Lewy Body-like Pathology and Loss of Dopaminergic Neurons in Midbrain Organoids Derived from Familial Parkinson's Disease Patient.

Progressive accumulation of α-Synuclein (αSyn) in Lewy bodies (LBs) and loss of dopaminergic (DA) neurons are the hallmark pathological features of Parkinson's disease (PD). Although currently available in vitro and in vivo models have provided crucial information about PD pathogenesis, the mechanistic link between the progressive accumulation of αSyn into LBs and the loss of DA neurons is still unclear. To address this, it is critical to model LB formation and DA neuron loss, the two key neuropathological aspects of PD, in a relevant in vitro system. In this study, we developed a human midbrain-like organoid (hMBO) model of PD. We demonstrated that hMBOs generated from induced pluripotent stem cells (hiPSCs), derived from a familial PD (fPD) patient carrying αSyn gene (SNCA) triplication accumulate pathological αSyn over time. These cytoplasmic inclusions spatially and morphologically resembled diverse stages of LB formation and were composed of key markers of LBs. Importantly, the progressive accumulation of pathological αSyn was paralleled by the loss of DA neurons and elevated apoptosis. The model developed in this study will complement the existing in vitro models of PD and will provide a unique platform to study the spatiotemporal events governing LB formation and their relation with neurodegeneration. Furthermore, this model will also be beneficial for in vitro screening and the development of therapeutic compounds.

Development of a novel pharmacophore model to screen specific inhibitors for the serine-threonine protein phosphatase calcineurin.

Calcineurin (CaN) is a calcium/calmodulin-dependent serine/threonine phosphatase with a crucial role in cellular homeostasis. It is also the target of the Food and Drug Administration (FDA) approved immunosuppressant drugs FK506 and cyclosporine A. Recent work from our group and others indicated that an uncontrolled increase in CaN activity causes synaptic dysfunction and neuronal death in various models of neurodegenerative diseases associated with calcium dysregulation. Furthermore, pharmacological normalization of CaN activity can prevent disease progression in animal models. However, none of the FDA-approved CaN inhibitors bind CaN directly, leading to adverse side effects. The development of direct CaN inhibitors is required to reduce off-target effects, but its highly conserved active site and similar mechanism of action with other protein serine/threonine phosphatases impose a significant challenge. In this work, we developed a novel pharmacophore model to screen for CaN-specific inhibitors. Then, we performed a virtual screen for molecules having the pharmacophore model. We also show that the molecules identified in this screen can inhibit CaN with a low micromolar IC50. Interestingly, the inhibitors identified from the screen do not inhibit phosphoprotein phosphatase 2A, a member of the serine/threonine phosphatase family that shares 43% sequence identity with the CaN active site. The pharmacophore model that we developed and validated in this work may help to accelerate the development of specific CaN inhibitors.

Extensive accumulation of misfolded protein aggregates during natural aging and senescence.

Accumulation of misfolded protein aggregates is a hallmark event in many age-related protein misfolding disorders, including some of the most prevalent and insidious neurodegenerative diseases. Misfolded protein aggregates produce progressive cell damage, organ dysfunction, and clinical changes, which are common also in natural aging. Thus, we hypothesized that aging is associated to the widespread and progressive misfolding and aggregation of many proteins in various tissues. In this study, we analyzed whether proteins misfold, aggregate, and accumulate during normal aging in three different biological systems, namely senescent cells, Caenorhabditis elegans, and mouse tissues collected at different times from youth to old age. Our results show a significant accumulation of misfolded protein aggregates in aged samples as compared to young materials. Indeed, aged samples have between 1.3 and 2.5-fold (depending on the biological system) higher amount of insoluble proteins than young samples. These insoluble proteins exhibit the typical characteristics of disease-associated aggregates, including insolubility in detergents, protease resistance, and staining with amyloid-binding dye as well as accumulation in aggresomes. We identified the main proteins accumulating in the aging brain using proteomic studies. These results show that the aged brain contain large amounts of misfolded and likely non-functional species of many proteins, whose soluble versions participate in cellular pathways that play fundamental roles in preserving basic functions, such as protein quality control, synapsis, and metabolism. Our findings reveal a putative role for protein misfolding and aggregation in aging.

Protocol for controlled cortical impact in human cerebral organoids to model traumatic brain injury.

Modeling traumatic brain injury (TBI) has been a challenge. Rodent and cellular models have provided relevant contributions despite their limitations. Here, we present a protocol for a TBI model based on the controlled cortical impact (CCI) performed on human cerebral organoids (COs), self-assembled 3D cultures that recapitulate features of the human brain. Here, we generate COs from iPSCs obtained from reprogrammed fibroblasts. For complete details on the use and execution of this protocol, please refer to Ramirez et al. (2021).

Modeling Traumatic Brain Injury in Human Cerebral Organoids.

Traumatic brain injury (TBI) is a head injury that disrupts the normal brain structure and function. TBI has been extensively studied using various in vitro and in vivo models. Most of the studies have been done with rodent models, which may respond differently to TBI than human nerve cells. Taking advantage of the recent development of cerebral organoids (COs) derived from human induced pluripotent stem cells (iPSCs), which resemble the architecture of specific human brain regions, here, we adapted the controlled cortical impact (CCI) model to induce TBI in human COs as a novel in vitro platform. To adapt the CCI procedure into COs, we have developed a phantom brain matrix, matching the mechanical characteristics of the brain, altogether with an empty mouse skull as a platform to allow the use of the stereotactic CCI equipment on COs. After the CCI procedure, COs were histologically prepared to evaluate neurons and astrocyte populations using the microtubule-associated protein 2 (MAP2) and the glial fibrillary acidic protein (GFAP). Moreover, a marker of metabolic response, the neuron-specific enolase (NSE), and cellular death using cleaved caspase 3 were also analyzed. Our results show that human COs recapitulate the primary pathological changes of TBI, including metabolic alterations related to neuronal damage, neuronal loss, and astrogliosis. This novel approach using human COs to model TBI in vitro holds great potential and opens new alternatives for understanding brain abnormalities produced by TBI, and for the development and testing of new therapeutic approaches.

Induced Pluripotent Stem Cell-Derived Dopaminergic Neurons from Familial Parkinson's Disease Patients Display α-Synuclein Pathology and Abnormal Mitochondrial Morphology.

Accumulation of α-synuclein (α-syn) into Lewy bodies (LBs) and mitochondrial abnormalities are the two cardinal pathobiological features of Parkinson's disease (PD), which are associated with the loss of dopaminergic neurons. Although α-syn accumulates in many different cellular and mouse models, these models generally lack LB features. Here, we generated midbrain dopaminergic (mDA) neuronal cultures from induced pluripotent stem cells (iPSCs) derived from familial PD (fPD) patients and healthy controls. We show that mDA neuronal cultures from fPD patients with A53T mutation and α-syn gene (SNCA) triplication display pathological α-syn deposits, which spatially and morphologically resemble LBs. Importantly, we did not find any apparent accumulation of pathological α-syn in mDA neuronal culture derived from a healthy donor. Furthermore, we show that there are morphological abnormalities in the mitochondrial network in mDA neuronal cultures from fPD patients. Consequently, these cells were more susceptible to mitochondrial damage compared with healthy donor-derived mDA neuronal cultures. Our results indicate that the iPSC-derived mDA neuronal culture platform can be used to investigate the spatiotemporal appearance of LBs, as well as their composition, architecture, and relationship with mitochondrial abnormalities.

Mitochondrial transplant to replenish damaged mitochondria: A novel therapeutic strategy for neurodegenerative diseases?

Neurodegenerative diseases are currently some of the most debilitating and incurable illness, including highly prevalent disorders, such as Alzheimer's and Parkinson's disease. Despite impressive advances in understanding the molecular basis of neurodegenerative diseases, several clinical trials have failed in identifying drugs that successfully delay or stop disease progression. New targets are likely necessary to successfully combat these devastating diseases. In this chapter, we review the evidence indicating that impairment in the cellular energy machinery in the form of mitochondrial damage and dysfunction may be at the root of neurodegeneration. We also propose that transplant of functional isolated mitochondria may overcome the energetic damage and delay the progression of neurodegenerative diseases.

Identification of Multicolor Fluorescent Probes for Heterogeneous Aß Deposits in Alzheimer's Disease.

Accumulation of amyloid-beta (Aß) into amyloid plaques and hyperphosphorylated tau into neurofibrillary tangles (NFTs) are pathological hallmarks of Alzheimer's disease (AD). There is a significant intra- and inter-individual variability in the morphology and conformation of Aß aggregates, which may account in part for the extensive clinical and pathophysiological heterogeneity observed in AD. In this study, we sought to identify an array of fluorescent dyes to specifically probe Aß aggregates, in an effort to address their diversity. We screened a small library of fluorescent probes and identified three benzothiazole-coumarin derivatives that stained both vascular and parenchymal Aß deposits in AD brain sections. The set of these three dyes allowed the visualization of Aß deposits in three different colors (blue, green and far-red). Importantly, two of these dyes specifically stained Aß deposits with no apparent staining of hyperphosphorylated tau or α-synuclein deposits. Furthermore, this set of dyes demonstrated differential interactions with distinct types of Aß deposits present in the same subject. Aß aggregate-specific dyes identified in this study have the potential to be further developed into Aß imaging probes for the diagnosis of AD. In addition, the far-red dye we identified in this study may serve as an imaging probe for small animal imaging of Aß pathology. Finally, these dyes in combination may help us advance our understanding of the relation between the various Aß deposits and the clinical diversity observed in AD.

Discriminating α-synuclein strains in Parkinson's disease and multiple system atrophy.

Synucleinopathies are neurodegenerative diseases that are associated with the misfolding and aggregation of α-synuclein, including Parkinson's disease, dementia with Lewy bodies and multiple system atrophy1. Clinically, it is challenging to differentiate Parkinson's disease and multiple system atrophy, especially at the early stages of disease2. Aggregates of α-synuclein in distinct synucleinopathies have been proposed to represent different conformational strains of α-synuclein that can self-propagate and spread from cell to cell3-6. Protein misfolding cyclic amplification (PMCA) is a technique that has previously been used to detect α-synuclein aggregates in samples of cerebrospinal fluid with high sensitivity and specificity7,8. Here we show that the α-synuclein-PMCA assay can discriminate between samples of cerebrospinal fluid from patients diagnosed with Parkinson's disease and samples from patients with multiple system atrophy, with an overall sensitivity of 95.4%. We used a combination of biochemical, biophysical and biological methods to analyse the product of α-synuclein-PMCA, and found that the characteristics of the α-synuclein aggregates in the cerebrospinal fluid could be used to readily distinguish between Parkinson's disease and multiple system atrophy. We also found that the properties of aggregates that were amplified from the cerebrospinal fluid were similar to those of aggregates that were amplified from the brain. These findings suggest that α-synuclein aggregates that are associated with Parkinson's disease and multiple system atrophy correspond to different conformational strains of α-synuclein, which can be amplified and detected by α-synuclein-PMCA. Our results may help to improve our understanding of the mechanism of α-synuclein misfolding and the structures of the aggregates that are implicated in different synucleinopathies, and may also enable the development of a biochemical assay to discriminate between Parkinson's disease and multiple system atrophy.

Induction of IAPP amyloid deposition and associated diabetic abnormalities by a prion-like mechanism.

Although a large proportion of patients with type 2 diabetes (T2D) accumulate misfolded aggregates composed of the islet amyloid polypeptide (IAPP), its role in the disease is unknown. Here, we show that pancreatic IAPP aggregates can promote the misfolding and aggregation of endogenous IAPP in islet cultures obtained from transgenic mouse or healthy human pancreas. Islet homogenates immunodepleted with anti-IAPP-specific antibodies were not able to induce IAPP aggregation. Importantly, intraperitoneal inoculation of pancreatic homogenates containing IAPP aggregates into transgenic mice expressing human IAPP dramatically accelerates IAPP amyloid deposition, which was accompanied by clinical abnormalities typical of T2D, including hyperglycemia, impaired glucose tolerance, and a substantial reduction on ß cell number and mass. Finally, induction of IAPP deposition and diabetic abnormalities were also induced in vivo by administration of IAPP aggregates prepared in vitro using pure, synthetic IAPP. Our findings suggest that some of the pathologic and clinical alterations of T2D might be transmissible through a similar mechanism by which prions propagate in prion diseases.

Prion-like characteristics of the bacterial protein Microcin E492.

Microcin E492 (Mcc) is a pore-forming bacteriotoxin. Mcc activity is inhibited at the stationary phase by formation of amyloid-like aggregates in the culture. Here we report that, in a similar manner as prions, Mcc naturally exists as two conformers: a ß-sheet-rich, protease-resistant, aggregated, inactive form (Mccia), and a soluble, protease-sensitive, active form (Mcca). The exogenous addition of culture medium containing Mccia or purified in vitro-generated Mccia into the culture induces the rapid and efficient conversion of Mcca into Mccia, which is maintained indefinitely after passaging, changing the bacterial phenotype. Mccia prion-like activity is conformation-dependent and could be reduced by immunodepleting Mccia. Interestingly, an internal region of Mcc shares sequence similarity with the central domain of the prion protein, which is key to the formation of mammalian prions. A synthetic peptide spanning this sequence forms amyloid-like fibrils in vitro and is capable of inducing the conversion of Mcca into Mccia in vivo, suggesting that this region corresponds to the prion domain of Mcc. Our findings suggest that Mcc is the first prokaryotic protein with prion properties which harnesses prion-like transmission to regulate protein function, suggesting that propagation of biological information using a prion-based conformational switch is an evolutionary conserved mechanism.

Prion-Like Protein Aggregates and Type 2 Diabetes.

Type 2 diabetes (T2D) is a highly prevalent metabolic disease characterized by chronic insulin resistance and ß-cell dysfunction and loss, leading to impaired insulin release and hyperglycemia. Although the mechanism responsible for ß-cell dysfunction and death is not completely understood, recent findings suggest that the accumulation of misfolded aggregates of the islet amyloid polypeptide (IAPP) in the islets of Langerhans may play an important role in pancreatic damage. Misfolding and aggregation of diverse proteins and their accumulation as amyloid in different organs is the hallmark feature in a group of chronic, degenerative diseases termed protein misfolding disorders (PMDs). PMDs include highly prevalent human illnesses such as Alzheimer's and Parkinson's disease, as well as more than 25 rarer disorders. Among them, prion diseases are unique because the pathology can be transmitted by a proteinaceous infectious agent, termed a prion, which induces disease by propagating protein misfolding and aggregation. This phenomenon has a striking resemblance to the process of protein misfolding and aggregation in all of the PMDs, suggesting that misfolded aggregates have an intrinsic potential to be transmissible. Indeed, recent studies have shown that the pathological hallmarks of various PMDs can be induced in vivo under experimental conditions by inoculating tissue extracts containing protein aggregates into animal models. In this review, we describe our current understanding of the molecular mechanism underlying the prion-like transmission of protein aggregates and its possible role in T2D.

Delaying aging in Caenorhabditis elegans with protein aggregation inhibitors.

Recent evidence suggests that during aging there is widespread accumulation of aggregated insoluble proteins, even in the absence of pathological conditions. Pharmacological manipulation of protein aggregation might be helpful to unveil the involvement of protein aggregates during aging, as well as to develop novel strategies to delay aging. Here we investigated the effect of known protein aggregation inhibitors on the lifespan and health-span of Caenorhabditis elegans. For this purpose, we selected various structurally diverse anti-aggregation compounds and screened them in liquid and solid medium for their ability to alter the rate of aging in vivo. Our results show that treatment of C. elegans with diverse aggregation inhibitors significantly increases the animal lifespan and health-span. These findings indicate that protein misfolding and aggregation may play an important role in cellular dysfunction during aging, opening a novel approach to increase longevity and enhance the quality of life during aging.

Development of a Fluorescent Quenching Based High Throughput Assay to Screen for Calcineurin Inhibitors.

Currently there is no effective treatment available for major neurodegenerative disorders associated to protein misfolding, including Alzheimer's and Parkinson's disease. One of most promising therapeutic approaches under development focuses on inhibiting the misfolding and aggregation pathway. However, it is likely that by the time clinical symptoms appear, there is a large accumulation of misfolded aggregates and a very substantial damage to the brain. Thus, it seems that at the clinical stage of the disease it is necessary also to develop strategies aiming to prevent the neuronal damage produced by already formed misfolded aggregates. Chronic activation of calcineurin (CaN), a type IIB phosphatase, has been implicated as a pivotal molecule connecting synaptic loss and neuronal damage to protein misfolding. The fact that the crystal structure of CaN is also well established makes it an ideal target for drug discovery. CaN activity assays for High Throughput Screening (HTS) reported so far are based on absorbance. In this article we report the development of a fluorescent quenching based CaN activity assay suitable for robotic screening of large chemical libraries to find novel inhibitors. The assay yielded a Z score of 0.84 with coefficient of variance ≤ 15%. Our results also show that this assay can be used to identify CaN inhibitors with a wide range of potencies.

Type 2 diabetes as a protein misfolding disease.

Type 2 diabetes (T2D) is a highly prevalent and chronic metabolic disorder. Recent evidence suggests that formation of toxic aggregates of the islet amyloid polypeptide (IAPP) might contribute to ß-cell dysfunction and disease. However, the mechanism of protein aggregation and associated toxicity remains unclear. Misfolding, aggregation, and accumulation of diverse proteins in various organs is the hallmark of the group of protein misfolding disorders (PMDs), including highly prevalent illnesses affecting the central nervous system (CNS) such as Alzheimer's disease (AD) and Parkinson's disease (PD). In this review we discuss the current understanding of the mechanisms implicated in the formation of protein aggregates in the endocrine pancreas and associated toxicity in the light of the long-standing knowledge from neurodegenerative disorders associated with protein misfolding.

Role of protein misfolding and proteostasis deficiency in protein misfolding diseases and aging.

The misfolding, aggregation, and tissue accumulation of proteins are common events in diverse chronic diseases, known as protein misfolding disorders. Many of these diseases are associated with aging, but the mechanism for this connection is unknown. Recent evidence has shown that the formation and accumulation of protein aggregates may be a process frequently occurring during normal aging, but it is unknown whether protein misfolding is a cause or a consequence of aging. To combat the formation of these misfolded aggregates cells have developed complex and complementary pathways aiming to maintain protein homeostasis. These protective pathways include the unfolded protein response, the ubiquitin proteasome system, autophagy, and the encapsulation of damaged proteins in aggresomes. In this paper we review the current knowledge on the role of protein misfolding in disease and aging as well as the implication of deficiencies in the proteostasis cellular pathways in these processes. It is likely that further understanding of the mechanisms involved in protein misfolding and the natural defense pathways may lead to novel strategies for treatment of age-dependent protein misfolding disorders and perhaps aging itself.

Kosmotropic anions promote conversion of recombinant prion protein into a PrPSc-like misfolded form.

Prions are self-propagating proteins involved in transmissible spongiform encephalopaties in mammals. An aberrant conformation with amyloid-like features of a cell surface protein, termed prion protein (PrP), is thought to be the essential component of the infectious particle, though accessory co-factor molecules such as lipids and nucleotides may be involved. The cellular co-factors and environmental conditions implicated in PrP misfolding are not completely understood. To address this issue, several studies have been done inducing misfolding of recombinant PrP (recPrP) into classical amyloid structures using partially denaturing conditions. In this work, we report that misfolding of recPrP into PrP(Sc)-like aggregates can be induced by simply incubating the protein in the presence of kosmotropic salts at concentrations that are known to retain or increase the stability of the protein. We used a simple experimental reaction (protein, buffer and salts) submitted to agitation/incubation cycles at physiological temperature and pH. The formation of protease resistant-recPrP was time and salt-concentration dependent and required the presence of kosmotropic anions such as F(-) or SO(4)(-2). The molecular weights of the protease resistant recPrP fragments are reminiscent of those found in degradation assays of bona fide PrP(Sc). The aggregates also exhibited PrP(Sc)-like ultrastructural features including rod-shape morphology under electron microscope, high beta-sheet content and thioflavin-T positive signal. The formation of recPrP aggregates with PrP(Sc) biochemical features under conditions closer to physiological in the absence of organic co-factor molecules provides a simple setup that may prove helpful to understand the molecular mechanism of PrP misfolding.

Role of calcineurin in neurodegeneration produced by misfolded proteins and endoplasmic reticulum stress.

A hallmark event in neurodegenerative diseases is the accumulation of misfolded aggregated proteins in the brain leading to neuronal dysfunction and disease. Compelling evidence suggests that misfolded proteins damage cells by inducing endoplasmic reticulum (ER) stress and alterations in calcium homeostasis. Changes in cytoplasmic calcium concentration lead to unbalances on several signaling pathways. Recent data suggest that calcium-mediated hyperactivation of calcineurin (CaN), a key phosphatase in the brain, triggers synaptic dysfunction and neuronal death, the two central events responsible for brain degeneration in neurodegenerative diseases. Therefore, blocking CaN hyper-activation might be a promising therapeutic strategy to prevent brain damage in neurodegenerative diseases.

Calcineurin inhibition at the clinical phase of prion disease reduces neurodegeneration, improves behavioral alterations and increases animal survival.

Prion diseases are fatal neurodegenerative disorders characterized by a long pre-symptomatic phase followed by rapid and progressive clinical phase. Although rare in humans, the unconventional infectious nature of the disease raises the potential for an epidemic. Unfortunately, no treatment is currently available. The hallmark event in prion diseases is the accumulation of a misfolded and infectious form of the prion protein (PrP(Sc)). Previous reports have shown that PrP(Sc) induces endoplasmic reticulum stress and changes in calcium homeostasis in the brain of affected individuals. In this study we show that the calcium-dependent phosphatase Calcineurin (CaN) is hyperactivated both in vitro and in vivo as a result of PrP(Sc) formation. CaN activation mediates prion-induced neurodegeneration, suggesting that inhibition of this phosphatase could be a target for therapy. To test this hypothesis, prion infected wild type mice were treated intra-peritoneally with the CaN inhibitor FK506 at the clinical phase of the disease. Treated animals exhibited reduced severity of the clinical abnormalities and increased survival time compared to vehicle treated controls. Treatment also led to a significant increase in the brain levels of the CaN downstream targets pCREB and pBAD, which paralleled the decrease of CaN activity. Importantly, we observed a lower degree of neurodegeneration in animals treated with the drug as revealed by a higher number of neurons and a lower quantity of degenerating nerve cells. These changes were not dependent on PrP(Sc) formation, since the protein accumulated in the brain to the same levels as in the untreated mice. Our findings contribute to an understanding of the mechanism of neurodegeneration in prion diseases and more importantly may provide a novel strategy for therapy that is beneficial at the clinical phase of the disease.

De novo generation of infectious prions in vitro produces a new disease phenotype.

Prions are the proteinaceous infectious agents responsible for Transmissible Spongiform Encephalopathies. Compelling evidence supports the hypothesis that prions are composed exclusively of a misfolded version of the prion protein (PrP(Sc)) that replicates in the body in the absence of nucleic acids by inducing the misfolding of the cellular prion protein (PrP(C)). The most common form of human prion disease is sporadic, which appears to have its origin in a low frequency event of spontaneous misfolding to generate the first PrP(Sc) particle that then propagates as in the infectious form of the disease. The main goal of this study was to mimic an early event in the etiology of sporadic disease by attempting de novo generation of infectious PrP(Sc)in vitro. For this purpose we analyzed in detail the possibility of spontaneous generation of PrP(Sc) by the protein misfolding cyclic amplification (PMCA) procedure. Under standard PMCA conditions, and taking precautions to avoid cross-contamination, de novo generation of PrP(Sc) was never observed, supporting the use of the technology for diagnostic applications. However, we report that PMCA can be modified to generate PrP(Sc) in the absence of pre-existing PrP(Sc) in different animal species at a low and variable rate. De novo generated PrP(Sc) was infectious when inoculated into wild type hamsters, producing a new disease phenotype with unique clinical, neuropathological and biochemical features. Our results represent additional evidence in support of the prion hypothesis and provide a simple model to study the mechanism of sporadic prion disease. The findings also suggest that prion diversity is not restricted to those currently known, and that likely new forms of infectious protein foldings may be produced, resulting in novel disease phenotypes.