSyn dynamics at synapses. In addition, intracerebral injection of aSyn pre-formed fibrils into KI mice induced the formation of aSyn-GFP inclusions with a distribution pattern similar to that observed in wildtype mice, albeit with attenuated kinetics due to the GFP tag. We anticipate that this new mouse model will facilitate in vitro and in vivo studies requiring in situ detection of endogenous aSyn, therefore providing new insights into aSyn function in health and disease. Significance StatementAlpha-synuclein (aSyn) participates in synaptic vesicle function and represents a major component of the Lewy pathology found in Parkinsons and related neurodegenerative diseases. The function of aSyn and the sequence of events leading to its aggregation and neurotoxicity are not fully understood. Here we present a new mouse model in which Enhanced Green Fluorescence Protein (GFP) has been knocked-in at the C-terminal of the Snca gene. The resulting fusion protein shows identical expression and localization to that of wildtype animals, is functional, and is incorporated into pathological aggregates in vitro and in vivo. This new tool allows for monitoring aSyn under a variety of physiological and pathological conditions, and may uncover additional insights into its function and dysfunction.

neuroscience↗