Glucocerebrosidase activity, cathepsin D and monomeric α-synuclein interactions in a stem cell derived neuronal model of a PD associated GBA1 mutation.

The presence of GBA1 gene mutations increases risk for Parkinson's disease (PD), but the pathogenic mechanisms of GBA1 associated PD remain unknown. Given that impaired α-synuclein turnover is a hallmark of PD pathogenesis and cathepsin D is a key enzyme involved in α-synuclein degradation in neuronal cells, we have examined the relationship of glucocerebrosidase (GCase), cathepsin D and monomeric α-synuclein in human neural crest stem cell derived dopaminergic neurons. We found that normal activity of GCase is necessary for cathepsin D to perform its function of monomeric α-synuclein removal from neurons. GBA1 mutations lead to a lower level of cathepsin D protein and activity, and higher level of monomeric α-synuclein in neurons. When GBA1 mutant neurons were treated with GCase replacement or chaperone therapy; cathepsin D protein levels and activity were restored, and monomeric α-synuclein decreased. When cathepsin D was inhibited, GCase replacement failed to reduce monomeric α-synuclein levels in GBA1 mutant neurons. These data indicate that GBA1 gene mutations increase monomeric α-synuclein levels via an effect on lysosomal cathepsin D in neurons

Endo-lysosomal TRP mucolipin-1 channels trigger global ER Ca2+ release and Ca2+ influx.

Transient receptor potential (TRP) mucolipins (TRPMLs), encoded by the MCOLN genes, are patho-physiologically relevant endo-lysosomal ion channels crucial for membrane trafficking. Several lines of evidence suggest that TRPMLs mediate localised Ca(2+) release but their role in Ca(2+) signalling is not clear. Here, we show that activation of endogenous and recombinant TRPMLs with synthetic agonists evoked global Ca(2+) signals in human cells. These signals were blocked by a dominant-negative TRPML1 construct and a TRPML antagonist. We further show that, despite a predominant lysosomal localisation, TRPML1 supports both Ca(2+) release and Ca(2+) entry. Ca(2+) release required lysosomal and ER Ca(2+) stores suggesting that TRPMLs, like other endo-lysosomal Ca(2+) channels, are capable of 'chatter' with ER Ca(2+) channels. Our data identify new modalities for TRPML1 action

The lysosomotrope, GPN, mobilises Ca2+ from acidic organelles

Lysosomes are acidic Ca2+ stores often mobilised in conjunction with endoplasmic reticulum (ER) Ca2+ stores. GPN is a widely used lysosomotropic agent that evokes cytosolic Ca2+ signals in many cells. But whether these signals are due to a primary action on lysosomes is unclear in light of recent evidence showing GPN mediates direct ER Ca2+ release through changes in cytosolic pH. Here, we show that GPN evoked rapid increases in cytosolic pH but slower Ca2+ signals. NH4Cl evoked comparable changes in pH but failed to affect Ca2+ The V-type ATPase inhibitor, bafilomycin A1, increased lysosomal pH over a period of hours. Acute treatment modestly affected lysosomal pH and potentiated Ca2+ signals evoked by GPN. In contrast, chronic treatment led to more profound changes in luminal pH and selectively inhibited GPN-action. GPN blocked Ca2+ responses evoked by the novel NAADP-like agonist, TPC2-A1-N. GPN-evoked Ca2+ signals were thus better correlated with associated pH changes in the lysosome compared to the cytosol and coupled to lysosomal Ca2+ release. We conclude that Ca2+ signals evoked by GPN most likely derive from acidic organelles

The remote assessment of parkinsonism supporting ongoing development of interventions in Gaucher disease

Mutations in GBA which are causative of Gaucher disease in their biallelic form, are the most common genetic risk factor for Parkinson's disease (PD). The diagnosis of PD relies upon clinically defined motor features which appear after irreversible neurodegeneration. Prodromal symptoms of PD may provide a means to predict latent pathology, years before the onset of motor features. Previous work has reported prodromal features of PD in GBA mutation carriers, however this has been insufficiently sensitive to identify those that will develop PD. The Remote Assessment of Parkinsonism Supporting Ongoing Development of Interventions in Gaucher Disease (RAPSODI GD) study assesses a large cohort of GBA mutation carriers, to aid development of procedures for earlier diagnosis of PD

Mitochondria and Quality Control Defects in a Mouse Model of Gaucher Disease-Links to Parkinson's Disease

Mutations in the glucocerebrosidase (gba) gene cause Gaucher disease (GD), the most common lysosomal storage disorder, and increase susceptibility to Parkinson’s disease (PD). While the clinical and pathological features of idiopathic PD and PD related to gba (PD-GBA) mutations are very similar, cellular mechanisms underlying neurodegeneration in each are unclear. Using a mouse model of neuronopathic GD, we show that autophagic machinery and proteasomal machinery are defective in neurons and astrocytes lacking gba. Markers of neurodegeneration—p62/SQSTM1, ubiquitinated proteins, and insoluble α-synuclein—accumulate. Mitochondria were dysfunctional and fragmented, with impaired respiration, reduced respiratory chain complex activities, and a decreased potential maintained by reversal of the ATP synthase. Thus a primary lysosomal defect causes accumulation of dysfunctional mitochondria as a result of impaired autophagy and dysfunctional proteasomal pathways. These data provide conclusive evidence for mitochondrial dysfunction in GD and provide insight into the pathogenesis of PD and PD-GBA

Sex-Specific Microglial Responses to Glucocerebrosidase Inhibition: Relevance to GBA1-Linked Parkinson’s Disease

Microglia are heterogenous cells characterized by distinct populations each contributing to specific biological processes in the nervous system, including neuroprotection. To elucidate the impact of sex-specific microglia heterogenicity to the susceptibility of neuronal stress, we video-recorded with time-lapse microscopy the changes in shape and motility occurring in primary cells derived from mice of both sexes in response to pro-inflammatory or neurotoxic stimulations. With this morpho-functional analysis, we documented distinct microglia subpopulations eliciting sex-specific responses to stimulation: male microglia tended to have a more pro-inflammatory phenotype, while female microglia showed increased sensitivity to conduritol-B-epoxide (CBE), a small molecule inhibitor of glucocerebrosidase, the enzyme encoded by the GBA1 gene, mutations of which are the major risk factor for Parkinson’s Disease (PD). Interestingly, glucocerebrosidase inhibition particularly impaired the ability of female microglia to enhance the Nrf2-dependent detoxification pathway in neurons, attenuating the sex differences observed in this neuroprotective function. This finding is consistent with the clinical impact of GBA1 mutations, in which the 1.5–2-fold reduced risk of developing idiopathic PD observed in female individuals is lost in the GBA1 carrier population, thus suggesting a sex-specific role for microglia in the etiopathogenesis of PD-GBA1

Systemic exosomal siRNA delivery reduced alpha-synuclein aggregates in brains of transgenic mice.

Alpha-synuclein (α-Syn) aggregates are the main component of Lewy bodies, which are the characteristic pathological feature in Parkinson's disease (PD) brain. Evidence that α-Syn aggregation can be propagated between neurones has led to the suggestion that this mechanism is responsible for the stepwise progression of PD pathology. Decreasing α-Syn expression is predicted to attenuate this process and is thus an attractive approach to delay or halt PD progression. We have used α-Syn small interfering RNA (siRNA) to reduce total and aggregated α-Syn levels in mouse brains. To achieve widespread delivery of siRNAs to the brain we have peripherally injected modified exosomes expressing Ravies virus glycoprotein loaded with siRNA. Normal mice were analyzed 3 or 7 days after injection. To evaluate whether this approach can decrease α-Syn aggregates, we repeated the treatment using transgenic mice expressing the human phosphorylation-mimic S129D α-Syn, which exhibits aggregation. In normal mice we detected significantly reduced α-Syn messenger RNA (mRNA) and protein levels throughout the brain 3 and 7 days after treatment with RVG-exosomes loaded with siRNA to α-Syn. In S129D α-Syn transgenic mice we found a decreased α-Syn mRNA and protein levels throughout the brain 7 days after injection. This resulted in significant reductions in intraneuronal protein aggregates, including in dopaminergic neurones of the substantia nigra. This study highlights the therapeutic potential of RVG-exosome delivery of siRNA to delay and reverse brain α-Syn pathological conditions

Arsenic in the cerebrospinal fluid of a patient receiving arsenic trioxide for relapsed acute promyelocytic leukemia with CNS involvement

We report on a 42-year-old patient whose relapse of acute promyelocytic leukaemia (APL) included meningeal infiltration. Since he had previously experienced ATRA syndrome, he received arsenic trioxide (ATO) plus intrathecal therapy with cytarabine, prednisone, and methotrexate. We measured the concentration of arsenic in his cerebrospinal fluid (CSF). Arsenic showed a peak CSF concentration of 0.008mg/l (0.11mumol/l) and a nadir of 0.002mg/l (0.027mumol/l), both representing about 14% of blood levels. ATO thus crosses the blood-CSF-barrier when administered intravenously, but the concentration in CSF is probably not sufficient for treatment of meningeal leukemia