After an initial lag phase of 7 hours, there is a steady increase of the average intensity recorded. The concentration of protein required to detect the aggregation using the label-free method is a factor 50 smaller than that used for the fluorescent method presented here. In addition, no lag phase was observed during the cantilever measurements, whereas there was a seven hour lag phase observed in the fluorescence measurements.
However, if the aim is to determine the binding kinetics of the aggregation then such a stop flow situation could lead to incorrect conclusions as the rate that is obtained can heavily depend on the diffusion rate of the protein towards the surface of the cantilever within the fluidic chamber.
The sensitivity of a microcantilever for mass sensing allows detection of a very small mass of protein from the liquid flowed through the chamber the current sensitivity of our device lies in the subnanogram regime in liquids.
This can be advantageous when working with particularly expensive molecules or when the aim of the experiment is to detect molecules which are in very low concentrations in a natural, physiological environment.
This represents an improvement over other measurements of protein aggregation using microcantilevers reported in the literature [ 35 ]. As discussed in the Introduction, the static method is not suitable for determining the kinetics of the aggregation process. Further work will focus on determining the aggregation rates on the surface of the cantilever for a range of solution conditions and protein concentrations. This is an open access article distributed under the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Article of the Year Award: Outstanding research contributions of , as selected by our Chief Editors. Read the winning articles. Journal overview. Special Issues. Academic Editor: Maria Tenje.
Received 15 Jun Accepted 15 Aug Published 19 Oct Abstract Early detection of protein aggregation is of great importance in the field of neurodegenerative diseases. Materials and Methods 2.
Cantilever Measurements 2. Preparation of Cantilever Array Microcantilevers can respond to virtually any stimulus as is shown by the wide range of sensing applications that they have been applied to.
Figure 1. Schematic showing the functionalisation of the cantilever array. The reference cantilever is coated with a self-assembled OH monolayer. All remaining binding sites are blocked with BSA. Figure 2. Schematic of the experimental device and measurement procedure. A LabVIEW program controls a frequency generator which sends a sinusoidal frequency signal to the piezo actuator. The cantilever is swept through a range of frequencies, and the response of the cantilever is detected using optical beam deflection.
The peaks of the frequency spectrum correspond to the flexural resonance modes of the cantilever. The frequency spectra generated during the experiment are then processed to give a frequency versus time plot, and hence the change in bound mass versus time can be determined. Figure 3. Graph of bound mass on the surface of the cantilever versus time.
The frequency spectra recorded during the experiment were postprocessed using NOSEtools software to obtain the resulting plot of bound mass versus time. The scatter plot shows the raw data with the reference cantilever subtracted , and the line shows the median box filter of the raw data box size The left axis shows the bound mass on the surface of the cantilever, and the right axis shows the corresponding differential frequency shift.
Figure 4. Scaled Intensity versus time for thioflavin T intensity measurements. The intensity was scaled by the blank measurement, and the Thioflavin T reference was subtracted from the test intensity measurement. The data shown is the average of the intensity from the two wells.
References J. View at: Google Scholar M. Lotharius and P. View at: Google Scholar L. Golbe, G. Di Iorio, V. Bonavita, D. Miller, and R. View at: Google Scholar R. Kuhn, T. View at: Google Scholar K. Wakabayashi, K. Tanji, F. Mori, and H. Spillantini, M. Schmidt, V. Lee, J. Trojanowski, R.
Jakes, and M. Spillantini, R. Crowther, R. Jakes, M. Hasegawa, and M. Baba, S. Nakajo, P. Tu et al. Crowther, S. Daniel, and M. Weinreb, W. Zhen, A. Poon, K. Conway, and P. Eliezer, E. Kutluay, R. Bussell, and G. For example, proteinase K digestion is often used to assess and define the signature of different prion-like protein strains as different conformers display different accessibility to proteases. Aberrant protease activities in MSA patients compared to control could exacerbate this process, and this will be investigated in future studies.
For some cleavages, more than one protease has been implicated. These neo-epitopes are also prominent candidates to develop new and specific CSF and blood biomarker assays. All data generated or analyzed during this study are included in this published article and its supplementary information files. J Biol Chem — Mov Disord — Am J Pathol — Cell Death Dis 6:e Article Google Scholar.
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Nature — Nat Rev Neurol — Brain Pathol — Acta Neuropathol Commun Mol Neurodegen Acta Neuropathol. Article PubMed Google Scholar. Alzheimers Dement — FEBS Lett — Nat Rev Mol Cell Biol — Sci Rep Acta Neuropathol — Sci Transl Med eear Nat Genet — Muntane G, Ferrer I, Martinez-Vicente M alpha-synuclein phosphorylation and truncation are normal events in the adult human brain. Neuroscience — J Neuropathol Exp Neurol — Neurobiol Aging — Biochim Biophys Acta — Science — Brain — Moreover, select cytokine release rates as well as phagocytosis efficiency are altered by elevated human aSyn expression in mice and cultured cells Several authors have described a low level of SNCA gene expression and the presence of aSyn protein in monocytes and lymphocytes under normal conditions , Altered SNCA gene expression and aberrant aSyn metabolism have been reported downstream of virulent microbe exposure including in the brain, neuronal cultures, and the human gastrointestinal tract , , , , , A direct role for aSyn in host defenses, for example against viral and bacterial pathogens, and its regulation downstream of inflammation are two aspects of its interaction with the immune system.
These results suggest a conserved role for the protein in such processes, possibly including a signaling function. For example, modified forms of aSyn can also initiate neuroinflammatory responses e.
This is widely considered to occur as a consequence of disease processes, such as the release of pathogenic aSyn species from dysfunctional neurons, but also from intact cells In addition to its presence in primary immune cells and thus, the possibility of a direct involvement in host immunity, the relatively high expression level of SNCA in developing erythroblasts and megakaryocytes as well its high protein concentration in their mature progeny, i.
These may include aspects of iron homeostasis, lipid composition and curvature of cell membranes, vesicle formation and the release of contents by such vesicles Each of these important processes could indirectly also affect immune responses.
Of note, aSyn expression in primary cells of hematological origin have also provided a valuable model to study aSyn-modulated biological processes. Examples of this include: i to study its endogenous metabolism including the formation of oligomers , ; ii to explore the relation between monomeric and multimeric species of aSyn vis a vis lipid binding; iii to probe monomer-to-multimer ratios in peripheral cells as a model platform for the pathogenesis of aSyn-linked disease; 97 , , , iv to utilize the presence of aSyn proteins in hematological cells, plasma, serum and CSF for the exploration of potential biomarkers in aSyn-related pathological conditions, such as PD, DLB, or MSA , , , ; and v to interrogate aSyn metabolism in peripheral tissues in the context of microbiota on epidermal and epithelial surfaces of mammalian hosts , , , , , While the topic of exploring aSyn in immunological and hematological functions represents only a small file within the currently active aSyn research portfolio, the implications for the pathogenesis of these disorders—as well as their potential therapies—may be greater than is presently appreciated.
From these collective insights, three avenues of future research activities have become apparent. One, because SNCA gene is highly expressed outside the brain including at sites of host-environment interactions e.
Two, the function of aSyn proteins in the initiation and regulation of inflammatory responses and, vice versa, of the effects of inflammation on aSyn metabolism should be further studied both within and outside the brain.
A possible role for aSyn metabolism including the generation of immunogenic peptides may lie not only in dysregulated immune responses following neural cell death disease progression, but importantly, could be linked to disease initiation, as suggested by the early detection of reactive T-cell clones in PD subjects.
Defining these roles and associated processes may inform new therapeutic targets for intervention. Three, any change in relative abundance and half-life of post-translationally modified forms of aSyn that occurs during immunological responses should be further studied, including in the context of biomarker studies that relate to the diagnosis of PD, DLB and MSA, as well as during their progression. As of , clinical trials designed to lower aSyn levels and to alter the levels of higher-order oligomer formation are actively being pursued.
Select compounds and biologics have already entered phase-II trials. These concerns regarding the possibility of adverse events need to be considered in the design, safety monitoring and reporting of outcomes in clinical trials henceforth.
On the other hand, levels of distinct species of aSyn in peripheral cells, including those of hematological origin and related to the immune system, may serve as surrogate markers for disease-modifying treatments targeting PD, DLB and MSA. Last but not least, comprehensive epidemiological studies throughout the human lifespan should match the level of scrutiny usually applied to ongoing genome interrogations.
Thus, it would be of interest to collect further evidence of the association of typical, late-onset synucleinopathy disorders, such as PD, with altered incidence rates of communicable, microbial illnesses earlier in life as part of our exposome , such as hepatitis B and C It is intriguing that one year into the pandemic, a handful of case reports have been published to date that have described cases of parkinsonism in subjects afflicted by COVID19, at least one of which has shown spontaneous improvement , , Cellular models of aSyn pathology are often based on expression or extracellular addition of aSyn and they have recently been reviewed , In most healthy neurons in the brain, aSyn is highly concentrated in nerve terminals despite being translated on ribosomes in the cell body.
This demonstrates the efficiency of aSyn axonal transport by mechanisms that are not fully understood. The physiological subcellular localization is changed in pathological states where aSyn inclusions exist in axons and cell bodies, while yet poorly defined oligomeric species may occur in other compartments or organelles as well. When modeling synucleinopathies in cells, it should be kept in mind that specialized compartments like nerve terminals and axons do not exist in most cell models using mitotic cell lines, while even primary cultures of rodent nerve cells and human iPSC-derived neurons may not present distinct pre- and postsynaptic compartments when not fully differentiated and polarized.
The pre-synapse is a special domain with respect to proteostatic mechanisms given functional lysosomes are not present in the nerve terminals or distal axons.
Cellular defense mechanisms against pathogenic aSyn at the nerve terminal will likely include i a presynaptic chaperone system that unwinds aberrant aSyn states into native conformations, ii protein catabolism carried out by proteasomes, which can degrade native aSyn , , , iii disposal of protein aggregates by encapsulating them in autophagosomes that are subsequently fused with the lysosomal compartment upon retrograde axonal transport, and iv release into the extracellular space, as delineated above.
The axon of mature polarized neurons has a special organization of its microtubule system in which all microtubules point their plus-ends toward the distal pre-synapse , This differs from most cells and non-polarized neurons in culture, where microtubules are organized with mixed orientation. The polarized nature of axonal microtubules is required for efficient axonal transport.
Deficiencies in the autophagic-lysosomal system may lead to a local build-up of seeding-competent aSyn species. The significance of this system is underscored by the many genetic risk factors for PD and other synucleinopathies, whose gene products are involved in lysosomal biogenesis and autophagic function. The dysfunction of these gene products seems to be of special relevance for aSyn catabolism Mechanistic insight into how aSyn is chaperoned and catabolized in nerve terminals and axons is lacking and these processes will be difficult to model in cells not possessing these specialized nerve cell structures.
Recent studies suggest Lewy bodies are actively built by the cell as multi-organellar assemblies, and that such inclusions may be modeled in primary neuronal cultures seeded with preformed a-syn aggregates However, it should be kept in mind that aSyn-rich inclusions are not required for aSyn aggregates to exert toxicity in cell models , Such dysfunctional states may exist for years and, potentially, start as presynaptic dysfunctions with subsequent loss of synapses.
This may all occur before the initially affected neuron is dead. Modeling these topologically and temporally separated processes is complex and may not be achievable with one single-cell model. However, cell models are amenable to large screening efforts, and thus useful in the initial steps of drug discovery. Therefore, it will be important to continue developing and characterizing models that replicate as faithfully as possible phenotypes and mechanisms of the dysfunctions that occur in nerve cells and in nervous tissue as recently demonstrated 57 , Does the model reflect an effect of aggregated aSyn stress or merely aSyn overload?
Most models express aSyn under the regulation of strong promoters, leading to high aSyn levels in the cell body, and such high protein expression may cause toxicity per se The method of applying preformed aSyn aggregates to template aggregation of physiological aSyn levels has been used successfully in many labs and circumvents the need for high aSyn expression. The use of aggregate inhibitors to rescue the phenotype can also increase the confidence in the observed phenotypes as being truly aggregate-dependent , Do the aSyn constructs used in the models reflect what happens to native aSyn in the brain?
Single amino acid mutations, e. This exquisite sensitivity to acquire aberrant functions with even small changes should be kept in mind when evaluating the many models relying on tagging aSyn with reporter proteins that are often larger and compactly folded than the aSyn protein itself. What specific aSyn subcellular localization do our cell models reflect?
Can we draw conclusions relevant to presynaptic or nuclear events, and is this important for the questions of interest? In summary, our continued progress will depend on careful consideration of what specific defects and mechanisms in synucleinopathies we attempt to model—and how well they are represented in our cell models.
Animal models have been used extensively to test the function and toxicity of aSyn in detail as they are relatively fast to generate and easy to alter genetically. From the very early discovery of the protein from the electric organ of the Pacific electric ray Torpedo californica 36 , analysis of animal material enabled the study of potential physiological functions of the protein as shown already in the very first studies using Rattus norvegicus Before the discovery of aggregated aSyn in LBs , animal models of PD were mainly based on toxin-induced impairment of the dopaminergic nigrostriatal pathway, which causes a rapid dopamine depletion that mimics advanced disease stages However, these models miss key pathological events of PD and, more importantly, do not mimic its progressive nature.
Deciphering the role of aSyn in PD has led to the development of animal models mimicking central pathological features of such as aSyn-associated neuronal loss and aSyn aggregation Such models are referred to as disease gene-based models or etiologic models.
Several aSyn transgenic mouse models have been produced and various promoters have been used to drive the expression of the transgene, leading to different results After first being described in cell culture, the relevance of SNCA overexpression and its mutations in the process of protein aggregation was also validated in animal models Since then, animal models have been used for both deciphering disease processes such as protein degradation or metabolism as well as for testing therapeutic strategies.
In addition, animals are used to mimic high levels of aSyn that lead to cell death. Viral vector-mediated transgenesis offers a valid alternative to conventional transgenic animals, as recombinant viruses can i be injected specifically into brain structures of interest, ii be easily adjusted for expression levels, iii be targeted to either neurons or glia, and iv start to express their payload at any desired age of the animal Finally, animal models focus on studying mechanisms involved in the spread and toxicity of preformed aSyn fibrils.
While originally investigated in mouse brain , this phenomenon was also reproduced in multiple other animal models including rats and non-human primates In these models, cell-to-cell transmission and propagation of misfolded aSyn, could mirror the spread of human pathology, as deduced from the neuropathological observations.
In addition, other approaches to develop animal models, such as injection of brain extracts containing aSyn aggregates from transgenic mice or patients with aSyn pathology into the brain, muscles, peritoneal cavity, or the circulatory system of aSyn-overexpressing or wild-type rodents may also support a prion-like cascade in the development of synucleinopathies 67 , In general, some considerations must be acknowledged when using these models.
Along the same line, the use of diverse homogenates and the subsequent inflammatory response they trigger needs to be considered with care. Nevertheless, these models are commonly used, highly reproducible, and thus important for the further exploration of aSyn pathobiology. First, it needs to be clear that models of synucleinopathies are not necessarily models of PD.
None of the animal models described above perfectly recapitulates PD neuropathology and replicates the clinical syndrome. Each model mimics certain aspects of aSyn biology and pathology, and the use of each model depends on the question being investigated. Thus, scientists should first know the strengths and weaknesses of each model, before selecting the model s most suitable to address the experimental question of interest.
One should clearly highlight which disease aspect is recapitulated by the model and limit the conclusions accordingly. As PD patients are rather heterogeneous with respect to disease onset, progression, symptoms, and neuropathology, diversifying animal models may help us to restage distinct aspects of PD, and therefore, to develop personalized therapies. Moreover, combining different pathways of pathogenesis such as by creating animals with more than one genetic or environmental risk may enhance their PD-type pathologies, and thus approximate the phenotypic expressivity seen in humans.
It needs to be clear what is to be modeled and that models are only approximations of the human condition. While the protein coding sequence of human and rodent SNCA is highly conserved, several regulatory regions influencing SNCA expression levels and transcript isoforms differ between species Hence, humanized models are essential as they best recapitulate expression and splicing isoforms of human SNCA and other genes in rodents.
Here, we clearly need to improve our definition of a humanized model differentiating between models with only one human gene such as aSyn and those with multiple human genes covering other PD-related proteins. A consensus in the field is needed on which phenotypic read-outs should be prioritized when characterizing a model of PD.
So far, a strong focus was put on motor features, while non-motor features were largely neglected. Indeed, some transgenic models show numerous early non-motor features of PD with impairments in gastrointestinal function, olfaction, and sleep , which could serve as useful prodromal disease markers. In addition to motor deficits and pathology, these non-motor features should be included in preclinical studies to measure therapeutic efficacy.
For viral vector-based models, a clear drawback is the necessity to stereotaxically inject the vectors into each individual animal.
Preps from different sources may vary in terms of protein expression by 10 to fold, even though the vector genome and the vector genome titer are identical.
Given that almost all known patho- physiological effects of the synucleins are concentration-dependent, i. Hence, aSyn expression levels need to be tightly controlled. In general, there is a critical need for standardizing the tools we use to characterize the animals. Furthermore, consistency in experimental factors is likely to enhance experimental data reproducibility.
With respect to transgenic models, several observations have shown that the phenotype can change over time. Hence, guidelines on good practices to avoid phenotype vanishing and discrepant phenotypes between labs are needed.
The role of the genetic background in a model must be considered when generating a model. Several transgenic mouse lines are based on a background expressing endogenous murine aSyn, thereby complicating the interpretation of findings in these lines. Another limitation of rodent models for modeling neurodegeneration is their lack of neuromelanin. The pigment neuromelanin accumulates over time in the SNpc dopaminergic neurons of macaques but not in rodents. Non-melanized neurons have been shown to be less vulnerable to neurodegeneration than melanized neurons both in MPTP-treated primates , as well as in PD patients One might argue that, perhaps, only primate dopamine neurons are vulnerable enough to degenerate and, hence, rodent dopamine neurons in general are less suited, because of their higher plasticity, to model human degenerative processes.
A final, more general recommendation applicable to a wide range of research fields is that negative results need to be shared and published.
To really move the field forward, it is crucial to share results that are unexpected or contradictory to previous studies to enable a proper interpretation of relevant findings.
To date there are no reagents that have surpassed the utility of antibodies in biomedical science. Given their high specificity and affinity to the target antigen s , antibodies have found their way from a simple detection reagent to some of the most promising immunotherapeutic agents in neurodegenerative diseases.
Several antibodies have been developed targeting different regions as well as various forms of aSyn. Antibodies were generated against purified LBs , its non-amyloid component NAC , and to N-terminal and the C-terminal regions of the full-length protein , Given the potential pathological cell-to-cell transmission of aSyn see sections above the concept of therapeutically targeting this mechanism with antibodies has been tested preclinically and is being evaluated in ongoing clinical studies.
Both active and passive immunotherapeutic approaches have been shown to reduce aSyn pathology in rodent models , , Clinical trials were initiated in with PRX, a humanized version of mouse monoclonal antibody, 9E4 , and in with BIIB, a human derived aSyn antibody , , both of these antibodies are currently in Phase 2 clinical trials. Table 1 shows the status of all aSyn immunotherapies that we are currently aware of. How do we link aSyn pathology and progression of neuropathology to clinical symptoms?
Intuitively the earlier the better, but this may require prohibitively long and expensive trials in prodromal disease. Owing to the complex heterogeneity of aSyn see above , it is not known what forms of aSyn should be targeted. In general, they have all been shown or claimed to bind aggregated aSyn. However, a direct comparison across these clinical antibodies is lacking and represents an addressable gap for the field.
Whether the antibodies target a truly relevant pathological species, which is present extracellularly remains to be determined. The ongoing clinical studies may, somewhat empirically, define this but the number of other variables is extensive and likely confounding.
How do we measure target engagement? Can we get sufficient antibody into the brain? One of the biggest tasks in finding a treatment strategy for diseases affecting the brain is overcoming the blood brain barrier BBB.
Given the high molecular weight of antibodies, it is even a greater challenge to make them cross the BBB. To overcome these hurdles, antibodies have been engineered into smaller fragments without hindering their affinity or specificity to the target antigen. Some of these engineered recombinant antibody fragments, including single-chain variable fragments scFv , diabodies, triabodies, minibodies and single-domain antibodies, are currently being explored for their efficiency compared to the full-length antibodies.
Various scFvs were generated using monomeric aSyn , synthetic libraries and naive human scFv libraries Single-domain antibodies like nanobodies have also been developed against aSyn , Nanobodies were also found to reduce aSyn oligomer-induced cellular toxicity making them potential candidates for immunotherapeutic agents Despite these challenges, clinical development continues for at least five aSyn-targeted immunotherapies.
Although there have been considerable advances in understanding the structure of aSyn, we still lack thorough knowledge on how aSyn develops into distinct pathological phenotypes in synucleinopathies, and this possesses a great challenge for antibody-mediated immunotherapy. Validating and improving these assays would aid in the identification, isolation, and clinical characterization of a particular strain of aSyn, potentially allowing for stratification of patients for immunotherapy with strain-specific antibodies.
Identification of aSyn in extracellular biological fluids has helped researchers to better understand the pathogenesis of synucleinopathies. Hence a clear and better understanding of aSyn in the extracellular form would be beneficial. The target for immunotherapy using any given antibody should also be properly studied.
As aggregation of proteins is observed across a spectrum of neurodegenerative disorders, lessons should be learnt from the recent failures, and one potential success, from the antibody-based immunotherapy trials for AD.
The presence of concomitant pathologies in neurodegenerative disorders makes it even more challenging to identify the right target for an efficient therapy. Therefore, immunotherapy using a cocktail of antibodies may yield a more successful intervention in a complex disease such as PD. Alternatively, bispecific antibodies, targeting two different antigens could be also used as therapeutics agents once the combination of target molecules has been properly identified and validated.
Regardless of the developments in disease understanding and treatments, antibodies will continue to be a critical tool and potential therapeutic. The diagnostic accuracy of clinically diagnosed synuclein aggregation disorders has been very poor, especially in the early stages of the disease , which hampers the success of early clinical trials.
The presence of aSyn in cerebrospinal fluid CSF was proven by mass spectrometry and followed by the development and validation of several enzyme linked immunoassays ELISA for its quantification , This decrease was also prevalent in newly diagnosed, de novo PD subjects Since the prodromal state of aSyn disorders is well-defined with isolated REM sleep behavior disorder iRBD , hyposmia and other non-motor symptoms , several cohorts e.
In addition to the results on total aSyn, an assay for oligomeric aSyn showed promising results, that need further and independent validation. Data from the quantification of aSyn phosphorylated at serine by various ELISA show inconclusive results with either elevated pSaSyn or unchanged levels in PD compared to controls This antibody-free approach was inspired from the prion field and has been developed by several independent groups.
Thereby aggregation-inducing aSyn seeds present in biospecimens are monitored by sequential amplification in vitro and detected with thioflavin-T, resulting in a fluorescent signal that is proportional to the concentration of the aSyn seed in the biospecimens. While the quantitative and high-throughput nature of these assays also in better accessible biological fluids i. In addition, studies on aSyn detection in peripheral tissue, such as skin, colon and submandibular gland showed diagnostic potential for PD and for prodromal subjects with iRBD , , With the current methods of aSyn detection in tissue and biological fluids, no conclusive intraindividual pattern of aSyn distribution across the fluids and tissues analyzed has been found yet, but the immunohistochemistry analysis of aSyn in the submandibular gland and skin showed promising results pointing towards a promising peripheral diagnostic biomarker even in this multicenter setting Although there have been considerable efforts with building longitudinal cohorts of aSyn disorders, including prodromal conditions and improving technology, clear panels of reliably measurable and well-validated biomarkers for state, rate, fate, trait are lacking.
In most biomarker studies e. Some reasons for the variability, besides blood-contamination and possibly other minor aspects of SOP could lay in the genetic background as has been recently shown with lower CSF aSyn levels in PD subjects with GBA mutations , However, other reasons for the variability are largely unknown. Possibly, the existing clinical heterogeneity with different pheno- and progression types of PD, that is partially due to comorbidities and possibly also due to comedication and other reasons beyond the disease itself complicate the analyses.
Several publications have suggested different phenotypes, but its validation is difficult due to the heterogeneity of the data captured and the statistical power even in larger cohorts like PPMI. Therefore, additional biomarkers e. For clinical trials, markers of state for stratification are needed and markers of rate to objectively measure progression of the disease.
So far, only dopamine transporter SPECT scans, but no biological measures, have been FDA approved to stratify PD from other diseases like essential tremor for clinical trials but no biological measure. Serum NfL showed an interesting increase over time, which correlated with UPDRS and some cognitive measures in one multicenter cohort but this still needs replication and validation.
The role of other modified forms of aSyn PTMs e. Future investigations will show if fragments of aSyn can be more specific a biomarker than the total levels. The method still needs optimization to become reliably quantitative and for high-throughput measurements. PD subjects suggesting different underlying strains of aSyn 70 , 88 , needs further validation. The challenge for the future is the improvement of this technology and its application to blood and peripheral tissues, which will be interesting for studies like the above mentioned S4 study.
Exosomes are an intriguing matrix to consider since they contain intracellular components within biological fluids.
So far, exosomal aSyn has been reported in some cohorts in the CSF and also recently in a small study in plasma Still the extraction methods add variability to an anyway variable protein in biological fluids and need standardization. Once we have more specific aSyn markers, exosomal aSyn will help us to understand intracellular processes and may also lead to more accurate biomarkers in extracellular fluids.
In the future, we will also have additional biomarkers being identified by multiplex platforms, such as the proximity ligation assays, the aptamer approaches or even antibody-independent mass spectrometry techniques.
The inclusion of prodromal subjects in current PD biomarker efforts is essential. The Michael J. The collected data and samples from PPMI 2. How can we turn all this aSyn research into breakthrough medicines for PD patients? Ultimately, the nature and extent of the aSyn-PD connection can only be determined by carefully designed clinical experiments in humans.
Since the discovery of a genetic link between SNCA and PD 1 , there have been a large number of possible mechanisms proposed to explain this, and additional subsequent genetic links. Most are compelling and are consistent with selected aspects of PD pathology e.
However, none of the proposed mechanisms have been tested in PD itself. These issues fall into four interdependent categories:. This patient population should progress rapidly and relatively uniformly, so that slowing of progression can be measured in a smaller, shorter trial. Identification of such a population should drive target selection.
The lackluster results of both studies may result directly from patient heterogeneity. In hindsight, it may have been useful to restrict patients to individuals with a specific SNCA snp profile or expression level.
Natural history studies to test the relevance of this approach have not yet been done, but we do recommend addressing this gap in the near future to increase the probability of success for future aSyn therapeutics. Target selection: Selection of a therapeutic strategy that is likely to produce a large and measurable effect in the chosen patient population, given that aSyn-driven pathogenesis is likely to be multifactorial. It is preferable to target upstream events like SNCA expression or, possibly, aSyn aggregation, as opposed to downstream effects, since any given downstream abnormality is unlikely to account for all or even most of the SNCA effect.
It is optimal to base target selection on the genetics of disease which point directly at causality, as opposed to pathological findings Lewy bodies, Lewy neurites, etc. Thus, it is important to determine the relationship between a measurable clinical endpoint and the targeted synuclein biochemistry. Pharmacodynamic marker selection: To disprove any therapeutic hypothesis, it is necessary to assure that the therapeutic dose is sufficient to produce the desired biochemical effect.
This requires that target engagement be measured in human brain. An example would be an imaging agent that is selective for a particular pathological form of synuclein. If a brain marker is not available, a marker in CSF, such as a specific form of aSyn associated with the disease, may suffice. So, what exactly does that mean for an aSyn drug? Before discussing issues of trial design in more detail, it is critical to distinguish two features of PD: age-at-onset more precisely, age-at-diagnosis and rate of symptom progression.
A disease-modifying drug could delay PD diagnosis, slow PD symptom progression, or both. Thus, LRRK2 inhibitors, which may correct the effect of the mutations, will be difficult to test in a progression trial. One such study demonstrated that these two pools are largely separate, with only a single gene that clearly affects both: GBA1 A disconnect between SNCA risk-associated snps and disease progression was noted previously These findings suggest that age-at-diagnosis and symptom progression could be driven by different processes, so a single therapy is unlikely to affect both, and trials must be designed accordingly.
These patients will be most likely to respond to an aSyn-targeted therapy. PD is not a single entity with a single cause, but multiple distinct entities with diverse causes that happen to share some symptoms bradykinesia, tremor, stiffness, and postural instability. Other symptoms include cognitive loss, depression, REM sleep disorder, and autonomic failure. Each of these symptoms could have a different underlying cause since they do not always co-occur.
The study of genetic subtypes of PD e. The fact that LRRK2 mutations are highly penetrant but progress very slowly , while GBA1-PD mutations have low penetrance and progress rapidly supports the notion that these two subtypes of PD are likely to be driven by completely different underlying biochemistries, despite the fact that a large number of studies in cell culture and animal models purport to link GBA1-PD and LRRK2-PD.
Even though the significance of the population-wide association of SNCA snps with PD is clear very low p -values , the effect is relatively small on a per individual basis. Genetically defined subtypes of SNCA-PD, listed below, may be an exception to this generalization and may thus be attractive therapeutics targets. Diverse clinical phenotypes have been reported, even within single families , Given the scarcity of these families, it is difficult to determine the rate of progression of these forms or even whether specific genotypes e.
A second form of autosomal-dominant PD, derived from multiplication of the SNCA gene and overexpression of WT synuclein, has also been characterized Delaying disease onset is very difficult to test clinically, and is also challenging, since dosing may have to commence before symptom onset see below. Although there are anecdotal reports that multiplication patients progress faster than idiopathic PD patients, no published study has confirmed this, much less determined the magnitude of the difference in progression rate , CNV3 Finally, there are reports of great variability within families Together, these findings suggest that the effect of aSyn expression on the rate of PD symptom progression may be too small to justify a progression trial.
Therefore, demonstration of slowed progression by a therapeutic intervention aimed at reversing the effect of the snp will require a large, long, and expensive clinical trial. Furthermore, the typical odds ratios for these SNCA snps are in the range of 1.
One would not necessarily expect those patients to respond to a SNCA snp-targeting therapy. The average progression rates of patients carrying different SNCA snps may be distinguishable over five years of observation , but executing a five-year progression trial with hundreds of patients is prohibitively expensive.
It may be possible to refine a SNCA snp-carrying population to decrease variability in progression rate and efforts to do so must be prioritized. Using a combination of SNCA snps to identify fast progressing patients does not seem to be feasible Retrospective analysis of clinical progression in a 20 patient GBA1-PD population suggests that one SNCA snp may affect the rate of progression of GBA1-PD, but a natural history study of a larger patient population is necessary to confirm this finding This population may be hard to identify in sufficient numbers, but it will be much more homogeneous and faster progressing than the groups discussed above, so the required numbers are greatly reduced.
Reducing synuclein expression. Risk-associated snps appear to affect synuclein expression in blood. If these snps correlate to aSyn expression levels in the brain, then reduction of SNCA translation by an antisense or RNA-directed strategy may be a viable therapeutic strategy.
It is critical to be able to compare synuclein expression in brain of each SNCA snp and correlate with the effect on PD onset and progression.
This will allow one to estimate whether it is possible to significantly modify disease progression by reducing synuclein level. In addition, it will be critical to determine whether the reduction of synuclein will have a negative consequence, especially given the wealth of data that suggests an important role for aSyn in normal biology such as in synaptic vesicle formation, docking, and release, as described above. Modulating PTMs of aSyn. The lack of convincing evidence from GWAS studies is troubling, since one would expect that the genes encoding the enzymes that catalyze these modifications would be implicated.
Reducing a specific oligomeric form of aSyn. Many studies have attempted to link a specific oligomeric form of synuclein to PD, without success. This is very difficult to do for reasons better summarized elsewhere; one key point is that data from human brain is limited and virtually all of it comes from patients with advanced disease. Furthermore, identification and distinction of oligomeric forms is often based on antibodies that may not be selective Much of the activity around oligomeric aSyn is driven by the hope that an aSyn imaging agent, analogous to the PiB reagent that has been used for AD research, that can provide a pharmacodynamic marker for drug action in the brain.
However, the AD experience reduced image intensity does not correlate to improved cognition should remind us that putting such an emphasis on a brain biomarker, when the significance of the species that is recognized by the imaging agent is unclear, can be counterproductive. Reducing a downstream toxic effect of aSyn. Therefore, a drug that influences only one pathway will have a smaller effect size than a drug focused on several pathways.
Measuring target engagement in the brain is critical to disprove a hypothesis but is not necessary to develop a drug. Once a target and a potentially responsive subpopulation have been identified, it is optimal to develop a tool to measure the effective manipulation of that target in patients.
This step is often seen as a requirement in pharma to offer the possibility of explaining a failed trial failure to hit target of failure of idea? However, many successful CNS drugs have been developed without proof of target engagement Tecfidera, for example.
Designing a clinical trial to detect a drug effect in delaying age-at-diagnosis, since that is the genetically validated effect of SNCA. The possibility that PD onset and progression are driven by distinct processes is a critical consideration in the design of a clinical trial.
Diagnosis of PD occurs years after peripheral constipation and brainstem REM behavior disorder symptoms are clear, and after clear dopaminergic cell loss can be detected by imaging , It is also becoming evident that motor and cognitive symptoms that are characteristic of PD can be detected, and their progression measured, in this prodromal phase Further studies are necessary to enable clinical studies in prodromal PD It is also unclear when the process es driving diagnosis fade and those driving progression take over.
The trial design suggestions made here may not be quickly adapted, as one might tend to recruit a large number of mild-to-moderate idiopathic PD patients with the hope that large numbers can slow average progression enough to produce a low p -value.
However, this approach comes with noticeable challenges: 1 most of the patients may not respond to the drug, 2 earlier dosing during prodromal period is likely to be required, and 3 the effect of synuclein on symptom progression is not yet clear. In conclusion, identifying knowledge gaps and defining research challenges and opportunities will require not only a deep understanding of what we know, but also the courage to admit what we do not know.
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