Progressive dopaminergic alterations and mitochondrial abnormalities in LRRK2 G2019S knock-in mice.

Mutations in the LRRK2 gene represent the most common genetic cause of late onset Parkinson's disease. The physiological and pathological roles of LRRK2 are yet to be fully determined but evidence points towards LRRK2 mutations causing a gain in kinase function, impacting on neuronal maintenance, vesicular dynamics and neurotransmitter release. To explore the role of physiological levels of mutant LRRK2, we created knock-in (KI) mice harboring the most common LRRK2 mutation G2019S in their own genome. We have performed comprehensive dopaminergic, behavioral and neuropathological analyses in this model up to 24months of age. We find elevated kinase activity in the brain of both heterozygous and homozygous mice. Although normal at 6months, by 12months of age, basal and pharmacologically induced extracellular release of dopamine is impaired in both heterozygous and homozygous mice, corroborating previous findings in transgenic models over-expressing mutant LRRK2. Via in vivo microdialysis measurement of basal and drug-evoked extracellular release of dopamine and its metabolites, our findings indicate that exocytotic release from the vesicular pool is impaired. Furthermore, profound mitochondrial abnormalities are evident in the striatum of older homozygous G2019S KI mice, which are consistent with mitochondrial fission arrest. We anticipate that this G2019S mouse line will be a useful pre-clinical model for further evaluation of early mechanistic events in LRRK2 pathogenesis and for second-hit approaches to model disease progression.

DNAJC13 p.Asn855Ser mutation screening in Parkinson's disease and pathologically confirmed Lewy body disease patients.

BACKGROUND: Recently, a novel mutation in exon 24 of DNAJC13 gene (p.Asn855Ser, rs387907571) has been reported to cause autosomal dominant Parkinson's disease (PD) in a multi-incident Mennonite family. METHODS: In the present study the mutation containing exon of the DNAJC13 gene has been sequenced in a Caucasian series consisting of 1938 patients with clinical PD and 838 with pathologically diagnosed Lewy body disease (LBD). RESULTS: Our sequence analysis did not identify any coding variants in exon 24 of DNAJC13. Two previously described variants in intron 23 (rs200204728 and rs2369796) were observed. CONCLUSION: Our results indicate that the region surrounding the DNAJC13 p.Asn855Ser substitution is highly conserved and mutations in this exon are not a common cause of PD or LBD among Caucasian populations.

Alpha-synuclein-enhanced green fluorescent protein fusion proteins form proteasome sensitive inclusions in primary neurons.

Alpha-synuclein accumulates in the brains of sporadic Parkinson's disease patients as a major component of Lewy bodies, and mutations in alpha-synuclein are associated with familial forms of Parkinson's disease. The pathogenic mechanisms that precede and promote the aggregation of alpha-synuclein into Lewy bodies in neurons remain to be determined. Here, we constructed a series of alpha-synuclein-enhanced green fluorescent protein (alpha-synucleinEGFP, SynEGFP) fusion proteins to address whether the Parkinson's disease-associated mutations alter the subcellular distribution of alpha-synuclein, and to use as a tool for experimental manipulations to induce aggregate formation. When transfected into mouse cultured primary neurons, the 49-kDa alpha-synucleinEGFP fusion proteins are partially truncated to a approximately 27-kDa form. This non-fluorescent carboxy-terminally modified fusion protein spontaneously forms inclusions in the neuronal cytoplasm. A marked increase in the accumulation of inclusions is detected following treatment with each of three proteasome inhibitors, n-acetyl-leu-leu-norleucinal, lactacystin and MG132. Interestingly, Ala30Pro alpha-synucleinEGFP does not form the cytoplasmic inclusions that are characteristic of wild-type and Ala53Thr alpha-synucleinEGFP, supporting the idea that the Ala30Pro alpha-synuclein protein conformation differs from wild-type alpha-synuclein. Similar inclusions are formed if alpha-synuclein carboxy-terminus is modified by the addition of a V5/6xHistidine epitope tag. By contrast, overexpression of unmodified alpha-synuclein does not lead to aggregate formation. Furthermore, synphilin-1, an alpha-synuclein interacting protein also found in Lewy bodies, colocalizes with the carboxy-terminally truncated alpha-synuclein fusion protein in discrete cytoplasmic inclusions.Our finding that manipulations of the carboxy-terminus of alpha-synuclein lead to inclusion formation may provide a model for studies of the pathogenic mechanisms of alpha-synuclein aggregation in Lewy bodies.

Subcellular localization of alpha-synuclein in primary neuronal cultures: effect of missense mutations.

Numerous recent observations have implicated alpha-synuclein in the pathogenesis of several neurodegenerative diseases, including Parkinson's disease, Alzheimer's disease, dementia with Lewy bodies and multiple-system atrophy. Two missense mutations in the gene for alpha-synuclein have been identified in some cases of familial Parkinson's disease and it is thought that these may disrupt the normal structure of the protein and thus promote aggregation into Lewy body filaments. Here, we examine the subcellular localization of alpha-synuclein in primary cortical neurons maintained in a monolayer culture. The protein has widespread expression throughout neurons, including the nucleus, and has a discete localization in the neurites of more mature neurons, reminiscent of synaptic specializations. Interestingly, in a subpopulation of cortical neurons transfected at 13 days in vitro, we find that alpha-synuclein appears to aggregate into distinct punctate inclusions in the cytoplasm and proximal neurites. Unlike Lewy bodies, these structures are not ubiquitin positive. These regions of alpha-synuclein accumulation are observed following transfections with wild-type, Ala30Pro or Ala53Thr alpha-synuclein; neither mutation alters their frequency.

Molecular chaperones as rational drug targets for Parkinson's disease therapeutics.

Parkinson's disease is a neurodegenerative movement disorder that is caused, in part, by the loss of dopaminergic neurons within the substantia nigra pars compacta of the basal ganglia. The presence of intracellular protein aggregates, known as Lewy bodies and Lewy neurites, within the surviving nigral neurons is the defining neuropathological feature of the disease. Accordingly, the identification of specific genes mutated in families with Parkinson's disease and of genetic susceptibility variants for idiopathic Parkinson's disease has implicated abnormalities in proteostasis, or the handling and elimination of misfolded proteins, in the pathogenesis of this neurodegenerative disorder. Protein folding and the refolding of misfolded proteins are regulated by a network of interactive molecules, known as the chaperone system, which is composed of molecular chaperones and co-chaperones. The chaperone system is intimately associated with the ubiquitin-proteasome system and the autophagy-lysosomal pathway which are responsible for elimination of misfolded proteins and protein quality control. In addition to their role in proteostasis, some chaperone molecules are involved in the regulation of cell death pathways. Here we review the role of the molecular chaperones Hsp70 and Hsp90, and the cochaperones Hsp40, BAG family members such as BAG5, CHIP and Hip in modulating neuronal death with a focus on dopaminergic neurodegeneration in Parkinson's disease. We also review current progress in preclinical studies aimed at targetting the chaperone system to prevent neurodegeneration. Finally, we discuss potential future chaperone-based therapeutics for the symptomatic treatment and possible disease modification of Parkinson's disease.

Mapping of the alpha 4 subunit gene (GABRA4) to human chromosome 4 defines an alpha 2-alpha 4-beta 1-gamma 1 gene cluster: further evidence that modern GABAA receptor gene clusters are derived from an ancestral cluster.

We demonstrated previously that an alpha 1-beta 2-gamma 2 gene cluster of the gamma-aminobutyric acid (GABAA) receptor is located on human chromosome 5q34-q35 and that an ancestral alpha-beta-gamma gene cluster probably spawned clusters on chromosomes 4, 5, and 15. Here, we report that the alpha 4 gene (GABRA4) maps to human chromosome 4p14-q12, defining a cluster comprising the alpha 2, alpha 4, beta 1, and gamma 1 genes. The existence of an alpha 2-alpha 4-beta 1-gamma 1 cluster on chromosome 4 and an alpha 1-alpha 6-beta 2-gamma 2 cluster on chromosome 5 provides further evidence that the number of ancestral GABAA receptor subunit genes has been expanded by duplication within an ancestral gene cluster. Moreover, if duplication of the alpha gene occurred before duplication of the ancestral gene cluster, then a heretofore undiscovered subtype of alpha subunit should be located on human chromosome 15q11-q13 within an alpha 5-alpha x-beta 3-gamma 3 gene cluster at the locus for Angelman and Prader-Willi syndromes.

Interaction of alpha-synuclein and synphilin-1: effect of Parkinson's disease-associated mutations.

alpha-Synuclein is a major component of Lewy bodies, a neuropathological feature of Parkinson's disease. Two alpha-synuclein mutations, Ala53Thr and Ala30Pro, are associated with early onset, familial forms of the disease. Recently, synphilin-1, a protein found to interact with alpha-synuclein by yeast two hybrid techniques, was detected in Lewy bodies. In this study we report the interaction of alpha-synuclein and synphilin-1 in human neuroglioma cells using a sensitive fluorescence resonance energy transfer technique. We demonstrate that the C-terminus of alpha-synuclein is closely associated with the C-terminus of synphilin-1. A weak interaction occurs between the N-terminus of alpha-synuclein and synphilin-1. The familial Parkinson's disease associated mutations of alpha-synuclein (Ala53Thr and Ala30Pro) also demonstrate a strong interaction between their C-terminal regions and synphilin-1. However, compared with wild-type alpha-synuclein, significantly less energy transfer occurs between the C-terminus of Ala53Thr alpha-synuclein and synphilin-1, suggesting that the Ala53Thr mutation alters the conformation of alpha-synuclein in relation to synphilin-1.

Alpha-synuclein has an altered conformation and shows a tight intermolecular interaction with ubiquitin in Lewy bodies.

Alpha-synuclein, a protein in which two mutations have been identified that cause autosomal dominant Parkinson's disease, is thought to serve as a nidus for the development of a Lewy body. We hypothesized that alpha-synuclein would display different intra- and intermolecular associations in Lewy bodies than it does in its normal intracellular compartments. Using sensitive fluorescence resonance energy transfer (FRET) techniques, we found evidence that alpha-synuclein is more compact and in closer association with other alpha-synuclein molecules in Lewy bodies than it is in the neuropil. In addition, we found evidence of a close, direct intermolecular interaction between the N terminus of alpha-synuclein and ubiquitin. These observations provide support for the hypothesis that in Lewy bodies alpha-synuclein adopts an altered three-dimensional structure and undergoes N-terminal ubiquitination.

A minimal promoter for the GABA(A) receptor alpha6-subunit gene controls tissue specificity.

The ability of nerve cells to regulate the expression of specific neurotransmitter receptors is of central importance to nervous system function, but little is known about the DNA elements that mediate neuron specific gene expression. The type A gamma-aminobutyric acid (GABA(A)) receptor alpha6-subunit gene, which is expressed exclusively in cerebellar granule cells, presents a unique opportunity to study the cis elements involved in restricting gene expression to a distinct neuronal population. In an effort to identify the regulatory elements that govern cerebellar granule cell-specific gene expression, the proximal 5' flanking regions for the human, rat, and mouse alpha6 genes were cloned and sequenced, and a major transcriptional initiation site was identified in the rodent genes. Functional analysis of rat alpha6 gene-reporter constructs in primary neuronal cultures reveals that a 155-bp TATA-less promoter region (-130 to +25 bp) constitutes a minimal promoter that can drive cerebellar granule cell-specific expression. Internal deletion and decoy competition studies demonstrate that the minimal promoter contains a 60-bp region (-130 to -70 bp) that is critical for enhanced promoter activity in cerebellar granule cells. Activity of the compromised promoter containing the deletion cannot be rescued by placing the 60-bp region downstream of the reporter gene, demonstrating that it is not a classical enhancer but rather a positionally dependent regulator. An additional cerebellar-specific activating sequence is located between -324 and -130 bp, and a downstream negative regulatory region (+158 to +294) has been shown to be active in fibroblasts but inactive in cerebellar granule cells. Taken together, the results suggest a possible mechanism for the control of cerebellar granule cell-specific expression of the GABA(A) receptor alpha6 subunit gene.

Membrane association and protein conformation of alpha-synuclein in intact neurons. Effect of Parkinson's disease-linked mutations.

Two missense mutations (Ala-30 --> Pro and Ala-53 --> Thr) in the gene encoding alpha-synuclein are associated with rare autosomal dominant forms of familial Parkinson's disease. In addition, alpha-synuclein is an abundant component of Lewy bodies in sporadic Parkinson's disease and diffuse Lewy body disease. However, the normal conformation of alpha-synuclein, its cellular localization in neurons, and the effects of the mutations remain to be determined. In the present study, we examine these questions using sensitive fluorescence resonance energy transfer techniques. Transient transfection of alpha-synuclein expression constructs into primary cortical neurons and counterstaining with the lipophilic fluorescent marker, DiI, demonstrates a close association between alpha-synuclein and cellular membranes. Both the N- and C-terminal regions of alpha-synuclein are tightly associated with membranes. A weak interaction also occurs between the N and C termini themselves. The Parkinson's disease-associated mutations have no effect on membrane interaction; however, the Ala-30 --> Pro mutation alters the three-dimensional conformation of alpha-synuclein, as measured by significantly increased fluorescence resonance energy transfer between the N and C termini.

Neuritic alterations and neural system dysfunction in Alzheimer's disease and dementia with Lewy bodies.

Alzheimer's disease (AD) and dementia with Lewy bodies (DLB) are neurodegenerative disorders that share progressive dementia as the common major clinical symptom. Damages to memory-related brain structures are the likely pathological correlate, and in both illnesses deposition of amyloidogenic proteins are present mainly within these limbic structures. Amyloid-beta-positive plaques and phospho-tau-positive neurofibrillary tangles are the main feature of AD and alpha-synuclein-positive Lewy bodies and Lewy neurites are found in DLB. Interestingly the associated proteins also interfere with synaptic function and synaptic plasticity. Here, we propose that the same neuronal circuits are disturbed within the hippocampal formation in AD and DLB and that in both diseases the associated proteins might lead to changes in synaptic plasticity and function. Thus both classic neuropathological changes and cellular dysfunctions might contribute to the cognitive impairments in AD and DLB.

A close association of torsinA and alpha-synuclein in Lewy bodies: a fluorescence resonance energy transfer study.

TorsinA, a novel protein in which a mutation causes dominant, early onset torsion dystonia, may serve as a chaperone for misfolded proteins that require refolding or degradation. It has been hypothesized that misfolded alpha-synuclein, a protein in which two mutations cause autosomal dominantly inherited Parkinson's disease, serves as a nidus for the development of a Lewy body. We hypothesized that torsinA plays a role in the cellular processing of alpha-synuclein. We demonstrate that anti-torsin antibodies stain Lewy bodies and Lewy neurites in the substantia nigra and cortex. Using sensitive fluorescent resonance energy transfer (FRET) techniques, we find evidence of a close association between torsinA and alpha-synuclein in Lewy bodies.