Aplp1 interacts with Lag3 to facilitate transmission of pathologic α-synuclein.

Pathologic α-synuclein (α-syn) spreads from cell-to-cell, in part, through binding to the lymphocyte-activation gene 3 (Lag3). Here we report that amyloid ß precursor-like protein 1 (Aplp1) interacts with Lag3 that facilitates the binding, internalization, transmission, and toxicity of pathologic α-syn. Deletion of both Aplp1 and Lag3 eliminates the loss of dopaminergic neurons and the accompanying behavioral deficits induced by α-syn preformed fibrils (PFF). Anti-Lag3 prevents the internalization of α-syn PFF by disrupting the interaction of Aplp1 and Lag3, and blocks the neurodegeneration induced by α-syn PFF in vivo. The identification of Aplp1 and the interplay with Lag3 for α-syn PFF induced pathology deepens our insight about molecular mechanisms of cell-to-cell transmission of pathologic α-syn and provides additional targets for therapeutic strategies aimed at preventing neurodegeneration in Parkinson's disease and related α-synucleinopathies.

Lymphocyte-Activation Gene 3 Facilitates Pathological Tau Neuron-to-Neuron Transmission.

The spread of prion-like protein aggregates is a common driver of pathogenesis in various neurodegenerative diseases, including Alzheimer's disease (AD) and related Tauopathies. Tau pathologies exhibit a clear progressive spreading pattern that correlates with disease severity. Clinical observation combined with complementary experimental studies has shown that Tau preformed fibrils (PFF) are prion-like seeds that propagate pathology by entering cells and templating misfolding and aggregation of endogenous Tau. While several cell surface receptors of Tau are known, they are not specific to the fibrillar form of Tau. Moreover, the underlying cellular mechanisms of Tau PFF spreading remain poorly understood. Here, it is shown that the lymphocyte-activation gene 3 (Lag3) is a cell surface receptor that binds to PFF but not the monomer of Tau. Deletion of Lag3 or inhibition of Lag3 in primary cortical neurons significantly reduces the internalization of Tau PFF and subsequent Tau propagation and neuron-to-neuron transmission. Propagation of Tau pathology and behavioral deficits induced by injection of Tau PFF in the hippocampus and overlying cortex are attenuated in mice lacking Lag3 selectively in neurons. These results identify neuronal Lag3 as a receptor of pathologic Tau in the brain，and for AD and related Tauopathies, a therapeutic target.

Pathological Tau transmission initiated by binding lymphocyte-activation gene 3.

The spread of prion-like protein aggregates is believed to be a common driver of pathogenesis in many neurodegenerative diseases. Accumulated tangles of filamentous Tau protein are considered pathogenic lesions of Alzheimer's disease (AD) and related Tauopathies, including progressive supranuclear palsy, and corticobasal degeneration. Tau pathologies in these illnesses exhibits a clear progressive and hierarchical spreading pattern that correlates with disease severity1,2. Clinical observation combined with complementary experimental studies3,4 have shown that Tau preformed fibrils (PFF) are prion-like seeds that propagate pathology by entering cells and templating misfolding and aggregation of endogenous Tau. While several receptors of Tau are known, they are not specific to the fibrillar form of Tau. Moreover, the underlying cellular mechanisms of Tau PFF spreading remains poorly understood. Here, we show that the lymphocyte-activation gene 3 (Lag3) is a cell surface receptor that binds to PFF, but not monomer, of Tau. Deletion of Lag3 or inhibition of Lag3 in primary cortical neurons significantly reduces the internalization of Tau PFF and subsequent Tau propagation and neuron-to-neuron transmission. Propagation of Tau pathology and behavioral deficits induced by injection of Tau PFF in the hippocampus and overlying cortex are attenuated in mice lacking Lag3 selectively in neurons. Our results identify neuronal Lag3 as a receptor of pathologic Tau in the brain, and for AD and related Tauopathies a therapeutic target.

α-Synuclein fibril-specific nanobody reduces prion-like α-synuclein spreading in mice.

Pathogenic α-synuclein (α-syn) is a prion-like protein that drives the pathogenesis of Lewy Body Dementia (LBD) and Parkinson's Disease (PD). To target pathogenic α-syn preformed fibrils (PFF), here we designed extracellular disulfide bond-free synthetic nanobody libraries in yeast. Following selection, we identified a nanobody, PFFNB2, that can specifically recognize α-syn PFF over α-syn monomers. PFFNB2 cannot inhibit the aggregation of α-syn monomer, but can significantly dissociate α-syn fibrils. Furthermore, adeno-associated virus (AAV)-encoding EGFP fused to PFFNB2 (AAV-EGFP-PFFNB2) can inhibit PFF-induced α-syn serine 129 phosphorylation (pS129) in mouse primary cortical neurons, and prevent α-syn pathology spreading to the cortex in the transgenic mice expressing human wild type (WT) α-syn by intrastriatal-PFF injection. The pS129 immunoreactivity is negatively correlated with the expression of AAV-EGFP-PFFNB2. In conclusion, PFFNB2 holds a promise for mechanistic exploration and therapeutic development in α-syn-related pathogenesis.

Poly(ADP-ribose) binding and macroH2A mediate recruitment and functions of KDM5A at DNA lesions.

The histone demethylase KDM5A erases histone H3 lysine 4 methylation, which is involved in transcription and DNA damage responses (DDRs). While DDR functions of KDM5A have been identified, how KDM5A recognizes DNA lesion sites within chromatin is unknown. Here, we identify two factors that act upstream of KDM5A to promote its association with DNA damage sites. We have identified a noncanonical poly(ADP-ribose) (PAR)-binding region unique to KDM5A. Loss of the PAR-binding region or treatment with PAR polymerase (PARP) inhibitors (PARPi's) blocks KDM5A-PAR interactions and DNA repair functions of KDM5A. The histone variant macroH2A1.2 is also specifically required for KDM5A recruitment and function at DNA damage sites, including homology-directed repair of DNA double-strand breaks and repression of transcription at DNA breaks. Overall, this work reveals the importance of PAR binding and macroH2A1.2 in KDM5A recognition of DNA lesion sites that drive transcriptional and repair activities at DNA breaks within chromatin that are essential for maintaining genome integrity.

In Time and Space: Laser Microirradiation and the DNA Damage Response.

Maintenance of genomic integrity depends on the spatiotemporal recruitment and regulation of DNA damage response and repair proteins at DNA damage sites. These highly dynamic processes have been widely studied using laser microirradiation coupled with fluorescence microscopy. Laser microirradiation has provided a powerful methodology to identify and determine mechanisms of DNA damage response pathways. Here we describe methods used to analyze protein recruitment dynamics of fluorescently tagged or endogenous proteins to laser-induced DNA damage sites using laser scanning and fluorescence microscopy. We further describe multiple applications employing these techniques to study additional processes at DNA damage sites including transcription. Together, we aim to provide robust visualization methods employing laser-microirradiation to detect and determine protein behavior, functions and dynamics in response to DNA damage in mammalian cells.

KDM5A Regulates a Translational Program that Controls p53 Protein Expression.

The p53 tumor suppressor pathway is frequently inactivated in human cancers. However, there are some cancer types without commonly recognized alterations in p53 signaling. Here we report that histone demethylase KDM5A is involved in the regulation of p53 activity. KDM5A is significantly amplified in multiple types of cancers, an event that tends to be mutually exclusive to p53 mutation. We show that KDM5A acts as a negative regulator of p53 signaling through inhibition of p53 translation via suppression of a subgroup of eukaryotic translation initiation genes. Genetic deletion of KDM5A results in upregulation of p53 in multiple lineages of cancer cells and inhibits tumor growth in a p53-dependent manner. In addition, we have identified a regulatory loop between p53, miR-34, and KDM5A, whereby the induction of miR-34 leads to suppression of KDM5A. Thus, our findings reveal a mechanism by which KDM5A inhibits p53 translation to modulate cancer progression.

Recruitment of ubiquitin-activating enzyme UBA1 to DNA by poly(ADP-ribose) promotes ATR signalling.

The DNA damage response (DDR) ensures cellular adaptation to genotoxic insults. In the crowded environment of the nucleus, the assembly of productive DDR complexes requires multiple protein modifications. How the apical E1 ubiquitin activation enzyme UBA1 integrates spatially and temporally in the DDR remains elusive. Using a human cell-free system, we show that poly(ADP-ribose) polymerase 1 promotes the recruitment of UBA1 to DNA. We find that the association of UBA1 with poly(ADP-ribosyl)ated protein-DNA complexes is necessary for the phosphorylation replication protein A and checkpoint kinase 1 by the serine/threonine protein kinase ataxia-telangiectasia and RAD3-related, a prototypal response to DNA damage. UBA1 interacts directly with poly(ADP-ribose) via a solvent-accessible and positively charged patch conserved in the Animalia kingdom but not in Fungi. Thus, ubiquitin activation can anchor to poly(ADP-ribose)-seeded protein assemblies, ensuring the formation of functional ataxia-telangiectasia mutated and RAD3-related-signalling complexes.

The Aeromonas caviae AHA0618 gene modulates cell length and influences swimming and swarming motility.

Aeromonas caviae is motile via a polar flagellum in liquid culture, with a lateral flagella system used for swarming on solid surfaces. The polar flagellum also has a role in cellular adherence and biofilm formation. The two subunits of the polar flagellum, FlaA and FlaB, are posttranslationally modified by O-linked glycosylation with pseudaminic acid on 6-8 serine and threonine residues within the central region of these proteins. This modification is essential for the formation of the flagellum. Aeromonas caviae possesses the simplest set of genes required for bacterial glycosylation currently known, with the putative glycosyltransferase, Maf1, being described recently. Here, we investigated the role of the AHA0618 gene, which shares homology (37% at the amino acid level) with the central region of a putative deglycosylation enzyme (HP0518) from the human pathogen Helicobacter pylori, which also glycosylates its flagellin and is proposed to be part of a flagellin deglycosylation pathway. Phenotypic analysis of an AHA0618 A. caviae mutant revealed increased swimming and swarming motility compared to the wild-type strain but without any detectable effects on the glycosylation status of the polar flagellins when analyzed by western blot analysis or mass spectroscopy. Bioinformatic analysis of the protein AHA0618, demonstrated homology to a family of l,d-transpeptidases involved in cell wall biology and peptidoglycan cross-linking (YkuD-like). Scanning electron microscopy (SEM) and fluorescence microscopy analysis of the wild-type and AHA0618-mutant A. caviae strains revealed the mutant to be subtly but significantly shorter than wild-type cells; a phenomenon that could be recovered when either AHA0618 or H. pylori HP0518 were introduced. We can therefore conclude that AHA0618 does not affect A. caviae behavior by altering polar flagellin glycosylation levels but is likely to have a role in peptidoglycan processing at the bacterial cell wall, consequently altering cell length and hence influencing motility.