Evaluation of Strategies for Measuring Lysosomal Glucocerebrosidase Activity.

Mutations in GBA1, which encode for the protein glucocerebrosidase (GCase), are the most common genetic risk factor for Parkinson's disease and dementia with Lewy bodies. In addition, growing evidence now suggests that the loss of GCase activity is also involved in onset of all forms of Parkinson's disease, dementia with Lewy bodies, and other dementias, such as progranulin-linked frontal temporal dementia. As a result, there is significant interest in developing GCase-targeted therapies that have the potential to stop or slow progression of these diseases. Despite this interest in GCase as a therapeutic target, there is significant inconsistency in the methodology for measuring GCase enzymatic activity in disease-modeling systems and patient populations, which could hinder progress in developing GCase therapies. In this review, we discuss the different strategies that have been developed to assess GCase activity and highlight the specific strengths and weaknesses of these approaches as well as the gaps that remain. We also discuss the current and potential role of these different methodologies in preclinical and clinical development of GCase-targeted therapies. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

CAF-1 and Rtt101p function within the replication-coupled chromatin assembly network to promote H4 K16ac, preventing ectopic silencing.

Replication-coupled chromatin assembly is achieved by a network of alternate pathways containing different chromatin assembly factors and histone-modifying enzymes that coordinate deposition of nucleosomes at the replication fork. Here we describe the organization of a CAF-1-dependent pathway in Saccharomyces cerevisiae that regulates acetylation of histone H4 K16. We demonstrate factors that function in this CAF-1-dependent pathway are important for preventing establishment of silenced states at inappropriate genomic sites using a crippled HMR locus as a model, while factors specific to other assembly pathways do not. This CAF-1-dependent pathway required the cullin Rtt101p, but was functionally distinct from an alternate pathway involving Rtt101p-dependent ubiquitination of histone H3 and the chromatin assembly factor Rtt106p. A major implication from this work is that cells have the inherent ability to create different chromatin modification patterns during DNA replication via differential processing and deposition of histones by distinct chromatin assembly pathways within the network.

Progranulin mutations result in impaired processing of prosaposin and reduced glucocerebrosidase activity.

Frontotemporal dementia (FTD) is a common neurogenerative disorder characterized by progressive degeneration in the frontal and temporal lobes. Heterozygous mutations in the gene encoding progranulin (PGRN) are a common genetic cause of FTD. Recently, PGRN has emerged as an important regulator of lysosomal function. Here, we examine the impact of PGRN mutations on the processing of full-length prosaposin to individual saposins, which are critical regulators of lysosomal sphingolipid metabolism. Using FTD-PGRN patient-derived cortical neurons differentiated from induced pluripotent stem cells, as well as post-mortem tissue from patients with FTLD-PGRN, we show that PGRN haploinsufficiency results in impaired processing of prosaposin to saposin C, a critical activator of the lysosomal enzyme glucocerebrosidase (GCase). Additionally, we found that PGRN mutant neurons had reduced lysosomal GCase activity, lipid accumulation and increased insoluble α-synuclein relative to isogenic controls. Importantly, reduced GCase activity in PGRN mutant neurons is rescued by treatment with saposin C. Together, these findings suggest that reduced GCase activity due to impaired processing of prosaposin may contribute to pathogenesis of FTD resulting from PGRN mutations.

LRRK2 kinase activity regulates lysosomal glucocerebrosidase in neurons derived from Parkinson's disease patients.

Mutations in LRRK2 and GBA1 are common genetic risk factors for Parkinson's disease (PD) and major efforts are underway to develop new therapeutics that target LRRK2 or glucocerebrosidase (GCase). Here we describe a mechanistic and therapeutic convergence of LRRK2 and GCase in neurons derived from patients with PD. We find that GCase activity was reduced in dopaminergic (DA) neurons derived from PD patients with LRRK2 mutations. Inhibition of LRRK2 kinase activity results in increased GCase activity in DA neurons with either LRRK2 or GBA1 mutations. This increase is sufficient to partially rescue accumulation of oxidized dopamine and alpha-synuclein in PD patient neurons. We have identified the LRRK2 substrate Rab10 as a key mediator of LRRK2 regulation of GCase activity. Together, these results suggest an important role of mutant LRRK2 as a negative regulator of lysosomal GCase activity.

Modulation of Gene Silencing by Cdc7p via H4 K16 Acetylation and Phosphorylation of Chromatin Assembly Factor CAF-1 in Saccharomyces cerevisiae.

CAF-1 is an evolutionarily conserved H3/H4 histone chaperone that plays a key role in replication-coupled chromatin assembly and is targeted to the replication fork via interactions with PCNA, which, if disrupted, leads to epigenetic defects. In Saccharomyces cerevisiae, when the silent mating-type locus HMR contains point mutations within the E silencer, Sir protein association and silencing is lost. However, mutation of CDC7, encoding an S-phase-specific kinase, or subunits of the H4 K16-specific acetyltransferase complex SAS-I, restore silencing to this crippled HMR, HMRae** Here, we observed that loss of Cac1p, the largest subunit of CAF-1, also restores silencing at HMRae**, and silencing in both cac1Δ and cdc7 mutants is suppressed by overexpression of SAS2 We demonstrate Cdc7p and Cac1p interact in vivo in S phase, but not in G1, consistent with observed cell cycle-dependent phosphorylation of Cac1p, and hypoacetylation of chromatin at H4 K16 in both cdc7 and cac1Δ mutants. Moreover, silencing at HMRae** is restored in cells expressing cac1p mutants lacking Cdc7p phosphorylation sites. We also discovered that cac1Δ and cdc7-90 synthetically interact negatively in the presence of DNA damage, but that Cdc7p phosphorylation sites on Cac1p are not required for responses to DNA damage. Combined, our results support a model in which Cdc7p regulates replication-coupled histone modification via a CAC1-dependent mechanism involving H4 K16ac deposition, and thereby silencing, while CAF-1-dependent replication- and repair-coupled chromatin assembly per se are functional in the absence of phosphorylation of Cdc7p consensus sites on CAF-1.

Cell cycle regulation of silent chromatin formation.

Identical genes in two different cells can stably exist in alternate transcriptional states despite the dynamic changes that will occur to chromatin at that locus throughout the cell cycle. In mammals, this is achieved through epigenetic processes that regulate key developmental transitions and ensure stable patterns of gene expression during growth and differentiation. The budding yeast Saccharomyces cerevisiae utilizes silencing to control the expression state of genes encoding key regulatory factors for determining cell-type, ribosomal RNA levels and proper telomere function. Here, we review the composition of silent chromatin in S. cerevisiae, how silent chromatin is influenced by chromatin assembly and histone modifications and highlight several observations that have contributed to our understanding of the interplay between silent chromatin formation and stability and the cell cycle. This article is part of a Special Issue entitled: Histone chaperones and Chromatin assembly.