Correction: Mutations in LRRK2 linked to Parkinson disease sequester Rab8a to damaged lysosomes and regulate transferrin-mediated iron uptake in microglia.

[This corrects the article DOI: 10.1371/journal.pbio.3001480.].

Mutations in LRRK2 linked to Parkinson disease sequester Rab8a to damaged lysosomes and regulate transferrin-mediated iron uptake in microglia.

Mutations in leucine-rich repeat kinase 2 (LRRK2) cause autosomal dominant Parkinson disease (PD), while polymorphic LRRK2 variants are associated with sporadic PD. PD-linked mutations increase LRRK2 kinase activity and induce neurotoxicity in vitro and in vivo. The small GTPase Rab8a is a LRRK2 kinase substrate and is involved in receptor-mediated recycling and endocytic trafficking of transferrin, but the effect of PD-linked LRRK2 mutations on the function of Rab8a is poorly understood. Here, we show that gain-of-function mutations in LRRK2 induce sequestration of endogenous Rab8a to lysosomes in overexpression cell models, while pharmacological inhibition of LRRK2 kinase activity reverses this phenotype. Furthermore, we show that LRRK2 mutations drive association of endocytosed transferrin with Rab8a-positive lysosomes. LRRK2 has been nominated as an integral part of cellular responses downstream of proinflammatory signals and is activated in microglia in postmortem PD tissue. Here, we show that iPSC-derived microglia from patients carrying the most common LRRK2 mutation, G2019S, mistraffic transferrin to lysosomes proximal to the nucleus in proinflammatory conditions. Furthermore, G2019S knock-in mice show a significant increase in iron deposition in microglia following intrastriatal LPS injection compared to wild-type mice, accompanied by striatal accumulation of ferritin. Our data support a role of LRRK2 in modulating iron uptake and storage in response to proinflammatory stimuli in microglia.

Extracellular clusterin limits the uptake of α-synuclein fibrils by murine and human astrocytes.

The progressive neuropathological damage seen in Parkinson's disease (PD) is thought to be related to the spreading of aggregated forms of α-synuclein. Clearance of extracellular α-synuclein released by degenerating neurons may be therefore a key mechanism to control the concentration of α-synuclein in the extracellular space. Several molecular chaperones control misfolded protein accumulation in the extracellular compartment. Among these, clusterin, a glycoprotein associated with Alzheimer's disease, binds α-synuclein aggregated species and is present in Lewy bodies, intraneuronal aggregates mainly composed by fibrillary α-synuclein. In this study, using murine primary astrocytes with clusterin genetic deletion, human-induced pluripotent stem cell (iPSC)-derived astrocytes with clusterin silencing and two animal models relevant for PD we explore how clusterin affects the clearance of α-synuclein aggregates by astrocytes. Our findings showed that astrocytes take up α-synuclein preformed fibrils (pffs) through dynamin-dependent endocytosis and that clusterin levels are modulated in the culture media of cells upon α-synuclein pffs exposure. Specifically, we found that clusterin interacts with α-synuclein pffs in the extracellular compartment and the clusterin/α-synuclein complex can be internalized by astrocytes. Mechanistically, using clusterin knock-out primary astrocytes and clusterin knock-down hiPSC-derived astrocytes we observed that clusterin limits the uptake of α-synuclein pffs by cells. Interestingly, we detected increased levels of clusterin in the adeno-associated virus- and the α-synuclein pffs- injected mouse model, suggesting a crucial role of this chaperone in the pathogenesis of PD. Overall, our observations indicate that clusterin can limit the uptake of extracellular α-synuclein aggregates by astrocytes and, hence, contribute to the spreading of Parkinson pathology.

Sequential screening nominates the Parkinson's disease associated kinase LRRK2 as a regulator of Clathrin-mediated endocytosis.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are an established cause of inherited Parkinson's disease (PD). LRRK2 is expressed in both neurons and glia in the central nervous system, but its physiological function(s) in each of these cell types is uncertain. Through sequential screens, we report a functional interaction between LRRK2 and Clathrin adaptor protein complex 2 (AP2). Analysis of LRRK2 KO tissue revealed a significant dysregulation of AP2 complex components, suggesting LRRK2 may act upstream of AP2. In line with this hypothesis, expression of LRRK2 was found to modify recruitment and phosphorylation of AP2. Furthermore, expression of LRRK2 containing the R1441C pathogenic mutation resulted in impaired clathrin-mediated endocytosis (CME). A decrease in activity-dependent synaptic vesicle endocytosis was also observed in neurons harboring an endogenous R1441C LRRK2 mutation. Alongside LRRK2, several PD-associated genes intersect with membrane-trafficking pathways. To investigate the genetic association between Clathrin-trafficking and PD, we used polygenetic risk profiling from IPDGC genome wide association studies (GWAS) datasets. Clathrin-dependent endocytosis genes were found to be associated with PD across multiple cohorts, suggesting common variants at these loci represent a cumulative risk factor for disease. Taken together, these findings suggest CME is a LRRK2-mediated, PD relevant pathway.

Transcriptome analysis of LRRK2 knock-out microglia cells reveals alterations of inflammatory- and oxidative stress-related pathways upon treatment with α-synuclein fibrils.

Several previous studies have linked the Parkinson's disease (PD) gene LRRK2 to the biology of microglia cells. However, the precise ways in which LRRK2 affects microglial function have not been fully resolved. Here, we used the RNA-Sequencing to obtain transcriptomic profiles of LRRK2 wild-type (WT) and knock-out (KO) microglia cells treated with α-synuclein pre-formed fibrils (PFFs) or lipopolysaccharide (LPS) as a general inflammatory insult. We observed that, although α-synuclein PFFs and LPS mediate overlapping gene expression profiles in microglia, there are also distinct responses to each stimulus. α-Synuclein PFFs trigger alterations of oxidative stress-related pathways with the mitochondrial dismutase Sod2 as a strongly differentially regulated gene. We validated SOD2 at mRNA and protein levels. Furthermore, we found that LRRK2 KO microglia cells reported attenuated induction of mitochondrial SOD2 in response to α-synuclein PFFs, indicating a potential contribution of LRRK2 to oxidative stress-related pathways. We validate several genes in vivo using single-cell RNA-Seq from acutely isolated microglia after striatal injection of LPS into the mouse brain. Overall, these results suggest that microglial LRRK2 may contribute to the pathogenesis of PD via altered oxidative stress signaling.

In vivo quantification of glial activation in minipigs overexpressing human α-synuclein.

Parkinson's disease is characterized by a progressive loss of substantia nigra (SN) dopaminergic neurons and the formation of Lewy bodies containing accumulated alpha-synuclein (α-syn). The pathology of Parkinson's disease is associated with neuroinflammatory microglial activation, which may contribute to the ongoing neurodegeneration. This study investigates the in vivo microglial and dopaminergic response to overexpression of α-syn. We used positron emission tomography (PET) and the 18 kDa translocator protein radioligand, [11 C](R)PK11195, to image brain microglial activation and (+)-α-[11 C]dihydrotetrabenazine ([11 C]DTBZ), to measure vesicular monoamine transporter 2 (VMAT2) availability in Göttingen minipigs following injection with recombinant adeno-associated virus (rAAV) vectors expressing either mutant A53T α-syn or green fluorescent protein (GFP) into the SN (4 rAAV-α-syn, 4 rAAV-GFP, 5 non-injected control minipigs). We performed motor symptom assessment and immunohistochemical examination of tyrosine hydroxylase (TH) and transgene expression. Expression of GFP and α-syn was observed at the SN injection site and in the striatum. We observed no motor symptoms or changes in striatal [11 C]DTBZ binding potential in vivo or striatal or SN TH staining in vitro between the groups. The mean [11 C](R)PK11195 total volume of distribution was significantly higher in the basal ganglia and cortical areas of the α-syn group than the control animals. We conclude that mutant α-syn expression in the SN resulted in microglial activation in multiple sub- and cortical regions, while it did not affect TH stains or VMAT2 availability. Our data suggest that microglial activation constitutes an early response to accumulation of α-syn in the absence of dopamine neuron degeneration.

Toxic effects of human and rodent variants of alpha-synuclein in vivo.

In Parkinson's disease, abnormal alpha-synuclein (asyn) accumulation leads to the formation of soluble oligomeric species thought to be toxic to cells as well as intraneuronal inclusions. To date, the precise mechanisms leading to aggregation of asyn in the brain is not well-understood. Previous studies in yeast, drosophila, and transgenic mice suggested that a non-A beta component depleted version of human asyn [h-asyn(D70-83)] or human beta-synuclein (h-bsyn), naturally lacking this centrally located hydrophobic region, are less prone to form aggregates in vitro and are expected to be less toxic compared to h-asyn in vivo, although not all experimental studies unequivocally support the latter view. To address this outstanding issue, we directly compared the neurotoxicity of human asyn against that of h-asyn(D70-83), h-bsyn as well as rat asyn using an adeno-associated viral vector to express these proteins in a dose-response study where the vector load was varied over two orders of magnitude. By quantifying the neurodegeneration of rat substantia nigra dopamine neurons here we show that h-asyn, h-bsyn, and h-asyn(D70-83) display comparable neurotoxicity across the vector doses tested. On the other hand, rat asyn and GFP control vectors displayed a different profile, where no detectable neurodegeneration was seen except at the highest vector titer. Thus, the two main conclusions of our study are that (i) deletion of the central hydrophobic region in h-asyn is not sufficient to alter its neurotoxic properties and (ii) expression of the widely used GFP control protein can cause measurable neurodegeneration at high titers.

Assessment of brain metabolite correlates of adeno-associated virus-mediated over-expression of human alpha-synuclein in cortical neurons by in vivo (1) H-MR spectroscopy at 9.4 T.

In this study, we used proton-localized spectroscopy ((1) H-MRS) for the acquisition of the neurochemical profile longitudinally in a novel rat model of human wild-type alpha-synuclein (α-syn) over-expression. Our goal was to find out if the increased α-syn load in this model could be linked to changes in metabolites in the frontal cortex. Animals injected with AAV vectors encoding for human α-syn formed the experimental group, whereas green fluorescent protein expressing animals were used as the vector-treated control group and a third group of uninjected animals were used as naïve controls. Data were acquired at 2, 4, and 8 month time points. Nineteen metabolites were quantified in the MR spectra using LCModel software. On the basis of 92 spectra, we evaluated any potential gender effect and found that lactate (Lac) levels were lower in males compared to females, while the opposite was observed for ascorbate (Asc). Next, we assessed the effect of age and found increased levels of GABA, Tau, and GPC+PCho. Finally, we analyzed the effect of treatment and found that Lac levels (p = 0.005) were specifically lower in the α-syn group compared to the green fluorescent protein and control groups. In addition, Asc levels (p = 0.05) were increased in the vector-injected groups, whereas glucose levels remained unchanged. This study indicates that the metabolic switch between glucose-lactate could be detectable in vivo and might be modulated by Asc. No concomitant changes were found in markers of neuronal integrity (e.g., N-acetylaspartate) consistent with the fact that α-syn over-expression in cortical neurons did not result in neurodegeneration in this model. We acquired the neurochemical profile longitudinally in a rat model of human wild-type alpha-synuclein (α-syn) over-expression in cortical neurons. We found that Lactate levels were reduced in the α-syn group compared to the control groups and Ascorbate levels were increased in the vector-injected groups. No changes were found in markers of neuronal integrity consistent with the fact that α-syn over-expression did not result in frank neurodegeneration.

Ser129 phosphorylation of endogenous α-synuclein induced by overexpression of polo-like kinases 2 and 3 in nigral dopamine neurons is not detrimental to their survival and function.

Phosphorylation of the α-synuclein (α-syn) protein at Ser129 [P(S129)-α-syn] was found to be the most abundant form in intracellular inclusions in brains from Parkinson's disease (PD) patients. This finding suggests that P(S129)-α-syn plays a central role in the pathogenesis of PD. However, it is at present unclear whether P(S129)-α-syn is pathogenic driving the neurodegenerative process. Rodent studies using neither the phosphomimics of human α-syn nor co-expression of human wild-type α-syn and kinases phosphorylating α-syn at Ser129 gave consistent results. One major concern in interpreting these findings is that human α-syn was expressed above physiological levels inducing neurodegeneration in rat nigral neurons. In order to exclude this confounding factor, we took a different approach and increased the phosphorylation level of endogenous α-syn. For this purpose, we took advantage of recombinant adeno-associated viral (rAAV) vectors to deliver polo-like kinase (PLK) 2 or PLK3 in the substantia nigra and investigated whether increased levels of P(S129)-α-syn compromised the function and survival of nigral dopaminergic neurons. Interestingly, we observed that hyperphosphorylated α-syn did not induce nigral dopaminergic cell death, as assessed at 1 and 4months. Furthermore, histological analysis did not show any accumulation of α-syn protein or formation of inclusions. Using in vivo microdialysis, we found that the only measurable functional alteration was the depolarisation-induced release of dopamine, while the in vivo synthesis rate of DOPA and dopamine baseline release remained unaltered. Taken together, our results suggest that phosphorylation of α-syn at Ser129 does not confer a toxic gain of function per se.

Hippocampal Lewy pathology and cholinergic dysfunction are associated with dementia in Parkinson's disease.

The neuropathological substrate of dementia in patients with Parkinson's disease is still under debate, particularly in patients with insufficient alternate neuropathology for other degenerative dementias. In patients with pure Lewy body Parkinson's disease, previous post-mortem studies have shown that dopaminergic and cholinergic regulatory projection systems degenerate, but the exact pathways that may explain the development of dementia in patients with Parkinson's disease remain unclear. Studies in rodents suggest that both the mesocorticolimbic dopaminergic and septohippocampal cholinergic pathways may functionally interact to regulate certain aspects of cognition, however, whether such an interaction occurs in humans is still poorly understood. In this study, we performed stereological analyses of the A9 and A10 dopaminergic neurons and Ch1, Ch2 and Ch4 cholinergic neurons located in the basal forebrain, along with an assessment of α-synuclein pathology in these regions and in the hippocampus of six demented and five non-demented patients with Parkinson's disease and five age-matched control individuals with no signs of neurological disease. Moreover, we measured choline acetyltransferase activity in the hippocampus and frontal cortex of eight demented and eight non-demented patients with Parkinson's disease, as well as in the same areas of eight age-matched controls. All patients with Parkinson's disease exhibited a similar 80-85% loss of pigmented A9 dopaminergic neurons, whereas patients with Parkinson's disease dementia presented an additional loss in the lateral part of A10 dopaminergic neurons as well as Ch4 nucleus basalis neurons. In contrast, medial A10 dopaminergic neurons and Ch1 and Ch2 cholinergic septal neurons were largely spared. Despite variable Ch4 cell loss, cortical but not hippocampal cholinergic activity was consistently reduced in all patients with Parkinson's disease, suggesting significant dysfunction in cortical cholinergic pathways before frank neuronal degeneration. Patients with Parkinson's disease dementia were differentiated by a significant reduction in hippocampal cholinergic activity, by a significant loss of non-pigmented lateral A10 dopaminergic neurons and Ch4 cholinergic neurons (30 and 55% cell loss, respectively, compared with neuronal preservation in control subjects), and by an increase in the severity of α-synuclein pathology in the basal forebrain and hippocampus. Overall, these results point to increasing α-synuclein deposition and hippocampal dysfunction in a setting of more widespread degeneration of cortical dopaminergic and cholinergic pathways as contributing to the dementia occurring in patients with pure Parkinson's disease. Furthermore, our findings support the concept that α-synuclein deposition is associated with significant neuronal dysfunction in the absence of frank neuronal loss in Parkinson's disease.

Boosting chaperone-mediated autophagy in vivo mitigates α-synuclein-induced neurodegeneration.

α-Synuclein levels are critical to Parkinson's disease pathogenesis. Wild-type α-synuclein is degraded partly by chaperone-mediated autophagy, and aberrant α-synuclein may act as an inhibitor of the pathway. To address whether the induction of chaperone-mediated autophagy may represent a potential therapy against α-synuclein-induced neurotoxicity, we overexpressed lysosomal-associated membrane protein 2a, the rate-limiting step of chaperone-mediated autophagy, in human neuroblastoma SH-SY5Y cells, rat primary cortical neurons in vitro, and nigral dopaminergic neurons in vivo. Overexpression of the lysosomal-associated membrane protein 2a in cellular systems led to upregulation of chaperone-mediated autophagy, decreased α-synuclein turnover, and selective protection against adenoviral-mediated wild-type α-synuclein neurotoxicity. Protection was observed even when the steady-state levels of α-synuclein were unchanged, suggesting that it occurred through the attenuation of α-synuclein-mediated dysfunction of chaperone-mediated autophagy. Overexpression of the lysosomal receptor through the nigral injection of recombinant adeno-associated virus vectors effectively ameliorated α-synuclein-induced dopaminergic neurodegeneration by increasing the survival of neurons located in the substantia nigra as well as the axon terminals located in the striatum, which was associated with a reduction in total α-synuclein levels and related aberrant species. We conclude that induction of chaperone-mediated autophagy may provide a novel therapeutic strategy in Parkinson's disease and related synucleinopathies through two different mechanisms: amelioration of dysfunction of chaperone-mediated autophagy and lowering of α-synuclein levels.

Specific Detection of Physiological S129 Phosphorylated α-Synuclein in Tissue Using Proximity Ligation Assay.

BACKGROUND: Synucleinopathies are a group of neurodegenerative disorders that are pathologically characterized by intracellular aggregates called Lewy bodies. Lewy bodies are primarily composed of α-synuclein (asyn) protein, which is mostly phosphorylated at serine 129 (pS129) when aggregated and therefore used as a marker for pathology. Currently commercial antibodies against pS129 asyn stain aggregates well but in healthy brains cross react with other proteins, thus making it difficult to specifically detect physiological pS129 asyn. OBJECTIVE: To develop a staining procedure that detects endogenous and physiological relevant pS129 asyn with high specificity and low background. METHODS: We used the fluorescent and brightfield in situ proximity ligation assay (PLA) to specifically detect pS129 asyn in cell culture, mouse, and human brain sections. RESULTS: The pS129 asyn PLA specifically stained physiological and soluble pS129 asyn in cell culture, mouse brain sections, and human brain tissue without significant cross-reactivity or background signal. However, this technique was not successful in detecting Lewy bodies in human brain tissue. CONCLUSION: We successfully developed a novel PLA method that can, in the future, be used on in vitro and in vivo samples as a tool to explore and better understand the cellular localization and function of pS129 asyn in health and disease.

Stereotaxic Intracranial Delivery of Chemicals, Proteins or Viral Vectors to Study Parkinson's Disease.

Parkinson's disease (PD) is a progressive disorder traditionally defined by resting tremor and akinesia, primarily due to loss of dopaminergic neurons in the substantia nigra. Affected brain areas display intraneuronal fibrillar inclusions consisting mainly of alpha-synuclein (asyn) proteins. No animal model thus far has recapitulated all characteristics of this disease. Here, we describe the use of stereotaxic injection to deliver chemicals, proteins, or viral vectors intracranially in order to mimic various aspects of PD. These methods are well-established and widely used throughout the PD field. Stereotaxic injections are incredibly flexible; they can be adjusted in concentration, age of animal used for injection, brain area targeted and in animal species used. Combinations of substances allow for rapid variations to assess treatments or alter severity of the pathology or behavioral deficits. By injecting toxins into the brain, we can mimic inflammation and/or a severe loss of dopaminergic neurons resulting in substantial motor phenotypes. Viral vectors can be used to transduce cells to mimic genetic or mechanistic aspects. Preformed fibrillar asyn injections best recapitulate the progressive phenotype over an extended period of time. Once these methods are established, it can be economical to generate a new model compared to creating a new transgenic line. However, this method is labor intensive as it requires 30 minutes to four hours per animal depending on the model used. Each animal will have a slightly different targeting and therefore create a diverse cohort which on one hand can be challenging to interpret results from; on the other hand, help mimic a more realistic diversity found in patients. Mistargeted animals can be identified using behavioral or imaging readouts, or only after being sacrificed leading to smallercohort size after the study has already been concluded. Overall, this method is a rudimentary but effective way to assess a diverse set of PD aspects.

LAG3 is not expressed in human and murine neurons and does not modulate α-synucleinopathies.

While the initial pathology of Parkinson's disease and other α-synucleinopathies is often confined to circumscribed brain regions, it can spread and progressively affect adjacent and distant brain locales. This process may be controlled by cellular receptors of α-synuclein fibrils, one of which was proposed to be the LAG3 immune checkpoint molecule. Here, we analysed the expression pattern of LAG3 in human and mouse brains. Using a variety of methods and model systems, we found no evidence for LAG3 expression by neurons. While we confirmed that LAG3 interacts with α-synuclein fibrils, the specificity of this interaction appears limited. Moreover, overexpression of LAG3 in cultured human neural cells did not cause any worsening of α-synuclein pathology ex vivo. The overall survival of A53T α-synuclein transgenic mice was unaffected by LAG3 depletion, and the seeded induction of α-synuclein lesions in hippocampal slice cultures was unaffected by LAG3 knockout. These data suggest that the proposed role of LAG3 in the spreading of α-synucleinopathies is not universally valid.

LRRK2 mediates tubulation and vesicle sorting from lysosomes.

Genetic variation around the LRRK2 gene affects risk of both familial and sporadic Parkinson's disease (PD). However, the biological functions of LRRK2 remain incompletely understood. Here, we report that LRRK2 is recruited to lysosomes after exposure of cells to the lysosome membrane-rupturing agent LLOME. Using an unbiased proteomic screen, we identified the motor adaptor protein JIP4 as an LRRK2 partner at the lysosomal membrane. LRRK2 can recruit JIP4 to lysosomes in a kinase-dependent manner via the phosphorylation of RAB35 and RAB10. Using super-resolution live-cell imaging microscopy and FIB-SEM, we demonstrate that JIP4 promotes the formation of LAMP1-negative tubules that release membranous content from lysosomes. Thus, we describe a new process orchestrated by LRRK2, which we name LYTL (LYsosomal Tubulation/sorting driven by LRRK2), by which lysosomal tubulation is used to release vesicles from lysosomes. Given the central role of the lysosome in PD, LYTL is likely to be disease relevant.

Two C-terminal sequence variations determine differential neurotoxicity between human and mouse α-synuclein.

BACKGROUND: α-Synuclein (aSyn) aggregation is thought to play a central role in neurodegenerative disorders termed synucleinopathies, including Parkinson's disease (PD). Mouse aSyn contains a threonine residue at position 53 that mimics the human familial PD substitution A53T, yet in contrast to A53T patients, mice show no evidence of aSyn neuropathology even after aging. Here, we studied the neurotoxicity of human A53T, mouse aSyn, and various human-mouse chimeras in cellular and in vivo models, as well as their biochemical properties relevant to aSyn pathobiology. METHODS: Primary midbrain cultures transduced with aSyn-encoding adenoviruses were analyzed immunocytochemically to determine relative dopaminergic neuron viability. Brain sections prepared from rats injected intranigrally with aSyn-encoding adeno-associated viruses were analyzed immunohistochemically to determine nigral dopaminergic neuron viability and striatal dopaminergic terminal density. Recombinant aSyn variants were characterized in terms of fibrillization rates by measuring thioflavin T fluorescence, fibril morphologies via electron microscopy and atomic force microscopy, and protein-lipid interactions by monitoring membrane-induced aSyn aggregation and aSyn-mediated vesicle disruption. Statistical tests consisted of ANOVA followed by Tukey's multiple comparisons post hoc test and the Kruskal-Wallis test followed by a Dunn's multiple comparisons test or a two-tailed Mann-Whitney test. RESULTS: Mouse aSyn was less neurotoxic than human aSyn A53T in cell culture and in rat midbrain, and data obtained for the chimeric variants indicated that the human-to-mouse substitutions D121G and N122S were at least partially responsible for this decrease in neurotoxicity. Human aSyn A53T and a chimeric variant with the human residues D and N at positions 121 and 122 (respectively) showed a greater propensity to undergo membrane-induced aggregation and to elicit vesicle disruption. Differences in neurotoxicity among the human, mouse, and chimeric aSyn variants correlated weakly with differences in fibrillization rate or fibril morphology. CONCLUSIONS: Mouse aSyn is less neurotoxic than the human A53T variant as a result of inhibitory effects of two C-terminal amino acid substitutions on membrane-induced aSyn aggregation and aSyn-mediated vesicle permeabilization. Our findings highlight the importance of membrane-induced self-assembly in aSyn neurotoxicity and suggest that inhibiting this process by targeting the C-terminal domain could slow neurodegeneration in PD and other synucleinopathy disorders.

Longitudinal monoaminergic PET imaging of chronic proteasome inhibition in minipigs.

Impairment of the ubiquitin proteasome system has been implicated in Parkinson's disease. We used positron emission tomography to investigate longitudinal effects of chronic intracerebroventricular exposure to the proteasome inhibitor lactacystin on monoaminergic projections and neuroinflammation. Göttingen minipigs were implanted in the cisterna magna with a catheter connected to a subcutaneous injection port. Minipigs were imaged at baseline and after cumulative doses of 200 and 400 µg lactacystin, respectively. Main radioligands included [11C]-DTBZ (vesicular monoamine transporter type 2) and [11C]-yohimbine (α2-adrenoceptor). [11C]-DASB (serotonin transporter) and [11C]-PK11195 (activated microglia) became available later in the study and we present their results in a smaller subset of animals for information purposes only. Striatal [11C]-DTBZ binding potentials decreased significantly by 16% after 200 µg compared to baseline, but the decrease was not sustained after 400 µg (n = 6). [11C]-yohimbine volume of distribution increased by 18-25% in the pons, grey matter and the thalamus after 200 µg, which persisted at 400 µg (n = 6). In the later subset of minipigs, we observed decreased [11C]-DASB (n = 5) and increased [11C]-PK11195 (n = 3) uptake after 200 µg. These changes may mimic monoaminergic changes and compensatory responses in early Parkinson's disease.

A novel multiplex assay for simultaneous quantification of total and S129 phosphorylated human alpha-synuclein.

BACKGROUND: Alpha-synuclein (asyn) has been shown to play an important role in the neuropathology of Parkinson's disease (PD). In the diseased brain, classic intraneuronal inclusions called Lewy bodies contain abnormal formations of asyn protein which is mostly phosphorylated at serine 129 (pS129 asyn). This suggests that post-translational modifications may play a role in the pathogenic process. To date, several uniplex assays have been developed in order to quantify asyn not only in the brain but also in cerebrospinal fluid and blood samples in order to correlate asyn levels to disease severity and progression. Notably, only four assays have been established to measure pS129 asyn specifically and none provide simultaneous readout of the total and pS129 species. Therefore, we developed a sensitive high-throughput duplex assay quantifying total and pS129 human asyn (h-asyn) in the same well hence improving accuracy as well as saving time, consumables and samples. RESULTS: Using our newly established duplex assay we measured total and pS129 h-asyn in vitro showing that polo-like kinase 2 (PLK2) can phosphorylate asyn up to 41 % in HEK293 cells and in vivo the same kinase phosphorylated h-asyn up to 17 % in rat ventral midbrain neurons. Interestingly, no increase in phosphorylation was observed when PLK2 and h-asyn were co-expressed in rat striatal neurons. Furthermore, using this assay we investigated h-asyn levels in brain tissue samples from patients with PD as well as PD dementia and found significant differences in pS129 h-asyn levels not only between disease tissue and healthy control samples but also between the two distinct disease states especially in hippocampal tissue samples. CONCLUSIONS: These results demonstrate that our duplex assay for simultaneous quantification is a useful tool to study h-asyn phosphorylation events in biospecimens and will be helpful in studies investigating the precise causative link between post-translational modification of h-asyn and PD pathology.

Characterization of cognitive deficits in rats overexpressing human alpha-synuclein in the ventral tegmental area and medial septum using recombinant adeno-associated viral vectors.

Intraneuronal inclusions containing alpha-synuclein (a-syn) constitute one of the pathological hallmarks of Parkinson's disease (PD) and are accompanied by severe neurodegeneration of A9 dopaminergic neurons located in the substantia nigra. Although to a lesser extent, A10 dopaminergic neurons are also affected. Neurodegeneration of other neuronal populations, such as the cholinergic, serotonergic and noradrenergic cell groups, has also been documented in PD patients. Studies in human post-mortem PD brains and in rodent models suggest that deficits in cholinergic and dopaminergic systems may be associated with the cognitive impairment seen in this disease. Here, we investigated the consequences of targeted overexpression of a-syn in the mesocorticolimbic dopaminergic and septohippocampal cholinergic pathways. Rats were injected with recombinant adeno-associated viral vectors encoding for either human wild-type a-syn or green fluorescent protein (GFP) in the ventral tegmental area and the medial septum/vertical limb of the diagonal band of Broca, two regions rich in dopaminergic and cholinergic neurons, respectively. Histopathological analysis showed widespread insoluble a-syn positive inclusions in all major projections areas of the targeted nuclei, including the hippocampus, neocortex, nucleus accumbens and anteromedial striatum. In addition, the rats overexpressing human a-syn displayed an abnormal locomotor response to apomorphine injection and exhibited spatial learning and memory deficits in the Morris water maze task, in the absence of obvious spontaneous locomotor impairment. As losses in dopaminergic and cholinergic immunoreactivity in both the GFP and a-syn expressing animals were mild-to-moderate and did not differ from each other, the behavioral impairments seen in the a-syn overexpressing animals appear to be determined by the long term persisting neuropathology in the surviving neurons rather than by neurodegeneration.

Rapid, reproducible transduction of select forebrain regions by targeted recombinant virus injection into the neonatal mouse brain.

Viral vectors can mediate long-term gene expression in different regions of the brain. Recombinant adeno-associated virus (rAAV) and Lenti virus (LV) have both gained prominence due to their ability to achieve specific transduction of various neuronal populations. Whilst widespread gene delivery has been obtained by targeted injection of rAAV in various brain structures, LV has also been utilized for infection of stem cell populations for cell lineage tracing. Both viral vector systems are most commonly used for gene delivery in mature brains, but the great potential of somatic gene delivery into the neonate brain has not been systematically exploited. Here we provide a systematic guideline for efficient stereotaxic virus delivery into different neuronal populations of the neonate brain. We demonstrate region specific recombination of a 'stop-floxed' Rosa26 reporter allele upon targeted injection of rAAV vectors expressing Cre-recombinase at postnatal day zero (P0). In addition, utilizing LV, we show efficient transduction of P0 subventricular zone stem cells with subsequent labeling of approximately 20% of migrating neuroblasts along the rostral migratory stream (RMS) into the olfactory bulb. In summary, we report on an optimized protocol for facile, reproducible, high-throughput virus-based gene transfer into neonatal brains of wild-type and genetically altered mice, which allows targeted transduction of different brain regions and distinct neuronal populations.