GBA1 gene encodes for the lysosomal membrane protein glucocerebrosidase (GCase). GBA1 heterozygous mutations profoundly impair GCase activity and are currently recognized as an important risk factor for the development of Parkinson's disease (PD). Deficits in lysosomal degradation pathways may contribute to pathological α-synuclein accumulation, thereby favoring dopaminergic neuron degeneration and associated microglial activation. However, the precise mechanisms by which GCase deficiency may influence PD onset and progression remain unclear. In this work we used conduritol-β-epoxide (CBE), a potent inhibitor of GCase, to induce a partial, systemic defect of GCase activity comparable to that associated with heterozygous GBA1 mutations, in mice. Chronic (28 days) administration of CBE (50 mg/kg, i.p.) was combined with administration of a classic PD-like inducing neurotoxin, such as MPTP (30 mg/kg, i.p. for 5 days). The aim was to investigate whether a pre-existing GCase defect may influence the effects of MPTP in terms of nigrostriatal damage, microglia activation and α-synuclein accumulation. Pre-treatment with CBE had tendency to enhance MPTP-induced neurodegeneration in striatum and caused significant increase of total α-synuclein expression in substantia nigra. Microglia was remarkably activated by CBE alone, without further increases when combined with MPTP. Overall, we propose this model as an additional tool to study pathophysiological processes of PD in the presence of GCase defects.

Heterozygous mutations in GBA1 gene, encoding for lysosomal enzyme glucocerebrosidase (GCase), are a major risk factor for sporadic Parkinson's disease (PD). Defective GCase has been reported in fibroblasts of GBA1-mutant PD patients and pharmacological chaperone ambroxol has been shown to correct such defect. To further explore this issue, we investigated GCase and elements supporting GCase function and trafficking in fibroblasts from sporadic PD patients--with or without heterozygous GBA1 mutations--and healthy subjects, in basal conditions and following in vitro exposure to ambroxol. We assessed protein levels of GCase, lysosomal integral membrane protein-2 (LIMP-2), which mediates GCase trafficking to lysosomes, GCase endogenous activator saposin (Sap) C and parkin, which is involved in degradation of defective GCase. We also measured activities of GCase and cathepsin D, which cleaves Sap C from precursor prosaposin. GCase activity was reduced in fibroblasts from GBA1-mutant patients and ambroxol corrected this defect. Ambroxol increased cathepsin D activity, GCase and Sap C protein levels in all groups, while LIMP-2 levels were increased only in GBA1-mutant PD fibroblasts. Parkin levels were slightly increased only in the PD group without GBA1 mutations and were not significantly modified by ambroxol. Our study confirms that GCase activity is deficient in fibroblasts of GBA1-mutant PD patients and that ambroxol corrects this defect. The drug increased Sap C and LIMP-2 protein levels, without interfering with parkin. These results confirm that chemical chaperone ambroxol modulates lysosomal markers, further highlighting targets that may be exploited for innovative PD therapeutic strategies.

Patients with Parkinson's disease (PD) are often characterized by functional gastrointestinal disorders. Such disturbances can occur at all stages of PD and precede the typical motor symptoms of the disease by many years. However, the morphological alterations associated with intestinal disturbances in PD are undetermined. This study examined the remodelling of colonic wall in 6-hydroxydopamine (6-OHDA)-induced PD rats.

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare leukodystrophy caused by mutations in the gene encoding MLC1, a membrane protein mainly expressed in astrocytes in the central nervous system. Although MLC1 function is unknown, evidence is emerging that it may regulate ion fluxes. Using biochemical and proteomic approaches to identify MLC1 interactors and elucidate MLC1 function we found that MLC1 interacts with the vacuolar ATPase (V-ATPase), the proton pump that regulates endosomal acidity. Because we previously showed that in intracellular organelles MLC1 directly binds Na, K-ATPase, which controls endosomal pH, we studied MLC1 endosomal localization and trafficking and MLC1 effects on endosomal acidity and function using human astrocytoma cells overexpressing wild-type (WT) MLC1 or MLC1 carrying pathological mutations. We found that WT MLC1 is abundantly expressed in early (EEA1(+), Rab5(+)) and recycling (Rab11(+)) endosomes and uses the latter compartment to traffic to the plasma membrane during hyposmotic stress. We also showed that WT MLC1 limits early endosomal acidification and influences protein trafficking in astrocytoma cells by stimulating protein recycling, as revealed by FITC-dextran measurement of endosomal pH and transferrin protein recycling assay, respectively. WT MLC1 also favors recycling to the plasma-membrane of the TRPV4 cation channel which cooperates with MLC1 to activate calcium influx in astrocytes during hyposmotic stress. Although MLC disease-causing mutations differentially affect MLC1 localization and trafficking, all the mutated proteins fail to influence endosomal pH and protein recycling. This study demonstrates that MLC1 modulates endosomal pH and protein trafficking suggesting that alteration of these processes contributes to MLC pathogenesis.

The endoplasmic reticulum (ER) stress-mediated pathway is involved in a wide range of human neurodegenerative disorders. Hence, molecules that regulate the ER stress response represent potential candidates as drug targets to tackle these diseases. In previous studies we demonstrated that upon acetylation the reticulon-1C (RTN-1C) variant of the reticulon family leads to inhibition of histone deacetylase (HDAC) enzymatic activity and endoplasmic reticulum stress-dependent apoptosis. Here, by microarray analysis of the whole human genome we found that RTN-1C is able to specifically regulate gene expression, modulating transcript clusters which have been implicated in the onset of neurodegenerative disorders. Interestingly, we show that some of the identified genes were also modulated in vivo in a brain-specific mouse model overexpressing RTN-1C. These data provide a basis for further investigation of RTN-1C as a potential molecular target for use in therapy and as a specific marker for neurological diseases.