Rigor and reproducibility in human brain organoid research: Where we are and where we need to go.

Human brain organoid models have emerged as a promising tool for studying human brain development and function. These models preserve human genetics and recapitulate some aspects of human brain development, while facilitating manipulation in an in vitro setting. Despite their potential to transform biology and medicine, concerns persist about their fidelity. To fully harness their potential, it is imperative to establish reliable analytic methods, ensuring rigor and reproducibility. Here, we review current analytical platforms used to characterize human forebrain cortical organoids, highlight challenges, and propose recommendations for future studies to achieve greater precision and uniformity across laboratories.

A novel iPSC model reveals selective vulnerability of neurons in multiple sulfatase deficiency.

Multiple sulfatase deficiency (MSD) is an ultra-rare, inherited lysosomal storage disease caused by mutations in the gene sulfatase modifying factor 1 (SUMF1). MSD is characterized by the functional deficiency of all sulfatase enzymes, leading to the storage of sulfated substrates including glycosaminoglycans (GAGs), sulfolipids, and steroid sulfates. Patients with MSD experience severe neurological impairment, hearing loss, organomegaly, corneal clouding, cardiac valve disease, dysostosis multiplex, contractures, and ichthyosis. Here, we generated a novel human model of MSD by reprogramming patient peripheral blood mononuclear cells to establish an MSD induced pluripotent stem cell (iPSC) line (SUMF1 p.A279V). We also generated an isogenic control iPSC line by correcting the pathogenic variant with CRISPR/Cas9 gene editing. We successfully differentiated these iPSC lines into neural progenitor cells (NPCs) and NGN2-induced neurons (NGN2-iN) to model the neuropathology of MSD. Mature neuronal cells exhibited decreased SUMF1 gene expression, increased lysosomal stress, impaired neurite outgrowth and maturation, reduced sulfatase activities, and GAG accumulation. Interestingly, MSD iPSCs and NPCs did not exhibit as severe of phenotypes, suggesting that as neurons differentiate and mature, they become more vulnerable to loss of SUMF1. In summary, we demonstrate that this human iPSC-derived neuronal model recapitulates the cellular and biochemical features of MSD. These cell models can be used as tools to further elucidate the mechanisms of MSD pathology and for the development of therapeutics.

Reproducible Differentiation of Human Pluripotent Stem Cells into Two-Dimensional Cortical Neuron Cultures with Checkpoints for Success.

The patterning of excitatory cortical neurons from human pluripotent stem cells (hPSCs) is a desired technique for the study of neurodevelopmental disorders, as neurons can be created and compared from control hPSC lines, hPSC lines generated from patients, and CRISPR-modified hPSC lines. Therefore, this technique allows for the examination of disease phenotypes and assists in the development of potential new therapeutics for neurodevelopmental disorders. Many protocols, however, are optimized for use with specific hPSC lines or within a single laboratory, and they often provide insufficient guidance on how to identify positive stages in the differentiation or how to troubleshoot. Here, we present an efficient and reproducible directed differentiation protocol to generate two-dimensional cultures of hPSC-derived excitatory cortical neurons without intermediary embryoid body formation. This novel protocol is supported by our data generated with five independent hPSC lines and in two independent laboratories. Importantly, as neuronal differentiations follow a long time course to reach maturity, we provide extensive guidance regarding morphological and flow cytometry checkpoints allowing for early indications of successful differentiation. We also include extensive troubleshooting tips and support protocols to assist the operator. The goal of this protocol is to assist others in the successful differentiation of excitatory cortical neurons from hPSCs. © 2023 Wiley Periodicals LLC. Basic Protocol: Directed differentiation of hPSCs into excitatory cortical neurons Support Protocol 1: Harvesting and fixing cells for flow cytometry analyses Support Protocol 2: Performing flow cytometry analyses Support Protocol 3: Thawing NPCs from a cryopreserved stock Alternate Protocol 1: Continuing Expansion of NPCs Alternate Protocol 2: Treatment of neurons with Ara-C to ablate radial glia Support Protocol 4: Experimental methods for validation of excitatory cortical neurons.

Cross-site reproducibility of human cortical organoids reveals consistent cell type composition and architecture.

Background: Reproducibility of human cortical organoid (hCO) phenotypes remains a concern for modeling neurodevelopmental disorders. While guided hCO protocols reproducibly generate cortical cell types in multiple cell lines at one site, variability across sites using a harmonized protocol has not yet been evaluated. We present an hCO cross-site reproducibility study examining multiple phenotypes. Methods: Three independent research groups generated hCOs from one induced pluripotent stem cell (iPSC) line using a harmonized miniaturized spinning bioreactor protocol. scRNA-seq, 3D fluorescent imaging, phase contrast imaging, qPCR, and flow cytometry were used to characterize the 3 month differentiations across sites. Results: In all sites, hCOs were mostly cortical progenitor and neuronal cell types in reproducible proportions with moderate to high fidelity to the in vivo brain that were consistently organized in cortical wall-like buds. Cross-site differences were detected in hCO size and morphology. Differential gene expression showed differences in metabolism and cellular stress across sites. Although iPSC culture conditions were consistent and iPSCs remained undifferentiated, primed stem cell marker expression prior to differentiation correlated with cell type proportions in hCOs. Conclusions: We identified hCO phenotypes that are reproducible across sites using a harmonized differentiation protocol. Previously described limitations of hCO models were also reproduced including off-target differentiations, necrotic cores, and cellular stress. Improving our understanding of how stem cell states influence early hCO cell types may increase reliability of hCO differentiations. Cross-site reproducibility of hCO cell type proportions and organization lays the foundation for future collaborative prospective meta-analytic studies modeling neurodevelopmental disorders in hCOs.

Generation of a human Tropomyosin 1 knockout iPSC line.

The CHOPWT17_TPM1KOc28 iPSC line was generated to interrogate the functions of Tropomyosin 1 (TPM1) in primary human cell development. This line was reprogrammed from a previously published wild type control iPSC line.

Gain and loss of function variants in EZH1 disrupt neurogenesis and cause dominant and recessive neurodevelopmental disorders.

Genetic variants in chromatin regulators are frequently found in neurodevelopmental disorders, but their effect in disease etiology is rarely determined. Here, we uncover and functionally define pathogenic variants in the chromatin modifier EZH1 as the cause of dominant and recessive neurodevelopmental disorders in 19 individuals. EZH1 encodes one of the two alternative histone H3 lysine 27 methyltransferases of the PRC2 complex. Unlike the other PRC2 subunits, which are involved in cancers and developmental syndromes, the implication of EZH1 in human development and disease is largely unknown. Using cellular and biochemical studies, we demonstrate that recessive variants impair EZH1 expression causing loss of function effects, while dominant variants are missense mutations that affect evolutionarily conserved aminoacids, likely impacting EZH1 structure or function. Accordingly, we found increased methyltransferase activity leading to gain of function of two EZH1 missense variants. Furthermore, we show that EZH1 is necessary and sufficient for differentiation of neural progenitor cells in the developing chick embryo neural tube. Finally, using human pluripotent stem cell-derived neural cultures and forebrain organoids, we demonstrate that EZH1 variants perturb cortical neuron differentiation. Overall, our work reveals a critical role of EZH1 in neurogenesis regulation and provides molecular diagnosis for previously undefined neurodevelopmental disorders.

Mapping PTBP2 binding in human brain identifies SYNGAP1 as a target for therapeutic splice switching.

Alternative splicing of neuronal genes is controlled partly by the coordinated action of polypyrimidine tract binding proteins (PTBPs). While PTBP1 is ubiquitously expressed, PTBP2 is predominantly neuronal. Here, we define the PTBP2 footprint in the human transcriptome using brain tissue and human induced pluripotent stem cell-derived neurons (iPSC-neurons). We map PTBP2 binding sites, characterize PTBP2-dependent alternative splicing events, and identify novel PTBP2 targets including SYNGAP1, a synaptic gene whose loss-of-function leads to a complex neurodevelopmental disorder. We find that PTBP2 binding to SYNGAP1 mRNA promotes alternative splicing and nonsense-mediated decay, and that antisense oligonucleotides (ASOs) that disrupt PTBP binding redirect splicing and increase SYNGAP1 mRNA and protein expression. In SYNGAP1 haploinsufficient iPSC-neurons generated from two patients, we show that PTBP2-targeting ASOs partially restore SYNGAP1 expression. Our data comprehensively map PTBP2-dependent alternative splicing in human neurons and cerebral cortex, guiding development of novel therapeutic tools to benefit neurodevelopmental disorders.

Generation of a human Tropomyosin 1 knockout iPSC line.

The CHOPWT17_TPM1KOc28 iPSC line was generated to interrogate the functions of Tropomyosin 1 ( TPM1 ) in primary human cell development. This line was reprogrammed from a previously published wild type control iPSC line.

Drug screening identifies tazarotene and bexarotene as therapeutic agents in multiple sulfatase deficiency.

Multiple sulfatase deficiency (MSD, MIM #272200) results from pathogenic variants in the SUMF1 gene that impair proper function of the formylglycine-generating enzyme (FGE). FGE is essential for the posttranslational activation of cellular sulfatases. MSD patients display reduced or absent sulfatase activities and, as a result, clinical signs of single sulfatase disorders in a unique combination. Up to date therapeutic options for MSD are limited and mostly palliative. We performed a screen of FDA-approved drugs using immortalized MSD patient fibroblasts. Recovery of arylsulfatase A activity served as the primary readout. Subsequent analysis confirmed that treatment of primary MSD fibroblasts with tazarotene and bexarotene, two retinoids, led to a correction of MSD pathophysiology. Upon treatment, sulfatase activities increased in a dose- and time-dependent manner, reduced glycosaminoglycan content decreased and lysosomal position and size normalized. Treatment of MSD patient derived induced pluripotent stem cells (iPSC) differentiated into neuronal progenitor cells (NPC) resulted in a positive treatment response. Tazarotene and bexarotene act to ultimately increase the stability of FGE variants. The results lay the basis for future research on the development of a first therapeutic option for MSD patients.

Characterization of cellular protective effects of ATP13A2/PARK9 expression and alterations resulting from pathogenic mutants.

Mutations in ATP13A2, which encodes a lysosomal P-type ATPase of unknown function, cause an autosomal recessive parkinsonian syndrome. With mammalian cells, we show that ATP13A2 expression protects against manganese and nickel toxicity, in addition to proteasomal, mitochondrial, and oxidative stress. Consistent with a recessive mode of inheritance of gene defects, disease-causing mutations F182L and G504R are prone to misfolding and do not protect against manganese and nickel toxicity because they are unstable as a result of degradation via the endoplasmic reticulum-associated degradation (ERAD)-proteasome system. The protective effects of ATP13A2 expression are not due to inhibition of apoptotic pathways or a reduction in typical stress pathways, insofar as these pathways are still activated in challenged ATP13A2-expressing cells; however, these cells display a dramatic reduction in the accumulation of oxidized and damaged proteins. These data indicate that, contrary to a previous suggestion, ATP13A2 is unlikely to convey cellular resilience simply by acting as a lysosomal manganese transporter. Consistent with the recent identification of an ATP13A2 recessive mutation in Tibetan terriers that develop neurodegeneration with neuronal ceroid lipofucinoses, our data suggest that ATP13A2 may function to import a cofactor required for the function of a lysosome enzyme(s).

E46K human alpha-synuclein transgenic mice develop Lewy-like and tau pathology associated with age-dependent, detrimental motor impairment.

Synucleinopathies are a group of neurodegenerative disorders associated with the formation of aberrant amyloid inclusions composed of the normally soluble presynaptic protein α-synuclein (α-syn). Parkinson disease is the most well known of these disorders because it bears α-syn pathological inclusions known as Lewy bodies (LBs). Mutations in the gene for α-syn, including the E46K missense mutation, are sufficient to cause Parkinson disease as well as other synucleinopathies like dementia with LBs. Herein, we describe transgenic mice expressing E46K human α-syn in CNS neurons that develop detrimental age-dependent motor impairments. These animals accumulate age-dependent intracytoplasmic neuronal α-syn inclusions that parallel disease and recapitulate the biochemical, histological, and morphological properties of LBs. Surprisingly, the morphology of α-syn inclusions in E46K human α-syn transgenic mice more closely resemble LBs than the previously described transgenic mice (line M83) that express neuronal A53T human α-syn. E46K human α-syn mice also develop abundant neuronal tau inclusions that resemble neurofibrillary tangles. Subsequent studies on the ability of E46K α-syn to induce tau inclusions in cellular models suggest that both direct and indirect mechanisms of protein aggregation are probably involved in the formation of the tau inclusions observed here in vivo. Re-evaluation of presymptomatic transgenic mice expressing A53T human α-syn reveals that the formation of α-syn inclusions in mice must be synchronized; however, inclusion formation is diffuse within affected areas of the neuroaxis such that there was no clustering of inclusions. Collectively, these findings provide insights in the mechanisms of formation of these aberrant proteinaceous inclusions and support the notion that α-syn aggregates are involved in the pathogenesis of human diseases.

Induction of intracellular tau aggregation is promoted by α-synuclein seeds and provides novel insights into the hyperphosphorylation of tau.

Intracytoplasmic proteinaceous inclusions, primarily composed of tau or α-synuclein (α-syn), are predominant pathological features of Alzheimer's disease (AD) and Parkinson's disease (PD), respectively. However, the coexistence of these pathological aggregates is identified in many neurodegenerative disorders, including spectrum disorders of AD and PD. Whereas α-syn can spontaneously polymerize into amyloidogenic fibrils, in vitro, tau polymerization requires an inducing agent. The current study presents a human-derived cellular model, in which recombinant, preformed α-syn fibrils cross-seed intracellular tau to promote the formation of neurofibrillary tangle-like aggregates. These aggregates were hyperphosphorylated, Triton insoluble, and thioflavin-S positive, either comingling with endogenously expressed α-syn aggregates or induced by only exogenously applied recombinant α-syn fibrils. Furthermore, filamentous, amyloidogenic tau took over the cellular soma, displacing the nucleus and isolating or displacing organelles, likely preventing cellular function. Although a significant proportion of wild-type tau formed these cellular inclusions, the P301L mutation in tau increased aggregation propensity resulting from α-syn seeds to over 50% of total tau protein. The role of phosphorylation on the development of these tau aggregates was investigated by coexpressing glycogen synthase kinase 3 ß or microtubule-associated protein/microtubule affinity-regulating kinase 2. Expression of either kinase inhibited the formation of α-syn-induced tau aggregates. Analyses of phosphorylation sites suggest that multiple complex factors may be associated with this effect and that Triton-soluble versus Triton-insoluble tau may be independently targeted by kinases. The current work not only provides an exceptional cellular model of tau pathology, but also examines α-syn-induced tau inclusion formation and provides novel insights into hyperphosphorylation observed in disease.

Characterization of kinases involved in the phosphorylation of aggregated α-synuclein.

α-Synuclein (α-syn) is the major component of pathological inclusions characteristic of several neurodegenerative disorders, such as Parkinson's disease. The major posttranslational modification of α-syn is phosphorylation at S129, and previous studies estimate that approximately 90% of α-syn in proteinaceous, pathological inclusions is phosphorylated at this site. α-Syn can be phosphorylated by polo-like kinases (PLKs) 1-3 and casein kinases (CK) 1 and 2; however, the kinases associated with the hyperphosphorylation of aggregated α-syn are still under debate. Using a high-efficiency cellular model of α-syn aggregate formation, we found that selective inhibitors for CK2 and PLKs each partially inhibited S129 phosphorylation of soluble (nonaggregated) α-syn, but only PLK inhibitors modestly attenuated the phosphorylation of aggregated α-syn. In addition, none of the kinase inhibitors used had a substantial effect on the propensity of α-syn to aggregate. Overexpression of all PLKs promoted robust phosphorylation of soluble α-syn, but none altered the propensity of α-syn to aggregate. Overexpression of only PLK2 increased phosphorylation of aggregated α-syn at S129, which likely is due to increased phosphorylation of soluble α-syn, which then was incorporated into aggregates. Overexpression of PLK1 and treatment with BI2536 resulted in a significant reduction of phosphorylated, aggregated α-syn protein, beyond that of BI2536 treatment alone. These studies suggest that phosphorylation of α-syn is independent of α-syn aggregate formation, that PLK1 is involved in the phosphorylation of aggregated α-syn at S129 in this system, and that mechanisms resulting in hyperphosphorylation of aggregated α-syn appear to be independent of those responsible for the phosphorylation of soluble α-syn.

A novel, high-efficiency cellular model of fibrillar alpha-synuclein inclusions and the examination of mutations that inhibit amyloid formation.

Intracytoplasmic alpha-synuclein (alpha-syn) amyloidogenic inclusions are a major pathological feature of Parkinson's disease, dementia with Lewy body disease and multiple systems atrophy. The mechanisms involved in the formation and inhibition of these aggregates are areas of intense investigation. The present study characterizes a novel cellular model for the study of alpha-syn aggregation, incorporating nucleation-dependent aggregation and a new function for calcium phosphate precipitation. Cultured cells were readily induced to develop large, cytoplasmic alpha-syn filamentous aggregates that were hyperphosphorylated, often ubiquitinated and thioflavin positive. These cellular aggregates formed in the majority of transfected cells and recruited approximately half of endogenously expressed alpha-syn. Using this system, we examined single-point mutations that inhibit alpha-syn amyloid formation in vitro. Three mutations (V66P, T72P and T75P) significantly hindered alpha-syn aggregation in this cell model. The T75P mutant, which could abrogate amyloid formation of wild-type alpha-syn in vitro, did not prevent wild-type alpha-syn cellular aggregates. These studies suggest that the propensity of alpha-syn to form cellular aggregates may be more pronounced than in isolated in vitro studies. This novel high-efficiency cellular model of alpha-syn aggregation is a valuable system that may be used to further understand alpha-syn aggregation and allow for the generation of future therapeutics.

Residue Glu83 plays a major role in negatively regulating alpha-synuclein amyloid formation.

Alpha-synuclein (alpha-syn) amyloid filaments are the major ultrastructural component of pathological inclusions that define several neurodegenerative disorders, including Parkinson disease and other disorders that are collectively termed synucleinopathies. Since the aggregation of alpha-syn is associated with the etiology of these diseases, defining the molecular elements that influence this process may have important therapeutics implication. The deletions of major portions of the hydrophobic region of alpha-syn (Delta74-79 and Delta71-82) impair the ability to form amyloid. However, mutating residue E83 to an A restored the ability of these proteins to form amyloid. Additionally supporting an inhibitory role of residue E83 on amyloid formation, mutating this residue to an A enhanced amyloid formation in the presence of small molecule inhibitors, such as dopamine and EGCG. Our data, therefore, suggest that the presence and placement of the highly charged E83 residue plays a significant inhibitory role in alpha-syn amyloid formation and these findings provide important insights in the planning of therapeutic agents that may be capable of preventing alpha-syn amyloid formation.

Characterization of hydrophobic residue requirements for alpha-synuclein fibrillization.

alpha-Synuclein is the major component of pathological inclusions characteristic of diseases like Parkinson's disease, dementia with Lewy bodies, and multiple-system atrophy. A role for alpha-synuclein in neurodegenerative diseases is further supported by point mutations and duplication and triplication of the alpha-synuclein gene (SNCA) that are causative of these disorders. The middle hydrophobic region of the alpha-synuclein protein, also termed the "non-Abeta component of Alzheimer's disease amyloid plaque (NAC)" domain, is required for alpha-synuclein to polymerize into amyloid filaments, which are the major components of alpha-synuclein pathological inclusions. In this study, we assessed the importance of specific stretches of hydrophobic residues in driving the intrinsic ability of alpha-synuclein to polymerize. Several small deletions, even one with as few as two amino acid residues (A76 and V77), dramatically impaired the ability of alpha-synuclein to polymerize into mature amyloidogenic fibrils, and instead, it preferentially formed oligomers. However, this inhibition of filament assembly was clearly dependent on the spatial context, since similar and larger hydrophobic deletions in other parts of the NAC domain reduced only the rate of fibril formation, without abrogating filament assembly. Further, mutation of residue E83 to an A rescued the ability of mutant Delta76-77 alpha-synuclein to polymerize. These findings support the notion that while both the location and hydrophobicity of protein segments are important elements that affect the propensity to form amyloid fibrils, the intrinsic ability of a polypeptide to fold structurally into amyloid is also critical.

Leucine-rich repeat kinase 2 expression leads to aggresome formation that is not associated with alpha-synuclein inclusions.

Mutations in leucine-rich repeat kinase-2 (LRRK2) are the most common known cause of Parkinson disease, but how this protein results in the pathobiology of Parkinson disease is unknown. Moreover, there is variability in pathology among cases, and alpha-synuclein (alpha-syn) neuronal inclusions are often present, but whether LRRK2 is present in these pathological inclusions is controversial. This study characterizes novel LRRK2 antibodies, some of which preferentially recognize an aggregated form of LRRK2, as observed in cell culture models. Large perinuclear aggregates containing LRRK2 were promoted by proteasome inhibition and prevented by microtubule polymerization inhibition. Furthermore, they were vimentin- and gamma-tubulin- but not lamp1-immunoreactive, suggesting that these structures fit the definition of aggresomes. Inhibition of heat shock protein 90 led to the degradation of only the soluble/cytosolic pool of LRRK2, suggesting that the aggresomes formed independent of the stability provided by the heat shock protein 90. Although these novel anti-LRRK2 antibodies identified aggregates in model cell systems, they did not immunostain pathological inclusions in human brains. Furthermore, coexpression of LRRK2 and alpha-syn did not recruit alpha-syn into aggresomes in cultured cells, even in the presence of proteasome inhibition. Thus, although LRRK2 is a model system for aggresome formation, LRRK2 is not present in alpha-syn pathological inclusions.

Clinical and pathological characteristics of patients with leucine-rich repeat kinase-2 mutations.

Mutations in LRRK2 are the single most common known cause of Parkinson's disease (PD). Two new PD patients with LRRK2 mutation were identified from a cohort with extensive postmortem assessment. One of these patients harbors the R793M mutation and presented with the typical clinical and pathological features of PD. A novel L1165P mutation was identified in a second patient. This patient had the classical and pathological features of PD, but additionally developed severe neuropsychological symptoms and dementia associated with abundant neurofibrillary tangles in the hippocampal formation; features consistent with a secondary diagnosis of tangle-predominant dementia. alpha-Synuclein-containing pathological inclusions in these patients also were highly phosphorylated at Ser-129, similar to other patients with idiopathic PD. These two PD patients also were characterized by the presence of occasional cytoplasmic TDP-43 inclusions in the temporal cortex, a finding that was not observed in three other patients with the G2019S mutation in LRRK2. These findings extend the clinical and pathological features that may be associated with LRRK2 mutations.

Molecular mechanisms of alpha-synuclein neurodegeneration.

alpha-Synuclein is an abundant highly charged protein that is normally predominantly localized around synaptic vesicles in presynaptic terminals. Although the function of this protein is still ill-defined, genetic studies have demonstrated that point mutations or genetic alteration (duplications or triplications) that increase the number of copies of the alpha-synuclein (SCNA) gene can cause Parkinson's disease or the related disorder dementia with Lewy bodies. alpha-Synuclein can aberrantly polymerize into fibrils with typical amyloid properties, and these fibrils are the major component of many types of pathological inclusions, including Lewy bodies, which are associated with neurodegenerative diseases, such as Parkinson's disease. Although there is substantial evidence supporting the toxic nature of alpha-synuclein inclusions, other modes of toxicity such as oligomers have been proposed. In this review, some of the evidence for the different mechanisms of alpha-synuclein toxicity is presented and discussed.

Specificity and regulation of casein kinase-mediated phosphorylation of alpha-synuclein.

alpha-Synuclein (alpha-syn) is the major component of pathologic inclusions that characterize neurodegenerative disorders such as Parkinson disease, dementia with Lewy body disease, and multiple system atrophy. The present study uses novel phospho-specific antibodies to assess the presence and regulation of phosphorylated Ser87 and Ser129 in alpha-syn in human brain samples and in a transgenic mouse model of alpha-synucleinopathies. By immunohistochemistry, alpha-syn phosphorylated at Ser129, but not at Ser87, was abundant in alpha-syn inclusions. Under normal conditions, Ser129 phosphorylation, but not Ser87 phosphorylation, was detected at low levels in the soluble biochemical fractions in human alpha-syn transgenic mice and stably transfected cultured cells. Therefore, a role for Ser87 phosphorylation in alpha-synucleinopathies is unlikely, and in vitro assays showed that phosphorylation at this site would inhibit polymerization. In vitro studies also indicated that hyperphosphorylation of Ser129 alpha-syn in pathologic inclusions may be due in part to the intrinsic properties of aggregated alpha-syn to act as substrates for kinases but not phosphatases. Further studies in transgenic mice and cultured cells suggest that cellular toxicity, including proteasomal dysfunction, increases casein kinase 2 activity, which results in elevated Ser129 alpha-syn phosphorylation. These data provide novel explanations for the presence of hyperphosphorylated Ser129 alpha-syn in pathologic inclusions.