A minimalistic co-culture platform for alpha-synuclein spreading in human dopaminergic neurons

In this study, we characterized the bioenergetic response of the Lund human mesencephalic (LUHMES) cell line and a mouse astrocyte cell line to oxidative stress. Extracellular hydrogen peroxide (H2O2) levels and bioenergetic response were investigated in these cell lines after exposure to paraquat (PQ), a redox cycling compound that causes oxidative stress in cells. We used extracellular flux analysis to measure mitochondrial function in adherent astrocytes and LUHMES cells. Extracellular H2O2 was measured fluorometrically. H2O2 levels increased in both cell lines after exposure to 5 µM PQ for 18 h; however, the extent of H2O2 increase with astrocytes was significantly lower than that with LUHMES cells (33% vs. 67%). Measurements of basal mitochondrial respiration showed that PQ almost completely eliminated oxygen consumption rate (OCR) in astrocytes and significantly reduced it in LUHMES cells. Notably, OCR in LUHMES cells was higher than that in astrocytes, indicating that neuronal cells maintain higher oxidative metabolism than glial cells, which is also consistent with higher energy demands of the neuronal cells. Moreover, LUHMES cells exhibited a higher amount of adenosine triphosphate (ATP) being produced by oxidative phosphorylation than by glycolysis. In contrast, astrocytes demonstrated a higher glycolytic capacity and glycolytic reserve than LUHMES cells and higher ATP production rate by glycolysis than its production by mitochondrial oxidative metabolism. Collectively, this study showed the differential bioenergetic responses between astrocytes and LUHMES cells in responding to oxidative stress and the findings may provide insights into the mitochondrial reserve capacity in neurons and astrocytes in responding to oxidative stress. (First online: Mar 30, 2021)

Parkinson’s disease (PD) is a complex neurodegenerative disorder that manifests through a broad ran ge of motor and non - motor symptoms. Alpha - synuclein (aSyn) is the major protein component of Lewy bodies and Lewy neurites, considered the pathological hallmarks in PD and other synucleinopathies. Some familial forms of PD can be caused by duplication , triplication, or missense mutations in the gene encoding for aSyn. However, the precise molecular mechanisms linking aSyn to the disease are still elusive. Although it has been shown that aSyn plays a role in transcriptional deregulation, the effect of spe cific aSyn mutants associated with familial forms of PD, such as the A30P mutant, remains unclear. This thesis compiles two studies that contain our major findings focused on the role of aSyn on transcriptional deregulation in PD. In the first study, we ai med to investigate the impact of aSyn on transcriptional deregulation using Lund Human Mesencephalic (LUHMES) cells as a model, since they can be differentiated into dopaminergic neurons. To achieve this, we generated two cell lines expressing wild - type (W T) or mutant A30P aSyn and performed gene expression analysis using RNA - sequencing. We observed that both WT and A30P aSyn induced robust transcriptional deregulation, including changes in expression of DNA damage/repair genes. Interestingly, increased DNA damage and was only observed in WT aSyn dopaminergic neurons. Furthermore, WT aSyn affected mitochondrial ROS (miROS) handling, unlike A30P aSyn. In these cells, aSyn expression decreased expression of acetylated histone 3 (acH3) levels that were restored by treatment with sodium butyrate (NaB) , a histone deacetylase inhibitor (HDACi). Interestingly, NaB was able to rescue the DNA damage induced by aSyn expression, possibly by upregulation of DNA repair genes observed upon the treatment. Moreover, treatme nt with NaB was shown to ameliorate miROS handling in WT aSyn cells. In the second study, our main goal was to investigate the role of aSyn on transcriptional deregulation in transgenic mice models of PD. For this purpose , we used transgenic mice overexpr essing human WT aSyn and A30P and conducted gene 16 expression studies. We observed that A30P aSyn promotes stronger transcriptional deregulation and increases DNA binding when compared to endogenous aSyn, consistently with the results obtained previously in LUHMES cells. Importantly, we identified several biological processes affected by A30P mutant aSyn, such as ER - associated pathways. Interestingly, COL4A2, a pro - apoptotic gene, was found to be upregulated in both A30P aSyn transgenic mice and in dopaminerg ic neurons expressing A30P aSyn. Finally, we observed that aSyn A30P alters Golgi morphology and increases endoplasmic reticulum (ER) stress in dopaminergic cells. Our findings suggest that aSyn can impact on transcriptio n , both in dopaminergic neurons an d in transgenic mouse models of PD, and that A30P aSyn has a stronger effect than WT aSyn. These studies provide novel insight into the mechanism underlying aSyn - toxicity, including gene deregulation, histone modification, DNA damage, miROS handling and Golgi - ER systems . Ultimately, our studies open novel avenues for future therapeutic intervention in PD and other synucleinopathies.

BackgroundAlzheimer’s disease (AD) is a common neurodegenerative disorder that results in the accumulation of amyloid‐beta, neurofibrillary tangles, and progressive cognitive decline. Despite extensive research into the pathophysiology of AD and potential treatments, a definitive cure remains elusive. Appropriate in vitro cell models are crucial for understanding pathophysiology and drug screening for AD. Cell lines like HEK293, HeLa, SH‐SY5Y, and PC have been used to study AD pathology. However, these models lack neuronal cell characteristics and do not express endogenous tau, a microtubule‐associated protein implicated in AD pathophysiology. The Lund Human Mesencephalic (LUHMES) cell line is a promising in vitro neuronal cell model that expresses endogenous tau protein, allowing a deeper exploration of tau pathophysiology and the assessment of biotherapeutics.MethodThe data was collected through the PubMed database. Review articles were selected based on the following keywords: “in vitro tauopathy,” “LUHMES and neurodegenerative disease,” “LUHMES and tauopathy,” “LUHMES and Alzheimer’s disease,” and “neuron cell line.” Articles utilized for the purpose of this study were primary research articles published in academic journals between 2014 and 2023.ResultThe LUHMES cell line is used to model known tauopathies, like AD and progressive supranuclear palsy (PSP), yet few studies have harnessed their potential. One study revealed that PP242, a dual inhibitor of mTORC1 and mTORC2, could reverse fenazaquin‐induced tau aggregation in LUHMES cells. Another study discovered that activating PERK, an endoplasmic reticulum stress sensor, reduced tau phosphorylation and aggregation in LUHMES cells. As human‐origin cells with neuronal properties, LUHMES cells provide an essential model for studying tau pathology in AD. The insights gathered from these LUHMES‐based studies highlight the utility and relevance of these cells in propelling our understanding of tauopathy forward.ConclusionThe LUHMES are an important neuronal cell line that can be utilized for in vitro experiments to study tauopathy and evaluate biotherapeutics for their ability to modulate tau pathology. These cells have endogenous tau expression, a key characteristic differentiating this line from more commonly used cell lines, such as HEK293 and SH‐SY5Y. Significant progress toward evaluating effective treatments can be made by using the LUHMES cell line to study AD‐related tauopathy pathology.

Many human cell types are ciliated, including neural progenitors and differentiated neurons. Ciliopathies are characterized by defective cilia and comprise various disease states, including brain phenotypes, where the underlying biological pathways are largely unknown. Our understanding of neuronal cilia is rudimentary, and an easy-to-maintain, ciliated human neuronal cell model is absent. The Lund human mesencephalic (LUHMES) cell line is a ciliated neuronal cell line derived from human fetal mesencephalon. LUHMES cells can easily be maintained and differentiated into mature, functional neurons within one week. They have a single primary cilium as proliferating progenitor cells and as postmitotic, differentiating neurons. These developmental stages are completely separable within one day of culture condition change. The sonic hedgehog (SHH) signaling pathway is active in differentiating LUHMES neurons. RNA-sequencing timecourse analyses reveal molecular pathways and gene-regulatory networks critical for ciliogenesis and axon outgrowth at the interface between progenitor cell proliferation, polarization and neuronal differentiation. Gene expression dynamics of cultured LUHMES neurons faithfully mimic the corresponding in vivo dynamics of human fetal midbrain. In LUHMES cells, neuronal cilia biology can be investigated from proliferation through differentiation to mature neurons.

Lewy bodies (LBs), α-synuclein-enriched intracellular inclusions, are a hallmark of Parkinson's disease (PD) pathology, yet a cellular model for LB formation remains elusive. Recent evidence indicates that immune dysfunction may contribute to the development of PD. In this study, we found that induced pluripotent stem cell (iPSC)-derived human dopaminergic (DA) neurons form LB-like inclusions after treatment with α-synuclein preformed fibrils (PFFs) but only when coupled to a model of immune challenge (interferon-γ or interleukin-1β treatment) or when co-cultured with activated microglia-like cells. Exposure to interferon-γ impairs lysosome function in DA neurons, contributing to LB formation. The knockdown of LAMP2 or the knockout of GBA in conjunction with PFF administration is sufficient for inclusion formation. Finally, we observed that the LB-like inclusions in iPSC-derived DA neurons are membrane bound, suggesting that they are not limited to the cytoplasmic compartment but may be formed due to dysfunctions in autophagy. Together, these data indicate that immune-triggered lysosomal dysfunction may contribute to the development of PD pathology.

Sept4, a Component of Presynaptic Scaffold and Lewy Bodies, Is Required for the Suppression of α-Synuclein Neurotoxicity

Glucocerebrosidase (GBA1) pathogenic variants are strongly associated with Parkinson disease (PD); however, insufficient data exist on the prevalence of PD among patients with Gaucher disease type 1 (GD1) (biallelic pathogenic GBA1 variants). Also, penetrance estimates in patients with GD are lower than expected given their severely diminished enzymatic activity. We aimed to estimate the age-specific risk of PD in patients with GD1, overall and by GBA1 genotype. Participants were patients with GD1 in the International Collaborative Gaucher Group Gaucher Registry, a global GD database, as of February 2024. We longitudinally collected data on clinical diagnosis of PD and dementia with Lewy bodies (DLB) and report of motor (rest tremor, falls) and nonmotor (cognitive impairment, REM sleep behavior disorder, loss of sense of smell, autonomic dysfunction) signs/symptoms. In addition to a conservative physician-based PD and DLB diagnosis, we created a liberal definition of possible parkinsonian syndrome (pPS; ≥2 signs/symptoms, PD, or DLB) to test whether previous low penetrance estimates stem from underdiagnosis. Patients were classified as pPS at earliest of the following dates: PD diagnosis, DLB diagnosis, or report of second sign/symptom. We separately estimated age-specific prevalence of PD and pPS using Kaplan-Meier survival curves. Among 1,618 patients with GD1 (median age at last follow-up 47.8 years; 53% female), 51 were diagnosed with PD and 86 as pPS. The age-specific prevalence (95% CI) of PD and pPS was 4.0% (2.7-5.7) and 6.0% (4.5-7.9) at 60 years and 12.2% (8.6-17.0) and 22.9% (17.1-30.1) at 80 years, respectively. Patients with 2 mild pathogenic GBA1 variants had a qualitatively lower prevalence of PD and pPS vs patients with one mild variant. In this large cohort of 1,618 patients, approximately one-in-nine patients with GD1 were diagnosed with PD and more than one-in-five patients were diagnosed with PD/DLB or experienced movement disorder symptoms by 80 years. Most patients were from North America and Europe; generalizability to other regions is unknown. Our finding that most patients remain free of PD despite very low residual enzyme activity informs the hypothesis that acid ß-glucosidase levels directly predict risk of PD.

Parkinson’s disease (PD) is the most common age-related neurodegenerative movement disorder that affects 1-2% of the general population over the age of 65 and rising to 4-5% over 80 years of age. It is estimated that 6.3 million people worldwide have Parkinson’s disease and 1.2 million in the European Union (EU) alone. As the disease progresses, patients frequently develop a mask-like facial expression, festinating gait and cognitive impairment and the final clinical symptoms are muscle rigidity, bradykinesia, resting tremor and postural instability in addition to non-motor problems that result in a marked reduction in quality of life. The motor symptoms of PD arise from the progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc) leading to a deficiency of the neurotransmitter dopamine (DA) in the striatum. While dopamine replacement therapy is initially effective in treating the motor symptoms of PD, there is no known cure or neuroprotective therapy for this debilitating disease. Although the pathology underlying the disease is well defined, it is still unclear why DAergic neurons degenerate and die. PD is generally considered as a sporadic disease of unknown etiology, however in the last two decades it has become clear that heritability plays an important role in the pathogenesis of PD. Of the different genes associated with familial disease, mutations in two genes encoding for α-synuclein and leucine-rich repeat kinase 2 (LRRK2) proteins cause autosomal dominant PD. One of the neuropathological hallmarks of idiopathic and some familial forms of PD are Lewy bodies, which are intracytoplasmic inclusions that are enriched with fibrillar α- synuclein protein. Notably, LRRK2 mutation carriers with PD predominantly develop Lewy body pathology suggesting that LRRK2 may lie upstream of α-synuclein aggregation. These observations and other in vivo studies have suggested a common pathogenic pathway with LRRK2 potentially regulating the aggregation and neuronal toxicity of α-synuclein. However, the relationship between LRRK2 and α-synuclein in the mammalian brain is presently unclear and requires further evaluation, especially within midbrain dopaminergic neurons that degenerate in PD. The aim of this thesis work was to delineate a potential pathogenic interplay between these two PD-related gene products in mediating neurodegeneration in vivo. Understanding how and where the functional pathways of LRRK2 and α-synuclein converge will provide important insight into the common mechanisms underlying neurodegeneration in PD.[...]

The Role of NAC in Amyloidogenesis in Alzheimer's Disease

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the progressive degeneration of the dopaminergic nigrostriatal pathway and the abnormal appearance of intracellular inclusions named Lewy bodies (LBs). Over the past few years, the discovery of genes involved in hereditary forms of the disease led to new insights into the pathogenesis of PD. Mutations in the α-synuclein gene have been associated with autosomal dominant PD, and mutations in parkin with autosomal recessive-juvenile parkinsonism (AR-JP). α-synuclein has emerged as a key protein in the pathogenesis of PD, as it appears to be the major structural component of LBs and its accumulation seems to play a prominent role in sporadic PD. To date, genetic PD models based on α-synuclein conventional transgenesis in rodents have been unsuccessful in recapitulating the main features of the human pathology, especially the loss of nigral dopamine neurons. The lack of clear dopaminergic cellular degeneration in these transgenic animal models is likely to be attributed to insufficient expression levels of α-synuclein in the substantia nigra. Injection of viral vectors constitutes an alternative approach for the development of genetic models, as viral vectors allow high gene expression levels in a localized brain region and can be applied in mammalian species other than mice. In the present study, the viral-mediated expression of different α-synuclein forms (normal or mutated) was explored in the substantia nigra of rats. Following the full characterization of this new genetic model, several neuroprotective strategies based on the delivery of potential neuroprotective factors such as the neurotrophic factor GDNF (glial cell line-derived neurotrophic factor), the E3 ligase parkin or the chaperone Hsp104 were evaluated in this α-synuclein rat model. As HIV-1-derived lentiviral vectors can efficiently transduce neurons in the brain, these retroviral vectors were used to overexpress normal or mutated forms of α-synuclein in the substantia nigra of adult rats. In contrast to α-synuclein transgenic mice, the lentiviral-based model developed a progressive and selective loss of nigral dopaminergic neurons associated with a dopaminergic denervation of the striatum in animals expressing either wild-type or mutant forms of human α-synuclein. The neuronal degeneration correlated with the appearance of ?α-synuclein-positive inclusions. This viral-based model thus constitutes an excellent genetic model for testing molecules that can interfere with the neurodegenerative process induced by α-synuclein. Delivery of GDNF, a potent neuroprotective factor for the survival of dopaminergic neurons, is currently the most promising strategy for the treatment of PD. The neuroprotective properties of GDNF were therefore evaluated in the lentiviral-based model expressing mutated human α-synuclein. Although a robust expression of GDNF was observed in the whole nigrostriatal pathway due to retrograde and/or anterograde transport, nigral GDNF delivery did not prevent the α-synuclein-induced neurodegeneration. As loss of function of parkin leads to dopaminergic cell loss in Autosomal Recessive-Juvenile Parkinsonism (AR-JP) patients, parkin may represent a critical survival factor for dopaminergic neurons. The neuroprotective role of the E3 ubiquitin ligase parkin was assessed in the viral-based model overexpressing mutated human α-synuclein. Co-expression of parkin with α-synuclein prevented the nigrostriatal degeneration and increased the number of hyperphosphorylated α-synuclein inclusions. These results suggest that parkin may also play a key role in the formation of aggregates. Abnormal folding and aggregation of α-synuclein is now recognized as a critical issue in the pathology of PD. Chaperones can block or reverse the incorrect folding of misfolded proteins, and increase the clearance of aggregates. To enhance the elimination of aggregated α-synuclein and to understand the importance of inclusions in the α-synuclein pathogenesis, we expressed a protein disaggregase, the yeast chaperone Hsp104, in the lentiviral-based model of PD. This chaperone can facilitate the clearance of misfolded proteins from an aggregate state. Expression of Hsp104 reduced the dopaminergic cell loss induced by lentiviralmediated expression of PD-linked mutated α-synuclein. This neuroprotective effect correlated with a reduction in the formation of hyperphosphorylated α-synuclein inclusions, indicating that enhancing clearance of protein aggregates through the disaggregase activity of Hsp104 may constitute a novel therapeutic strategy for PD and other diseases caused by misfolded protein accumulation. The present thesis demonstrates that lentiviral vectors expressing human α-synuclein constitute a powerful and flexible tool to mimic the neuropathological features of PD. Although not effective in the lentiviral-based model of PD, GDNF may still represent a promising molecule to promote the sprouting of dopaminergic axons. On the contrary, the E3 ligase parkin and the chaperone Hsp104 have demonstrated potent neuroprotective properties against the accumulation of the toxic mutated α-synuclein. Therapeutic approaches aiming at increasing parkin or chaperone levels in the brain may therefore open new perspectives for the treatment of PD.

In this review I outline the arguments as to whether we should consider Parkinson disease one or more than one entity and discuss genetic findings from Mendelian and whole-genome association analysis in that context. I discuss what the demonstration of disease spread implies for our analysis of the genetic and epidemiologic risk factors for disease and outline the surprising fact that we now have genetically identified on the order of half our risk for developing the disease.

Parkinson's disease (PD) is a chronic neurodegenerative disorder affecting older individuals. There is inconsistent evidence about the prevalence and incidence of PD in China at present. The aim of the meta-analysis was to estimate the prevalence and incidence of PD and its relation to age, gender, and stage in China. The literature search was conducted using PubMed, EMBASE, Chinese Biological Medical Literature database (CBM), Chinese National Knowledge Infrastructure database (CNKI), Chinese Wanfang and Chongqing VIP database for studies investigating the prevalence and incidence of PD in China from the commencement of the database until August 2012; both English and Chinese publications were included. We estimated the prevalence and incidence of PD using meta-analysis. Thirteen eligible articles were collected. The results showed that the pooled prevalence and incidence of PD were 2 per 100,000 population and 797 per 100,000 person-years. A higher prevalence of PD was found in males than in females (OR1.29, 95% CI 1.05-1.57). The prevalence of PD increased with age: the highest was 1,663 per 100,000 in those aged 80 and older. The overall prevalence of PD is lower in China than in developed countries, but the incidence is higher than in some developed countries. Overall, the prevalence of PD appears to increase with age and there are sex differences evident in Chinese individuals.

Non-motor features of Parkinson's disease (PD) and dementia with Lewy bodies (DLB), such as auditory hallucinations (AH), contribute to disease burden but are not well understood. Systematic review and random-effects meta-analyses of studies reporting AH associated with PD or DLB. Prevalence of visual hallucinations (VH) in identified studies meeting eligibility criteria were included in meta-analyses, facilitating comparison with AH. Synthesis of qualitative descriptions of AH was performed. PubMed, Web of Science and Scopus databases were searched for primary journal articles, written in English, published from 1970 to 2017. Studies reporting AH prevalence in PD or DLB were screened using PRISMA methods. Searches identified 4542 unique studies for consideration, of which, 26 met inclusion criteria. AH pooled prevalence in PD was estimated to be 8.9% [95% confidence interval (CI) 5.3-14.5], while in DLB was estimated to be 30.8% (±23.4 to 39.3). Verbal hallucinations, perceived as originating outside the head, were the most common form of AH. Non-verbal AH were also common while musical AH were rare. VH were more prevalent, with an estimated pooled prevalence in PD of 28.2% (±19.1 to 39.5), while in DLB they were estimated to be 61.8% (±49.1 to 73.0). Meta-regression determined that the use of validated methodologies to identify hallucinations produced higher prevalence estimates. AH and VH present in a substantial proportion of PD and DLB cases, with VH reported more frequently in both conditions. Both AH and VH are more prevalent in DLB than PD. There is a need for standardised use of validated methods to detect and monitor hallucinations.

LRP10 in α-synucleinopathies

Lewy bodies (LBs) are pathological hallmarks of Parkinson disease (PD) but also occur in Alzheimer disease (AD) and dementia of LBs. Alpha-synuclein, the major component of LBs, is observed in the brain of Down syndrome (DS) patients with AD. Dyrk1A, a dual specificity tyrosine-regulated kinase (Dyrk) family member, is the mammalian ortholog of the Drosophila minibrain (Mnb) gene, essential for normal postembryonic neurogenesis. The Dyrk1A gene resides in the human chromosome 21q22.2 region, which is associated with DS anomalies, including mental retardation. In this study, we examined whether Dyrk1A interacts with alpha-synuclein and subsequently affects intracellular alpha-synuclein inclusion formation in immortalized hippocampal neuronal (H19-7) cells. Dyrk1A selectively binds to alpha-synuclein in transformed and primary neuronal cells. Alpha-synuclein overexpression, followed by basic fibroblast growth factor-induced neuronal differentiation, resulted in cell death. We observed that accompanying cell death was increased alpha-synuclein phosphorylation and intracytoplasmic aggregation. In addition, the transfection of kinase-inactive Dyrk1A or Dyrk1A small interfering RNA blocked alpha-synuclein phosphorylation and aggregate formation. In vitro kinase assay of anti-Dyrk1A immunocomplexes demonstrated that Dyrk1A could phosphorylate alpha-synuclein at Ser-87. Furthermore, aggregates formed by phosphorylated alpha-synuclein have a distinct morphology and are more neurotoxic compared with aggregates composed of unmodified wild type alpha-synuclein. These findings suggest alpha-synuclein inclusion formation regulated by Dyrk1A, potentially affecting neuronal cell viability.