Reduced LRRK2 in association with retromer dysfunction in post-mortem brain tissue from LRRK2 mutation carriers

Missense mutations in leucine-rich repeat kinase 2 (LRRK2) are pathogenic for familial Parkinson's disease. However, it is unknown whether levels of LRRK2 protein in the brain are altered in patients with LRRK2-associated Parkinson's disease. Because LRRK2 mutations are relatively rare, accounting for approximately 1% of all Parkinson's disease, we accessioned cases from five international brain banks to investigate levels of the LRRK2 protein, and other genetically associated Parkinson's disease proteins. Brain tissue was obtained from 17 LRRK2 mutation carriers (12 with the G2019S mutation and five with the I2020T mutation) and assayed by immunoblot. Compared to matched controls and idiopathic Parkinson's disease cases, we found levels of LRRK2 protein were reduced in the LRRK2 mutation cases. We also measured a decrease in two other proteins genetically implicated in Parkinson's disease, the core retromer component, vacuolar protein sorting associated protein 35 (VPS35), and the lysosomal hydrolase, glucocerebrosidase (GBA). Moreover, the classical retromer cargo protein, cation-independent mannose-6-phosphate receptor (MPR300, encoded by IGF2R), was also reduced in the LRRK2 mutation cohort and protein levels of the receptor were correlated to levels of LRRK2. These results provide new data on LRRK2 protein expression in brain tissue from LRRK2 mutation carriers and support a relationship between LRRK2 and retromer dysfunction in LRRK2-associated Parkinson's disease brain

The carboxy-terminal fragment of α1A calcium channel preferentially aggregates in the cytoplasm of human spinocerebellar ataxia type 6 Purkinje cells

Spinocerebellar ataxia type 6 (SCA6) is an autosomal dominant neurodegenerative disease caused by a small polyglutamine (polyQ) expansion (control: 4–20Q; SCA6: 20–33Q) in the carboxyl(C)-terminal cytoplasmic domain of the α1A voltage-dependent calcium channel (Cav2.1). Although a 75–85-kDa Cav2.1 C-terminal fragment (CTF) is toxic in cultured cells, its existence in human brains and its role in SCA6 pathogenesis remains unknown. Here, we investigated whether the small polyQ expansion alters the expression pattern and intracellular distribution of Cav2.1 in human SCA6 brains. New antibodies against the Cav2.1 C-terminus were used in immunoblotting and immunohistochemistry. In the cerebella of six control individuals, the CTF was detected in sucrose- and SDS-soluble cytosolic fractions; in the cerebella of two SCA6 patients, it was additionally detected in SDS-insoluble cytosolic and sucrose-soluble nuclear fractions. In contrast, however, the CTF was not detected either in the nuclear fraction or in the SDS-insoluble cytosolic fraction of SCA6 extracerebellar tissues, indicating that the CTF being insoluble in the cytoplasm or mislocalized to the nucleus only in the SCA6 cerebellum. Immunohistochemistry revealed abundant aggregates in cell bodies and dendrites of SCA6 Purkinje cells (seven patients) but not in controls (n = 6). Recombinant CTF with a small polyQ expansion (rCTF-Q28) aggregated in cultured PC12 cells, but neither rCTF-Q13 (normal-length polyQ) nor full-length Cav2.1 with Q28 did. We conclude that SCA6 pathogenesis may be associated with the CTF, normally found in the cytoplasm, being aggregated in the cytoplasm and additionally distributed in the nucleus

Preferential recruitment of ataxin-3 independent of expanded polyglutamine: an immunohistochemical study on Marinesco bodies

In an immunohistochemical study of Marinesco bodies—a neuronal intranuclear inclusion often seen in neurons of the substantia nigra of patients with hepatic encephalopathy—it was shown that one of the polyglutamine proteins, ataxin-3, is preferentially recruited into this inclusion, whereas other polyglutamine proteins (ataxin-2 and TATA box-binding protein) are not. This suggests that recruitment of each of the polyglutamine proteins may be differently regulated. Because this nuclear inclusion is thought to be formed in response to cellular stress, as occurs in hepatic encephalopathy, even in the absence of an expanded CAG/polyglutamine repeat, recruitment of ataxin-3 and ubiquitin into Marinesco bodies may represent a cellular response to noxious external stimuli unrelated to expanded CAG/polyglutamine.


Behavioral disorder, dementia, ataxia, and rigidity in a large family with TATA box-binding protein mutation.

BACKGROUND: Spinocerebellar ataxia type 17 is an autosomal dominant cerebellar ataxia caused by a CAG repeat expansion in the TATA box-binding protein gene. Ataxia is typically the first sign whereas behavioral symptoms occur later. OBJECTIVE: To characterize the unusual phenotypic expression of a large spinocerebellar ataxia type 17 kindred. DESIGN: Clinical, neuropathological, and molecular genetic characterization of a 4-generation family with 16 affected patients. RESULTS: Behavioral symptoms and frontal impairment dominated the early stages preceding ataxia, rigidity, and dystonic movements. Neuropathological examination showed cortical, subcortical, and cerebellar atrophy. Purkinje cell loss and gliosis, pseudohypertrophic degeneration of the inferior olive, marked neuronal loss and gliosis in the caudate nucleus, and in the medial thalamic nuclei were salient features together with neuronal intranuclear inclusions stained with anti-TATA box-binding protein and antipolyglutamine antibodies. The disease was caused by a stable 52 CAG repeat expansion of the TATA box-binding protein gene, although there was apparent variability in the age of onset. CONCLUSION: The characteristics of this family broaden the clinical picture of spinocerebellar ataxia type 17: initial presenile dementia with behavioral symptoms should be added to ataxia, rigidity, and dystonic movements, which are more commonly encountered

LRRK2 Levels and Phosphorylation in Parkinson's Disease Brain and Cases With Restricted Lewy Bodies

Background: Leucine rich repeat kinase 2 (LRRK2) is a promising target for the treatment of Parkinson's disease; however, little is known about the expression of LRRK2 in human brain and if/how LRRK2 protein levels are altered in Parkinson's disease. Objectives: We measured the protein levels of LRRK2 as well as its phosphorylation on serines 910, 935, and 973 in the postmortem brain tissue of Parkinson's disease patients and aged controls with and without Lewy bodies. Methods: LRRK2 and its phosphorylation were measured by immunoblot in brain regions differentially affected in Parkinson's disease (n = 30) as well as subjects with Lewy bodies restricted to the periphery and lower brain stem (n = 25) and matched controls without pathology (n = 25). Results: LRRK2 levels were increased in cases with restricted Lewy bodies, with a 30% increase measured in the substantia nigra. In clinical Parkinson's disease, levels of LRRK2 negatively correlated to disease duration and were comparable with controls. LRRK2 phosphorylation, however, particularly at serine 935, was reduced with clinical Parkinson's disease with a 36% reduction measured in the substantia nigra. Conclusions: Our data show that LRRK2 phosphorylation is reduced with clinical PD, whereas LRRK2 expression is increased in early potential prodromal stages. These results contribute to a better understanding of the role of LRRK2 in idiopathic Parkinson's disease and may aid efforts aimed at therapeutically targeting the LRRK2 protein. © 2016 International Parkinson and Movement Disorder Societ

LRRK2 levels and phosphorylation in Parkinson's disease brain and cases with restricted Lewy bodies

Background: Leucine rich repeat kinase 2 (LRRK2) is a promising target for the treatment of Parkinson's disease; however, little is known about the expression of LRRK2 in human brain and if/how LRRK2 protein levels are altered in Parkinson's disease. Objectives: We measured the protein levels of LRRK2 as well as its phosphorylation on serines 910, 935, and 973 in the postmortem brain tissue of Parkinson's disease patients and aged controls with and without Lewy bodies. Methods: LRRK2 and its phosphorylation were measured by immunoblot in brain regions differentially affected in Parkinson's disease (n = 30) as well as subjects with Lewy bodies restricted to the periphery and lower brain stem (n = 25) and matched controls without pathology (n = 25). Results: LRRK2 levels were increased in cases with restricted Lewy bodies, with a 30% increase measured in the substantia nigra. In clinical Parkinson's disease, levels of LRRK2 negatively correlated to disease duration and were comparable with controls. LRRK2 phosphorylation, however, particularly at serine 935, was reduced with clinical Parkinson's disease with a 36% reduction measured in the substantia nigra. Conclusions: Our data show that LRRK2 phosphorylation is reduced with clinical PD, whereas LRRK2 expression is increased in early potential prodromal stages. These results contribute to a better understanding of the role of LRRK2 in idiopathic Parkinson's disease and may aid efforts aimed at therapeutically targeting the LRRK2 protein

NSF, Unc-18-1, dynamin-1 and HSP90 are inclusion body components in neuronal intranuclear inclusion disease identified by anti-SUMO-1-immunocapture

© Springer-Verlag 2008Neuronal intranuclear inclusion disease, a progressive ataxia that may be familial or sporadic, is characterized by numerous neuronal intranuclear inclusion bodies similar to those found in polyglutamine repeat diseases. Previously, we found that the intranuclear inclusion bodies are intensely immunopositive for SUMO-1, a protein which covalently conjugates to other proteins in a similar way to ubiquitin. To identify the SUMO-1-associated proteins in the inclusion bodies, we isolated intranuclear inclusion bodies from fresh, frozen brain tissue of a case with familial neuronal intranuclear inclusion disease and solubilized the proteins. SUMO-1-associated inclusion body proteins were then immunocaptured using an anti-SUMO-1 antibody. The proteins, NSF, dynamin-1 and Unc-18-1 (rbSEC1), involved in membrane trafficking of proteins, and the chaperone HSP90, were identified following anti-SUMO-1-immunocapture by using tandem mass spectrometry and database searching. Immunohistochemistry of brain sections and crude brain homogenates of three cases of familial neuronal intranuclear inclusion disease confirmed the presence of these proteins in intranuclear inclusions.Dean L. Pountney, Mark J. Raftery, Fariba Chegini, Peter C. Blumbergs, Wei Ping Ga