α-Synuclein interacts directly but reversibly with psychosine: implications for α-synucleinopathies

Aggregation of α-synuclein, the hallmark of α-synucleinopathies such as Parkinson´s disease, occurs in various glycosphingolipidoses. Although α-synuclein aggregation correlates with deficiencies in the lysosomal degradation of glycosphingolipids (GSL), the mechanism(s) involved in this aggregation remains unclear. We previously described the aggregation of α-synuclein in Krabbe´s disease (KD), a neurodegenerative glycosphingolipidosis caused by lysosomal deficiency of galactosyl-ceramidase (GALC) and the accumulation of the GSL psychosine. Here, we used a multi-pronged approach including genetic, biophysical and biochemical techniques to determine the pathogenic contribution, reversibility, and molecular mechanism of aggregation of α-synuclein in KD. While genetic knock-out of α-synuclein reduces, but does not completely prevent, neurological signs in a mouse model of KD, genetic correction of GALC deficiency completely prevents α-synuclein aggregation. We show that psychosine forms hydrophilic clusters and binds the C-terminus of α-synuclein through its amino group and sugar moiety, suggesting that psychosine promotes an open/aggregation-prone conformation of α-synuclein. Dopamine and carbidopa reverse the structural changes of psychosine by mediating a closed/aggregation-resistant conformation of α-synuclein. Our results underscore the therapeutic potential of lysosomal correction and small molecules to reduce neuronal burden in α-synucleinopathies, and provide a mechanistic understanding of α-synuclein aggregation in glycosphingolipidoses.Fil: Abdelkarim, Hazem. University of Illinois; Estados UnidosFil: Marshall, Michael S.. University of Illinois; Estados UnidosFil: Scesa, Giuseppe. University of Illinois; Estados UnidosFil: Smith, Rachael A.. University of Illinois; Estados UnidosFil: Rue, Emily. University of Illinois; Estados UnidosFil: Marshall, Jeffrey. University of Illinois; Estados UnidosFil: Elackattu, Vince. University Of Illinois Chicago; Estados UnidosFil: Stoskute, Monika. University Of Illinois Chicago; Estados UnidosFil: Issa, Yazan. University Of Illinois Chicago; Estados UnidosFil: Santos, Marta. University Of Illinois Chicago; Estados UnidosFil: Nguyen, Duc. University Of Illinois Chicago; Estados UnidosFil: Hauck, Zane. University Of Illinois Chicago; Estados UnidosFil: Van Breemen, Richard B.. University Of Illinois Chicago; Estados UnidosFil: Celej, Maria Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; ArgentinaFil: Gaponenko, Vadim. University Of Illinois Chicago; Estados UnidosFil: Bongarzone, Ernesto R.. University Of Illinois Chicago; Estados Unido

Analysis of age-related changes in psychosine metabolism in the human brain

<div><p>α-Synuclein aggregation has been linked to Gaucher’s disease (GD) and Krabbe’s disease (KD), lysosomal conditions affecting glycosphingolipid metabolism. α-Synuclein pathology has been directly attributed to the dysregulation of glycosphingolipids in both conditions, specifically to increased galactosylsphingosine (psychosine) content in the context of KD. Furthermore, the gene (<i>GALC</i>) coding for the psychosine degrading enzyme galactosylceramidase (GALC), has recently been identified as a risk loci for Parkinson’s disease. However, it is unknown if changes in psychosine metabolism and GALC activity in the context of the aging human brain correlate with Parkinson’s disease. We investigated psychosine accumulation and GALC activity in the aging brain using fresh frozen post-mortem tissue from Parkinson’s (PD, n = 10), Alzheimer’s (AD, n = 10), and healthy control patients (n = 9), along with tissue from neuropsychiatric patients (schizophrenia, bipolar disorder and depression, n = 15 each). An expanded mutational analysis of PD (n = 20), AD (n = 10), and healthy controls (n = 30) examined if PD was correlated with carriers for severe <i>GALC</i> mutations. Psychosine content within the cerebral cortex of PD patients was elevated above control patients. Within all patients, psychosine displayed a significant (p<0.05) and robust regional distribution in the brain with higher levels in the white matter and substantia nigra. A mutational analysis revealed an increase in the incidence of severe <i>GALC</i> mutations within the PD patient population compared to the cohorts of Alzheimer’s patients and healthy controls tested. In addition to α-synuclein pathology identified in the KD brain, control patients identified as GALC mutational carriers or possessing a GALC pathogenic variant had evidence of α-synuclein pathology, indicating a possible correlation between α-synuclein pathology and dysregulation of psychosine metabolism in the adult brain. Carrier status for <i>GALC</i> mutations and prolonged exposure to increased psychosine could contribute to α-synuclein pathology, supporting psychosine metabolism by galactosylceramidase as a risk factor for Parkinson’s disease.</p></div

GALC mutational analysis.

<p>GALC mutational analysis.</p

Immunohistological staining for α-synuclein.

<p>Immunohistological staining for α-synuclein was performed in brain tissue from 3 infantile KD patients (A-C), a late-onset KD patient (D), 2 patients found to be <i>GALC</i> mutation/variant carriers with no neurological disorders previously diagnosed (E,F), a Parkinson’s patient (G), and a healthy control with no <i>GALC</i> mutations (H). Accumulations of α-synuclein were observed in all Krabbe patients (A-D), the Parkinson’s brain (G), and also within the brains of <i>GALC</i> mutation/variant carriers (E,F). Accumulations in the infantile brains were abundant but smaller in size than the Lewy bodies observed in the Parkinson’s brain and the α-synuclein accumulations found in late-onset KD and <i>GALC</i> mutation carrier tissue. No significant α-synuclein accumulation was observed in the healthy diagnosed patient without GALC mutations (H). A secondary control experiment confirmed the specificity of the secondary antibody (I). Scale Bars: 100 μm.</p

GALC mutational effect on psychosine accumulation.

<p>Severe <i>GALC</i> mutations were identified in 2 patients diagnosed with Parkinson’s disease (PD). Psychosine levels in tissue from these two patients were elevated compared to that measured in tissue from PD patients without a severe <i>GALC</i> mutation.</p

Patient demographics.

<p>Patient demographics.</p

Psychosine content and GALC activity in neurodegenerative cohort.

<p>Psychosine concentration in white (WM) and gray (GM) cortical matter, substantia nigra, and caudate was examined in patients diagnosed with Parkinson’s, Alzheimer’s, Krabbe, or healthy controls. A-D) Psychosine was found to have a robust distribution, elevated in the white matter and substantia nigra of all patients. E-H) Psychosine in white and gray matter of Parkinson’s brains trended upwards compared to AD and healthy controls but did not reach significance. GALC activity in white (WM) and gray (GM) cortical matter, substantia nigra, and caudate was examined in patients diagnosed with Parkinsons’s, Alzheimer’s, Krabbe’s, or healthy controls. I-L) GALC activity was found to have a robust distribution, with lower levels found in the white matter and substantia nigra of all patients. M-P) GALC activity in white matter of Parkinson’s brains trended downwards compared to AD and healthy controls but did not reach significance. Q) Total cortical psychosine (summation of white and gray matter of cortex) found higher levels (p = 0.0554) of psychosine in Parkinson’s tissue compared to healthy controls. R) A total cortical GALC activity (summation of white and gray matter of cortex) did not find significantly different levels of GALC activity in Parkinson’s tissue compared to healthy controls. S) Correlation of psychosine to GALC activity as measured within the total cortical tissue of each group. (panel A-P: one way ANOVA with Tukey’s multiple comparison test, *p<0.05. **p<0.01, ***p<0.001, ****p<0.0001, panel Q,R: t-test).</p

Psychosine content and GALC activity in neuropsychiatric cohort.

<p>Psychosine content and GALC activity was measured in white (WM) and gray (GM) matter of the cortex in patients diagnosed with schizophrenia, bipolar disorder, depression, or as age-matched healthy controls. A,D) The same robust distribution of psychosine and GALC activity was observed in this cohort as found in the older neurodegenerative cohort. B,C,E,F) No significant changes in psychosine content or GALC activity were observed between any disease state. (One way ANOVA with Tukey’s multiple comparison test, *p<0.05. **p<0.01, ***p<0.001, ****p<0.0001).</p

Psychosine content and GALC activity correlated with aging in mid-aged cohort.

<p>Psychosine content (A,B) and GALC activity (C,D) in cortical brain tissue from a combined cohort of mid-aged neuropsychiatric patients and healthy control patient tissue was compared to age at time of death. Linear regression revealed a significant positive correlation between psychosine content in the white matter (A) and age in this cohort, but a similar positive correlation did not reach significance in grey matter (B). However, a significant negative correlation between GALC activity and age was present in both white (C) and grey (D) matter. (Statistical test of slope significantly non-zero, *p<0.05).</p