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Dementia is a progressive and debilitating neurological disease that affects millions of people worldwide. Identifying the minimally invasive biomarkers associated with dementia that could provide insights into the disease pathogenesis, improve early diagnosis, and facilitate the development of effective treatments is pressing. Proteomic studies have emerged as a promising approach for identifying the protein biomarkers associated with dementia. This pilot study aimed to investigate the plasma proteome profile and identify a panel of various protein biomarkers for dementia. We used a high-throughput proximity extension immunoassay to quantify 1090 proteins in 122 participants (22 with dementia, 64 with mild cognitive impairment (MCI), and 36 controls with normal cognitive function). Limma-based differential expression analysis reported the dysregulation of 61 proteins in the plasma of those with dementia compared with controls, and machine learning algorithms identified 17 stable diagnostic biomarkers that differentiated individuals with AUC = 0.98 ± 0.02. There was also the dysregulation of 153 plasma proteins in individuals with dementia compared with those with MCI, and machine learning algorithms identified 8 biomarkers that classified dementia from MCI with an AUC of 0.87 ± 0.07. Moreover, multiple proteins selected in both diagnostic panels such as NEFL, IL17D, WNT9A, and PGF were negatively correlated with cognitive performance, with a correlation coefficient (r2) ≤ −0.47. Gene Ontology (GO) and pathway analysis of dementia-associated proteins implicated immune response, vascular injury, and extracellular matrix organization pathways in dementia pathogenesis. In conclusion, the combination of high-throughput proteomics and machine learning enabled us to identify a blood-based protein signature capable of potentially differentiating dementia from MCI and cognitively normal controls. Further research is required to validate these biomarkers and investigate the potential underlying mechanisms for the development of dementia.

The aging population contributes to an increase in the prevalence of neurodegenerative diseases, such as Parkinson's disease (PD). Due to the progressive nature of these diseases and an incomplete understanding of their pathophysiology, current drugs are inefficient, with a limited efficacy and major side effects. In this study, CRISPR-Cas9 RNA-proteins (RNP) composed of a Cas9 nuclease and single-guide RNA were delivered with a non-viral targeted delivery system to rescue the PD-associated phenotype in neuronal cells. Here, we fused the cell-penetrating amphipathic peptide, PepFect14 (PF14), with a short fragment of the rabies virus glycoprotein (C2) previously shown to have an affinity towards nicotinic acetylcholine receptors expressed on neuronal cells and on the blood-brain barrier. The resultant peptide, C2-PF14, was used to complex with and deliver RNPs to neuronal cells. We observed that RNP/C2-PF14 complexes formed nanosized, monodispersed, and nontoxic nanoparticles that led to a specific delivery into neuronal cells. α-Synuclein (α-syn) plays a major role in the pathology of PD and is considered to be a target for therapy. We demonstrated that CRISPR/Cas9 RNP delivered by C2-PF14 achieved α-syn gene (

Neuropathologically, Parkinson's disease (PD) and dementia with Lewy bodies (DLB) are characterized by the accumulation of insoluble aggregates of α-synuclein (α-syn) in the Lewy bodies (LBs). In addition to full-length α-syn fibrils, C-terminally truncated α-syn is also abundant in the LBs that acts as seeds and facilitates the aggregation of the full-length α-syn

Background Alpha-synuclein (α-syn) preformed fibril (PFF)-induced pathology can be used to study the features and progression of synucleinopathies, such as Parkinson's disease. Intrastriatal injection of mouse α-syn PFFs produce accumulation of α-syn pathology in both mice and rats. Previous studies in mice have revealed that greater sequence homology between the α-syn amino acid sequence used to produce PFFs with that of the endogenous host α-syn increases α-syn pathology in vivo. New methods Based on the prediction that greater sequence homology will result in more α-syn pathology, PFFs generated from recombinant rat α-syn (rPFFs) were used instead of PFFs produced from recombinant mouse α-syn (mPFFs), which are normally used in the model. Rats received unilateral intrastriatal injections of either rPFFs or mPFFs and accumulation of α-syn phosphorylated at serine 129 (pSyn) was examined at 1-month post-surgery. Results Rats injected with mPFFs exhibited abundant accumulation of α-syn inclusions in the substantia nigra and cortical regions, whereas in rats injected with rPFFs had significantly fewer SNpc neurons containing pSyn inclusions (≈60% fewer) and little, if any, pSyn inclusions were observed in the cortex. Conclusions Our results suggest that additional factors beyond the degree of sequence homology between host α-syn and injected recombinant α-syn impact efficiency of seeding and subsequent inclusion formation. More practically, these findings caution against the use of rPFFs in the rat preformed fibril model.

Nanobodies (Nbs), the single-domain antigen-binding fragments of dromedary heavy-chain antibodies (HCAb), are excellent candidates as therapeutic and diagnostic tools in synucleinopathies because of their small size, solubility and stability. Here, we constructed an immune nanobody library specific to the monomeric form of alpha-synuclein (α-syn). Phage display screening of the library allowed the identification of a nanobody, Nbα-syn01, specific for α-syn. Unlike previously developed nanobodies, Nbα-syn01 recognized the N-terminal region which is critical for in vitro and in vivo aggregation and contains many point mutations involved in early PD cases. The affinity of the monovalent Nbα-syn01 and the engineered bivalent format BivNbα-syn01 measured by isothermal titration calorimetry revealed unexpected results where Nbα-syn01 and its bivalent format recognized preferentially α-syn fibrils compared to the monomeric form. Nbα-syn01 and BivNbα-syn01 were also able to inhibit α-syn-seeded aggregation in vitro and reduced α-syn-seeded aggregation and toxicity in cells showing their potential to reduce α-syn pathology. Moreover, both nanobody formats were able to recognize Lewy-body pathology in human post-mortem brain tissue from PD and DLB cases. Additionally, we present evidence through structural docking that Nbα-syn01 binds the N-terminal region of the α-syn aggregated form. Overall, these results highlight the potential of Nbα-syn01 and BivNbα-syn01 in developing into a diagnostic or a therapeutic tool for PD and related disorders.

Aggregation of the protein α‐synuclein (α‐syn) into insoluble intracellular assemblies termed Lewy bodies (LBs) is thought to be a critical pathogenic event in LB diseases such as Parkinson’s disease and dementia with LBs. In LB diseases, the majority of α‐syn is phosphorylated at serine 129 (pS129), suggesting that this is an important disease‐related post‐translational modification (PTM). However, PTMs do not typically occur in isolation and phosphorylation at the proximal tyrosine 125 (pY125) residue has received considerable attention and has been inconsistently reported to be present in LBs. Furthermore, the proximity of Y125 to S129 means that some pS129 antibodies may have epitopes that include Y125, in which case phosphorylation of Y125 will impede recognition of α‐syn. This would potentially lead to underestimating LB pathology burdens if pY125 occurs alongside pS129. To address the apparent controversy in the literature regarding the detection of pY125, we investigated its presence in the LB pathology. We generated pS129 antibodies whose epitope includes or does not include Y125 and compared the extent of α‐syn pathology recognized in mouse models of α‐synucleinopathies, human brain tissue lysates and fixed post‐mortem brain tissues. Our study demonstrated no difference in α‐syn pathology recognized between pS129 antibodies, irrespective of whether Y125 was part of the epitope or not. Furthermore, evaluation with pY125 antibodies whose epitope does not include S129 demonstrated no labeling of LB pathology. This study reconciles disparate results in the literature and demonstrates pY125 is not a key component of LB pathology in murine models or human tissues in idiopathic LB diseases.

α-Synuclein (α-syn) phosphorylation at serine 129 (pS129–α-syn) is substantially increased in Lewy body disease, such as Parkinson’s disease (PD) and dementia with Lewy bodies (DLB). However, the pathogenic relevance of pS129–α-syn remains controversial, so we sought to identify when pS129 modification occurs during α-syn aggregation and its role in initiation, progression and cellular toxicity of disease. Using diverse aggregation assays, including real-time quaking-induced conversion (RT-QuIC) on brain homogenates from PD and DLB cases, we demonstrated that pS129–α-syn inhibits α-syn fibril formation and seeded aggregation. We also identified lower seeding propensity of pS129–α-syn in cultured cells and correspondingly attenuated cellular toxicity. To build upon these findings, we developed a monoclonal antibody (4B1) specifically recognizing nonphosphorylated S129–α-syn (WT–α-syn) and noted that S129 residue is more efficiently phosphorylated when the protein is aggregated. Using this antibody, we characterized the time-course of α-syn phosphorylation in organotypic mouse hippocampal cultures and mice injected with α-syn preformed fibrils, and we observed aggregation of nonphosphorylated α-syn followed by later pS129–α-syn. Furthermore, in postmortem brain tissue from PD and DLB patients, we observed an inverse relationship between relative abundance of nonphosphorylated α-syn and disease duration. These findings suggest that pS129–α-syn occurs subsequent to initial protein aggregation and apparently inhibits further aggregation. This could possibly imply a potential protective role for pS129–α-syn, which has major implications for understanding the pathobiology of Lewy body disease and the continued use of reduced pS129–α-syn as a measure of efficacy in clinical trials.

Autism spectrum disorder (ASD) is an umbrella term that encompasses several disabling neurodevelopmental conditions. These conditions are characterized by impaired manifestation in social and communication skills with repetitive and restrictive behaviors or interests. Thus far, there are no approved biomarkers for ASD screening and diagnosis; also, the current diagnosis depends heavily on a physician’s assessment and family’s awareness of ASD symptoms. Identifying blood proteomic biomarkers and performing deep blood proteome profiling could highlight common underlying dysfunctions between cases of ASD, given its heterogeneous nature, thus laying the foundation for large-scale blood-based biomarker discovery studies. This study measured the expression of 1196 serum proteins using proximity extension assay (PEA) technology. The screened serum samples included ASD cases (n = 91) and healthy controls (n = 30) between 6 and 15 years of age. Our findings revealed 251 differentially expressed proteins between ASD and healthy controls, of which 237 proteins were significantly upregulated and 14 proteins were significantly downregulated. Machine learning analysis identified 15 proteins that could be biomarkers for ASD with an area under the curve (AUC) = 0.876 using support vector machine (SVM). Gene Ontology (GO) analysis of the top differentially expressed proteins (TopDE) and weighted gene co-expression analysis (WGCNA) revealed dysregulation of SNARE vesicular transport and ErbB pathways in ASD cases. Furthermore, correlation analysis showed that proteins from those pathways correlate with ASD severity. Further validation and verification of the identified biomarkers and pathways are warranted.

Krabbe disease (KD) is an infantile neurodegenerative disorder resulting from pathogenic variants in the GALC gene which causes accumulation of the toxic sphingolipid psychosine. GALC variants are associated with increased risk of Lewy body diseases (LBD), an umbrella term for age-associated neurodegenerative diseases in which the protein α-synuclein aggregates into Lewy bodies. To explore whether α-synuclein in KD has pathological similarities to that in LBD, we compared post-mortem KD tissue to that of infant control cases and identified alterations to α-synuclein localisation and expression of modifications associated with LBD. To determine whether α-synuclein in KD displayed pathogenic properties associated with LBD we evaluated its seeding capacity using the real-time quaking-induced conversion assay. Strikingly, seeded aggregation of α-synuclein resulted in the formation of fibrillar aggregates similar to those observed in LBD, confirming the prion-like capacity of KD-derived α-synuclein. These observations constitute the first report of prion-like α-synuclein in the brain tissue of infants and challenge the putative view that α-synuclein pathology is merely an age-associated phenomenon, instead suggesting it can result from alterations to biological processes such as sphingolipid homeostasis. Our findings have important implications for understanding the mechanisms underlying Lewy body formation in LBD.

Parkinson’s disease is one of the most common movement disorders and is characterized by dopaminergic cell loss and the accumulation of pathological α-synuclein, but its precise pathogenetic mechanisms remain elusive. To develop disease-modifying therapies for Parkinson’s disease, an animal model that recapitulates the pathology and symptoms of the disease, especially in the prodromal stage, is indispensable. As subjects with α-synuclein gene (SNCA) multiplication as well as point mutations develop familial Parkinson’s disease and a genome-wide association study in Parkinson’s disease has identified SNCA as a risk gene for Parkinson’s disease, the increased expression of α-synuclein is closely associated with the aetiology of Parkinson’s disease. In this study we generated bacterial artificial chromosome transgenic mice harbouring SNCA and its gene expression regulatory regions in order to maintain the native expression pattern of α-synuclein. Furthermore, to enhance the pathological properties of α-synuclein, we inserted into SNCA an A53T mutation, two single-nucleotide polymorphisms identified in a genome-wide association study in Parkinson’s disease and a Rep1 polymorphism, all of which are causal of familial Parkinson’s disease or increase the risk of sporadic Parkinson’s disease. These A53T SNCA bacterial artificial chromosome transgenic mice showed an expression pattern of human α-synuclein very similar to that of endogenous mouse α-synuclein. They expressed truncated, oligomeric and proteinase K-resistant phosphorylated forms of α-synuclein in the regions that are specifically affected in Parkinson’s disease and/or dementia with Lewy bodies, including the olfactory bulb, cerebral cortex, striatum and substantia nigra. Surprisingly, these mice exhibited rapid eye movement (REM) sleep without atonia, which is a key feature of REM sleep behaviour disorder, at as early as 5 months of age. Consistent with this observation, the REM sleep-regulating neuronal populations in the lower brainstem, including the sublaterodorsal tegmental nucleus, nuclei in the ventromedial medullary reticular formation and the pedunculopontine nuclei, expressed phosphorylated α-synuclein. In addition, they also showed hyposmia at 9 months of age, which is consistent with the significant accumulation of phosphorylated α-synuclein in the olfactory bulb. The dopaminergic neurons in the substantia nigra pars compacta degenerated, and their number was decreased in an age-dependent manner by up to 17.1% at 18 months of age compared to wild-type, although the mice did not show any related locomotor dysfunction. In conclusion, we created a novel mouse model of prodromal Parkinson’s disease that showed RBD-like behaviour and hyposmia without motor symptoms.

Background Neurofilament light chain is a marker of axonal damage and is of interest as a biofluid biomarker for PD. The objective of this study was to investigate whether CSF or serum neurofilament contributes to a combination of CSF biomarkers in defining the optimal biomarker panel for discriminating PD patients from healthy controls. In addition, we aimed to assess whether CSF and/or serum neurofilament levels are associated with clinical measures of disease severity. Methods We measured neurofilament light chain levels in CSF and/or serum of 139 PD patients and 52 age‐matched healthy controls. We used stepwise logistic regression analyses to test whether neurofilament contributes to a biomarker CSF panel including total, oligomeric, and phosphorylated α‐synuclein and Alzheimer's disease biomarkers. Measures of disease severity included disease duration, UPDRS‐III, Hoehn & Yahr stage, and MMSE. Results After correcting for age, CSF neurofilament levels were 42% higher in PD patients compared with controls (P

Neurodegenerative disorders of the aging population are characterized by progressive accumulation of neuronal proteins such as α-synuclein (α-syn) in Parkinson’s Disease (PD) and Amyloid ß (Aß) and Tau in Alzheimer’s disease (AD) for which no treatments are currently available. The ability to regulate the expression at the gene transcription level would be beneficial for reducing the accumulation of these proteins or regulating expression levels of other genes in the CNS. Short interfering RNA molecules can bind specifically to target RNAs and deliver them for degradation. This approach has shown promise therapeutically in vitro and in vivo in mouse models of PD and AD and other neurological disorders; however, delivery of the siRNA to the CNS in vivo has been achieved primarily through intra-cerebral or intra-thecal injections that maybe less amenable for clinical translation; therefore, alternative approaches for delivery of siRNAs to the brain is needed. Recently, we described a small peptide from the envelope protein of the rabies virus (C2-9r) that was utilized to deliver an siRNA targeting α-syn across the blood brain barrier (BBB) following intravenous injection. This approach showed reduced expression of α-syn and neuroprotection in a toxic mouse model of PD. However, since receptor-mediated delivery is potentially saturable, each allowing the delivery of a limited number of molecules, we identified an alternative peptide for the transport of nucleotides across the BBB based on the apolipoprotein B (apoB) protein targeted to the family of low-density lipoprotein receptors (LDL-R). We used an 11-amino acid sequence from the apoB protein (ApoB(11)) that, when coupled with a 9-amino acid arginine linker, can transport siRNAs across the BBB to neuronal and glial cells. To examine the value of this peptide mediated oligonucleotide delivery system for PD, we delivered an siRNA targeting the α-syn (siα-syn) in a transgenic mouse model of PD. We found that ApoB(11) was effective (comparable to C2-9r) at mediating the delivery of siα-syn into the CNS, co-localized to neurons and glial cells and reduced levels of α-syn protein translation and accumulation. Delivery of ApoB(11)/siα-syn was accompanied by protection from degeneration of selected neuronal populations in the neocortex, limbic system and striato-nigral system and reduced neuro-inflammation. Taken together, these results suggest that systemic delivery of oligonucleotides targeting α-syn using ApoB(11) might be an interesting alternative strategy worth considering for the experimental treatment of synucleinopathies.

α-Synuclein is the major component of Lewy bodies and a candidate biomarker for neurodegenerative diseases in which Lewy bodies are common, including Parkinson's disease and dementia with Lewy bodies. A large body of literature suggests that these disorders are characterized by reduced concentrations of α-synuclein in cerebrospinal fluid (CSF), with overlapping concentrations compared to healthy controls and variability across studies. Several reasons can account for this variability, including technical ones, such as inter-assay and inter-laboratory variation (reproducibility). We compared four immunochemical methods for the quantification of α-synuclein concentration in 50 unique CSF samples. All methods were designed to capture most of the existing α-synuclein forms in CSF (‘total’ α-synuclein). Each of the four methods showed high analytical precision, excellent correlation between laboratories (R2 0.83–0.99), and good correlation with each other (R2 0.64–0.93), although the slopes of the regression lines were different between the four immunoassays. The use of common reference CSF samples decreased the differences in α-synuclein concentration between detection methods and technologies. Pilot data on an immunoprecipitation mass spectrometry (IP-MS) method is also presented. Our results suggest that the four immunochemical methods and the IP-MS method measure similar forms of α-synuclein and that a common reference material would allow harmonization of results between immunoassays. (Figure presented.).

Parkinson's disease is a progressive neurodegenerative disorder characterized by altered striatal dopaminergic signalling that leads to motor and cognitive deficits. Parkinson's disease is also characterized by abnormal presence of soluble toxic forms of α-synuclein that, when clustered into Lewy bodies, represents one of the pathological hallmarks of the disease. However, α-synuclein oligomers might also directly affect synaptic transmission and plasticity in Parkinson's disease models. Accordingly, by combining electrophysiological, optogenetic, immunofluorescence, molecular and behavioural analyses, here we report that α-synuclein reduces N-methyl-d-aspartate (NMDA) receptor-mediated synaptic currents and impairs corticostriatal long-term potentiation of striatal spiny projection neurons, of both direct (D1-positive) and indirect (putative D2-positive) pathways. Intrastriatal injections of α-synuclein produce deficits in visuospatial learning associated with reduced function of GluN2A NMDA receptor subunit indicating that this protein selectively targets this subunit both in vitro and ex vivo. Interestingly, this effect is observed in spiny projection neurons activated by optical stimulation of either cortical or thalamic glutamatergic afferents. We also found that treatment of striatal slices with antibodies targeting α-synuclein prevents the α-synuclein-induced loss of long-term potentiation and the reduced synaptic localization of GluN2A NMDA receptor subunit suggesting that this strategy might counteract synaptic dysfunction occurring in Parkinson's disease.

Both bacterial viability and concentration are significant metrics for bacterial detection. Existing miniaturized and cost-effective single-mode sensor, pH or optical, can only be skilled at detecting single information viability or concentration. This paper presents an inverter-based CMOS ion-sensitive-field-effect-transistor (ISFET) sensor array, featuring bacterial pH detection which is an indicator of viability. The proposed design realizes pH detection using the native passivation layer of CMOS process as a sensing layer and configuring an inverter-based front-end as a capacitive feedback amplifier. This sensor array is assisted by temperature sensing and optical detection which reveals bacterial concentration. The optical detection is enabled using the leakage current of a reset switch as a response to a light source. While in reset mode, the inverter-based amplifier works as a temperature sensor that could help to reduce temperature influences on pH and optical detection. All the functionalities are realized using one single inverter-based amplifier, resulting in a compact pixel structure and largely relaxed design complexity for the sensor system. Fabricated in 0.18 $\mu$ m standard CMOS process, the proposed CMOS sensor array system achieves an amplified pH sensitivity of 221 mV/pH, an improved sensor resolution of 0.03 pH through systematic noise optimization, a linear optical response, and a maximum temperature error of 0.69 $^{\circ }$ C. The sensing capabilities of the proposed design are demonstrated through on-chip Escherichia coli (E. coli) detection. This study may be extended to a rapid and cost-effective platform that renders multiple information of bacterial samples.

Aggregated α-synuclein (αSyn) protein is a core pathological feature of Parkinson’s disease (PD) and dementia with Lewy bodies (DLB). Both PD and DLB demonstrate the presence of diverse intracellular α-synuclein (αSyn) species, including C-terminally truncated αSyn (C-αSyn), although it is unknown how C-αSyn species contribute to disease progression. Using recombinant C-αSyn and PD and DLB brain lysates as seeds in the real-time quaking-induced conversion (RT-QuIC) assay, we explored how C-αSyn may be involved in disease stratification. Comparing the seeding activity of aqueous-soluble fractions to detergent-soluble fractions, and using αSyn 1-130 as substrate for the RT-QuIC assay, the temporal cortex seeds differentiated PD and DLB from healthy controls. In contrast to the temporal cortex, where PD and DLB could not be distinguished, αSyn 1-130 seeded by the detergent-soluble fractions from the PD frontal cortex demonstrated greater seeding efficiency compared to the DLB frontal cortex. Moreover, proteinase K-resistant (PKres) fragments from the RT-QuIC end products using C-αSyn 1-130 or C-αSyn 1-115 were more obvious in the frontal cortex compared to the temporal cortex. Morphological examinations of RT-QuIC end products showed differences in the size of the fibrils between C-αSyn 1-130 and C-αSyn 1-115, in agreement with the RT-QuIC results. These data show that C-αSyn species can distinguish PD from DLB and suggest diversity in αSyn species across these synucleinopathies, which could play a role in disease progression.

The accumulation and aggregation of α-synuclein (α-syn) is the main pathologic event in Parkinson’s disease (PD), dementia with Lewy bodies, and multiple system atrophy. α-Syn-seeded fibril formation and its induced toxicity occupy a major role in PD pathogenesis. Thus, assessing compounds that inhibit this seeding process is considered a key towards the therapeutics of synucleinopathies. Using biophysical and biochemical techniques and seeding-dependent cell viability assays, we screened a total of nine natural compounds of alkaloid origin extracted from Chinese medicinal herbs. Of these compounds, synephrine, trigonelline, cytisine, harmine, koumine, peimisine, and hupehenine exhibited in vitro inhibition of α-syn-seeded fibril formation. Furthermore, using cell viability assays, six of these compounds inhibited α-syn-seeding-dependent toxicity. These six potent inhibitors of amyloid fibril formation and toxicity caused by the seeding process represent a promising therapeutic strategy for the treatment of PD and other synucleinopathies.

Autism spectrum disorder (ASD) is a multifactorial neurodevelopmental disorder characterized by impairments in two main areas: social/communication skills and repetitive behavioral patterns. The prevalence of ASD has increased in the past two decades, however, it is not known whether the evident rise in ASD prevalence is due to changes in diagnostic criteria or an actual increase in ASD cases. Due to the complexity and heterogeneity of ASD, symptoms vary in severity and may be accompanied by comorbidities such as epilepsy, attention deficit hyperactivity disorder (ADHD), and gastrointestinal (GI) disorders. Identifying biomarkers of ASD is not only crucial to understanding the biological characteristics of the disorder, but also as a detection tool for its early screening. Hence, this review gives an insight into the main areas of ASD biomarker research that show promising findings. Finally, it covers success stories that highlight the importance of precision medicine and the current challenges in ASD biomarker discovery studies.

In early-stage Parkinson&prime, s disease (PD), cognitive impairment is common, and a variety of cognitive domains including memory, attention, and executive functioning may be affected. Cerebrospinal fluid (CSF) biomarkers are potential markers of cognitive functioning. We aimed to explore whether CSF &alpha, synuclein species, neurofilament light chain, amyloid-&beta, 42, and tau are associated with cognitive performance in early-stage PD patients. CSF levels of total-&alpha, synuclein and phosphorylated-&alpha, synuclein, neurofilament light chain, amyloid-&beta, 42, and total-tau and phosphorylated-tau were measured in 26 PD patients (disease duration &le, 5 years and Hoehn and Yahr stage 1&ndash, 2.5). Multivariable linear regression models, adjusted for age, gender, and educational level, were used to assess the relationship between CSF biomarker levels and memory, attention, executive and visuospatial function, and language performance scores. In 26 early-stage PD patients, attention and memory were the most commonly affected domains. A higher CSF phosphorylated-&alpha, synuclein/total-&alpha, synuclein ratio was associated with better executive functioning (s&beta, = 0.40). Higher CSF neurofilament light was associated with worse memory (s&beta, = &minus, 0.59), attentional (s&beta, 0.32), and executive functioning (s&beta, 0.35). Reduced CSF amyloid-&beta, 42 levels were associated with poorer attentional functioning (s&beta, = 0.35). Higher CSF phosphorylated-tau was associated with worse language functioning (s&beta, 0.33). Thus, CSF biomarker levels, in particular neurofilament light, were related to the most commonly affected cognitive domains in early-stage PD. This indicates that CSF biomarker levels may identify early-stage PD patients who are at an increased risk of developing cognitive impairment.

Neurons of the nucleus basalis of Meynert (nbM) are vulnerable to Lewy body formation and neuronal loss, which is thought to underlie cognitive dysfunction in Lewy body dementia (LBD). There is continued debate about whether Lewy bodies exert a neurodegenerative effect by affecting mitochondria, or whether they represent a protective mechanism. Therefore, the present study sought to determine whether the nbM is subject to mitochondrial dysfunctional in LBD and the association of Lewy body formation with such changes. Post-mortem nbM tissue was stained for Complex I or IV and quantitated relative to porin with immunofluorescence using confocal microscopy of individual cells from LBD (303 neurons, 8 cases), control (362 neurons, 8 cases) and asymptomatic incidental LBD (iLBD) cases (99 neurons, 2 cases). Additionally, α-synuclein, tau and amyloid-β pathology were analysed using quantitative immunohistochemistry, and respiratory chain markers were compared in cells with Lewy bodies (N = 134) and unaffected cells (N = 272). The expression of Complex I normalised to mitochondrial mass was significantly lower in LBD compared to control and iLBD cases and this was unrelated to local neuropathological burdens but trended toward a relationship with neuronal loss. Furthermore, Complex I expression was higher in cells with Lewy bodies compared to adjacent cells without α-synuclein aggregates. These findings suggest that Complex I deficits in the nbM occur in symptomatic LBD cases and may relate to neuronal loss, but that contrary to the view that Lewy body formation underlies neuronal dysfunction and damage in LBD, Lewy bodies are associated with higher Complex I expression than neurons without Lewy bodies. One could speculate that Lewy bodies may provide a mechanism to encapsulate damaged mitochondria and/or α-synuclein oligomers, thus protecting neurons from their cytotoxic effects.

In Parkinson’s disease, synucleinopathy is hypothesized to spread from the enteric nervous system, via the vagus nerve, to the CNS. Here, we compare, in baboon monkeys, the pathological consequences of either intrastriatal or enteric injection of α-synuclein-containing Lewy body extracts from patients with Parkinson’s disease. This study shows that patient-derived α-synuclein aggregates are able to induce nigrostriatal lesions and enteric nervous system pathology after either enteric or striatal injection in a non-human primate model. This finding suggests that the progression of α-synuclein pathology might be either caudo-rostral or rostro-caudal, varying between patients and disease subtypes. In addition, we report that α-synuclein pathological lesions were not found in the vagal nerve in our experimental setting. This study does not support the hypothesis of a transmission of α-synuclein pathology through the vagus nerve and the dorsal motor nucleus of the vagus. Instead, our results suggest a possible systemic mechanism in which the general circulation would act as a route for long-distance bidirectional transmission of endogenous α-synuclein between the enteric and the central nervous systems. Taken together, our study provides invaluable primate data exploring the role of the gut-brain axis in the initiation and propagation of Parkinson’s disease pathology and should open the door to the development and testing of new therapeutic approaches aimed at interfering with the development of sporadic Parkinson’s disease.

Aims: Lewy body diseases are neuropathologically characterized by the abnormal accumulation of α-synuclein (α-syn) protein within vulnerable neurons. Although studies have evaluated α-syn in post mortem brain tissue, previous findings have been limited by typically employing pan-α-syn antibodies that may not recognize disease-relevant forms of protein. We investigated the presence of α-syn species present in post mortem brain tissues from Lewy body disease and Alzheimer's disease. Methods: Soluble and insoluble/aggregated α-syn from frontal cortex of post mortem brain tissues form Parkinson's disease (PD), dementia with Lewy bodies (DLB), Alzheimer's disease (AD) and aged control cases were sequentially extracted using buffers with increasing detergent concentrations. Enzyme-linked immunosorbent assay (ELISA) was used to quantify the levels of total-, oligomeric- and phosphorylated-Ser129-α-syn (t-, o- and pS129-α-syn). ELISA data were validated by western blot and compared to histological data from the same region of the contralateral hemisphere. Results: There was no difference in t-α-syn levels between groups in the aqueous-soluble, detergent-soluble or urea-soluble tissue fractions. However, aqueous-soluble non-phosphorylated o-α-syn was increased not only in PD and DLB but also in AD without neocortical Lewy bodies. In PD and AD, pS129-α-syn was increased in the detergent-soluble tissue fragment and, in AD, this was positively correlated with the burden of tau pathology. Increased levels of urea-soluble pS129-α-syn were demonstrated only in DLB tissue lysates but this did not correlate with Lewy body pathological burden. Conclusions: Taken together, these findings suggest that DLB have elevated levels of insoluble pS129-α-syn, but that increased levels of aqueous-soluble o-α-syn and detergent-soluble pS129-α-syn are also observed in PD and AD, suggesting different changes to α-syn across the spectrum of neurodegenerative proteopathies. (Less)

Background: The objective of this study was to investigate the discriminating value of a range of CSF α‐synuclein species for dementia with Lewy bodies compared with Alzheimer's disease, PD, and cognitively normal controls. Methods: We applied our recently published enzyme‐linked immunosorbent assays to measure the CSF levels of total α‐synuclein, oligomeric α‐synuclein, and phosphorylated α‐synuclein in dementia with Lewy bodies (n = 42), Alzheimer's disease (n = 39), PD (n = 46), and controls (n = 78). General linear models corrected for age and sex were performed to assess differences in α‐synuclein levels between groups. We used backward‐elimination logistic regression analysis to investigate the combined discriminating value of the different CSF α‐synuclein species and Alzheimer's disease biomarkers. Results: CSF levels of total α‐synuclein were lower in dementia with Lewy bodies and PD compared with Alzheimer's disease as well as controls (P 0.40, P

Parkinson's disease biomarkers are needed to increase diagnostic accuracy, to objectively monitor disease progression and to assess therapeutic efficacy as well as target engagement when evaluating novel drug and therapeutic strategies. This article summarizes perianalytical considerations for biomarker studies (based on immunoassays) in Parkinson's disease, with emphasis on quantifying total α-synuclein protein in biological fluids. Current knowledge and pitfalls are discussed, and selected perianalytical variables are presented systematically, including different temperature of sample collection and types of collection tubes, gradient sampling, the addition of detergent, aliquot volume, the freezing time, and the different thawing methods. We also discuss analytical confounders. We identify gaps in the knowledge and delineate specific areas that require further investigation, such as the need to identify posttranslational modifications of α-synuclein and antibody-independent reference methods for quantification, as well as the analysis of potential confounders, such as comorbidities, medication, and phenotypes of Parkinson's disease in larger cohorts. This review could be used as a guideline for future Parkinson's disease biomarker studies and will require regular updating as more information arises in this growing field, including new technical developments as they become available. In addition to reviewing best practices, we also identify the current technical limitations and gaps in the knowledge that should be addressed to enable accurate and quantitative assessment of α-synuclein levels in the clinical setting. © 2017 International Parkinson and Movement Disorder Society.

Braak and Del Tredici have proposed that typical Parkinson disease (PD) has its origins in the olfactory bulb and gastrointestinal tract. However, the role of the olfactory system has insufficiently been explored in the pathogeneses of PD and Alzheimer disease (AD) in laboratory models. Here, we demonstrate applications of a new method to process mouse heads for microscopy by sectioning, mounting, and staining whole skulls (‘holocranohistochemistry’). This technique permits the visualization of the olfactory system from the nasal cavity to mitral cells and dopamine-producing interneurons of glomeruli in the olfactory bulb. We applied this method to two specific goals: first, to visualize PD- and AD-linked gene expression in the olfactory system, where we detected abundant, endogenous α-synuclein and tau expression in the olfactory epithelium. Furthermore, we observed amyloid-β plaques and proteinase-K-resistant α-synuclein species, respectively, in cranial nerve-I of APP- and human SNCA-over-expressing mice. The second application of the technique was to the modeling of gene–environment interactions in the nasal cavity of mice. We tracked the infection of a neurotropic respiratory-enteric-orphan virus from the nose pad into cranial nerves-I (and -V) and monitored the ensuing brain infection. Given its abundance in the olfactory epithelia, we questioned whether α-synuclein played a role in innate host defenses to modify the outcome of infections. Indeed, Snca-null mice were more likely to succumb to viral encephalitis versus their wild-type littermates. Moreover, using a bacterial sepsis model, Snca-null mice were less able to control infection after intravenous inoculation with Salmonella typhimurium. Together, holocranohistochemistry enabled new discoveries related to α-synuclein expression and its function in mice. Future studies will address: the role of Mapt and mutant SNCA alleles in infection paradigms; the contribution of xenobiotics in the initiation of idiopathic PD; and the safety to the host when systemically targeting α-synuclein by immunotherapy. Electronic supplementary material The online version of this article (doi:10.1007/s00702-017-1726-7) contains supplementary material, which is available to authorized users.

The majority of α-synuclein (α-syn) within Lewy bodies (LBs) has been reported to be phosphorylated at serine 129 (pS129-α-syn), suggesting a central role for phosphorylation in the pathogenesis of Parkinson's disease (PD) and related synucleinopathies. Various studies have investigated the effect of α-syn phosphorylation but have failed to reach a consensus as to whether this modification accelerates or inhibits α-syn aggregation. Nevertheless, pS129-α-syn is a reliable marker of α-syn aggregates and is widely evaluated in biomarkers and post-mortem studies. While several antibodies specific for pS129-α-syn exist, their reactivity with non-specific antigens appears to be a common challenge. To gain valuable insights into the role of α-syn phosphorylation in disease pathogenesis, antibodies that are highly specific to pS129-α-syn are necessary. In this study, we describe the generation of three mouse monoclonal antibodies (mAbs; 5B9, 6H5 and 9G1) using hybridoma technology. These were thoroughly characterized and validated in combination with our previously generated mAb (PS129), and the commercial ab51253 (Abcam). We demonstrated that our mAbs are highly specific for pS129-α-syn and do not cross react with wild-type α-syn. Results from staining of post-mortem human brain tissue showed that our mAbs detect pS129-α-syn pathology in patients with synucleinopathies. This study highlights three new antibodies as excellent and highly specific research tools to explore the role of pS129-α-syn inclusions in synucleinopathies.

Machine learning–based approach unravels distinct pathological signatures induced by patient-derived α-synuclein seeds in monkeys., Dopaminergic neuronal cell death, associated with intracellular α-synuclein (α-syn)–rich protein aggregates [termed “Lewy bodies” (LBs)], is a well-established characteristic of Parkinson’s disease (PD). Much evidence, accumulated from multiple experimental models, has suggested that α-syn plays a role in PD pathogenesis, not only as a trigger of pathology but also as a mediator of disease progression through pathological spreading. Here, we have used a machine learning–based approach to identify unique signatures of neurodegeneration in monkeys induced by distinct α-syn pathogenic structures derived from patients with PD. Unexpectedly, our results show that, in nonhuman primates, a small amount of singular α-syn aggregates is as toxic as larger amyloid fibrils present in the LBs, thus reinforcing the need for preclinical research in this species. Furthermore, our results provide evidence supporting the true multifactorial nature of PD, as multiple causes can induce a similar outcome regarding dopaminergic neurodegeneration.

Animal models that accurately recapitulate the accumulation of alpha-synuclein (α-syn) inclusions, progressive neurodegeneration of the nigrostriatal system and motor deficits can be useful tools for Parkinson's disease (PD) research. The preformed fibril (PFF) synucleinopathy model in rodents generally displays these PD-relevant features, however, the magnitude and predictability of these events is far from established. We therefore sought to optimize the magnitude of α-syn accumulation and nigrostriatal degeneration, and to understand the time course of both. Rats were injected unilaterally with different quantities of α-syn PFFs (8 or 16 μg of total protein) into striatal sites selected to concentrate α-syn inclusion formation in the substantia nigra pars compacta (SNpc). Rats displayed an α-syn PFF quantity-dependent increase in the magnitude of ipsilateral SNpc inclusion formation at 2 months and bilateral loss of nigral dopamine neurons at 6 months. Unilateral 16 μg PFF injection also resulted in modest sensorimotor deficits in forelimb adjusting steps associated with degeneration at 6 months. Bilateral injection of 16 μg α-syn PFFs resulted in symmetric bilateral degeneration equivalent to the ipsilateral nigral degeneration observed following unilateral 16 μg PFF injection (~50% loss). Bilateral PFF injections additionally resulted in alterations in several gait analysis parameters. These α-syn PFF parameters can be applied to generate a reproducible synucleinopathy model in rats with which to study pathogenic mechanisms and vet potential disease-modifying therapies.

Parkinson's disease is the second most common neurodegenerative disorder after Alzheimer's disease and is estimated to affect approximately 1-4% of individuals aged over 60 years old. Although considerable efforts have been invested into developing disease-modifying therapies for Parkinson's disease, such efforts have been confounded by the difficulty in accurately diagnosing Parkinson's disease during life to enable accurate patient stratification for clinical trialling of candidate therapeutics. Therefore, the search for effective biomarkers that can be accurately evaluated during life with non-invasive means is a pressing issue in the field. Since the discovery of α-synuclein (α-syn) as a protein linked to a familial form of Parkinson's disease, later identified as the major protein component of the neuropathological hallmark of idiopathic Parkinson's disease, considerable interest has focused on this protein and its distinct conformers. We describe here the progress that has been made in the area of Parkinson's disease biomarker discovery with a focus on α-synuclein. In particular, we highlight the novel assays that have been employed and the increasing complexity in evaluating α-synuclein with regard to the considerable diversity of conformers that exist in the biofluids and peripheral tissues under disease conditions. "This article is part of the Special Issue Synuclein."

Synucleinopathies including Parkinson's disease, dementia with Lewy bodies and multiple system atrophy are characterized by the abnormal accumulation and propagation of α-synuclein (α-syn) pathology in the central and peripheral nervous system as Lewy bodies or glial cytoplasmic inclusions. Several antibodies against α-syn have been developed since it was first detected as the major component of Lewy bodies and glial cytoplasmic inclusions. Over the years, researchers have generated specific antibodies that alleviate the accumulation of intracellular aggregated α-syn and associated pathology in cellular and preclinical models of synucleinopathies. So far, antibodies have been the first choice as tools for research and diagnosis and currently, a wide variety of antibody fragments have been developed as an alternative to full-length antibodies for increasing its therapeutic usefulness. Recently, conformation specific antibody-based approaches have been found to be promising as therapeutic strategies, both to block α-syn aggregation and ameliorate the resultant cytotoxicity, and as diagnostic tools. In this review, we summarize different α-syn specific antibodies and provide their usefulness in tackling synucleinopathies. This article is part of the Special Issue "Synuclein".

Background Parkinson's disease (PD) diagnosis is mainly based on clinical criteria, with a high risk of misdiagnosis. The identification of reliable biomarkers for disease diagnosis and progression has a key role for developing disease-modifying therapies. In this article, we investigated the longitudinal changes of CSF α-synuclein species in early PD patients and explored the potential use of these species as surrogate biomarkers for PD progression. Methods We used our newly developed enzyme-linked immunosorbent assay systems for measuring different forms of α-synuclein, such as oligomeric-α-synuclein, phosphorylated-α-synuclein at serine 129, or total-α-synuclein in CSF from the longitudinal Deprenyl and Tocopherol Antioxidative Therapy for Parkinsonism study cohort (n = 121). CSF Alzheimer's disease biomarkers (total-tau, phosphorylated-tau, Aβ40, and Aβ42) were also measured for this cohort. Results Interestingly, total-α-synuclein and oligomeric-α-synuclein levels significantly increased during the 2-year Deprenyl and Tocopherol Antioxidative Therapy for Parkinsonism study follow-up period, whereas phosphorylated-α-synuclein at serine 129 levels showed a longitudinal decrease. We have also noted an association between a change of the oligomeric-α-synuclein/total-α-synuclein ratio and a worsening of motor signs, in particular in the postural-instability and gait-difficulty dominant PD group. A strong positive correlation between the changes in CSF total-α-synuclein and oligomeric-α-synuclein during the 2-year Deprenyl and Tocopherol Antioxidative Therapy for Parkinsonism study was also noted (r = 0.84, P

Parkinson's disease (PD) is a debilitating neurodegenerative disease characterized by tremor, rigidity, bradykinesia, and postural instability, for which there is no effective treatment available till date. Here, we report the development of nonviral vectors specific for neuronal cells that can deliver short interfering RNA (siRNA) against the α-synuclein gene (SNCA), and prevent PD-like symptoms both in vitro and in vivo. These vectors not only help siRNA duplexes cross the blood–brain barrier in mice, but also stabilize these siRNAs leading to a sustainable 60–90% knockdown of α-synuclein protein. Mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine rapidly develop PD-like symptoms which were significantly alleviated when SNCA was knocked down using our vectors. Together, our data not only confirm the central role of α-synuclein in the onset of PD, but also provide a proof of principle that these nonviral vectors can be used as novel tools to design effective strategies to combat central nervous system diseases.