Alpha-synuclein regulates the repair of genomic DNA double-strand breaks in a DNA-PKcs-dependent manner.

α-synuclein (αSyn) is a presynaptic and nuclear protein that aggregates in important neurodegenerative diseases such as Parkinson's Disease (PD), Parkinson's Disease Dementia (PDD) and Lewy Body Dementia (LBD). Our past work suggests that nuclear αSyn may regulate forms of DNA double-strand break (DSB) repair in HAP1 cells after DNA damage induction with the chemotherapeutic agent bleomycin1. Here, we report that genetic deletion of αSyn specifically impairs the non-homologous end-joining (NHEJ) pathway of DSB repair using an extrachromosomal plasmid-based repair assay in HAP1 cells. Importantly, induction of a single DSB at a precise genomic location using a CRISPR/Cas9 lentiviral approach also showed the importance of αSyn in regulating NHEJ in HAP1 cells and primary mouse cortical neuron cultures. This modulation of DSB repair is dependent on the activity of the DNA damage response signaling kinase DNA-PKcs, since the effect of αSyn loss-of-function is reversed by DNA-PKcs inhibition. Using in vivo multiphoton imaging in mouse cortex after induction of αSyn pathology, we find an increase in longitudinal cell survival of inclusion-bearing neurons after Polo-like kinase (PLK) inhibition, which is associated with an increase in the amount of aggregated αSyn within inclusions. Together, these findings suggest that αSyn plays an important physiologic role in regulating DSB repair in both a transformed cell line and in primary cortical neurons. Loss of this nuclear function may contribute to the neuronal genomic instability detected in PD, PDD and DLB and points to DNA-PKcs and PLK as potential therapeutic targets.

Aggregated Alpha-Synuclein Inclusions within the Nucleus Predict Impending Neuronal Cell Death in a Mouse Model of Parkinsonism.

Alpha-synuclein (aSyn) is a 14 kD protein encoded by the SNCA gene that is expressed in vertebrates and normally localizes to presynaptic terminals and the nucleus. aSyn forms pathological intracellular aggregates that typify a group of important neurodegenerative diseases called synucleinopathies. Previous work in human tissue and model systems indicates that some of these aggregates can be intranuclear, but the significance of aSyn aggregation within the nucleus is not clear. We used a mouse model that develops aggregated aSyn nuclear inclusions. Using aSyn preformed fibril injections in GFP-tagged aSyn transgenic mice, we were able to induce the formation of nuclear aSyn inclusions and study their properties in fixed tissue and in vivo using multiphoton microscopy. In addition, we analyzed human synucleinopathy patient tissue to better understand this pathology. Our data demonstrate that nuclear aSyn inclusions may form through the transmission of aSyn between neurons, and these intranuclear aggregates bear the hallmarks of cytoplasmic Lewy pathology. Neuronal nuclear aSyn inclusions can form rod-like structures that do not contain actin, excluding them from being previously described nuclear actin rods. Longitudinal, in vivo multiphoton imaging indicates that certain morphologies of neuronal nuclear aSyn inclusions predict cell death within 14 days. Human multiple system atrophy cases contain neurons and glia with similar nuclear inclusions, but we were unable to detect such inclusions in Lewy body dementia cases. This study suggests that the dysregulation of a nuclear aSyn function associated with nuclear inclusion formation could play a role in the forms of neurodegeneration associated with synucleinopathy.

Phosphorylation of the aggregate-forming protein alpha-synuclein on serine-129 inhibits its DNA-bending properties.

Alpha-synuclein (aSyn) is a vertebrate protein, normally found within the presynaptic nerve terminal and nucleus, which is known to form somatic and neuritic aggregates in certain neurodegenerative diseases. Disease-associated aggregates of aSyn are heavily phosphorylated at serine-129 (pSyn), while normal aSyn protein is not. Within the nucleus, aSyn can directly bind DNA, but the mechanism of binding and the potential modulatory roles of phosphorylation are poorly understood. Here we demonstrate using a combination of electrophoretic mobility shift assay and atomic force microscopy approaches that both aSyn and pSyn can bind DNA within the major groove, in a DNA length-dependent manner and with little specificity for DNA sequence. Our data are consistent with a model in which multiple aSyn molecules bind a single 300 base pair (bp) DNA molecule in such a way that stabilizes the DNA in a bent conformation. We propose that serine-129 phosphorylation decreases the ability of aSyn to both bind and bend DNA, as aSyn binds 304 bp circular DNA forced into a bent shape, but pSyn does not. Two aSyn paralogs, beta- and gamma-synuclein, also interact with DNA differently than aSyn, and do not stabilize similar DNA conformations. Our work suggests that reductions in aSyn's ability to bind and bend DNA induced by serine-129 phosphorylation may be important for modulating aSyn's known roles in DNA metabolism, including the regulation of transcription and DNA repair.

In vivo aggregation of presynaptic alpha-synuclein is not influenced by its phosphorylation at serine-129.

Abnormal aggregation of the α-synuclein protein is a key molecular feature of Parkinson's disease and other neurodegenerative diseases. The precise mechanisms that trigger α-synuclein aggregation are unclear, and it is not known what role aggregation plays in disease pathogenesis. Here we use an in vivo zebrafish model to express several different forms of human α-synuclein and measure its aggregation in presynaptic terminals. We show that human α-synuclein tagged with GFP can be expressed in zebrafish neurons, localizing normally to presynaptic terminals and undergoing phosphorylation at serine-129, as in mammalian neurons. The visual advantages of the zebrafish system allow for dynamic in vivo imaging to study α-synuclein, including the use of fluorescence recovery after photobleaching (FRAP) techniques to probe protein mobility. These experiments reveal three distinct terminal pools of α-synuclein with varying mobility, likely representing different subpopulations of aggregated and non-aggregated protein. Human α-synuclein is phosphorylated by an endogenous zebrafish Polo-like kinase activity, and there is a heterogeneous population of neurons containing either very little or extensive phosphorylation throughout the axonal arbor. Both pharmacological and genetic manipulations of serine-129 show that phosphorylation of α-synuclein at this site does not significantly affect its mobility. This suggests that serine-129 phosphorylation alone does not promote α-synuclein aggregation. Together our results show that human α-synuclein can be expressed and measured quantitatively in zebrafish, and that disease-relevant post-translational modifications occur within neurons. The zebrafish model provides a powerful in vivo system for measuring and manipulating α-synuclein function and aggregation, and for developing new treatments for neurodegenerative disease.

Genetic deletion of Polo-like kinase 2 reduces alpha-synuclein serine-129 phosphorylation in presynaptic terminals but not Lewy bodies.

Phosphorylation of alpha-synuclein at serine-129 is an important marker of pathologically relevant, aggregated forms of the protein in several important human diseases, including Parkinson's disease, Dementia with Lewy bodies, and Multiple system atrophy. Although several kinases have been shown to be capable of phosphorylating alpha-synuclein in various model systems, the identity of the kinase that phosphorylates alpha-synuclein in the Lewy body remains unknown. One member of the Polo-like kinase family, PLK2, is a strong candidate for being the Lewy body kinase. To examine this possibility, we have used a combination of approaches, including biochemical, immunohistochemical, and in vivo multiphoton imaging techniques to study the consequences of PLK2 genetic deletion on alpha-synuclein phosphorylation in both the presynaptic terminal and preformed fibril-induced Lewy body pathology in mouse cortex. We find that PLK2 deletion reduces presynaptic terminal alpha-synuclein serine-129 phosphorylation, but has no effect on Lewy body phosphorylation levels. Serine-129 mutation to the phosphomimetic alanine or the unphosphorylatable analog aspartate does not change the rate of cell death of Lewy inclusion-bearing neurons in our in vivo multiphoton imaging paradigm, but PLK2 deletion does slow the rate of neuronal death. Our data indicate that inhibition of PLK2 represents a promising avenue for developing new therapeutics, but that the mechanism of neuroprotection by PLK2 inhibition is not likely due to reducing alpha-synuclein serine-129 phosphorylation and that the true Lewy body kinase still awaits discovery.

Trans-synaptic and retrograde axonal spread of Lewy pathology following pre-formed fibril injection in an in vivo A53T alpha-synuclein mouse model of synucleinopathy.

It is necessary to develop an understanding of the specific mechanisms involved in alpha-synuclein aggregation and propagation to develop disease modifying therapies for age-related synucleinopathies, including Parkinson's disease and Dementia with Lewy Bodies. To adequately address this question, we developed a new transgenic mouse model of synucleinopathy that expresses human A53T SynGFP under control of the mouse prion protein promoter. Our characterization of this mouse line demonstrates that it exhibits several distinct advantages over other, currently available, mouse models. This new model allows rigorous study of the initial location of Lewy pathology formation and propagation in the living brain, and strongly suggests that aggregation begins in axonal structures with retrograde propagation to the cell body. This model also shows expeditious development of alpha-synuclein pathology following induction with small, in vitro-generated alpha-synuclein pre-formed fibrils (PFFs), as well as accelerated cell death of inclusion-bearing cells. Using this model, we found that aggregated alpha-synuclein somatic inclusions developed first in neurons, but later showed a second wave of inclusion formation in astrocytes. Interestingly, astrocytes appear to survive much longer after inclusion formation than their neuronal counterparts. This model also allowed careful study of peripheral-to-central spread of Lewy pathology after PFF injection into the hind limb musculature. Our results clearly show evidence of progressive, retrograde trans-synaptic spread of Lewy pathology through known neuroanatomically connected pathways in the motor system. As such, we have developed a promising tool to understand the biology of neurodegeneration associated with alpha-synuclein aggregation and to discover new treatments capable of altering the neurodegenerative disease course of synucleinopathies.

Alpha-synuclein is a DNA binding protein that modulates DNA repair with implications for Lewy body disorders.

Alpha-synuclein is a presynaptic protein that forms abnormal cytoplasmic aggregates in Lewy body disorders. Although nuclear alpha-synuclein localization has been described, its function in the nucleus is not well understood. We demonstrate that alpha-synuclein modulates DNA repair. First, alpha-synuclein colocalizes with DNA damage response components within discrete foci in human cells and mouse brain. Removal of alpha-synuclein in human cells leads to increased DNA double-strand break (DSB) levels after bleomycin treatment and a reduced ability to repair these DSBs. Similarly, alpha-synuclein knock-out mice show increased neuronal DSBs that can be rescued by transgenic reintroduction of human alpha-synuclein. Alpha-synuclein binds double-stranded DNA and helps to facilitate the non-homologous end-joining reaction. Using a new, in vivo imaging approach that we developed, we find that serine-129-phosphorylated alpha-synuclein is rapidly recruited to DNA damage sites in living mouse cortex. We find that Lewy inclusion-containing neurons in both mouse model and human-derived patient tissue demonstrate increased DSB levels. Based on these data, we propose a model whereby cytoplasmic aggregation of alpha-synuclein reduces its nuclear levels, increases DSBs, and may contribute to programmed cell death via nuclear loss-of-function. This model could inform development of new treatments for Lewy body disorders by targeting alpha-synuclein-mediated DNA repair mechanisms.

Curcumin Treatment Improves Motor Behavior in α-Synuclein Transgenic Mice.

The curry spice curcumin plays a protective role in mouse models of neurodegenerative diseases, and can also directly modulate aggregation of α-synuclein protein in vitro, yet no studies have described the interaction of curcumin and α-synuclein in genetic synucleinopathy mouse models. Here we examined the effect of chronic and acute curcumin treatment in the Syn-GFP mouse line, which overexpresses wild-type human α-synuclein protein. We discovered that curcumin diet intervention significantly improved gait impairments and resulted in an increase in phosphorylated forms of α-synuclein at cortical presynaptic terminals. Acute curcumin treatment also caused an increase in phosphorylated α-synuclein in terminals, but had no direct effect on α-synuclein aggregation, as measured by in vivo multiphoton imaging and Proteinase-K digestion. Using LC-MS/MS, we detected ~5 ng/mL and ~12 ng/mL free curcumin in the plasma of chronic or acutely treated mice, with a glucuronidation rate of 94% and 97%, respectively. Despite the low plasma levels and extensive metabolism of curcumin, these results show that dietary curcumin intervention correlates with significant behavioral and molecular changes in a genetic synucleinopathy mouse model that mimics human disease.

Progressive aggregation of alpha-synuclein and selective degeneration of lewy inclusion-bearing neurons in a mouse model of parkinsonism.

Aggregated alpha-synuclein inclusions are found where cell death occurs in several diseases, including Parkinson's disease, dementia with Lewy bodies, and multiple-system atrophy. However, the relationship between inclusion formation and an individual cell's fate has been difficult to study with conventional techniques. We developed a system that allows for in vivo imaging of the same neurons over months. We show that intracerebral injection of preformed fibrils of recombinant alpha-synuclein can seed aggregation of transgenically expressed and endogenous alpha-synuclein in neurons. Somatic inclusions undergo a stage-like maturation, with progressive compaction coinciding with decreased soluble somatic and nuclear alpha-synuclein. Mature inclusions bear the post-translational hallmarks of human Lewy pathology. Long-term imaging of inclusion-bearing neurons and neighboring neurons without inclusions demonstrates selective degeneration of inclusion-bearing cells. Our results indicate that inclusion formation is tightly correlated with cellular toxicity and that seeding may be a pathologically relevant mechanism of progressive neurodegeneration in many synucleinopathies.

Presynaptic alpha-synuclein aggregation in a mouse model of Parkinson's disease.

Parkinson's disease and dementia with Lewy bodies are associated with abnormal neuronal aggregation of α-synuclein. However, the mechanisms of aggregation and their relationship to disease are poorly understood. We developed an in vivo multiphoton imaging paradigm to study α-synuclein aggregation in mouse cortex with subcellular resolution. We used a green fluorescent protein-tagged human α-synuclein mouse line that has moderate overexpression levels mimicking human disease. Fluorescence recovery after photobleaching (FRAP) of labeled protein demonstrated that somatic α-synuclein existed primarily in an unbound, soluble pool. In contrast, α-synuclein in presynaptic terminals was in at least three different pools: (1) as unbound, soluble protein; (2) bound to presynaptic vesicles; and (3) as microaggregates. Serial imaging of microaggregates over 1 week demonstrated a heterogeneous population with differing α-synuclein exchange rates. The microaggregate species were resistant to proteinase K, phosphorylated at serine-129, oxidized, and associated with a decrease in the presynaptic vesicle protein synapsin and glutamate immunogold labeling. Multiphoton FRAP provided the specific binding constants for α-synuclein's binding to synaptic vesicles and its effective diffusion coefficient in the soma and axon, setting the stage for future studies targeting synuclein modifications and their effects. Our in vivo results suggest that, under moderate overexpression conditions, α-synuclein aggregates are selectively found in presynaptic terminals.

A survey of new temperature-sensitive, embryonic-lethal mutations in C. elegans: 24 alleles of thirteen genes.

To study essential maternal gene requirements in the early C. elegans embryo, we have screened for temperature-sensitive, embryonic lethal mutations in an effort to bypass essential zygotic requirements for such genes during larval and adult germline development. With conditional alleles, multiple essential requirements can be examined by shifting at different times from the permissive temperature of 15°C to the restrictive temperature of 26°C. Here we describe 24 conditional mutations that affect 13 different loci and report the identity of the gene mutations responsible for the conditional lethality in 22 of the mutants. All but four are mis-sense mutations, with two mutations affecting splice sites, another creating an in-frame deletion, and one creating a premature stop codon. Almost all of the mis-sense mutations affect residues conserved in orthologs, and thus may be useful for engineering conditional mutations in other organisms. We find that 62% of the mutants display additional phenotypes when shifted to the restrictive temperature as L1 larvae, in addition to causing embryonic lethality after L4 upshifts. Remarkably, we also found that 13 out of the 24 mutations appear to be fast-acting, making them particularly useful for careful dissection of multiple essential requirements. Our findings highlight the value of C. elegans for identifying useful temperature-sensitive mutations in essential genes, and provide new insights into the requirements for some of the affected loci.