Missense mutations in leucine-rich repeat kinase 2 (LRRK2) cause familial Parkinson's disease (PD). However, a potential role of wild-type LRRK2 in idiopathic PD (iPD) remains unclear. Here, we ...developed proximity ligation assays to assess Ser1292 phosphorylation of LRRK2 and, separately, the dissociation of 14-3-3 proteins from LRRK2. Using these proximity ligation assays, we show that wild-type LRRK2 kinase activity was selectively enhanced in substantia nigra dopamine neurons in postmortem brain tissue from patients with iPD and in two different rat models of the disease. We show that this occurred through an oxidative mechanism, resulting in phosphorylation of the LRRK2 substrate Rab10 and other downstream consequences including abnormalities in mitochondrial protein import and lysosomal function. Our study suggests that, independent of mutations, wild-type LRRK2 plays a role in iPD. LRRK2 kinase inhibitors may therefore be useful for treating patients with iPD who do not carry LRRK2 mutations.
•Zebrafish motor activity varies in antiphase with sinusoidally-modulated light.•Mean swimming speed is inversely related to illuminance by a power function.•Motor activity is also influenced by the ...proportional rate of change of illuminance.•The motor response to illuminance is dependent on dopaminergic function.•The illuminance-activity assay may provide a useful 96-well screening tool.
Larval zebrafish show stereotyped motor responses to changes in ambient illumination. The responses can be evaluated in 96-well plates, and are used widely to assess neurological function in zebrafish models. However, the square-wave (on/off) light stimuli commonly employed in these studies do not allow analysis of the relationship between motor activity and illumination intensity or its rate of change. To address this limitation, we measured larval zebrafish motor function while ambient illumination was modulated sinusoidally. Motor activity varied robustly and reproducibly in antiphase with illumination. The relationship between mean swimming speed (dependent variable) and illuminance (independent variable) was described most closely by a power function, and was influenced dynamically by the proportional rate of change of illuminance. Several predictions from this model were verified experimentally by testing responses to sinusoidal illumination waveforms that were amplitude-, phase-, or offset-modulated, or transformed by a power function. At concentrations ≤5 μM, the dopamine D2 receptor inverse agonist haloperidol selectively abrogated the motor response to decreasing Illuminance without altering baseline activity in bright light, suggesting that dopamine is essential for illuminance-dependent motor function. These data contribute to understanding the environmental determinants of motor activity in zebrafish larvae, suggest experimental opportunities to elucidate underlying neural mechanisms, and potentially provide an assay of dopaminergic function for chemical and genetic screening applications.
Abstract As a consequence of the widespread use of zebrafish in developmental biology studies, an extensive array of experimental tools and techniques has been assembled; it has recently become ...apparent that these might be exploited in the analysis of human neurodegenerative diseases. A surprising degree of functional conservation has been demonstrated between human genes implicated in neurodegenerative diseases and their zebrafish orthologues. In zebrafish models of recessive parkinsonism, Parkin or Pink1 knockdown gave rise to specific loss of dopamine neurons; in a zebrafish model of recessive spinal muscular atrophy, loss of Smn1 function caused specific motor axonal defects. In addition, pathological features of several dominant diseases were replicated by transgenic over-expression of mutant human proteins, including Tau, Huntingtin, and SOD1. In some cases, conservation of relevant cellular pathways was sufficient that disease-specific posttranslational changes to the respective proteins were found in the zebrafish models. These data collectively suggest that the zebrafish can be an appropriate setting in which to model the molecular events underlying human neuropsychiatric disease. Consequently, novel findings yielded by studies in zebrafish models may be applicable to human diseases; this is an exciting prospect, in view of the many potential uses of zebrafish models, for example, screening for lead therapeutic compounds, rapid functional assessments of putative modifier genes, and live observation of pathogenic mechanisms in vivo.
•Zebrafish show stereotypical motor responses to changes in illumination.•We characterized the spectral properties of the visual motor response (VMR).•Light at 399nm, 458nm, 514nm or 632nm elicited ...the characteristic phases of the VMR.•Components of the VMR were differentially elicited by discrete light wavebands.•Distinct photoreceptor populations likely mediate different components of the VMR.
Larval zebrafish react to changes in ambient illumination with a series of stereotyped motor responses, called the visual motor response (VMR). The VMR has been used widely in zebrafish models to analyze how genetic or environmental manipulations alter neurological function. Prior studies elicited the VMR using white light. In order to elucidate the underlying afferent pathways and to identify light wavelengths that elicit the VMR without also activating optogenetic reagents, we employed calibrated narrow-waveband light sources to analyze the spectral properties of the response. Narrow light wavebands with peaks between 399nm and 632nm triggered the characteristic phases of the VMR, but there were quantitative differences between responses to different light wavelengths at the same irradiant flux density. The O-bend component of the VMR was elicited readily at dark onset following illumination in 399nm or 458nm light, but was less prominent at the transition from 632nm light to dark. Conversely, stable motor activity in light was observed at 458nm, 514nm, and 632nm, but not at 399nm. The differential effect of discrete light wavebands on components of the VMR suggests they are driven by distinct photoreceptor populations. Furthermore, these data enable the selection of light wavebands to drive the VMR in a separate channel to the activation of optogenetic reagents and photosensitizers.
DJ-1 is a redox-sensitive protein with several putative functions important in mitochondrial physiology, protein transcription, proteasome regulation, and chaperone activity. High levels of DJ-1 ...immunoreactivity are reported in astrocytes surrounding pathology associated with idiopathic Parkinson's disease, possibly reflecting the glial response to oxidative damage. Previous studies showed that astrocytic over-expression of DJ-1 in vitro prevented oxidative stress and mitochondrial dysfunction in primary neurons. Based on these observations, we developed a pseudotyped lentiviral gene transfer vector with specific tropism for CNS astrocytes in vivo to overexpress human DJ-1 protein in astroglial cells. Following vector delivery to the substantia nigra and striatum of adult Lewis rats, the DJ-1 transgene was expressed robustly and specifically within astrocytes. There was no observable transgene expression in neurons or other glial cell types. Three weeks after vector infusion, animals were exposed to rotenone to induce Parkinson's disease-like pathology, including loss of dopaminergic neurons, accumulation of endogenous α-synuclein, and neuroinflammation. Animals over-expressing hDJ-1 in astrocytes were protected from rotenone-induced neurodegeneration, and displayed a marked reduction in neuronal oxidative stress and microglial activation. In addition, α-synuclein accumulation and phosphorylation were decreased within substantia nigra dopaminergic neurons in DJ-1–transduced animals, and expression of LAMP-2A, a marker of chaperone mediated autophagy, was increased. Together, these data indicate that astrocyte-specific overexpression of hDJ-1 protects neighboring neurons against multiple pathologic features of Parkinson's disease and provides the first direct evidence in vivo of a cell non-autonomous neuroprotective function of astroglial DJ-1.
•DJ-1 is a redox-sensitive protein, upregulated in astrocytes in Parkinson's disease.•Using a pseudotyped lentiviral vector, DJ-1 was overexpressed in astrocytes in vivo.•Rats expressing astrocytic DJ-1 were protected from rotenone model of PD.•Astrocytic DJ-1 attenuated rotenone-induced oxidative stress and neuroinflammation.•Astrocyte DJ-1 expression decreased α-synuclein accumulation in dopaminergic neurons.
α-Synuclein accumulation and mitochondrial dysfunction have both been strongly implicated in the pathogenesis of Parkinson's disease (PD), and the two appear to be related. Mitochondrial dysfunction ...leads to accumulation and oligomerization of α-synuclein, and increased levels of α-synuclein cause mitochondrial impairment, but the basis for this bidirectional interaction remains obscure. We now report that certain posttranslationally modified species of α-synuclein bind with high affinity to the TOM20 (translocase of the outer membrane 20) presequence receptor of the mitochondrial protein import machinery. This binding prevented the interaction of TOM20 with its co-receptor, TOM22, and impaired mitochondrial protein import. Consequently, there were deficient mitochondrial respiration, enhanced production of reactive oxygen species, and loss of mitochondrial membrane potential. Examination of postmortem brain tissue from PD patients revealed an aberrant α-synuclein-TOM20 interaction in nigrostriatal dopaminergic neurons that was associated with loss of imported mitochondrial proteins, thereby confirming this pathogenic process in the human disease. Modest knockdown of endogenous α-synuclein was sufficient to maintain mitochondrial protein import in an in vivo model of PD. Furthermore, in in vitro systems, overexpression of TOM20 or a mitochondrial targeting signal peptide had beneficial effects and preserved mitochondrial protein import. This study characterizes a pathogenic mechanism in PD, identifies toxic species of wild-type α-synuclein, and reveals potential new therapeutic strategies for neuroprotection.
Recent studies delineate a pathway involving familial Parkinson's disease (PD)-related proteins PINK1 and Parkin, in which PINK1-dependent mitochondrial accumulation of Parkin targets depolarized ...mitochondria towards degradation through mitophagy. The pathway has been primarily characterized in cells less dependent on mitochondria for energy production than neurons. Here we report that in neurons, unlike other cells, mitochondrial depolarization by carbonyl cyanide m-chlorophenyl hydrazone did not induce Parkin translocation to mitochondria or mitophagy. PINK1 overexpression increased basal Parkin accumulation on neuronal mitochondria, but did not sensitize them to depolarization-induced Parkin translocation. Our data suggest that bioenergetic differences between neurons and cultured cell lines contribute to these different responses. In HeLa cells utilizing usual glycolytic metabolism, mitochondrial depolarization robustly triggered Parkin-mitochondrial translocation, but this did not occur in HeLa cells forced into dependence on mitochondrial respiration. Declining ATP levels after mitochondrial depolarization correlated with the absence of induced Parkin-mitochondrial translocation in both HeLa cells and neurons. However, intervention allowing neurons to maintain ATP levels after mitochondrial depolarization only modestly increased Parkin recruitment to mitochondria, without evidence of increased mitophagy. These data suggest that changes in ATP levels are not the sole determinant of the different responses between neurons and other cell types, and imply that additional mechanisms regulate mitophagy in neurons. Since the Parkin-mitophagy pathway is heavily dependent on bioenergetic status, the unique metabolic properties of neurons likely influence the function of this pathway in the pathogenesis of PD.
The retinal pigment epithelium (RPE) is a specialized monolayer of pigmented cells within the eye that is critical for maintaining visual system function. Diseases affecting the RPE have dire ...consequences for vision, and the most prevalent of these is atrophic (dry) age-related macular degeneration (AMD), which is thought to result from RPE dysfunction and degeneration. An intriguing possibility for treating RPE degenerative diseases like atrophic AMD is the stimulation of endogenous RPE regeneration; however, very little is known about the mechanisms driving successful RPE regeneration in vivo. Here, we developed a zebrafish transgenic model (rpe65a:nfsB-eGFP) that enabled ablation of large swathes of mature RPE. RPE ablation resulted in rapid RPE degeneration, as well as degeneration of Bruch's membrane and underlying photoreceptors. Using this model, we demonstrate for the first time that zebrafish are capable of regenerating a functional RPE monolayer after RPE ablation. Regenerated RPE cells first appear at the periphery of the RPE, and regeneration proceeds in a peripheral-to-central fashion. RPE ablation elicits a robust proliferative response in the remaining RPE. Subsequently, proliferative cells move into the injury site and differentiate into RPE. BrdU incorporation assays demonstrate that the regenerated RPE is likely derived from remaining peripheral RPE cells. Pharmacological disruption using IWR-1, a Wnt signaling antagonist, significantly reduces cell proliferation in the RPE and impairs overall RPE recovery. These data demonstrate that the zebrafish RPE possesses a robust capacity for regeneration and highlight a potential mechanism through which endogenous RPE regenerate in vivo.
α-Synuclein plays a central role in the pathogenesis of Parkinson's disease (PD); interventions that decrease its expression appear neuroprotective in PD models. Successful translation of these ...observations into effective therapies will be dependent on the safety of suppressing α-synuclein expression in the adult brain. We investigated long-term α-synuclein knockdown in the adult rat CNS. 8-month old animals received either AAV-shSnca (an RNA interference vector targeting the Snca mRNA transcript) or AAV-shCtrl (a control vector) unilaterally into the substantia nigra. No signs of systemic toxicity or motor dysfunction were observed in either experimental group over 12 months. Viral transgene expression persisted to 12 months post-inoculation, at which point Snca mRNA expression in substantia nigra dopaminergic neurons of animals that received AAV-shSnca was decreased by ≈90%, and α-synuclein immunoreactivity by >70% relative to the control side. Stereological quantification of Nissl-labeled neurons showed no evidence of neurodegeneration in the substantia nigra 12 months after inoculation with either vector, and we observed abundant dopaminergic neurons with minimal α-synuclein immunoreactivity that appeared otherwise unremarkable in the AAV-shSnca group. Despite the absence of neurodegeneration, some loss of TH expression was evident in nigral neurons after transduction with either vector, presumably a non-specific consequence of vector delivery, cellular transduction, or expression of shRNA or GFP. We conclude that long-term α-synuclein knockdown in the substantia nigra does not cause significant functional deficits in the ascending dopaminergic projection, or neurodegeneration. These findings are encouraging that it may be feasible to target α-synuclein expression therapeutically in PD.
•shRNA targeting Snca was delivered to nigral dopamine neurons using an AAV vector.•Snca mRNA was reduced by 90% and α-synuclein expression by >70% at 12 months.•No motor deficit or neuronal loss was observed after α-synuclein knockdown.•Long-term α-synuclein knockdown in the adult brain does not cause neurodegeneration.
The major obstacle to a cure for HIV infection is the persistence of replication-competent viral reservoirs during antiretroviral therapy. HIV-specific chimeric antigen receptor (CAR) T cells have ...been developed to target latently infected CD4
T cells that express virus either spontaneously or after intentional latency reversal. Whether HIV-specific CAR-T cells can recognize and eliminate the follicular dendritic cell (FDC) reservoir of HIV-bound immune complexes (ICs) is unknown. We created HIV-specific CAR-T cells using human peripheral blood mononuclear cells (PBMCs) and a CAR construct that enables the expression of CD4 (domains 1 and 2) and the carbohydrate recognition domain of mannose binding lectin (MBL) to target native HIV Env (CD4-MBL CAR). We assessed CAR-T cell cytotoxicity using a carboxyfluorescein succinimidyl ester (CFSE) release assay and evaluated CAR-T cell activation through interferon gamma (IFN-γ) production and CD107a membrane accumulation by flow cytometry. CD4-MBL CAR-T cells displayed potent lytic and functional responses to Env-expressing cell lines and HIV-infected CD4
T cells but were ineffective at targeting FDC bearing HIV-ICs. CD4-MBL CAR-T cells were unresponsive to cell-free HIV or concentrated, immobilized HIV-ICs in cell-free experiments. Blocking intercellular adhesion molecule-1 (ICAM-1) inhibited the cytolytic response of CD4-MBL CAR-T cells to Env-expressing cell lines and HIV-infected CD4
T cells, suggesting that factors such as adhesion molecules are necessary for the stabilization of the CAR-Env interaction to elicit a cytotoxic response. Thus, CD4-MBL CAR-T cells are unable to eliminate the FDC-associated HIV reservoir, and alternative strategies to eradicate this reservoir must be sought.
Efforts to cure HIV infection have focused primarily on the elimination of latently infected CD4
T cells. Few studies have addressed the unique reservoir of infectious HIV that exists on follicular dendritic cells (FDCs), persists
during antiretroviral therapy, and likely contributes to viral rebound upon cessation of antiretroviral therapy. We assessed the efficacy of a novel HIV-specific chimeric antigen receptor (CAR) T cell to target both HIV-infected CD4
T cells and the FDC reservoir
Although CAR-T cells eliminated CD4
T cells that express HIV, they did not respond to or eliminate FDC bound to HIV. These findings reveal a fundamental limitation to CAR-T cell therapy to eradicate HIV.
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