Fluid and tissue biomarkers in Parkinson's disease: Immunodetection or seed amplification? Central or peripheral?

Over the last two decades there have been meaningful developments on biomarkers of neurodegenerative diseases, extensively (but not solely) focusing on their proteinopathic nature. Accordingly, in Alzheimer's disease determination of levels of total and phosphorylated tau (τ and p-τ, usually p-τ181) along with amyloid-beta1-42 (Aß1-42) by immunodetection in cerebrospinal fluid (CSF) and currently even in peripheral blood, have been widely accepted and introduced to routine diagnosis. In the case of Parkinson's disease, α-synuclein as a potential biomarker (both for diagnosis and progression tracking) has proved more elusive under the immunodetection approach. In recent years, the emergence of the so-called seed amplification assays is proving to be a game-changer, with mounting evidence under different technical approaches and using a variety of biofluids or tissues, yielding promising diagnostic accuracies. Currently the least invasive but at once more reliable source of biosamples and techniques are being sought. Here we overview these advances.

Altered sleep and neurovascular dysfunction in alpha-synucleinopathies: the perfect storm for glymphatic failure.

Clinical and cognitive progression in alpha-synucleinopathies is highly heterogeneous. While some patients remain stable over long periods of time, other suffer early dementia or fast motor deterioration. Sleep disturbances and nocturnal blood pressure abnormalities have been identified as independent risk factors for clinical progression but a mechanistic explanation linking both aspects is lacking. We hypothesize that impaired glymphatic system might play a key role on clinical progression. Glymphatic system clears brain waste during specific sleep stages, being blood pressure the motive force that propels the interstitial fluid through brain tissue to remove protein waste. Thus, the combination of severe sleep alterations, such as REM sleep behavioral disorder, and lack of the physiological nocturnal decrease of blood pressure due to severe dysautonomia may constitute the perfect storm for glymphatic failure, causing increased abnormal protein aggregation and spreading. In Lewy body disorders (Parkinson's disease and dementia with Lewy bodies) the increment of intraneuronal alpha-synuclein and extracellular amyloid-ß would lead to cognitive deterioration, while in multisystemic atrophy, increased pathology in oligodendroglia would relate to the faster and malignant motor progression. We present a research model that may help in developing studies aiming to elucidate the role of glymphatic function and associated factors mainly in alpha-synucleinopathies, but that could be relevant also for other protein accumulation-related neurodegenerative diseases. If the model is proven to be useful could open new lines for treatments targeting glymphatic function (for example through control of nocturnal blood pressure) with the objective to ameliorate cognitive and motor progression in alpha-synucleinopathies.

TDP-43 and ER Stress in Neurodegeneration: Friends or Foes?

Nuclear depletion, abnormal modification, and cytoplasmic aggregation of TAR DNA-binding protein 43 (TDP-43) are linked to a group of fatal neurodegenerative diseases called TDP-43 proteinopathies, which include amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Although our understanding of the physiological function of TDP-43 is rapidly advancing, the molecular mechanisms associated with its pathogenesis remain poorly understood. Accumulating evidence suggests that endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) are important players in TDP-43 pathology. However, while neurons derived from autopsied ALS and FTLD patients revealed TDP-43 deposits in the ER and displayed UPR activation, data originated from in vitro and in vivo TDP-43 models produced contradictory results. In this review, we will explore the complex interplay between TDP-43 pathology, ER stress, and the UPR by breaking down the evidence available in the literature and addressing the reasons behind these discrepancies. We also highlight underexplored areas and key unanswered questions in the field. A better synchronization and integration of methodologies, models, and mechanistic pathways will be crucial to discover the true nature of the TDP-43 and ER stress relationship and, ultimately, to uncover the full therapeutic potential of the UPR.

Aß40 displays amyloidogenic properties in the non-transgenic mouse brain but does not exacerbate Aß42 toxicity in Drosophila.

BACKGROUND: Self-assembly of the amyloid-ß (Aß) peptide into aggregates, from small oligomers to amyloid fibrils, is fundamentally linked with Alzheimer's disease (AD). However, it is clear that not all forms of Aß are equally harmful and that linking a specific aggregate to toxicity also depends on the assays and model systems used (Haass et al., J Biol. Chem 269:17741-17748, 1994; Borchelt et al., Neuron 17:1005-1013, 1996). Though a central postulate of the amyloid cascade hypothesis, there remain many gaps in our understanding regarding the links between Aß deposition and neurodegeneration. METHODS: In this study, we examined familial mutations of Aß that increase aggregation and oligomerization, E22G and ΔE22, and induce cerebral amyloid angiopathy, E22Q and D23N. We also investigated synthetic mutations that stabilize dimerization, S26C, and a phospho-mimetic, S8E, and non-phospho-mimetic, S8A. To that end, we utilized BRI2-Aß fusion technology and rAAV2/1-based somatic brain transgenesis in mice to selectively express individual mutant Aß species in vivo. In parallel, we generated PhiC31-based transgenic Drosophila melanogaster expressing wild-type (WT) and Aß40 and Aß42 mutants, fused to the Argos signal peptide to assess the extent of Aß42-induced toxicity as well as to interrogate the combined effect of different Aß40 and Aß42 species. RESULTS: When expressed in the mouse brain for 6 months, Aß42 E22G, Aß42 E22Q/D23N, and Aß42WT formed amyloid aggregates consisting of some diffuse material as well as cored plaques, whereas other mutants formed predominantly diffuse amyloid deposits. Moreover, while Aß40WT showed no distinctive phenotype, Aß40 E22G and E22Q/D23N formed unique aggregates that accumulated in mouse brains. This is the first evidence that mutant Aß40 overexpression leads to deposition under certain conditions. Interestingly, we found that mutant Aß42 E22G, E22Q, and S26C, but not Aß40, were toxic to the eye of Drosophila. In contrast, flies expressing a copy of Aß40 (WT or mutants), in addition to Aß42WT, showed improved phenotypes, suggesting possible protective qualities for Aß40. CONCLUSIONS: These studies suggest that while some Aß40 mutants form unique amyloid aggregates in mouse brains, they do not exacerbate Aß42 toxicity in Drosophila, which highlights the significance of using different systems for a better understanding of AD pathogenicity and more accurate screening for new potential therapies.

PhotoGal4: A Versatile Light-Dependent Switch for Spatiotemporal Control of Gene Expression in Drosophila Explants.

We present here PhotoGal4, a phytochrome B-based optogenetic switch for fine-tuned spatiotemporal control of gene expression in Drosophila explants. This switch integrates the light-dependent interaction between phytochrome B and PIF6 from plants with regulatory elements from the yeast Gal4/UAS system. We found that PhotoGal4 efficiently activates and deactivates gene expression upon red- or far-red-light irradiation, respectively. In addition, this optogenetic tool reacts to different illumination conditions, allowing for fine modulation of the light-dependent response. Importantly, by simply focusing a laser beam, PhotoGal4 induces intricate patterns of expression in a customized manner. For instance, we successfully sketched personalized patterns of GFP fluorescence such as emoji-like shapes or letterform logos in Drosophila explants, which illustrates the exquisite precision and versatility of this tool. Hence, we anticipate that PhotoGal4 will expand the powerful Drosophila toolbox and will provide a new avenue to investigate intricate and complex problems in biomedical research.

Bringing Light to Transcription: The Optogenetics Repertoire.

The ability to manipulate expression of exogenous genes in particular regions of living organisms has profoundly transformed the way we study biomolecular processes involved in both normal development and disease. Unfortunately, most of the classical inducible systems lack fine spatial and temporal accuracy, thereby limiting the study of molecular events that strongly depend on time, duration of activation, or cellular localization. By exploiting genetically engineered photo sensing proteins that respond to specific wavelengths, we can now provide acute control of numerous molecular activities with unprecedented precision. In this review, we present a comprehensive breakdown of all of the current optogenetic systems adapted to regulate gene expression in both unicellular and multicellular organisms. We focus on the advantages and disadvantages of these different tools and discuss current and future challenges in the successful translation to more complex organisms.

Short Aß peptides attenuate Aß42 toxicity in vivo.

Processing of amyloid-ß (Aß) precursor protein (APP) by γ-secretase produces multiple species of Aß: Aß40, short Aß peptides (Aß37-39), and longer Aß peptides (Aß42-43). γ-Secretase modulators, a class of Alzheimer's disease therapeutics, reduce production of the pathogenic Aß42 but increase the relative abundance of short Aß peptides. To evaluate the pathological relevance of these peptides, we expressed Aß36-40 and Aß42-43 in Drosophila melanogaster to evaluate inherent toxicity and potential modulatory effects on Aß42 toxicity. In contrast to Aß42, the short Aß peptides were not toxic and, when coexpressed with Aß42, were protective in a dose-dependent fashion. In parallel, we explored the effects of recombinant adeno-associated virus-mediated expression of Aß38 and Aß40 in mice. When expressed in nontransgenic mice at levels sufficient to drive Aß42 deposition, Aß38 and Aß40 did not deposit or cause behavioral alterations. These studies indicate that treatments that lower Aß42 by raising the levels of short Aß peptides could attenuate the toxic effects of Aß42.

secHsp70 as a tool to approach amyloid-ß42 and other extracellular amyloids.

Self-association of amyloidogenic proteins is the main pathological trigger in a wide variety of neurodegenerative disorders. These aggregates are deposited inside or outside the cell due to hereditary mutations, environmental exposures or even normal aging. Cumulative evidence indicates that the heat shock chaperone Hsp70 possesses robust neuroprotection against various intracellular amyloids in Drosophila and mouse models. However, its protective role against extracellular amyloids was largely unknown as its presence outside the cells is very limited. Our recent manuscript in PNAS revealed that an engineered form of secreted Hsp70 (secHsp70) is highly protective against toxicity induced by extracellular deposition of the amyloid-ß42 (Aß42) peptide. In this Extra View article, we extend our analysis to other members of the heat shock protein family. We created PhiC31-based transgenic lines for human Hsp27, Hsp40, Hsp60 and Hsp70 and compared their activities in parallel against extracellular Aß42. Strikingly, only secreted Hsp70 exhibits robust protection against Aß42-triggered toxicity in the extracellular milieu. These observations indicate that the ability of secHsp70 to suppress Aß42 insults is quite unique and suggest that targeted secretion of Hsp70 may represent a new therapeutic approach against Aß42 and other extracellular amyloids. The potential applications of this engineered chaperone are discussed.

Holdase activity of secreted Hsp70 masks amyloid-ß42 neurotoxicity in Drosophila.

Alzheimer's disease (AD) is the most prevalent of a large group of related proteinopathies for which there is currently no cure. Here, we used Drosophila to explore a strategy to block Aß42 neurotoxicity through engineering of the Heat shock protein 70 (Hsp70), a chaperone that has demonstrated neuroprotective activity against several intracellular amyloids. To target its protective activity against extracellular Aß42, we added a signal peptide to Hsp70. This secreted form of Hsp70 (secHsp70) suppresses Aß42 neurotoxicity in adult eyes, reduces cell death, protects the structural integrity of adult neurons, alleviates locomotor dysfunction, and extends lifespan. SecHsp70 binding to Aß42 through its holdase domain is neuroprotective, but its ATPase activity is not required in the extracellular space. Thus, the holdase activity of secHsp70 masks Aß42 neurotoxicity by promoting the accumulation of nontoxic aggregates. Combined with other approaches, this strategy may contribute to reduce the burden of AD and other extracellular proteinopathies.

Anti-Aß single-chain variable fragment antibodies exert synergistic neuroprotective activities in Drosophila models of Alzheimer's disease.

Both active and passive immunotherapy protocols decrease insoluble amyloid-ß42 (Aß42) peptide in animal models, suggesting potential therapeutic applications against the main pathological trigger in Alzheimer's disease (AD). However, recent clinical trials have reported no significant benefits from humanized anti-Aß42 antibodies. Engineered single-chain variable fragment antibodies (scFv) are much smaller and can easily penetrate the brain, but identifying the most effective scFvs in murine AD models is slow and costly. We show here that scFvs against the N- and C-terminus of Aß42 (scFv9 and scFV42.2, respectively) that decrease insoluble Aß42 in CRND mice are neuroprotective in Drosophila models of Aß42 and amyloid precursor protein neurotoxicity. Both scFv9 and scFv42.2 suppress eye toxicity, reduce cell death in brain neurons, protect the structural integrity of dendritic terminals in brain neurons and delay locomotor dysfunction. Additionally, we show for the first time that co-expression of both anti-Aß scFvs display synergistic neuroprotective activities, suggesting that combined therapies targeting distinct Aß42 epitopes can be more effective than targeting a single epitope. Overall, we demonstrate the feasibility of using Drosophila as a first step for characterizing neuroprotective anti-Aß scFvs in vivo and identifying scFv combinations with synergistic neuroprotective activities.

Modeling the complex pathology of Alzheimer's disease in Drosophila.

Alzheimer's disease (AD) is the leading cause of dementia and the most common neurodegenerative disorder. AD is mostly a sporadic disorder and its main risk factor is age, but mutations in three genes that promote the accumulation of the amyloid-ß (Aß42) peptide revealed the critical role of amyloid precursor protein (APP) processing in AD. Neurofibrillary tangles enriched in tau are the other pathological hallmark of AD, but the lack of causative tau mutations still puzzles researchers. Here, we describe the contribution of a powerful invertebrate model, the fruit fly Drosophila melanogaster, to uncover the function and pathogenesis of human APP, Aß42, and tau. APP and tau participate in many complex cellular processes, although their main function is microtubule stabilization and the to-and-fro transport of axonal vesicles. Additionally, expression of secreted Aß42 induces prominent neuronal death in Drosophila, a critical feature of AD, making this model a popular choice for identifying intrinsic and extrinsic factors mediating Aß42 neurotoxicity. Overall, Drosophila has made significant contributions to better understand the complex pathology of AD, although additional insight can be expected from combining multiple transgenes, performing genome-wide loss-of-function screens, and testing anti-tau therapies alone or in combination with Aß42.

Alpha-synuclein transcript isoforms in three different brain regions from Parkinson's disease and healthy subjects in relation to the SNCA rs356165/rs11931074 polymorphisms.

Mutations in the alpha-synuclein (SNCA) gene cause autosomal dominant Parkinson's disease (PD). Common SNCA polymorphisms have been associated with the risk of developing PD. Abnormal expression and post-translational modification of SNCA has been found in PD-brains. In addition to a full length transcript (SNCA-140) there are three short isoforms (SNCA-98, -112, and -126) that could be prone to aggregation. The association between SNCA polymorphisms and PD could be explained through an increased expression of these alternative transcripts. Our aim was to measure the different SNCA transcripts in the substantia nigra (SN), cerebellum (CB), and occipital cortex (OC) from PD-patients (n=9) and healthy subjects (n=6). In addition, we determined whether two SNCA polymorphisms (SNPs rs356165 and rs11931074) were related to differences in transcript isoform expression. PD brain tissues showed higher levels of the three short transcripts in the SN, but only SNCA-112 and SNCA-98 were significantly increased in the CB of patients vs. controls (p=0.02, p=0.03). The genotyping of a large cohort of PD-patients and controls showed that haplotype rs356165-A+rs11931074-G had a protective effect (OR=0.71; CI=0.59-0.83), while the G-T haplotype increased the risk for PD (OR=1.44; CI=1.06-1.96). We did not find significant differences for the SNCA levels between the haplotypes. In conclusion, we found statistically significant higher levels of the SNCA-112 and SNCA-98 transcripts in the CB of PD brains, and a trend toward higher levels of the short transcript isoforms in the SN of PD brains.

Mutational screening of PARKIN identified a 3' UTR variant (rs62637702) associated with Parkinson's disease.

PRKN mutations have been linked to Parkinson's disease (PD). Most of the mutational screenings have focused on the coding exons. The 3' untranslated region (UTR) could also harbor functionally relevant nucleotide changes. We performed a mutational screening of PRKN in a cohort of early-onset PD patients (n = 235) from Spain. We found 16 mutations (five new): 16 patients (7 %) carried two mutations and only one mutation was found in 28 (12 %). Patients with two mutations had significantly lower mean age (30 ± 9 years) compared to patients with one (40 ± 7) or no mutation (42 ± 7). We found a total of 15 nucleotide variants (three new) in the 3' UTR region. The frequency of carriers of the rare rs62637702 G allele (*94A/G) was significantly lower among the patients compared to healthy controls (n = 418) (0.03 vs. 0.004; p < 0.001), suggesting a protective role for this allele. In order to investigate the basal effect of this variant, we performed luciferase assays. No different basal activity was observed between the two alleles. In conclusion, the rs62637702 polymorphism was associated with PD. This could be a surrogate marker for disease risk, in linkage disequilibrium with other non-identified functional variant.

A search for SNCA 3' UTR variants identified SNP rs356165 as a determinant of disease risk and onset age in Parkinson's disease.

Alpha-synuclein gene (SNCA) polymorphisms have been associated with the common sporadic form of Parkinson's disease (PD). We searched for DNA variants at the SNCA 3' UTR through single strand conformation analysis and direct sequencing in a cohort of Spanish PD patients and controls. We have genotyped the rs356165 SNCA 3' UTR polymorphism in a total of 1,135 PD patients and 772 healthy controls from two Spanish cohorts (Asturias and Navarre). We identified six SNCA 3' UTR variants. Single nucleotide polymorphism (SNP) rs356165 was significantly associated with PD risk in the Spanish cohort (p = 0.0001; odd ratio = 1.37, 95%CI = 1.19-1.58). This SNP was also significantly associated with early age at onset of PD. Our work highlights rs356165 as an important determinant of the risk of developing PD and early age at onset and encourages future research to identify a functional effect on SNCA expression.

Replication of MAPT and SNCA, but not PARK16-18, as susceptibility genes for Parkinson's disease.

Recent genome-wide association studies of Parkinson's disease have nominated 3 new susceptibility loci (PARK16-18) and confirmed 2 known risk genes (MAPT and SNCA) in populations of European ancestry. We sought to replicate these findings. We genotyped single-nucleotide polymorphisms in each of these genes/loci in 1445 Parkinson's disease patients and 1161 controls from northern Spain. Logistic regression was used to test for association between genotype and Parkinson's disease under an additive model, adjusting for sex, age, and site. We also performed analyses stratified by age at onset. Single-nucleotide polymorphisms in MAPT (rs1800547; P = 3.1 × 10(-4) ) and SNCA (rs356219; P = 5.5 × 10(-4) ) were significantly associated with Parkinson's disease. However, none of the markers in PARK16-18 associated with Parkinson's disease in the overall sample, or in any age stratum, with P values ranging from .09 to .88. Although our data further validate MAPT and SNCA as Parkinson's disease susceptibility genes, we failed to replicate PARK16, PARK17, and PARK18. Potential reasons for the discordance between our study and previous genome-wide association studies include effects of population structure, power, and population-specific environmental interactions. Our findings suggest that additional studies of PARK16-18 are necessary to establish the role of these loci in modifying risk for Parkinson's disease in European-derived populations. © 2011 Movement Disorder Society.

FGF20 rs12720208 SNP and microRNA-433 variation: no association with Parkinson's disease in Spanish patients.

DNA variation at the FGF20 gene has been associated with Parkinson's disease (PD). In particular, SNP rs12720208 in the 3' untranslated region (3' UTR) was linked to PD-risk through a mechanism that would implicate a differential binding to microRNA-433 (miR-433). The reduction of the affinity of miR-433 to the 3' UTR would result in increased FGF20 expression and upregulation of alpha-synuclein, which could in turn promote dopaminergic neurons degeneration. We genotyped the rs12720208 SNP in a total of 512 PD patients and 258 healthy controls from Spain, and searched for miR-433 variants in the patients. We did not find significant differences in allele and genotype frequencies between patients and controls. None of the patients had miR-433 variants. In conclusion, our work did not confirm the association between rs12720208 and PD, or an effect of miR-433 variants on this disease.

Analysis of the Micro-RNA-133 and PITX3 genes in Parkinson's disease.

MicroRNAs are small RNA sequences that negatively regulate gene expression by binding to the 3' untranslated regions of mRNAs. MiR-133b has been implicated in Parkinson's disease (PD) by a mechanism that involves the regulation of the transcription factor PITX3. The variation in these genes could contribute to the risk of developing PD. We searched for DNA variants in miR-133 and PITX3 genes in PD patients and healthy controls from Spain. We found common DNA variants in the three miR-133 genes. Genotyping of a first set of patients (n = 777) and controls (n = 650) showed a higher frequency of homozygous for a miR-133b variant (-90 del A) in PD-patients (6/575; 1%) than in healthy controls (0/650) (P = 0.03). However, this association was not confirmed in a second set of patients (1/250; 0.4%) and controls (2/210; 1%). No common PITX3 variants were associated with PD, although a rare missense change (G32S) was found in only one patient and none of the controls. In conclusion, we report the variation in genes of a pathway that has been involved in dopaminergic neuron differentiation and survival. Our work suggests that miR-133 and PITX3 gene variants did not contribute to the risk for PD.

Mutational screening of the mortalin gene (HSPA9) in Parkinson's disease.

Mortalin is a mitochondrial chaperone of the heat shock protein 70 family. Mortalin plays a central role in mitochondrial biogenesis through its capacity to direct the import of nuclear-encoded proteins into the mitochondria. As mitochondrial dysfunction has been involved in Parkinson's disease (PD), changes in mortalin function and expression could manifest as a higher risk of developing PD. In agreement with this, mortalin expression was decreased in the mitochondrial fraction of neurons from the substantia nigra of PD patients. We hypothesised that DNA variants in the mortalin gene (HSPA9) could contribute to the risk of developing PD. We analysed the 17 HSPA9 coding exons in 330 PD patients and 250 controls. In addition to several polymorphisms, found in patients and controls, three variants were found in 3 patients but none of the controls: two missense (R126 > W and P509 > S) and a 17 bp insertion in intron 8 (predicted to affect RNA splicing). Our study suggests that putative mutations in the mortalin, although rare, could contribute to the risk of developing PD.