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Loss of activity of the lysosomal glycosidase β-glucocerebrosidase (GCase) causes the lysosomal storage disease Gaucher disease (GD) and has emerged as the greatest genetic risk factor for the development of both Parkinson disease (PD) and dementia with Lewy bodies. There is significant interest into how GCase dysfunction contributes to these diseases, however, progress toward a full understanding is complicated by presence of endogenous cellular factors that influence lysosomal GCase activity. Indeed, such factors are thought to contribute to the high degree of variable penetrance of GBA mutations among patients. Robust methods to quantitatively measure GCase activity within lysosomes are therefore needed to advance research in this area, as well as to develop clinical assays to monitor disease progression and assess GCase-directed therapeutics. Here, we report a selective fluorescence-quenched substrate, LysoFQ-GBA, which enables measuring endogenous levels of lysosomal GCase activity within living cells. LysoFQ-GBA is a sensitive tool for studying chemical or genetic perturbations of GCase activity using either fluorescence microscopy or flow cytometry. We validate the quantitative nature of measurements made with LysoFQ-GBA using various cell types and demonstrate that it accurately reports on both target engagement by GCase inhibitors and the GBA allele status of cells. Furthermore, through comparisons of GD, PD, and control patient-derived tissues, we show there is a close correlation in the lysosomal GCase activity within monocytes, neuronal progenitor cells, and neurons. Accordingly, analysis of clinical blood samples using LysoFQ-GBA may provide a surrogate marker of lysosomal GCase activity in neuronal tissue.

Lysosomal dysfunction is an emerging feature in the pathology of Parkinson's disease and Dementia with Lewy bodies. Mutations in the GBA gene, encoding the enzyme Glucocerebrosidase (GCase), have been identified as a genetic risk factor for these synucleinopathies. As a result, there has been a growing interest in the involvement of GCase in these diseases. This GCase activity assay is based on the catalytic hydrolysis of 4-methylumbelliferyl β-D-glucopyranoside that releases the highly fluorescent 4-methylumbelliferyl (4-MU). The final assay protocol was tested for the following parameters: Lower limit of quantification (LLOQ), precision, parallelism, linearity, spike recovery, number of freeze-thaw events, and sample handling stability. The GCase activity assay is within acceptable criteria for parallelism, precision and spike recovery. The LLOQ of this assay corresponds to an enzymatic activity of generating 0.26 pmol 4-MU/min/ml. The enzymatic activity was stable when samples were processed and frozen at - 80 °C within 4 h after the lumbar puncture procedure. Repetitive freeze-thaw events significantly decreased enzyme activity. We present the validation of an optimized in vitro GCase activity assay, based on commercially available components, to quantify its enzymatic activity in human cerebrospinal fluid and the assessment of preanalytical factors.

Neuronal loss in numerous neurodegenerative disorders has been linked to protein aggregation and oxidative stress. Emerging data regarding overlapping proteinopathy in traditionally distinct neurodegenerative diseases suggest that disease-modifying treatments targeting these pathological features may exhibit efficacy across multiple disorders. Here, we describe proteinopathy distinct from classic synucleinopathy, predominantly comprised of the anti-oxidant enzyme superoxide dismutase-1 (SOD1), in the Parkinson's disease brain. Significant expression of this pathology closely reflected the regional pattern of neuronal loss. The protein composition and non-amyloid macrostructure of these novel aggregates closely resembles that of neurotoxic SOD1 deposits in SOD1-associated familial amyotrophic lateral sclerosis (fALS). Consistent with the hypothesis that deposition of protein aggregates in neurodegenerative disorders reflects upstream dysfunction, we demonstrated that SOD1 in the Parkinson's disease brain exhibits evidence of misfolding and metal deficiency, similar to that seen in mutant SOD1 in fALS. Our data suggest common mechanisms of toxic SOD1 aggregation in both disorders and a potential role for SOD1 dysfunction in neuronal loss in the Parkinson's disease brain. This shared restricted proteinopathy highlights the potential translation of therapeutic approaches targeting SOD1 toxicity, already in clinical trials for ALS, into disease-modifying treatments for Parkinson's disease.

Parkinson's disease (PD) patients progressively accumulate intracytoplasmic inclusions formed by misfolded α-synuclein known as Lewy bodies (LBs). LBs also contain other proteins that may or may not be relevant in the disease process. To identify proteins involved early in LB formation, we performed proteomic analysis of insoluble proteins in a primary neuron culture model of α-synuclein pathology. We identified proteins previously found in authentic LBs in PD as well as several novel proteins, including the microtubule affinity-regulating kinase 1 (MARK1), one of the most enriched proteins in this model of LB formation. Activated MARK proteins (MARKs) accumulated in LB-like inclusions in this cell-based model as well as in a mouse model of LB disease and in LBs of postmortem synucleinopathy brains. Inhibition of MARKs dramatically exacerbated α-synuclein pathology. These findings implicate MARKs early in synucleinopathy pathogenesis and as potential therapeutic drug targets. SIGNIFICANCE STATEMENT Neurodegenerative diseases are diagnosed definitively only in postmortem brains by the presence of key misfolded and aggregated disease proteins, but cellular processes leading to accumulation of these proteins have not been well elucidated. Parkinson's disease (PD) patients accumulate misfolded α-synuclein in LBs, the diagnostic signatures of PD. Here, unbiased mass spectrometry was used to identify the microtubule affinity-regulating kinase family (MARKs) as activated and insoluble in a neuronal culture PD model. Aberrant activation of MARKs was also found in a PD mouse model and in postmortem PD brains. Further, inhibition of MARKs led to increased pathological α-synuclein burden. We conclude that MARKs play a role in PD pathogenesis., (Copyright © 2017 the authors 0270-6474/17/375870-15$15.00/0.)

Background: Accumulating evidence has demonstrated that the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a part of Lewy body inclusions and involves the pathogenesis of Parkinson's disease (PD). However, it remains unknown whether or not genetic variation at the GAPDH locus contributes to the risk for PD., Methods: A total of 302 sporadic PD patients and 377 control subjects were recruited in our study for assessing two single nucleotide polymorphisms (rs3741918 and rs1060619) in the GAPDH gene. Both allelic association and additive models were used to analyze association between GAPDH variants and risk for PD., Results: Both polymorphisms were significantly associated with risk for PD after correction by Bonferroni multiple testing. The minor allele of rs3741918 was associated with decreased risk of sporadic PD (allelic contrast, OR = 0.74, 95% CI: 0.59-0.93, corrected P = 0.028; additive model, OR = 0.73, 95% CI: 0.58-0.92, corrected P = 0.018). While for the rs1060619 locus, the minor allele conferred increased risk for PD (allelic contrast, OR = 1.41, 95% CI: 1.14-1.75, corrected P = 0.007; additive model, OR = 1.43, 95% CI: 1.15-1.79, corrected P = 0.002)., Conclusion: Our study indicates that GAPDH variants confer susceptibility to sporadic PD in a Chinese Han population, which is consistent with the role of GAPDH protein in neuronal apoptosis. To our knowledge, this is the first study of genetic association between GAPDH locus and risk for PD in the Chinese population.

Background: Lysosomal dysfunction is thought to be a prominent feature in the pathogenetic events leading to Parkinson's disease (PD). This view is supported by the evidence that mutations in GBA gene, coding the lysosomal hydrolase β-glucocerebrosidase (GCase), are a common genetic risk factor for PD. Recently, GCase activity has been shown to be decreased in substantia nigra and in cerebrospinal fluid of patients diagnosed with PD or dementia with Lewy Bodies (DLB). Here we measured the activity of GCase and other endo-lysosomal enzymes in different brain regions (frontal cortex, caudate, hippocampus, substantia nigra, cerebellum) from PD (n = 26), DLB (n = 16) and age-matched control (n = 13) subjects, screened for GBA mutations. The relative changes in GCase gene expression in substantia nigra were also quantified by real-time PCR. The role of potential confounders (age, sex and post-mortem delay) was also determined., Findings: Substantia nigra showed a high activity level for almost all the lysosomal enzymes assessed. GCase activity was significantly decreased in the caudate (-23%) and substantia nigra (-12%) of the PD group; the same trend was observed in DLB. In both groups, a decrease in GCase mRNA was documented in substantia nigra. No other lysosomal hydrolase defects were determined., Conclusion: The high level of lysosomal enzymes activity observed in substantia nigra, together with the selective reduction of GCase in PD and DLB patients, further support the link between lysosomal dysfunction and PD pathogenesis, favoring the possible role of GCase as biomarker of synucleinopathy. Mapping the lysosomal enzyme activities across different brain areas can further contribute to the understanding of the role of lysosomal derangement in PD and other synucleinopathies.

Many neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease, are characterized by abnormal accumulations of aggregated proteins. Brains in these diseases also show accumulation of autophagic vesicles in the neuronal cytoplasm, suggesting impairment of the autophagic process. As autophagy involves de novo membrane production and vesicle fusion, extensive changes in lipid molecules are necessary. However, the involvement of signaling lipid-modifying enzymes in autophagy and their roles in neurodegenerative diseases are not clear. Using specific inhibitor, we show that loss of phospholipase D1 (PLD1) activity resulted in an accumulation of microtubule-associated protein light chain 3 (LC3), p62, and polyubiquitinated proteins, signs representing malfunction in autophagic flux. Fluorescence and electron microscopic analyses demonstrated impaired fusion of autophagosomes with lysosomes, resulting in accumulation of autophagosomes. Within the cells with impaired autophagic flux, α-synuclein aggregates accumulated in autophagosomes. Knockdown of PLD1 expression using small interfering RNA also resulted in impaired autophagic flux and accumulation of α-synuclein aggregates in autophagosomes. Neuronal toxicity caused by α-synuclein accumulation was rescued by overexpression of PLD1; however, expression of activity-deficient mutant, PLD1-KRM, showed reduced rescue effects. Finally, we demonstrated that both PLD activity and expression levels were reduced in brain tissues of dementia with Lewy bodies (DLB) patients, whereas the amounts of α-synuclein and p62 were increased in the same tissue samples. Collectively, these results suggest that insufficient PLD activity, and therefore, the changes in phospholipid compositions within membranes, might be an important contributor to impaired autophagic process and protein accumulation in Lewy body diseases.

FIG4 is a phosphatase that regulates intracellular vesicle trafficking along the endosomal-lysosomal pathway. Mutations of FIG4 lead to the development of Charcot-Marie-Tooth disease type 4J and amyotrophic lateral sclerosis (ALS). Moreover, ALS-associated proteins (transactivation response DNA protein 43 (TDP-43), fused in sarcoma (FUS), optineurin, ubiquilin-2, charged mutivesicular body protein 2b (CHMP2B) and valosin-containing protein) are involved in inclusion body formation in several neurodegenerative diseases. Using immunohistochemistry, we examined the brains and spinal cords of patients with various neurodegenerative diseases, including sporadic TDP-43 proteinopathy (ALS and frontotemporal lobar degeneration). TDP-43 proteinopathy demonstrated no FIG4 immunoreactivity in neuronal inclusions. However, FIG4 immunoreactivity was present in Pick bodies in Pick's disease, Lewy bodies in Parkinson's disease and dementia with Lewy bodies, neuronal nuclear inclusions in polyglutamine and intranuclear inclusion body diseases, and Marinesco and Hirano bodies in aged control subjects. These findings suggest that FIG4 is not incorporated in TDP-43 inclusions and that it may have a common role in the formation or degradation of neuronal cytoplasmic and nuclear inclusions in several neurodegenerative diseases., (© 2013 Japanese Society of Neuropathology.)

α-synuclein (α-Syn) is a chaperone-like protein that is highly implicated in Parkinson's disease (PD) as well as in dementia with Lewy bodies (DLB). Rare forms of PD occur in individuals with mutations of α-Syn or triplication of wild type α-Syn, and in both PD and DLB the intraneuronal inclusions known as Lewy bodies contain aggregated α-Syn that is highly phosphorylated on serine 129. In neuronal cells and in the brains of α-Syn overexpressing transgenic mice, soluble α-Syn stimulates the activity of protein phosphatase 2A (PP2A), a major serine/threonine phosphatase. Serine 129 phosphorylation of α-Syn attenuates its stimulatory effects on PP2A and also accelerates α-Syn aggregation; however, it is unknown if aggregation of α-Syn into Lewy bodies impairs PP2A activity. To assess for this, we measured the impact of α-Syn aggregation on PP2A activity in vitro and in vivo. In cell-free assays, aggregated α-Syn had ∼50% less PP2A stimulatory effects than soluble recombinant α-Syn. Similarly in DLB and α-Syn triplication brains, which contain robust α-Syn aggregation with high levels of serine 129 phosphorylation, PP2A activity was also ∼50% attenuated. As α-Syn normally stimulates PP2A activity, our data suggest that overexpression of α-Syn or sequestration of α-Syn into Lewy bodies has the potential to alter the phosphorylation state of key PP2A substrates; raising the possibility that all forms of synucleinopathy will benefit from treatments aimed at optimizing PP2A activity., (Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.)

Histone deacetylase 6 (HDAC6) plays a crucial role in aggresome formation, resulting in the clearance of misfolded proteins. Previous studies have shown that HDAC6 is concentrated in Lewy bodies (LBs) in Parkinson's disease (PD) and dementia with LBs (DLB) (Cell 115: 727-738, 2003). We performed immunohistochemical and ultrastructural investigations on the brains of patients with various neurodegenerative disorders. Anti-HDAC6 antibody faintly immunostained the cytoplasm of neuronal and glial cells in control subjects. In PD and DLB, almost all of the cortical, brainstem-type and peripheral LBs were intensely immunolabeled with anti-HDAC6. In multiple system atrophy (MSA), the vast majority of glial cytoplasmic inclusions (GCIs) were also positive for HDAC6. Immunoelectron microscopy revealed that the reaction product was localized to the filamentous structures in LBs and GCIs. Various neuronal and glial inclusions in neurodegenerative disorders other than LB disease and MSA were HDAC6-negative. These findings suggest that accumulation of HDAC6 is specific to α-synucleinopathy and that both LBs and GCIs may represent cytoprotective responses to sequester toxic proteins., (© 2011 Japanese Society of Neuropathology.)

Parkinson's disease (PD) and Dementia with Lewy Bodies (DLB) are characterized pathologically by intraneuronal inclusions called Lewy bodies (LBs) and Lewy neurites. A major component of these inclusions is the protein α-synuclein, which is natively unfolded but forms oligomers and insoluble fibrillar aggregates under pathological conditions. Although α-synuclein is known to undergo several posttranslational modifications, the contribution of SUMOylation to α-synuclein aggregation and the pathogenesis of α-synucleinopathies have not been elucidated. Here, we provide evidence that aggregates and inclusions formed as a result of impaired proteasome activity contain SUMOylated α-synuclein. Additionally, SUMO1 is present in the halo of LBs colocalizing with α-synuclein in the brains of PD and DLB patients. Interestingly, SUMOylation does not affect the ubiquitination of α-synuclein. These findings suggest that proteasomal dysfunction results in the accumulation of SUMOylated α-synuclein and subsequently its aggregation, pointing to the contribution of this posttranslational modification to the pathogenesis of inclusion formation in α-synucleinopathies., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Mutations in the gene encoding the lysosomal enzyme glucocerebrosidase, known to cause Gaucher disease (GD), are a risk factor for the development of Parkinson disease (PD) and related disorders. This association is based on the concurrence of parkinsonism and GD, the identification of glucocerebrosidase mutations in cohorts with PD from centers around the world, and neuropathologic findings. The contribution of glucocerebrosidase to the development of parkinsonian pathology was explored by studying seven brain samples from subjects carrying glucocerebrosidase mutations with pathologic diagnoses of PD and/or Lewy body dementia. Three individuals had GD and four were heterozygous for glucocerebrosidase mutations. All cases had no known family history of PD and the mean age of disease onset was 59 years (range 42-77). Immunofluorescence studies on brain tissue samples from patients with parkinsonism associated with glucocerebrosidase mutations showed that glucocerebrosidase was present in 32-90% of Lewy bodies (mean 75%), some ubiquitinated and others non-ubiquitinated. In samples from seven subjects without mutations, <10% of Lewy bodies were glucocerebrosidase positive (mean 4%). This data demonstrates that glucocerebrosidase can be an important component of α-synuclein-positive pathological inclusions. Unraveling the role of mutant glucocerebrosidase in the development of this pathology will further our understanding of the lysosomal pathways that likely contribute to the formation and/or clearance of these protein aggregates.

Lewy bodies (LBs), the pathological hallmark of Lewy body disease (LBD), contain α-synuclein, as well as other proteins. In this study, we examined the relationship of α-synuclein to two rate-limiting enzymes in neurotransmitter synthesis, tyrosine hydroxylase (TH) and choline acetyltransferase (ChAT). Double-labeling immunohistochemistry for α-synuclein and TH revealed TH immunoreactivity within LBs in catecholaminergic neurons in the substantia nigra and locus coeruleus, but not within LBs in cholinergic neurons in the pedunculopontine nucleus and nucleus basalis of Meynert. In contrast, ChAT immunoreactivity within LBs was detected in cholinergic, but not within LBs in catecholaminergic neurons. The amygdala was devoid of TH and ChAT positive LBs, although a few Lewy neurites contained ChAT immunoreactivity. Further analysis revealed two distinct patterns of neurotransmitter immunoreactivity within LBs. One pattern had diffuse co-localization of TH or ChAT with α-synuclein as in cortical-type LBs, while the other had intense TH or ChAT immunoreactivity in the LB core surrounded by a peripheral rim of α-synuclein as in brainstem-type LBs. Levels of both TH and ChAT were higher in brainstem-type LBs than in the cytoplasm of the same neuron or in neurons from the same case devoid of LBs. Given the fact that LB-containing neurons have decreases in cytoplasmic TH and ChAT immunoreactivity, these results suggest LBs may disrupt cholinergic and catecholaminergic neurotransmitter production by sequestration of the rate-limiting enzymes for acetylcholine and catecholamine synthesis.

Mutation of the leucine-rich repeat kinase 2 (LRRK2) gene is the most frequent genetic cause of Parkinson disease (PD). To understand the role of LRRK2 in the neuropathology of PD, we investigated the protein expression in a healthy brain and brains from patients with PD and its subcellular localization in dopaminergic neurons. LRRK2 was found to be widely expressed in healthy adult brain, including areas involved in PD. By double fluorescent staining, we found that endogenous LRRK2 is colocalized with the endoplasmic reticulum (ER) markers Neurotrace and KDEL in human dopaminergic neurons. Labeling of brain sections with anti-LRRK2 and anti-α-synuclein antibodies revealed localization of LRRK2 in the core of 24% of Lewy bodies (LBs) in the substantia nigra and 11% of LBs in the locus coeruleus in idiopathic PD patients. The percentage was increased to 50% in both areas in a patient with the G2019S LRRK2 mutation. The finding of ER localization suggests the possibility that LRRK2 is involved in the ER stress response and could account for the susceptibility to neuronal degeneration of LRRK2 mutation carriers. The localization of LRRK2 protein in the core of a subset of LBs demonstrates the contribution of LRRK2 to LB formation and disease pathogenesis.

Background: Mutations in the glucocerebrosidase (GBA) gene are associated with Lewy body (LB) disorders., Objective: To determine the relationship of GBA mutations and APOE4 genotype to LB and Alzheimer disease (AD) pathological findings., Design: Case-control study., Setting: Academic research., Participants: The 187 subjects included patients with primary neuropathological diagnoses of LB disorders with or without AD changes (95 cases), randomly selected patients with AD (without significant LB pathological findings; 60 cases), and controls with neither LB nor AD pathological findings (32 cases)., Main Outcome Measures: GBA mutation status, APOE4 genotype, LB pathological findings (assessed according to the third report of the Dementia With Lewy Body Consortium), and Alzheimer plaque and tangle pathological findings (rated by criteria of Braak and Braak, the Consortium to Establish a Registry for Alzheimer Disease, and the National Institute on Aging-Reagan Institute)., Results: GBA mutations were found in 18% (34 of 187) of all subjects, including 28% (27 of 95) of those with primary LB pathological findings compared with 10% (6 of 60) of those with AD pathological findings and 3% (1 of 32) of those without AD or LB pathological findings (P=.001). GBA mutation status was significantly associated with the presence of cortical LBs (odds ratio, 6.48; 95% confidence interval, 2.45-17.16; P<.001), after adjusting for sex, age at death, and presence of APOE4. GBA mutation carriers were significantly less likely to meet AD pathological diagnostic (National Institute on Aging-Reagan Institute intermediate or high likelihood) criteria (odds ratio, 0.35; 95% confidence interval, 0.15-0.79; P=.01) after adjustment for sex, age at death, and APOE4., Conclusion: GBA mutations may be associated with pathologically "purer" LB disorders, characterized by more extensive (cortical) LB, and less severe AD pathological findings and may be a useful marker for LB disorders.

We immunohistochemically examined the expression of glycogen synthase kinase-3beta (GSK-3beta) in the brains of Parkinson's disease (PD) patients. GSK-3beta was localized in punctate structures in the cytosol of subsets of neurons in the midbrain and upper pons. GSK-3beta was also localized in Lewy bodies (LBs) as was phosphorylated GSK-3beta (Ser9) (pGSK-3beta (Ser9)). Both GSK-3beta and pGSK-3beta (Ser9) were localized specifically in the halo of LBs. The core of LBs was negative for GSK-3beta, while pGSK-3beta (Ser9) was present in only a small number of LB cores. Cortical LBs were positive for pGSK-3beta (Ser9) but not for GSK-3beta. Neither GSK-3beta nor pGSK-3beta (Ser9) was present in glial cytoplasmic inclusions (GCIs) in the brains of multiple system atrophy (MSA) patients. Our results suggest that GSK-3beta plays a role in the pathogenesis of PD but not in that of MSA.

Ubiquitin-positive intraneuronal inclusions are a consistent feature of the major human neurodegenerative diseases, suggesting that dysfunction of the ubiquitin proteasome system is central to disease etiology. Research using inhibitors of the 20S proteasome to model Parkinson's disease is controversial. We report for the first time that specifically 26S proteasomal dysfunction is sufficient to trigger neurodegenerative disease. Here, we describe novel conditional genetic mouse models using the Cre/loxP system to spatially restrict inactivation of Psmc1 (Rpt2/S4) to neurons of either the substantia nigra or forebrain (e.g., cortex, hippocampus, and striatum). PSMC1 is an essential subunit of the 26S proteasome and Psmc1 conditional knock-out mice display 26S proteasome depletion in targeted neurons, in which the 20S proteasome is not affected. Impairment of specifically ubiquitin-mediated protein degradation caused intraneuronal Lewy-like inclusions and extensive neurodegeneration in the nigrostriatal pathway and forebrain regions. Ubiquitin and alpha-synuclein neuropathology was evident, similar to human Lewy bodies, but interestingly, inclusion bodies contained mitochondria. We support this observation by demonstrating mitochondria in an early form of Lewy body (pale body) from Parkinson's disease patients. The results directly confirm that 26S dysfunction in neurons is involved in the pathology of neurodegenerative disease. The model demonstrates that 26S proteasomes are necessary for normal neuronal homeostasis and that 20S proteasome activity is insufficient for neuronal survival. Finally, we are providing the first reproducible genetic platform for identifying new therapeutic targets to slow or prevent neurodegeneration.

The manifestation of Lewy bodies (LB) in the brain is a hallmark of Parkinson's disease. Here, we present a comprehensive analysis of protein elements in Lewy bodies by comparative mass spectrometry. Cortical LB inclusions were enriched by sucrose gradient centrifugation from postmortem brains, and a negative control sample was prepared from specimen without LB pathology. Whereas approximately 550 proteins were identified in the LB-enriched sample by mass spectrometry, quantitative comparison with the control sample revealed that approximately 40 proteins were co-enriched with alpha-synuclein, the major component in Lewy bodies. As expected, the list of proteins included previously reported constituents, such as those involved in protein folding, membrane trafficking and oxidative stress. More interestingly, we discovered in the LB-enriched sample several kinases (MAPKK1/MEK1, protein kinase C, and doublecortin-like kinase), a novel deubiquitinating enzyme (otubain 1), and numerous ubiquitin ligases (KPC and SCF). The proteomic studies provide enzyme candidates to investigate the regulation of alpha-synuclein and/or other LB proteins, which may contribute to the formation of Lewy bodies and the toxicity of alpha-synuclein in the related neurodegenerative disorders.

We reported previously that phosphorylation by casein kinase II (CKII) regulates the interaction between alpha-synuclein and its binding partner synphilin-1, and that both CKII alpha and beta subunits co-localize with alpha-synuclein in cytoplasmic inclusions in transfected cells. In this study, we extended these observations to the brains of patients with Parkinson's disease (PD) and examined whether CKII subunits are present in Lewy bodies. Immunohistochemical studies on PD brains harboring Lewy bodies revealed a positive stain for CKII beta but not for CKII alpha. In addition, CKII beta subunits co-localized with alpha-synuclein in most Lewy bodies. These findings suggest that CKII beta subunits may play a role in the formation of intracytoplasmic inclusions in human alpha-synucleinopathies either through phosphorylation events or through a separate mechanism linked to the beta subunit itself.

Mutations in the LRRK2 gene cause autosomal dominant, late-onset parkinsonism, which presents with pleomorphic pathology including alpha-synucleopathy. To promote our understanding of the biological role of LRRK2 in the brain we examined the distribution of LRRK2 mRNA and protein in postmortem human brain tissue from normal and neuropathological subjects. In situ hybridization and immunohistochemical analysis demonstrate the expression and localization of LRRK2 to various neuronal populations in brain regions implicated in Parkinson's disease (PD) including the cerebral cortex, caudate-putamen and substantia nigra pars compacta. Immunofluorescent double labeling studies additionally reveal the prominent localization of LRRK2 to cholinergic-, calretinin- and GABA(B) receptor 1-positive, dopamine-innervated, neuronal subtypes in the caudate-putamen. The distribution of LRRK2 in brain tissue from sporadic PD and dementia with Lewy bodies (DLB) subjects was also examined. In PD brains, LRRK2 immunoreactivity localized to nigral neuronal processes is dramatically reduced which reflects the disease-associated loss of dopaminergic neurons in this region. However, surviving nigral neurons occasionally exhibit LRRK2 immunostaining of the halo structure of Lewy bodies. Moreover, LRRK2 immunoreactivity is not associated with Lewy neurites or with cortical Lewy bodies in sporadic PD and DLB brains. These observations indicate that LRRK2 is not a primary component of Lewy bodies and does not co-localize with mature fibrillar alpha-synuclein to a significant extent. The localization of LRRK2 to key neuronal populations throughout the nigrostriatal dopaminergic pathway is consistent with the involvement of LRRK2 in the molecular pathogenesis of familial and sporadic parkinsonism.

G-protein coupled receptor kinases (GRKs) constitute a serine/threonine kinase family playing a major role in agonist-induced phosphorylation and desensitization of G-protein coupled receptors. Recently, GRK2 and GRK5 have been demonstrated to phosphorylate alpha-synuclein (Ser129) and other synuclein isoforms. We studied colocalization of GRK2, GRK5, alpha-synuclein, and tau in neurodegenerative disorders characterized by fibrillary tau inclusions and/or alpha-synuclein-enriched Lewy bodies. We found that Lewy bodies were negative for both GRK2 and GRK5 in Lewy body disease (LBD) and LBD mixed with Alzheimer disease (AD + LBD). Instead, GRK2 but not GRK5 colocalized with 40% to 50% of neurofibrillary tangles in AD + LBD and AD brains. In disorders with less prominent alpha-synucleinopathy, neuronal and glial fibrillary tau deposits known to contain distinct subsets of tau isoforms were also positive for GRK2. These deposits included tufted astrocytes and coiled bodies in progressive supranuclear palsy, astrocytic plaques in corticobasal degeneration, and Pick bodies in Pick disease. In addition, paired helical filaments isolated from AD and AD + LBD brains were found to immunogold-label for GRK2, suggesting that GRK2 could be a potential tau kinase associated with fibrillary tau. Our studies indicate that GRK2 is a novel component of neuronal and glial fibrillary tau deposits with no preference in tau isoform binding. GRK2 may play a role in hyperphosphorylation of tau in tauopathies.

Sporadic Parkinson's disease (sPD) is a common neurodegenerative disorder, characterized by selective degeneration of dopaminergic neurons in the substantia nigra. Although the pathogenesis of the disease remains undetermined, phosphorylation of alpha-synuclein and its oligomer formation seem to play a key role. However, the protein kinase(s) involved in the phosphorylation in the pathogenesis of sPD has not been identified. Here, we found that G-protein-coupled receptor kinase 5 (GRK5) accumulated in Lewy bodies and colocalized with alpha-synuclein in the pathological structures of the brains of sPD patients. In cotransfected cells, GRK5 phosphorylated Ser-129 of alpha-synuclein at the plasma membrane and induced translocation of phosphorylated alpha-synuclein to the perikaryal area. GRK5-catalyzed phosphorylation also promoted the formation of soluble oligomers and aggregates of alpha-synuclein. Genetic association study revealed haplotypic association of the GRK5 gene with susceptibility to sPD. The haplotype contained two functional single-nucleotide polymorphisms, m22.1 and m24, in introns of the GRK5 gene, which bound to YY1 (Yin Yang-1) and CREB-1 (cAMP response element-binding protein 1), respectively, and increased transcriptional activity of the reporter gene. The results suggest that phosphorylation of alpha-synuclein by GRK5 plays a crucial role in the pathogenesis of sPD.

Parkinson disease (PD) and dementia with Lewy bodies (DLB) are characterized by the accumulation of abnormal alpha-synuclein and ubiquitin in protein aggregates conforming Lewy bodies and Lewy neurites. Ubiquitin C-terminal hydrolase-1 (UCHL-1) disassembles polyubiquitin chains to increase the availability of free monomeric ubiquitin to the ubiquitin proteasome system (UPS) thus favoring protein degradation. Since mutations in the UCHL-1 gene, reducing UPS activity by 50%, have been reported in autosomal dominant PD, and UCHL-1 inhibition results in the formation of alpha-synuclein aggregates in mesencephalic cultured neurons, the present study was initiated to test UCHL-1 mRNA and protein levels in post-mortem frontal cortex (area 8) of PD and DLB cases, compared with age-matched controls. TaqMan PCR assays, and Western blots demonstrated down-regulation of UCHL-1 mRNA and UCHL-1 protein in the cerebral cortex in DLB (either in pure forms, not associated with Alzheimer disease: AD, and in common forms, with accompanying AD changes), but not in PD, when compared with age-matched controls. Interestingly, UCHL-1 mRNA and protein expressions were reduced in the medulla oblongata in the same PD cases. Moreover, UCHL-1 protein was decreased in the substantia nigra in cases with Lewy body pathology. UCHL-1 down-regulation was not associated with reduced protein levels of several proteasomal subunits, including 20SX, 20SY, 19S and 11Salpha. Yet UCHL-3 expression was reduced in the cerebral cortex of PD and DLB patients. Together, these observations show reduced UCHL-1 expression as a contributory factor in the abnormal protein aggregation in DLB, and points UCHL-1 as a putative therapeutic target in the treatment of DLB.

We studied the distribution pattern of pathology and cholinergic deficits in the subnuclei of the amygdaloid complex (AC) in five patients with Alzheimer's disease (AD), eight with dementia with Lewy bodies (DLB) and five normal controls. In controls, the basal nucleus contained the highest choline acetyltransferase activity; the activity in the lateral and central nuclei and those in the cortical, medial and accessory basal nuclei were comparable. In AD, there was a significant decrease in choline acetyltransferase activity in the accessory basal and lateral nuclei, in DLB a significant decrease was observed in the accessory basal, lateral and cortical nuclei. Compared to controls the hyperphosphorylated tau-pathology burden was significantly higher in the basal, central and medial nuclei in AD and in the central, cortical, lateral and medial nuclei in DLB. The amyloid plaque burden was significantly higher in the accessory basal, basal, lateral and cortical nuclei in AD and in all nuclei in DLB. The alpha-synuclein burden was significantly higher in all nuclei in both AD and DLB. Compared to AD alpha-synuclein burden was higher in all nuclei in DLB. There were no correlations between the distribution pattern of hyperphosphorylated tau-pathology, amyloid plaques and alpha-synuclein-positive structures, and choline acetyltransferase activity, except the lateral nucleus in DLB. In conclusion we found no relationship between the pattern of cholinergic deficits and the distribution pattern of lesions in the AC of patients with AD or DLB. Cholinergic deficits were more prominent in the nuclei of basolateral (BL) group in AD, whereas the nuclei of both BL and corticomedial groups were involved in DLB, which may be due to the involvement of both basal forebrain and brainstem cholinergic nuclei in the latter.

Ubiquitin is one of the major components of Lewy bodies (LB), the pathological hallmark of Parkinson's disease (PD). Here, we identified that a phosphorylated form of IkappaBalpha (pIkappaBalpha), an inhibitor of NF-kappaB, and SCF(beta-TrCP), the ubiquitin ligase of pIkappaBalpha, are components of LB in brains of PD patients. In vitro studies identified those proteins in the ubiquitin- and alpha-synuclein (known as the major component of LB)-positive LB-like inclusions generated in dopaminergic SH-SY5Y cells treated with MG132, a proteasome inhibitor. Intriguingly, IkappaBalpha migration into such ubiquitinated inclusions in cells treated with MG132 was inhibited by a cell-permeable peptide known to block phosphorylation of IkappaBalpha, although this peptide did not influence cell viability under proteasomal inhibition. Our results indicate that phosphorylation of IkappaBalpha plays a role in the formation of IkappaBalpha-containing inclusions caused by proteasomal dysfunction, and that the generation of such inclusion is independent of cell death caused by impairment of proteasome.

Neurodegenerative disorders such as Parkinson's disease (PD) and 'dementia with Lewy bodies' (DLB) are characterized pathologically by selective neuronal death and the appearance of intracytoplasmic protein aggregates (Lewy bodies). The process by which these inclusions are formed and their role in the neurodegenerative process remain elusive. In this study, we demonstrate a close relationship between Lewy bodies and aggresomes, which are cytoplasmic inclusions formed at the centrosome as a cytoprotective response to sequester and degrade excess levels of potentially toxic abnormal proteins within cells. We show that the centrosome/aggresome-related proteins gamma-tubulin and pericentrin display an aggresome-like distribution in Lewy bodies in PD and DLB. Lewy bodies also sequester the ubiquitin-activating enzyme (E1), the proteasome activators PA700 and PA28, and HSP70, all of which are recruited to aggresomes for enhanced proteolysis. Using novel antibodies that are specific and highly sensitive to ubiquitin-protein conjugates, we revealed the presence of numerous discrete ubiquitinated protein aggregates in neuronal soma and processes in PD and DLB. These aggregates appear to be being transported from peripheral sites to the centrosome where they are sequestered to form Lewy bodies in neurons. Finally, we have shown that inhibition of proteasomal function or generation of misfolded proteins cause the formation of aggresome/Lewy body-like inclusions and cytotoxicity in dopaminergic neurons in culture. These observations suggest that Lewy body formation may be an aggresome-related event in response to increasing levels of abnormal proteins in neurons. This phenomenon is consistent with growing evidence that altered protein handling underlies the etiopathogenesis of PD and related disorders.

Brain tissue from 44 patients with Parkinson's disease (PD) and 36 age-matched controls was examined for choline acetyltransferase (ChAT) activity, and for densities of D1 and D2 dopamine receptors. Brain samples were examined for Alzheimer' disease (AD) type changes and for Lewy bodies (LBs), and for apolipoprotein E genotype. Patients were evaluated for the stage of cognitive impairment using Reisberg's global deterioration scale. ChAT activity in PD was reduced in all brain areas examined, being 51% of the control mean in the hippocampus (P<0.001), 57% in the prefrontal cortex (P< 0.001) and 64% in the temporal cortex (P<0.001). The number of LBs had a significant negative correlation with ChAT activity in both prefrontal (r=-0.33, P<0.05) and temporal cortex (r=-0.32, P<0.05). The reduction in ChAT activity in the prefrontal cortex had a significant negative correlation (r=-0.38, P=0.012) with the extent of cognitive impairment. When the CERAD class 'C' was excluded, cognitive impairment correlated significantly with both prefrontal ChAT activity (r=-0.52, P=0.0051) and the density of D1 dopamine receptors in the caudate nucleus (r=-0.40, P=0.037). The number of D1 and D2 dopamine receptors was reduced in both caudate nucleus and putamen in PD patients without neuroleptics as compared to controls. An increased D2 receptor number was found in the caudate nucleus and putamen in PD patients treated with neuroleptics. The present study showed that cognitive decline in PD is associated with reduced ChAT activity in the prefrontal cortex and the D1 dopamine receptor number in the caudate nucleus, even in the absence of AD-type pathology.

Parkinson's disease (PD) is a common neurodegenerative disorder characterized by the progressive accumulation in selected neurons of protein inclusions containing alpha-synuclein and ubiquitin. Rare inherited forms of PD are caused by autosomal dominant mutations in alpha-synuclein or by autosomal recessive mutations in parkin, an E3 ubiquitin ligase. We hypothesized that these two gene products interact functionally, namely, that parkin ubiquitinates alpha-synuclein normally and that this process is altered in autosomal recessive PD. We have now identified a protein complex in normal human brain that includes parkin as the E3 ubiquitin ligase, UbcH7 as its associated E2 ubiquitin conjugating enzyme, and a new 22-kilodalton glycosylated form of alpha-synuclein (alphaSp22) as its substrate. In contrast to normal parkin, mutant parkin associated with autosomal recessive PD failed to bind alphaSp22. In an in vitro ubiquitination assay, alphaSp22 was modified by normal but not mutant parkin into polyubiquitinated, high molecular weight species. Accordingly, alphaSp22 accumulated in a non-ubiquitinated form in parkin-deficient PD brains. We conclude that alphaSp22 is a substrate for parkin's ubiquitin ligase activity in normal human brain and that loss of parkin function causes pathological alphaSp22 accumulation. These findings demonstrate a critical biochemical reaction between the two PD-linked gene products and suggest that this reaction underlies the accumulation of ubiquitinated alpha-synuclein in conventional PD.

The distribution of casein kinase 1 delta (Cki delta) was studied by immunohistochemistry and correlated with other pathological hallmarks in Alzheimer's disease (AD), Down syndrome (DS), progressive supranuclear palsy (PSP), parkinsonism dementia complex of Guam (PDC), Pick's disease (PiD), pallido-ponto-nigral degeneration (PPND), Parkinson's disease (PD), dementia with Lewy bodies (DLB), amyotrophic lateral sclerosis (ALS), and elderly controls. Cki delta was found to be associated generally with granulovacuolar bodies and tau-containing neurofibrillary tangles in AD, DS, PSP, PDC, PPND, and controls, and Pick bodies and ballooned neurons in PiD. It was not associated with tau-containing inclusions in astroglia and oligodendroglia in PPND, PSP, and PDC. It was also not associated with tau-negative Lewy bodies in PD and DLB, Hirano bodies in PDC, Marinesco bodies in PD, AD, and controls and "skein"-like inclusions in anterior motor neurons in ALS. The colocalization of the kinase Cki delta and its apparent substrate tau suggests a function for Cki delta in the abnormal processing of tau.

We investigated the cyclin-dependent kinase (Cdk) 5 distribution pattern in diffuse Lewy body disease brains using immunohistochemistry. Cdk5 immunoreactivity was detected in both brainstem-type Lewy bodies (LBs) and cortical LBs. The number of Cdk5-positive LBs was less than that of ubiquitin- or alpha-synuclein-positive LBs, and more than that of phosphorylated neurofilament-positive LBs. Immunoelectron microscopy revealed Cdk5-immunolabeled granulo-filamentous components in LBs and LB-related neurites. These data suggest that Cdk5 may be associated with LB formation.

Heme oxygenase-1 is a cellular stress protein expressed in brain and other tissues in response to oxidative challenge and other noxious stimuli. In the present study, immunohistochemistry was used to assess HO-1 expression in various postmortem human brain specimens derived from PD and control subjects. In the substantia nigra of both PD and control specimens, moderate HO-1 immunoreactivity was consistently observed in neuromelanin-containing (dopaminergic) neurons. Lewy bodies in PD nigra neurons exhibited intense HO-1 immunostaining in their peripheries. In both PD and control specimens, neuronal HO-1 staining was faint or nondetectable in the other brain regions surveyed. The fraction of GFAP-positive astroglia expressing HO-1 in PD substantia nigra (77.1 +/- 12.3) was significantly greater than that observed in the substantia nigra of control subjects (18.7 +/- 7.1; P = 0.0015). In the other regions examined, percentages of GFAP-positive astroglia coexpressing HO-1 were relatively low and did not differ significantly (P > 0.05) between control and PD specimens. Upregulation of HO-1 in the substantia nigra of PD subjects supports the view that the affected tissue is experiencing chronic oxidative stress. In addition, excessive cellular levels of heme-derived free iron and carbon monoxide resulting from HO-1 overactivity may contribute to the pathogenesis of PD.

We examined the immunohistochemical localization of p35nck5a, the regulatory subunit of cyclin-dependent kinase 5 (cdk5), in brains obtained postmortem from patients with Parkinson's disease (PD) and controls. We found p35nck5a immunoreactivity in Lewy bodies (LB) in the substantia nigra, locus ceruleus, and neocortex of brains from patients with PD. In addition, p35nck5a was colocalized with cdk5 immunoreactivity in LB. Cdk5 is the kinase most likely to be responsible for the phosphorylation of neurofilament proteins of LB, which is a crucial step for the formation of the insoluble LB fibrils. Since p35nck5a regulates the catalytic activity of cdk5 by forming a heterodimer with cdk5, the present results strongly support the hypothesis that a cdk5-p35nck5a complex is involved in the formation of LB fibrils.

Lewy body-like inclusion (LI) is a cytoplasmic inclusion body deposited in anterior horn cells of patients with amyotrophic lateral sclerosis (ALS), in which there are phosphorylated neurofilament proteins (NFP). Since phosphorylation of NFP occurs normally in the axon, aberrant phosphorylation of NFP should take place in these cell bodies. To clarify the mechanism of LI formation it is crucial to identify the kinase responsible for the phosphorylation. We found cyclin-dependent kinase 5 (cdk5) immunoreactivity in most of LI in an ALS patient. Since cdk5 phosphorylates NFP, cdk5 is a candidate kinase for NFP in LI and might be involved in the cytoskeletal disorganization in LI-containing neurons.

This report concerns a case of sporadic amyotrophic lateral sclerosis (ALS) with dementia and Lewy body-like hyaline inclusions (LBHIs). The patient was a 70-year-old woman who initially showed memory disturbance and later developed bulbar palsy, muscle atrophy and weakness. The total clinical course was 51 months. The postmortem examination revealed superficial sponginess and subcortical gliosis in the frontotemporal cortices. Ubiquitin-positive intraneuronal inclusions were found in small cortical neurons of the frontotemporal lobe. Neuronal loss was marked in the spinal anterior horn with degeneration of the pyramidal tracts. The anterior horn cells had ubiquitin-immunoreactive skein-like inclusions and Bunina bodies. LBHIs were present in the lumbar horn; ultrastructurally they were composed of randomly arranged thick filamentous structures studded with granules. The LBHIs were intensely stained with anti-ubiquitin antibody. As in familial ALS and in certain cases of sporadic ALS, some of these inclusions reacted with an antibody against Cu/Zn superoxide dismutase, the enzyme whose gene was recently found to be mutated in some forms of familial ALS.

Recent evidence, based upon immunocytochemical and histochemical analysis of brain cortical tissue from alzheimer's disease patients, has suggested that altered activity and/or distribution of the lysosomal proteases cathepsins B and D may be implicated in the abnormal protein processing pathway resulting in formation of the neurotoxic amyloid A4 peptide, characteristic of this neurodegenerative disorder. We have therefore compared, via biochemical assay techniques using conventional or specially synthesised (corresponding to protein cleavage points of relevant to A4 peptide formation) fluorogenic substrates, the levels of activity of the lysosomal proteases cathepsins B, D, H and L, and dipeptidyl aminopeptidases I and II in frontal cortex (grey/white matter) from control and Alzheimer's disease patients. For comparative purposes, activity levels of the above enzymes were also determined in frontal cortex tissue from cases with Lewy body dementia and Parkinson's disease, and in caudate tissue from control and Huntington's disease cases. There was no significant difference in activity for any protease types in tissue from control cases and cases with Alzheimer's disease, Lewy body dementia or Parkinson's disease, with the exception of reduced dipeptidyl aminopeptidase II activity in Lewy body dementia and Parkinson's cases. We have therefore been unable to confirm a potential role for lysosomal cathepsins in the characteristic neurodegeneration associated with Alzheimer's disease; however the finding of significant increases in activity of dipeptidyl aminopeptidase II, cathepsin H and cathepsin D specifically in cases with Huntington's disease is of particular note. We therefore suggest the potential role of the latter enzymes in the pathogenesis of Huntington's disease requires further investigation.

In recent years much has been speculated about a pathogenic role of mitochondrial defects in Parkinson's disease. Ozawa et al. (BBRC 176, 938-946, 1991) have described an A/T transversion at nucleotide 7237 of mitochondrial DNA affecting cytochrome-c-oxidase (complex IV) of the respiratory chain that could contribute to the pathogenesis of PD. Employing PCR based genomic sequencing and restriction enzyme analysis on 19 cases of Lewy-body parkinsonism, we exclude this mutation as a common cause of Parkinson's disease. This demonstrates the need for systematic sequencing of the mitochondrial genome in a large number of histologically verified cases of Parkinson's disease.

Copper-zinc superoxide dismutase (SOD1)-like immunoreactivity has been demonstrated in Lewy body-like inclusions (LIs) in brain tissues from patients with familial and sporadic amyotrophic lateral sclerosis. Using immunocytochemistry, we studied Lewy bodies (LBs), the original inclusions from which the term LI was derived, in five patients with Parkinson disease (PD). Surprisingly, many LBs were immunostained by an antibody against SOD1. There were two types of staining pattern: a diffuse pattern, and a peripheral pattern with an unstained core. An immunoelectron microscopic study demonstrated that the immunoreactive products were restricted to the fibrillary profiles, sparing the unstructured core. Our results showed that SOD1-like immunoreactivity occurred frequently in LBs and LIs, suggesting that a common cytopathological process is responsible for the formation of LB-type neuronal intracytoplasmic inclusions. Our results also suggest that SOD1 plays a role in the neurodegeneration associated with PD.

Cu/Zn superoxide dismutase (SOD)-like immunoreactivity (LI) was found within Lewy body-like inclusions (LBIs) in the spinal cords of patients with sporadic amyotrophic lateral sclerosis (ALS) by using an antibody to human Cu/ZnSOD. LBIs were detected in the anterior horn cells in 10 of 20 patients with sporadic ALS. In each of these patients, 7 to 60% of LBIs showed Cu/ZnSOD-LI. No Cu/ZnSOD-LI was detected in intact neurons and glia in the 20 ALS patients, as well as in these cells in 10 normal control individuals. The skein-like inclusions and Bunina bodies, which were found in all of the 20 ALS cases, showed no Cu/ZnSOD-LI. Thus, Cu/ZnSOD appears to play roles in the production and/or degradation process of LBIs.

The ATP-ubiquitin-dependent proteolytic pathway (ubiquitin pathway) is believed to be involved in the formation of various neuronal inclusion bodies including Lewy bodies (LBs), a pathological hallmark of Parkinson disease and diffuse Lewy body disease (DLBD). Since multicatalytic proteinase (MCP) is involved in the ubiquitin pathway, an investigation of whether MCP is involved in neuronal inclusion bodies would provide a clue to the mechanism underlying the formation of neuronal inclusion bodies as well as to the pathogenesis of degenerative neurological disorders. In this study, we investigated detailed immunolocalization of MCP in LBs in DLBD brains using light and electron microscopy. We raised three different monoclonal antibodies against purified human MCP. Each of them recognized different sets of MCP subunits on Western blotting. Immunohistochemically, anti-MCP antibodies recognized all ubiquitin-positive cortical LBs in situ as well as those isolated from frozen DLBD cortices, suggesting that MCP is present in LBs as a whole molecule exhibiting protease activity. In electron microscopy, MCP immunoreactivity (MCP-IR) was exclusively localized on a characteristic oval structure with an approximate diameter of 100 nm. This structure was distributed throughout the LBs and was devoid of ubiquitin immunoreactivity. Treatment of isolated LBs with 2% SDS, but not with 0.5% Triton X-100, removed this structure from LBs in which fibrous materials predominated. Ubiquitin immunoreactivity was also decreased in isolated LBs treated with 2% SDS, suggesting that the fibrous structures in LBs were not ubiquitinated in situ. Thus, it is suggested that LBs are subjected to a proteolytic process in which MCP plays a role via processing of specific components of LBs.

We measured the concentrations of the monoamines, their precursors, and their metabolites, and the activity of choline acetyltransferase (ChAT) in basal ganglia and cortical regions of postmortem brains from cases with histologically verified pure Alzheimer's disease (AD), AD with diffusely distributed Lewy bodies (Lewy body variant [LBV]), and normal controls. Dopamine and homovanillic acid (HVA) were severely depleted in basal ganglia of the LBV cases but were not significantly altered in pure AD cases; tyrosine hydroxylase levels in putamen were also significantly reduced in LBV but not AD cases. These reductions in basal ganglia dopamine and HVA suggest that LBV cases have a level of dopamine depletion similar to Parkinson's disease (PD). Additionally, ChAT activity in caudate and norepinephrine concentration in putamen were significantly reduced in the LBV group, which may have contributed to the absence of resting tremor and the milder presentation of parkinsonian features in this group compared with classic PD. In frontal, parietal, and temporal cortex, activity of ChAT in the LBV group was significantly reduced compared with controls and lower than in pure AD.

A non-lysosomal multicatalytic proteinase (MCP) is immunohistochemically visualized in Lewy bodies and loosely arranged globose tangles but not in the majority of compact neurofibrillary tangles in brains with cortical diffuse Lewy body disease. Virtually all Lewy bodies are immunoreactive to MCP, exhibiting various staining patterns where they are moderately diffuse, intense in the central core or intense in the peripheral rim. MCP may be involved in the formation of these inclusions.

Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) is one of the predominant protein kinases in the brain. We found that CaM kinase II immunoreactivity was concentrated in the peripheral halos of Lewy bodies (LBs) in Parkinson's disease and Lewy body-like hyaline inclusions (LBHIs) in amyotrophic lateral sclerosis. An immunoelectron microscopic examination of LBs revealed that the filaments at the periphery of LBs were decorated with immunopositive deposits. Since CaM kinase II has a broad substrate specificity and can phosphorylate neurofilaments and other cytoskeletal proteins, it may play some role in the formation of LBs and LBHIs through the aberrant phosphorylation of the cytoskeletal elements in these inclusions.