Glucocerebrosidase gene therapy prevents α-synucleinopathy of midbrain dopamine neurons

AbstractDiminished lysosomal function can lead to abnormal cellular accumulation of specific proteins, including α-synuclein, contributing to disease pathogenesis of vulnerable neurons in Parkinson's disease (PD) and related α-synucleinopathies. GBA1 encodes for the lysosomal hydrolase glucocerebrosidase (GCase), and mutations in GBA1 are a prominent genetic risk factor for PD. Previous studies showed that in sporadic PD, and in normal aging, GCase brain activity is reduced and levels of corresponding glycolipid substrates are increased. The present study tested whether increasing GCase through AAV-GBA1 intra-cerebral gene delivery in two PD rodent models would reduce the accumulation of α-synuclein and protect midbrain dopamine neurons from α-synuclein-mediated neuronal damage. In the first model, transgenic mice overexpressing wildtype α-synuclein throughout the brain (ASO mice) were used, and in the second model, a rat model of selective dopamine neuron degeneration was induced by AAV-A53T mutant α-synuclein. In ASO mice, intra-cerebral AAV-GBA1 injections into several brain regions increased GCase activity and reduced the accumulation of α-synuclein in the substantia nigra and striatum. In rats, co-injection of AAV-GBA1 with AAV-A53T α-synuclein into the substantia nigra prevented α-synuclein-mediated degeneration of nigrostriatal dopamine neurons by 6months. These neuroprotective effects were associated with altered protein expression of markers of autophagy. These experiments demonstrate, for the first time, the neuroprotective effects of increasing GCase against dopaminergic neuron degeneration, and support the development of therapeutics targeting GCase or other lysosomal genes to improve neuronal handling of α-synuclein

Elucidating the Regulatory Role of 3D Genome Folding During Neural Differentiation and Synaptic Activation

The causal link between the three-dimensional conformation of the genome and spatiotemporal control of gene regulation has long been studied in the form of enhancer-promoter interactions. Only recently have advances in molecular biology and next generation sequencing allowed higher-order chromatin folding to be queried genome-wide at ultra-high-resolution. In this thesis we leverage Chromosome Conformation Capture Carbon Copy (5C) along with RNA-seq and ChIP-seq to elucidate how the genome is reconfigured during neural development, cellular reprogramming, and synaptic activation. We observe that the first step in neural differentiation is accompanied by a bulk decommissioning of nearly half of the architectural protein CTCF’s binding sites in the pluripotent genome, a trend which continues throughout terminal neuronal differentiation and results in the dissolution of many chromatin loops present in embryonic stem cells (ESCs). We identify another zinc finger protein, Yin Yang 1 (YY1), at the base of looping interactions between neural progenitor cell (NPC) specific genes and enhancers; siRNA knockdown of YY1 specifically disrupts interactions between key NPC enhancers and their target genes. Additionally, we find that many of the CTCF sites that are decommissioned during neural lineage commitment are not efficiently restored during cellular reprogramming of NPCs to induced pluripotent stem cells (iPSCs). CTCF sites that do not successfully regain binding in iPSCs underlie incompletely reprogrammed chromatin architecture, resulting in an iPSC genome folding and transcriptional signature that resembles an intermediate state between ESCs and NPCs. Culture in 2i media conditions restores the CTCF binding, genome folding, and gene expression of iPSCs to patterns resembling those of ESCs. Finally, we find that a large subset of chromatin loops surrounding select neuronal activity response genes (ARGs) are induced de novo during cortical neuron activation. We observe a striking correlation between the number, length, and kinetics of loops an ARG forms and how much time that ARG takes to be upregulated in response to neuronal activity. Additionally, we find that common single nucleotide variants (SNVs) associated with Autism Spectrum Disorder connect activity-inducible enhancers to upregulated genes, whereas Schizophrenia SNVs anchor pre-existing loops connecting activity-decommissioned enhancers to activity-downregulated genes. Altogether this work begins to elucidate how the 3-D genome orchestrates cellular state and function decisions during mammalian brain development from the earliest neural lineage commitment through the refinement of connections between terminally differentiated neurons

A Nurr1 Agonist Causes Neuroprotection in a Parkinson’s Disease Lesion Model Primed with the Toll-Like Receptor 3 dsRNA Inflammatory Stimulant Poly(I:C)

Dopaminergic neurons in the substantia nigra pars compacta (SNpc) are characterized by the expression of genes required for dopamine synthesis, handling and reuptake and the expression of these genes is largely controlled by nuclear receptor related 1 (Nurr1). Nurr1 is also expressed in astrocytes and microglia where it functions to mitigate the release of proinflammatory cytokines and neurotoxic factors. Given that Parkinson’s disease (PD) pathogenesis has been linked to both loss of Nurr1 expression in the SNpc and inflammation, increasing levels of Nurr1 maybe a promising therapeutic strategy. In this study a novel Nurr1 agonist, SA00025, was tested for both its efficiency to induce the transcription of dopaminergic target genes in vivo and prevent dopaminergic neuron degeneration in an inflammation exacerbated 6-OHDA-lesion model of PD. SA00025 (30mg/kg p.o.) entered the brain and modulated the expression of the dopaminergic phenotype genes TH, VMAT, DAT, AADC and the GDNF receptor gene c-Ret in the SN of naive rats. Daily gavage treatment with SA00025 (30mg/kg) for 32 days also induced partial neuroprotection of dopaminergic neurons and fibers in rats administered a priming injection of polyinosinic-polycytidylic acid (poly(I:C) and subsequent injection of 6-OHDA. The neuroprotective effects of SA00025 in this dopamine neuron degeneration model were associated with changes in microglial morphology indicative of a resting state and a decrease in microglial specific IBA-1 staining intensity in the SNpc. Astrocyte specific GFAP staining intensity and IL-6 levels were also reduced. We conclude that Nurr1 agonist treatment causes neuroprotective and anti-inflammatory effects in an inflammation exacerbated 6-OHDA lesion model of PD

Disease-Associated Short Tandem Repeats Co-localize with Chromatin Domain Boundaries

More than 25 inherited human disorders are caused by the unstable expansion of repetitive DNA sequences termed short tandem repeats (STRs). A fundamental unresolved question is why some STRs are susceptible to pathologic expansion, whereas thousands of repeat tracts across the human genome are relatively stable. Here, we discover that nearly all disease-associated STRs (daSTRs) are located at boundaries demarcating 3D chromatin domains. We identify a subset of boundaries with markedly higher CpG island density compared to the rest of the genome. daSTRs specifically localize to ultra-high-density CpG island boundaries, suggesting they might be hotspots for epigenetic misregulation or topological disruption linked to STR expansion. Fragile X syndrome patients exhibit severe boundary disruption in a manner that correlates with local loss of CTCF occupancy and the degree of FMR1 silencing. Our data uncover higher-order chromatin architecture as a new dimension in understanding repeat expansion disorders

Sustained systemic glucocerebrosidase inhibition induces brain α-Synuclein aggregation, microglia and complement C1q activation in mice

Aims: Loss-of-function mutations in GBA1, which cause the autosomal recessive lysosomal storage disease, Gaucher disease (GD), are also a key genetic risk factor for the α-synucleinopathies, including Parkinson's disease (PD) and dementia with Lewy bodies. GBA1 encodes for the lysosomal hydrolase glucocerebrosidase and reductions in this enzyme result in the accumulation of the glycolipid substrates glucosylceramide and glucosylsphingosine. Deficits in autophagy and lysosomal degradation pathways likely contribute to the pathological accumulation of α-synuclein in PD. In this report we used conduritol-β-epoxide (CBE), a potent selective irreversible competitive inhibitor of glucocerebrosidase, to model reduced glucocerebrosidase activity in vivo, and tested whether sustained glucocerebrosidase inhibition in mice could induce neuropathological abnormalities including α-synucleinopathy, and neurodegeneration. Results: Our data demonstrate that daily systemic CBE treatment over 28 days caused accumulation of insoluble α-synuclein aggregates in the substantia nigra, and altered levels of proteins involved in the autophagy lysosomal system. These neuropathological changes were paralleled by widespread neuroinflammation, upregulation of complement C1q, abnormalities in synaptic, axonal transport and cytoskeletal proteins, and neurodegeneration. Innovation: A reduction in brain GCase activity has been linked to sporadic PD and normal aging, and may contribute to the susceptibility of vulnerable neurons to degeneration. This report demonstrates that systemic reduction of GCase activity using chemical inhibition, leads to neuropathological changes in the brain reminiscent of α-synucleinopathy. Conclusions: These data reveal a link between reduced glucocerebrosidase and the development of α-synucleinopathy and pathophysiological abnormalities in mice, and support the development of GCase therapeutics to reduce α-synucleinopathy in PD and related disorders

Local Genome Topology Can Exhibit an Incompletely Rewired 3D-Folding State during Somatic Cell Reprogramming

Pluripotent genomes are folded in a topological hierarchy that reorganizes during differentiation. The extent to which chromatin architecture is reconfigured during somatic cell reprogramming is poorly understood. Here we integrate fine-resolution architecture maps with epigenetic marks and gene expression in embryonic stem cells (ESCs), neural progenitor cells (NPCs), and NPC-derived induced pluripotent stem cells (iPSCs). We find that most pluripotency genes reconnect to target enhancers during reprogramming. Unexpectedly, some NPC interactions around pluripotency genes persist in our iPSC clone. Pluripotency genes engaged in both fully-reprogrammed and persistent-NPC interactions exhibit over/undershooting of target expression levels in iPSCs. Additionally, we identify a subset of poorly reprogrammed interactions that do not reconnect in iPSCs and display only partially recovered, ESC-specific CTCF occupancy. 2i/LIF can abrogate persistent-NPC interactions, recover poorly reprogrammed interactions, reinstate CTCF occupancy, and restore expression levels. Our results demonstrate that iPSC genomes can exhibit imperfectly rewired 3D-folding linked to inaccurately reprogrammed gene expression

ALS-associated peripherin spliced transcripts form distinct protein inclusions that are neuroprotective against oxidative stress

Intracellular proteinaceous inclusions are well-documented hallmarks of the fatal motor neuron disorder amyotrophic lateral sclerosis (ALS). The pathological significance of these inclusions remains unknown. Peripherin, a type III intermediate filament protein, is upregulated in ALS and identified as a component within different types of ALS inclusions. The formation of these inclusions may be associated with abnormal peripherin splicing, whereby an increase in mRNA retaining introns 3 and 4 (Per-3,4) leads to the generation of an aggregation-prone isoform, Per-28. During the course of evaluating peripherin filament assembly in SW-13 cells, we identified that expression of both Per-3,4 and Per-28 transcripts formed inclusions with categorically distinct morphology: Per-3,4 was associated with cytoplasmic condensed/bundled filaments, small inclusions (< 10 μM), or large inclusions (≥ 10 μM); while Per-28 was associated with punctate inclusions in the nucleus and/or cytoplasm. We found temporal and spatial changes in inclusion morphology between 12 and 48 h post-transfected cells, which were accompanied by unique immunofluorescent and biochemical changes of other ALS-relevant proteins, including TDP-43 and ubiquitin. Despite mild cytotoxicity associated with peripherin transfection, Per-3,4 and Per-28 expression increased cell viability during H2O2-mediated oxidative stress in BE(2)-M17 neuroblastoma cells. Taken together, this study shows that ALS-associated peripherin isoforms form dynamic cytoplasmic and intranuclear inclusions, effect changes in local endogenous protein expression, and afford cytoprotection against oxidative stress. These findings may have important relevance to understanding the pathophysiological role of inclusions in ALS

SA00025 induced anti-inflammatory activity in the 6-OHDA lesion model primed with dsRNA inflammatory stimulant (poly(I:C)).

<p>There was a significant increase in the amount IBA-1 +ve microglia in the SNpc scored as rating 1 (ramified and resting) and a significant decrease in microglia scored at rating 3 (bushy and reactive) with 32 days of Nurr1 agonist treatment, compared to vehicle (A). Representative photomicrographs show that Nurr1 agonist treatment caused a reduction in IBA-1 +ve microglia that were bushy and reactive and an increase in IBA-1 +ve microglia that were ramified and resting in the SNpc (B). Representative photomicrographs show that Nurr1 agonist treatment also caused a reduction in the immunofluorescent intensity of IBA-1 +ve microglia (green) and GFAP +ve astrocytes (C). Optical density analysis indicated that Nurr1 agonist treatment caused a significant decrease in IBA-1 (D) and GFAP (E) staining in the SNpc, compared to vehicle conditions. Significance is annotated as p<0.05*, 2- way ANOVA. N = 6–8/ group. Graphs are expressed at mean ± SEM. Scale bars = 100μm.</p